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https://f1000research.com/articles/11-1530/v1	19 Dec 22	{ "type": "Research Article", "title": "Concatenated 16S rRNA sequence analysis improves bacterial taxonomy", "authors": [ "Bobby Paul" ], "abstract": "Background: Microscopic, biochemical, molecular, and computer-based approaches are extensively used to identify and classify bacterial populations. Advances in DNA sequencing and bioinformatics workflows have facilitated sophisticated genome-based methods for microbial taxonomy although sequencing of the 16S rRNA gene is widely employed to identify and classify the bacterial community as a cost-effective and single-gene approach. However, the 16S rRNA sequence-based species identification accuracy is limited by multiple copies of the gene and their higher sequence identity between closely related species. The availability of a large volume of bacterial whole-genome data provided an opportunity to develop comprehensive species-specific 16S rRNA reference libraries. Methods: The 16S rRNA copies were retrieved from the whole genomes in the complete stage at the Genome database. With defined rules, four 16S rRNA gene copy variants were concatenated to develop a species-specific reference library. The sequence similarity search was performed with a web-based BLAST program, and MEGA software was used to construct the phylogenetic tree. Results: Using this approach, species-specific 16S rRNA gene libraries were developed for four closely related Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae). Sequence similarity and phylogenetic analysis using concatenated 16S rRNA copies yielded better resolution than single gene copy approaches. Conclusions: The approach is very effective in classifying genetically related species and may reduce misclassification of bacterial species and genome assemblies.", "keywords": [ "bacterial nomenclature", "bacterial taxonomy", "concatenated phylogeny", "species-specific barcode reference library" ], "content": "Introduction\n\nThe 16S ribosomal RNA (16S rRNA) encoding region is extensively studied to identify and classify bacterial species. The 16S rRNA is a conserved component of the 30S small subunit of a prokaryotic ribosome. The gene is ~1500 base pair (bp) long, and it consists of nine variable regions (Reller et al. 2007; Sabat et al. 2017). For decades, the sequence of the 16S rRNA gene has been used as a potential molecular marker in culture-independent methods to identify and classify diverse bacterial communities (Clarridge, 2004; Johnson et al. 2019). The 16S rRNA sequences are currently being used as an accurate and rapid method to study bacterial evolution, phylogenetic relationships, populations in an environment, and quantification of abundant taxa (Vetrovsky and Baldrian, 2013; Srinivasan et al. 2015; Peker et al. 2019).\n\nDespite the wide range of applications, a few shortcomings limit the accuracy of results derived through the 16S rRNA sequence analysis. One such aspect is that the 16S rRNA gene has poor discriminatory power at the species level (Winand et al. 2020), and the copy number can vary from 1 to 15 or even more (Vetrovsky and Baldrian, 2013; Winand et al. 2020). The presence of multiple variable copies of this gene makes distinct data for a species. Hence, gene copy normalization (GCN) is necessary prior to sequence analysis. However, studies show that the GCN approach does not improve the 16S rRNA sequence analyses in real scenarios and suggests a comprehensive species-specific catalogue of gene copies (Starke et al. 2021). Secondly, the intra-genomic variations between the 16S rRNA gene copies were observed in several bacterial genome assemblies (Paul et al. 2019). Only a minority of the bacterial genomes harbor identical 16S rRNA gene copies, and sequence diversity increases with increasing copy numbers (Vetrovsky and Baldrian, 2013). Further, currently available 16S rRNA-based bioinformatics approaches are not always amenable to classify bacterium at the species level due to high inter-species sequence similarities (Peker et al. 2019; Deurenberg et al. 2017).\n\nA few other issues are also related to the sequencing and bioinformatics analysis of 16S rRNA gene regions. These include the purity of bacterial isolates, the quality of isolated DNA, and the possibility of chimeric molecules (Janda and Abbott, 2007; Church et al. 2020). Base-call errors can also mislead the sequence identity and phylogenetic inferences (Alachiotis et al. 2013). The other concerns on sequence-based analysis, comparison, and species identification include the number of base ambiguities processed, gaps generated during sequence comparison, and algorithm (local or global) used for the sequence alignment. The local alignment algorithm is extensively used for sequence similarity-based species identification. Several studies were conducted to identify the best variable region or combination of variable regions for bacterial classification, and a consensus remains to be implemented (Janda and Abbott, 2007; Johnson et al. 2019; Winand et al. 2020). Usage of misclassified sequence as a reference and improper bioinformatics workflows mislead the bacterial taxonomy. Further, the growth of bioinformatics and genetic data has placed genome-based microbial classification with researchers with little or no taxonomic experience, which may also mislead the bacterial taxonomy (Baltrus, 2016).\n\nA few bacterial identification systems with high resolution have been developed using the sequence of polymerase chain reaction (PCR) amplified ∼4.5 kb long 16S–23S rRNA regions (Benítez-Páez and Sanz, 2017; Sabat et al. 2017; Kerkhof et al. 2017). However, these approaches have a few limitations, such as the lack of reference 16S–23S rRNA sequence databases and complementary bioinformatics resources for reliable species identification (Sabat et al. 2017). The recent advancements in bioinformatics workflows (Winand et al. 2020; Schloss, 2020) and reference databases such as SILVA, EzBioCloud (Quast et al. 2013; Yoon, 2017) improved 16S rRNA-based bacterial taxonomy. However, a few recent genome-based studies highlighted the misclassification incidences in bacterial species and genome assemblies (Steven et al. 2017; Martínez-Romero, et al. 2018; Mateo-Estrada et al. 2019; Bagheri et al. 2020).\n\nNowadays, conventional and high throughput sequencers can amplify all the nine variable regions of the 16S rRNA gene. Although, many 16S rRNA-based bacterial identification studies lack a complete set of variable regions (Stackebrandt et al. 2021). The classical and high throughput sequencing technologies produce a large volume of whole-genome data. There is an urgent need to translate the genomic data for convenient microbiome analyses that ensure clinical practitioners can readily understand and quickly implement it (Church et al. 2020). Hence, the study intended to demonstrate a workflow to develop species-specific concatenated 16S rRNA reference libraries and its analysis. The species-specific libraries can yield better resolution in sequence similarity and phylogeny based bacterial classification approaches.\n\n\nMethods\n\nSequence alignment of 16S rRNA copies at the intra-genomic level shows a higher degree of variability in species belonging to the Firmicutes and Proteobacteria (Vetrovsky and Baldrian, 2013; Ibal et al. 2019). Hence, the study used eight 16S rRNA copies (Underlying data: Supplementary data 1 (Paul, 2022)) retrieved from the whole genome of Enterobacter asburiae strain ATCC 35953 (NZ_CP011863.1). The BLAST+ 2.13.0 (RRID:SCR_004870; Altschul et al. 1990) and Clustal Omega 1.2.4 (RRID:SCR_001591; Sievers et al. 2011) sequence alignment algorithms were used to estimate intra-genomic variability between the 16S rRNA gene copies. Phylogenetic relatedness between intra-genomic 16S rRNA copies were estimated using the Maximum Likelihood method (Tamura-Nei model; 500 bootstrap replicates) with MEGA software (version 11; RRID: SCR_000667; Kumar et al. 2018).\n\nPrevious studies have reported that several bacterial species share more than 99% sequence identity in the 16S rRNA encoding region. Hence, the 16S rRNA-based bacterial identification methods failed to discriminate such genetically related species (Deurenberg et al. 2017; Devanga-Ragupathi et al. 2018). It has been reported that Streptococcus mitis and Streptococcus pneumoniae are almost indistinguishable from each other based on the sequence similarity of their 16S rRNA regions (Reller et al. 2007; Lal et al. 2011). To develop species-specific barcode reference libraries, the study used 16S rRNA gene copies from whole-genome assemblies of four closely related species of Streptococcus (S. gordonii, S. mitis, S. oralis, and S. pneumoniae).\n\nMore than 463,000 whole-genome assemblies are currently available for prokaryotes at the Genome database (RRID:SCR_002474; https://www.ncbi.nlm.nih.gov/genome). Most microbial genomes were sequenced with high throughput sequencing technologies such as Illumina/Ion-Torrent (short read sequencing) and PacBio/Nanopre (long read sequencing). Further, many of these whole-genome assemblies are derived through a hybrid assembly of short and long read sequence data. The large volume of high throughput data can be effectively used to develop advanced genome-based approaches for microbial systematics. The genomic data is available in four assembly completion levels (contig, scaffold, chromosome, and complete). However, the study used only the genomes assemblies in the 'complete' stage to retrieve 16S rRNA gene copies.\n\nThe study retrieved full-length 16S rRNA gene copies from 16 genome assemblies belonging to four Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae). The detailed information on the dataset used to develop species-specific concatenated reference libraries is provided in Table 1 and the sequences are provided in the underlying data (Supplementary data 2 (Paul, 2022)). To maintain equal length, sequences were trimmed out beyond the universal primer pair fD1-5'-GAG TTT GAT CCT GGC TCA-3' and rP2-5'-ACG GCT AAC TTG TTA CGA CT-3' (Weisburg et al. 1991) for full-length 16S rDNA amplification. The study used MEGA 11 software to perform multiple sequence alignment and identify the intra-species parsimony informative (Parsim-info) variable sites. A species-specific barcode reference library covering entire Parsim-info variable sites was constructed by concatenating four 16S rRNA gene copies representing four different strains of a species. The rationale behind the selection of four copies for a species-specific barcode reference library is: (i) a maximum of four variations can be found on a single site, and (ii) earlier studies have shown that the mean 16S rRNA copies per genome is four (Vetrovsky and Baldrian, 2013).\n\nThe study analyzed a few cases to demonstrate the classical sequence similarity and phylogenetic analysis using concatenated species-specific 16S rRNA reference libraries. The study used nine Sanger sequenced 16S rRNA gene copies showing higher sequence similarity with multiple species of Streptococcus retrieved from the GenBank database (RRID:SCR_002760). The web based BLAST2 (version 2.13.0) program for aligning two or more sequences was used to estimate the maximum score, total alignment score, and sequence identity. A single copy of the 16S rRNA region derived through Sanger sequencing or retrieved from a whole-genome assembly can be considered as ‘Query sequence’. The concatenated species-specific reference libraries must be provided in the ‘Subject sequence’ section. To perform phylogenetic analysis, it is mandatory that the target sequence (length = n bp) has to be concatenated four times (length = 4 × n bp), appending next to the last base. Phylogenetic relatedness was estimated using the Maximum Likelihood method (Tamura-Nei model; 500 bootstrap replicates) with MEGA 11 software.\n\n\nResults\n\nHistorically, the 16S rRNA gene sequences were used to identify known and new bacterial species. However, this method is impacted by several factors such as amplification efficiency, poor discriminatory power at the species level, multiple polymorphic 16S rRNA gene copies, and improper bioinformatics workflows for the data analysis. The E. asburiae genome had eight 16S rRNA gene copies that showed a mean identity of 99.29% in sequence alignment using Clustal Omega (global alignment), whereas BLAST (local alignment) analysis resulted in an average of 99% identity between the copies (Table 2). Hence, the selection of an appropriate algorithm has a significant role in the estimation of percent identity, and a vital role in sequence-based species delineation. Global sequence alignment programs generally perform better for highly identical sequence pairs, and the algorithm considers all the bases for the estimation of sequence identity. The multiple sequence alignment showed 22 variable sites in 16S rRNA gene copies of the E. asburiae genome (Figure 1).\n\nPercent identity given below the diagonal line is calculated with Clustal Omega software (Mean identity: 99.29%) and those above the diagonal line were calculated with the BLASTN program (Mean identity: 99.00%). Genome coordinates of 16S rRNA copies: R1: 2686082–2687660 (1579 bp); R2: 3148265–3149814 (1550 bp); R3: 3313470–3315019 (1550 bp); R4: 3583942–3585481 (1540 bp); R5:3684745–3686294 (1550 bp); R6: 3771751–3773300 (1550 bp); R7: 3968538–3970087 (1550 bp); R8: 4647650–4649199 (1550 bp)\n\nThe evolutionary relationship between species is usually represented in a phylogenetic tree drawn using a single barcode gene, multiple genes, or whole genomes. However, bacterial species nomenclature is mainly designated based on the confidence obtained from the phylogenetic tree derived through single copy 16S rRNA analysis. To highlight how the intra-genomic 16S rRNA variations influence the species delineation, a phylogenetic tree was constructed using eight 16S rRNA gene copies of E. asburiae reference genome showing multiple nodes (Figure 2). The sequence similarity and phylogeny-based analysis indicate that the intra-genomic variations in 16S rRNA copies may mislead the bacterial taxonomy in single gene copy approaches.\n\nThe node label denotes the coordinate of 16S rRNA regions in the genome.\n\nThe study selected four Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae) to construct species-specific concatenated 16S rRNA reference libraries. The study used 16S rRNA copies retrieved from four whole genome assemblies in the ‘complete’ stage to construct a species-specific barcode library. Four copies of the 16S rRNA gene are required to construct the concatenated library for a species. The details of constructed species-specific libraries are listed in Table 1 and the sequence is provided in the underlying data (Supplementary data 3 (Paul, 2022)). The 16S rRNA sequence analysis shows 24 Parsim-info variable sites for S. oralis, 11 variations in S. mitis, seven variations in S. gordonii, and six variations found in S. pneumoniae. The observed intra-species Parsim-info variable sites are residing on both conserved and variable regions of the 16S rRNA gene.\n\nThe study used full-length 16S rRNA copies from four different strains to highlight the variations at the species level. However, a large volume of partial 16S rRNA sequences are available in the public genetic databases. In such cases, a species-specific concatenated 16S rRNA reference library can be developed with partial sequences. Intra-species variation on 16S rRNA gene copies influences the sequence based bacterial taxonomy. Hence, the concatenated 16S rRNA approach yields better resolution than single copy analysis in classical sequence similarity and phylogeny based species identification approaches.\n\nThe study compared nine 16S rRNA sequences representing Streptococcus species (Table 3) with species-specific concatenated reference libraries. Concatenated sequence analysis gives better resolution in sequence similarity search and phylogenetic analysis. The sequence accession numbers GU470907.1 and KF933785.1 classified as S. mitis showed a higher maximum and total alignment score with S. oralis than S. mitis (Table 3). Whereas the sequence (OM368574.1; classified as S. mitis) showed a higher sequence alignment score with S. pneumoniae. Figure 3A shows a maximum likelihood tree of the nine 16S rRNA gene sequences with four concatenated species-specific reference libraries. The concatenated GU470907.1 and KF933785.1 sequences showed a phylogenetic relationship with S. oralis and sequence OM368574.1 was genetically related to S. pneumoniae. These results indicate that the species-specific concatenated 16S rRNA reference libraries have great potential in the taxonomic classification. Hence, the study suggests the usage of concatenated variable 16S rRNA copies for sequence similarity and phylogeny-based species identification. A species-specific reference library with concatenated 16S rRNA gene copies provides better resolution in phylogenetic analysis than the single copy inference.\n\nA) Phylogenetic analysis of randomly selected 16S rRNA sequences classified as Streptococcus species.\n\nB) Concatenated 16S rRNA phylogeny of Streptococcus mitis sequence (Accession Number GU470907.1) showed 100% identity with Streptococcus oralis genome (Accession Number CP034442.1) in a BLAST based sequence similarity search. The node name highlighted in shapes (●, ■, ▲, ◆) represents the four species-specific reference libraries.\n\n\nDiscussion\n\nSequencing and analysis of the 16S rRNA encoding region is considered a conventional and robust method for identifying and classifying the bacterial species. The barcode gene is widely used in sequence similarity, phylogeny, and metagenome-based species identification. However, the accuracy of bacterial taxonomy based on 16S rRNA barcode regions is limited by the intra-genomic heterogeneity of multiple 16S rRNA gene copies and significant sequence identity of this gene between closely related taxa. Further, discrimination of closely related species identification through sequences of the 16S rRNA gene is a challenge, and it may lead to species misidentification (Boudewijns et al. 2006; Church et al. 2020). About 15% of the genomes have only a single copy of the 16S rRNA gene, and only a minority of bacterial genomes harbour identical 16S rRNA gene copies (Vetrovsky and Baldrian, 2013). The 16S rRNA gene copies can vary from 1 to 15 in a genome, and the copy number of variations is taxon specific (Vetrovsky and Baldrian, 2013). Sequence diversity increases with the increasing 16S rRNA copy numbers. The 16S rRNA sequence variation can even be found at intra-genomic level or in different strains of a species as well. Amplification of limited number of variable regions cannot achieve the taxonomic resolution achieved by sequencing the entire gene (Johnson et al. 2019). Usage of misclassified 16S rRNA sequences as a reference and inappropriate bioinformatics workflows also mislead the taxonomic assignment. To overcome these challenges, it is important to translate high throughput microbial genomic data into meaningful, actionable information that clinicians can readily understand and quickly implement for bacterial identification. Hence, the study intended to develop a species-specific catalogue of concatenated 16S rRNA gene copies that can yield better inference in sequence similarity and phylogenetic analysis.\n\nSeveral bioinformatics resources are extensively used for the 16S rRNA sequence analysis and bacterial identification. However, several researchers report the sequence similarity derived through a local alignment algorithm. Earlier reports have suggested that the species belonging to the taxa Gammaproteobacteria show higher intra-species variability (Vetrovsky and Baldrian, 2013). Hence, the study estimated the percent identity of intra-genomic 16S rRNA gene copies of Enterobacter asburiae using local and global alignment algorithms. The reference genome of E. asburiae has eight 16S rRNA gene copies in its genome. The BLAST and Clustal sequence alignment algorithms yielded marginally varying results for the intra-genomic 16S rRNA gene copies. Local alignment algorithms may not consider base mismatches at the sequence ends for calculating percent identity, while global alignment algorithms consider entire bases. Therefore, global sequence alignment is best for estimating intra and inter-species identity for single gene copies. However, BLAST can calculate the total alignment score with multiple paralogue regions. Hence, web-based BLAST2 is suggested for estimating the sequence similarity using concatenated barcode reference libraries.\n\nThe GenBank (Leray et al. 2019) and NCBI 16S RefSeq database for bacteria (Winand et al. 2020) are reliable for species-level identification and classification. However, few earlier studies have highlighted the misclassification of species and genome assemblies in public genetic databases (Parks et al. 2018; Varghese et al. 2015). For example, the 16S rRNA sequence accession number (Ac. No.) LT707617.1 shows the organism as Streptococcus mitis. Conventional BLAST-based sequence similarity search shows the highest identity of 99.60% with S. mitis 16S rRNA sequence (Ac. No. AB002520.1). However, the 16S rRNA sequence (Ac. No. LT707617.1) did not show significant similarity with other 16S rRNA reference sequences available for S. mitis. Further, the sequence also shows 99.44% identity with reference 16S rRNA sequences of S. gordonii. Hence, the study performed a sequence alignment of the sequence (Acc. No. LT707617.1) against species-specific concatenated 16S rRNA reference libraries for S. gordonii (S.gordonii-Ref-I), and S. mitis (S.mitis-Ref-I). The alignment resulted in a significant identity of 99.44% with S.gordonii-Ref-I (2279 maximum and 9041 total alignment score) than S.mitis-Ref-I (97.13% identity with 2119 maximum and 8449 total alignment score). Single copy BLAST results may show only a minor fraction of the difference in percent identity and maximum or total alignment score for closely related species. However, sequence similarity estimation using species-specific concatenated reference libraries shows marginal difference in total alignment score, as it is aligned against four copies. Hence, 16S rRNA analysis with a species-specific concatenated barcode reference library will give better accuracy for bacterial classification than approaches using a single copy.\n\nSeveral 16S rRNA sequences show 100% identity with multiple species, which is the major challenge in sequence-based species identification. For example, the 16S rRNA sequence from S. mitis (Ac. No. GU470907.1; 1522 bp) shares 100% identity with the 16S rRNA gene from S. oralis strain ATCC 35037 genome (Ac. No. CP034442.1). Hence, the sequence (GU470907.1) aligned against the species-specific concatenated reference libraries for S. oralis (S.oralis-Ref-I), and S. mitis (S.mitis-Ref-I). The result showed 100% identity with S. oralis (2787 maximum and 10936 total alignment score), and 99.14% identity with S. mitis (2715 maximum and 10796 total alignment score). Further, a phylogenetic tree of GU470907.1 (1509 × 4 = 6036 bp) with reference libraries S.mitis-Ref-I, and S.oralis-Ref-I was plotted. The maximum likelihood-based phylogenetic tree showed that the S. mitis (GU470907.1) sequence is more closely related to S. oralis than S. mitis (Figure 3B). Concatenated 16S rRNA-based estimation of sequence similarity and a phylogenetic inference provides better resolution than single-gene approaches. These results show that concatenated 16S rRNA approach is very effective in discriminating even genetically related bacterial species. Further, other studies also highlighted that the phylogenetic tree inferred from vertically inherited protein sequence concatenation provided higher resolution than those obtained from a single copy (Ciccarelli et al. 2006; Thiergart et al. 2014).\n\nRecent phylogenetic studies using concatenated multi-gene sequence data highlighted the importance of incorporating variation in gene histories, which will improve the traditional phylogenetic inferences (Devulder et al. 2005; Johnston et al. 2019). Further, one type of analysis should not be relied upon, instead, and to a certain extent, integrated bioinformatics approaches can avoid misclassification. As a cost-effective approach, the study combined substantial variations in 16S rRNA gene copies from a species to examine the performance of the single gene concatenation approach. Analyses using a concatenated 16S rRNA gene approach have some advantages: (i) the gene is present in all the bacterial species, (ii) the gene is weakly affected by horizontal gene transfer, (iii) the approach is very cost-effective, (iv) there is a large volume of reference genomic data available for several bacterial species, (v) it is effective in discriminating closely related bacterial species, (vi) the analyses can be performed in a computer with minimum configuration, and (vii) the analyses can be employed with available tools for sequence similarity and molecular phylogeny.\n\n\nConclusions\n\nThe concatenated 16S rRNA analyses drew the following suggestions:\n\n• Full-length 16S rRNA gene amplification provides better accuracy than inference from a partial gene with a limited number of variable regions.\n\n• Prior to the analysis, trim the bases beyond the primer ends and correct the base-call errors, which will avoid several mismatches in the sequence alignment.\n\n• Estimation of mean 16S rRNA identity at the intra-species level helps to classify the species having a higher degree of intra-genomic 16S rRNA heterogeneity.\n\n• Use full-length 16S rRNA gene copies from whole-genome assemblies (in 'complete' stage) rather than partial sequences available from the public genetic databases to construct species-specific concatenated 16S rRNA libraries and further downstream analysis.\n\n• Distinct four 16S rRNA gene copies cover all the Parsim-Info variable sites and can be used to construct a concatenated species-specific reference library.\n\n• The total alignment score can be considered if the query sequence shows more or less the same percent identity with multiple species.\n\n• Do not rely only on sequence similarity; make a final decision based on the phylogenetic inference.", "appendix": "Data availability\n\nZenodo: Underlying data for ‘Concatenated 16S rRNA sequence analysis improves bacterial taxonomy’. https://www.doi.org/10.5281/zenodo.7384708 (Paul, 2022)\n\nThis project contains the following underlying data:\n\n• Supplementary data 1: The 16S rRNA copies retrieved from the whole genome of Enterobacter asburiae strain ATCC 35953.\n\n• Supplementary data 2: Full-length 16S rRNA gene copies retrieved from 16 genome assemblies belonging to four Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae).\n\n• Supplementary data 3: Species-specific concatenated 16S rRNA libraries constructed for four Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\nGenBank: Streptococcus gordonii strain FDAARGOS 1454 chromosome, complete genome. Accession number CP077224.1. https://www.ncbi.nlm.nih.gov/nuccore/CP077224.1\n\nGenBank: Streptococcus gordonii strain NCTC7869, chromosome 1, complete genome. Accession number LR134291.1. https://www.ncbi.nlm.nih.gov/nuccore/LR134291.1\n\nGenBank: Streptococcus gordonii strain KCOM 1506 (=ChDC B679), complete genome. Accession number CP012648.1. https://www.ncbi.nlm.nih.gov/nuccore/CP012648.1\n\nGenBank: Streptococcus gordonii strain NCTC9124, chromosome 1, complete genome. Accession number LR594041.1. https://www.ncbi.nlm.nih.gov/nuccore/LR594041.1\n\nGenBank: Streptococcus mitis B6, complete genome. Accession number NC_013853.1. https://www.ncbi.nlm.nih.gov/nuccore/NC_013853.1\n\nGenBank: Streptococcus mitis strain KCOM 1350 (= ChDC B183), complete genome. Accession number CP012646.1. https://www.ncbi.nlm.nih.gov/nuccore/CP012646.1\n\nGenBank: Streptococcus mitis strain SVGS_061 chromosome, complete genome. Accession number CP014326.1. https://www.ncbi.nlm.nih.gov/nuccore/CP014326.1\n\nGenBank: Streptococcus mitis NCTC 12261 chromosome, complete genome. Accession number CP028414.1. https://www.ncbi.nlm.nih.gov/nuccore/CP028414.1\n\nGenBank: Streptococcus oralis strain NCTC11427, chromosome 1, complete genome. Accession number LR134336.1. https://www.ncbi.nlm.nih.gov/nuccore/LR134336.1\n\nGenBank: Streptococcus oralis strain 34 chromosome, complete genome. Accession number CP079724.1. https://www.ncbi.nlm.nih.gov/nuccore/CP079724.1\n\nGenBank: Streptococcus oralis strain FDAARGOS_886 chromosome, complete genome. Accession number CP065706.1. https://www.ncbi.nlm.nih.gov/nuccore/CP065706.1\n\nGenBank: Streptococcus oralis subsp. dentisani strain F0392 chromosome, complete genome. Accession number CP034442.1. https://www.ncbi.nlm.nih.gov/nuccore/CP034442.1\n\nGenBank: Streptococcus pneumoniae strain 475 chromosome, complete genome. Accession number CP046355.1. https://www.ncbi.nlm.nih.gov/nuccore/CP046355.1\n\nGenBank: Streptococcus pneumoniae NU83127 DNA, complete genome. Accession number AP018936.1. https://www.ncbi.nlm.nih.gov/nuccore/AP018936.1\n\nGenBank: Streptococcus pneumoniae NCTC7465, chromosome 1, complete genome. Accession number LN831051.1. https://www.ncbi.nlm.nih.gov/nuccore/LN831051.1\n\nGenBank: Streptococcus pneumoniae strain 6A-10 chromosome, complete genome. Accession number CP053210.1. https://www.ncbi.nlm.nih.gov/nuccore/CP053210.1\n\nGenBank: Streptococcus mitis strain 127R, partial 16S rRNA gene. Accession number AJ295848.1. https://www.ncbi.nlm.nih.gov/nuccore/AJ295848.1\n\nGenBank: Streptococcus mitis clone 2C4, 16S rRNA gene. Accession number AM157428.1. https://www.ncbi.nlm.nih.gov/nuccore/AM157428.1\n\nGenBank: Streptococcus mitis strain NS51, partial 16S rRNA gene. Accession number NR_028664.1. https://www.ncbi.nlm.nih.gov/nuccore/NR_028664.1\n\nGenBank: Streptococcus mitis bv. 2 strain F0392, partial 16S rRNA gene. Accession number GU470907.1. https://www.ncbi.nlm.nih.gov/nuccore/GU470907.1\n\nGenBank: Streptococcus mitis strain ChDC B553, partial 16S rRNA gene. Accession number KF933785. https://www.ncbi.nlm.nih.gov/nuccore/KF933785.1\n\nGenBank: Streptococcus mitis strain FC6528, partial 16S rRNA gene. Accession number OM368574.1. https://www.ncbi.nlm.nih.gov/nuccore/OM368574.1\n\nGenBank: Streptococcus pneumoniae strain FC6532, partial 16S rRNA gene. Accession number OM368578.1. https://www.ncbi.nlm.nih.gov/nuccore/OM368578.1\n\nGenBank: Streptococcus pneumoniae clone 4V4, 16S rRNA gene. Accession number AM157442. https://www.ncbi.nlm.nih.gov/nuccore/AM157442.1\n\nGenBank: Streptococcus oralis subsp. dentisani strain 7747, partial 16S rRNA gene. Accession number NR_117719. https://www.ncbi.nlm.nih.gov/nuccore/NR_117719.1\n\nGenBank: Enterobacter asburiae strain ATCC 35953 chromosome, complete genome. Accession number NZ_CP011863. https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP011863.1\n\nGenBank: Streptococcus mitis strain HAC11, isolate #11, partial 16S rRNA gene. Accession number LT707617. https://www.ncbi.nlm.nih.gov/nuccore/LT707617.1\n\nGenBank: Streptococcus mitis strain NCTC 3165, MAFF 911479, 16S rRNA gene. Accession number AB002520.1. https://www.ncbi.nlm.nih.gov/nuccore/AB002520.1\n\n\nAcknowledgements\n\nThe author would like to thank DST-FIST, the Government of India, TIFAC-CORE in Pharmacogenomics and Manipal Academy of Higher Education (MAHE), Manipal for the support and facilities provided.\n\n\nReferences\n\nAlachiotis N, Vogiatzi E, Pavlidis P, et al.: Chromatogate: a tool for detecting base mis-calls in multiple sequence alignments by semi-automatic chromatogram inspection. Comput. Struct. Biotechnol. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSteven B, Hesse C, Soghigian J, et al.: Simulated rRNA/DNA ratios show potential to misclassify active populations as dormant. Appl. Environ. Microbiol. 2017; 83: e00696–e00617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThiergart T, Landan G, Martin WF: Concatenated alignments and the case of the disappearing tree. BMC Evol. Biol. 2014; 14: 212–266. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVarghese NJ, Mukherjee S, Ivanova N, et al.: Microbial species delineation using whole genome sequences. Nucleic Acids Res. 2015; 43: 6761–6771. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVetrovsky T, Baldrian P: The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses. PLoS One. 2013; 8: e57923. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "158444", "date": "10 Feb 2023", "name": "Siddaramappa Shivakumara", "expertise": [ "Reviewer Expertise Bacterial Genomics", "Bacterial Taxonomy", "Comparative Genomics", "Pathogenomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “Concatenated 16S rRNA sequence analysis improves bacterial taxonomy [version 1]” by Bobby Paul is generally well written and reports interesting results. The methods are appropriate and the analyses meet the quality standards.\nHowever, there are some concerns about the clarity and presentation of results. Major revisions in the text are required to improve clarity and comprehensibility. My suggestions/critiques for revising the text in different sections of the manuscript are provided. Author’s attention has also been drawn to technical concerns, which need to be carefully addressed.\nThe Discussion section is rather lengthy; it should be shortened and include only relevant results/studies (present and previous). I would be happy to provide detailed critiques on the Discussion section after the manuscript goes through the first round of revision.\nIs the work clearly and accurately presented and does it cite the current literature? Some statements/results are missing citations and references. These have been highlighted and need to be revised.\nAre the conclusions drawn adequately supported by the results? Some conclusions are unsubstantiated, and require relevant data and proper interpretation. These have been highlighted and need to be revised.\n\nIntroduction\n\"The 16S ribosomal RNA (16S rRNA) encoding region is extensively studied to identify and classify bacterial species. The 16S rRNA is a conserved component of the 30S small subunit of a prokaryotic ribosome\" Please revise this as \"The DNA region encoding the 16S ribosomal RNA (16S rRNA) is extensively studied, and used to identify and classify bacterial species. The 16S rRNA is a conserved component of the small subunit (30S) of the prokaryotic ribosome\".\nThe gene is ~1500 base pair (bp) long, and it consists of nine variable regions. Please revise this as \"The gene encoding the 16S rRNA is base pair (bp) long, and it consists of nine variable regions\"\n\"For decades, the sequence of the 16S rRNA gene has been used as a potential molecular marker in culture-independent methods to identify and classify diverse bacterial communities (Clarridge, 2004; Johnson et al. 2019)\" Please revise this as \"The sequence of the 16S rRNA gene has been extensively used as a molecular marker in culture-independent methods to identify and classify diverse bacterial communities\"\n\"The 16S rRNA sequences are currently being used as an accurate and rapid method to study bacterial evolution, phylogenetic relationships, populations in an environment, and quantification of abundant taxa\" Please revise this as \"Bacterial 16S rRNA sequences are currently being used to study the evolution, phylogenetic relationships, and environmental abundanceof various taxa\".\n\"Despite the wide range of applications, a few shortcomings limit the accuracy of results derived through the 16S rRNA sequence analysis. One such aspect is that the 16S rRNA gene has poor discriminatory power at the species level (Winand et al. 2020), and the copy number can vary from 1 to 15 or even more (Vetrovsky and Baldrian, 2013; Winand et al. 2020).\" Please revise this as \"Although 16S rRNA sequence analyses are the mainstay of taxonomic studies of bacteria, there are some limitations. For example, the 16S rRNA gene has poor discriminatory power at the species level (Winand et al. 2020), and the copy number is highly variable (Vetrovsky and Baldrian, 2013; Winand et al. 2020). \".\n\n\"The presence of multiple variable copies of this gene makes distinct data for a species.\" The above statement is a little confusing, and should be revised. Their presence in itself may not \"make distinct data\".\n\"Hence, gene copy normalization (GCN) is necessary prior to sequence analysis.\" Please revise this as \"Therefore, gene copy normalization (GCN) may be necessary prior to sequence analysis.\n\"However, studies show that the GCN approach does not improve the 16S rRNA sequence analyses in real scenarios and suggests a comprehensive species-specific catalogue of gene copies (Starke et al. 2021)\". Please revise this as \"However, GCN may not improve the 16S rRNA sequence analyses in all scenarios, and comprehensive, species-specific catalogues of 16S rRNA gene copies may be necessary (Starke et al. 2021).\n\"Secondly, the intra-genomic variations between the 16S rRNA gene copies were observed in several bacterial genome assemblies (Paul et al. 2019).\" Please revise this as \"Furthermore, intra-species variations in the number of copies of the16S rRNA gene were observed in several bacterial genome assemblies\"\n\"Only a minority of the bacterial genomes harbor identical 16S rRNA gene copies, and sequence diversity increases with increasing copy numbers (Vetrovsky and Baldrian, 2013). \" Please revise this as \"Only a few bacterial species contain identical 16S rRNA gene copies, and sequence diversity increases with increasing copy numbers of 16S rRNA genes\".\n\"Further, currently available 16S rRNA-based bioinformatics approaches are not always amenable to classify bacterium at the species level due to high inter-species sequence similarities (Peker et al. 2019; Deurenberg et al. 2017).\" Please revise this as \"The high levels of similiarty of the 16S rRNA genes across some bacterial species poses a major challege for taxonomic studies using bioinformatics methods\".\n“A few other issues are also related to the sequencing and bioinformatics analysis of 16S rRNA gene regions. These include the purity of bacterial isolates, the quality of isolated DNA, and the possibility of chimeric molecules (Janda and Abbott, 2007; Church et al. 2020)” Please revise this as Factors such as purity of bacterial cultures, quality of the purified DNA samples, and potential DNA chimeras should be carefully considered while sequencing and analysis of 16S rRNA genes.\n“Base-call errors can also mislead the sequence identity and phylogenetic inferences (Alachiotis et al. 2013). Please revise this as “Sequencing errors can lead to misidentification of bacteria and phylogenetic anomalies”.\n“The other concerns on sequence-based analysis, comparison, and species identification include the number of base ambiguities processed, gaps generated during sequence comparison, and algorithm (local or global) used for the sequence alignment.” Please revise this as “Other concerns include sequence ambiguities, gaps generated during DNA sequencing and sequence comparisons, and choosing the appropriate algorithm (local or global) for sequence alignment.”.”\n“The local alignment algorithm is extensively used for sequence similarity-based species identification. Several studies were conducted to identify the best variable region or combination of variable regions for bacterial classification, and a consensus remains to be implemented (Janda and Abbott, 2007; Johnson et al. 2019; Winand et al. 2020).” Please revise this as “Since the local alignment algorithm is extensively used for sequence similarity-based comparisons, it is important to carefully consider whether a single variable region or a combination of variable regions of the 16S rRNA gene would be ideal for bacterial classification.”\n“Usage of misclassified sequence as a reference and improper bioinformatics workflows mislead the bacterial taxonomy. Please revise this as “Using erroneous 16S rRNA sequences as references and improper bioinformatics workflows can mislead bacterial identification”.\n\n\"Further, the growth of bioinformatics and genetic data has placed genome-based microbial classification with researchers with little or no taxonomic experience, which may also mislead the bacterial taxonomy (Baltrus, 2016)”. Kindly revise the above statement because it is too complex and confusing.\n“A few bacterial identification systems with high resolution have been developed using the sequence of polymerase chain reaction (PCR) amplified ∼4.5 kb long 16S–23S rRNA regions (Benítez-Páez and Sanz, 2017; Sabat et al. 2017; Kerkhof et al. 2017).” Please revise this as “Other methods for bacterial identification include the sequencing and analysis of the polymerase chain reaction (PCR) amplified ∼4.5 kb 16S–23S rRNA regions (Benítez-Páez and Sanz, 2017; Sabat et al. 2017; Kerkhof et al. 2017).”\n“However, these approaches have a few limitations, such as the lack of reference 16S–23S rRNA sequence databases and complementary bioinformatics resources for reliable species identification (Sabat et al. 2017)”. Please revise this as “However, the 16S–23S rRNA sequence-based method is less practical due to the lack of appropriate reference sequence databases and reliable tools/methods for sequence analysis”\n“The recent advancements in bioinformatics workflows (Winand et al. 2020; Schloss, 2020) and reference databases such as SILVA, EzBioCloud (Quast et al. 2013; Yoon, 2017) improved 16S rRNA-based bacterial taxonomy. However, a few recent genome-based studies highlighted the misclassification incidences in bacterial species and genome assemblies (Steven et al. 2017; Martínez-Romero, et al. 2018; Mateo-Estrada et al. 2019; Bagheri et al. 2020)”. The first part here talks about 16S rRNA-based bacterial taxonomy, and the second part talks about genome assemblies. It is not clear what the connection is. Kindly revise these two sentences. A suggestion for revision is shown below (although even this suggestion fails to convey the connection): “Although improvements in reference databases (such as SILVA and EzBioCloud) and bioinformatics workflows have facilitated 16S rRNA-based bacterial taxonomy, recent genome-based studies have indicated that incidences of misclassification of bacterial species and erroneous genome assemblies.”\n“Nowadays, conventional and high throughput sequencers can amplify all the nine variable regions of the 16S rRNA gene”. Please revise as “The entire 16S rRNA gene (~1500 bp) can be amplified and sequenced using the conventional or high throughput sequencing methods”.\n“Although, many 16S rRNA-based bacterial identification studies lack a complete set of variable regions (Stackebrandt et al. 2021)”. Please revise as “However, many 16S rRNA sequence-based bacterial identification studies do not seem to include all of these nine variable regions (Stackebrandt et al. 2021)”.\n“The classical and high throughput sequencing technologies produce a large volume of whole-genome data. There is an urgent need to translate the genomic data for convenient microbiome analyses that ensure clinical practitioners can readily understand and quickly implement it (Church et al. 2020)”. Please revise as “Due to the large volume of whole-genome data that is being produced by high throughput sequencing technologies, there is an urgent need to develop methods for analyzing this data for accurate identification of bacteria. It is also important to envisage that clinical practitioners would be able to understand these methods and implement them quickly (Church et al. 2020)”.\n“Hence, the study intended to demonstrate a workflow to develop species-specific concatenated 16S rRNA reference libraries and its analysis. The species-specific libraries can yield better resolution in sequence similarity and phylogeny based bacterial classification approaches”. Please revise as “This study aimed to develop a workflow for accurate identification of bacteria using concatenated, species-specific 16S rRNA sequences. It was hoped that the species-specific libraries would yield much better resolution in sequence similarity- and phylogeny-based bacterial classification”.\nMethods Estimation of variations in intra-genomic 16S rRNA gene copies “Sequence alignment of 16S rRNA copies at the intra-genomic level shows a higher degree of variability in species belonging to the Firmicutes and Proteobacteria (Vetrovsky and Baldrian, 2013; Ibal et al. 2019)”. Please revise this as “It has been reported that sequence alignment of 16S rRNA alleles at the intra-genomic level shows a higher degree of variability in species belonging to the Firmicutes and Proteobacteria (Vetrovsky and Baldrian, 2013; Ibal et al. 2019)”.\n“Hence, the study used eight 16S rRNA copies (Underlying data: Supplementary data 1 (Paul, 2022)) retrieved from the whole genome of Enterobacter asburiae strain ATCC 35953 (NZ_CP011863.1)”. Please revise this as “Therefore, this study used eight 16S rRNA alleles (Underlying data: Supplementary data 1 (Paul, 2022)) retrieved from the complete genome of Enterobacter asburiae strain ATCC 35953 (NZ_CP011863.1)”.\n“The BLAST+ 2.13.0 (RRID:SCR_004870; Altschul et al. 1990) and Clustal Omega 1.2.4 (RRID:SCR_001591; Sievers et al. 2011) sequence alignment algorithms were used to estimate intra-genomic variability between the 16S rRNA gene copies”. Please revise this as “To estimate intra-genomic variability between these 16S rRNA alleles, BLAST+ 2.13.0 (RRID:SCR_004870; Altschul et al. 1990) and Clustal Omega 1.2.4 (RRID:SCR_001591; Sievers et al. 2011) sequence alignment algorithms were used”.\n“Phylogenetic relatedness between intra-genomic 16S rRNA copies were estimated using the Maximum Likelihood method (Tamura-Nei model; 500 bootstrap replicates) with MEGA software (version 11; RRID: SCR_000667; Kumar et al. 2018).” Please revise this as “Phylogenetic analysis of these 16S rRNA alleles were performed using the maximum likelihood method (Tamura-Nei model; 500 bootstrap replicates) and the MEGA software (version 11; RRID: SCR_000667; Kumar et al. 2018)”.\nConstruction of species-specific concatenated 16S rRNA reference libraries\n“Previous studies have reported that several bacterial species share more than 99% sequence identity in the 16S rRNA encoding region”. Please revise this as “Previous studies have reported that the genes encoding 16S rRNA from several bacterial species share >99% sequence identity”. [ALSO PROVIDE A REFERENCE HERE]\n“Hence, the 16S rRNA-based bacterial identification methods failed to discriminate such genetically related species (Deurenberg et al. 2017; Devanga-Ragupathi et al. 2018)”. Please revise this as “Therefore, the 16S rRNA-based methods failed to correctly identify bacterial species that are genetically closely related (Deurenberg et al. 2017; Devanga-Ragupathi et al. 2018)”.\n“It has been reported that Streptococcus mitis and Streptococcus pneumoniae are almost indistinguishable from each other based on the sequence similarity of their 16S rRNA regions (Reller et al. 2007; Lal et al. 2011)”. Please revise this as It has been reported that 16S rRNA-based methods cannot distinguish between Streptococcus mitis and Streptococcus pneumoniae due to the high sequence similarity (Reller et al. 2007; Lal et al. 2011).\n“To develop species-specific barcode reference libraries, the study used 16S rRNA gene copies from whole-genome assemblies of four closely related species of Streptococcus (S. gordonii, S. mitis, S. oralis, and S. pneumoniae)”. Please DELETE the above, because it is repeated below.\n“More than 463,000 whole-genome assemblies are currently available for prokaryotes at the Genome database (RRID:SCR_002474; https://www.ncbi.nlm.nih.gov/genome)”. Please revise this as ‘More than 463,000 whole-genome sequences are currently (please provide a date here) available for prokaryotes (please specify if this is only for bacteria, or also includes archaea) in the Genome database (RRID:SCR_002474; https://www.ncbi.nlm.nih.gov/genome)”.\n“Most microbial genomes were sequenced with high throughput sequencing technologies such as Illumina/Ion-Torrent (short read sequencing) and PacBio/Nanopre (long read sequencing”). Please revise this as “Many of these genomes were sequenced using high throughput sequencing technologies such as Illumina/Ion-Torrent (short read sequencing) and PacBio/Nanopre (long read sequencing)”. “Further, many of these whole-genome assemblies are derived through a hybrid assembly of short and long read sequence data”. Please revise this as “Furthermore, most of these whole-genome sequences were obtained after a hybrid assembly of short and long read sequence data”.\n“The large volume of high throughput data can be effectively used to develop advanced genome-based approaches for microbial systematics”. Please revise this as “This extensive, high throughput data can be effectively used to develop advanced genome-based methods for microbial systematics.”\nThe genomic data is available in four assembly completion levels (contig, scaffold, chromosome, and complete). However, the study used only the genomes assemblies in the 'complete' stage to retrieve 16S rRNA gene copies. Please revise this as Although the genomic data is available in four levels (contig, scaffold, chromosome, and complete), this study used only the complete genomes to retrieve 16S rRNA genes.\n“The study retrieved full-length 16S rRNA gene copies from 16 genome assemblies belonging to four Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae”). Please revise this as “To develop species-specific barcode reference libraries, this study retrieved full-length 16S rRNA genes from 16 complete genome sequences belonging to four Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae)\".\n“The detailed information on the dataset used to develop species-specific concatenated reference libraries is provided in Table 1 and the sequences are provided in the underlying data (Supplementary data 2 (Paul, 2022))” Please revise this as “Details of the dataset used to develop species-specific concatenated reference libraries are provided in Table 1, and the sequences are provided in the underlying data (Supplementary data 2 (Paul, 2022))”\n“To maintain equal length, sequences were trimmed out beyond the universal primer pair fD1-5'-GAG TTT GAT CCT GGC TCA-3' and rP2-5'-ACG GCT AAC TTG TTA CGA CT-3' (Weisburg et al. 1991) for full-length 16S rDNA amplification”. Please revise this as “Sequences were trimmed beyond the universal primer pair (fD1-5'-GAG TTT GAT CCT GGC TCA-3' and rP2-5'-ACG GCT AAC TTG TTA CGA CT-3', which are used for full-length 16S rDNA amplification, Weisburg et al. 1991) to maintain uniform length [PLEASE MENTION THIS IN BASE PAIRS]\"\n“The study used MEGA 11 software to perform multiple sequence alignment and identify the intra-species parsimony informative (Parsim-info) variable sites”. Please revise this as “To perform multiple sequence alignment and identify the intra-species parsimony informative (Parsim-info) variable sites, the MEGA 11 software was used”.\n“A species-specific barcode reference library covering entire Parsim-info variable sites was constructed by concatenating four 16S rRNA gene copies representing four different strains of a species”. Please revise this as “A species-specific barcode reference library that covers the entire Parsim-info variable sites was constructed by concatenating four 16S rRNA gene copies from four different strains of a species”.\n“The rationale behind the selection of four copies for a species-specific barcode reference library is: (i) a maximum of four variations can be found on a single site, and (ii) earlier studies have shown that the mean 16S rRNA copies per genome is four (Vetrovsky and Baldrian, 2013)”. Please revise this as “The rationale for the selection of four copies for constructing a species-specific barcode reference library was: (i) a maximum of four variations can be found at a single site, and (ii) earlier studies have shown that the mean 16S rRNA copies per genome is four (Vetrovsky and Baldrian, 2013)”.\nDemonstration of concatenated 16S rRNA in sequence similarity and phylogeny\n“The study analyzed a few cases to demonstrate the classical sequence similarity and phylogenetic analysis using concatenated species-specific 16S rRNA reference libraries”. Please revise this as “This study analyzed a few cases to demonstrate (i) the classical sequence similarity and (ii) phylogenetic analysis using concatenated species-specific 16S rRNA reference libraries”.\n“The study used nine Sanger sequenced 16S rRNA gene copies showing higher sequence similarity with multiple species of Streptococcus retrieved from the GenBank database (RRID:SCR_002760)”. Please revise this as “Nine 16S rRNA gene copies (sequenced using the Sanger method) showing higher sequence similarity to the 16S rRNA genes of multiple species of Streptococcus were retrieved from GenBank database (RRID:SCR_002760)”.\n“The web based BLAST2 (version 2.13.0) program for aligning two or more sequences was used to estimate the maximum score, total alignment score, and sequence identity”. Please revise this as “The web based BLAST2 (version 2.13.0) program for aligning two or more sequences was used to estimate the maximum score, total alignment score, and sequence identity of these nine 16S rRNA”.\n“A single copy of the 16S rRNA region derived through Sanger sequencing or retrieved from a whole-genome assembly can be considered as ‘Query sequence”. Please revise this as “A single 16S rRNA gene (sequenced using the Sanger method or retrieved from a whole-genome assembly was the ‘Query sequence’”. [IT IS NOT CLEAR WHETHER THIS GENE WAS FROM Streptococcus. PLEAASE CLARIFY THIS]\n“The concatenated species-specific reference libraries must be provided in the ‘Subject sequence’ section”. Please revise this as “The concatenated species-specific reference libraries were provided in the ‘Subject sequence’ window”.\n“To perform phylogenetic analysis, it is mandatory that the target sequence (length = n bp) has to be concatenated four times (length = 4 × n bp), appending next to the last base”. Please revise this as “To perform phylogenetic analysis, it is mandatory that the target sequence (length = n bp) be concatenated four times (length = 4 × n bp)”.\n“Phylogenetic relatedness was estimated using the Maximum Likelihood method (Tamura-Nei model; 500 bootstrap replicates) with MEGA 11 software”. Please revise this as “Phylogenetic analysis was performed as indicated above/”\nResults Intra-genomic 16S rRNA variations in Enterobacter asburiae\nHistorically, the 16S rRNA gene sequences were used to identify known and new bacterial species. Please revise this as Historically, sequences of the 16S rRNA genes have been used to identify known and new bacterial species.\nHowever, this method is impacted by several factors such as amplification efficiency, poor discriminatory power at the species level, multiple polymorphic 16S rRNA gene copies, and improper bioinformatics workflows for the data analysis. Please revise this as However, efficiency of PCR-based amplification, poor discrimination at the species level, multiple polymorphic 16S rRNA gene copies, and improper bioinformatics workflows for the data analysis can impact the identification. [PLEASE PROVIDE A REFERENCE HERE]\nThe E. asburiae genome had eight 16S rRNA gene copies that showed a mean identity of 99.29% in sequence alignment using Clustal Omega (global alignment), whereas BLAST (local alignment) analysis resulted in an average of 99% identity between the copies (Table 2). Please revise this as The genome of E. asburiae contains eight copies of the 16S rRNA gene. Analysis using Clustal Omega (global alignment) and BLAST (local alignment) showed that the sequences of these eight alleles had average identities of 99.29 and 99%, respectively (Table 2).\nHence, the selection of an appropriate algorithm has a significant role in the estimation of percent identity, and a vital role in sequence-based species delineation. Please revise this as Therefore, choosing the appropriate algorithm/tool is critical for the estimation of sequence identities and sequence-based species delineation.\nGlobal sequence alignment programs generally perform better for highly identical sequence pairs, and the algorithm considers all the bases for the estimation of sequence identity. The multiple sequence alignment showed 22 variable sites in 16S rRNA gene copies of the E. asburiae genome (Figure 1). Please revise this as For analyzing sequence pairs that are highly identical, global sequence alignment programs/tools seem to be more appropriate because they consider all the nucleotides for the estimation of sequence identity.\nThe multiple sequence alignment showed 22 variable sites in 16S rRNA gene copies of the E. asburiae genome (Figure 1). Please revise this as Multiple sequence alignment [PLEASE MENTION THE TOOL/ALGORITHM HERE, AND ALSO IN THE LEGEND OF FIGURE 1] of the sequences of the eight alleles of the 16S rRNA gene in the genome of E. asburiae showed 22 variable sites (Figure 1).\nThe evolutionary relationship between species is usually represented in a phylogenetic tree drawn using a single barcode gene, multiple genes, or whole genomes. Please revise this as The evolutionary relationship between species is usually represented using a phylogenetic tree based on the analysis of a single gene, multiple genes, or whole genomes.\nHowever, bacterial species nomenclature is mainly designated based on the confidence obtained from the phylogenetic tree derived through single copy 16S rRNA analysis. Please revise this as However, bacterial identification and classification is mainly based on the phylogenetic analysis of single copies of 16S rRNA genes\nTo highlight how the intra-genomic 16S rRNA variations influence the species delineation, a phylogenetic tree was constructed using eight 16S rRNA gene copies of E. asburiae reference genome showing multiple nodes (Figure 2). Please revise this as A phylogenetic tree was constructed to understand how variations in the sequences of the eight alleles of the 16S rRNA gene in the genome of E. asburiae influence species delineation (Figure 2).\nThe sequence similarity and phylogeny-based analysis indicate that the intra-genomic variations in 16S rRNA copies may mislead the bacterial taxonomy in single gene copy approaches. IT IS NOT CLEAR TO ME HOW THIS ANALYSIS “indicate(s) that the intra-genomic variations in 16S rRNA copies may mislead the bacterial taxonomy in single gene copy approaches”. KINDLY ELABORATE ON THE SAME.\nSpecies-specific concatenated 16S rRNA libraries\nThe study selected four Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae) to construct species-specific concatenated 16S rRNA reference libraries. Please revise this as This study selected four species of Streptococcus (S. gordonii, S. mitis, S. oralis, and S. pneumoniae) to construct species-specific concatenated reference libraries based on 16S rRNA gene sequences obtained from complete genomes.\nThe study used 16S rRNA copies retrieved from four whole genome assemblies in the ‘complete’ stage to construct a species-specific barcode library. Please DELETE the above sentence.\nFour copies of the 16S rRNA gene are required to construct the concatenated library for a species. Please revise this as Sequences from four copies of the 16S rRNA gene are required to construct a concatenated library for a species [PLEASE PROVIDE A REFERENCE HERE]. The details of constructed species-specific libraries are listed in Table 1 and the sequence is provided in the underlying data (Supplementary data 3 (Paul, 2022)). Please revise this as The details of species-specific libraries are listed in Table 1 and the sequences are provided in the underlying data (Supplementary data 3 (Paul, 2022)). THIS REFERENCE IS INCOMPLETE IN THE LIST, AND THE LINK IS NOT WORKING. PLEASE CHECK ANC CORRECT. ALSO, IN THE TITLE OF TABLE 1, “for the development of concatenated” SHOULD BE REVISED AS “for the construction of concatenated”\nThe 16S rRNA sequence analysis shows 24 Parsim-info variable sites for S. oralis, 11 variations in S. mitis, seven variations in S. gordonii, and six variations found in S. pneumoniae. Please revise this as Analysis using the sequences of 16S rRNA genes showed 24, 11, 7, and 6 Parsim-info variable sites for S. oralis, S. mitis, S. gordonii, and S. pneumoniae, respectively. [PLEASE PROVIDE/SHOW THE DATA FOR THIS]\nThe observed intra-species Parsim-info variable sites are residing on both conserved and variable regions of the 16S rRNA gene. Please revise this as The intra-species [PLEASE CHECK IF THIS SHOULD BE “inter-species”] Parsim-info variable sites were located in both the conserved and variable regions of the 16S rRNA gene. [PLEASE PROVIDE/SHOW THE DATA FOR THIS]\nThe study used full-length 16S rRNA copies from four different strains to highlight the variations at the species level. Please revise this as This study used full-length sequences of 16S rRNA genes from four different species to check the variations at the species level.\nHowever, a large volume of partial 16S rRNA sequences are available in the public genetic databases. Please revise this as However, a large number of partial sequences of 16S rRNA genes are available in the public genetic databases.\nIn such cases, a species-specific concatenated 16S rRNA reference library can be developed with partial sequences. Please revise this as In such cases, a species-specific concatenated reference library can be constructed using partial sequences.\nIntra-species variation on 16S rRNA gene copies influences the sequence based bacterial taxonomy. Please revise this as Intra-species [PLEASE CHECK IF THIS SHOULD BE “inter-species”] variations in the sequences of 16S rRNA gene copies influences the sequence-based bacterial identification. [PLEASE PROVIDE/SHOW THE DATA FOR THIS, OR SUBSTANTIATE THE CONCLUSION]\nHence, the concatenated 16S rRNA approach yields better resolution than single copy analysis in classical sequence similarity and phylogeny based species identification approaches. Please revise this as Therefore, concatenation of the sequences of 16S rRNA genes/alleles provides much better resolution compared to analysis using sequences from a single copy of the 16S rRNA gene. PLEASE NOTE: IN THE ABSENCE OF DATA, THIS STATEMENT REMAINS UNSUBSTANTIATED.\nDemonstration of concatenated 16S rRNA based species identification\nThe study compared nine 16S rRNA sequences representing Streptococcus species (Table 3) with species-specific concatenated reference libraries. Please revise this as This study compared sequences of nine 16S rRNA genes from different species of Streptococcus (Table 3) using species-specific concatenated reference libraries.\nConcatenated sequence analysis gives better resolution in sequence similarity search and phylogenetic analysis. Please revise this as Concatenated sequences provide much better resolution in sequence similarity search and phylogenetic analysis.\nThe sequence accession numbers GU470907.1 and KF933785.1 classified as S. mitis showed a higher maximum and total alignment score with S. oralis than S. mitis (Table 3). Please revise this as Two sequences (accession numbers GU470907.1 and KF933785.1) from S. mitis had a higher maximum and total alignment score to sequences from S. oralis than S. mitis (Table 3). PLEASE PROVIDE THE ACCESSION NUMBERS FROM S. ORALIS THAT PRODUCED THIS RESULT.\nWhereas the sequence (OM368574.1; classified as S. mitis) showed a higher sequence alignment score with S. pneumoniae. Please revise this as Furthermore, yet another sequence from S. mitis (accession number OM368574.1) had a higher alignment score to sequences from S. pneumoniae. PLEASE PROVIDE THE ACCESSION NUMBERS FROM S. PNEUMONIAE THAT PRODUCED THIS RESULT.\nFigure 3A shows a maximum likelihood tree of the nine 16S rRNA gene sequences with four concatenated species-specific reference libraries. Please revise this as The maximum likelihood phylogenetic tree based on four concatenated species-specific reference libraries and the sequences of nine 16S rRNA genes is shown in Figure 3A. [THE LEGEND IS INSUFFICIENT; PLEASE PROVIDE MORE DETAILS IN THE LEGEND. ALSO, FIGURE 3B HAS NOT BEEN CALLED IN THE RESULTS SECTION, IT HAS BEEN CALLED DIRECTLY IN THE DISCUSSION SECTION]\nThe concatenated GU470907.1 and KF933785.1 sequences showed a phylogenetic relationship with S. oralis and sequence OM368574.1 was genetically related to S. pneumoniae. Please revise this as [BASED ON THE ACCESSION NUMBERS, GU470907.1 and KF933785.1 CANNOT BE CONCATENATED SEQUENCES, PLEASE CHECK AND CORRECT] Two sequences (accession numbers GU470907.1 and KF933785.1) from S. mitis appeared to be phylogenetically closer to S. oralis, and yet another sequence from S. mitis (accession number OM368574.1) from S. mitis was closer to S. pneumoniae. [PLEASE NOTE: THE BOOTSTRAP VALUES ARE RATHER LOW; THEREFORE THE RESULTS NEED TO BE INTERPRETED CAREFULLY]\nThese results indicate that the species-specific concatenated 16S rRNA reference libraries have great potential in the taxonomic classification. Please revise this as These results further confirm that species-specific concatenated 16S rRNA reference libraries provide much better taxonomic resolution.\nHence, the study suggests the usage of concatenated variable 16S rRNA copies for sequence similarity and phylogeny-based species identification. Please revise this as Therefore, this study recommends using concatenated sequences of 16S rRNA genes for sequence similarity- and phylogeny-based species identification.\nA species-specific reference library with concatenated 16S rRNA gene copies provides better resolution in phylogenetic analysis than the single copy inference. THE SENTENCE ABOVE MAY BE DELETED BECAUSE A SIMILAR STATEMENT HAS ALREADY BEEN MADE.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9493", "date": "03 Apr 2023", "name": "Bobby Paul", "role": "Author Response", "response": "Dear Sir, Thank you very much for your critical review and valuable suggestions for manuscript improvement. I have revised the manuscript by addressing all the suggestions and resubmitted it for your consideration. I sincerely hope that the modified manuscript is sufficiently improved, and kindly respond if you have any further questions or comments. Thank you Kind Regards Bobby Paul" } ] } ]	1	https://f1000research.com/articles/11-1530
https://f1000research.com/articles/12-1090/v1	01 Sep 23	{ "type": "Research Article", "title": "In silico molecular docking, ADME study and synthesis of new 1,3-diazetidin-2-one derivatives with high anti-proliferative activity", "authors": [ "Farah Haidar Abdulredha", "Monther Faisal Mahdi", "Ayad Kareem Khan", "Monther Faisal Mahdi", "Ayad Kareem Khan" ], "abstract": "Background: Cancer and inflammation are strongly connected; tumor growth and spread are also greatly influenced by inflammation. Nitrogen-based heterocycle analogs are excellent suppliers of pharmaceuticals. Quaternary rings play a bigger role in drug development as bioactive scaffolds. For improved tolerance and synergistic benefits, heterocyclic nitrogen rings are present in many anticancer medications. Understanding how to bind to the EGFR and its prospective impacts on cancer cells, expect to construct new heterocyclic compounds that may help produce potent anticancer medicines with a high safety profile. Methods: Novel 1,3-diazetidin-2-one derivatives were designed, synthesized from mefenamic acid, and their cytotoxic activity against a lung cancer cell line (A549) was initially tested in vitro. These compounds were anchored to the crystal structure of the epidermal growth factor receptor (PDB code 1M17) in a molecular docking study to determine their binding affinity at the active site. The newly synthesized derivatives were verified and confirmed by elemental analysis and spectroscopic data (FT-IR, 1H-NMR, and 13C-NMR). In addition, physicochemical, drug-like, and toxicological predictions were performed for these derivatives. Results: Based on a molecular docking study, all compounds (M4a-e) demonstrated superior PLPfitness (84.70, 85.89, 91.90, 88.61, and 92.77, respectively) to erlotinib (76.20). The anti-proliferation evaluation of the A549 cell line revealed that compounds M4c and M4e had exceptional and promising anti-proliferative activity on this cell line to treat lung cancer, with IC50 values of 1.75 µm and 2.05 µm at 72 hours, respectively, making them significantly more active than the reference erlotinib, which had an IC50 value of 11.5 µm at 72 hours. Conclusions: The cytotoxicity investigation and the molecular docking study showed a robust association with the novel compounds (M4a-e). Suggest a comprehensive pharmacological survey to understand how these newly created chemicals combat cancer fully.", "keywords": [ "A549", "Diazaitidine", "Mefenamic Acid", "Molecular Docking", "Synthesis" ], "content": "Introduction\n\nLung cancer is the leading cause of cancer-related death worldwide.1 Cancer treatment causes severe physical and mental exhaustion due to the high toxicity and ineffectiveness of commercially available anticancer drugs.2 Efficient targeting has led to the development of more potent and less toxic anticancer drugs in recent years.3\n\nCancer and inflammation are tightly connected, tumor development and growth are greatly influenced by inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs), among other anti-inflammatory medications, have been proven in several clinical investigations to affect the tumor microenvironment by preventing cell migration, promoting apoptosis, and increasing chemosensitivity.4 There has been a lot of focus on the role that inflammation plays in the onset and aggressiveness of several malignancies, including non-small cell lung cancer (NSCLC). Numerous studies have shown that NSAIDs help prevent cancer in animal models, in part because they can reduce the activity of the enzyme cyclooxygenase (COX). It has been demonstrated that cyclooxygenases, particularly COX-2, are important during various phases of carcinogenesis. Erlotinib, an EGFR tyrosine kinase inhibitor, was combined with a number of nonsteroidal anti-inflammatory medicines (NSAIDs) to create numerous novel anticancer medications.5 Nuclear transcription factors that have been shown to be overexpressed in cancer cells are inhibited by NSAIDs. Nuclear factor kappa B (NF-B) controls the expression of inflammatory and proliferative enzymes and proteins such as COX-2 and cyclin D1. Therefore, blocking the COX-2 enzyme may reduce the risk of cancer development, progression, and spread. Thus, inflammation can provide key mutations and a suitable environment for tumor growth.6\n\nDue to their role in the treatment of inflammation and their cytotoxic activities, NSAIDs have become a popular target for drug design technology to create substances with improved COX-2 selectivity and fewer side effects.7\n\nMefenamic acid (MFA) is an anthranilic acid-class NSAIDs.8 In an era where other (NSAIDs) have proliferated, the clinical application of mefenamic acid has typically waned. It is a member of the fenamates and shares comparable modes of action and general toxicities with others (NSAIDs).9 However, mefenamic acid has distinct in vitro properties that may separate this medication from others. The basic sciences have created an opening for using mefenamic acid alone or in conjunction with other medicines for some novel indications. For instance, the effect of mefenamic acid on persistent, progressive brain disease needs additional exploration.10 Cellular mechanisms of action could be primarily utilized to influence cellular and then macroscopic transformation in various novel applications.11\n\nMefenamic acid has been demonstrated to be effective against human breast cancer (MCF-7), human bladder cancer (T24), human lung cancer (A-549), and other cancers by inducing apoptosis in human hepatoma cells (CHANG and HuH-7).12\n\nA. Altai et al. successfully prepared two Ag(I) complexes containing mefenamic acid. Flow cytometry was used to study the apoptotic effects of complex and intracellular ROS production in MCF-7 cells. These XTT and LDH assays demonstrate that both complexes exhibit potent anti-proliferative activity with higher selectivity for cancer cells compared to normal cell selectivity.13\n\nA major source of therapeutic medicines in medicinal chemistry is heterocyclic analogs based on nitrogen. Because biological targets and nitrogen easily form hydrogen bonds. Quaternary rings are becoming more crucial in drug development initiatives as bioactive scaffolds. They contribute to the advancement of physicochemical qualities as well as structural innovation to previously unknown regions of chemical space.14\n\nSantos and co. completed an important step in the production of a series of 1,2-diazetidin-3-ones by the intramolecular N-H insertion of rhodium carotenoids. These substances have a mild growth-inhibitory effect on breast cancer cells.15\n\nThe strategy of this work was to incorporate this heterocyclic four-membered ring system into mefenamic acid to synthesize new derivatives and evaluate their anti-proliferative activity against the A549 cell line using the MTT method, with an emphasis on understanding how to bind to the EGFR and its possible effects on cancer cells, hoping to design new heterocyclic compounds that may help in the development of new potent anticancer agents with high safety.\n\n\nMethods\n\nFT-IR spectra were recorded with [Tensor 27 Brucker (USA), Shimadzu FT-IR KBr disc (Japan)], melting points were measured with glass capillaries on a Stuart melting point apparatus (UK), proton NMR (400.13, 300.81). MHz) and carbon NMR spectra (100.62, 75.65 MHz) were determined by Brucker (USA) using DMSO as solvent. Mefenamic acid and various aryl aldehydes from Hyperchem (China), SOCL2 from Thomas Baker (India) and phenylisocyanate from Sigma-Aldrich (Germany).\n\nMefenamic acid was dissolved in 15 ml of dry methanol at a ratio of 4.3 mmoles, and the mixture was chilled to a temperature between 0 and -5°C. To guarantee full solubilization, a small amount of tetrahydrofuran solvent was added. Thionyl chloride was gradually added to the solution dropwise until 0.32 ml (4.5 mmol) had been added. The mixture was heated in a water bath at 40°C for three hours, followed by eight hours of reflux stirring at 65°C. It was evaporated and then treated with 25 ml of ice water after it had warmed to room temperature. The product is recrystallized from 100% ethanol to produce a pure product, using a silica gel-coated TLC plate to monitor the reaction.16 The purity of the synthesized compounds was assessed through recrystallization and confirmed by determining a sharp melting point. Physical properties, Rf values, FT-IR, and NMR spectra interpretations of the synthesized compounds are illustrated in Table 1.\n\nCompound [M2] was synthesized by dissolving 1 mmol of compound [M1] in 15 mL of absolute ethanol. Add an excess of 0.25 mL (5 mmol) of hydrazine hydrate, 99.99%, to the solution. The mixture was refluxed for 15 hours. After cooling, ice distilled water were added to precipitate the solution. Recrystallization from absolute ethanol gave compound [M2] a light yellow powder. Monitor the reaction with silica gel-coated TLC plates.17\n\nEach aromatic aldehyde (1 mmol) was combined with 10 mL of 100% ethanol before adding 2-3 drops of glacial acetic acid to begin the synthesis of the compounds [M3a-e]. The mixture was then swirled for 30 minutes at room temperature. Then mix 1 mmol of compound [M2] with 15 ml of 100% ethanol for 30 minutes at room temperature, reflux the mixture for 3 to 6 hours at 75 °C. After filtering, washing with cold water, recrystallization from pure ethanol. Follow the development of the reaction on a TLC plate with silica coating.18\n\nAnhydrous 1,4-dioxane (25 ml) was first mixed with 1 mmol of (M3a-e), and then 1.5 mmol of phenyl isocyanate was slowly added dropwise while maintaining a temperature between 0 and -5°C. The resultant mixture was refluxed for (5-7) hours after being agitated for 3 hours at room temperature. The resultant solid was washed with a solution of ethyl acetate: petroleum ether (1:1), filtered, and dried after the solvent had evaporated at room temperature. The process of recrystallizing was done from pure ethanol.14\n\nThe synthesis method of the compound M3a-e and M4a-e is shown in Figure 1.\n\nGOLD Suite (v. 2021.2.0), a licensed CCDC tool, was utilized for the docking technique to examine the possible binding of certain 1,3-diazetidin-2-one derivatives to EGFR. A nonproprietary alternative that can be suggested are AutoDock and AutoDock Vina. Additionally, using the CCDC Hermes Visualizer program (version 2021.2.0), bond lengths, brief contacts, hydrogen bond interactions, and interactions between proteins and ligands were estimated. Nonproprietary alternative that can be suggested in this instance are PyMOL, jmol and ChimeraX. The ligands from Chem Sketch were transformed into SMILE names using the Swiss ADME program, and the physicochemical characteristics and pharmacokinetic statistics of molecular atoms were predicted. Additionally, using BOILED EGG,19 the lipophilicity and polarity of molecules are estimated.20 The component amino acids of the EGFR protein crystal structure now exist in ionized and tautomeric forms because hydrogen atoms have taken the role of water molecules in the structure. With default settings for all parameters, CheBio3D (version 16.0), MM2 force field, and CHEMPLP fitness algorithm were used to assess each proposed solution. Nonproprietary alternatives to CheBio3D are Avogadro, MarvinSketch, and BKChem. Additionally, the hydrogen atoms' dependence on distance and angle was taken into account when calculating the steric complementarity between the protein and its ligand. The candidate solutions are then evaluated, and the binding mode, docking position, and binding energy for each candidate solution are determined by a piecewise linear potential function. These findings assess how our created compounds interact with the EGFR's amino acid residues.21\n\nCell culture\n\nA human lung cancer cell line (A549) from ATCC has been purchased by Al Mustansiriyah University Cell Bank Tissue Culture Research Center and is currently housed at the Faculty of Pharmacy.\n\nStorage and resuscitation of cell line\n\nFor 24 hours, cells were kept frozen in liquid nitrogen (-80°C). Add 10 ml of fresh media after thawing at 37 °C, and the cells were separated via a centrifuge. The cells are then transfered to a 75 cm2 culture flask after resuspending them in 25 ml of fresh media.22\n\nCell maintenance\n\nThe cells are incubated at 37°C in 95% humidified air and 5% CO2, A549 cells were cultured in RPMI 1640 medium. The medium was supplemented with antibiotics such as penicillin-streptomycin-amphotericin B100X and 1% L-glutamine. An addition of 10% of fetal bovine serum (FBS) was made to enhance the medium. In sterile 75 cm2 flasks, the cells were grown and when 90% confluency was reached, they were subcultured by washing with 5 ml of phosphate-buffered saline (PBS) and detached from the bottom of the flask using trypsin solution that was incubated for 2 min at 37°C. Using a hemocytometer, check the cell count after transferring a cell suspension to a 50 ml conical tube and centrifuging at 1200 rpm for 3 minutes. After decanting the supernatant, resuspend cell pellets in supplemented growth medium. Complete growth medium should also be added in equal volume.23\n\nCell viability by MTT colorimetric assay\n\nIn an effort to uncover new information, we conducted an experiment to evaluate how synthetic chemicals [M4a-e] would impact the survival of lung cancer cell lines by administering the MTT colorimetric assay. Our approach involved dispensing 100 μl of a cell suspension, with a concentration of (5×103) cells/well, into each individual well of a 96-well flat-bottom tissue culture plate and leaving them undisturbed for a total of 24, 48, or 72 hours. After the first 24 hours, we introduced each chemical, with a 5 μm concentration, to the cells. Incubate the culture in a medium containing 10 μl of MTT solution (5 mg/ml MTT powder in PBS) at 37°C for two hours after the recovery period (24 h, 48 h, and 72 h). Cast aside the medium after 2 hours, and attribute 100 μl DMSO to each well, and put it in a dark room at room temperature for around 15-20 minutes. Using a Multiscan Reader, the optical density of each plate (well) was measured through a transmitted wavelength range of 560-600 nm. To calculate the cell growth inhibition (percent cytotoxicity), the formula [Percent inhibition = (A-B/A)100] was implemented, with [A and B] representing the tested optical density substance and the standard (erlotinib), respectively. Performances were completed in duplicate.24\n\nDetermination of the half maximal inhibitory concentration (IC50)\n\nUsing the dose response curve, the IC50 of the test compound can be calculated. For in vitro MTT assays, IC50 is the concentration of the test chemical [M4a-e] required to reduce cell viability by 50%. Using the results of the in vitro MTT assay, IC50 values were calculated for the study drug 72 hours after exposure to the cells. The concentration ranges of the substances [N4a-c] used for the calculation of IC50 values were (500, 250, 125, 12.5, 62.5, 31.25, 15.62, 7.81, 3.90 and 1, 95 μm).\n\n\nResults and Discussion\n\nThe identification of the synthesized compounds was made through the examination of their melting point and Rf values. The FT-IR analysis revealed that the hydroxyl group of mefenamic acid had disappeared, as seen from the absence of its broad peak at 2500-3200 cm-1. Instead, a signal was detected at 1732 cm-1 for compound (M1), indicating the presence of a carbonyl group of ester (-C=O) in place of the carboxylic acid (C=O) found in mefenamic acid. The stretching of the ester was observed at 1224 cm-1 for compound (M1), while the singlets of (-OCH3) of ester were detected at 3.87 (δ) in the 1H-NMR results. The broad peak of the hydroxyl group at 12.99 (δ) for mefenamic acid had disappeared. In the 13C-NMR results, a peak at 168.71 (δ) was observed for the carbonyl of the ester in (M1), while the methyl group showed a peak at 52.19 (δ).\n\nInfrared analysis (FT-IR) of compound (M2) revealed the disappearance of a broad band at (1732 cm-1) associated with stretching of the carbonyl (C=O) of the (M1) ester and the appearance of a New bands are assigned to stretches of the amide carbonyl (C=O) and to the (-NHNH2) group of (M2) at (3351, 3329, 3188 cm-1). The 1H-NMR spectrum showed a broad singlet at 4.31 (δ) for the NH2 proton of the hydrazide and a singlet at 9.52 (δ) for the NH proton of the hydrazide (M2). 13C-NMR showed that the methyl ester peak disappeared and the amide carbonyl at (172.73) (δ) became visible.\n\nThe FT-IR characteristic absorption bands of (M3a-e) showed υNH stretching of amide at (3344-3325 cm-1) and combination band of υC=O stretching of amide and υC=N stretching at (1664-1622 cm-1). 1H-NMR spectra of compounds (M3a-e) showed singlet for N=CH-Ar (imine proton) at (8.34-8.88) (δ), and disappearance of the broad singlet for NH2 protons of hydrazide. 13C-NMR showed the peak of (-CH) imine at (~143-150) (δ).\n\nThe characteristic FT-IR absorption band of N-H stretching of secondary amides appears at (3325, 3286 cm-1) as two peaks due to hydrogen bonding; in the presence of hydrogen bonding, the N-H stretching splits into two peaks: one relatively low frequency peak and a higher frequency peak. Lower frequency peaks correspond to N-H stretching in hydrogen-bonded NH groups, while higher frequency peaks correspond to N-H stretching in non-hydrogen-bonding NH groups. In addition, υC=O at (1745, 1730 cm-1) is a diazetidine ring, υC=O at (1650, 1641 cm-1) is an amide ring, at (3136, 3102 cm-1), the -CH segment of the diazetidine ring. 1H-NMR results show that the characteristic single peak of the imine proton has disappeared, while the (-CH) proton of the diazetidine ring is at (6.96-6.99) (δ) was increased. 13C-NMR shows that the characteristic single peak of imine carbon disappears, the (-CH) peak of diazetidinine is at (61.86-66.82) (δ), and the carbonyl peak of diazetidinine is at (152 –161) (δ).\n\nA genetic algorithm called GOLD was used to dock flexible ligands to protein binding sites, predicting the optimal molecular interaction between the expected compound (M4a-e) and the active binding site of the EGFR protein.24 The PLP fitness scores rank inhibitory activities, and docking studies suggest that all predicted compounds have excellent binding energies with active receptor pockets, potentially showing promising activity with EGFR proteins. The preliminary molecular docking perfectly correlated with subsequent cytotoxicity (in-vitro) studies for compounds (M4a-e) against lung cancers. Amino acids of the epidermal growth factor receptor (PDB: 1M17) interacted with the novel compounds through hydrogen and short contact bonds, including VAL 702, LYS 721, ASP831, THR830, LEU820, MET769, LEU768, GLY772, THR766, LEU764, CYS773, CYS721, and CYS 773. All anticipated final compounds exhibited higher binding energies than the standard drug erlotinib, ranging from (84.70) to (92.77), with a PLP fitness value of (76.20) as shown in Table 2.\n\n Numbers in parentheses indicate the amount of the bond.\n\nThe use of in silico tools to predict the pharmacokinetic properties of potential drug candidates during the lead generation and optimization stages has been shown to increase the chances of surviving the high turnover rates of drug discovery. To expedite the drug discovery process, efforts have been made to integrate pharmacokinetic and developmental considerations early in the research process, with a focus on identifying compounds with greater potential to optimize binding.25 All predicted compounds had a bioavailability of 0.55 and a TPSA of less than 140 A0, except compounds M4c and M4e, which had a bioavailability of 0.17, indicating that they entered the systemic circulation. These compounds do not cross the blood-brain barrier (BBB). In addition, none of the compounds had an affinity for P-gp, the protein responsible for preventing chemotherapy drugs from being taken up by cells. Since these compounds are P-gp non-substrates, they are not excreted from the cell by efflux transporters.26 In addition, all predicted compounds complied with the Lipinski rule of five, with the exception of M4c and M4e, which had two violations, as shown in Table 3.\n\nBased on the anti-proliferative evaluation27 on the A549 cell line, we found that the compounds M4c and M4e had outstanding and promising anticancer activity against this cell line for the treatment of lung cancer with IC50 values of 1.75 and 2.05 μm, respectively, after 72 hours of administration, they are more active than the reference product erlotinib with IC50 (11.5) μm after 72 hours. The results of the cytotoxicity studies of the terminal compounds (M4c and M4e) were consistent with the predictions of the molecular docking studies. All synthesized compounds showed excellent anti-proliferative activity except compound (M4d) with IC50 (11.33) μm comparable to erlotinib (11.5) μm and compound (M4b) with slightly lower IC50 (14, 01) μm, higher than that of erlotinib as shown in Figure 2, and A549 cells were also observed by microscopy after 48 hours after exposure to 62.5 μm synthetic compounds (M4a-e). Morphological changes, compared with A549 cells (control cells) in Figure 3.\n\n(a)=M4a, (b)=M4b, (c)=M4c, (d)=M4d, (e)=M4e.\n\na: control, b: M4a, c: M4b, d: M4c, e: M4d, f: M4e.\n\n\nConclusion\n\nA series of novel 1,3-diazetidin-2-one derivatives have been successfully characterized and evaluated using in silico methods including ADME studies. The results showed that all compounds, were strongly passively absorbed from the gastrointestinal tract and conformed to Lipinski's \"rule of five except compounds M4c and M4e, with two violations. Molecular docking studies of the final compounds (M4a-e) revealed a significant interaction with the EGFR protein, which outperformed the reference drug erlotinib. The anti-proliferative activity of the synthesized compounds against the lung cancer cell line A549 was evaluated. All synthesized compounds showed excellent anti-proliferative activity except compound M4d, which had an IC50 value (11.33) μm comparable to that of erlotinib (11.5) μm and compound M4b exhibited a slightly higher IC50 than erlotinib after 72 hours. New Compound M4a-e shows excellent correlation between cytotoxicity and molecular docking studies.", "appendix": "Data availability\n\nZenodo: Underlying data, https://doi.org/10.5281/zenodo.8049181\n\nThis project contains the following underlying data:\n\n• 001_Farah.xlsx (cytotoxicity data of MTT assay).\n\nZenodo: FT-IR, HNMR, and CNMR charts/spectra, https://doi.org/10.5281/zenodo.8176123\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nFrankell AM, Dietzen M, Al Bakir M, et al.: The evolution of lung cancer and impact of subclonal selection in TRACERx. Nat. 2023 [cited 2023 May 10]; 616(7957): 525–533. Reference Source\n\nAl-tuwaijri HM, Al-abdullah ES, El-rashedy AA, et al.: Anticancer Agents: Design, Synthesis, and In Silico Studies.2023.\n\nSabnis AJ, Bivona TG: Principles of Resistance to Targeted Cancer Therapy: Lessons from Basic and Translational Cancer Biology. Trends Mol. Med. 2019 [cited 2023 May 21]; 25(3): 185–197. Reference Source\n\nRahman MM, Islam F, Afsana Mim S, et al.: Multifunctional Therapeutic Approach of Nanomedicines against Inflammation in Cancer and Aging. J. Nanomater. 2022; 2022.\n\nZhang Y, Tortorella MD, Liao J, et al.: Synthesis and Evaluation of Novel Erlotinib-NSAID Conjugates as More Comprehensive Anticancer Agents. ACS Med. Chem. Lett. 2015; 6(10): 1086–1090.\n\nSuri A, Sheng X, Schuler KM, et al.: The effect of celecoxib on tumor growth in ovarian cancer cells and a genetically engineered mouse model of serous ovarian cancer.7(26).\n\nStockmann P, Gozzi M, Kuhnert R, et al.: New keys for old locks: carborane-containing drugs as platforms for mechanism-based therapies. Chem. Soc. Rev. 2019 [cited 2023 May 21]; 48(13): 3497–3512. Reference Source\n\nRashid MH, Sarsam SW, Al-sabea N: A spectrophotometric method for the determination of Mefenamic acid in pharmaceutical dosage form.2017; 17(2): 2–8.\n\nKrawczyk MS, Sroka A, Majerz I: The crystal structure and intermolecular interactions in fenamic acids–acridine complexes. Molecules. 2021; 26(10).\n\nPatel SS, Tripathi R, Chavda VK, et al.: Anticancer Potential of Mefenamic Acid Derivatives with Platelet-Derived Growth Factor Inhibitory Property. Anti Cancer Agents Med. Chem. 2020; 20(8): 998–1008.\n\nRev E, Pharmacol C: The potential and promise of mefenamic acid.2013; 6(3): 289–305.\n\nUseini L, Mojić M, Laube M, et al.: Carboranyl Analogues of Mefenamic Acid and Their Biological Evaluation. ACS Omega. 2022 [cited 2023 May 21]; 7(28): 24282–24291. Publisher Full Text\n\nAltay A, Caglar S, Caglar B, et al.: Novel silver(I) complexes bearing mefenamic acid and pyridine derivatives: Synthesis, chemical characterization and in vitro anticancer evaluation. Inorg. Chim. Acta. 2019; 493(January): 61–71. Publisher Full Text\n\nSuć Sajko J, Jerić I: Synthesis of Nβ-Substituted 1,2-Diazetidin-3-ones by the Ugi Reaction Comprising Chiral α-Hydrazino Acids. J. Org. Chem. 2022; 87(11): 7076–7084.\n\nSantos MS, Nortcliffe A, Lewis W, et al.: Synthesis of Highly Substituted 1,2-Diazetidin-3-ones, Small-Ring Scaffolds for Drug Discovery. Chem - A Eur J. 2018; 24(33): 8325–8330.\n\nSabah AA, Al-rawi MS, Tomma JH: Study the Toxicity and Anticancer activity of Some New Amic Acid and Their Derivatives of Mefenamic acid.2020; 14(March): 642–648.\n\nAmmar YA, Khalifa MM, Eisa SI, et al.: New Naproxen Analogs: Synthesis, Docking and Anti-Inflammatory Evaluation. Polycycl. Aromat. Compd. 2022; 42(6): 3586–3605. Publisher Full Text\n\nMahdi MF, Al-smaism RF, Mahmood AI: Date of acceptance: 10-6-2015 Synthesis, Characterization of Some New 2-Azetidinone Derivatives.2015; 15(2).\n\nŞahin S, Dege N: (E)-N-(3-chlorophenyl)-1-(5-nitro-2-(piperidin-1-yl)phenyl)methanimine: X-Ray, DFT, ADMET, Boiled-Egg Model, Druggability, Bioavailabilty, and Human Cyclophilin D (CypD) Inhibitory Activity. J. Mol. Struct. 2022; 1250: 131744.\n\nOmran SM, Abd Razik BM, Mahdi MF: Density functional theory and molecular modeling studies of new 4-(Furan-2-yl) thiazol-2-amine derivatives as cyclooxygenase inhibitors. Egypt. J. Chem. 2021; 64(9): 4833–4841.\n\nNg HW, Zhang W, Shu M, et al.: Competitive molecular docking approach for predicting estrogen receptor subtype α agonists and antagonists. BMC Bioinformatics. 2014 [cited 2023 Apr 3]; 15 Suppl 11(Suppl 11). Reference Source\n\nShi Y, Tang B, Yu PW, et al.: Autophagy protects against oxaliplatin-induced cell death via ER stress and ROS in Caco-2 cells. PLoS One. 2012 [cited 2023 Feb 26]; 7(11). Reference Source\n\nKaplan A, Akalin Ciftci G, Kutlu HM: The apoptotic and genomic studies on A549 cell line induced by silver nitrate. Tumor Biol. 2017; 39(4).\n\nAbdul-Majeed SZ, Mahdi MF, Al-Mugdadi SFH: Pharmacological Evaluation of New 4, 5-dihydro-1H-Pyrazole-1-yl acetate Derivatives as Anti-inflammatory Agents. Int. J. Drug. Deliv. Technol. 2022; 12(4): 1733–1741.\n\nKamal IM, Chakrabarti S: MetaDOCK: A Combinatorial Molecular Docking Approach. ACS Omega. 2023 [cited 2023 May 12]; 8(6): 5850–5860. Publisher Full Text\n\nVogel HG, Maas J, Hock FJ, et al.: Drug Discovery and evaluation: Safety and pharmacokinetic assays. second ed.2013.\n\nHassanzadeh F, Jafari E, Zarabi M, et al.: Synthesis, cytotoxic evaluation, and molecular docking studies of some new 1, 3, 4-oxadiazole-based compounds. Res Pharm Sci. 2020; 15(5): 454–462." }	[ { "id": "203766", "date": "26 Sep 2023", "name": "Sujeet Kumar", "expertise": [ "Reviewer Expertise Cancer research. Biologically active small heterocyclic molecules." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript needs to be revised.\n\nThe title of all figures should be written in a more scientific way. Please refer to standard published articles to know how to label figures and tables.\n\nOnly five derivatives are prepared. It can't be called a series.\n\nLipinski violation data is missing.\n\nDocking figures for standard and potent molecules are missing. Please include it in the manuscript.\n\nCheck the structure of M1. I think Cl is missing in the structure after oxygen.\n\nEnglish editing and corrections are required.\n\nIrrelevant references (such as Ref 8, and Ref 9) can be avoided.\n\nWrite all references in one format (please check Ref 11).\n\nConclusion/summary: Kindly revise the manuscript thoroughly and re-submit.\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": "10323", "date": "06 Oct 2023", "name": "Farah Abdulredha", "role": "Author Response", "response": "I wanted to extend my sincere appreciation for the time and effort you dedicated to reviewing our paper. Your feedback has been invaluable to us, and I would like to express my gratitude for your valuable insights. I have carefully considered all the points you raised. I apologize for any confusion or oversight on our part in this regard. I would like to offer some clarification on this matter: [The manuscript needs to be revised]. I understand that you've suggested revisions to the manuscript, and I take your recommendations seriously. We are committed to enhancing the quality of our work, and your feedback will be instrumental in achieving that goal. I have already uploaded the revised manuscript to the journal system. [The title of all figures should be written in a more scientific way. Please refer to standard published articles to know how to label figures and tables]. Regarding the figure titles, we understand your concern and have taken steps to ensure that they align with the standards found in published scientific articles. [Only five derivatives are prepared. It can't be called a series]. Thank you for bringing attention to this. I agree with this comment. Therefore, I have deleted the word ‘series’. [Lipinski violation data is missing]. I have already included the Lipinski violation for each of the synthesized compounds in Table 3 throughout the entire manuscript; if you would like me to add more detail, please let me know. [Docking figures for standard and potent molecules are missing. Please include it in the manuscript]. The three-dimensional photos of all synthesized compounds were published before by Farah et al. DOI: 10.4103/JAPTR.JAPTR_116_23 [Check the structure of M1. I think Cl is missing in the structure after oxygen]. In synthesizing ester from carboxylic acid using thionyl chloride, chlorine will be released as an HCl gas side product according to the following mechanism. [English editing and corrections are required]. I agree with this and have incorporated your suggestion throughout the manuscript. & 9: [ Irrelevant references (such as Ref 8, and Ref 9) can be avoided], [Write all references in one format (please check Ref 11)]. I agree with this and have incorporated your suggestion throughout the manuscript. I look forward to hearing from you in due time regarding my submission and to respond to any further questions and comments you may have. Sincerely, Pharmacist Farah Haidar Abdulredha" } ] }, { "id": "234283", "date": "15 Feb 2024", "name": "Muhammad Usman Khan", "expertise": [ "Reviewer Expertise Computational 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\nIn this contribution entitled “In silico molecular docking, ADME study and synthesis of new 1,3-diazetidin-2-one derivatives with high anti-proliferative activity” Novel 1,3-diazetidin-2-one derivatives were designed, synthesized from mefenamic acid, and their cytotoxic activity against a lung cancer cell line (A549) was initially tested in vitro. The newly synthesized derivatives were verified and confirmed by elemental analysis and spectroscopic data (FT-IR, 1H-NMR, and 13C-NMR). In addition, physicochemical, drug-like, and toxicological predictions were performed for these derivatives. In my opinion, this work is very useful study and very much relevant to the journal’s scope. This research is properly handled, results are accurate and written with good graphics. It could be accepted after addressing these minor issues.\n\nAvoid abbreviation in abstract. Authors are advised to use general terms or specific name of particular compound. Avoid abbreviation in keywords. Introduction should clearly state the aim of study. All the units should have spaced from numbers. Tables should be aligned properly. The author should add one-word space before citing reference number in the whole manuscript. Extra spacing should be removed from the whole manuscript. A few typo errors have been noted. The typing errors need to be corrected. Please indicate the full name of each abbreviation at its first appearance in the draft. Enhance the introduction section of your study by incorporating the specified articles related to combined experimental-computational studies and comparing your results with those of other studies mentioned in the following articles. This comparison will provide valuable insights into the similarities and differences between your findings and the existing literature in the investigated field. The suggested articles are these:\nKhalid et al., (2020a) 1; Tariq et al.,(2020a) 2; Tariq et al., (2020b) 3; Khalid et al., (2020b) 4\n\n11. All Figures and Tables in the manuscript are needed to magnify and properly arrange to make it suitable for reading.\n\n12.There are errors, such as incorrect use of subscripts, mistyped molecular formulas, inconsistent capitalization within sentences, and missing articles where necessary. I suggest carefully rereading the article and correcting these issues to enhance the clarity and accuracy of the article\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/12-1090
https://f1000research.com/articles/11-686/v1	21 Jun 22	{ "type": "Research Article", "title": "EncephalApp Stroop Test for covert hepatic encephalopathy screening in Tunisian cirrhotic patients", "authors": [ "Lamine Hamzaoui", "Moufida Mahmoudi", "Ghanem Mohamed", "Hanene Elloumi", "Asma Laabidi", "Jalel Boubaker", "Mona Boudabbous", "Nabil Tahri", "Imen Jemni", "Leila Safer", "Taieb Jomni", "Hedi Douggui", "Dorra Trad", "Dalila Gargouri", "Shema Ayadi", "Radhouane Debbeche", "Sami Belhouchet", "R Marouani", "Imed Cheikh", "Mohamed Nabil Abdelli", "Moufida Mahmoudi", "Ghanem Mohamed", "Hanene Elloumi", "Asma Laabidi", "Jalel Boubaker", "Mona Boudabbous", "Nabil Tahri", "Imen Jemni", "Leila Safer", "Taieb Jomni", "Hedi Douggui", "Dorra Trad", "Dalila Gargouri", "Shema Ayadi", "Radhouane Debbeche", "Sami Belhouchet", "R Marouani", "Imed Cheikh", "Mohamed Nabil Abdelli" ], "abstract": "Background: Covert hepatic encephalopathy (CHE) is underdiagnosed and is difficult to detect. The EncephalApp Stroop test is validated for its screening. The aim of the study was to define Tunisian norms for the test based on healthy controls norms and to estimate the prevalence of CHE in cirrhotic Tunisian patients. Methods: A prospective, multicenter, cross‐sectional study was conducted. Ambulatory or hospitalized cirrhotic patients aged 40 years and over were recruited at 11 centers. Healthy subjects aged 40 years and over were recruited at 8 centers. We used a translated Arabic version of the streamlined EncephalApp Stroop test. The task has two components: “Off” and “On” state depending on the discordance or concordance of the stimuli. Results: 142 patients were included. The mean age was 57.26 years [40–86]. 40 (28.17%) of cirrhotic patients who were included were diagnosed as having a minimal hepatic encephalopathy or CHE. Among the ineligible patients, 22 had overt hepatic encephalopathy. If we consider these patients, the overall prevalence rate of CHE was around 24.39% in cirrhotic patients. It was more frequent in women (34.21% vs 25.96%), and in patients whose level of school education is between 6 and 13 years. Its prevalence does not appear to be affected by gender, MELD score, etiology of cirrhosis and age group of patients, as these variables were independent with respective p according to the chi-square test 0.413; 0.736; 0.663 and 0.1. The stroop times (On / Off and On + Off) correlated significantly with each other, are associated significantly and positively with age (respective Pearson coefficients: 0.578; 0.567 and 0.6). The more the age increases, the more the stroop response times increases (p > 10-3). Conclusions: EncephalApp Stroop test was an efficient screening tool for CHE in Tunisian cirrhotic patients.", "keywords": [ "Cirrhosis", "Tool", "Subclinical hepatic encephalopathy", "West Haven Criteria", "Medical apps" ], "content": "Abbreviations\n\nANT: Animal Naming test\n\nCFF: Critical Flicker Frequency\n\nCHE: covert hepatic encephalopathy\n\nCRT: Continuous Reaction Time (CRT)\n\nHE: hepatic encephalopathy\n\nICT: inhibitory control test\n\nISHEN: International Society for Hepatic Encephalopathy and Nitrogen Metabolism\n\nMELD: Model for End-Stage Liver Disease\n\nNCT-B: Number connection test- B (NCT-B)\n\nOHE: overt hepatic encephalopathy\n\nPHES: Psychometric Hepatic Encephalopathy Score\n\nSDT: serial dotting test\n\nSPT: standard psychometric test\n\nStE: Stroop Encephal-App\n\n\nIntroduction\n\nCovert hepatic encephalopathy (CHE) is a part of the spectrum of neurocognitive impairment in cirrhosis.1,2 Patients with CHE have slow alterations of their psychomotor and neuropsychiatric functions,3 without any clinical evidence of cognitive changes, making its diagnosis a veritable challenge.4–7 Despite representing the earliest and mildest form of hepatic encephalopathy (HE),3 CHE is associated with increased risks of progression to overt hepatic encephalopathy (OHE), hospitalization and mortality.3,5,8,9 It is also associated with impaired employment, driving capability and quality of life.8–14 CHE treatment can improve quality of life and prevent progression to OHE.15–18 However, this condition represents the most underdiagnosed form of HE and its treatment is not part of the standard of care.19,20 Currently, there is several validated testing modalities to detect this complication. Stroop Encephal-App (StE) test, a sensitive test for the detection of cognitive impairment in CHE,6,21,22 is an attractive option for point of care testing strategy of CHE in cirrhotic patients. It evaluates cognitive flexibility and psychomotor speed.21,22 It is a simple, short, valid and reliable test.6,21,22 The aims of the current study were to define Tunisian norms for the StE test based on healthy controls norms and to estimate the prevalence of CHE in cirrhotic Tunisian patients.\n\n\nMethods\n\nThis was a prospective, multicenter, cross-sectional study conducted from May 14, 2018, to October 2, 2020, at 11 centers across Tunisia. The study was designed and led by a steering committee that included academic investigators and representatives from AlphaSigma. A contract research organization (Poseidoncro) performed database development, centers monitoring and data analysis. The study was conducted in accordance with the International Council for Harmonization guidelines for Good Clinical Practice and the 1975 Declaration of Helsinki as well as local regulations and was approved by institutional review boards and ethics committees at each trial center. Written informed consent was obtained from all participants.\n\nAmbulatory or hospitalized cirrhotic patients aged 40 years and over were recruited at 11 centers. We excluded patients with history or evidence at screening of OHE . Other exclusion criteria were illiterate patient, inability to perform the test due to mental or physical disabilities, red-green color blindness, psychoactive medications use and history of transjugular intrahepatic portosystemic shunt or surgical portosystemic shunt.\n\nHealthy subjects aged 40 years and over were recruited at 8 centers among members of the medical and nursing staff, their relatives, and friends. Exclusion criteria were illiterate patients, inability to perform the test due to mental or physical disabilities, red-green color blindness and psychoactive medications use.\n\nWe used a translated Arabic version of the streamlined EncephalApp stroop test. The test was carried out on an android tablet with a 10-inch screen. Briefly, the task has two components: “Off” and “On” state depending on the discordance or concordance of the stimuli. Both components were administered after two training runs. Five correct runs were required for each component. The specific results of EncephalApp are as follows: 1) total time for five correct runs in the off state (Off time); 2) number of runs needed to complete the five correct Off runs; 3) total time for five correct runs in the on state (On time); and 4) number of runs needed to complete the five correct on runs. The Off Time+On Time was the ultimate measure used to diagnose the presence of CHE.\n\nThe subjects who responded to the eligibility criteria were sorted to take the test. The data was collected through a case report form in which we gathered information about sex, age, educational level, the etiology of the cirrhosis, the alcohol or tobacco use, and the Model for End-Stage Liver Disease (MELD) score, as well as the test results for each participant.\n\nA sample of 124 healthy subjects was deemed as representative of the general population with a 80% confidence level and a 95% confidence range. A non-proportionate quota sampling strategy was used to enroll the healthy subjects. Quotas were set regarding age categories, sex, and education level. Age, sex, and education level were selected for quota sampling because they are important predictors of Stroop test performance. To guarantee the presence of the required number of healthy subjects, and to make sure of the equitability of the samples between the different centers, we added 20 healthy subjects to the study. A total of 18 participants were recruited at each of the 8 centers following a quota sampling matrix of 3×2×3 cells.\n\n\nResults\n\nAll the norms defined in the Tunisian population after adjustment for age, gender, and education level are shown in Table 1.\n\nAmong 369 recruited cirrhotic patients, 142 were included and 227 were not eligible. Almost half of the cirrhotic patients recruited are illiterate (49.32%) (Table 2).\n\nRegions\n\nCirrhotic patients are distributed into 11 centers and 6 regions (Table 3).\n\nBaseline characteristics\n\nBaseline characteristics are detailed in Table 4. The population of cirrhosis patients meeting the study selection criteria is predominantly male with an M/F sex ratio of 2.74. The mean age was 57.26 years (± DS 9.34 years) [40-86]. The North region has the population with the highest average age of 60.048 years (± 7.24), while the region of Sfax has the youngest population with an average of 53.21 years (± 9.53) (Table 5). The patients were allocated on three age groups, as studied in the validation phase (Figure 1).\n\n40 (28.17%) of cirrhotic patients who were included and passed the Stroop test were diagnosed as having MHE/CHE. Among the ineligible patients, 22 had OHE and therefore most likely do not have CHE. If we consider these patients, the overall prevalence rate of CHE was around 24.39% in cirrhotic patients. The highest prevalence rate (29.73%) was recorded in the region of Tunis (Figure 2). CHE is more frequent in women (34.21% vs 25.96%), and in patients whose level of school education is between 6 and 13 years (70% vs 20% in patients with education level <6 years and 10% in patients with education level >13 years). CHE prevalence according to age groups is represented in Figure 3.\n\nPrevalence of CHE does not appear to be affected by gender, MELD score, etiology of cirrhosis and age group of patients, as these variables were independent with respective p according to the chi-square test 0.413; 0.736; 0.663 and 0.1.\n\nThe stroop times (On/Off and On + Off) correlated significantly with each other, are correlated significantly and positively with age (respective Pearson coefficients: 0.578; 0.567 and 0.6). The more the age increases, the more the stroop response times increases (p > 10-3). On the other hand, the level of education is negatively and significantly correlated with the stroop responses times (−0.611; −0.491 and −0.584 respectively; p ≤ 10-3).\n\n\nDiscussion\n\nCHE is a cognitive impairment in cirrhotic patients. It is characterized by abnormalities in attention and psychomotor speed.1 However, it is difficult to diagnose by using simple clinical examination.1,23 Its diagnosis is, also hampered by the lack of tests that can be applied at point of care without psychological expertise.23 CHE stills underdiagnosed. About 38% of American physicians never test for HE because of time, costs or requirement of trained staff and equipment.20 Another survey from India showed that only 6.3% of physicians screened their patients for minimal hepatic encephalopathy.19\n\nThe latest American and European Associations for the Study of Liver Diseases practice guidelines recommended that CHE should be diagnosed by neuropsychological, neurophysiological, or computerized testing.4 According to the International Society for Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) consensus, neuropsychological tests, such as Psychometric Hepatic Encephalopathy Score (PHES), Critical Flicker Frequency (CFF), Continuous Reaction Time (CRT), EncephalApp, and Animal Naming test (ANT) have had validation and could be recommended for investigating CHE. The combination of two tests and more to establish the diagnosis of CHE is discouraged.24\n\nSome CHE diagnostic tests like PHES are time consuming and necessitate trained physicians.21,25 The application of rapid and simple point-of-care tests like ANT or StE could be of great benefit for the screening of CHE.6,24,26 StE Assesses cognitive flexibility and psychomotor speed in paper-pencil and electronic formats through a conflict between word reading and color naming.22,27 Since 2013, Bajaj et al., found that the StE was able to detect cognitive dysfunction with good discriminative validity and test-retest reliability in cirrhosis.21 Later studies had confirmed the face and external validity and test-retest reliability of this test.6,22\n\nThe StE is easy to administer, quick to teach to subjects, and simple to score and interpret. Its use is appropriate for CHE screening.21,27 Moreover, when we consider the financial impact of untreated CHE, the cost effectiveness ratio is in favor of StE.28,29 This test is available on iTunes for free download in different languages.21 The major limitation of its use is the requirement a compatible device to be applicable.\n\nThe stroop test consists to measure psychomotor function (time required to complete the tasks), cognitive flexibility (On time - off time) and errors committed (number of runs required).21 It was found that psychomotor function was more predictive than errors committed and cognitive flexibility in differentiating patients with CHE and without CHE.30–32 The consistent differentiators between affected and unaffected patients with CHE are the indices of psychomotor speed and reaction time (Off time + On time) and not measures of cognitive flexibility (On time - Off times) or those of accuracy (number of runs required).32 In fact, it was demonstrated that On Time + Off Time was the best element to discriminate CHE patients among cirrhotic patients.21,22 The cutoff values retained for Off time + On time in the US cirrhotic patients were 274.9, 195.9 and 190 seconds based on PHEES, SPT or ICT.6,21,22 In Chinese patients, the cutoff value retained for Off time + On time was 186.63 seconds based on PHES.25 Additional studies are indicated to validate diagnostic cutoffs.24 The sensitivity and the specificity of StE was ranging from 72% to 88% and 54% to 81% respectively according to the reference test and to the study: PHSPT, standard psychometric test (SPT) or inhibitory control test (ICT).6,21,22,25 The first study conducted by Bajaj et al, showed that the EncephalApp had a sensitivity of 78% and specificity of 90% for diagnosing MHE when using the PHES as the reference standard.21 A subsequent study realized by the same team, found 80% sensitivity and 81% specificity for the diagnosis of CHE in patients without prior OHE.22 A US multicenter study reported that the EncephalApp had a sensitivity of 80% and of 70% and a specificity of 61% and 54% based on PHES and ICT respectively.6 A more recent Chinese study demonstrated 86% sensitivity and 59% specificity.25\n\nIn our study, because of the lack of Tunisian norms, we first recruited healthy controls for the diagnosis of CHE. The cutoff values were to retain the diagnosis of CHE and were obtained according to the values recorded in healthy subjects (+ two Standard deviation) after adjustment for age, gender, and education level. The lack of a reference standard test may constitute one limitation in our study although it have been demonstrated that the use of PHES and StE, whether alone or in combination, were found to be equivalent for screening CHE, which indicates that single testing with the StE is sufficient for screening CHE.33 Recently, a Chinese study suggested that the combination of StE and two subtests of PHES (Number connection test- B (NCT-B) and serial dotting test (SDT)) is a good diagnostic tool for CHE in cirrhotic patients with 97.7% sensitivity, 79.3 specificity and 86.9% accuracy. This combination is also time saving compared to PHES.7\n\nThe prevalence of CHE in cirrhotic patients varies between 20% and 80% around the world.5,19,34–38 Epidemiologic studies regarding CHE are lacking in Tunisia. There is no normalized and validated data for appropriate diagnostic test for MHE. Our study is the first one screening CHE in Tunisian cirrhotic patients using StE test. The prevalence of CHE was 24.39%.\n\nSeveral factors such as age, severity of liver disease, alcoholic etiology, history of OHE and portosystemic shunts could influence the prevalence of CHE.39 It was observed that age and education levels were correlated with the risk of CHE diagnosis based on StE. Gender influence on StE outcomes was however controversial.6,21,22,25 Patients with alcoholic liver disease were more likely to perform worse StE scores.21,40 These data indicates that contrary to the benefic effect of education on cognitive functions, age and alcohol could have a negative impact on cognitive ability.21,25 Smoking was also considered as a risk factor for MHE.41,42 In our study, CHE was more frequent in women, elderly patients and in patients whose level of school education is between 6 and 13 years compared to patients with education level <6 years and >13 years). None of our patients had alcoholic cirrhosis.\n\nConcerning the severity of the liver disease, it was established that the application is able de discriminate patients from the early to advanced stage of cirrhosis.14,43,44 The StE times were correlated with MELD scores21,22 and were worse in previous OHE patients.6,21,22,25 According to an American multicenter study, poor StE performance based on ICT predicted OHE independently of the MELD (Hazard ratio: 4.1).6 It has been observed that patients with previous OHE did not improve on repeated testing contrary to patients without previous OHE.43,44 The StE could also be considered for therapeutic trials.21\n\nIn our study, the prevalence of CHE does not appear to be affected by gender, MELD score, etiology of cirrhosis and age group of patients (p = 0.413; 0.736; 0.663 and 0.1 respectively). The stroop times (On/Off and On + Off) were significantly and positively correlated with age (respective Pearson coefficients: 0.567 and 0.6). The more the age increases, the more the stroop response times increases (p > 10-3). On the other hand, the level of education is negatively and significantly correlated with the stroop responses times (p ≤ 10-3).\n\nOur study is the first one estimating the prevalence of CHE in Tunisia. There are some limitations to the present study. Firstly, there is the lack cutoff values of StE based on a reference standard test and secondly, a relatively small sample size of patients was recruited in this study. Further multicenter studies are required to determine the cutoff values of StE for the diagnosis of CHE and the prevalence of CHE in Tunisian patients.\n\n\nConclusion\n\nEncephalApp Stroop test, validated in several studies, was an efficient screening tool for CHE in Tunisian cirrhotic 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.", "appendix": "References\n\nBajaj JS, Wade JB, Sanyal AJ: Spectrum of neurocognitive impairment in cirrhosis: Implications for the assessment of hepatic encephalopathy. Hepatology. 2009; 50(6): 2014–2021. PubMed Abstract \| Publisher Full Text\n\nFerenci P, Lockwood A, Mullen K, et al.: Hepatic encephalopathy–definition, nomenclature, diagnosis, and quantification: final report of the working party at the 11th World Congresses of Gastroenterology, Vienna, 1998. Hepatology. 2002; 35(3): 716–721. PubMed Abstract \| Publisher Full Text\n\nKaranfilian BV, Park T, Senatore F, et al.: Minimal Hepatic Encephalopathy. Clin. Liver Dis. 2020; 24(2): 209–218. Publisher Full Text\n\nVilstrup H, Amodio P, Bajaj J, et al.: Hepatic encephalopathy in chronic liver disease: 2014 Practice Guideline by the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver. Hepatology. 2014; 60(2): 715–735. PubMed Abstract \| Publisher Full Text\n\nPatidar KR, Thacker LR, Wade JB, et al.: Covert hepatic encephalopathy is independently associated with poor survival and increased risk of hospitalization. Am. J. Gastroenterol. 2014; 109(11): 1757–1763. PubMed Abstract \| Publisher Full Text\n\nAllampati S, Duarte-Rojo A, Thacker LR, et al.: Diagnosis of Minimal Hepatic Encephalopathy Using Stroop EncephalApp: A Multicenter US-Based, Norm-Based Study. Am. J. Gastroenterol. 2016; 111(1): 78–86. PubMed Abstract \| Publisher Full Text\n\nZeng X, Zhang L-Y, Liu Q, et al.: Combined Scores from the EncephalApp Stroop Test, Number Connection Test B, and Serial Dotting Test Accurately Identify Patients With Covert Hepatic Encephalopathy. Clin. Gastroenterol. Hepatol. 2020; 18(7): 1618–1625.e7. PubMed Abstract \| Publisher Full Text\n\nTapper EB, Parikh ND, Waljee AK, et al.: Diagnosis of Minimal Hepatic Encephalopathy: A Systematic Review of Point-of-Care Diagnostic Tests. Am. J. Gastroenterol. 2018; 113(4): 529–538. PubMed Abstract \| Publisher Full Text\n\nRidola L, Cardinale V, Riggio O: The burden of minimal hepatic encephalopathy: from diagnosis to therapeutic strategies. Ann. Gastroenterol. 2018; 31(2): 151–164. PubMed Abstract \| Publisher Full Text\n\nLabenz C, Baron JS, Toenges G, et al.: Prospective evaluation of the impact of covert hepatic encephalopathy on quality of life and sleep in cirrhotic patients. Aliment. Pharmacol. Ther. 2018; 48(3): 313–321. PubMed Abstract \| Publisher Full Text\n\nBajaj JS, Saeian K, Schubert CM, et al.: Minimal hepatic encephalopathy is associated with motor vehicle crashes: the reality beyond the driving test. Hepatology. 2009; 50(4): 1175–1183. PubMed Abstract \| Publisher Full Text\n\nShaw J, Bajaj JS: Covert Hepatic Encephalopathy: Can My Patient Drive?. J. Clin. Gastroenterol. 2017; 51(2): 118–126. PubMed Abstract \| Publisher Full Text\n\nSoriano G, Román E, Córdoba J, et al.: Cognitive dysfunction in cirrhosis is associated with falls: a prospective study. Hepatology. 2012; 55(6): 1922–1930. PubMed Abstract \| Publisher Full Text\n\nDhiman RK, Kurmi R, Thumburu KK, et al.: Diagnosis and prognostic significance of minimal hepatic encephalopathy in patients with cirrhosis of liver. Dig. Dis. Sci. 2010; 55(8): 2381–2390. PubMed Abstract \| Publisher Full Text\n\nBajaj JS, Heuman DM, Wade JB, et al.: Rifaximin improves driving simulator performance in a randomized trial of patients with minimal hepatic encephalopathy. Gastroenterology. 2011; 140(2): 478–487.e1. PubMed Abstract \| Publisher Full Text\n\nPrasad S, Dhiman RK, Duseja A, et al.: Lactulose improves cognitive functions and health-related quality of life in patients with cirrhosis who have minimal hepatic encephalopathy. Hepatology. 2007; 45(3): 549–559. PubMed Abstract \| Publisher Full Text\n\nSharma P, Sharma BC, Agrawal A, et al.: Primary prophylaxis of overt hepatic encephalopathy in patients with cirrhosis: an open labeled randomized controlled trial of lactulose versus no lactulose. J. Gastroenterol. Hepatol. 2012; 27(8): 1329–1335. PubMed Abstract \| Publisher Full Text\n\nSidhu SS, Goyal O, Mishra BP, et al.: Rifaximin improves psychometric performance and health-related quality of life in patients with minimal hepatic encephalopathy (the RIME Trial). Am. J. Gastroenterol. 2011; 106(2): 307–316. PubMed Abstract \| Publisher Full Text\n\nSharma P, Sharma BC: A survey of patterns of practice and perception of minimal hepatic encephalopathy: a nationwide survey in India. Saudi J. Gastroenterol. 2014; 20(5): 304–308. PubMed Abstract \| Publisher Full Text\n\nBajaj JS, Etemadian A, Hafeezullah M, et al.: Testing for minimal hepatic encephalopathy in the United States: An AASLD survey. Hepatology. 2007; 45(3): 833–834. PubMed Abstract \| Publisher Full Text\n\nBajaj JS, Thacker LR, Heuman DM, et al.: The Stroop smartphone application is a short and valid method to screen for minimal hepatic encephalopathy. Hepatology. 2013; 58(3): 1122–1132. PubMed Abstract \| Publisher Full Text\n\nBajaj JS, Heuman DM, Sterling RK, et al.: Validation of EncephalApp, Smartphone-Based Stroop Test, for the Diagnosis of Covert Hepatic Encephalopathy. Clin. Gastroenterol. Hepatol. 2015; 13(10): 1828–1835.e1. PubMed Abstract \| Publisher Full Text\n\nKappus MR, Bajaj JS: Covert hepatic encephalopathy: not as minimal as you might think. Clin. Gastroenterol. Hepatol. 2012; 10(11): 1208–1219. PubMed Abstract \| Publisher Full Text\n\nBajaj JS, Lauridsen M, Tapper EB, et al.: Important Unresolved Questions in the Management of Hepatic Encephalopathy: An ISHEN Consensus. Am. J. Gastroenterol. 2020; 115(7): 989–1002. PubMed Abstract \| Publisher Full Text\n\nZeng X, Li X-X, Shi P-M, et al.: Utility of the EncephalApp Stroop Test for covert hepatic encephalopathy screening in Chinese cirrhotic patients. J. Gastroenterol. Hepatol. 2019; 34(10): 1843–1850. PubMed Abstract \| Publisher Full Text\n\nCampagna F, Montagnese S, Ridola L, et al.: The animal naming test: An easy tool for the assessment of hepatic encephalopathy. Hepatology. 2017; 66(1): 198–208. PubMed Abstract \| Publisher Full Text\n\nLuo M, Mu R, Liu J-F, et al.: Novel computerized psychometric tests as primary screening tools for the diagnosis of minimal hepatic encephalopathy. World J. Clin. Cases. 2020; 8(16): 3377–3389. PubMed Abstract \| Publisher Full Text\n\nHuang E, Esrailian E, Spiegel BMR: The cost-effectiveness and budget impact of competing therapies in hepatic encephalopathy - a decision analysis. Aliment. Pharmacol. Ther. 2007; 26(8): 1147–1161. PubMed Abstract \| Publisher Full Text\n\nBajaj JS, Pinkerton SD, Sanyal AJ, et al.: Diagnosis and treatment of minimal hepatic encephalopathy to prevent motor vehicle accidents: a cost-effectiveness analysis. Hepatology. 2012; 55(4): 1164–1171. PubMed Abstract \| Publisher Full Text\n\nSchiff S, D’Avanzo C, Cona G, et al.: Insight into the relationship between brain/behavioral speed and variability in patients with minimal hepatic encephalopathy. Clin. Neurophysiol. 2014; 125(2): 287–297. PubMed Abstract \| Publisher Full Text\n\nWeissenborn K, Heidenreich S, Ennen J, et al.: Attention deficits in minimal hepatic encephalopathy. Metab. Brain Dis. 2001; 16(1-2): 13–19. Publisher Full Text\n\nLauridsen MM, Thiele M, Kimer N, et al.: The continuous reaction times method for diagnosing, grading, and monitoring minimal/covert hepatic encephalopathy. Metab. Brain Dis. 2013; 28(2): 231–234. PubMed Abstract \| Publisher Full Text\n\nDuarte-Rojo A, Allampati S, Thacker LR, et al.: Diagnosis of covert hepatic encephalopathy: a multi-center study testing the utility of single versus combined testing. Metab. Brain Dis. 2019; 34(1): 289–295. PubMed Abstract \| Publisher Full Text\n\nWang A-J, Peng A-P, Li B-M, et al.: Natural history of covert hepatic encephalopathy: An observational study of 366 cirrhotic patients. World J. Gastroenterol. 2017; 23(34): 6321–6329. PubMed Abstract \| Publisher Full Text\n\nSaxena N, Bhatia M, Joshi YK, et al.: Auditory P300 event-related potentials and number connection test for evaluation of subclinical hepatic encephalopathy in patients with cirrhosis of the liver: a follow-up study. J. Gastroenterol. Hepatol. 2001; 16(3): 322–327. PubMed Abstract \| Publisher Full Text\n\nSchomerus H, Hamster W: Quality of life in cirrhotics with minimal hepatic encephalopathy. Metab. Brain Dis. 2001; 16(1-2): 37–41. Publisher Full Text\n\nBajaj JS: Management options for minimal hepatic encephalopathy. Expert Rev. Gastroenterol. Hepatol. 2008; 2(6): 785–790. PubMed Abstract \| Publisher Full Text\n\nRomero-Gómez M, Córdoba J, Jover R, et al.: Value of the critical flicker frequency in patients with minimal hepatic encephalopathy. Hepatology. 2007; 45(4): 879–885. Publisher Full Text\n\nDhiman RK, Chawla YK: Minimal hepatic encephalopathy. Indian J. Gastroenterol. 2009; 28(1): 5–16. Publisher Full Text\n\nGunn C, Mackus M, Griffin C, et al.: A systematic review of the next-day effects of heavy alcohol consumption on cognitive performance. Addiction. 2018; 113(12): 2182–2193. PubMed Abstract \| Publisher Full Text\n\nSabia S, Elbaz A, Dugravot A, et al.: Impact of smoking on cognitive decline in early old age: the Whitehall II cohort study. Arch. Gen. Psychiatry. 2012; 69(6): 627–635.\n\nRathi S, Chopra M, Chouduri G, et al.: Prevalence of Minimal Hepatic Encephalopathy in Patients With Liver Cirrhosis: A Cross-Sectional, Clinicoepidemiological, Multicenter, Nationwide Study in India: The PREDICT Study. J. Clin. Exp. Hepatol. 2019; 9(4): 476–483. PubMed Abstract \| Publisher Full Text\n\nRiggio O, Ridola L, Pasquale C, et al.: Evidence of persistent cognitive impairment after resolution of overt hepatic encephalopathy. Clin. Gastroenterol. Hepatol. 2011; 9(2): 181–183. PubMed Abstract \| Publisher Full Text\n\nBajaj JS, Schubert CM, Heuman DM, et al.: Persistence of cognitive impairment after resolution of overt hepatic encephalopathy. Gastroenterology. 2010; 138(7): 2332–2340. PubMed Abstract \| Publisher Full Text" }	[ { "id": "141643", "date": "16 Aug 2022", "name": "Rym Ennaifer", "expertise": [ "Reviewer Expertise Hepatology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 multicentric cross sectional study. The methods are well detailed, and the number of patients included is sufficient. Covert encephalopathy is a topical subject in hepatology and difficult to diagnose. The diagnostic method (stroop test) studied is interesting but needs validation.\nConcerning the end of the discussion “Our study is the first one estimating the prevalence of CHE in Tunisia”, actually it is not the first Tunisian study, and here is the reference to cite: Encéphalopathie hépatique minime: un diagnostic précoce pour améliorer le pronostic Minimal hepatic encephalopathy: A better diagnostic to improve prognostic1\nIn this study, we used 5 diagnostic tests: Psychometric hepatic encephalopathy score » (PHES): digit symbol test, number connection test A, number connection test B, serial dotting test et line tracing test and the prevalence was 44%. It is very important to cite Tunisian numbers+++ and it was one of the earliest studies about covert encephalopathy in the literature.\n\nIs the work 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": "10123", "date": "01 Sep 2023", "name": "Lamine Hamzaoui", "role": "Author Response", "response": "- Prevalence of CHE in the other tunisian study was precised and the study was cited as a reference" } ] }, { "id": "146617", "date": "22 Aug 2022", "name": "Helene Larrue", "expertise": [ "Reviewer Expertise chronic liver disease", "portal hypertension" ], "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 the present study, the authors aimed both to define specific norms for Stroop Encephal-App test (StE) and to estimate the prevalence of covert hepatic encephalopathy (CHE) in cirrhotic Tunisian population. This was a prospective multicenter, cross-sectional study with healthy controls to help define norms.\nA total of 142 cirrhotic patients and 124 healthy subjects were recruited. Tunisian norms of the stroop on + off time variable was defined in healthy subjects, in subgroups by age, gender and study level.\nThe prevalence of CHE in cirrhotic patients was 28.17% and was more frequent in women (34.21 vs 25.96%) and in patients whose level of school education was between 6 – 13 years (70% vs 20% in patients with education level <6 years).\nThis work validated the use of EncephalApp Stroop test as an efficient tool to diagnose CME. Also this is the first study to estimate the prevalence of CHE in cirrhotic Tunisian population.\nThe major concern is the lack of definition of cutoff values of StE to define CHE as the authors didn’t use any reference test. So, we don’t know whether the cutoff values (based on healthy patients) used to classify patients as having CHE or not were right. Thus, neither of the study's objectives were properly met.\nIn addition, the sample size is small and near 61% of patients recruited weren’t included mainly because of illiteracy.\nIn the section “Prevalence of CHE”, it’s said CHE is more frequent in women… but there are no statistical tests proving it (no p-value).\nIn table 3, it’s not very clear the difference between local prevalence of CHE and global prevalence of CHE, please clarify this.\nIn the section “Analytic statistics”, it’s written “The stroop times correlated significantly with each other are correlated significantly and positively with age”, please rephrase.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10124", "date": "01 Sep 2023", "name": "Lamine Hamzaoui", "role": "Author Response", "response": "1. The major concern is the lack of definition of cutoff values of StE to define CHE as the authors didn’t use any reference test. So, we don’t know whether the cutoff values (based on healthy patients) used to classify patients as having CHE or not were right. Thus, neither of the study's objectives were properly met. Response: Thank you for this pertinent comment. Indeed, the lack of a standard reference test may be a limitation of our study, but as we clearly state in the discussion: \"the use of PHES and StE, alone or in combination, has been shown to be equivalent for screening for HEC, indicating that a single test with StE is sufficient for screening for HEC.33\" With regard to threshold values, as with all tests, it is vital to establish local thresholds, based on healthy subjects from the general population, which was done in our study and in all other studies using this tool (recent reference example: Cunha-Silva M, Neto FLP, de Araújo PS, Pazinato LV, Greca RD, Secundo TML, Imbrizi MR, Monici LT, Sevá-Pereira T, Mazo DF. EncephalApp Stroop Test validation for the screening of minimal hepatic encephalopathy in Brazil. Ann Hepatol. 2022 Jan-Feb;27(1):100543. doi: 10.1016/j.aohep.2021.100543. Epub 2021 Sep 24. PMID: 34571266.) 2. In addition, the sample size was small and almost 61% of patients recruited were not included, mainly due to illiteracy. Response: 2.1. The sample size (n=142) was well above the statistically required number of patients. The minimum population size was determined by the Schwarz formula: n= (i² x p x q) / α² i = 95% confidence level (i = 0.95) p = prevalence of hepatic encephalopathy in cirrhotics is estimated at 0.3 based on a previous study (ref A) q = 1-p (q= 0.7) α = margin of error at 5% (α = 0.05) n = (0.9025 x 0.7 x 0.3)/0.0025= 75,81 REF A: Fallahzadeh MA, Rahimi RS. Hepatic Encephalopathy: Current and Emerging Treatment Modalities. Clin Gastroenterol Hepatol. 2022 Aug;20(8S):S9-S19. doi: 10.1016/j.cgh.2022.04.034. PMID: 35940731. 2.2. There are several reasons for the high rate of illiteracy in our study. Firstly, the illiteracy rate is high in Tunisia (17,7% in 2022). According to figures from the National Institute of Statistics, the illiteracy rate among women and men in communal areas has reached 12.9%, while it has risen to 29.5% in non-communal areas. Moreover, the illiteracy rate rises as the average age increases, reaching 2.8% for the 10-14 age group, while it reaches 79.8% among the elderly (aged 80 and over). REF B However, our population tends to be older, from rural and disadvantaged areas, like most of the patients treated in Tunisian public hospitals, which explains the high illiteracy rate and therefore the non-inclusion rate. REF B: https://www.courrierinternational.com/article/societe-en-tunisie-les-2-millions-danalphabetes-representent-une-bombe-retardement-sociale 3. In the section “Prevalence of CHE”, it’s said CHE is more frequent in women… but there are no statistical tests proving it (no p-value). Response: Prevalence of CHE wasn’t affected by gender (p=0.413). The sentence « CHE is more frequent in women » was deleted 4. In table 3, it’s not very clear the difference between local prevalence of CHE and global prevalence of CHE, please clarify this. Response: The table reports the prevalence of CHE in different regions. It shows that the rates in the hospitals of Tunis (the capital) were higher than the other regions." } ] } ]	1	https://f1000research.com/articles/11-686
https://f1000research.com/articles/12-1086/v1	01 Sep 23	{ "type": "Research Article", "title": "Illuminating the English as a Foreign Language (EFL) teaching odyssey: unveiling the professional identities of pre-service teachers", "authors": [ "Thanh-Thao Le", "Hoang-Yen Phuong", "Anh-Thi Nguyen", "Trut-Thuy Pham", "Anh-Thu Huynh Thi", "Huong-Tra Nguyen", "Thanh-Thao Le", "Anh-Thi Nguyen", "Trut-Thuy Pham", "Anh-Thu Huynh Thi", "Huong-Tra Nguyen" ], "abstract": "Background: This study delves into the self-perceived professional identities of pre-service English as a Foreign Language (EFL) teachers within a Vietnamese context. The objective of this study was to augment the understanding of the evolution of teacher identity, which would subsequently provide valuable insights for the formulation of bespoke teacher education programs. Methods: This research was anchored in grounded theory, adhering to a qualitative research design. Reflective writing was the primary instrument for data collection, with 238 pre-service teachers, enrolled in an English Teacher Education training program. Data collection took place in the academic year 2022-2023 at a university in Can Tho city, Vietnam from February 1st until May 31st, 2023. Pre-service teachers were tasked with writing an 80-word reflection on their perceived professional identity. Participants were chosen through convenience sampling. Data analysis encompassed open coding and a constant comparative method, culminating in the construction of a theoretical framework. Results: The findings of this study revealed a continuum of self-perceived professional identities among 238 pre-service teachers (28 identities in total), encompassing flexible teachers (n=24), student-cantered teachers (n=21), reflective teachers (n=16), growth-oriented teachers (n=13), engaging teachers (n=12), supportive teachers (n=11), enthusiastic teachers (n=10), disciplinarian teachers (n=9), subject-matter expert teachers (n=9), resourceful teachers (n=8), balanced teachers (n=8), pragmatic teachers (n=7), relationship-builder teachers (n=7), sociable teachers (n=7), attentive teachers (n=7), real-world connector teachers (n=7), professional boundaries teachers (n=7), interactive teachers (n=6), traditional teachers (n=6), holistic teachers (n=6), contextually aware teachers (n=5), cheerful teachers (n=5), authentic teachers (n=5), appreciated teachers (n=5), compassionate communicator teachers (n=5), guided teachers (n=4), feedback-driven teachers (n=4), and consistent identity teachers (n=4). Conclusions: The study offers invaluable perspectives into the genesis of professional identities in the domain of teacher education within the Vietnamese EFL context and holds significant implications for the enhancement of teacher training methodologies and pedagogical development.", "keywords": [ "EFL pre-service teachers", "grounded theory", "professional identity", "reflective practice", "teacher education" ], "content": "Introduction\n\nThe quintessentiality of teacher professional identity in influencing educational practices, perceptions, and dialogues necessitates comprehension of its genesis and evolution for efficacious teacher training and pedagogical enhancement (Lap et al., 2022; Pillen et al., 2013; Trede et al., 2012). Particularly in English as a Foreign Language (EFL) pedagogy, burgeoning teachers’ self-construed identities bears significant implications as they strive to become adept EFL educators (Izadinia, 2013).\n\nIn the face of global integration, Vietnam, akin to numerous countries, acknowledges the exigency of English competency, resulting in a marked increase in the demand for capable EFL instructors and subsequent inception of countless EFL teaching programs nationwide (Vu and Peters, 2021). Such programs strive to empower pre-service teachers with the requisite acumen, capabilities, and dispositions to excel as EFL educators. Discerning how these nascent teachers perceive their professional identities is critical for refining teacher training initiatives and advancing English language pedagogy in Vietnam.\n\nPrevious scholarship has delved into diverse facets of EFL teacher identity, yet there exists a dearth of studies centred specifically on the self-construed professional identities of Vietnamese EFL pre-service teachers. This study endeavours to scrutinize these self-perceived professional identities within the Vietnamese EFL pre-service teacher population through the application of grounded theory. By probing these self-perceptions, this study aims to enrich the extant academic discourse on teacher identity development, yielding insights that could guide the formulation of bespoke and contextually pertinent teacher education programs.\n\nTeacher professional identity\n\nTeacher identity, a convoluted and multi-layered construct, has been dissected through varied lenses (Zembylas and Chubbuck, 2018), rendering a universal definition elusive due to its multidisciplinary theoretical considerations (Beauchamp and Thomas, 2009; Rodgers and Scott, 2008). Despite its inherent diversity, researchers gravitate towards definitions congruous with their contextual, purposeful, and theoretical underpinnings. Until the 1990s, the essentialist perspective, viewing identity as static personal attributes, was prevalent (Yazan, 2018), giving way to a sociocultural perspective underscoring identity’s dynamism (Beauchamp and Thomas, 2009). Teacher identity, an evolving process moulded by experiences and context, amalgamates diverse sub-identities reflecting teachers’ various life aspects (Beijaard et al., 2004; Miller, 2009; Sahling and De Carvalho, 2021). Comprehension of its dynamic, constructive, context-specific, multifarious, and societal nature is instrumental in discerning its role in teacher growth (Akkerman and Meijer, 2011). Identity significantly impacts teachers’ convictions, practices, dedication, motivation, and self-efficacy (Day et al., 2005; Freese, 2006; Xue, 2022).\n\nGrounded theory and its essential role in framework development for unveiling teacher’s professional identities\n\nGrounded theory, a qualitative research methodology, has been extensively employed in social sciences for the generation of theoretical constructs grounded in empirical evidence (Glaser and Strauss, 2017). This approach is particularly apt for exploring intricate social phenomena, and in this research context, it is used to construct a theoretical framework pertaining to teacher professional identities. Distinctly characterized by its inductive orientation, grounded theory allows theoretical emergence from data, circumventing preconceived or externally imposed theories (Oktay, 2012). It promotes a systematic, iterative data collection and analysis process, aimed at discerning patterns, categories, and data relationships. The method underscores constant comparison, theoretical sampling, and theoretical saturation to corroborate theoretical evolution rooted in empirical substantiation.\n\nConsidering the multifaceted nature of professional identities and the diverse influences shaping them (Chu, 2019; Schutz et al., 2018), grounded theory offers a nuanced exploration of these complexities through systematic data organization and analysis, facilitating key theme identification and relationship elucidation contributing to a comprehensive framework. To develop a theoretical scaffold for teacher professional identities, context-specific comprehension is requisite. Grounded theory aids in unearthing context-driven influences moulding professional identities through a close examination of teachers’ experiences, viewpoints, and interactions within their distinctive educational milieu (Sawatsky et al., 2018; Walker and Myrick, 2006). Such contextual understanding is vital for theory creation that resonates within the specified educational context.\n\nBy emphasizing data-derived theory formation, grounded theory ensures that the developed framework is anchored firmly within participants’ experiences and perspectives. Through meticulous data analysis via coding, constant comparison, and theoretical sampling, it allows researchers to devise a framework that truly mirrors realities and insights extracted from the data, thereby enhancing theoretical findings’ credibility and applicability (Walker and Myrick, 2006). Finally, grounded theory encapsulates an iterative, reflexive data collection, analysis, and theory development process (Charmaz, 2014). Through constant comparison, researchers persistently refine and amend their theoretical construct, assimilating emerging insights and perspectives. This iterative process enables a profound comprehension of the investigated phenomena and fosters a theoretical evolution responsive to the intricacies and subtleties of teacher professional identities.\n\n\nMethod\n\nThis study obtained ethical clearance from “Hội đồng Bảo vệ đề cương đại học của Khoa Ngoại ngữ” (Bachelor’s Thesis Proposal Defence Council of School of Foreign Languages), an institutional review board affiliated with Can Tho University situated in the Mekong Delta region of Vietnam, prior to commencing data collection. The approval was granted under the official reference number T2023-53. Written informed consent was obtained from all participants with the consent forms clearly outlining the study’s purpose, voluntary nature of participation, procedures involved, potential risks and benefits, and confidentiality assurances. Participants were given the opportunity to ask questions and address any concerns before granting their consent. Adherence to ethical guidelines protected the rights, well-being, and privacy of the participants.\n\nThis study employed a qualitative research design using the grounded theory approach to explore pre-service teachers’ self-perceived identities. In-depth interviews were conducted to gain insights into participants’ experiences, beliefs, and perspectives on their professional identities (Legard et al., 2003). The data analysis involved open coding and constant comparison, resulting in the development of a comprehensive theoretical framework. The sample size was determined based on theoretical saturation, ensuring adequate data collection. Trustworthiness was confirmed through strategies such as member checking and peer debriefing. This research design contributed to understanding teacher professional identities and their formation.\n\nThe study included 238 pre-service teachers who were enrolled in an English Teacher Education program administered by the school of foreign languages, under a well-known university, situated in Can Tho city, within the Mekong Delta region of Vietnam. The participants were selected through convenience sampling to ensure diverse representation of EFL teachers. To be eligible for participation, individuals had to be enrolled in a training program of English Teacher Education at a recognized university and be midway through their pre-service training. The cohort encompassed a range of backgrounds, including variations in age, gender, and cultural diversity, providing a comprehensive perspective on the self-perceptions of pre-service EFL teachers. Table 1 displays a summary of the participants’ demographic information.\n\nThe recruitment process involved collaborating with EFL program administrators and faculty to identify potential participants. Invitations to participate were extended to eligible candidates, accompanied by a detailed explanation of the study’s purpose, objectives, procedures, and informed consent forms. Individuals who were not willing or unable to give informed consent were excluded. Demographic data such as age, gender, educational history, prior teaching experience, and relevant cultural and linguistic background attributes were collected from the participants. The demographic data encompassed within the paper documents were acquired through the assistance of the educational institution wherein the pre-service teachers were undergoing their learning process. The sample size was determined based on practical considerations, resource availability, and data saturation, which occurred when no new insights emerged from additional participants.\n\nStringent measures were taken to ensure confidentiality and anonymity. Unique identifiers were used for each participant during result reporting, and all identifying information was securely stored and solely used for research purposes. To streamline the analysis process, the pre-service teachers’ papers were initially assigned numbers based on the order in which they were reviewed by the research team. Following this, to facilitate coding, the papers were sorted according to the respective identity themes, and the ID numbers were reassigned based on these themes. For example, if the first theme included 40 participants, the numbering for that theme would range from No.1 to No.40. The numbering for the subsequent theme would then commence from No.41 onwards.\n\nIn the current study, the participants engaged in a reflective exercise where they were explicitly instructed to express an 80-word self-analysis, in English, about how they perceive their own professional identity as teachers. Participants were provided with physical paper notebooks where they were asked to record their reflective writings. The settings were varied, depending on the comfort and convenience of the participants. However, most of these writings took place in a comfortable and quiet setting, such as the pre-service teacher’s own home, to promote deep and genuine reflection. Once the handwritten reflections were completed, participants were instructed to submit the physical notebooks to the research team. The team collected the notebooks at regular intervals, or via mail for teachers who were geographically distant. It was ensured that the notebooks were handled and stored with care to prevent any loss of information. The participants were not mandated to disclose their names or any personal identification details on the paper. If any identifying information was inadvertently included, it was either substituted with fictional names or removed in its entirety. In the end, the data anonymization process was utilized, replacing unique identifiers specific to each participant with numbered codes. This ensured that the reflective entries were separated from the identities of the participants, while preserving the authenticity and reliability of the collected data.\n\nThe deployment of reflective writing as a data accrual mechanism in this investigation was intentional and advantageous for numerous reasons. Firstly, reflective writing furnished a regimented and particular task for the pre-service teachers to participate in, granting them the ability to concentrate their cognitions and express their self-perceived professional identities within the confine of an 80-word introspection. This paradigm stimulated participants to condense their conceptions and succinctly convey their viewpoints.\n\nSecondly, reflective writing offered a platform for participants to engage in introspective self-evaluation. In the process of contemplating their professional identities, participants were stimulated to ponder their convictions, principles, ambitions, and hindrances in the context of their prospective roles as pedagogues. This mechanism facilitated an exploration of their latent thoughts and sentiments, nurturing a profound comprehension of themselves as burgeoning teachers.\n\nFurthermore, the scripted introspections acted as palpable and analysable data for the researchers. By amassing and scrutinizing the participants’ self-perceived identities, the researchers acquired discernment into the intricacies and subtleties of their professional aspirations and apprehensions. The examination of these introspections proffered a holistic comprehension of the participants’ standpoints, illuminating their distinctive conceptualizations of what encapsulates the essence of being a teacher.\n\nThe reflective writings given by the participants manifested as both tangible and analysable data that the researchers could scrutinize meticulously. Two complementary research methodologies including a thematic analysis and a grounded theory were implemented to examine these reflections with an aim to dissect the participants’ self-perceived identities.\n\nThematic analysis, as an analytic approach, facilitated the recognition and categorization of recurrent themes and patterns that emerged within the reflective writings (Braun and Clarke, 2012). This process involved careful reading and re-reading of the data, and coding significant features of the data in a systematic fashion. Following the coding process, themes were identified by clustering similar codes together and constructing a thematic ‘map’. These emergent themes provided profound insights into the participants’ aspirations and apprehensions regarding their evolving professional identities. They furnished a nuanced understanding of the thoughts, feelings, and expectations that these prospective teachers harboured in relation to their future roles.\n\nIn concert with thematic analysis, grounded theory was employed in the current study as a systematic qualitative research methodology that aimed to construct theory from the data itself (Glaser and Strauss, 2017; Oktay, 2012). In line with the principles of grounded theory, the researchers did not begin with a preconceived theory; instead, they allowed the theory to emerge from the data. This involved a process of constant comparison where each piece of data was compared with all others to identify similarities and differences. Through this iterative process of data collection, coding, and analysis, the researchers were able to inductively generate a theoretical framework that was truly grounded in the empirical data.\n\nThis theoretical framework successfully encapsulated the complexities of the participants’ self-perceived professional identities, as well as the myriad perspectives and experiences that influenced their personal interpretations of what it means to embark on a journey in the educational field. By leveraging these two analytic methodologies in tandem, the researchers achieved a nuanced, multi-layered understanding of the participants’ professional aspirations and concerns. They were able to highlight the unique conceptualizations that each participant held of their identities as teachers. Consequently, this comprehensive analysis of reflective writings illuminated the distinctive, individualized interpretations of the participants’ professional journey in education, shedding light on the diverse conceptions of the essence of becoming a teacher.\n\nThe decision to agree upon a number of categories was primarily due to the diversity and richness of the reflective writings. The reflective exercise resulted in a wide range of perceptions and identities, demonstrating the complexity and multifaceted nature of the teaching profession. Each category represents a unique aspect of teaching and provides a comprehensive overview of the various professional identities perceived by teachers in their roles. For instance, if a participant expressed being particularly focused on student needs and adapting his/her teaching style to suit those needs, he/she was classified under the ‘flexible teacher’ category. However, in terms of overlapping characteristics among the categories, it is important to note that these categories were not mutually exclusive. A teacher could, for example, have some sharing attributes of a ‘flexible teacher’ and a ‘student-centred teacher’. However, for the purpose of this study, the research team allocated each participant to the category that best represented the dominant theme of their reflection. As so, by segregating the data into these specific categories, the study was able to better understand the nuances of teacher identities, gain deeper insights into the teachers’ self-perceptions, and highlight the multifarious nature of the teaching profession. Furthermore, the decision to present one quote from a participant in each category was based on an intent to provide a succinct, illustrative snapshot of each identity theme while avoiding an overload of qualitative data.\n\n\nResults\n\nThe results of this study were derived from a detailed analysis of the reflective writings provided by the teacher participants. The study identified a total of 28 distinct categories of teacher professional identities, each showcasing a unique aspect of their perceived roles. These identities include: flexible teachers (n=24), student-cantered teachers (n=21), reflective teachers (n=16), growth-oriented teachers (n=13), engaging teachers (n=12), supportive teachers (n=11), enthusiastic teachers (n=10), disciplinarian teachers (n=9), subject-matter expert teachers (n=9), resourceful teachers (n=8), balanced teachers (n=8), pragmatic teachers (n=7), relationship-builder teachers (n=7), sociable teachers (n=7), attentive teachers (n=7), real-world connector teachers (n=7), professional boundaries teachers (n=7), interactive teachers (n=6), traditional teachers (n=6), holistic teachers (n=6), contextually aware teachers (n=5), cheerful teachers (n=5), authentic teachers (n=5), appreciated teachers (n=5), compassionate communicator teachers (n=5), guided teachers (n=4), feedback-driven teachers (n=4), and consistent identity teachers (n=4).\n\nA proportion of pre-service teachers (n=24 out of 238) identified themselves as ‘flexible teachers’ showcasing an aptitude for dynamic pedagogical adaptation in response to diverse educational settings. These pre-service teachers, keenly aware of the varied learning styles and abilities in their classrooms, demonstrated resilience, compassion, and a commitment to continuous professional development. Participant 19, for instance, stated, “The dynamic nature of education demands a willingness to evolve. My teaching strategies adapt to individual student needs, curriculum changes, and pedagogical trends. Embracing innovation and nurturing an inclusive environment are fundamental to my practice.” The teacher showed evident awareness of diverse learning styles and capabilities present in his/her classrooms.\n\nThis awareness led ‘flexible teachers’ to adopt a flexible approach in pedagogy that allowed them to effectively cater to individual student needs. It further suggests that they were capable of making necessary instructional adjustments and variations in teaching methods depending on their learners’ unique requirements. The characteristics associated with this theme—resilience, compassion, and a commitment to ongoing professional development—were also notably evident. Resilience, for example, was seen in their ability to navigate and adapt to the constantly changing dynamics of educational settings. Compassion was identified in their empathetic approach towards understanding and accommodating different learning styles and capabilities. Lastly, their commitment to continuous professional development was manifested in their willingness to keep up with curriculum changes and evolving pedagogical trends.\n\nA similar amount of pre-service teachers (n=21 out of 238) identified themselves as ‘student-centred teachers’ displaying a commitment to prioritizing student needs, fostering inclusive classrooms, and promoting student autonomy. These participants utilized varied instructional strategies and differentiated approaches, nurturing a cooperative and interactive learning environment. For example, Participant 33 stated, “Empowering students is central to my approach. I involve them actively in learning and adapt strategies to their needs, promoting engagement and ownership. Peer interaction and choice within the curriculum are key elements of my practice.” The student displayed a dedication to prioritizing student needs, cultivating inclusive classrooms, and fostering student autonomy.\n\nThe theme of ‘student-centredness’ was determined based on the teachers’ prioritization of student needs above all else. These teachers exhibited a strong commitment to ensuring that their classrooms were places where each student felt heard, valued, and supported. This active prioritization highlighted their role as facilitators of student learning, as opposed to traditional, more directive forms of teaching. The associated characteristics—cultivating inclusivity, promoting student autonomy, using varied instructional strategies, and fostering a cooperative and interactive learning environment—were inferred from the behaviours, attitudes, and practices of these pre-service teachers. Inclusivity was seen in their efforts to make sure that every student was included, valued, and supported in their classrooms. This commitment to inclusivity went beyond mere acceptance and involved creating an environment where diverse learners felt like they belonged. Promotion of student autonomy, another key characteristic, was reflected in their teaching strategies that encouraged students to take charge of their own learning. By prioritizing student choice and fostering self-reliance, these teachers facilitated a greater sense of ownership and engagement in their students. The use of varied instructional strategies and differentiated approaches emphasized their commitment to adapt their teaching to the diverse needs of their students. By offering varied and adaptive learning experiences, they were able to cater to different learning styles and paces, thus enhancing student engagement and learning outcomes. The fostering of a cooperative and interactive learning environment was another key characteristic, which was evident in their promotion of peer interaction and collaborative learning. They understood the value of social learning and capitalized on opportunities to facilitate productive interactions among students.\n\nA slightly smaller subset (n=16 out of 238) embodied a ‘reflective teacher’ persona, characterized by self-analysis, pedagogical adaptation, and a proclivity for continuous professional development. As Participant 48 elucidated, “I engage in continual introspection, evolving my teaching practice through lessons learnt from both triumphs and tribulations. This growth-oriented mindset, bolstered by actively seeking feedback and challenging traditional practices, is essential for pedagogical effectiveness.” This persona was marked by self-analysis, pedagogical adaptation, and a strong inclination for continuous professional development.\n\nThe theme of ‘reflectiveness’ was determined based on these teachers’ self-awareness and willingness to critically analyse their teaching practices. A reflective teacher is someone who is deeply self-aware and engages in regular self-analysis, scrutinizing their methods, understanding the implications of their actions, and acknowledging the potential for improvement. This ongoing self-analysis helps to inform their practice and contributes to their development as effective teachers. The characteristic of pedagogical adaptation associated with this theme signifies their readiness to modify their teaching strategies as required. Reflective teachers recognize that the effectiveness of a particular teaching approach can vary with different students, contexts, and subject matters. They are willing to adapt and evolve their pedagogical practices to better meet the needs of their students and the demands of their profession. A strong inclination towards continuous professional development is another characteristic tied to this theme. Reflective teachers are committed to lifelong learning. They view professional development not as a one-time activity, but as an ongoing process that is integral to their role. They seek feedback, learn from their triumphs and tribulations, and strive to refine their skills and knowledge continually. This shows their growth-oriented mindset and their commitment to enhancing their pedagogical effectiveness.\n\nA comparable fraction of pre-service teachers (n=13 out of 238) were identified as ‘growth-oriented teachers’ indicating a commitment to continuous improvement, self-reflection, and professional development. These teachers displayed high self-awareness, sought feedback, and viewed challenges as learning opportunities. To illustrate, Participant 63 stated, “As a teacher, my journey is focused on growth. Embracing challenges, using feedback, and continuous learning are key to my practice. I believe in exploring innovative teaching strategies and staying updated with educational trends to enhance my approach.” This self-identification was marked by a commitment to continuous improvement, self-reflection, and professional development.\n\nThe theme of ‘growth-orientation’ was established based on the pre-service teachers’ dedicated commitment to continuous improvement. These teachers displayed an ongoing desire to enhance their skills and pedagogical effectiveness, as well as a readiness to engage in self-reflection and actively seek professional development opportunities. They embody the principle that learning is a lifelong journey, and this journey should be marked by continuous growth and improvement. Associated with this theme are characteristics such as high self-awareness, active feedback seeking, and viewing challenges as opportunities for learning. High self-awareness allows these teachers to critically examine their practices, strengths, and areas for improvement. It is this awareness that feeds into their growth orientation, as it enables them to identify where and how they can grow. Actively seeking feedback is another characteristic tied to this theme. Feedback plays a crucial role in professional growth, providing valuable insights into effectiveness and areas that could be improved. By actively seeking feedback, these teachers demonstrate their dedication to improving their practice. Viewing challenges as learning opportunities is the third characteristic associated with the “growth-oriented” theme. Rather than perceiving challenges as hurdles, these teachers see them as catalysts for professional growth and development. This optimistic perspective motivates them to constantly seek out new learning experiences and embrace opportunities for growth.\n\nRoughly the same number of pre-service teachers (n=12 out of 238) were identified as ‘engaging teachers’ highlighting a commitment to stimulating, relevant, and personalized learning experiences. These individuals employed innovative teaching methodologies, multimedia resources, and real-world connections, fostering a positive and supportive classroom environment. As Participant 80 articulated, “The key to a vibrant classroom is connecting learning to real-life scenarios, employing interactive activities, and understanding student interests. It is not just about effective communication but creating an emotional bond and fostering a passion for learning.” The teacher emphasized a dedication to creating stimulating, relevant, and individualized learning experiences.\n\nThe theme of ‘engagement’ was determined by these teachers’ commitment to keeping their students actively involved in the learning process. They sought to make the educational experience not only informative but also captivating and personally relevant for each student. Engaging teachers understand that students learn best when they are fully absorbed in the material, hence they strive to make their lessons as intriguing and meaningful as possible. The associated characteristics—utilizing innovative teaching methodologies, multimedia resources, and real-world connections, along with fostering a positive and supportive classroom environment—were evident in the actions and strategies employed by these teachers. The use of innovative teaching methodologies suggests their readiness to venture beyond traditional pedagogical techniques. They aim to employ diverse strategies to cater to different learning styles, making the learning experience more effective and engaging for all students. Incorporating multimedia resources is another characteristic that is associated with this theme. The use of such resources can greatly enhance the delivery of lessons and make them more interactive and appealing to the students, thus promoting better understanding and retention of the material. Making real-world connections is a significant strategy used by these teachers. They link the concepts being taught to real-life situations to make learning more meaningful and relevant. This not only facilitates a deeper understanding of the material but also helps students appreciate the practical applicability of what they learn. Fostering a positive and supportive classroom environment is another key characteristic. These teachers strive to create an atmosphere where students feel comfortable to express their ideas, engage in discussions, and take risks in their learning. This supportive environment promotes confidence, stimulates curiosity, and encourages a love for learning.\n\nA few pre-service teachers (n=11 out of 238) were found to align with the ‘supportive teachers’ category characterized by their ability to inspire and motivate students towards academic success and personal growth. They exhibited a passion for teaching, used emotionally resonant communication strategies, and provided supportive feedback. Participant 90 reflected: “Through storytelling and real-life examples, I aim to ignite curiosity and instil self-belief in my students. It is rewarding to see their confidence grow as we celebrate their achievements, ultimately inspiring them to reach their full potential.” This categorization was marked by their ability to inspire and motivate students toward academic success and personal growth.\n\nThe theme of ‘supportiveness’ was ascertained based on these teachers’ dedication to fostering a nurturing and encouraging learning environment. Supportive teachers inspire their students to strive for academic achievement and personal development. They exhibit a passion for teaching and believe in their students’ ability to succeed, thereby inspiring confidence and fostering a positive attitude toward learning. Characteristics associated with this theme—such as using emotionally resonant communication strategies and providing supportive feedback—were discerned from the teachers’ interactions with their students and their feedback approach. Emotionally resonant communication strategies are methods that elicit emotional responses and promote engagement. These teachers understand that emotions can significantly influence learning, and therefore they incorporate storytelling and real-life examples to make learning experiences more engaging and personally meaningful. Providing supportive feedback is another significant characteristic of this theme. Supportive feedback is focused on improvement and growth rather than criticism. These teachers provide constructive, positive feedback that motivates students to continue working towards their goals and celebrates their progress. It also encourages students to see mistakes as opportunities for learning, which boosts their confidence and willingness to engage in challenging tasks.\n\nA handful of pre-service teachers (n=10 out of 238) were identified as ‘enthusiastic teachers’ demonstrating an infectious passion for their subject and teaching, marked by energetic delivery and engaging instructional techniques. They aimed to create an engaging learning environment, fostering active participation and a positive teacher-student relationship. Participant 100 succinctly encapsulated this philosophy, saying, “I utilize lively discussions and interactive activities to create an electrifying classroom atmosphere, inspiring students to actively engage. My enthusiasm for teaching is the fuel that drives my students’ interest and curiosity.” The teacher displayed a contagious passion for his/her subject matter and teaching, characterized by an energetic delivery style and engaging teaching techniques.\n\nThe theme of ‘enthusiasm’ was determined based on these teachers’ vibrant passion for teaching and their subject matter. Enthusiastic teachers exhibit a high level of energy and excitement towards teaching, which is often contagious and inspires students to become interested and engaged in the learning process. Their enthusiasm helps make the classroom environment lively and inviting, and it is this positivity that can ignite a passion for learning in their students. The associated characteristics—energetic delivery, engaging instructional techniques, and fostering active participation and a positive teacher-student relationship—are evident in the teaching methods these individuals employ. An energetic delivery refers to their dynamic and lively way of presenting information, keeping students engaged and interested. This is not just about speaking loudly or quickly; it is about using varied vocal intonation, body language, and visual aids to make the content more engaging and memorable. The use of engaging instructional techniques refers to their strategic use of various teaching methods designed to actively involve students in the learning process. These techniques can include discussions, problem-solving activities, collaborative work, or multimedia presentations. Fostering active participation is another significant characteristic of enthusiastic teachers. They encourage students to take an active role in their own learning, promoting interactive discussions and hands-on activities. They also strive to build positive relationships with their students, creating a supportive and engaging learning environment where students feel comfortable expressing their ideas and asking questions.\n\nA number of pre-service teachers (n=9 out of 238) were disclosed as ‘disciplinarian teachers’. Emphasizing adaptability and problem-solving, these participants were proficient in navigating pedagogical challenges. For instance, Participant 112 expressed, “In the face of disruptions or conflicts, my goal is to maintain a calm learning atmosphere. Clear routines help foster a positive and engaging classroom climate. Addressing behavioural issues and providing support are vital for creating a sense of belonging.” This self-categorization was characterized by an emphasis on adaptability, problem-solving, and proficiency in dealing with pedagogical challenges.\n\nThe theme of ‘disciplinarian’ was established based on these teachers’ commitment to maintaining order and discipline within their classrooms. Disciplinarian teachers understand the importance of a structured, well-managed classroom for effective learning. They are prepared to address any disruptions or conflicts that may occur, ensuring a calm and conducive learning environment. The associated characteristics—adaptability, problem-solving, and proficiency in navigating pedagogical challenges—were identified from the teachers’ actions and strategies for managing their classrooms. Adaptability refers to the teachers’ ability to adjust their plans or strategies based on the situation at hand. Disciplinarian teachers are skilled in modifying their approach in response to any disruptions or changes, ensuring that learning is not affected. Problem-solving is another key characteristic. These teachers are adept at addressing and resolving any issues or conflicts that may arise in the classroom. They employ effective strategies to manage behavioural issues, ensuring a smooth and uninterrupted learning process. Their proficiency in navigating pedagogical challenges refers to their ability to handle any teaching-related issues, such as dealing with diverse learning needs, managing student behaviour, or adapting to changes in curriculum or educational policies.\n\nAn identical fraction of pre-service teachers (n=9 out of 238) were highlighted as ‘subject-matter expert teachers’ stressing their deep comprehension and expertise in their teaching discipline. These teachers underscored the significance of maintaining comprehensive content knowledge and staying abreast of advancements in their field. This commitment to continuous learning was coupled with a focus on effectively conveying complex concepts using varied instructional strategies to aid student understanding. As Participant 117 voiced, “My comprehensive knowledge forms the foundation of my effectiveness. Continual learning and clear conveyance of current, relevant information to students are pivotal to my teaching approach.” The teacher emphasized his/her in-depth understanding and expertise in his/her teaching discipline.\n\nThe ‘subject-matter expert’ theme was defined based on these teachers’ extensive knowledge and expertise in their discipline. Subject-matter expert teachers possess a deep understanding of the content they teach, enabling them to answer students’ queries accurately and clarify complex concepts effectively. They value comprehensive content knowledge as a cornerstone of their teaching effectiveness. The associated characteristics — comprehensive content knowledge, staying updated on advancements in their field, and effectively conveying complex concepts — were identified from the teachers’ self-reports and instructional strategies. Comprehensive content knowledge refers to their deep understanding of the subject matter they teach. These teachers have a firm grasp of the key concepts, theories, and developments in their discipline, which is essential for delivering high-quality instruction. Staying abreast of advancements in their field signifies their commitment to continuous learning. Subject-matter expert teachers understand the importance of staying updated on the latest research and trends in their field to ensure their teaching is current and relevant. Effectively conveying complex concepts denotes their ability to break down difficult topics into understandable segments. They use varied instructional strategies to present these concepts, ensuring that students can grasp them effectively.\n\nA few pre-service teachers (n=8 out of 238) were identified as ‘resourceful teachers’ underscoring their capacity to optimally use available resources to augment teaching practices and student learning. These teachers displayed creative pedagogical methods and problem-solving skills, exemplified by Participant 128, who stated, “I strive to enrich lessons by seeking diverse resources, be it teaching materials or tech tools. Facing classroom challenges, I see opportunities for innovative solutions. Keeping abreast of new resources through continuous professional development is key to my practice.” The teacher emphasized their ability to make optimal use of available resources to enhance his/her teaching practices and facilitate student learning.\n\nThe theme of ‘resourcefulness’ was established based on these teachers’ adeptness at creatively leveraging diverse resources to supplement their teaching. Resourceful teachers are characterized by their capacity to seek out and utilize a variety of materials and tools, ensuring a rich, dynamic, and engaging learning environment. The associated characteristics—optimal use of resources, creative pedagogical methods, and problem-solving skills—were discerned from the teachers’ strategies and approaches to instruction. Optimal use of resources refers to these teachers’ ability to effectively utilize diverse teaching materials and technological tools to enhance learning experiences. They are skilled at identifying and incorporating a wide range of resources to support their instruction, ensuring their teaching remains engaging and effective. Creative pedagogical methods describe these teachers’ innovative approach to teaching. They devise unique teaching strategies that are adapted to the needs and interests of their students, ensuring active participation and effective learning. Problem-solving skills relate to these teachers’ ability to address and resolve any challenges they may encounter in the classroom. They view these challenges as opportunities for innovation and adaptation, continually seeking solutions that enhance the learning experience.\n\nAn equivalent subset of pre-service teachers (n=8 out of 238) were delineated as ‘balanced teachers’ underscoring their commitment to harmonizing various instructional elements to optimize students’ outcomes. Participant 136 illustrated this, stating, “My teaching approach balances academic rigor with a supportive environment, addressing both cognitive and socio-emotional needs of students. It is about balancing structure with flexibility, providing clear expectations while promoting student autonomy.” This self-description emphasized his/her commitment to harmonizing various instructional elements to maximize student outcomes.\n\nThe ‘balanced teacher’ theme was determined based on these teachers’ dedication to creating an equilibrium between different instructional elements in their teaching. Balanced teachers strive to harmonize academic rigor with a supportive environment, cognitive learning with socio-emotional development, and structure with flexibility. They believe that this balanced approach contributes to optimal student outcomes. The associated characteristics—balancing academic rigor and a supportive environment, addressing cognitive and socio-emotional needs, and combining structure with flexibility—were identified from the teachers’ self-reported teaching philosophies and strategies. Balancing academic rigor and a supportive environment refers to these teachers’ ability to maintain high academic standards while creating a nurturing and encouraging learning environment. They believe in challenging their students academically but also provide the support needed to help students meet these challenges. Addressing both cognitive and socio-emotional needs points to these teachers’ focus on the holistic development of their students. They aim not only to enhance their students’ intellectual capabilities but also to foster their socio-emotional well-being. Combining structure with flexibility signifies these teachers’ ability to provide clear expectations and guidelines while also allowing room for student autonomy and individual learning paths.\n\nA smaller number of pre-service teachers (n=7 out of 238) were identified as ‘pragmatic teachers’ highlighting their propensity for practicality and efficiency in their teaching strategies, tailoring to suit their specific educational setting. For instance, Participant 144 expressed, “A balanced integration of theory and practice in my teaching strategy is crucial. I prioritize practical, evidence-informed methods considering available resources and long-term implications. Overcoming challenges with effective solutions is my primary focus.” The teacher highlighted his/her tendency towards practicality and efficiency in his/her teaching strategies, tailored to suit his/her specific educational context.\n\nThe ‘pragmatic teacher’ theme was established based on these teachers’ predilection for practical, efficient teaching strategies. Pragmatic teachers prioritize methods that are both effective and suited to their teaching context, balancing theory and practice in a way that maximizes the benefit for their students. The associated characteristics—practicality, efficiency, and a balanced integration of theory and practice—were discerned from the teachers’ self-reports and observed instructional methods. Practicality refers to these teachers’ preference for teaching methods that are directly applicable and beneficial in their teaching context. They prioritize strategies that are feasible and have proven to be effective, ensuring that their teaching is rooted in practical considerations. Efficiency relates to these teachers’ focus on maximizing the impact of their teaching strategies. They aim to utilize resources effectively and minimize wastage, always seeking the most efficient ways to facilitate learning. A balanced integration of theory and practice signifies these teachers’ commitment to align theoretical knowledge with practical application. They understand the importance of marrying theoretical principles with hands-on experiences to provide a well-rounded learning experience for their students.\n\nA similar amount of pre-service teachers (n=7 out of 238) regarded themselves as ‘relationship-builder teachers’ underlining the enhancement of constructive and significant rapport with their learners. These pre-service teachers placed the establishment of trust, respect, and amicability at the forefront of their educational spaces, aspiring to generate an inclusive and supporting milieu. Participant 150 echoed this stance, sharing, “As a relation-building pedagogue, I prioritize strong bonds with learners in a secure and respectful environment. This understanding helps me customize teaching to their unique needs and interests.” This theme emphasizes the importance these teachers place on cultivating positive, meaningful relationships with their students.\n\nThe ‘relationship-builder teacher’ theme emerged from the value these teachers place on developing strong, supportive relationships with their students. These teachers understand that positive teacher-student relationships are foundational to successful teaching and learning and contribute to an inclusive and supportive classroom environment. The associated characteristics—building trust, fostering respect, and promoting amicability—were identified from the teachers’ self-reported practices and beliefs. Building trust is central to the “relationship-builder teacher” persona. These teachers believe that establishing a trusting relationship with their students is critical for creating a safe and effective learning environment where students feel comfortable to express their thoughts and take risks in their learning. Fostering respect relates to these teachers’ emphasis on promoting mutual respect in the classroom. They aim to cultivate a classroom culture where students feel valued and respected, thereby enhancing their motivation and engagement in learning. Promoting amicability refers to these teachers’ commitment to maintaining a friendly, approachable demeanour. They strive to be not just educators but also mentors and friends to their students, enhancing students’ comfort and openness in the classroom.\n\nThe same number of pre-service teachers (n=7 out of 238) were identified as ‘sociable teachers’ predicating on their proclivity for fostering positive relationships and social connectivity within their practice. Participant 157, for instance, stated, “I promote a warm classroom environment by building rapport with all stakeholders. Through empathy and open dialogue, I foster a sense of belonging and extend my collaborative efforts beyond the classroom to connect with educators and community members.” This theme centres on the teacher’s propensity to cultivate positive relationships and foster social connections within their teaching practice.\n\nThe ‘sociable teacher’ theme was deduced from these teachers’ inclination towards fostering a warm, collaborative classroom environment. These teachers believe that positive relationships and social connectivity are key to an effective, inclusive learning environment. The associated characteristics—relationship building, empathy, and collaboration—were discerned from the teachers’ self-reports and observed instructional methods. Relationship building refers to these teachers’ commitment to establishing strong, positive relationships with all stakeholders in the learning process. They prioritize building rapport with not just their students, but also parents, fellow educators, and community members. Empathy is a crucial attribute of the “sociable teacher”. These teachers show a deep understanding and appreciation for the perspectives and feelings of their students, thereby fostering a safe, supportive learning environment where students feel seen and understood. Collaboration signifies these teachers’ emphasis on collaborative learning and problem-solving. They believe in the power of collective intelligence and aim to foster a collaborative classroom culture where everyone works together to facilitate learning and solve problems.\n\nA handful of pre-service teachers (n=7 out of 238) were identified as ‘attentive teachers’ archetype accentuating the role of diligent observation in optimizing educational practices. These participants advocated for careful scrutiny of student behaviour, progress, and engagement, using these insights to customize pedagogical techniques in accordance with individual learning styles and needs. Participant 165 shared, “By attentively understanding students’ individual needs and areas for growth, I adapt my teaching strategies to align with their preferences. Prioritizing their socio-emotional well-being, I create a safe environment and offer timely guidance and support.” As observed, the teacher perceived the meticulous observation to be significantly important in improving his/her educational practices.\n\nThe ‘attentive teacher’ theme is derived from these teachers’ emphasis on careful observation of their students. These teachers recognize that meticulous observation and understanding of individual student behaviour, progress, and engagement can enable them to tailor their teaching methods to better suit individual learning styles and needs. The associated characteristics—meticulous observation, individualized instruction, and a focus on socio-emotional well-being—emerge from the teachers’ self-reported practices and beliefs. Meticulous observation refers to the teachers’ diligent scrutiny of student behaviour and progress. By attentively observing their students, these teachers gain valuable insights into students’ strengths, areas for growth, learning styles, and needs, which guide their instructional planning and delivery. Individualized instruction relates to the teachers’ commitment to tailoring their teaching strategies based on their understanding of each student’s unique learning preferences and needs. They believe that such customization enhances student engagement and learning outcomes. Focusing on students’ socio-emotional well-being shows the teachers’ recognition of the importance of attending to the whole child—not just their academic needs but also their emotional and social needs. They strive to create a safe and supportive learning environment where students feel valued and cared for.\n\nThe same number of pre-service teachers (n=7 out of 238) were identified as ‘real-world connector teachers’ advocating for the integration of real-world contexts and experiences into classroom learning. Such an approach aimed to bridge theoretical knowledge and practical application, engaging students in activities emulating real-life scenarios. Participant 176 remarked, “I leverage real-life examples to enhance learning relevance. This process enables students to comprehend the practical implications of their learning, thereby fostering critical thinking and problem-solving abilities.” The teacher highlighted integrating real-world contexts and experiences into classroom instruction.\n\nThe ‘real-world connector teacher’ theme emerges from the teachers’ teaching practices that bridge the gap between classroom learning and the outside world. By integrating real-world experiences and contexts into classroom learning, these teachers aim to make learning more relevant and engaging for their students. They believe that such pedagogical strategies can help students see the relevance of their learning to their lives outside the classroom, thereby deepening their understanding and enhancing their engagement. The characteristics associated with this theme include the use of real-life examples, engaging students in activities simulating real-life situations, and fostering critical thinking and problem-solving abilities. These characteristics arise from the teachers’ teaching practices and beliefs. The use of real-life examples reflects the teachers’ commitment to contextualizing classroom learning in the real world. By drawing on real-life scenarios, these teachers aim to illustrate the practical implications of theoretical knowledge, enhancing its relevance and applicability to students’ lives. The practice of engaging students in activities that emulate real-life situations underscores the teachers’ goal of making classroom learning more engaging and interactive. They believe that such activities can facilitate active learning, wherein students apply theoretical knowledge to practical situations, deepening their understanding and honing their problem-solving skills. The focus on fostering critical thinking and problem-solving abilities arises from the teachers’ recognition of the importance of these skills for students’ success in the real world. By engaging students in activities that emulate real-life situations, these teachers provide opportunities for students to apply their learning in meaningful contexts, thereby developing their critical thinking and problem-solving abilities.\n\nA few pre-service teachers (n=6 out of 238) were identified as ‘professional boundaries teachers’ prioritizing professional ethics and boundary delineation in their pedagogical interactions. They emphasized maintaining professionalism, confidentiality, and respect. As Participant 178 articulated, “In my role as an educator, I set clear expectations for student conduct, while ensuring approachability. Furthermore, I uphold professional relationships with my colleagues, recognizing and respecting their expertise and boundaries.” The teacher highlighted the importance of maintaining professional ethics and clear boundaries within his/her pedagogical roles.\n\nTeachers aligned with the ‘professional boundaries’ theme place a high priority on professionalism in their teaching practice. They recognize their role not only as educators but also as professionals in the academic environment, and this awareness shapes their interactions with students and colleagues. The characteristics associated with this theme, such as upholding professional ethics, maintaining confidentiality, and respecting boundaries, are direct reflections of their professional identity. Upholding professional ethics includes adherence to rules and regulations, maintaining the integrity of their roles as teachers, and promoting ethical behaviour among students. Maintaining confidentiality underscores the respect they show towards private information related to students or colleagues. It showcases their understanding of the sensitive nature of their position and the trust placed in them by students, parents, and colleagues. Respecting boundaries relates to the teachers’ understanding of their roles and the appropriate degree of involvement in their students’ personal lives or issues. They realize that while it is important to be approachable and supportive, it is equally important to maintain a professional distance to ensure that their support does not intrude on students’ personal lives or compromise their professional ethics.\n\nA similar amount of pre-service teachers (n=6 out of 238) were identified as ‘interactive teachers’. This underpins the significance of fostering active learner engagement and collaboration. They employed a variety of interactive pedagogical techniques, alongside effective communication skills and technology-enhanced learning approaches. Participant 184 encapsulated this, saying, “My classroom is a platform for open dialogue and dynamic learning. I use digital resources to boost interactivity, thereby deepening understanding through hands-on tasks, group activities, and virtual collaborations.” The excerpts encapsulated the teacher’s commitment to fostering active engagement and collaboration among learners. The characteristics associated with this theme—such as using interactive pedagogical techniques, having effective communication skills, and employing technology-enhanced learning approaches—were derived from these teacher’s emphasis on interaction and student participation.\n\nTeachers classified as ‘interactive teachers’ believe in creating an active and engaging learning environment where learners play a crucial role. These teachers perceive themselves as facilitators rather than dictators of knowledge, creating a space that encourages dialogue, exploration, and collaboration. Interactive pedagogical techniques associated with this theme can include group activities, discussions, hands-on tasks, role-play, and other strategies that require active learner participation. These techniques ensure that students are not passive recipients of knowledge but active participants in the learning process, fostering deeper understanding and active engagement. Effective communication is another significant characteristic associated with this theme. “Interactive teachers” understand the importance of clear, direct, and empathetic communication in creating an environment conducive to interaction and learning. They use communication as a tool to convey complex concepts in an understandable manner, resolve conflicts, and build trust and rapport with students. Lastly, the use of technology-enhanced learning approaches reflects the interactive teachers’ recognition of the role of technology in modern education. They employ digital resources to augment traditional teaching methods, harnessing the power of technology to promote interactivity, engagement, and collaborative learning.\n\nThe findings also identified some pre-service teachers (n=6 out of 238) as ‘traditional teachers’ showing a predilection for conventional pedagogical approaches and an emphasis on discipline, authority, and direct instruction. They appreciated the merits of longstanding teaching methods, viewing teachers as primary knowledge transmitters and prioritizing teacher-centred instruction, including lecture-based delivery and textbook-guided content. Participant 193, for instance, said: “My belief lies in the strength of traditional methods; they offer structure and impart foundational knowledge effectively. I see my role as a guide, leading students through established academic pathways.” The teacher demonstrated an appreciation for long-established teaching methods, positioning the teacher as the primary source of knowledge and favouring teacher-centred instructional strategies such as lecture-based teaching and textbook-guided content.\n\nThe preference for conventional pedagogical approaches that ‘traditional teachers’ display is indicative of their belief in the effectiveness of these methods, which have been used in educational contexts for centuries. These methods offer structure and consistency, factors that many traditional teachers believe are essential for effective learning. For example, lecture-based teaching enables the teacher to deliver a large amount of information within a short time frame, while textbook-guided content provides a predetermined structure that can guide both teaching and learning processes. The emphasis on discipline and authority is associated with this theme because traditional teachers often view the maintenance of order and discipline as a prerequisite for effective teaching and learning. Such a classroom environment, they believe, ensures that all students can focus on the content being delivered without distractions. This belief also reflects the traditional perception of the teacher’s role as an authority figure responsible for maintaining decorum and managing the classroom. The concept of the teacher as the primary source of knowledge and the prioritization of teacher-centred instruction reflects traditional teachers’ belief in their role as a guide. They see themselves as leading students through the established academic pathways, rather than facilitating a student-led discovery of these paths. This perspective reinforces the teacher’s role as an authority and an expert, with students primarily positioned as recipients of knowledge.\n\nA handful of pre-service teachers (n=6 out of 238) were identified as ‘holistic teachers’ who championed an integrated, whole-child approach to education. They stressed an inclusive learning environment that accommodates diverse needs. Commitment to cultivating self-awareness, empathy, and positive values was evident in their remark. The Holistic Teachers underscored community-building. Participant 199 said, “I champion an integrated, whole-child approach to education. Addressing academic, social-emotional, character, and personal growth, I foster students’ holistic development. Nurturing an inclusive learning environment, I embrace diversity and cultivate a sense of belonging. Incorporating social-emotional learning and character education, I promote collaboration and connectedness within the classroom.” Observably, the teacher is characterized by a dedication to an integrated, whole-child approach to education, an emphasis on inclusivity, cultivation of self-awareness, empathy and positive values, and a strong focus on community-building within the classroom.\n\nThe whole-child approach associated with ‘holistic’ teachers extends the educational focus beyond academics to encompass social, emotional, and personal growth. This approach stems from the belief that students are multi-faceted individuals whose various developmental aspects (e.g., cognitive, emotional, social, physical, and moral) are interrelated and equally significant. In turn, this informs holistic teachers’ teaching strategies and curriculum design, as they seek to engage students in learning experiences that promote holistic development. Inclusivity is another crucial characteristic of the holistic teaching theme. Holistic teachers understand and value the diversity of student backgrounds, abilities, learning styles, and needs. They strive to create a learning environment where all students feel respected, valued, and able to participate fully. This commitment to inclusivity means holistic teachers often employ differentiated instruction, offering multiple paths to learning to accommodate varied student needs. The commitment to cultivating self-awareness, empathy, and positive values reflects the holistic teachers’ recognition of the importance of socio-emotional learning and character education. They understand that these aspects contribute significantly to students’ overall well-being and success, both within and beyond the school setting. Thus, these teachers incorporate activities and discussions that promote self-reflection, emotional understanding, moral reasoning, and interpersonal skills. The focus on community-building is tied to holistic teachers’ emphasis on the social context of learning. They believe that a sense of connectedness and community can foster a positive learning atmosphere, increase student engagement, and promote collaborative learning. They aim to build classroom communities where students feel a sense of belonging and are encouraged to work cooperatively.\n\nA few pre-service teachers (n=5 out of 238) saw themselves as ‘contextually aware teachers’ emphasizing the necessity to comprehend and tailor their pedagogical methods to cater to the distinct requisites of their learners within their individual educational contexts. These pre-service teachers acknowledged the importance of taking into account cultural origins, diverse learning necessities, and educational environments when formulating their instructional approaches. Participant 204 embodied this viewpoint, sharing, “As a context-conscious pedagogue, I aim to recognize diverse learner needs and foster an inclusive, culturally sensitive, and equitable learning environment. Adapting my teaching methods to their specific context enhances their educational experience and promotes their growth.” Based on the excerpts as the representative of the ‘contextually aware teacher’ theme, this group is distinguished by a focus on understanding and adapting their teaching strategies to the specific needs of their students within their unique educational contexts. This approach includes recognizing cultural backgrounds, diverse learning needs, and the influence of the wider educational environment.\n\nThe recognition of cultural backgrounds is crucial to the theme of contextually aware teachers. They understand that cultural experiences shape students’ learning styles, perspectives, and levels of engagement. To be effective, teachers must respect and incorporate cultural diversity into their teaching, promoting multicultural education and fostering cultural sensitivity. This approach not only respects students’ cultural identities but also enriches the learning environment by exposing students to a diversity of experiences and perspectives. Addressing diverse learning needs is another critical aspect of contextually aware teaching. Students come from different educational backgrounds and have different learning styles, abilities, and interests. Contextually aware teachers understand this and strive to tailor their instructional approaches to meet these diverse needs. This can involve employing differentiated instruction strategies, providing additional support for students with specific learning needs, and using a variety of teaching methods to engage all learners. The broader educational environment is also a significant consideration for contextually aware teachers. They understand that each learning environment—whether a large urban school, a small rural classroom, an online learning platform, or a special education setting—has its unique characteristics and challenges. Thus, they adapt their teaching approaches to fit these different environments, optimizing the learning experiences of their students.\n\nA small subset of pre-service teachers (n=5 out of 238) were identified as ‘cheerful teachers’ highlighting their commitment to fostering a positive and joyful pedagogical environment. These pre-service teachers underscored the importance of a buoyant demeanour and utilized effective communication, humour, and engaging teaching techniques to stimulate student participation. Their perceived teaching philosophy aligned with the idea that a cheerful disposition enhances student motivation and fosters a sense of belonging. As Participant 210 noted, “Fostering a positive and joyful pedagogical environment is fundamental to my teaching approach. My buoyant demeanour exudes enthusiasm and optimism. Utilizing effective communication, humour, and engaging teaching techniques, I inspire student participation and motivation. Contributing to a supportive classroom environment, I foster a sense of belonging and empowerment.” The teacher was marked by his/her commitment to fostering a positive, joyful learning environment, underscored by his/her buoyant demeanour, effective communication, use of humour, and engaging teaching techniques. He/she believed that a cheerful atmosphere enhances student motivation and fosters a sense of belonging.\n\n‘Cheerful teachers’ buoyant demeanour sets the tone for the learning environment. Their joyful, positive attitude can be infectious, spreading to students and affecting their mood and engagement. When teachers are cheerful, they create a comfortable atmosphere that makes students feel more open to learning, asking questions, and participating in classroom activities. Effective communication is a hallmark of cheerful teachers. They are adept at conveying complex ideas in a clear, accessible manner. They listen actively to their students, giving them a voice and validating their contributions. Such positive interactions can help to build rapport, trust, and mutual respect, enhancing the student-teacher relationship. The use of humour and engaging teaching techniques is another characteristic of cheerful teachers. Humour can alleviate stress, break down barriers, and make learning more enjoyable. It can foster a positive class climate and improve students' attention, comprehension, and retention. Engaging teaching techniques, such as interactive activities, games, and collaborative projects, can further stimulate student interest, participation, and learning. Cheerful teachers also believe that a positive, joyful environment enhances student motivation and fosters a sense of belonging. When students feel welcome, valued, and part of a supportive community, they are more likely to engage in learning, strive to meet academic expectations, and develop a positive attitude towards school.\n\nA few pre-service teachers (n=5 out of 238) were discerned as ‘authentic teachers’ underlining their dedication to embodying genuineness and sincerity in their pedagogical approach. These pre-service teachers espoused personal authenticity and prioritized establishing a sincere rapport with their students, endorsing transparency and openness in their practices. As Participant 213 explained, “Personal authenticity and sincerity are integral to my teaching philosophy. I establish a genuine rapport with my students, building trust and mutual respect. Transparency and openness in my teaching practices foster an environment where students feel comfortable expressing themselves and engaging in meaningful discussions.” The characteristics of the teacher, based on the excerpts, that define this ‘authentic teacher’ archetype include a dedication to embodying genuineness and sincerity in their pedagogical approach, personal authenticity, prioritizing establishing a sincere rapport with students, and endorsing transparency and openness in their practices.\n\nThe authenticity of these teachers is rooted in their self-awareness and commitment to being genuine in their interactions with students. This characteristic encourages trust as students recognize their teachers’ authenticity and respond positively to their sincerity. Authentic teachers approach their students with respect and genuineness, showcasing their own passion and curiosity about the subjects they teach and thereby motivating students to follow suit. The emphasis on establishing a sincere rapport with students supports the creation of an inclusive and respectful learning environment. By making a conscious effort to understand their students, recognize their individuality, and value their perspectives, authentic teachers are able to establish meaningful connections with their students. This rapport enhances students’ sense of belonging and engagement in the classroom, thus promoting their active participation and academic success. The commitment of authentic teachers to transparency and openness in their practices further enhances the learning environment. They communicate their expectations clearly, provide constructive feedback, admit when they do not know something, and are open to learning from their students. This openness creates an atmosphere of mutual respect and trust, fostering an environment conducive to student participation, collaboration, and critical thinking.\n\nThe study identified a handful of pre-service teachers (n=5 out of 238) who self-identified as an ‘appreciated teacher’ demonstrating a strong perception of value and acknowledgment in their professional roles. They emphasized genuine care for their students, nurtured positive relationships, and facilitated a supportive, inclusive learning environment. Their commitment to continuous professional growth was evidenced by their reflective practices and active contributions to the teaching community. As Participant 211 stated, “Demonstrating a strong perception of value and acknowledgment in my professional role, I genuinely care for my students’ academic and personal development. Nurturing positive relationships, I create a supportive and inclusive learning environment where all students feel valued and respected. Engaging in continuous professional growth through reflection and feedback, I am dedicated to refining my teaching methods. Furthermore, I actively contribute to the broader educational community, collaborating with fellow educators to enhance the overall learning experience.” The excerpts show that the characteristics of Participant 211 include a strong perception of value and acknowledgment in his/her professional roles, a genuine care for his/her students, the nurturing of positive relationships, and the facilitation of a supportive, inclusive learning environment. He/she also committed to continuous professional growth as evidenced by his/her reflective practices and active contributions to the teaching community.\n\nThe ‘appreciated teacher’ theme is marked by a distinct sense of value and recognition in their role as educators. This acknowledgement comes from a range of sources: the tangible progress and success of students, positive feedback from learners and their parents, and recognition from colleagues and educational leadership. This perceived appreciation motivates these teachers and reinforces their sense of purpose and satisfaction in their profession. Their genuine care for students forms a central part of their teaching philosophy. By investing time and energy in understanding their students’ unique needs and aspirations, they can create individualized learning experiences that foster academic and personal development. This caring approach builds trust with students and boosts their motivation and engagement, as they feel understood and valued in the classroom. Appreciated teachers also prioritize the cultivation of positive relationships, both with students and with their peers. They seek to create a supportive and inclusive learning environment where all students feel valued, respected, and motivated to learn. This focus on fostering positive relationships can lead to higher levels of student engagement, improved academic outcomes, and a stronger sense of community within the classroom. These teachers are also committed to their own continuous professional growth, often reflected in their pursuit of continuous learning, seeking feedback, and engaging in reflective practices. They view each teaching experience as an opportunity for professional development, often adjusting and refining their teaching methods based on feedback and reflection. They see themselves as lifelong learners, mirroring the attitude they hope to instil in their students. Active participation and contribution to the broader educational community is another characteristic of “appreciated teachers”. They often collaborate with fellow educators, sharing resources, strategies, and insights to enhance collective teaching practices and the overall learning experience. This collaborative approach helps to enrich their own teaching practices while contributing to the advancement of the broader educational community.\n\nA few participants (n=4 out of 238), being identified as ‘compassionate communicator teachers’, emphasized empathy, understanding, and communication as cornerstones of their practice. Reflecting this stance, Participant 224 noted, “Empathy and understanding are integral to my teaching approach. I foster a supportive and inclusive learning environment, where every student’s voice is valued. Prioritizing effective communication strategies like active listening and clarity, I encourage open discussions and mutual respect. Addressing students’ emotional needs, I provide the necessary support, creating a safe space for them to share their thoughts and feelings.” Based on the excerpts of Participant 224, the hallmarks of this teacher archetype include a strong emphasis on empathy, understanding, and communication as fundamental elements of their pedagogical approach. These teachers aim to foster a supportive and inclusive learning environment where every student’s voice is valued. They prioritize effective communication strategies like active listening and clarity in instructions to foster open dialogue and mutual respect. They also underscore the importance of addressing students’ emotional needs, providing support, and creating a safe space for students to express their thoughts and feelings.\n\nThe association of these characteristics with the theme of ‘compassionate communicator teachers’ comes from their recognition of the power of effective communication in the teaching-learning process. They understand that students learn best when they feel heard, understood, and valued. This recognition drives their commitment to empathy and understanding as central tenets of their teaching philosophy. “Compassionate communicator teachers” place great importance on creating a supportive and inclusive learning environment. By fostering such an environment, they aim to make every student feel comfortable and confident to participate, express their opinions, ask questions, and share their feelings. This inclusivity enhances student engagement, fosters a sense of belonging, and cultivates a respectful and empathetic classroom culture. Effective communication is another key characteristic associated with this theme. These teachers use strategies such as active listening and clarity in instructions to enhance their interactions with students. Active listening allows them to understand students’ needs, concerns, and perspectives more accurately, fostering mutual respect and understanding. Providing clear instructions helps ensure that students understand their learning tasks, expectations, and goals, leading to increased student motivation and achievement. Furthermore, these teachers understand that students’ emotional needs are just as important as their academic needs. They acknowledge that students’ emotional well-being significantly impacts their ability to learn and engage with the learning material. By addressing these needs and providing necessary support, they create a safe and nurturing environment where students feel comfortable sharing their thoughts and feelings. This emotional support can enhance students’ overall well-being, motivation, and academic success.\n\nThe study also found that a few pre-service teachers (n=4 out of 238) were identified as ‘guided teachers’. They emphasized structured support and guidance in pedagogical approach, the pedagogical method of scaffolding, and progress tracking. Participant 232 articulated, “Structured support and guidance are key aspects of my teaching approach. I offer clear instructions and modelling to facilitate students’ understanding. Utilizing scaffolding techniques, I break down complex concepts into manageable components. Guided practice opportunities strengthen their skills, while I constantly monitor progress and provide timely feedback to ensure their success.” The excerpts of Participant 232 show that the primary features of a ‘guided teacher’ include an emphasis on structured support and guidance in their pedagogical approach, the use of scaffolding techniques in teaching, and the practice of progress tracking. These teachers see their role as facilitators who offer clear instructions and model tasks to aid students’ understanding. They break down complex concepts into manageable components using scaffolding techniques and provide students with opportunities for guided practice to strengthen their skills. These teachers also monitor students’ progress regularly and provide timely feedback to ensure their success.\n\nThe association of these characteristics with the ‘guided teachers’ theme arises from these teachers’ recognition of the importance of guided instruction in facilitating students’ learning. These teachers understand that clear instructions and modelling can significantly enhance students’ understanding and help them gain a better grasp of the learning material. The “guided teachers” theme is also characterized by the use of scaffolding techniques. Scaffolding is a pedagogical method that involves breaking down complex concepts into smaller, more manageable parts to facilitate understanding. This approach can aid students in mastering new concepts and skills and gradually becoming more independent in their learning. These teachers see scaffolding as a crucial strategy in their teaching, aligning with their commitment to providing structured support and guidance to their students. Another key characteristic associated with this theme is the practice of progress tracking. These teachers recognize the importance of monitoring students’ progress and providing timely feedback. By doing so, they can identify students’ strengths and areas for improvement, adapt their instruction accordingly, and ensure that students are on the right track towards achieving their learning goals. This approach reflects these teachers’ commitment to facilitating students’ success and their belief in the power of feedback in enhancing students’ learning.\n\nThe same number of pre-service teachers (n=4 out of 238), identified as ‘feedback-driven teachers’ underscored the primacy of timely, constructive feedback in pedagogical interactions. Participant 233 stated, “I firmly believe in the importance of timely, constructive feedback to guide my students’ progress and foster growth. Utilizing various feedback mechanisms, I provide a comprehensive understanding of their performance. I make it a point to illuminate their strengths and areas of improvement, motivating them to excel. Encouraging a feedback-receptive ethos, I promote self-reflection for continuous learning and development.” The defining characteristics of this teacher, based on the excerpts, are a strong belief in the importance of timely, constructive feedback and the use of various feedback mechanisms to enhance student performance.\n\nThese teachers view feedback as a crucial tool for guiding students’ progress and fostering their growth. They provide students with a comprehensive understanding of their performance, highlighting their strengths and areas for improvement. Moreover, these teachers encourage a feedback-receptive ethos in their classrooms, promoting self-reflection for continuous learning and development. The association of these characteristics with the ‘feedback-driven teachers’ theme stems from these teachers’ understanding of the central role that feedback plays in the learning process. These teachers recognize that timely, constructive feedback can significantly enhance students’ understanding and performance. By providing clear, actionable feedback, they can guide students towards improving their skills and knowledge, fostering their academic growth. These teachers also utilize various feedback mechanisms in their pedagogical practice. This approach ensures that they provide students with a comprehensive understanding of their performance, covering all aspects of their work. This comprehensive feedback approach can motivate students to excel, as it illuminates both their strengths and areas for improvement. Another key characteristic associated with this theme is the promotion of a feedback-receptive ethos in the classroom. By encouraging students to be open to feedback and promoting self-reflection, these teachers facilitate continuous learning and development. They understand that students who are receptive to feedback and capable of self-reflection are more likely to improve their performance and develop their skills.\n\nThe findings reveal that an identical fraction of participants (n=4 out of 238) were identified as ‘consistent identity teachers’ underlining the importance of maintaining an authentic and stable teaching persona. Participant 235 noted, “As an educator, I maintain an authentic and stable teaching persona, reflecting my true values and beliefs. By establishing trust and credibility with my students, I create a positive and supportive learning environment. Aligning my actions with core principles, I demonstrate consistency in instructional methods and behaviour. Engaging in reflective practice, I continuously uphold my teaching identity.” Observably, this teacher as the representative of the theme emphasized the importance of maintaining an authentic and stable teaching persona.\n\nThese teachers believe in the value of reflecting their true values and beliefs in their teaching, creating a positive and supportive learning environment through trust and credibility. They strive for consistency in instructional methods and behaviour, and they engage in reflective practice to continuously uphold their teaching identity. These characteristics are associated with the ‘consistent identity teachers’teachers” theme because they reflect an understanding of the influence of a teacher’s persona on their teaching practices and the learning environment they create. An authentic and stable teaching persona can help to establish trust and credibility with students, fostering a positive and supportive learning environment. Students are more likely to respond positively to teachers who they perceive as genuine and consistent, which can enhance the learning process. Moreover, these teachers believe in the importance of aligning their actions with their core principles. They understand that consistency in instructional methods and behaviour not only provides a predictable learning environment for students but also serves as a model of integrity and reliability. The commitment of these teachers to reflective practice is another key characteristic associated with this theme. Through reflection, they continually assess and refine their teaching identity, ensuring that it remains consistent with their values and beliefs and aligns with their teaching practices.\n\nTable 2 summarizes the findings of the current study. It includes the types of teacher professional identity, number of participants who self-perceived themselves as the aforementioned images, and the typical characteristics of these images.\n\n\n\n• Demonstrates dynamic pedagogical adaptation.\n\n• Shows resilience and compassion in diverse educational settings.\n\n• Commits to continuous professional development.\n\n• Embraces innovation in teaching strategies.\n\n• Nurtures an inclusive classroom environment.\n\n\n\n• Prioritizes student needs and fosters inclusive classrooms.\n\n• Promotes student autonomy and engagement.\n\n• Utilizes varied instructional strategies and differentiated approaches.\n\n• Encourages peer interaction and choice within the curriculum.\n\n• Creates a cooperative and interactive learning environment.\n\n\n\n• Engages in self-analysis and introspection.\n\n• Adapts pedagogical practices based on lessons learned from both successes and failures.\n\n• Embraces a growth-oriented mindset.\n\n• Actively seeks feedback to improve teaching practice.\n\n• Challenges traditional teaching methods.\n\n\n\n• Commits to continuous improvement, self-reflection, and professional development.\n\n• Seeks feedback and views challenges as learning opportunities.\n\n• Demonstrates self-awareness and a willingness to explore innovative teaching strategies.\n\n• Stays informed about educational trends to refine pedagogical approach.\n\n\n\n• Provides stimulating, relevant, and personalized learning experiences.\n\n• Uses innovative teaching methodologies and multimedia resources.\n\n• Establishes real-world connections to learning.\n\n• Fosters a positive and supportive classroom environment.\n\n• Builds emotional bonds with students and ignites a passion for learning.\n\n\n\n• Inspires and motivates students towards academic success and personal growth.\n\n• Exhibits a passion for teaching.\n\n• Uses emotionally resonant communication strategies.\n\n• Provides supportive feedback.\n\n• Ignites curiosity and instills self-belief in students.\n\n\n\n• Demonstrates an infectious passion for the subject and teaching.\n\n• Delivers lessons with energy and enthusiasm.\n\n• Uses engaging instructional techniques.\n\n• Creates an engaging learning environment.\n\n• Fosters a positive teacher-student relationship.\n\n\n\n• Demonstrates adaptability and problem-solving skills in classroom management.\n\n• Maintains a composed learning atmosphere amid disruptions or conflicts.\n\n• Implements clear routines and procedures.\n\n• Fosters a positive and engaging classroom climate.\n\n• Addresses behavioral issues and provides support to students.\n\n\n\n• Demonstrates deep comprehension and expertise in the teaching discipline.\n\n• Maintains comprehensive content knowledge.\n\n• Stays abreast of advancements in the field.\n\n• Effectively conveys complex concepts using varied instructional strategies.\n\n• Focuses on providing relevant and current information to students.\n\n\n\n• Optimally utilizes available resources to enhance teaching practices and student learning.\n\n• Demonstrates creativity in pedagogical methods.\n\n• Applies problem-solving skills to overcome classroom challenges.\n\n• Actively seeks diverse teaching materials and utilizes tech tools.\n\n• Engages in continuous professional development to stay updated on new resources.\n\n\n\n• Harmonizes various instructional elements to optimize student outcomes.\n\n• Balances academic rigor with a supportive classroom environment.\n\n• Addresses both cognitive and socio-emotional needs of students.\n\n• Provides clear expectations while promoting student autonomy.\n\n• Creates a structured yet flexible learning environment.\n\n\n\n• Demonstrates practicality and efficiency in teaching strategies.\n\n• Tailors teaching approaches to suit the specific educational setting.\n\n• Balances theory and practice in instructional methods.\n\n• Considers available resources and long-term implications.\n\n• Focuses on effective solutions to overcome challenges.\n\n\n\n• Focuses on establishing constructive and significant rapport with learners.\n\n• Places trust, respect, and amicability at the forefront.\n\n• Creates an inclusive and supporting learning environment.\n\n• Tailors instructional approaches based on individual requisites and interests.\n\n• Customizes the pedagogical approach to strengthen relationships.\n\n\n\n• Fosters positive relationships and social connectivity within the classroom.\n\n• Builds rapport with students, colleagues, and community members.\n\n• Advocates for a warm and inclusive classroom environment.\n\n• Values empathy and open dialogue.\n\n• Networks with fellow educators and community members.\n\n\n\n• Engages in diligent observation of student behavior, progress, and engagement.\n\n• Customizes pedagogical techniques based on individual learning styles and needs.\n\n• Uses insights from observation to enhance teaching strategies.\n\n• Prioritizes creating a safe environment and addressing socio-emotional needs.\n\n• Provides timely guidance and support.\n\n\n\n• Integrates real-world contexts and experiences into classroom learning.\n\n• Bridges theoretical knowledge and practical application.\n\n• Engages students in activities that emulate real-life scenarios.\n\n• Enhances learning relevance and practical implications.\n\n• Fosters critical thinking and problem-solving abilities.\n\n\n\n• Prioritizes professional ethics and boundary delineation in pedagogical interactions.\n\n• Maintains professionalism, confidentiality, and respect.\n\n• Sets clear expectations for student conduct while ensuring approachability.\n\n• Upholds professional relationships with colleagues.\n\n• Recognizes and respects colleagues’ expertise and boundaries.\n\n\n\n• Fosters active learner engagement and collaboration.\n\n• Uses a variety of interactive pedagogical techniques.\n\n• Demonstrates effective communication skills.\n\n• Utilizes technology-enhanced learning approaches.\n\n• Promotes hands-on tasks, group activities, and virtual collaborations.\n\n\n\n• Emphasizes conventional pedagogical approaches.\n\n• Values discipline, authority, and direct instruction.\n\n• Views teachers as primary knowledge transmitters.\n\n• Prioritizes teacher-centered instruction.\n\n• Believes in the strength of traditional methods and established academic pathways.\n\n\n\n• Champions an integrated, whole-child approach to education.\n\n• Addresses academic, social-emotional, character, and personal growth.\n\n• Nurtures an inclusive learning environment.\n\n• Incorporates social-emotional learning and character education.\n\n• Fosters collaboration and connectedness within the classroom.\n\n\n\n• Comprehends and tailors pedagogical methods to the distinct requisites of learners within their educational contexts.\n\n• Considers cultural origins, diverse learning necessities, and educational environments.\n\n• Provides equitable opportunities and accommodates the specific context of learners.\n\n• Enhances the educational experience by adapting instructional approaches.\n\n\n\n• Fosters a positive and joyful pedagogical environment.\n\n• Maintains a buoyant demeanor.\n\n• Utilizes effective communication, humor, and engaging teaching techniques.\n\n• Stimulates student participation and motivation.\n\n• Contributes to a supportive classroom environment.\n\n\n\n• Emphasizes personal authenticity and sincerity in the pedagogical approach.\n\n• Establishes a genuine rapport with students.\n\n• Prioritizes transparency and openness in teaching practices.\n\n• Fosters student-teacher rapport and comfort in self-expression.\n\n\n\n• Demonstrates a strong perception of value and acknowledgment in the professional role.\n\n• Cares genuinely for students and nurtures positive relationships.\n\n• Facilitates a supportive, inclusive learning environment.\n\n• Engages in continuous professional growth through reflection and feedback.\n\n• Contributes to the broader educational community.\n\n\n\n• Demonstrates empathy and understanding in the classroom.\n\n• Fosters a supportive and inclusive learning environment.\n\n• Prioritizes effective communication strategies, such as active listening and clarity.\n\n• Addresses students’ emotional needs and provides necessary support.\n\n• Creates a safe space for students to express thoughts and feelings.\n\n\n\n• Emphasizes structured support and guidance in instruction.\n\n• Provides clear instructions and modeling.\n\n• Utilizes scaffolding techniques to break down complex concepts.\n\n• Offers guided practice opportunities.\n\n• Monitors understanding and provides timely feedback.\n\n\n\n• Emphasizes the importance of timely, constructive feedback.\n\n• Guides student progress and fosters growth.\n\n• Utilizes multiple feedback mechanisms.\n\n• Illuminates strengths and areas of improvement.\n\n• Cultivates a feedback-receptive ethos and encourages self-reflection.\n\n\n\n• Maintains an authentic and stable teaching persona.\n\n• Establishes trust and credibility with students.\n\n• Aligns actions with core principles.\n\n• Demonstrates consistency in instructional methods and behavior.\n\n• Engages in reflective practice to uphold teaching identity.\n\n\nDiscussion\n\nAs previously indicated, given the complex nature of professional identities and the diverse array of factors that shape them (Chu, 2019; Schutz et al., 2018), grounded theory offers a systematic approach to organizing and analysing data, facilitating the identification of key themes and the exploration of relationships that contribute to a comprehensive framework. Specifically, the findings of this current study provide valuable insights into the various types of teachers observed among pre-service teachers in Vietnam, each characterized by distinct qualities and instructional approaches. The findings underscore the significance of tailoring teaching strategies to align with the cultural contexts and diverse learning needs of students, thereby addressing the unique requirements and preferences of educators in Vietnam. This study extends the existing body of knowledge by offering a comprehensive overview of teacher professional identities in Vietnam, further advancing the understanding of this subject matter.\n\nComparing these findings with previous studies can provide insights into the consistency and variation of these identities across different contexts and populations. In general, several identities identified in this study align with those found in previous research. For example, the presence of adaptable and flexible teachers, reflective teachers, and student-centred teachers has been documented in prior studies, highlighting the significance of self-reflection, adaptability, and student-centeredness in effective teaching (Mardiana, 2020; Munif et al., 2022; Sanders et al., 2014). The identification of engaging teachers, growth-oriented teachers, and flexible teachers in this study is consistent with the notion of creating dynamic and responsive learning environments that foster student engagement, growth mindset, and adaptability (Chance, 2008; Tschannen-Moran and Woolfolk Hoy, 2001). Additionally, the presence of sociable teachers and relationship-builder teachers aligns with previous research highlighting the importance of positive teacher-student relationships, open communication, and a supportive classroom environment (Pianta et al., 2012; Roorda et al., 2011). However, it is worth noting that some identified identities in this study, such as resourceful teachers and pragmatic teachers, have received less attention in previous research. While the concept of resourcefulness and the consideration of practicality align with aspects of effective teaching, further exploration and examination of these identities would contribute to the existing literature.\n\nIt is important to acknowledge that the identified teacher professional identities may vary across different studies due to factors such as cultural context and educational systems. Cultural context plays a significant role in shaping teacher identities (Flores and Day, 2006; Gu and Benson, 2015). Teaching practices and expectations can vary across cultures, and teachers may prioritize different aspects of their professional identity based on cultural values and norms. For example, Vietnamese culture places high value on discipline (Lokot et al., 2020), respect for authority (Kawaguchi-Suzuki et al., 2019), and academic achievement (Le et al., 2019). Consequently, teachers in Vietnam may prioritize aspects of their professional identity that align with these cultural values. Educational systems also contribute to the variation in teacher professional identities (Vähäsantanen, 2015). Different educational systems have distinct philosophies (Hassan and Jamaludin, 2010), pedagogical approaches (Sailor, 2017), and expectations for teachers (Gün and Baskan, 2014; Rubenstein, 2006). For instance, in the Vietnamese education system, there is a strong emphasis on academic achievement and standardized testing (Asadullah et al., 2020). Teachers are expected to deliver content effectively and ensure that students perform well in exams. This expectation may lead teachers to perceive themselves as subject-matter expert teachers, focusing on delivering curriculum content and preparing students for assessments. In contrast, in systems that emphasize holistic development and student well-being, teachers may identify more strongly with identities such as supportive teachers or relationship-builder teachers, prioritizing the social-emotional aspects of education. For example, there is also a growing recognition of the importance of holistic development and student well-being in the Vietnamese education system (Bodewig et al., 2014; Nguyen, 2019; Tran et al., 2022). Efforts are being made to shift towards a more student-centred approach and to promote social-emotional learning. As a result, some teachers in Vietnam may identify more strongly with identities such as supportive teachers or relationship-builder teachers, emphasizing the development of positive relationships with students and creating a supportive learning environment.\n\nThis study’s limitations warrant acknowledgment, despite its valuable insights. Firstly, the study exclusively focused on self-perceived professional identities of pre-service EFL teachers, neglecting a comprehensive exploration of additional factors that contribute to professional identity development, such as contextual influences, mentorship, and personal experiences. Secondly, relying solely on a reflective writing task for data collection introduces potential subjectivity and recall bias on the part of the participants. Future research should expand the scope of investigation to encompass a wider range of determinants contributing to professional identity development. This may involve examining contextual influences, such as cultural and institutional factors, and assessing the impact of mentorship and personal experiences on the formation of professional identity. Integrating these additional dimensions will lead to a more comprehensive understanding of the intricate interplay between diverse factors and the development of professional identity. To overcome limitations associated with exclusive reliance on a data collection instrument, researchers should consider adopting mixed method approaches for data collection. This entails combining reflection papers and/or in-depth interviews with quantitative methods like surveys or observations, enabling the acquisition of a diversified and triangulated dataset. By incorporating multiple data sources, researchers can enhance the validity and reliability of their findings, thereby mitigating potential subjectivity and recall bias.\n\nBesides, to advance the comprehension of pre-service teachers’ self-perceived professional identities, future studies could employ a longitudinal approach. Long-term investigations would facilitate the examination of developmental trajectories of professional identities throughout the entire teacher education program and early stages of teaching practice, providing valuable insights into stability, changes, and influential factors over time. Additionally, exploring the impact of contextual factors, including school culture, curriculum demands, and policy implications, would enrich understanding of how these external elements shape and interact with pre-service teachers’ professional identities. Furthermore, conducting comparative studies across diverse cultural and educational contexts would deepen understanding of the universal aspects versus unique characteristics of identity formation, potentially unveiling cultural variations and identifying best practices across educational systems. Lastly, investigating the influence of mentorship and support systems on pre-service teachers’ professional identity formation would be advantageous. Exploring the roles of mentors, cooperating teachers, and teacher educators in guiding and nurturing professional identity development could inform effective strategies for supporting pre-service teachers during their training and early career stages. Addressing these recommendations would contribute to a nuanced and comprehensive understanding of pre-service teachers’ self-perceived professional identities, thereby informing teacher education programs and policies with the aim of preparing highly competent and reflective educators.\n\nThe findings of this study have several implications for the development of contextually relevant teacher education programs in the field of EFL. By gaining insights into the self-perceived professional identities of Vietnamese pre-service EFL teachers, this research contributes to a comprehensive understanding of teacher identity formation and its influence on educational practices, perceptions, and dialogues.\n\nFirstly, the diverse range of self-perceived professional identities identified among the pre-service EFL teachers highlights the multifaceted nature of teacher identity. Understanding the various characteristics and qualities that these teachers associate with their professional identities can inform the design of teacher education programs that foster the development of these attributes. For example, programs can incorporate reflective practices, promote adaptability, and emphasize student-centred approaches to align with the identified professional identities.\n\nSecondly, the insights gained from this study can guide the formulation of tailored and contextually pertinent teacher education programs. By recognizing the unique cultural and educational contexts of Vietnamese EFL pre-service teachers, programs can incorporate elements that address their specific needs, challenges, and aspirations. This tailored approach can enhance the effectiveness and relevance of teacher training initiatives, leading to better-prepared educators who can navigate the complexities of the EFL context.\n\nAdditionally, the findings emphasize the importance of fostering certain qualities and characteristics in pre-service EFL teachers, such as engagement, growth-orientation, flexibility, and effectiveness. Teacher education programs can integrate strategies and activities that promote these attributes, such as providing opportunities for professional development, encouraging reflective practices, and facilitating collaboration and networking among pre-service teachers.\n\nFurthermore, the comprehensive theoretical framework developed through the grounded theory approach offers a valuable tool for understanding the formation of professional identities. This framework can serve as a basis for future research and provide a foundation for the design of interventions and support mechanisms aimed at enhancing pre-service teachers’ professional identity development. It is important to acknowledge that the implications of this study are specific to the Vietnamese EFL context and may have limited generalizability to other contexts. However, the findings provide a starting point for further exploration of professional identity development among pre-service EFL teachers in different cultural and educational settings.\n\n\nConclusion\n\nThis study aimed to scrutinize the self-perceived professional identities of Vietnamese pre-service teachers in the field of EFL education. The significance of teacher professional identity in shaping educational practices, perceptions, and dialogues underscores the need for a comprehensive understanding of its formation. By specifically exploring the self-construed professional identities of Vietnamese EFL pre-service teachers, this research contributes to the development of contextually relevant teacher education programs. Employing a qualitative research design, specifically grounded theory, this study used a reflective writing task requiring pre-service teachers to describe their self-perceived images to delve into their subjective experiences, beliefs, and perspectives on their professional identities. The data analysis involved open coding and constant comparison, resulting in the development of a comprehensive theoretical framework. Trustworthiness was ensured through member checking and peer debriefing of the sample of 238 pre-service teachers. Collaboration with EFL program administrators and faculty facilitated the recruitment process, and ethical guidelines were strictly followed to protect participants’ rights and privacy. The reflective writing task served as the primary method for data collection, enabling participants to express their subjective experiences, values, aspirations, and challenges related to their future teacher identities. The study revealed diverse self-perceived professional identities among the pre-service EFL teachers, including characteristics such as reflectiveness, adaptability, student-centeredness, engagement, growth-orientation, flexibility, effectiveness, resourcefulness, pragmatism, balance, and sociability. These findings contribute to the academic discourse on teacher identity development and inform the formulation of tailored and contextually pertinent teacher education programs.", "appendix": "Data availability\n\nFigshare: Data set of the study entitled “Illuminating the English as a Foreign Language teaching odyssey: unveiling the professional identities of pre-service teachers”. https://doi.org/10.6084/m9.figshare.23723508. (Thao et al., 2023)\n\nThe project contains the following underlying data:\n\n• Data set – professional identities.docs (238 anonymised reflective writing pieces from pre-service teachers as the data set of this study).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAkkerman SF, Meijer PC: A dialogical approach to conceptualizing teacher identity. Teach. Teach. Educ. 2011; 27(2): 308–319. Publisher Full Text\n\nAsadullah MN, Perera LDH, Xiao S: Vietnam’s extraordinary performance in the PISA assessment: A cultural explanation of an education paradox. J. Policy Model. 2020; 42(5): 913–932. Publisher Full Text\n\nBeauchamp C, Thomas L: Understanding teacher identity: An overview of issues in the literature and implications for teacher education. Camb. J. Educ. 2009; 39(2): 175–189. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrede F, Macklin R, Bridges D: Professional identity development: a review of the higher education literature. Stud. High. Educ. 2012; 37(3): 365–384. Publisher Full Text\n\nTschannen-Moran M, Hoy AW: Teacher efficacy: Capturing an elusive construct. Teach. Teach. Educ. 2001; 17(7): 783–805. Publisher Full Text\n\nVähäsantanen K: Professional agency in the stream of change: Understanding educational change and teachers’ professional identities. Teach. Teach. Educ. 2015; 47: 1–12. Publisher Full Text\n\nVu DV, Peters E: Vocabulary in English language learning, teaching, and testing in Vietnam: A review. Educ. Sci. 2021; 11(9): 563. Publisher Full Text\n\nWalker D, Myrick F: Grounded theory: An exploration of process and procedure. Qual. Health Res. 2006; 16(4): 547–559. Publisher Full Text\n\nXue Y: The Role of EFL Teachers’ Self-Efficacy and Emotional Resilience in Appraisal of Learners’ Success. Front. 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Publisher Full Text" }	[ { "id": "240542", "date": "13 Feb 2024", "name": "Muchamad Sholakhuddin Al Fajri", "expertise": [ "Reviewer Expertise EAP" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 introduction provides a clear overview of the research context and objectives. However, there are some areas where critical comments could be provided:\n1. While the introduction briefly mentions previous scholarship on EFL teacher identity, it would benefit from a more comprehensive literature review. Providing a synthesis of existing studies on teacher identity, particularly in the context of EFL pedagogy and within Vietnam, would help situate the current research within the broader scholarly discourse. Justification for the Study:\n2. The introduction emphasizes the importance of understanding professional identities of Vietnamese EFL pre-service teachers for refining teacher training initiatives and advancing English language pedagogy. However, it could be strengthened by providing more explicit justification for why this specific research gap is significant and how addressing it contributes to the field.\n3. While the research objectives are stated, they could be more clearly linked to the broader research questions or hypotheses. How do these objectives address the gaps identified in the literature review? Clarifying this connection would provide a stronger rationale for the study.\nThe method section of the study provides a detailed description of the research design, participant recruitment, data collection procedures, and data analysis techniques. Here are some critical comments on the method:\n\n1. The sample size of 238 pre-service teachers appears to be large, which could provide rich data for analysis. However, the justification for this sample size based on theoretical saturation could be elaborated further. Additionally, while convenience sampling may be practical, it could introduce bias, and its limitations should be acknowledged.\n\n2. The combination of thematic analysis and grounded theory provides a robust approach to analyzing the reflective writings. However, it would be helpful to provide more detail on how themes were identified and validated to ensure the rigor and trustworthiness of the analysis process. Categorization of Data:\n3. The decision to categorize participants' reflections into specific themes is appropriate for organizing and interpreting the data. However, the criteria used for assigning participants to each category should be clearly defined to ensure consistency and transparency in the analysis process. Additionally, acknowledging the potential for overlapping characteristics among categories is important for accurately capturing the complexity of teacher identities.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "240534", "date": "13 Feb 2024", "name": "Lizette Drusila Flores Delgado", "expertise": [ "Reviewer Expertise TESOL", "Teacher and learner identity", "teacher autonomy", "technology in EFL" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper provides valuable knowledge in the teacher identity field. Personally, I found it very interesting and will likely use their results to see how they work in my context. The authors provide support to their claims and it is clear that they conducted an in-depth analysis to reach their conclusions.\n\nI do think, however, that the methodology needs to be expanded. For example:\n* Were there any guidelines as to what they were supposed to write about? An 80 word reflection is short as to really expand into one's perceived identity, so I wonder if there were any questions that guided the reflection. * How did you come up with the 28 identities? I can see why you categorized each participant in each identity, as their descriptions and evidence are clear, but after reading the paper some times, I still do not know where those identities came from. Did they emerge from the data? What were you looking for that led to the emergence of those 28 identities? You do mention that \"the research team allocated each participant to the category that best represented the dominant theme of their reflection\", so this made me wonder if you already had the categories pre-established, or if they emerged from your data. It would make your study stronger if you clarify where those identities came from; be more specific so that your study can be replicated in another context. *Did you use any other source to validate data? Like triangulation? If not, you should include a paragraph mentioning how you ensure validity based on just an 80-word-reflection.\n\nI also believe that you should add literature on the different types of identity. From your methodology on, you do clarify that you are focusing on self-perceived identities of teachers, but nowhere in your literature review you mention this. As identity experts, we know there are different types of identities, but your readers may not know this. So please add a couple of paragraphs in your review of the literature where you explain the types of identity and how they work. Then, justify why you focused on perceived identity.\nAgain, I found this paper very interesting and easy to read due to its clarity, but making your methodology more specific will make your paper stronger.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1086
https://f1000research.com/articles/12-1085/v1	01 Sep 23	{ "type": "Study Protocol", "title": "Awareness regarding knowledge associated with polycystic ovarian syndrome (PCOS) among women in Wardha district- A cross-sectional study.", "authors": [ "Sejal Patil", "Gaurav Mude", "Gaurav Mude" ], "abstract": "Women can develop PCOS (polycystic ovarian syndrome) at a young age. It is a reasonably common endocrine disorder that results in hormonal imbalances and mostly raises androgen hormone production. In India, one in eight women had PCOS, according to a study from the year 2020. More than half of the 10 million people with PCOS are unaware that they have it, hence there is a need for improved public awareness of the condition. For the general public to realize the necessity, it is necessary to have awareness of having symptoms like irregular periods and pelvic pain. Despite the way that there is no solution for PCOS, there are medicines that could facilitate the pressure and difficulties brought about by side effects. The likelihood of an early diagnosis will increase as PCOS becomes more well-known. The adoption of a standardized questionnaire, which will guarantee consistency in data collection and minimize bias, is the study's key strength. A big sample size will also increase the study's accuracy and the generalizability of its findings. The study's drawbacks include the possibility of recollection bias since respondents might not fully recall their familiarity with PCOS. Additionally, because the study will only be conducted in one Indian district, the generalizability may be limited of the outcomes. Analyzing the level of PCOS knowledge and awareness among women in India's Wardha area is the goal of this research proposal. The discoveries of this study will be useful for public health initiatives aiming at raising PCOS awareness and, ultimately, lowering the prevalence of PCOS among women.", "keywords": [ "Polycystic ovarian syndrome", "endocrine", "androgen", "anovulation" ], "content": "Introduction\n\nThe most well-known endocrine condition in ladies and a critical supporter of anovulatory fruitlessness is polycystic ovary syndrome (PCOS). Because of the variety of its aggregates, PCOS displays a great many clinical side effects and chance variables. Feminine cycle inconsistencies, ovarian pimples, and hirsutism are ordinary side effects. PCOS influences ladies of all races and nationalities who are of conceptive age. Patients with PCOS could display many signs and side effects, making it trying to definitively grade the sickness. The measures of the ESRHE/ASRM Rotterdam agreement meeting,1 which extended the earlier NIH (National Institute of Health) order of 1990, are as of now used to analyze PCOS. It was predicated on something like two of the accompanying qualities: polycystic ovaries by ultrasonography, hyperandrogenism, and oligo-anovulation. A board of trustees of experts was laid out by the Androgen Overabundance Society (AES) in 2006 to examine all PCOS-related material delivered fully intent on smoothing out determination. The AES standards are for oligo/anovulation, polycystic ovaries, and clinical and additionally biochemical hyperandrogenism simultaneously.2\n\nThere are four assortments of PCOS:\n\n1. Insulin-resistant PCOS: Around 70% of ladies have this sort of PCOS, making it the most pervasive. Generally, insulin obstruction happens when the body has higher-than-typical measures of insulin or hyperinsulinemia. This happens when our cells become somewhat “numb” to the impacts of insulin, making the pancreas emit expanding measures of the chemical until the cells get the message. You can experience difficulty getting thinner, convey additional load in your stomach or midsection, sugar cravings, and have side effects like exhaustion or mind haze assuming you have this sort of PCOS. Expanded androgen levels, which bring about issues like additional hair, males experiences include going bald, and skin breaking out, are brought about by high insulin levels.\n\n2. Post-pill PCOS: Certain individuals who quit utilizing the oral preventative pill foster post-pill PCOS. In this kind, no side effects like skin break out, sporadic periods or extreme hair development existed before the pill was at any point begun. Because of the kind of engineered progestins that are available in oral contraceptives like Ginet, Yasmin, and Yaz, are engaged with this sort of PCOS. The ovary hosts a get-together when you quit utilizing the pill, and there is a characteristic expansion in androgens, which could make the exemplary side effects of PCOS; be that as it may, there is no insulin opposition in this assortment. This generally happens three to a half years subsequent to suspending the medication. With legitimate sustenance, way-of-life changes, and enhancements or home-grown prescription help, this sort can be dealt with all the more quickly despite the fact that it can require an investment to mend all alone.3\n\n3. Adrenal PCOS: Around 10% of people with the determination have this sort of PCOS, which is welcomed by an abnormal pressure response.3 Elevated degrees of testosterone and androstenedione are frequently not noticed; all things being equal, DHEA-S, one more sort of androgen delivered by the adrenal organs, will regularly be raised alone.\n\n4. Inflammatory PCOS: Persistent irritation in provocative PCOS makes the ovaries produce an excess of testosterone, which prompts actual uneasiness and trouble in ovulating. Migraines, joint distress, unexplained depletion, dermatitis, and IBS indicate irritation here of PCOS. Regularly, blood tests will uncover raised incendiary markers, like high CRP (C-responsive protein). Different tests, like fasting glucose and insulin, are inside the typical reach, however, they may periodically be influenced by aggravation.3\n\n50-70% of females with PCOS have insulin opposition, which expands their gamble of creating diabetes, metabolic condition, hypertension, dyslipidemia, and other comorbidities like hypertension and dyslipidemia.4 As per studies, ladies who have PCOS are bound to have higher carotid intima-media thickness and higher coronary vein calcium scores, also Ladies with PCOS are likewise bound to encounter psychological well-being sicknesses such as depression, uneasiness, bipolar, confusion, and voraciously consuming food problems. Indian ladies have a high PCOS commonness rate. Utilizing Public Establishments of Wellbeing (NIH) rules, the pooled predominance of PCOS was 5.8%.4 45% of females with PCOS express that they have never gotten direction on the most proficient method to deal with their way of life.5 As studies on PCOS have not been concluded previously in our Wardha District. So here is our aim to appraise the awareness of PCOS among the rural and semi-urban female population in District Wardha. With the help of its outcome, the campaign or health professional will try to educate and inform people about PCOS which will help in reducing the rate in Wardha district (India).\n\n\n\n• To collect the question-based data on women in the Wardha district.\n\n• To assess the knowledge of awareness regarding polycystic ovarian syndrome (PCOS).\n\n\nProtocol\n\nType of Study – Cross-Sectional study (observational study)\n\nDuration of study – 4 months\n\n• Conceptual research on polycystic ovarian syndrome will be done.\n\n• Prior to the initiation of the study, approval will be obtained from the ethics committee.\n\n• By using the random sampling method, the subject will be assigned.\n\n• The awareness research will be done through face-to-face interviews using a standardized questionnaire form.\n\n• Subjects will be provided informed consent, which will be filled out after explaining the purpose of the study.\n\n• Standardized Questionnaires will be given to the subject and will be filled by them.\n\n• The data collected will be tabulated and analyzed.\n\nThe study is conducted among the female general population of the Wardha district. The study is about the awareness of polycystic ovarian syndrome among women. The recruitment of the subject will be done independently and the awareness will be observed through a standardized questionnaire tool.\n\nMethod of data collection\n\n• Techniques of Sampling: Simple Random Sampling will be used.\n\n• Instrument: Researcher prepared a structured questionnaire consisting of MCQs.\n\nSteps of data collection\n\n• Step 1: Permission will be obtained prior from the ethics committee for the data collection.\n\n• Step 2: Introduction between the participants and the researcher.\n\n• Step 3: The participants will be assigned by using a random sampling method.\n\n• Step 4: ICF will be taken from the participants who are willing to participate, after explaining the study purpose.\n\n• Step 5: Standardized questionary will be filled out by the subjects with the help of face-to-face interviews.\n\nExpected dates of data collection\n\nThe data will be collected from March 2024 to July 2024.\n\nExpected dates of recruitment\n\nThe First subject is to be recruited on 1st March 2024 and 375 participants will be recruited by the end of July 2024.\n\nCross-sectional study\n\nSelection of subjects\n\nInclusion criteria\n\n• Age Range\n\n• Subjects aged above 15 to 45\n\n• Gender\n\n• Subjects only female will be included randomly.\n\n• Participants who are willing to participate after explaining the protocol.\n\nExclusion criteria\n\n1. Age below 15 and above 45 years\n\n2. The subject is under the category of vulnerable personnel patients in an emergency, ethnic minority group, homeless persons, minors, or others\n\n3. Participants not willing to participate\n\nSources of participants\n\nParticipants will be randomly assigned from the rural, semi-urban, and urban residential areas of Wardha district. Subjects will be recruited from various locations like hospitals, local institutions, homes, and from the campaigns in public spaces.\n\nVariables\n\nAs studies on PCOS have not been concluded previously in our Wardha District. So here is our aim to appraise the awareness of PCOS among the rural and semi-urban female population in District Wardha. With the help of its outcome, the campaign or health professional will try to educate and inform people about PCOS which will help in reducing the rate in Wardha district (India).\n\nData source and management\n\nThe recruitment of the subject will be done independently and the awareness will be observed through a standardized questionnaire tool. Participants will be randomly assigned from the rural, semi-urban, and urban residential areas of Wardha district. Subjects will be recruited from various locations like hospitals, local institutions, homes, and from the campaigns in public spaces.\n\nBias\n\nAs the patient will be recruited randomly selection bias will be avoided.\n\nSample size6:\n\nDaniel’s formula for sample size:\n\nZα/2 is the level of significance at 5% i.e., 95%\n\nConfidence interval = 1.96\n\nP = Aware of polycystic ovarian syndrome = 57.8% = 0.578\n\nd = Desired error of margin = 5% = 0.05\n\n\n\nHence, 375 subjects were needed in the study.\n\nQuantitative variables\n\nIn this study quantitative variables will be a percentage of female population such as who are aware of PCOS, who are aware about which organ system will be affected due to PCOS, aware about symptoms and indicators of PCOS and many more.\n\nMethod of data analysis\n\nMeasurable investigation including mean, standard deviation, recurrence, extent, and rate, and inferential measurements containing χ2 test, factor examination, and discriminant examination was utilized to dissect the information utilizing SPSS (factual bundle for sociologies) and Excel.\n\nIt will be published in an Index Journal.\n\nThe study yet to be started.\n\n\nResult\n\nAs the study is yet to be started, the result will be concluded after the data collection at the end the study.\n\n\nDiscussion\n\nPolycystic ovaries with high antral (2-8 mm) follicle numbers are the main attribute of PCOS. The average ovarian morphology is the result of the debilitated follicular turn of events and untimely discontinuance of follicular development after the aggravation of endocrine which includes hyperandrogenaemia, hyperinsulinemia, and Luteinizing hormone hypersecretion. Accordingly, more than 50% of patients have unpredictable periods, which is joined with anovulatory barrenness.7\n\nThe clinical show of PCOS changes broadly. Ladies with PCOS frequently look for care for feminine unsettling influences, clinical appearances of hyperandrogenism, and fruitlessness. Feminine aggravations usually seen in PCOS incorporate oligomenorrhea, amenorrhea, and delayed unpredictable feminine dying. Notwithstanding, 30% of ladies with PCOS will have ordinary menses. Around 85%-90% of ladies with oligomenorrhea have PCOS while between 30% to 40% of ladies suffering from amenorrhea can be a patient of PCOS.8\n\nBarrenness influences 40% of ladies with PCOS. PCOS is the most well-known reason for anovulatory barrenness. Ladies suffering from PCOS have an ordinary no. of early-stage follicles which fundamentally expand. Nonetheless, because of confusion in factors engaged with the typical follicular turn of events, follicular development becomes captured as follicles arrive at a width of 4 to 8 mm. However, a predominant follicle doesn’t create, ovulation doesn’t result. Likewise, unconstrained early termination happens all the more regularly in PCOS with frequencies going from 42% to 73%.8 PCOS may be a genetic disorder. PCOS can be considered a heritable problem.\n\nInsulin compensatory and opposition hyperinsulinemia are the main components of PCOS. The patients suffering from PCOS both with and without corpulence may contain higher commonness of insulin obstruction contrasted with solid controls, despite the fact that insulin opposition is more serious in fat subjects.8\n\nHyperandrogenism is a critical indicative component of polycystic ovarian syndrome influencing 60 to 100 percent of patients with both clinical (alopecia, hirsutism, and skin inflammation) and biochemical hyperandrogenism.8 Hyperandrogenism prompts constant anovulation and feminine aggravations in patients suffering from PCOS. More than 80% of ladies suffering from androgen abundance have PCOS.9 Hirsutism is a typical clinical show of hyperandrogenism happening in up to 70% of ladies with polycystic ovarian syndrome. Hirsutism is assessed with the help of a changed Ferriman-Gallwey scoring framework. This instrument is utilized to assess hair development at seven locales: upper lip, jawline/face, chest, back, midsection, arms, and thighs. More than 90% of typically bleeding ladies with hirsutism are distinguished through ultrasound to have polycystic ovaries. Also, PCOS happens in half of the ladies with less serious conveyance of undesirable hair development. Skin breaks out can be a marker of hyperandrogenism but less common in PCOS and also less unambiguous than hirsutism.\n\nWeight gain can be a clinical element of PCOS, and following a proper lifestyle has been effective in diminishing stomach fat, and body weight, lessening testosterone, further developing insulin opposition, and declining hirsutism in women with PCOS.8\n\nWeight reduction works on feminine abnormalities, side effects of androgen overabundance, and barrenness. The board of clinical signs of PCOS incorporates oral contraceptives for feminine anomalies and hirsutism. Spironolactone and finasteride are utilized to treat the side effects of androgen overabundance. Treatment choices for fruitlessness incorporate clomiphene, laparoscopic ovarian penetrating, and gonadotropins, and helped conceptive innovation. Letrozole and metformin might assume a significant part in ovulation enlistment. Appropriate conclusions and the executives of PCOS are vital for addressing patient worries yet additionally to forestall future metabolic, endocrine, mental, and cardiovascular inconveniences. Insulin-sharpening specialists are shown for most ladies with polycystic ovary disorder since they decidedly affect insulin opposition, feminine abnormalities, anovulation, hirsutism, and stoutness. Metformin has the most information supporting its adequacy. Rosiglitazone and pioglitazone are likewise successful in enhancing hirsutism and insulin opposition. Metformin and clomiphene, alone or in blend, are first-line specialists for ovulation acceptance. Insulin-sharpening specialists, oral contraceptives, spironolactone, and effective eflornithine can be utilized in patients with hirsutism.\n\nEflornithine hydrochloride in human skin has been endorsed as effective for treating facial hirsutism. It tends to be joined with laser treatment for further impact.\n\nOral preventative pills (OCPs) can be used for treatment. OCPs lessen hyperandrogenism by advancing direct regrettable criticism of luteinizing hormone (LH) emission, which brings about a diminished ovarian blend of androgens. One of the most up-to-date OCPs that may be more successful in lessening the development of new terminal hair and skin break out development is a recipe that contains a mix of nonandrogenic progestin, drospirenone, and ethinyl estradiol; subsequently, it is possibly great for the treatment of ladies with PCOS.10 Estrogen-progestin mix treatment (with the utilization of a mix OCP) stays the transcendent treatment for hirsutism and skin breakout in PCOS.11 Myoinositol is an over-the-counter food supplement that increments insulin awareness.12\n\nWay of life changes, including a weight-lessening diet and exercise, are suggested as the first-line treatment for all hefty ladies with PCOS. In any case, numerous stout ladies with PCOS find weight reduction hard to accomplish and keep up with. Additionally, around 10-30% of ladies with PCOS are lean, and weight reduction isn’t a possibility for them. Consequently, insulin-sharpening drugs are being utilized all the more habitually and have come to assume a significant part in the persistent treatment of PCOS.13\n\nPolycystic ovary condition (PCOS) and obesity are related with a more serious gamble of ovarian growths, where PCOS could be either the reason or as a result of an ovarian cancer.14 An instance of a 53-year-old postmenopausal lady introducing a monster ovarian cystic mucinous growth was accounted to weigh 24 kg. At the point when she was seen first at facility, she had gross stomach distension for 2 years, and she griped of excruciating forceful agony. Her figured tomography (CT) examine was done which came reminiscent of ovarian serous cystadenoma of enormous gigantic size 35 × 40 × 32 cm with moderate ascites.15 Obesity can be separated into metabolic syndrome (MetS) or metabolically unhealthy obesity (MUO) and metabolically healthy obesity (MHO). The clinical and metabolic aspects of women with MHO will be different in PCOS women with or without MetS/MUO and may contain high chances of having cardiovascular disease.16 In view of different clinical show, ladies with PCOS might introduce in gynecology, dermatology, or endocrinology OPD. The sequalae of PCOS arrives at past regenerative wellbeing, with an expanded gamble of cardiovascular illness and type 2 DM, mindfulness in regards to PCOS is significant for early finding and to forestall its sequalae.17 As per current science, the physiology connected with ovulation is constrained by chemicals of hypothalamo – pituitary – ovarian pivot. This pivot is upset in pcos. Different methodologies in ayurveda for patho-physiology in polycystic ovarian disorder reason that pcos is a puzzle. There is no finished comprehension of its essential pathophysiology according to present day science. No treatment is a panacea, as treatments have so far been designated at the side effects however not at the actual disorder.18 Uncontrolled steroidogenesis, insulin opposition, oxidative pressure, and genetic factors all assume a part in PCOS pathogenesis, which might start as soon as pre-birth life. Extra examination is expected to overcome any barrier between the numerous vulnerability factors that might assume a part in PCOS.19\n\nThis study is limited to only female population of age group 15 to 45 years of Wardha district. As in this study patient will be recruited randomly selection bias will be avoided.\n\nA cross-sectional study of 400 women between the ages of 18 and 30 who were either working or attending college in the city of Indore was conducted. Only 41% of the 400 women who took part in the study were familiar with the term PCOS. 46% of the participants knew which organ system this condition affected. The majority of people learn about this condition via friends or family. The majority of the women, 49%, were aware of the numerous PCOS symptoms and indicators.20 So here is our aim to appraise the awareness of PCOS among the rural and semi-urban female population in District Wardha. With the help of its outcome, the campaign or health professional will try to educate and inform people about PCOS which will help in reducing the rate in Wardha district (India).\n\nApproved by the Institutional Ethics Committee Ref. No. DMIHER (DU)/IEC/2023/597.\n\nDatta Meghe Institute of Higher Education And Research Sawangi (M) Wardha- 442107 Maharashtra, India.", "appendix": "Data availability\n\nZenodo. Sejal Patil Questionnaire. DOI: 10.5281/zenodo.8181193\n\nThis project contains the following underlying data:\n\n• Sejal Patil Questionnaire.docx (This document include questionnaire of study protocol.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nZenodo. Sejal Patil Informed consent form. DOI: 10.5281/zenodo.8181220\n\nThis project contains the following underlying data:\n\n• Sejal Patil Informed consent form.docx (This document contain Informed consent form related to my study)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nZenodo. STROBE checklist. DOI: 10.5281/zenodo.8210670\n\nThis project contains the following underlying data:\n\n• Sejal Patil STROBE checklist.docx (STROBE checklist guidelines)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nRotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group: Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod. 2004 Jan; 19: 41–47. PubMed Abstract \| Publisher Full Text\n\nDe Leo V, Musacchio MC, Cappelli V, et al.: Genetic, hormonal and metabolic aspects of PCOS: an update. Reprod Biol Endocrinol. 2016; 14: 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nhttp\n\nBharali MD, Rajendran R, Goswami J, et al.: Prevalence of Polycystic Ovarian Syndrome in India: A Systematic Review and Meta-Analysis. Cureus. 2022 Dec 9; 14(12): e32351. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAly JM, Decherney AH: Lifestyle Modification in PCOS.Publisher Full Text\n\nhttp\n\nKamenov Z, Gateva A: Inositol in PCOS. Molecules. 2020 Nov 27; 25(23): 5566. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSirmans SM, Pate KA: Epidemology, diagnosis, and management of polycystic ovary syndrome. Clin Epidemiol. 2014 Dec 31; 6: 1–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAzziz R, Sanchez LA, Knochenhauer ES, et al.: Androgen excess in women: experience with over 1000 consecutive patients. J Clin Endocrinol Metab. 2004; 89: 453–462. PubMed Abstract \| Publisher Full Text\n\nFalsetti L, Gambera A, Tisi G: Efficacy of the combination ethinyl oestradiol and cyptroterone acetate on endrocrine, clinical and ultrasonographic profile in polycystic ovarian syndrome. Hum Reprod. Publisher Full Text\n\nBadawy A, Elnashar A: Treatment options of polycystic ovary syndrome. Int J Womens Health. 2011; 3: 25–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRasquin Leon LI, Anastasopoulou C, Mayrin JV: Polycystic Ovarian Diseases.Reference Source\n\nSharma ST MBBS, Nestler JE MD: Prevention of diabetes and cardiovascular diseases in women with PCOS: Treatment with insulin sensitizers. Best Pract Res Clin Endocrinol Metab. 2006 Jun; 20(2): 245–260. Publisher Full Text\n\nThaweekul P MD, Thaweekul Y MD, Mairiang K MD: A huge ovarian mucinous cystadenoma associated with contralateral teratoma and polycystic ovary syndrome in an obese adolescent girl. Asia Pac J Clin Nutr. 2016 Dec; 25(4): 920–923. Accessed 2 June 2023. Publisher Full Text Reference Source\n\nHalani D, et al.: Postmenopausal woman with 24 kilograms ovarian mucinous cystadenoma: a rare case report. Pan Afr Med J. 2023; 44(42). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAcharya N, Acharya S, Shukla S, et al.: Polycystic Ovarian Syndrome (Pcos) in Obese Metabolic Phenotypes. Int J Curr Res Rev. 2020; 12(22 Special Issue): 13–15. Publisher Full Text\n\nJungar ML, Nair P, Gode S, et al.: PCOS: Clinical Picture of Pcos Patients in a Peri Urban Tertiary Care Hospital of Central India. J Crit Rev. 2020; 7(8): 1076–1080. Publisher Full Text\n\nAndhara R, Jadhav S, Shrinivas Y, et al.: Multiple Approaches in Ayurveda for Patho-physiology in Polycystic Ovarian Syndrome. J Med Pharm Allied Health Sci. 2021; 10(3): 3055–3058. Publisher Full Text\n\nDhapke G, Hawale D, Jha RK, et al.: Pcod-Polycystic Ovarian Disease: A Review Article. ECS Trans. 2022; 15973–15979. Reference Source\n\nPatel J, Rai S: Polycystic ovarian syndrome (PCOS) awareness among young women of central India. Int J Reprod Contracept Obstet Gynecol. Oct 2018; 7(10): 3960. Publisher Full Text" }	[ { "id": "218211", "date": "23 Dec 2023", "name": "Milan Perovic", "expertise": [ "Reviewer Expertise gestational diabetes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSejal Patil and Gaurav Mude have submitted a SPIRIT compliant Study Protocol entitled \"Awareness regarding knowledge associated with polycystic ovarian syndrome (PCOS) among women in Wardha district- A cross-sectional study.\". In general, this is a well written study protocol, but it could benefit from revision. Please see the specific comments to the authors.\n\nTitle is descriptive; it is identifying the study design, population, and interventions. However, I suggest the authors to put the trial acronym in the title. Trial identifier and registry name is missing. If you have not yet registered, please provide the name of intended registry. Furthermore, I believe if you add \"(India)\" after \"among women in Wardha district-\" it could be more informative for the majority of readers. You have written \"Grant information: The author(s) declared that no grants were involved in supporting this work\". Please provide information regarding other sources of funding if there are some: other types of financial, material, and other support to the study. In order to provide better justification for undertaking the trial, including summary of relevant studies, I suggest the authors to add something like this \"Furthermore, PCOS has important impact on human reproduction, both for infertility and high-risk pregnancies (reference: Gojnic-Dugalic M et al. (20211)).\n\nafter the sentence \"50-70% of females with PCOS have insulin opposition, which expands their gamble of creating diabetes, metabolic condition, hypertension, dyslipidemia, and other comorbidities like hypertension and dyslipidemia\". References should be improved! Reference number 3 and 6 are missing! References 5, 10, 12 are not cited well. Number of references is low. 5 out of 20 references are older than 10 years.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "307816", "date": "27 Aug 2024", "name": "Deepa Shanmugam", "expertise": [ "Reviewer Expertise PCOS", "infertility" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is complete with the aim of the research and study design clearly mentioned..\nIntroduction tells about the problem statement, background of the study, novelty, need for the study and rationale... The objectives are clearly explained In the methodology, the study design, study participants, methods of data collection and statistical analysis is described. The study design is based on the research question *Finally, the application of this research outcome in the population by creating awareness is mentioned, which adds to the strength..\nMajor comments:\n1. In the Introduction, the four consortments of PCOS is mentioned which needs authentication. The reference for the same needs to be added.. 2. It will be better to add Review of literature on the similar study, done at national and international level\nMinor comments: 1. Introduction is elaborate, can be more brief 2. Even though methodology is described clearly in the proposal, it can be depicted by a consortium diagram/ flowchart\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1085
https://f1000research.com/articles/12-1083/v1	01 Sep 23	{ "type": "Research Article", "title": "Epigenetic germline variants predict cancer prognosis and risk and distribute uniquely in topologically associating domains", "authors": [ "Shervin Goudarzi", "Meghana Pagadala", "Adam Klie", "James V Talwar", "Hannah Carter", "Shervin Goudarzi", "Meghana Pagadala", "Adam Klie", "James V Talwar" ], "abstract": "Background: Methylation quantitative trait loci (meQTLs) associate with different levels of local DNA methylation in cancers. Here, we investigated whether the distribution of cancer meQTLs reflected functional organization of the genome in the form of chromatin topologically associated domains (TADs) and evaluated whether cancer meQTLs near known driver genes have the potential to influence cancer risk or progression. Methods: Published cancer meQTLs were analyzed according to their location in transcriptionally active or inactive TADs and TAD boundary regions. Cancer meQTLs near known cancer genes were analyzed for association with cancer risk in the UKBioBank and prognosis in The Cancer Genome Atlas (TCGA). Results: In TAD boundary regions, the density of cancer meQTLs was higher near inactive TADs. Furthermore, we observed an enrichment of cancer meQTLs in active TADs near tumor suppressors, whereas there was a depletion of such meQTLs near oncogenes. Several meQTLs were associated with cancer risk in the UKBioBank, and we were able to reproduce breast cancer risk associations in the DRIVE cohort. Survival analysis in TCGA implicated a number of meQTLs in 13 tumor types. In 10 of these, polygenic cancer meQTL scores were associated with increased hazard in a CoxPH analysis. Risk and survival-associated meQTLs tended to affect cancer genes involved in DNA damage repair and cellular adhesion and reproduced cancer-specific associations reported in prior literature. Conclusions: This study provides evidence that genetic variants that influence local DNA methylation are affected by chromatin structure and can impact tumor evolution.", "keywords": [ "meQTLs", "TAD", "Cancer", "Polygenic Risk Score", "XGBoost", "Machine learning" ], "content": "Introduction\n\nCancer is a heterogeneous disease and common treatments like chemotherapy have only a 55% response rate.1 Precision medicine and biomarker analysis can tailor treatment options and optimize outcomes. Genetic factors, such as germline and somatic mutations, contribute to heterogeneous disease risk and progression. For example, germline variants in the BRCA2 gene can greatly increase the risk of developing breast and ovarian cancer.2 Epigenetic factors including DNA methylation, histone modification, and acetylation also play a key role in cancer progression. Recently, promising therapeutics have been developed that inhibit DNA methyltransferases (DNMTs), reducing tumor growth in breast cancer and highlighting the importance of DNA methylation and other epigenetic factors in carcinogenesis.2,3 However, the interplay between epigenetics and genetics in cancer risk and progression remains mostly elusive.\n\nMethylation quantitative trait loci, or meQTLs, are single nucleotide polymorphisms (SNPs) that significantly correlate with DNA methylation at CpG sites. These SNPs provide a bridge between genetic variation and corresponding epigenetic effects shown to correlate with cancer risk.4 Disruptions in DNA methylation are well-known in the context of cancer; DNA is frequently hypermethylated at promoter regions of tumor suppressor genes while hypomethylated at the promoters of oncogenes, and there is an inverse correlation with gene expression.5 Promoter hyper- and hypo-methylation has been of specific interest due to its role in regulating the expression of cancer genes including suppression of tumor suppressor genes like BRCA6 and the expression of oncogenes like L1NE1.7 Subsequently, germline SNPs that acted as meQTLs were shown to predict risk in many cancer types like breast and lung, regulating expression and methylation of genes like FBXO-18.4\n\nThe organization of the genome into 3D structures may further modify the potential of genetic variants to interact with epigenetic factors in a disease specific manner.8 Topologically associating domains (TADs) are isolated regions of highly-interacting and folded chromatin separated by insulator proteins. TADs are important for maintaining controlled patterns of local gene regulation and provide a framework for transcriptionally similar genes and SNPs to interact with one another.9 In fact, because TADs have been found to be highly stable across tissue types, they provide valuable context for understanding the genome’s functional landscape allowing the study of genetic variation in the context of 3D chromatin structure.10 Mutational burden of somatic mutations within the context of cancer demonstrated correlation with TADs.11 In addition, genes within TADs demonstrate correlated gene expression and histone modification,12,13 allowing us to group similar acting genes and SNPs, narrowing a search for potentially cancer related SNPs.\n\nIn this study, we integrate genetic correlates of DNA methylation across 23 cancer types (i.e. cancer meQTLs) and TAD domains to better understand how 3-D chromatin structure might determine the potential of meQTLs to influence cancer risk and survival. We focus on meQTLs near TADs containing key cancer-related genes. Analyzing the location and distribution of such variants across the genome, we find that methylation-related germline variants, or meQTLs, in cancer do not lie uniformly across the genome and the occurrence of TAD boundaries correlates with significant cancer meQTL presence. In addition, meQTLs closely related to cancer progression show specific nonrandom distribution in TAD domains. Then we assessed whether meQTLs near cancer genes could predict cancer survival and risk and found significant prediction power of these meQTLs across multiple cancer types. Our study suggests that the potential of meQTLs to contribute to cancer risk and progression depends in part on local genome architecture and chromatin state.\n\n\nResults\n\nWe identified 1100 TADs shared across 5 cell lines (GM12878, HMEC, HUVEC, IMR90, and NHEK) and categorized them into “Mixed”, “Inactive-1”, “Inactive-2”, “Active-1”, and “Active-2” groups using chromatin state information (Figure 1A). Combining the active and inactive groups resulted in 222 active, 626 inactive and 252 mixed TADs. DNA methylation is linked with TAD activity via nucleosome positioning and chromatin condensation14 as well as to regulation of gene expression, where promoter CpG methylation is associated with gene silencing.15 We compared our categorization of TAD activity with genome-wide DNA methylation in promoter regions defined based on the ENCODE Screen Pipeline. Promoters in active TADs showed overall lower levels of methylation whereas those in inactive TADs had a higher level of methylation (Kruskal-Wallis, p-value<0.001) (Figure 1B), supporting that promoter methylation silencing aligns with categorization of TADs into transcriptionally different groups, namely into “active” and “inactive”.\n\n(A) 5 state-based K-Means clustering of common TAD domains (n=1100) between 5 human cell lines (GM12878, HMEC, HUVEC, IMR90, and NHEK). Purple indicates TADs classified as a “Mixed”, Gray as “Inactive-1”, Light Blue as “Active-1”, Orange as “Active-2”, and Red as “Inactive-2”. Combining active and inactive categories leads to 222 Active, 626 Inactive, and 252 Mixed TADs. (B) On average, inactive TADs have higher DNA methylation levels than active TADs (p-value<0.001). These results are supported by previous literature concerning promoter methylation and transcriptional activity. (C) Number of meQTLs across inactive TADs versus active TADs are shown. meQTL counts per TAD were normalized by TAD length in base pairs. Active TADs show on average a larger normalized burden of meQTLs than inactive TADs (Student-t Test, p<0.05).\n\nNext we measured the overall burden of independent cancer meQTLs (i.e. meQTLs deemed to represent distinct haplotypes based on the level of linkage disequilibrium; LD) across TAD categories, normalized by TAD length in base pairs. To obtain independent meQTLs, we clumped related meQTLs based on linkage disequilibrium using PLINK. Out of the 1.2 million SNPs, 60,602 remained after LD pruning (Table 1). We observed a slightly increased number of cancer meQTLs in inactive domains relative to active regions (Student T-test, p-value<0.05; Figure 1C).\n\nEach row shows the total number of meQTLs after each analysis across each TAD type. The rows are as follows: all meQTLs without filtration, meQTLs in LD from PLINK clumping software (p<1×10-5) and meQTLs in LD with CpG probe in cancer driver gene promoter region.\n\nWe also evaluated cancer meQTLs at TAD boundaries, considering four categories of boundary based on the category of the flanking TADs: “Active-Boundary-Active”, “Inactive-Boundary-Inactive”, “Active-Boundary-Inactive”, and “Inactive-Boundary-Active”. To allow aggregation across variable length regions, we divided each boundary region into 40 equal genomic bins and calculated the number of meQTLs in each. We then compared the observed density of meQTLs to that obtained by randomizing flanking TAD categories 100 times. Comparing the density of meQTLs in each boundary category to the randomized equivalent, the active-active (student t-test, p<0.01), active-inactive (p<0.01), and inactive-active boundaries (p<0.01) all showed difference in distribution from random, while inactive-inactive (p=0.089) did not (Figure 2A-D). Distributions suggested an increase in density of clumped meQTLs when transitioning from active to inactive regions, and conversely, a decrease from inactive to active regions (Kruskal-Wallis ANOVA, p-value<0.05) when compared to the randomly shuffled distribution, but no shift in density for Active-Boundary-Active and Inactive-Boundary-Inactive categories (Figure 2B-D).\n\nThe binned average normalized meQTL burden distribution is shown across boundaries between consecutive TADs, grouped by transition category: active to active, active to inactive, inactive to active, and inactive to inactive. The start/end of the TADs for both active and inactive are shown red and blue, respectively. Distributions are smoothened by rolling average for visualization purposes. The graphs represent a unique distribution of meQTL burden across consecutive TADs as opposed to an even spread. The dotted brown line represents the distribution for shuffled random TADs to act as control. (A) Active-active (p=3.51×10-10), (B) active-inactive (p=3.45×10-46), and (C) inactive-active (p=1.65×10-25) boundaries all showed clear difference in distribution from random, while (D) inactive-inactive (p=0.089) did not.\n\nClumped cancer meQTls were further narrowed to those associated with the methylation status of CpG probes located within the promoter regions of cancer driver genes including oncogenes and tumor suppressor genes (TSGs) from the COSMIC database.16 In total, 103 oncogenes and 223 TSGs were used for this analysis, where only 67 of them contained meQTL-affecting CpG probes in their promoter regions (i.e. 49 TSGs and 18 oncogenes). Out of the 60,602 clumped meQTLs, 156 of them significantly affected CpG probes located in promoter regions of cancer driver genes (driver meQTLs; Table 1). Overall, we saw an overwhelming bias for driver meQTLs to occur in active regions, followed by boundary, and inactive (Figure 3A). To understand whether the observed distribution of driver meQTLs was expected, we selected equivalent numbers of meQTLs at random and evaluated their distribution across region types. We did this separately for meQTLs associated with oncogenes versus TSGs, as meQTLs might have different implications in the context of selection for gain versus loss of function. In the oncogene case, meQTLs were depleted relative to random in active TADs, and enriched relative to random in inactive TADs, with no difference in boundary regions. Conversely, for TSGs, there was a significant enrichment of cancer-related meQTLs in active TADs and boundary regions, but a depletion in inactive TADs (Figure 3B-C). These opposing trends could suggest genes with the potential to be oncogenes or tumor suppressors (i.e. growth promoting versus limiting) are under different constraints with respect to the propensity for methylation to accumulate in their promoter regions.\n\n(A) The number of driver meQTLs per MB are plotted, divided according to the category of TAD they are located in. Normalization was conducted by the total region size in each category. (B-C) Randomization analysis for burden of non-cancer meQTLs normalized by number of base pairs in each region was conducted to obtain the expected number of cancer meQTLs per MB. To model random expectation (B) 54 non-cancer meQTLs (i.e. number of oncogene-proximal meQTLs) and (C) 102 non-cancer meQTLs (i.e. number of TSG-proximal meQTLs) were sampled 1000 times for oncogenes and TSGs respectively. Bar graphs are drawn with standard errors. The actual observed cancer meQTL burden is shown as a red dot.\n\nWe next evaluated the potential for driver meQTLs to have clinical relevance. A principal component analysis (PCA) was first conducted on the 156 driver meQTLs across individuals in the TCGA. The principal components (PCs) that explained more than 1% of the variance were assessed for association with clinical covariates by linear regression. We noted some association of PCs with tumor type, age at diagnosis and tumor stage at diagnosis, suggesting that cancer meQTLs could have tumor-type specific implications for risk and prognosis. Interestingly, further examining the 10 meQTLs with the strongest loadings in PCs correlated with tumor type, we found that the meQTLs disproportionately affected oncogenes, suggesting that tumor types differ more in oncogene effects than in tumor suppressor effects of DNA methylation.\n\nWe first evaluated the driver meQTLs for cancer risk associations using the UKBioBank. In total, 86 of the 155 (1 SNP was not in the UKBioBank registry) driver meQTLs in the initial PheWAS analysis from UKBioBank patients showed a nominal association with one or more cancer ICD10 codes (p-value<0.05) with 5 SNPs passing a Benjamini-Hochberg FDR threshold of 0.05 (Table 2). In total, meQTLs were associated with risk of 15 different cancer types as described by ICD10 codes (Table 3). We focused on C50-C50 (malignant neoplasm of the breast) as this tumor type had a large sample size in UKBioBank (n=11,188) and other large cohorts exist to support validation studies.\n\nThe beta value is the correlation coefficient of the meQTLs with DNA methylation at the promoter region of the probe gene. The TAD type that the meQTL resides is also represented.\n\nThe ICD 10 code used by UKBioBank is shown alongside their definitions for the risk analysis.\n\nTo further assess the relevance of driver meQTLs to cancer risk, we used them to predict breast cancer status alongside clinical covariates using the approach described by Elgart et al.17 We first performed feature selection by LASSO on nominally significant driver meQTLs and available clinical factors (age, ancestry as represented by the top 10 genotype-derived PCs); LASSO regularization removed ancestry and some meQTLs. Selected features were then used to train an XGBoost classifier on 189,022 examples derived from UKBioBank breast cancer cases and non-cancer controls (Methods). The score resulting from the trained XGBoost model was used as the PRS. We applied the trained model to predict breast cancer status for individuals in the DRIVE dataset, comprising 26,374 breast cancer cases and 32,428 controls. The distribution of PRS values across cases was significantly higher than controls for the breast cancer outcome, as expected (Mann-Whitney U, p-value<0.001) (Figure 4A). In both UKBioBank and DRIVE datasets, the incidence of breast cancer was significantly higher among individuals in the upper 20% percentile of the PRS score versus the bottom 20% percentile (Fisher’s exact test, UKBioBank: p=4.25×10-7<0.001, DRIVE: p=1.47×10-13<0.001), suggesting that a higher burden of meQTLs impacts breast cancer risk (Figure 4B-C).\n\n(A) An XGBoost classifier trained to predict incidence of breast cancer in the UKBioBank, was applied to predict cancer risk in the DRIVE cohort. PRS scores provided by the model were higher for individuals diagnosed with breast cancer (Figure 8, Mann-Whitney U p=2.4×10-19). (B-C) Plots showing the odds ratio of a breast cancer diagnosis across 10% quantiles of the XGBoost predicted PRS in the UKBioBank and DRIVE cohorts respectively. Risk increased from a hazards ratio of ~0.8 to ~1.1 between 0th and 90th PRS percentiles, supporting that cancer meQTLs impact breast cancer risk. C50-C50: ICD10 code for malignant neoplasms of the breast.\n\nWe extracted feature importances from the UKBioBank-trained PRS to better understand the driver meQTLs underlying breast cancer risk (Figure 5). Overall, cancer meQTLs near 29 cancer genes were included in the model. The most predictive driver meQTL was associated MSH2, a gene associated with Lynch syndrome and increased risk of breast cancer.18 Polymorphic variation affecting the expression of EZH2, the second most informative feature, has also been linked to breast cancer risk.19 ASXL2 may be required for estrogen receptor alpha (ERa) activation in ERa positive breast cancers.20 Notably, EZH2 overexpression has been linked more strongly to triple negative breast cancer21 suggesting that the model includes features predictive of multiple subtypes.\n\nFeatures are ranked according to their contribution to classifier predictive performance. Total importances sum to 1.\n\nFinally, we evaluated the implications of driver meQTLs for prognosis. We first removed one meQTL, 2:209220238:C:G, that had a minor allele frequency <1% across TCGA samples, then conducted a Kaplan-Meier analysis for the remaining meQTLs separately for each tumor type with at least 100 samples. Out of the 155 SNPs, 21 passed the Benjamini-Hochberg adjusted FDR of less than 0.05 (Table 2). To assess overall contribution of driver meQTLs to survival, we built polygenic survival scores (PSS) using XGBoost and incorporated them into Cox proportional hazards (PH) models alongside relevant covariates. Here we only evaluated tumor types that had at least 5 SNPs implicated as nominally significant by Kaplan-Meier analysis (n=23 tumor types). Nominally significant driver meQTLs for each tumor type were subjected to selection by LASSO and used to train XGBoost models to predict binary survival outcome (binarized based on median time to an event) separately for each tumor type. Out of the 23 tumor types, 13 had a higher XGBoost classification AUC value when both SNPs and clinical were combined as compared with using only clinical covariates. These included BLCA, BRCA, PAAD, PRAD, UCEC, OV, STAD, SKCM, PCPG, LUSC, KIRC, HNSC and ESCA. This suggests that for these cases, meQTLs contributed survival-relevant information beyond the covariates (i.e. age, sex, tumor stage in some cases). For these tumor types, we trained XGBoost models using only meQTLs to obtain tumor-type specific polygenic survival scores (PSS) that were then included alongside covariates (tumor stage, age at diagnosis and sex) in Cox PH models to predict overall survival time in months (Methods).\n\nPSS values made a significant contribution to predicting overall survival time for all cancer types except BRCA and SKCM (Figure 6). PSS had the highest hazard ratios compared to other covariates for most cancer types, including: ESCA, BLCA, KIRC, LUSC, OV, PAAD, PCPG, PRAD, STAD, UCEC. Most covariates behaved as expected in the analysis with tumor stage having one of the highest odds ratios. However, it is difficult to assess the generalizability of the estimated effect sizes in the absence of independent validation cohorts with both genotype and survival measured in the same cancer types. Nonetheless, to further investigate the prognostic implications of driver meQTLs, we analyzed their feature importances in their respective XGBoost models (Figure 7). The number of meQTLs contributing to tumor type specific PSS ranged from 2 to 12, often with 1 or 2 meQTLs dominating the model.\n\nThe hazard ratios and 95% confidence intervals associated with various covariates are shown across 13 cancer types: BLCA, BRCA, PAAD, PRAD, UCEC, OV, STAD, SKCM, PCPG, LUSC, KIRC, HNSC, ESCA. Due to limitations in availability of data some tumor types lacked covariates like tumor stage. Sex was excluded for tumors that only occur in males or females. ER: Estrogen receptor, PR: Progesterone Receptor.\n\nA heatmap of the feature importances of SNPs for the cancer type specific XGBoost survival classifiers is shown. For each model across the 13 tumor types, the feature importances sum to 1 with red demonstrating larger importance of a SNP and blue demonstrating lesser importance.\n\nFocusing on the most informative tumor type-associated meQTLs, we investigated the relevance of the associated oncogenes to cancer progression. In many cases, the identified genes were supported by previous studies. For example, PTPRD loss in melanoma was shown to cause disruption of desmosomes, resulting in increased invasive potential.22 Polymorphisms in exonuclease ERCC2 have also been found to modify melanoma prognosis23 and have been linked to prostate cancer progression as well.24 In pancreatic cancer, RFWD3 expression quantitative trait loci (eQTLs) are associated with survival.25 RFWD3 is an E3 protein ubiquitin ligase important for DNA damage and has been shown to stabilize TP53 in response to DNA damage.26 We note that RFWD3 meQTLs were among the informative features for many other tumor types as well (Figure 7). RAC1 has previously been shown to determine the metastatic potential of renal cell carcinoma (KIRC).27 Reduced expression of CDKN1B is a known risk factor for PCPG and is common in this disease but usually cannot be explained by somatic alterations, though cases of allelic imbalance have been noted.28 CASP9 promoter polymorphisms confer increased risk of breast cancer29 and higher expression of CASP9 was associated with better survival.30 Downregulation of ERCC5 is associated with longer progression free survival in ovarian cancer treated with platinum therapy31 as is the case for OV in TCGA. In head and neck cancer, the most informative driver meQTL was associated with ETNK1, a cancer gene more commonly associated with myeloid neoplasms32 though there is increasing evidence that it may contribute to dysregulation of phospholipid metabolism in multiple tumor types.33\n\n\nDiscussion\n\nThere is an increasing appreciation that both genome structure34–38 and common genetic variants39–46 modify to the potential for carcinogenesis. However, the interplay between these factors is not well understood. To start to understand this, we investigated the relationship between the cancer meQTLs recently reported by Gong et al., and 3D genome structure in the form of TADs. To determine the relevance to cancer, we further investigated cancer meQTLs near driver genes for potential to modify cancer risk and progression. We took advantage of a recently introduced modeling strategy that first performs feature selection on a set of nominally associated SNPs, then trains a non-linear XGBoost model based on those features.17 Feature importances can be extracted from the trained model to gain insight as to which features were most influential, suggesting biological hypotheses that can be further investigated.\n\nWe observed higher levels of promoter methylation in inactive versus active TADs, slightly more meQTLs in active TADs and higher densities of meQTLs in boundary regions proximal to inactive versus active TADs. Furthermore, analyzing meQTL distribution across TAD boundaries revealed a non-uniform pattern, suggesting that TAD boundaries affected distributional burden of meQTLs. It is of note that TAD boundaries conserved across cell types are reportedly highly enriched for evolutionary constraint and complex trait heritability.10 Interestingly, we found that meQTLs associated with driver genes showed patterns of enrichment or depletion in a manner dependent on the activity state of the TAD in which the meQTLs occurred. Investigating cancer meQTLs, which are polymorphic sites that associate with differences in the level of DNA methylation found in tumors, showed depletion for germline meQTLs affecting oncogenes but enrichment for such meQTLs affecting tumor suppressor genes in active TADs. This could suggest that the potential to modulate tumor suppressor gene expression through methylation is evolutionarily advantageous whereas modulating oncogene expression by promoter methylation may be less so. These trends point to evolutionary constraints on the distribution of meQTLs imposed by 3D genome architectures and that could set the stage for genomic vulnerabilities to later malignancy.\n\nFocusing on meQTLs near known driver genes, we evaluated the potential of meQTLs to modify cancer risk or progression. We found a number of meQTLs associated with survival in the UKBioBank and were able to validate a polygenic score constructed from these meQTLs in the independent DRIVE cohort. The inclusion of genes linked to distinct breast cancer subtypes among the features that most contributed to classifier performance suggests that cancer meQTLs may differentially affect risk of developing different forms of breast cancer and raises the possibility that subtype-specific meQTL-based risk classifiers may outperform a generic model. The meQTLs most strongly predictive of prognosis tended to occur near cancer genes that were also associated with risk or prognosis in the same tumor type. However, we saw cases such as ETNK1 in head and neck cancer, where meQTLs implicated a gene that has not been considered a factor promoting progression. This could point to a new therapeutic opportunity in this disease. Further studies are merited to determine whether the observed associations result from meQTLs being in linkage with eQTLs or coding variants that contribute to risk or progression, or whether meQTLs themselves make it easier or more difficult for genes to be modulated through DNA methylation. Interestingly, we noted multiple independent meQTLs for the same cancer gene were informative in predictive models. This suggests that at least in some cases, the cumulative burden of meQTLs near driver genes could further alter gene function to exacerbate risk or progression. While we focused on cancer genes, other studies have more broadly implicated meQTLs in cancer survival, supporting expanded analyses in the future.\n\nThere are a few limitations for this study. First, the meQTLs utilized for this study are derived from a study of tumors46 which could be biased toward detecting meQTLs associated with DNA methylation events that are positively selected in tumors. Second, once focusing on specific tumor types, the number of samples available to predict prognosis is relatively small, and some samples were missing tumor stage or age at diagnosis data, key clinical features for survival prediction. In addition, we lacked independent cohorts to validate the generalizability of polygenic survival scores based on meQTLs, which could lead to overfitting in some of our results as suggested by the large hazard ratios observed in CoxPH analysis. This validation should be a priority as suitable data sets become available. We also made a few assumptions. We only considered common TADs across multiple human cell lines which could have potentially removed some important cell-type specific TAD domains, though our methodology follows what other studies11,47 have done. For predicting prognosis, we made the assumption that TAD domains from healthy human cell lines would also apply to cancer patients and thus avoided events where TAD structure could change. We justified our decision through previous studies determining TAD domains are overwhelmingly similar across cancer and noncancer patients.47 In future studies, it would be of interest to study meQTL trends in normal tissue samples to see if enrichment patterns associated with cancer genes are driven by selection in tumors, or highlight evolutionary constraints more broadly associated with human health that coincidentally are advantageous for tumor development.\n\nThis study investigated the relationship between epigenetic factors like chromatin structure and DNA methylation and genetic variation in the context of cancer, and established the potential for cancer gene associated meQTLs to uncover cancer-specific modifiers of risk and progression.\n\n\nMethods\n\nTCGA meQTLs data were obtained from Gong et al.46 TCGA outcome and survival data alongside RNA-seq expression data were obtained from the pan-can atlas, Liu et al.48 Illumina 450k DNA methylation data were also obtained from the TCGA pan-cancer atlas.48 The promoter data was obtained from the ENCODE Screen pipeline.49,50\n\nGenotypes and ICD10 codes were obtained for 394,034 samples across 40 ICD 10 codes from the UK BioBank.51 For the C50-C50 analysis, only exclusive cases and controls were considered: patients who were only diagnosed with the breast neoplasm were compared with controls who were not diagnosed for any neoplasm. This reduced the sample size to 189,022 for the breast cancer risk analysis.\n\nDiscovery, Biology, and Risk of Inherited Variants in Breast Cancer (DRIVE) (dbGaP Study Accession: phs001265.v1.p1)52 was used to validate the risk outcome analysis of our XGBoost model. There were 60,231 breast cancer cases and controls with genotype data alongside outcome, age, and ancestry principal components.\n\nTopologically associating domain (TAD) regions from the GM12878, HMEC, HUVEC, IMR90, and NHEK cell lines were downloaded from Rao et al.12 and only common TAD domains using a 20% overlap algorithm described previously across all 5 cancer cell lines were considered for the rest of the analysis. TAD domains were characterized into 5 clusters: “Active-1”, “Active-2”, “Inactive-1”, “Inactive-2”, and “Mixed” through K-means clustering and use of a 15-chromatin state model derived from the Roadmap Epigenomics Project.53 For most of the analysis, the two active and two inactive groups were combined for simpler visualization and mixed regions were ignored due to their biological ambiguity. The boundary of each TAD was considered as the 50 kb region upstream and downstream of TAD endpoints (i.e. 100 kb long boundaries) with the exception of consecutive TADs that had a region in between smaller than 100k base pairs. For those cases, the boundary was considered as the proximal half of the region for each of the two TADs. This TAD boundary definition using a 100 kb boundary ±50 kb upstream and downstream from the start and end of a TAD-is supported by previous literature.10\n\nDNA methylation levels were compared to TAD domains as follows. DNA methylation levels were summarized at promoters identified by the ENCODE’s SCREEN pipeline for in human hg38. We compared the methylation beta values (i.e. the proportion of methylated region) using TCGA’s DNA methylation data, and averaged these beta values for all promoter regions across Active 1, Active 2, Inactive 1, Inactive 2, and Mixed regions. The hypothesis that methylation levels in promoter regions of actively transcribed TADs would be lower than in inactive TADs was tested by a Kruskal-Wallis test.\n\nWe retrieved 1,236,142 unique cis-meQTLs across 23 cancer types from the Pancan-meQTL database.46 meQTLs were further clumped by linkage-disequilibrium (LD) to obtain independent associations using the PLINK54 clumping function using association p-values derived from the Pancan-meQTL database as input and default parameters (p1=0.0001, p2=0.01, r2=0.5, kb=250). These clumped, independent meQTLs were used for all subsequent analyses. First, the burden of clumped meQTLs across Active, Inactive, and Mixed TAD regions was measured. The burden was normalized by the length in base pairs of each region. To understand how meQTLs are distributed across the genome and whether TADs have an effect on the distribution of meQTLs, we analyzed the distributional burden of meQTLs within consecutive TADs. We compared the average meQTL density across different TAD transitions (i.e. Active-Boundary-Active, Active-Boundary-Inactive, Inactive-Boundary-Active and Inactive-Boundary-Inactive) by binning the genome between two TADs into 40 equal-sized bins and calculating average burden of meQTLs within these bins normalized by the bin size in base pairs. Resulting graphs were smoothed by a rolling average for visualization purposes. To evaluate whether the distribution reflected an association with transitions in TAD activity status, we shuffled the labels (i.e. “Active”, “Inactive”, etc.) of the TADs while preserving the number of transition categories (i.e. “Active-Active”, “Inactive-Active”, etc.) 100 times and ran the distribution analysis again on these randomly shuffled TADs by taking an average over all trials. Significance was assessed by comparing the observed difference in density between the TADs to the 100 average randomized trials using a student t-test.\n\nClumped meQTLs were annotated according to LD with CpG probes located in the promoter regions of cancer driver genes including oncogenes and tumor suppressor genes (TSGs) from the COSMIC database.16 A total of 231 oncogenes and TSGs were used for this analysis and promoter regions used were those identified by ENCODE’s SCREEN pipeline.55 To evaluate whether active/inactive TADs or boundary regions harboring cancer genes showed enrichment or depletion for meQTLs, we conducted a randomization analysis with 1000 trials. In each trial, we chose a random sample of meQTLs associated with non-cancer genes with matching minor allele frequency (±5%) to the set cancer-gene associated meQTLs, while also matching the number of randomly sampled meQTLs. We then mapped genes with meQTLs to active or inactive TADs and TAD boundaries, summed the meQTLs in each and normalized by the size of the region. The standard error was plotted alongside the true burden to see if the burden across TADs is significantly different from random.\n\nWe conducted a principal component analysis of TCGA genotype at the 156 meQTLs in European ancestry samples (n=8217), evaluating association of meQTL genotype-based PCs with clinical covariates. meQTL SNPs were quantified by the number of minor alleles carried (0, 1, 2). PCs explaining more than 1% of the genotypic variance across individuals were regressed with clinical variables including sex, age at diagnosis, tumor stage, and tumor type.\n\nFor both risk and survival analysis, we used a synthesis of LASSO regularization as a feature selector and XGBoost classifier as the machine learning predictor, described fully in Elgart et al.17 Specifically, after a preliminary association analysis, SNPs achieving a nominal p-value<0.05 were further selected by LASSO, and the selected SNPs were used to train an XGBoost model on a predictive task (e.g. cancer versus no cancer for risk, or high survival or low survival at median overall survival time), using a set of training samples. The probabilities achieved from the XGBoost classifier were then used to create a polygenic risk score (PRS) or polygenic survival score (PSS). Predictive performance was evaluated using cross validation for survival analysis and using an independent cohort of matched tumor types for the risk analysis.\n\nTo determine the association of meQTLs with risk of developing cancer, we conducted a phenome-wide association study (PheWAS) for each meQTL using the PLATO56 software. The genotype and phenotype data of 487,409 patients harboring the 156 cancer-related clumped meQTLs was retrieved from the UKBioBank51 and genotype at each meQTL was evaluated for association with all cancer phenotypes while controlling for covariates including age and ancestry. Individuals with multiple cancer diagnoses were excluded from the analysis, leaving 189,022 patients for risk analysis.\n\nNominally significant SNPs (p-value<0.05) were used for polygenic risk modeling with LASSO plus XGBoost. Out of the resulting tumor types where meQTLs were associated with risk we pursued breast (ICD-10: C50-C50) due to the abundance of validation data. Of the 189,022 UKBioBank individuals analyzed, 177,834 and 11,188 patients were non-cancer controls and breast cancer cases, respectively. An initial 10% quantile plot from the PheWAS analysis in UKBioBank was created using the PRS with the odds ratio for C50-C50 to compare the odds ratio of the 0th quantile PRS group to the 90th quantile PRS group.\n\nTo create a polygenic risk score (PRS) we utilized the approach described above under “Machine-learning for meQTL-based risk and survival prediction” section. Out of the tumor types that had nominally significant (p<0.05) risk-related SNPs (i.e.C64-C68, C40-C41, C69-C72, C00-C14, C15-C26, C81-C96, C50-C50, C43-C44, C45-C49, C76-C80, C60-C63, C51-C58, C97-C97, C73-C75, C30-C39), we chose to validate this relationship on an external cohort, DRIVE, on the C50-C50 or the breast cancer outcome due to an abundance of validation data. Similar to the survival analysis, we considered SNPs nominally associated with cancer risk using the associations from the PheWAS (p<0.05) for the rest of the analysis. We included other covariates including age and the first 10 principal components to represent population substructure in UKBioBank. Due to the class imbalance of the UKBioBank cohort (10,840 cases, 94,871 controls), we oversampled the cases to obtain a 1:1 case control ratio, resulting in a dataset size of 189,742 rows. Furthermore, we only included samples without any neoplasm diagnosis as controls to minimize confounding by other tumor types.\n\nWe first trained our XGBoost classification model on the entirety of the UKBioBank dataset. First the UKBioBank cohort (i.e. training cohort) was inputted into a LASSO regression model with α=0.001 (based on Ref. 17) to predict the intended phenotype. SNPs were further filtered to remove those that had a LASSO coefficient of 0. The modified cohort was used to train an XGBoost model on the filtered feature set using the entire UKBioBank cohort (n_estimators=500, learning_rate=0.1, max_depth=9). The probability of trees voting for either class (i.e. 0: no cancer, 1:cancer) was used as a polygenic risk score. We validated the breast cancer risk association of meQTLs alongside covariates using the Discovery, Biology, and Risk of Inherited Variants in Breast Cancer (DRIVE52) validation cohort. This validation cohort consists of 32,428 controls and 26,374 breast cancer cases for a total of 58,802 patients. Before validating, we mapped the MAF values of the SNPs in UKBioBank and DRIVE, and removed SNPs with MAF values of 2 standard deviations away from one another. PRS scores were predicted based on individual genotypes in DRIVE using the UKBioBank-trained XGBoost model (as described in Ref. 17). We compared score distributions across case and control in DRIVE using a Mann-Whitney U test. We also compared the incidence of breast cancer by partitioning the UKBioBank and DRIVE probabilities into 10% quantiles on PRS score. We plotted the 10% quantiles using the min-max normalized XGBoost-derived PRS scores.\n\nSurvival was modeled separately for each of 20 tumor types in TCGA (BLCA, CESC, KIRC, KIRP, PAAD, BRCA, HNSC, LGG, SKCM, PRAD, OV, UCEC, THCA, LUAD, LUSC, COAD, STAD, LIHC, SARC). Cancer meQTLs were included in predictive modeling if they were present with at least 1% minor allele frequency in the specific tumor type, and nominally significant in Kaplan-Meier analysis. Tumor types where fewer than 5 meQTLs showed a nominal association with overall survival or had less than 100 patients in TCGA were excluded from the analysis. For the remaining tumor types, we divided the analysis into three categories: clinical group containing only clinical features including sex, age, and tumor stage in certain cancer types (i.e. only cancer types >100 patients with non-null tumor stage contained stage as a covariate), control group and SNPs, and SNPs exclusively. For each of the categories, SNPs were selected by LASSO then used the complete dataset to train an XGBoost model, using 5-fold cross validation to estimate the generalization error and generate an AUC value. Specifically, for each individual we simplified the genotypes to a binary feature valued 1 if the patient had the heterozygous or homozygous meQTL allele and 0 if they didn’t. Binarized genotypes were then z-score normalized and input into a LASSO regularization model (α=0.001). Features with a LASSO coefficient of 0 (i.e. non-informative features) were removed and the LASSO-filtered SNP set was used to train an XGBoost classifier (n_estimators=500, learning_rate=0.1, max_depth=9) to predict binarized median overall survival (OS, 1=low survival<median survival, 0=high survival>median survival). Cancer types with a higher AUC value in the clinical+SNP group compared to the clinical group were only considered for the SNP only analysis. A higher AUC on the combined group could suggest that SNPs bring additive information. The output of the SNP-only XGBoost model used a non-linear polygenic survival score (PSS). Before inputting into the Cox, the PSS was scaled using the min-max algorithm and outliers were removed using a 1.5*(interquartile range) threshold.\n\nThe following tumor types and their corresponding XGBoost regression or PSS scores were used in the Cox model: BLCA, BRCA, CESC, HNSC, KIRC, LUSC, STAD, UCEC. We used Cox proportional hazards models to evaluate the meQTL-based PSS as a predictor of survival interval. We combined the PSS with clinical features including sex, age at diagnosis and tumor stage in a multivariable Cox-proportional hazards model to predict OS, and evaluated the hazard ratios and 95% confidence intervals for each covariate.\n\n\nAuthor contributions\n\nOriginal concept by SG and MP. HC supervised the project. SG performed computational data processing and analysis. MP, AK, JT provided support with data set preparation and contributed to computer code. SG, HC wrote the manuscript.", "appendix": "Data availability\n\nData were obtained from public sources including The Cancer Genome Atlas (TCGA; dbGaP: phs000178.v11.p8) and Discovery, Biology, and Risk of Inherited Variants in Breast Cancer (DRIVE; dbGaP: phs001265.v1.p1). dbGaP requires an application to access data; applicants will need to create an eRA Commons account and begin a project request. Senior Investigators and NIH Investigators are eligible to apply to access.\n\nWe use data from the UKBiobank resource under application number 37671 for this work. All bona fide researchers can apply to use the UK Biobank resource for health-related research that is in the public interest. Further information on the application process is available from the UK Biobank website.\n\nmeQTLs were obtained from Gong et al. 46 (http://bioinfo.life.hust.edu.cn/Pancan-meQTL/). TADs were obtained from Rao et al. 12 (https://doi.org/10.1016/j.cell.2014.11.021).\n\n\nAcknowledgements\n\nWe would like to acknowledge Rany M Salem for providing access to UKBioBank data and TJ Sears for helpful scientific discussion. This research has been conducted using the UK Biobank Resource under Application Number 37671. The results shown here are also based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. OncoArray genotyping and phenotype data harmonization for the Discovery, Biology, and Risk of Inherited Variants in Breast Cancer (DRIVE) breast-cancer case control samples was supported by X01 HG007491 and U19 CA148065 and by Cancer Research UK (C1287/A16563). Genotyping was conducted by the Center for Inherited Disease Research (CIDR), Centre for Cancer Genetic Epidemiology, University of Cambridge, and the National Cancer Institute. The following studies contributed germline DNA from breast cancer cases and controls: the Two Sister Study (2SISTER), Breast Oncology Galicia Network (BREOGAN), Copenhagen General Population Study (CGPS), Cancer Prevention Study 2 (CPSII), The European Prospective Investigation into Cancer and Nutrition (EPIC), Melbourne Collaborative Cohort Study (MCCS), Multiethnic Cohort (MEC), Nashville Breast Health Study (NBHS), Nurses Health Study (NHS), Nurses Health Study 2 (NHS2), Polish Breast Cancer Study (PBCS), Prostate Lung Colorectal and Ovarian Cancer Screening Trial (PLCO), Studies of Epidemiology and Risk Factors in Cancer Heredity (SEARCH), The Sister Study (SISTER), Swedish Mammography Cohort (SMC), Women of African Ancestry Breast Cancer Study (WAABCS), Women’s Health Initiative (WHI).\n\n\nReferences\n\nIyer JG, et al.: Response rates and durability of chemotherapy among 62 patients with metastatic Merkel cell carcinoma. Cancer Med. 2016; 5: 2294–2301. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoudarzi S, Hcarter: cartercompbio/meQTLs: Initial release (v1.0.0). Zenodo. 2023. Publisher Full Text" }	[ { "id": "233320", "date": "17 Jan 2024", "name": "Charu Mehta", "expertise": [ "Reviewer Expertise My area of research is gene regulation. I am able to assess significant parts of this manuscript", "however", "I am not a statistician or computational biologist", "so I cannot speak to the soundness of their methods." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"To obtain independent meQTLs, we clumped related meQTLs based on linkage disequilibrium using PLINK. Out of the 1.2 million SNPs, 60,602 remained after LD pruning (Table 1).\"\nComment: Cite the database used as a source of meQTLs? In the discussion, authors cited a database (46) but it is unclear if this is the same database they used to identify meQTLs.\n\n\"(A) 5 state-based K-Means clustering of common TAD domains (n=1100) between 5 human cell lines (GM12878, HMEC, HUVEC, IMR90, and NHEK). Purple indicates TADs classified as a “Mixed”, Gray as “Inactive-1”, Light Blue as “Active-1”, Orange as “Active-2”, and Red as “Inactive-2”. Combining active and inactive categories leads to 222 Active, 626 Inactive, and 252 Mixed TADs.\"\n\nComment: what are the x- and y-axes labels in 1A? Where are the five cell types indicated?\n\nComment: What does ‘other’ mean in Table 1?\n\n\"Distributions suggested an increase in density of clumped meQTLs when transitioning from active to inactive regions, and conversely, a decrease from inactive to active regions (Kruskal-Wallis ANOVA, p-value<0.05) when compared to the randomly shuffled distribution, but no shift in density for Active-Boundary-Active and Inactive- Boundary-Inactive categories (Figure 2B-D).\"\n\nComment: So what does any of this suggest??? expand?\n\n\"In total, 103 oncogenes and 223 TSGs were used for this analysis, where only 67 of them contained meQTL-affecting CpG probes in their promoter regions (i.e. 49 TSGs and 18 oncogenes).\"\n\nComment: This suggests CpGs affect meQTLs but it’s the other way round.\n\n\"Clumped cancer meQTls were further narrowed to those associated with the methylation status of CpG probes located within the promoter regions of cancer driver genes including oncogenes and tumor suppressor genes (TSGs) from the COSMIC database.” Comment: Also clarify if the correlation between methylation status of CpG vs meQTLs is also observed in normal tissues or only cancer tissues?\n\n\"Overall, cancer meQTLs near 29 cancer genes were included in the model. The most predictive driver meQTL was associated MSH2, a gene associated with Lynch syndrome and increased risk of breast cancer.\n\nPolymorphic variation affecting the expression of EZH2, the second most informative feature, has also been linked to breast cancer risk.\n\nASXL2 may be required for estrogen receptor alpha (ERa) activation in ERa positive breast cancers. Notably, EZH2 overexpression has been linked more strongly to triple negative breast suggesting that the model includes features predictive of multiple subtypes.”\n\nComment: 1. Since some of these meQTLs lie close to genes involved in epigenetic modifications --- have you looked if these are in the enhancer or otherwise defined regulatory domains? 2. Are these genes (MSH2, EZH2, ASXL2) known to be upregulated or downregulated in these risk cases? Does that agree with the prediction according to meQTLs?\n\nIs the work 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "233315", "date": "08 Feb 2024", "name": "Chiara Herzog", "expertise": [ "Reviewer Expertise epigenetics and cancer risk 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\nThe study by Goudarzi et al. provides interesting and new insights into the association of meQTLs and TAD regions the genome, and investigates the capacity of meQTLs to predict cancer status and survival.\nOverall the study is well done and clearly presented but I have a few comments and suggestions for improvement.\nMajor: - In the introduction and discussion the authors state “This study investigated the relationship between epigenetic factors like chromatin structure and DNA methylation and genetic variation in the context of cancer”. While the authors indeed investigate methylation and TADs in the first part of the manuscript, the majority of the predictors focus on genetic loci and not epigenetics or their interaction. Arguably, one of the most interesting aspects of meQTLs in their capacity for risk prediction are their modulated methylation levels and potential to reflect the integration of genetic and dynamic nonheritable factors (such as due to aging or lifestyle factors), but this was not looked at in detail. Could the authors comment on how meQTLs might be modulated by nonheritable factors as well as genetic factors, and e.g. look into methylation at these sites in cancers or samples preceding cancer? - Along these lines, it might in the future be interesting to develop dynamic cancer risk predictors as opposed to static tools (such as the PRSs), which might be enabled by nongenetic ‘omics’. Could the authors discuss the potential of these and how their findings might contribute to this (i.e. how meQTLs might contribute to dynamic risk monitoring)?\nMinor:\n- The authors describe a PCA but do not show any figures or supporting data. Could the authors either add a statement that no data are shown in the text, or (preferred) provide these data in the supplementary information? - Previous breast cancer PRSs (not based on meQTL) such as the PRS313 have already shown that they may be biased towards certain subtypes - it might be worth mentioning these prior models (e.g. Mavaddat et al 2019) when discussing the current study’s findings in context. - What was the ROC AUC (and 95% CI) of the cancer risk score (Figure 4A)? - Can the authors explain the discrepancy of the more ‘linear’ increase in risk in the UKBB compared to DRIVE (Figure 4b versus 4c)? - Figure 6/TCGA survival: It might also be interesting to look at recurrence-free survival in addition to overall survival. - In the section Oncogenes and tumor suppressor gene-related (…): capitalise L in ‘Clumped cancer mQTls’ - Figures:\n\n- Figure 1b: Could the authors also indicate in the Figure legend that this is a Kruskal-Wallis p value.\n\n- Figure 1c - For interpretability, it might be helpful to add at least y axis grid lines behind box plots. The effects may be significant and is visualised with the violin density plot, but is difficult to see using box plot.\n\n- Figure 2 - takes some time to understand upon first reading. It might be helpful to label the blue bars in B and C with a legend ‘expected’ and the red dot as ‘observed’ to make it easier to grasp quickly.\n\n- Figure 4: The text in the caption for A refers to a ‘Figure 8’ that does not exist. Please check this.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1083
https://f1000research.com/articles/12-1082/v1	01 Sep 23	{ "type": "Study Protocol", "title": "Histomorphometric analysis of angiogenesis in oral submucous fibrosis and oral squamous cell carcinoma associated with oral submucous fibrosis", "authors": [ "Alka Hande", "Archana Sonone", "Madhuri Gawande", "Swati Patil", "Aayushi Pakhale", "Shelley Durge", "Archana Sonone", "Madhuri Gawande", "Swati Patil", "Aayushi Pakhale", "Shelley Durge" ], "abstract": "Background: Oral submucous fibrosis (OSMF), now worldwide acknowledged as the disease of “Southeast Asia and Indian subcontinent”, has the utmost malignant transformation prevalence amongst oral precancerous disorders. Increased vascularity that is neoangiogenesis has been observed in the superficial connective tissue region of pre-cancerous lesions showing dysplasia. This could be an initial occurrence of the carcinogenesis process. The aim of this study will be to analyse neoangiogenesis by examining immunoexpression of CD105 in OSMF, and OSCC with OSMF cases. Methods: The project will comprise 30 normal oral mucosa samples (group I), 30 clinicopathologically diagnosed cases of OSMF (group II), and on the basis of association of OSMF, 30 surgically operated and histopathologically diagnosed cases of OSCC associated with OSMF (group III). Hematoxylin and eosin stains will be used for routine staining procedures and immunohistochemistry for the expression of CD105. Oral epithelial dysplasia (OED) cases of OSMF will be categorized into two groups, low-risk epithelial dysplasia (LRED) and high-risk epithelial dysplasia (HRED). The micro vessel density (MVD), total microvessel area (TVA), and mean microvessel area (MVA) are within and surrounding the tumor tissue sections immunostained with the CD105 antibody will be determined. ANOVA will be applied for evaluation of the mean scores of MVD, TVA, and MVA of groups II and III. The obtained score will be compared with different parameters of OSCC (TNM stage, lymph node metastasis, and histopathological grades). Conclusions: We postulate the progressively increased vascularity with the disease progression from LRED to HRED and further its transformation to invasive oral squamous cell carcinoma This increased vascularity will be evident by enhanced MVD, TVA and mean MVA, which is expressed by CD105 immunoexpression. This observation will emphasize the significance of neoangiogenesis in cases of OSMF with epithelial dysplasia and its further progression to OSCC.", "keywords": [ "Oral submucous fibrosis", "Oral squamous cell carcinoma", "neoangiogenesis", "CD105", "Immunohistochemistry" ], "content": "Introduction\n\nThe practice of betel nut chewing and its various custom-made preparations like pan masala, gutkha, khaini, or kharra is extensive in Southeast Asian countries including India. The chewing of these betel nut preparations has been acknowledged as one of the primary risk factors leading to oral submucous fibrosis (OSMF). It is a persistent progressive disease frequently affecting the oral and oropharyngeal mucosa and at times the superior third of mucosa of the esophagus.1 Histologically, OSMF is characterized by the loss of epithelial rete ridges and further atrophy of the epithelium with juxta epithelial inflammatory reaction. There is increased fibrosis of the underlying connective tissue and submucosal tissue, reduced vascularity, and hyalinization with the progression of the disease, which leads to stiffness of the oral mucous membrane and constrained opening of the mouth.2–4\n\nCommonly oral precancerous disorders are clinically identifiable diseases that further progress to oral cancer. In general, the malignant transformation of it begins with single-cell atypia subject to genetic mutation and/or various carcinogenic factors like tobacco, areca nut, betel quid, virus, and alcohol. The malignant potential of betel nut without tobacco has been recognized and classified as a group one human carcinogen as per a cancer monograph on betel-quid and areca nut chewing and some areca nut derived nitrosoamines by II International Agency for Research on Cancer.5 OSMF, now worldwide acknowledged as the disease of Southeast Asia and the Indian subcontinent, has the highest malignant transformation incidence. Paymaster in 1956 first described the carcinomatous potential of OSMF.6 The oral premalignant lesions showing epithelial dysplasia are at 15 times higher risk of malignant alterations so microscopic recognition of epithelial dysplasia is helpful for the assessment of malignant transformation in OSMF.7 Oral epithelial dysplasia has been displayed in 7 to 43% of cases of OSMF, as reported in previous studies,8,9 although the prevalence of malignant transformation of OSMF is between 7 and 12%.10\n\nPrediction of the clinical behavior of oral potentially malignant disorders (OPMDs) is complex because histological characterization may not always provide prognostic judgment.11 The most primitive and possibly utmost substantial properties expressed by means of pre-neoplastic cell populaces are their capability to bring forth a neovascular response that is neoangiogenesis. In numerous pre-malignant conditions together with lesions in the bladder, hyperplastic lesions in the pancreas, and intraepithelial carcinoma of the breast, angiogenic expression has been observed.11 Increased vascularity that is neoangiogenesis has been observed in the superficial connective tissue region of premalignant lesions showing dysplasia.7 This observation is further evidenced by the considerably additional quantity of newly formed endothelial-lined capillaries in the superficial stromal tissue of epithelial dysplastic lesions. Correspondingly there is an increase in VEGF secretion after the epithelial-mesenchymal transition (EMT) by epithelial cells, which eventually results in an invasive carcinoma. Thus, manifestation of neoangiogenesis may possibly be predictive of malignant potential, which is attained prematurely in the carcinogenic progression and may perhaps be of prognostic implication in its evaluation.12\n\nTo understand the etiopathogenesis, progression of the disease, and malignant transformation in OSMF various immunohistochemical biomarkers have been studied. The association of clinical grades/stages of OSMF with molecular biomarkers is a crucial area of research. Nevertheless, the association of biomolecular expression with grades of oral epithelial dysplasia (OED) has been overlooked in cases of OSMF. OED exhibits a significant role to suggest a malignant transformation of OSMF. However due to atrophic epithelium, the evaluation of OED in OSMF is challenging. In other OPMDs, the principles advocated for OED grading are mainly based on alterations in the proliferating epithelium. Taking into consideration the characteristic “atrophic epithelium” of OSMF, these criteria will not be suitable for the assessment of OED in OSMF. In view of this, the application of biomarkers may be useful for the OED grading in OSMF.\n\nNeoangiogenesis is considered to be an imperative characteristic in epithelial dysplastic lesions. It facilitates the requirement of nutrition for the growth and development of dysplastic cells. There are numerous angiogenic factors that are formed by the cells undergoing dysplastic changes, as well as tissue stromal cells like fibroblasts, macrophages, mast cells, and tumor cells. Further progression of dysplastic cells to invasive tumor cells and their metastasis is governed by these angiogenic factors.13 CD105 (Endoglin) is a transmembrane phosphorylated glycoprotein, a fundamental constituent of the TGF-β receptor signaling pathway. The proliferation and differentiation of cells is modulated by this pleiotropic cytokine, which is fundamental for neoangiogenesis and vascular growth. Multiplying endothelial cells show upregulation of CD105, and so is considered as a principal marker of proliferation of endothelial cells of newly formed blood vessels that is neoangiogenesis.14\n\nThe aim of this study will be to appraise the molecular basis of neoangiogenesis in terms of expression of CD105 in the extracellular matrix of OSMF, and OSCC with OSMF cases.\n\n\n\n1. To evaluate CD105 expression in OSMF cases\n\n2. To evaluate CD105 expression in OSCC associated with OSMF cases\n\n3. To compare CD105 expression in OSMF and OSCC associated with OSMF cases\n\n\n\n1. Clinicopathological and histopathological features of OSCC with OSMF cases.\n\n2. Quantitative measurement of CD105 expression (micro vessel density (MVD), total microvessel area (TVA), and mean microvessel area (MVA)) in OSMF and OSCC with OSMF.\n\n\nMethods\n\nIn this project, a total of 90 cases will be divided into three groups as follows: i) Group I, 30 cases with normal oral mucosa; ii) Group II, 30 cases with oral submucous fibrosis; and iii) Group III, 30 cases with oral squamous cell carcinoma with oral submucous fibrosis. Appropriate details from the STROBE checklist have been included in this study protocol.28\n\nOverall, 30 samples with normal oral mucosa retrieved during minor surgical procedures like removal of pericoronal flap, disimpaction of third molar will be used as controls. On the basis of appropriate clinical symptoms of OSMF like, inability to endure hot and spicy food beverages as well as difficulty in mouth opening, 30 clinicopathologically diagnosed cases of OSMF will be selected. On examination, the clinical signs show conspicuous fibrotic bands with rubbery blanching of the oral mucous membrane. The 30 samples of OSCC with OSMF that have undergone surgical treatment and have been histopathologically and clinically diagnosed will be included in the study.\n\nCases who had undergone presurgical radiotherapy and/or chemotherapy, history of second primary or local and distant recurrence and patients with any other systemic diseases and with coexisting malignancies were excluded from the study.\n\nWe have received approval from The Institutional Ethical Committee [DMIMS (DU)/IEC/2022/761, dated 14/02/2022] of Datta Meghe Institute of Higher Education and Research, (DU), Sawangi(M), Wardha, Maharashtra, India. This project will be undertaken at the Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital.\n\nThe study will comprise 30 normal oral mucosa samples (group I) and 30 clinicopathologically diagnosed cases of OSMF (group II).15 The included cases of OSMF will be histologically evaluated. OED in OSMF will be recorded on the basis of its presence or absence. Furthermore, cases will be categorized into two groups, low-risk epithelial dysplasia (LRED) and high-risk epithelial dysplasia (HRED).16 On the basis of the association of OSMF, 30 cases of histopathologically diagnosed, surgically treated OSCC cases of various histopathological grades will be retrieved from the archives of the department (group III). Three oral pathologists independently performed histopathological grading of all OSCC cases using Broders grading system in a blinded manner. The archival tissue section of 4 μm will be obtained for groups I, II, and III. Hematoxylin and eosin stains will be used for routine staining procedures and immunohistochemistry for the expression of CD105.\n\nParaffin blocks with tumor tissue and normal tissue will be selected. For de-paraffinization, sections will be placed in the xylene solution. Sections will be rehydrated by subjecting them to descending concentrations of alcohol. In order to wash sections, tap water will be used. The washing time for sections in distilled water should be 60 seconds. After washing all the sections, they will be transferred to a Coplin jar containing the retrieval buffer solution. The solution that is used for antigen retrieval is composed of 30 ml of retrieval solution in 1,500 ml of distilled water for 15-20 minutes in the pressure cooker. Cooling will be done at room temperature.\n\nSections will be dipped once in distilled water. Sections will be washed with Tris buffer solution for at least 5 minutes at room temperature. This step will be repeated three times. For peroxidase blocking, a mixture of 3-5% hydrogen peroxide and methanol will be used for 30 minutes. Tris buffer solution will be used for washing the sections three times for five minutes each. CD105 will be applied at room temperature for 1 hour. Once again washing of sections will be done in Tris buffer solution three times for five minutes each. Envision technique will be performed by utilizing a labeled polymer for 30 minutes at room temperature. To wash the sections, Tris buffer solution will be used three times for five minutes.\n\nThe application of the 3,3′-diaminobenzidine (DAB) substrate will be done for 15 to 20 minutes. The working DAB solution is comprised of the following, 1 ml of DAB buffer and 25 ml of DAB concentrate. Washing of sections will be done by Tris buffer, for 15-20 minutes. Sections will be cleaned in distilled water. For counterstaining, Mayer’s hematoxylin will be used, which is done for five minutes. Again, the washing of sections will be done under tap water. Sections are then dried, following which they are mounted in DPX and examined under a microscope.\n\nCD105 immunopositive tissue sections will be observed under a light microscope (Leica) at ×100 magnification and further by ×400 magnification.\n\nTo determine the MVD within and surrounding the tumor, the tissue sections immunostained with the CD105 antibody (Diluted 1:30, Monoclonal Mouse Anti-Human CD105, Endoglin, Clone SN6 h; Product code: M3527, Dako, North America Inc.) will be inspected under a light microscope (Leica) at ×400 magnification, as recommended by Weidner et al.17 The scanning of tissue sections will be carried out at ×100 to choose two fields with the highest MVD (“hot spots”) and further microvessels will be counted at ×400 magnification.\n\nThe identification of every independently stained microvessel in each particular field will be done. Furthermore, the outline of the recognized microvessel will be traced by an image analysis system to determine the MVD, TVA and MVA within and surrounding the tumor tissue sections.\n\nRetrospective cohort study.\n\nUsing the single proportion formula and the 2% prevalence of OSMF and OSCC cases in the Oral Pathology and Microbiology Outpatient Department, the sample size was determined as follows18:\n\nWhere,\n\n“Zα/22: The level of significance at 5%, i.e., 95% confidence interval = 1.96.\n\np: sample showing positive E-cadherin expression focally in small group cells in the basal layer of epithelium = 35% = 0.35.\n\nE: error of margin = 10% = 0.10.\n\nn=1.962×0.35×1−0.35/0.102\n\nn=87.39\n\nn=90”\n\nThe present study will determine the expression of CD105 by immunohistochemistry in OSMF and OSCC associated with OSMF.\n\nWe postulate the progressively increased vascularity with the disease progression from LRED to HRED and further its transformation to invasive squamous cell carcinoma. This increased vascularity will be evident by enhanced MVD, TVA and MVA, which is expressed by CD105 immunoexpression. This observation emphasizes the significance of neoangiogenesis in cases of OSMF with epithelial dysplasia and its further progression to OSCC.\n\nThis study will be published in an indexed journal.\n\nNot yet started.\n\n\nDiscussion\n\nNeoangiogenesis is an essential and fundamental process in development and progression, as well as in the formation of granulation tissue and healing of the wound. Moreover, it is also a crucial stride in the conversion of benign to aggressive, malignant tumors. In oral precancerous disorders the cells undergo alterations during the progression of carcinogenic process. One of the initial and possibly most considerable characteristics expressed by pre-cancerous cells is their capability to generate a neovascular response that is angiogenesis. Therefore, it could be assumed that the expression of neoangiogenic activity may possibly be representative of malignant potential that is attained prematurely in the carcinogenic process.\n\nThe abnormal alterations of the microvessels in 27 patients of OSMF were investigated by immunohistochemistry. Microvessel quantity and microvessel area were deliberated by an image analysis system. They observed enhanced microvessel quantity in the primary stage of OSMF and reduced MVA in the intermediate and late stages. From these study results they recommended that hyperplasia of microvessels takes place in the early stages of OSMF.19 They reported that the typical tissue reaction consequential to hypoxia does not seem to work in this disease. The enhanced vascular dilatation predominantly present in tissue is indicative of tissue adaptability to compensate for hypoxia due to extensive fibrosis.20\n\nThe microvessel and mast cell density in patients of various stages of OSMF and normal mucosal tissue was investigated by immunohistochemistry and further associated with the disease progression. The molecular markers, anti-mast cell tryptase and anti-factor VIII related von Willebrand Factor for endothelial cells were used. They observed significantly enhanced microvessel and mast cell density in various stages of OSMF cases. They also observed a positive association between microvessel and mast cell density. They concluded that enhanced microvessel and mast cell density plays a greater role in the pathophysiology of OSMF.21\n\nThe submucosal vasculature in the early and advanced stages of OSMF and WDSCC was assessed by morphometric analysis. Murgod et al.,22 investigated 30 histopathologically confirmed cases of OSMF from early and advanced clinical stages and WDSCC for assessment of microvascular density and microvascular luminal diameter. They observed significantly enhanced expression in the context of microvascular density and microvascular luminal diameter. The microvascular density and microvascular luminal diameter were considerably better in primary stages of OSMF and WDSCC as compared to normal mucosa, and also in an advanced stage of OSMF as well as in WDSCC in comparison to primary stages of OSMF. The enhanced vascularity from normal to precancerous and cancer tissue highlights the significance of neoangiogenesis in tumor development and progression.22\n\nImmunoexpression of CD105, E-cadherin, and VEGF in different stages of OSMF were investigated through semi-quantitative analysis in a previous study.23 They observed upregulation of CD105, E-cadherin, and VEGF expression gradually increasing from without epithelial dysplasia tissue toward advanced grades of dysplasia. They observed that the direct correlation between lesser expression of the E-cadherin molecule and enhanced CD105 expression suggests neoangiogenic attributes of OSMF with an increase in dysplastic grades. This could be used to assess the malignant potential of OSMF.23\n\nCD105 expression in 51 patients of OSCC was investigated by immunohistochemistry. MVD was evaluated by measuring the number of CD105-immunostained blood vessels. They revealed that CD105 immunoexpression is enhanced in OSCC as compared to healthy normal oral mucosal tissue. They concluded that “CD105 has a considerable function in the growth and progression of OSCC, which may be reasonably explicit as compared to other endothelial markers”.24\n\nIn a previous study, MVD in 27 HNSCC patients was evaluated by immunohistochemistry against CD34 and CD105 antibodies. They observed significantly higher MVD in the T3-T4 stage (advanced clinical stage) of tumors. In the case of tumors with positive lymph nodes, MVD was considerably elevated. On the correlation of MVD with survival data it was observed that the cases with an elevated MVD had a considerably reduced disease-free survival, whereas MVD expressed by CD34 had no correlation with a status of survival. They concluded that “increased expression of CD105 is considered as the single self-determining tumor recurrence marker”.25\n\nCD105 expression and MVD in 19 surgically operated specimens of OSCC were evaluated by immunohistochemistry. The MVD was determined by the “hot spot method”. They observed higher expression of MVD at the central and invading front of OSCC. Furthermore, MVD was higher in the peritumoral region as compared to the intratumoral region and the difference was statistically significant. Thus, they concluded that “enhanced expression of CD105 in the tumor is considered as an imperative predictive marker for the outcome in OSCC”.26\n\nExpression of VEGF and CD105 in 54 cases of OSCC were investigated by immunohistochemistry. They observed increased VEGF expression in OSCC specimens in contrast to premalignant and normal healthy oral tissues. Furthermore, VEGF expression is decreased in poorly differentiated oral squamous cell carcinoma (PDOSCC) as compared to moderately differentiated oral squamous cell carcinoma (MDOSCC) and well differentiated oral squamous cell carcinoma (WDOSCC), and MVA was elevated in OSCC in comparison to potentially malignant lesions and healthy tissues of the oral mucosa. They concluded that “VEGF and CD105 may be considered as reliable markers of tumor angiogenesis and progression in OSCC”.27\n\nThe study planned to be conducted is an in vitro, cross-sectional study. Therefore, a long term prospective follow up study with a larger sample size is recommended.\n\nIt will be possible to see the progressively increased vascularity evident by CD105 immunoexpression with the disease progression from LRED to HRED and further its transformation to invasive squamous cell carcinoma.\n\nA review of the literature reveals an association between CD105 expression and epithelial dysplasia in OSMF. Furthermore, on the basis of neoangiogenesis evaluated by CD105 immunoexpression with MVD, MVA, and TVA, the progression of epithelial dysplasia towards malignancy might be discernible. The management of immunotherapy for different patients might be considered based on the CD105 immunoexpression status.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: STROBE checklist for ‘Histomorphometric analysis of angiogenesis in oral submucous fibrosis and oral squamous cell carcinoma associated with oral submucous fibrosis’. https://doi.org/10.5281/zenodo.7895818. 28\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe acknowledge the support of laboratory technicians from the Department of Oral & Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College & Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe), Wardha. We also acknowledge Dr. Padmashri Kalmegh for her support in the plagiarism check of the manuscript.\n\n\nReferences\n\nRajalalitha P, Vali S: Molecular pathogenesis of oral submucous fibrosis: acollagen metabolic disorder. J. Oral Pathol. Med. 2005; 34(6): 321–328. PubMed Abstract \| Publisher Full Text\n\nAfroz N, Hasan SA, Naseem S: Oral submucous fibrosis a distressing disease with malignant potential. Indian J. Community Med. 2006; 31(4): 270–271.\n\nChole RH, Gondivkar SM, Gadbail AR, et al.: Review of drug treatment of oral submucous fibrosis. Oral Oncol. 2012; 48(5): 393–398. Publisher Full Text\n\nEkanayaka RP, Tilakaratne WM: Oral submucous fibrosis: review on mechanisms of pathogenesis and malignant transformation. J. Carcinog. Mutagen. 2013; S5: 002. Publisher Full Text\n\nInternational Agency for Research on Cancer: Betel-quid and areca nut chewing and some areca nut derived nitrosoamines. IARC Monogr. Eval. Carcinog. Risks Hum. 2004; 85: 123–129.\n\nDesai RS, Mamatha GS, Khatri MJ, et al.: Immunohistochemical expression of CD34 for characterization and quantification of mucosal vasculature and its probable role in malignant transformation of atrophic epithelium in oral submucous fibrosis. Oral Oncol. 2010; 46(7): 553–558. PubMed Abstract \| Publisher Full Text\n\nMadhavannirmal R, Veeravarmal V, Bhavani S, et al.: Evaluation of Micro-Vessel Density (MVD) and Vascular Endothelial Growth Factor (VEGF) as Possible Indicator of Malignant Transformation in Oral Submucous Fibrosis. J. Dent. Med. Sci. (IOSR-JDMS). 2016; 15(11): 72–77.\n\nHazarey VK, Erlewad DM, Mundhe K, et al.: Oral submucous fibrosis: a study of 1000 cases from central India. J. Oral Pathol. Med. 2007; 36: 12–17. PubMed Abstract\n\nTilakaratne WM, Klinikowski MF, Saku T, et al.: Oral submucous fibrosis: on aetiology and pathogenesis. Oral Oncol. 2006; 42: 561–568. Publisher Full Text\n\nRay JG, Ranganathan K, Chattopadhyay A: Malignant transformation of oral submucous fibrosis: overview of histopathological aspects. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2016; 122(2): 200–209. PubMed Abstract \| Publisher Full Text\n\nTipoe GL, Jin Y, White FH: The relationship between vascularity and cell proliferation in human normal and pathological lesions of the oral cheek epithelium. Eur. J. Cancer B Oral Oncol. 1996; 32B(1): 24–31. PubMed Abstract \| Publisher Full Text\n\nPolverini PJ: The pathophysiology of angiogenesis. Crit. Rev. Oral Biol. Med. 1995; 6(3): 230–247. Publisher Full Text\n\nGupta S, Gupta V, Tyagi N, et al.: Analysis of Role of Angiogenesis in Epithelial Dysplasia: An Immunohistochemical Study. J. Clin. Diagn. Res. 2017; 11(12): EC29-EC34. Publisher Full Text\n\nNassiri F, Cusimano MD, Scheithauer BW, et al.: Endoglin (CD105): A review of its role in angiogenesis and tumor diagnosis, progression and therapy. Anticancer Res. 2011; 31(6): 2283–2290. PubMed Abstract\n\nPindborg JJ, Murti PR, Bhonsle RB, et al.: Oral submucous fibrosis as a precancerous condition. Scand. J. Dent. Res. 1984; 92: 224–229. PubMed Abstract\n\nWarnakulasuriya S, Reibel J, Bouquot J, et al.: Oral epithelial dysplasia classification systems: predictive value, utility, weaknesses and scope for improvement. J. Oral Pathol. Med. 2008; 37: 127–133. PubMed Abstract \| Publisher Full Text\n\nWeidner N, Semple JP, Welch WR, et al.: Tumor angiogenesis and metastasis-- correlation in invasive breast carcinoma. N. Engl. J. Med. 1991; 324(1): 1–8. Publisher Full Text\n\nCochran WG: Sampling Techniques. 3rd ed.New York: John Wiley & Sons; 1977.\n\nFang CY, Han WN, Fong DY: A morphometric study on the microvessel in oral submucous fibrosis. Hunan Yi Ke Da Xue Bao. 2000; 25(1): 55–57.\n\nRajendran R, Paul S, Mathews PP, et al.: Characterization and quantification of mucosal vasculature in oral submucous fibrosis. Indian J. Dent. Res. 2005; 16(3): 83–91. PubMed Abstract\n\nSabarinath B, Sriram G, Saraswathi TR, et al.: Immunohistochemical evaluation of mast cells and vascular endothelial proliferation in oral submucousfibrosis. Indian J. Dent. Res. 2011; 22(1): 116–121. PubMed Abstract \| Publisher Full Text\n\nMurgod VV, Kale AD, Angadi PV, et al.: Morphometric analysis of the mucosal vasculature in oral submucous fibrosis and its comparison with oral squamous cell carcinoma. J. Oral Sci. 2014; 56(2): 173–178. PubMed Abstract \| Publisher Full Text\n\nAnura A, Das RK, Pal M, et al.: Correlated analysis of semi-quantitative immunohistochemical features of E-cadherin, VEGF and CD105 in assessing malignant potentiality of oral submucous fibrosis. Pathol. Res. Pract. 2014; 210(12): 1054–1063. PubMed Abstract \| Publisher Full Text\n\nSchimming R, Marmé D: Endoglin (CD105) expression in squamous cell carcinoma of the oral cavity. Head Neck. 2002; 24(2): 151–156. Publisher Full Text\n\nMartone T, Rosso P, Albera R, et al.: Prognostic relevance of CD105+ microvessel density in HNSCC patient outcome. Oral Oncol. 2005; 41(2): 147–155. PubMed Abstract \| Publisher Full Text\n\nMărgăritescu C, Simionescu C, Pirici D, et al.: Immunohistochemical characterization of tumoral vessels in oral squamous cell carcinoma. Romanian J. Morphol. Embryol. 2008; 49(4): 447–458. PubMed Abstract\n\nMărgăritescu C, Pirici D, Stîngă A, et al.: VEGF expression and angiogenesis in oral squamous cell carcinoma: an immunohistochemical and morphometric study. Clin. Exp. Med. 2010; 10(4): 209–214. PubMed Abstract \| Publisher Full Text\n\nHande A, Sonone A, Gawande M, et al.: Histomorphometric analysis of Angiogenesis in Oral Submucous Fibrosis & Oral Squamous Cell Carcinoma associated with Oral Submucous Fibrosis. [Dataset]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "226651", "date": "15 Dec 2023", "name": "Namrata Patil", "expertise": [ "Reviewer Expertise MOLECULAR BIOLOGY", "NEO -ANGIOGENESIS", "TUMOR MARKERS" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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, all the morphometric analysis and details have been explained. the manuscript is properly readable, plagiarism is not noted, ethical policies have been followed. The study design is properly framed. The article is fulfilling all aspects and can be considered for publication. The source of data is appropriate.\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": "265480", "date": "31 May 2024", "name": "Mohit Sharma", "expertise": [ "Reviewer Expertise Oral Cancer", "Epigenetics", "Oral Potentially Malignant Disorders", "Salivary Gland tumors" ], "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 practice of betel nut chewing, which is common in Southeast Asian countries, has been identified as a significant risk factor for the development of oral submucous fibrosis (OSMF). OSMF is a progressive disease that affects the oral and oropharyngeal mucosa, as well as the esophagus. The disease is characterized by the loss of epithelial rete ridges, atrophy of the epithelium, increased fibrosis in connective tissue, reduced vascularity, and hyalinization, which leads to stiffness of the oral mucous membrane and limited mouth opening. OSMF is also associated with a high risk of malignant transformation to oral cancer, with oral epithelial dysplasia playing a significant role in assessing this potential transformation.\nThe study aims to evaluate the expression of CD105, a marker of angiogenesis, in OSMF and oral squamous cell carcinoma (OSCC) associated with OSMF. The objectives include evaluating CD105 expression in OSMF cases, in OSCC cases associated with OSMF, and comparing CD105 expression in OSMF and OSCC associated with OSMF. The study will also assess clinicopathological and histopathological features of OSCC with OSMF cases, as well as quantitatively measure CD105 expression in OSMF and OSCC with OSMF.\nThe study design involves a total of 90 cases divided into three groups: normal oral mucosa, OSMF, and OSCC with OSMF. The inclusion criteria specify the selection of samples with normal oral mucosa, clinically diagnosed cases of OSMF, and histopathologically diagnosed cases of OSCC with OSMF. Cases that have undergone presurgical radiotherapy and/or chemotherapy, as well as those with systemic diseases or coexisting malignancies, were excluded from the study.\nImmunohistochemistry will be performed to evaluate CD105 expression in the extracellular matrix of OSMF and OSCC with OSMF cases. This will involve the use of paraffin blocks containing tumor tissue and normal tissue, de-paraffinization, and immunostaining with the CD105 antibody.\nMorphometric analysis of tumor vessels, measured by microvessel density (MVD), total microvessel area (TVA), and mean microvessel area (MVA), will be conducted to assess the level of CD105 expression in OSMF and OSCC with OSMF. Statistical analysis will be performed to determine the expected outcome of the study, which aims to identify the expression of CD105 by immunohistochemistry in OSMF and OSCC associated with OSMF.\nIn conclusion, the study aims to provide insights into the association between CD105 expression and epithelial dysplasia in OSMF, as well as the potential use of CD105 immunoexpression as a marker for assessing malignant potential and guiding immunotherapy for patients with OSMF and OSCC associated with OSMF.\n\nMajor Points\n1. The exclusion of moderate dysplasia and applying binary dysplasia classification to the malignant transformation of OSF raises concerns about the validity of the findings. Moderate dysplasia represents an important intermediate stage in the progression to malignancy and cannot be disregarded without justification. By excluding this category, the study may overlook critical insights into the dynamics of neoangiogenesis during the early stages of carcinogenesis in OSMF. In the context of OSMF, excluding moderate dysplasia seems questionable, as dysplasia may promote fibrosis and vice versa.\n2. A study evaluated the expression of TGF-β1 protein in 30 cases of oral squamous cell carcinoma (OSCC) to understand its role in oral carcinogenesis. Results showed increased TGF-β1 expression in different oral epithelial dysplasia and OSCC stages compared to normal mucosa. TGF-β1 expression was markedly enhanced in both OSCC cells and stromal cells. The study concluded that enhanced TGF-β1 expression could be responsible for transforming oral premalignant lesions to OSCC, aggressive tumor growth, metastasis, and resistance to treatment [refer 2] .....this TGF-β1 released from dysplastic epithelium can promote fibrosis. It has also been shown in a study exploring the Relationship between Fibrosis Thickness and Epithelial Dysplasia in Oral Submucous Fibrosis. that Increased fibrosis thickness correlated with a higher incidence of epithelial dysplasia. Advancement of fibrosis heightens the risk of epithelial dysplasia development in OSMF patients. Prevention of fibrosis progression may reduce the risk of dysplasia and oral squamous cell carcinoma [refer3] Thus, a two-way loop exists between fibrosis and epithelial dysplasia in the malignant transformation of Oral submucous fibrosis, this evident in very high rate of malignant transformation rate.\n3. Moreover, the assumption that the epithelium in OSMF is always in the atrophic stage is unsubstantiated and may introduce bias into the analysis [refer 4]. Anura et al explores the role of cellular competitiveness in oral submucous fibrosis (OSF) conditions, using computer-assisted neighbourhood analysis in quantitative immunohistochemistry (IHC) framework. It found that different states of OSF condition may be associated with different forms of competitiveness within epithelial neighboring cells, which may shape the present and future of the pre-malignant condition. The findings suggest that atrophic epithelium is associated with stress-driven competitive environments, while cells with high c-Myc+ give rise to hyperplastic epithelium. They stated that both hyperplastic and atropic epitltlium may co-exist in the same tissue. The heterogeneous nature of OSMF suggests that epithelial changes can vary widely among cases, making it essential to consider multiple phenotypic presentations rather than imposing a predetermined assumption.\n4. Coexisting oral leukoplakia and erythroplakia further complicate the interpretation of findings, and have a higher malignant transformation rate in the background of OSMF. These conditions have distinct pathophysiological characteristics that may confound the assessment of neoangiogenesis, specifically in OSMF and OSCC with OSMF. The study's conclusions may lack specificity and clinical relevance without appropriate stratification or control for these variables.Also, the proposal of a grading system without considering the potential harm of biopsies promoting fibrosis and malignant transformation raises ethical concerns.\n5. In summary, the article's methodological limitations, flawed interpretation of results, and inadequate consideration of confounding variables, ethical concerns undermine its scientific rigor and relevance. As such, it cannot be accepted for publication in its current form.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] } ]	1	https://f1000research.com/articles/12-1082
https://f1000research.com/articles/12-509/v1	17 May 23	{ "type": "Research Article", "title": "COVID-19 prevention efforts at Al-Zahraa University in the eyes of the students: A cross-sectional study from Iraq", "authors": [ "Hassan Al-Kazzaz" ], "abstract": "Background: For institutions in Iraq, the new coronavirus disease 2019 (COVID-19) pandemic has become a problem. In terms of the dissemination of COVID-19, Al Zahraa University is particularly risky due to the presence of susceptible, high-risk populations there. Methods: A total of 182 undergraduates at the Health and Medical Technology College took part. A cross-sectional, questionnaire-based study was done to evaluate the COVID-19 preventative measures for students at Al-Zahra University for Women. The study questionnaire received a 100% response rate, and the data were collected and analyzed.\nResults: It showed that of the participants, 53 (29.1%) were 20 years old, and 151 (82.9%) lived in Karbala. 120 (65.9%) students did not think the university applied preventive measures against COVID-19, and 94 (52%) thought the university ensured mask-wearing on campus. Regarding social distancing, 161 (88.4%) believed it was not enforced. 158 (86.8%) and 116 (63.7%) of the participants, respectively, thought that there were no preventive measures against Covid-19 in the campus cafeteria or no preventive measures when using university transportation.\nConclusions: During the COVID-19 pandemic, Al-Zahraa University's execution of prevention and control measures were weak. Interventions are urgently needed to improve the effectiveness of its preventive measures. The inadequacy of COVID-19 safety measures highlights the need for national guidance.", "keywords": [ "COVID19", "vaccine", "prevention efforts", "students", "Al-Zahraa University", "Iraq" ], "content": "Introduction\n\nAlmost every country’s higher education institution closed in the first half of 2020 because of the coronavirus disease 2019 (COVID-19) pandemic. University officials have had to decide if and how to safely reintroduce students, workers, and professors to campus.1\n\nThe pandemic has revolutionized the way people throughout the world study and teach. Online tools such as webinars, enabled by WebEx and its counterparts, are currently being used to replace the traditional face-to-face method of tuition.2\n\nTechnology has pervaded every aspect of our lives, and education is no different. The latent purpose of maintaining social distancing is likely to be one of the primary reasons for students enrolling in online courses during the pandemic era.3\n\nThe World Health Organization identified a number of basic measures which were undertaken up to Sep 2021 that must be adopted in the effort to control and contain the virus. The procedures involved areas such as hand hygiene, cough etiquette, surface cleaning, social distancing and recommendations around large gatherings.4\n\nThe pandemic, with its impact on morbidity and mortality rates, has affected the lives of people all over the world. As such, understandings of human behaviour and an ongoing assessment of knowledge, are crucial in the overall attempt to contain it.5\n\nFear, worry, and tension are all natural reactions to perceived or actual risks, uncertainty, and the unknown. People’s terror in the face of the pandemic is thus understandable.6\n\nIn the field of nursing, studies indicate that nurses face issues regarding management, the work environment, family cross-infection, the risk of self-infection, assault, emotional & physical energy drains, and psychological stress. Providing them with the training to work in isolation units is essential, keeping in mind that they may suffer from psychological exhaustion.7 Immunization programmes and coverage are critical for protecting people of all ages from the debilitating and potentially life-threatening impacts of infectious illnesses.8 Protection from infectious disease is one of the most valuable benefits any country can offer its citizens. Vaccination is a preventive method contributing to lower mortality and morbidity rates.9\n\nThe rapid spread of the pandemic has caused deep concern among healthcare professionals worldwide. The most up-to-date information on the COVID-19 outbreak should be available to all healthcare professionals.10\n\nThe knowledge, attitudes, and practices (KAP) concerning COVID-19 will determine a society’s willingness to accept behavioral change initiatives introduced by health authorities.11 It has been noted that the imposition of lockdowns to help control the spread of COVID-19 resulted in unusual behaviour by some consumers, affecting the ability of large corporations to operate in the market.12 Hoarding of food and toilet paper is one instance of unusual retail consumer behavior that was documented.\n\nThe safety of the students and staff is a top priority at Al-Zahraa University in Karbala, Iraq.\n\nThis article focuses on the efficacy of the Covid-19 prevention and control measures at the university, through a study looking at the students’ thoughts. Social distancing, environmental disinfection procedures, awareness campaigns, and medical care are just a few of the topics explored. In this way, the study seeks to provide an insight into pandemic response strategies in Iraqi higher education – with a view to enabling improvements where applicable.\n\n\nMethods\n\nA total of 182 health and medical technology students from the X-ray section of Al-Zahraa University for Women took part in this cross-sectional, questionnaire-based study. A questionnaire was designed and then distributed to the students through a Google classroom. These classroom sites serve as official communication routes between academic schools and students. The study questionnaire received a 100% response rate, and the data were collected and analyzed.\n\nThe research and data collection took place between January and September 2021. An academic class representative was active in disseminating the questionnaire link to the students. The responses were retrieved as Excel files from the electronic form of a questionnaire (a Google form) and imported into S.P.S.S. version 23 for analysis. The questionnaire related to COVID-19 prevention efforts at Al-Zahraa University for Women and employed a multiple choice format. The questionnaire was comprised of three tables, with a total of 18 questions. The demographic data obtained during the study included the age, marital status and current living situation of the students.\n\nPrior to the research, ethical approval (permit no. HREC 39) was granted by the independent ethics committee of Al-Zahraa University for Women in Karbala, Iraq.\n\nVerbal consent to participate was obtained from each student after informing them in writing about the study’s objectives, the time involved, the security of their answers, their freedom to participate or withdraw, and the future benefits to their community.\n\n\nResults\n\nTable 1 shows the participants’ demographic characteristics. 53 (29.1%) were 20 years old; 145 (79.67%) were unmarried, 33 (18.13%) were married, and 4 (2.19%) separated. The research shows that 151 (82.9%) were resident in Karbala, while 31 (17.1%) lived outside Karbala.\n\nTable 2 shows that those who had had a family member diagnosed with COVID-19 numbered 105 (57.6%). It was found that 48 (26.3%) of the participants had a vaccine refusal history, while 105 (57.6%) had refused COVID-19 vaccination. 21 (11.5%) had a family member who had died because of COVID-19.\n\nAs Table 3 shows, 120 (65.9%) students did not think the university applied preventive measures against COVID-19. In addition, in this study, 94 (52%) believed that the university made students wear masks on campus. Regarding social distancing on campus, 161 (88.4%) indicated that it was not enforced. Checking students’ temperatures at the entrance to campus is a COVID-19 prevention measure, yet only 60 (32.9%) students thought it was accomplished. Most students, 158 (86.8%), felt that the teaching staff were committed to COVID-19 prevention. Did the university sanitize the environment before allowing students on site? The answer was Yes for 100 (54.9%) students. In addition, 87(47.8%) of the participants felt that the institution would ensure the isolation of suspected COVID-19 patients. Soap and hand sanitizers were thought to be available in the university’s bathrooms by 98 (53.8%) students. 158 (86.6%) and 116 (63.7%) of the participants, respectively, believed that there were no preventive measures against the coronavirus in the campus cafeteria or on university transportation. 66 (36.2%) students thought electric fans were not available in university classrooms. 63 (34.6%) indicated that the university did not conduct awareness campaigns to prevent COVID-19. Was a vaccine or a nasal swab required for female students to enter the university? 162 (89.0%) answered No. When asked if they accepted remote e-learning or preferred in-person attendance, 110 (60.43%) participants responded that they didn’t support in-person attendance.\n\n\nDiscussion\n\nAcross the world, there was a migration to online learning because of school and university closures – an intense process for many institutions, students, instructors and parents, demanding expertise and speed to prevent losing instructional time.13\n\nThe researcher could not locate similar studies in the Middle East and other countries because the online switch has led to a lack of academic studies about campus management of Covid-19 prevention procedures.\n\nIn this study, 62 (34%) of the students thought that preventive measures were applied at the university.\n\nIn Shi et al.’s study,14 the average overall implementation rate of COVID-19 preventive and control measures was 80.0% in Chinese nursing homes – although this figure is in a care home context rather than an educational institution, it is a lot more than in our research.\n\nThe responses did not show strict COVID-19 prevention measures at Al-Zahraa University. 158 (86.8%) of the participants believed the teaching staff were committed to such measures – however, 158 (86.8%) did not think they were applied in the university cafeteria, and 88 (48.35%) did not think mask-wearing was enforced. 82 (45%) believed that the university management did not sanitize the campus before students arrived.\n\nAccording to Freeman et al.’s study,15 most large educational institutions in the US have implemented masking (n = 93) (100%) and physical distancing rules (99%). De-densification of classrooms was another preventive strategy used (61%). The findings of the Freeman study are unlike those of this research.\n\nOur study had several strengths. A 100% response rate was received on the survey for the study. Our study was cost-effective. Online questionnaires are a great approach for collecting a lot of data from a body of students while avoiding the possibility of contracting COVID-19.\n\nOur study also had some limitations. The first limitation is the small number of participants. The second is that for practical reasons the study did not include other universities in Iraq.\n\n\nConclusions\n\nDuring the COVID-19 pandemic, Al-Zahraa University’s execution of prevention and control measures was found to be weak. The safety of students and employees is a significant priority at the university. Interventions are urgently needed to improve the effectiveness with which social distancing, environmental disinfection procedures, awareness campaigns and medical care are implemented.", "appendix": "Data availability\n\nZenodo: Underlying data for ‘In the eyes of the students, Covid-19 prevention efforts at Al-Zahraa University’, https://doi.org/10.5281/zenodo.6979137. 16\n\nThis project contains the following underlying data:\n\n‐ Spreadsheet: In the eyes of the students, COVID-19 prevention efforts at Al-Zahraa University.xlsx\n\n‐ Questionnaire.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0).\n\n\nAcknowledgements\n\nI thank all the students who contributed to this study.\n\n\nReferences\n\nLopman B, Liu CY, Le Guillou MPHA, et al.: A model of COVID-19 transmission and control on university campuses. medRxiv. July 24, 2020. 2020.06.23.20138677. Publisher Full Text\n\nAnis SNM, Ibrahim MA, Tahir LM, et al.: COVID-19 and campus experience: Survey on online learning and time spent during the movement control order (MCO) among Malaysian postgraduates. J Adv Res Dyn Control Syst. 2020; 12(7 Special Issue): 2929–2933. Publisher Full Text\n\nSaxena C, Baber H, Kumar P: Examining the moderating effect of perceived benefits of maintaining social distance on e-learning quality during the COVID-19 pandemic.2020; 49(4): 532–554. Publisher Full Text\n\nDe Oliveira AC, Lucas TC, Iquiapaza RA: What has the COVID-19 pandemic taught us about preventive measures? Texto Context - Enferm. 2020; 29: 1–15. Publisher Full Text\n\nKhasawneh AI, Humeidan AA, Alsulaiman JW, et al.: Medical students and COVID-19: Knowledge, attitudes, and precautionary measures. A descriptive study from Jordan. Front Public Health. 2020; 8: 253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiradar V, Dalvi P: Impact of COVID 19 on child health: Parents’ perspective. Int J Nurs Educ Res. 2020; 8(4): 463–467. Publisher Full Text\n\nSharma A, Mohanan K: Obstacles faced by nurses working in a Covid-19 unit: A developing country viewpoint. Asian J Nurs Educ Res. 2020; 10(4): 459–462. Publisher Full Text\n\nSaraswathi KN, Lissa J: A study to assess the knowledge on selected optional vaccine among mothers of under five children in selected immunization centers at Mysore with view to develop information booklet. Asian J Nur Edu Res. 4(4): 513–515.\n\nMaheswari K: Knowledge of mothers regarding newer vaccines and vaccines for preventable diseases. Int J Adv Nurs Manag. 2015; 3(2): 107–108.\n\nFernandes MM, Thakur JR, Manisha M, et al.: A Study to assess knowledge regarding Covid-19 among nursing students. Asian J Nurs Educ Res. 11(1): 65–67. Publisher Full Text\n\nBraver A, Khaire S: Knowledge, attitude and practices towards the Covid-19 outbreak in Maharashtra State - ProQuest. Accessed August 27, 2022. Reference Source\n\nShyam Prasad S, Praharaj P: Indian consumers’ behaviour during Covid-19: An exploratory study. Asian J Manag. 2021; 12(2): 215–220. Publisher Full Text\n\nVarela DG, Fedynich A: Leading schools from a social distance: Surveying South Texas school district leadership during the Covid-19 pandemic. Natl Forum Educ Adm Superv J. 2020; 38(4): 1–10.\n\nShi M, Zhang F, He X, et al.: Are preventive measures adequate? An evaluation of the implementation of Covid-19 prevention and control measures in nursing homes in China. BMC Health Serv. Res. 2021; 21(1): 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreeman S, Nguyen T-V, Beliveau J, et al.: Covid-19 response strategies at large institutes of higher education in the United States: A landscape analysis, Fall 2020. J Adolesc Health. 2021; 68(4): 683–685. PubMed Abstract \| Publisher Full Text\n\nAl-Kazzaz HH: In the eyes of the students, COVID19 prevention efforts at Al Zahraa University.2022. Publisher Full Text" }	[ { "id": "186189", "date": "25 Jul 2023", "name": "Asita Elengoe", "expertise": [ "Reviewer Expertise Virology", "Statistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease proofread the manuscript.\n\nIntroduction: Please elaborate on the COVID-19 prevention measures at other universities.\n\nIntroduction: Please include more points on preventive measures in universities.\n\nIntroduction: Please mention the problem of the statement clearly.\n\nIntroduction: Please write the objective of the study more clearly.\n\nMethods: How do you validate the questionnaire?\n\nMethods: May I please know whether the questionnaire was prepared by yourself or modified from the previous study?\n\nMethods: Have you done a pre-test?\n\nMethods: In which language you prepared the questionnaire?\n\nWhat type of statistical test you carried out? eg. Chi-square, independent-t-test, etc?\n\nDiscussion: Lack of previous studies.\n\nDiscussion: Please compare your results with other studies.\n\nConclusion: Not clear\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Yes", "responses": [ { "c_id": "9971", "date": "01 Aug 2023", "name": "HASSAN al-kazzaz", "role": "Author Response", "response": "Dear reviewer Thank you so much for your valuable notes . I will work on it and let you know soon. Best, Hassan" }, { "c_id": "10116", "date": "31 Aug 2023", "name": "HASSAN al-kazzaz", "role": "Author Response", "response": "Dear Sir, Thank you so much for your valuable notes. I had corrected the article per your request. If you have any other notes, I will be more than happy to do it. All the best, Hassan" }, { "c_id": "12782", "date": "09 Nov 2024", "name": "HASSAN al-kazzaz", "role": "Author Response", "response": "As for proofread the manuscript : Abstract Background: The COVID-19 pandemic has become a significant issue for institutions in Iraq. Al Zahraa University is particularly vulnerable due to the presence of susceptible, high-risk populations. Methods: A total of 182 undergraduate students from the Health and Medical Technology College participated in this study. We conducted a cross-sectional, questionnaire-based survey to evaluate the COVID-19 preventive measures in place for students at Al Zahraa University for Women. The questionnaire achieved a 100% response rate, and the collected findings were analyzed. Results: The findings indicated that 53 participants (29.1%) were 20 years old, with 151 students (82.9%) residing in Karbala. A significant number, 120 students (65.9%), believed the university did not implement adequate preventive measures against COVID-19. Furthermore, 94 participants (52%) felt the university enforced mask-wearing on campus. Regarding social distancing, 161 students (88.4%) thought it was not enforced effectively. Additionally, 158 participants (86.8%) and 116 participants (63.7%) believed that there were no preventive measures in the campus cafeteria or during university transportation, respectively. Conclusions: During the COVID-19 pandemic, Al-Zahraa University's execution of prevention and control measures were weak. Interventions are urgently needed to improve the effectiveness of its preventive measures. The inadequacy of COVID-19 safety measures highlights the need for national guidance. Introduction In the first half of 2020, nearly all higher education institutions worldwide were compelled to close in response to the coronavirus disease 2019 (COVID-19) pandemic. University officials faced the critical task of determining the safest methods to reintroduce students, faculty, and staff to campus. Effective planning for educational interventions during the COVID-19 pandemic necessitates a comprehensive awareness of the health risks associated with the virus, along with considerations of knowledge, perceptions, and preventive behaviors. The pandemic has fundamentally altered the landscape of education, prompting a global shift toward remote learning. Online tools, such as webinars facilitated by platforms such as WebEx and similar technologies, have supplanted traditional face-to-face instruction. The increasing integration of technology into various facets of life extends to the educational sphere. The necessity for social distancing has served as a primary motivator for students opting to enroll in online courses throughout the pandemic. As of September 2021, the World Health Organization identified several essential measures that must be adopted to effectively control and contain the virus. These measures encompass hand hygiene, cough etiquette, surface disinfection, social distancing, and guidelines regarding large gatherings. The pandemic's repercussions on morbidity and mortality rates have had a profound global impact, necessitating a detailed understanding of human behavior and ongoing assessments of knowledge as integral components of virus containment efforts. Natural reactions such as fear, anxiety, and stress are common responses to perceived risks and uncertainties. Consequently, public apprehension during the pandemic is an understandable phenomenon. Within the nursing profession, studies indicate that nurses encounter a myriad of challenges, including management difficulties, adverse work environments, the risk of cross-infection within families, self-infection concerns, emotional and physical strain, and psychological stress. It is essential to provide these professionals with adequate training to work effectively in isolation units, particularly in light of their potential for psychological exhaustion. Immunization programs and coverage play a pivotal role in safeguarding individuals of all ages from severe and potentially life-threatening infectious diseases. Offering protection against such diseases is among the most significant benefits that a nation can provide its citizens. Vaccination stands as an essential preventive measure that contributes to reducing mortality and morbidity rates. The rapid proliferation of the pandemic has generated substantial concern among healthcare professionals globally. It is imperative that timely and accurate information regarding the COVID-19 outbreak is made readily available to all healthcare practitioners. The knowledge, attitudes, and practices (KAP) concerning COVID-19 significantly influence society’s readiness to accept behavioral change initiatives introduced by health authorities. The implementation of lockdown measures intended to curb the spread of COVID-19 has resulted in atypical consumer behaviors, which have impacted the operational capacity of large corporations. Noteworthy instances of such behaviors include panic buying of essential items such as food and toilet paper. At Al-Zahraa University in Karbala, Iraq, the safety of students and staff remains the highest priority. This article investigates the effectiveness of COVID-19 prevention and control measures within the university, utilizing a study focused on student perspectives. The research explores various topics, including social distancing practices, environmental disinfection procedures, awareness campaigns, and medical support. The objective of the study is to provide valuable insights into pandemic response strategies in Iraqi higher education and to identify opportunities for enhancement. Methods A total of 182 health and medical technology students from the X-ray section at Al-Zahraa University for Women participated in this cross-sectional, questionnaire-based study. A questionnaire was designed and distributed to the students via Google Classroom, which serves as the official communication channel between academic institutions and students. The study achieved a 100% response rate, and the data were collected and analyzed. Data collection took place from January to September 2021. An academic class representative played a key role in disseminating the link to the questionnaire among the students. Responses were retrieved as Excel files from the electronic questionnaire (a Google Form) and were imported into SPSS version 23 for analysis. This study utilized rates and percentages to evaluate and compare the findings with those of similar studies. The questionnaire focused on COVID-19 prevention efforts at Al-Zahraa University for Women and consisted of multiple-choice questions. It was developed based on previous studies and a review of various articles and research. The questionnaire included three sections, with a total of 18 questions. It was made available in both Arabic and English. The demographic data collected during the study included students’ age, marital status, and current living situation. Ethics and consent Before the research, ethical approval (permit no. HREC 39) was granted by the independent ethics committee of Al-Zahraa University for Women in Karbala, Iraq. Verbal consent to participate was obtained from each student after informing them in writing about the study’s objectives, the time involved, the security of their answers, their freedom to participate or withdraw, and the future benefits to their community. Discussion The global shift to online learning was prompted by the closures of schools and universities, creating a challenging situation for institutions, students, instructors, and parents. This transition required expertise and quick implementation to minimize instructional time loss. The results of this experience may inform the development of a COVID-19 preventive program aimed at enhancing essential university safety measures and reducing the virus's spread. However, the researcher struggled to find similar studies in the Middle East and other regions, mainly because the transition to online education has resulted in a lack of academic investigations into campus management concerning COVID-19 prevention practices. In this study, 62 students (34%) thought preventive measures were being effectively implemented at their university. In contrast, Albaqawi's study found that only 45 students (3.7%) knew someone in their family with a confirmed COVID-19 case. Shi et al.'s study reported an average overall implementation rate of COVID-19 preventive and control measures at 80.0% in Chinese nursing homes, although this is a different context than educational institutions, and is significantly higher than what was observed in our research. The findings indicate that strict COVID-19 prevention measures were lacking at Al-Zahraa University. While 158 participants (86.8%) believed that the teaching staff were committed to safety measures, the same number disagreed that these measures were enforced in the university cafeteria. Additionally, 88 participants (48.35%) felt that mask-wearing was not adequately enforced, and 82 participants (45%) believed that the university management failed to sanitize the campus before students' arrival. In contrast, Freeman et al. found that most large educational institutions in the US implemented masking (100%) and physical distancing rules (99%). Class de-densification was also a strategy employed by 61% of these institutions. These findings starkly contrast with those of our research, highlighting the need for university administrations to adhere to COVID-19 preventive guidelines. Strengths Our study had several strengths. A 100% response rate was received on the survey for the study. Our study was cost-effective. Online questionnaires are a great approach for collecting a lot of data from a body of students while avoiding the possibility of contracting COVID-19. Limitations Our study also had some limitations. The first limitation is the small number of participants. The second is that for practical reasons the study did not include other universities in Iraq. Conclusion During the COVID-19 pandemic, Al-Zahraa University’s execution of prevention and control measures was found to be weak. The safety of students and employees is a significant priority at the university. Interventions are urgently needed to improve the effectiveness with which social distancing, environmental disinfection procedures, awareness campaigns and medical care are implemented." } ] } ]	1	https://f1000research.com/articles/12-509
https://f1000research.com/articles/12-1078/v1	31 Aug 23	{ "type": "Research Article", "title": "Potential transformation of seagrass (Syringodium isoetifolium) into a bioactive food ingredient using different extraction techniques", "authors": [ "Bambang Susilo", "Oke Oktavianty", "Farida Rahayu", "Midia Lestari Wahyu Handayani", "Abd Rohim", "Oke Oktavianty", "Farida Rahayu", "Midia Lestari Wahyu Handayani", "Abd Rohim" ], "abstract": "Background: Syringodium isoetifolium is a seagrass that grows abundantly in Indonesian territorial waters and has been known to be of high significance not only for the seawater ecosystem, but also for human beings (as food, nutritional and pharmaceutical products). In this study, the bioactive constituent of Syringodium isoetifolium was extracted using several different techniques to recover a maximum yield. Methods: Extraction was carried out by conventional and non-conventional (Microwave-assisted extraction, ultrasound-assisted extraction-bath system, and ultrasound-assisted extraction- (UAE) probe system) techniques with green solvents (water, 50% ethanol, and 100% ethanol). Results: As a result, 50% ethanol and water extracts exhibited a significantly higher yield. Total phenol content was significantly higher for 50% ethanol extract. Different extraction techniques (using 50% ethanol solvent) showed that the UAE-probe was the best technique since it yielded the highest total phenol (17.37 ± 2.16 mg GAE/g) and the richest bioactive compounds (Choline, betaine, 3,5-di-tert-butyl-4-hydroxybenzoic acid, 7-Hydroxycoumarine, 4-Methoxycinnamic acid, Zearalenone, Caffeic acid, Levalbuterol, Phloretin, Dihydrocaffeic acid, Quercetin-3β-D-glucoside and Quercetin). Interestingly, choline was the most abundant compound in the extract obtained with different extraction techniques. In this in silico assay, choline from seagrass extract was shown as an anti-inflammatory. The interaction pathway of the choline compound with receptors (TNF-α, IL-1β, and IL6) had a higher binding affinity value than the inhibitor-receptor interaction (i.e. -3.4, -3.0, and -2.8 kcal/mol). The cytotoxicity test on TIG-1 cells showed that the extract did not have a toxic effect on them. Conclusions: These findings support the potential use of Syringodium isoetifolium as a bioactive food ingredient.", "keywords": [ "Extraction technique", "Phenolic compounds", "Phytochemical", "Syringodium isoetifolium", "Ultrasound" ], "content": "Introduction\n\nIndonesia's vast coastline is a habitat for seagrass, which grows to form seagrass meadows. Syringodium isoetifolium, Thalassia hemprichii, Cymodocea rotundata, Halophila ovalis, Enhalus acoroides, Cymodocea serrulata, Halodule pinifolia, and Halodule uninervis are species that are often found in seagrass beds in Indonesian waters (Kiswara et al., 2009; van Katwijk et al., 2011). The species S. isoetifolium, Cymodocea rotundata, and Cymodocea serrulata have a growth period that tends to be faster (Kiswara et al., 2009). Recently, Li et al. (2021) succeeded in seeding E. acoroides for the restoration of seagrass beds in the tropics. Therefore, seagrass is a marine plant that has the potential to be produced on a large scale. However, the high production potential of seagrass has not been followed by its postharvest utilization to produce high economic value products. So far, seagrass beds have been allowed to grow wild and play an important role not only in coral reef ecosystem and maintain the sustainability of the marine biota, but also in maintaining other marine ecosystem’s balance (van Katwijk et al., 2011).\n\nOnly the leaves of seagrass that has optimal growth can be harvested so that it does not cause the plant’s death. In addition, there is no need to replant after harvesting seagrass leaves. Seagrass leaves have a dark green color and a strong aroma, and are rich phytochemical compounds such as phenolics. Phenolic compounds are molecules with high diversity formed by the attachment of one or more –OH groups to the phenyl ring. Phenolics are divided into several classes including flavonoids and other benzopyran derivatives, phenolic acids and ester derivatives, quinones, lignins, lignans, tannins, stilbenes, curcuminoids, chalcones, and essential oils (Chiorcea-Paquim et al., 2020).\n\nAs seagrass has a deep green color and a quite strong aroma, it is predicted that it is rich in phytochemical compounds such as phenolics. A previous study by Rengasamy et al., 2019 reported that S. isoetifolium has a potential as a natural antioxidant and clinical enzyme inhibitor that can be applied in pharmaceutical cosmeceutical industries. Moreover, S. isoetifolium could also be a good candidate as a food complement, feed, and biomedical field due to its rich biochemical profile (Bharatharathna & Santhanam, 2019).\n\nThe extraction technique plays an important role in extracting nutrient content and phytochemical constituents. Efficient extraction techniques that are fast, inexpensive, economical in materials and solvents, high-yield, environmentally friendly, and do not damage the structure and functional properties of the target compounds, were chosen. Conventional extraction is usually carried out by maceration, which involves immersing the material with a solvent at a certain temperature for a certain time with agitation assistance (Abd Aziz et al., 2021); meanwhile, modern extraction techniques are carried out using microwave-assisted extraction (MAE) and ultrasonics. The principle of MAE is to use energy from microwaves to heat a mixture of sample material and extraction solvent, which is induced by microwave irradiation (Lefebvre et al., 2021). Microwave power is strongly absorbed by the interior of plant cells due to their water content. The pressure on the cell wall is generated by swelling of the plant cell when water evaporates, which pushes the cell wall from within to stretch and eventually rupture. This event can release metabolites from the broken cells into the surrounding solvent (Bachtler & Bart, 2021). On the other hand, the principle of UAE is to use energy from ultrasonic waves. At a certain frequency and amplitude, it creates cavitation bubbles, which when they reach an unstable point, then release high temperatures and pressures by the blasting process. This phenomenon can damage cell walls and promote the release of metabolites from the plant (Lefebvre et al., 2021). These different extraction techniques affect the quantity and bioactivity of the target compounds that are crucial to study.\n\nTo our knowledge, a recent study by Kalaivani et al. (2021) has reported the biological activities such as antibacterial, antifungal, antimicrobial, antifouling, and anticancer properties of S. isoetifolium. This research was conducted to develop the potential use of S. isoetifolium as a food bioactive contituents. To get the best phytochemical and nutrient compounds from S. isoetifolium, four different techniques, namely maceration, MAE, and UAE-bath (UAE with bath) and UAE-probe system were applied. Meanwhile, in this study, the phenol extract was analyzed and phytochemical compounds were screened by liquid chromatography–high resolution mass spectrometry (LC-HRMS). While the potential development is proven by in silico study and cell toxicity.\n\n\nMethods\n\nAll experiments were carried out at Universitas Brawijaya, Indonesia. Fresh S. isoetifolium was harvested from Kondang Merak Beach, East Java, Indonesia (-8.3964160388196N, 112.51923979683157E) and then directly washed with fresh water to remove attached dirt and salt particles. Afterward, the seagrass was dried using an oven at 40°C to a constant weight (13.87 ± 0.20% moisture) and then with a blender. The dried powder (297.00 μm) was used for extraction. Chemicals used for extraction and analysis comprised 2,2,1-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu phenol reagent, sodium carbonate (Na2CO3), gallic acid, Aquadest, and absolute ethanol, from Meck, Singapore. All chemicals used were analytical grade except water and ethanol for LC-HRMS analysis, which were HPLC-grade.\n\nPreparation\n\nWater, ethanol 50%, and ethanol 100% were applied for extraction with different techniques. So far, they have been considered green solvents because they have been proven safe (non-toxic) for either phytochemical extraction or food usage (Ahmad et al., 2021; Molino et al., 2018). In addition, green solvents have a positive environmental impact due to their lower eco-toxicity and are biodegradable due to a strong solvency (Ahmad et al., 2021). Also, these solvents are highly efficient to completely extract the molecules in various polarities (Farooq et al., 2021; Harscoat-Schiavo et al., 2021; Lao et al., 2017).\n\nExtraction process\n\nSeagrass powder in each extraction solvent (viz. water, ethanol 50%, and ethanol 100%;1:20 w/v) was extracted with different techniques, i.e. MAE (Anton Paar - Multiwave Pro), UAE-Probe (Branson 250/450), UAE-Bath (Soltec - Sonica 2400 EP Ultrasonic cleaner) and conventional maceration (Waterbath shaker – Memmert WNB 45). MAE followed the Albuquerque et al. (2017) method with a slight modification in extraction time. MAE settings were: 50°C, stirrer at high speed, a 5 min duration, and factory set (Maximum microwave power 700 W; Maximum pressure 18.0 bar; Maximum pressure increase rate 0.5 bar/s). UAE-Bath followed the optimum method by Albuquerque et al., 2017) with the following equipment settings: off-temperature control, 60 min duration, and factory set (40 kHz frequency, 260 W peak ultrasonic power). UAE-Probe followed (Albuquerque et al., 2017) method by modifying the maximum time. UAE-Probe settings were: 70% amplitude, off-temperature control, 5 min duration, continuous duty cycle, and factory set (19-20 kHz horn frequency, 400 W kinetic energy). Conventional based on the optimum method by (Albuquerque et al., 2017), extraction was processed in a water bath shaker, agitated at medium speed, at 30°C for 1440 min. Each treatment was done in triplicate.\n\nDry extract generated\n\nThe extracted mixture was filtered with filter paper and then centrifugated at 3000 rpm, 25°C, for 5 minutes to separate the solute and the material residue. The extract solution was dried with a vacuum oven at 40°C to achieve constant weight. The water, 50% ethanol, and 100% ethanol dry extracts were yielded for each extraction technique. The dry extracts were then stored at -20°C for the next analysis. The yield of dry extracts was calculated using Equation ((1) (Aboulghazi et al., 2022).\n\nAnalysis of total phenolic content followed a Folin-Ciocalteau method (Lim et al., 2019). Briefly, 30 μl of Folin-Ciocalteu reagent (1.0 N) and 60 μL of diluted samples were mixed in a 96-well microplate, then incubated for 5 min. Afterward, 150 μL of 20% sodium carbonate solution was added and incubated in a dark place at room temperature for 40 min. The absorbance of the supernatant was measured at 730 nm using Microplate Reader after centrifugation for 8 min (1600×g). The total phenolic content of the samples was calculated with a standard curve using gallic acid (mg GAE/g w.b.).\n\nIdentification of phenolic compounds contained in 50% ethanol extract of S. isoetifolium using different extraction techniques was carried out using a non-targeted screening method (Susilo et al., 2020) by dissolving 100 μL of seagrass extract in 1400 μLof water and ethanol depending on the solvent used for extraction. Samples were filtered with a 0.22 m minisart RC and then injected into the LC-HRMS apparatus. The 10 μL samples were processed automatically by using a hypersil gold aQ 50 mm × 1 mm × 1.9 μm (length x diameter x particle size) column with positive polarity conditions, flow rate 40 L/min, oven column temperature 30°C with elution gradient as follows: 0–2 min 5% B, 15–22 min 60% - 95% B, stabilized at 95% B for 3 min then dropped to 5% B for 5 min. The chromatogram data generated from the injection process was analyzed using Compound Discoverer 3.2 software based on the Mzcloud online library.\n\nLigand and protein preparation\n\nThe ligands used in this study consisted of choline compounds and inhibitors of each protein. All ligands were obtained from the PubChem web server in Sybil Data Files (SDF) format. The ligands were converted into a 3D structure in Protein Data Bank (PDB) format with BIOVIA Discovery Studio 2019. Choline is known to have anti-inflammatory activity, and in this study, it was hoped that it could bind to TNF-α, IL-1β, and IL6, which function as pro-inflammatory agents. The 3D structures of TNF-α (2AZ5), IL-1β (1ITB), and IL6 (1P9M) were obtained from the RCSB PDB web server. The receptors were then prepared to remove water molecules and ligands with BIOVIA Discovery Studio 2019.\n\nMolecular docking analysis and ligand-receptor interactions\n\nLigand-receptor interactions were analyzed by molecular docking using AutoDock Vina integrated in PyRx version 0.9.5. The molecular docking process was carried out using a specific docking method based on the active site of each receptor (Boulanger et al., 2003; Halim et al., 2015; He et al., 2005; Zia et al., 2020). Docking results, bond positions, and amino acid residues formed between the ligand-receptors were analyzed using the 2019 BIOVIA Discovery Studio.\n\nMolecular dynamic simulation\n\nMolecular dynamic (MD) was used as a simulation method to analyze the physical movements of atoms and molecules. The Yet Another Scientific Artificial Reality Application (YASARA) has been used for MD simulations. This simulation was carried out to compare the interaction of ligands and inhibitors in binding to receptors. The parameters in the simulation correspond to the physiological conditions of the cell at 37°C, 1 atm, pH 7.4, and 0.9% salt content for 50 ns with autosave every 25 ps. The simulation was run by the md_run macro program, and the results were displayed by the md_analyze and md_analyzebindenergy programs (Bagheri & Fatemi, 2018; Deeba et al., 2017; Krieger & Vriend, 2015).\n\nIn this study, the MTT assay based on Miasih et al. (2022) was applied with slight modification. The extract of S. isoetifolium was treated in TIG-1 cells. Finally, the absorbance was measured using a microplate reader at a wavelength of 595 nm. The results were used to construct a graph of cell viability percentage against extract concentrations.\n\nThe quantitative data served as average values ± standard deviation. Analysis of variance (ANOVA) used a Nested design with Minitab 18 software. Furthermore, the Tukey test with p < 0.05 revealed a significant difference.\n\n\nResults and discussion\n\nUsing 100% ethanol, 50% ethanol, and water as solvents aims to extract phytochemical compounds with high polarity to low polarity. The extraction results are presented in Table 1, the water and 50% ethanol solvent produced the highest yield while 100% ethanol solvent produced a significant yield for each extraction technique. It was expected, due to the phytochemical compounds (such as dihydrocaffeic acid, quercetin, caffeic acid, levalbuterol, phloretin, and zearalenone; see in Table 3) of S. isoetifolium having high polarity. In other plants (Blepharis linariifolia Pers., Guiera senegalensis JF Gmel. and Limnophila aromatica), 50% ethanol solvent also showed a higher yield than 100% ethanol, 75% ethanol, water, acetone, and dichloromethane (Dirar et al., 2019; Do et al., 2014). Many studies have stated the advantages of non-conventional extraction methods compared to conventional ones, namely high yields (Ameer et al., 2017). However, the yield of S. isoetifolium extraction with various non-conventional and conventional techniques showed no significant difference. The extraction yields depends on the extraction time (Đurović et al., 2018).\n\nPhenolics are molecules with high diversity defined by the attachment of one or more –OH groups to the phenyl ring. Phenolics are divided into several classes including flavonoids and other benzopyran derivatives, phenolic acids and ester derivatives, quinones, lignins, lignans, tannins, stilbenes, curcuminoids, chalcones, and essential oils (Chiorcea-Paquim et al., 2020). For each extraction technique, extraction of S. isoetifolium with 50% ethanol as a solvent produced the highest total phenol, meanwhile, with 100% ethanol solvent, phenol could not be obtained (Table 1). The polarity of the phenolic compounds is medium so extraction with 50% ethanol in many plants (Moringa oleifera L. leaves, rambutan peel, Cyperus rotundus L., and pomegranate) yielded the highest total phenol compared to extraction with 100% ethanol, 80% ethanol, 70% ethanol , water, acetone, and dichloromethane (Dirar et al., 2019; Harscoat-Schiavo et al., 2021; Phuong et al., 2020; Wu et al., 2020). The total phenol content in S. isoetifolium extract was influenced by the extraction technique used. UAE-probe yielded a significantly higher total phenol (17.37 ± 2.16 mg GAE/g) compared to UAE-bath, MAE, and conventional (Table 1). Đurović et al. (2018) reported that the total phenol content of yellow soybean seeds extracts, which was extracted using ultrasound-probe, was not different from that of ultrasound-bath and MAE. This is due to the different samples extracted, the structure of yellow soybean seeds being harder than S. isoetifolium leaves, as well as the different processing conditions of the ultrasound-probe (20 kHz, 30% of maximum amplitude, 10 min duration, the temperature was held constant at 25 ± 1°C) and UAE-probe (50/60 kHz, 70% amplitude, 5 min, at room temperature and the temperature was increased but not held).\n\nPhenolic compounds are non-polymeric phytochemical compounds that present bioactivity as antioxidants. Their main structure is an aromatic ring or phenol unit containing at least one hydroxyl group (Akyol et al., 2016; Lao et al., 2017; Yang et al., 2018). The complex phenolic structure causes a wide variety of molecular weights, and there are about 10,000 different phenolic derivatives (Chiorcea-Paquim et al., 2020). Caffeic acid, quercetin, coumarin, vanillic acid, benzoic acid, cinnamic acids, phloretin, vanillin, and benzopyran derivatives are examples of phenolic compounds (Akyol et al., 2016; Chiorcea-Paquim et al., 2020; Xiong et al., 2019). In this study, the extraction of S. isoetifolium with 50% ethanol produced the highest total phenol; therefore the use 50% ethanol for each extraction technique identified its phenolic compounds content. As can be seen in Table 2, UAE-probe yielded the most phenolic compounds (10 types), including 3,5-di-tert-Butyl-4-hydroxybenzoic acid, 7-hydroxycoumarine, 4-methoxycinnamic acid, zearalenone, caffeic acid, levalbuterol, phloretin, dihydrocaffeic acid, quercetin-3β-D-glucoside, and quercetin. Meanwhile UAE-bath, MAE, and conventional extraction yielded fewer phenolic compounds: 5, 5, and 6 types, respectively. Specifically, zearalenone, caffeic acid, phloretin, dihydrocaffeic acid, and quercetin were detected only by UAE-probe. The advantages of the UAE for the extraction of phenolic compounds have also been proven in other plants. The yield of ellagic acid and quercetin extraction from rambutan peel with UAE was significantly higher than conventional extraction (Phuong et al., 2020). Extraction yield of quercetin, gallic acid, vicenin-2, p-Hydroxybenzoic acid, orientin, rutin, hyperoside, kaempferol-3-O-rutinoside, isorhamnetin 3-O-glucoside, rosmarinic acid, apigenin, and kaempferol on Moringa oleifera L. Leaves with UAE was higher than with conventional extaction (maceration and stirring-assisted extraction) (Wu et al., 2020). Interestingly, choline was abundantly present in all extraction techniques, especially with UAE-probe. Choline is an essential nutrient that plays an important role for neurotransmitter synthesis (acetylcholine), cell-membrane signaling (phospholipids), lipid transport (lipoproteins), and methyl-group metabolism (homocysteine reduction). Moreover, choline is also required to make the phospholipids phosphatidylcholine, lysophosphatidylcholine, choline plasmalogen, and sphingomyelin – essential components for all membranes (Zeisel & da Costa, 2009). Betaine also presents in all extraction techniques, except MAE. According to findings from a previous study by Cho et al. (2006) and Detopoulou et al. (2008), a combination of dietary intake rich in choline and betaine are associated with lower concentrations of all of the inflammatory markers, including C-reactive protein (CRP), homocysteine, interleukin-6, and tumor necrosis factor.\n\nLigand-receptor interactions\n\nMolecular docking is used to determine the interaction between the receptor and the ligand, indicated by the binding affinity value and the amino acid residues involved (Figure 1). Since the presence of choline is abundant, this study used a ligand consisting of choline compounds and inhibitors from each protein. The inhibitor of each protein is a compound used as a control, to compare the strength of the interaction between choline-receptors and inhibitor-receptors. Molecular docking results show that the interaction of choline compounds with receptors (TNF-α, IL-1β, and IL6) had a higher binding affinity value than the inhibitor-receptor interaction, namely -3.4, -3.0, and -2.8 kcal/mol. The binding affinity between the inhibitor and TNF-α was the highest compared to the other inhibitor-receptors, with a value of -9.1 kcal/mol. The higher the binding affinity value, the weaker the bond between the ligand-receptor (Table 3).\n\n(A) Choline-TNF-α, (B) Inhibitor-TNF-α, (C) Choline-IL-1β, (D) Inhibitor-IL-1β, (E) Choline-IL6, and (F) Inhibitor-IL6. Choline and inhibitors are presented as yellow and red sticks, respectively. TNF-α, IL-1β, and IL6 are presented as ribbon structures with the different blue color.\n\nMolecular docking results were then visualized to determine the binding site of the ligand and receptor. Visualization with Discovery Studio showed that the choline compound had the same binding site as the inhibitor on each protein. The presence of the same binding site indicates that the choline compound can have the same function as the control, even though it has a relatively high binding affinity value. The same binding site between choline compounds and inhibitors in each protein was indicated by several amino acid residues that interact through hydrogen and hydrophobic interactions. Tyr59, Ser60, Gln61, Tyr119, Leu120, Gly121, and Tyr151 are seven amino acid residues involved in the interaction of choline-TNF-α and inhibitor-TNF-α. However, there was a slight difference: Ser60 in the choline-TNF-α reaction interacts through hydrogen bonds, whereas in the inhibitor-TNF-α reaction, it interacts through hydrophobic bonds. Tyr119 is an amino acid residue that plays an essential role in ligand-receptor interactions (Zia et al., 2020).\n\nLeu62, Lys63, Glu64, Lys65, Asn66, Leu67, Tyr68, Pro87, Tyr90, and Pro91 are 10 amino acid residues involved in the interaction between choline-IL-1β and inhibitor-IL-1β. Asn66 and Tyr68 are two amino acid residues that interact through hydrogen bonds in the inhibitor-IL-1β interaction while interacting through hydrophobic bonds in the choline-IL-1β interaction. Ser176 is an amino acid residue that interacts via hydrogen bonds in the reaction of choline-IL6 and inhibitor-IL6. Four other amino acid residues involved in the reaction of the two ligands with IL6 interact via hydrophobic bonds, namely Cys73, Phe74, Gln75, and Ala180. Amino acid residues involved in the ligand-receptor interaction complex through hydrogen bonds and hydrophobic bonds can increase the affinity of the ligand bonds in proteins (Chen et al., 2016). It is known that the formed hydrophobic interactions in ligand-receptor complex play an important role in stabilizing the ligand-protein bond and helping to increase the affinity of the ligand bond with protein (Varma et al., 2010).\n\nMolecular dynamic simulation results\n\nThe stability of the ligand complex with each pro-inflammatory protein is known based on molecular dynamic (MD) simulations. As seen in Figure 2, the parameters used in this simulation were the RMSD of the ligand-receptor complex, RMSF, and the number of hydrogen bonds of the ligand-receptor complex. MD analysis showed that the choline-TNF-α complex and the inhibitor-TNF-α had the same stability up to 20 ns simulation, indicated by an RMSD value of 3 Å. The stability of the choline-TNF-α complex fluctuated with RMSD values, reaching 3.5 Å at several simulation times. Unlike the inhibitor-TNF-α complex, which experienced relatively high fluctuations during the simulation time of around 29 ns with an RMSD value of 4 Å, the simulation stabilized again after 30 ns with an RMSD value of less than 3 Å. The simulation of the choline-IL-1β complex was very unstable, as indicated by very high fluctuations and an average RMSD value of more than 4 Å. These results are inversely proportional to the stable inhibitor-IL-1β complex from start to end of the simulation, indicated by the average RMSD value <3 Å. The choline-IL6 and inhibitor-IL6 complex was unstable compared to other interactions, indicated by an RMSD value of >3 Å. However, when paying attention to the choline-IL6 complex, it had a higher level of stability than the IL6-inhibitor, indicated by a lower RMSD value. Previous studies have shown that the ligand-receptor complex, which is stable during simulation, has an RMSD value of 3 Å (Martínez, 2015; Wargasetia et al., 2021).\n\nDuring simulation, the stability of the ligand-protein interaction complex is indicated by the RMSD value. RMSF value is based on fluctuations in the amino acid residues and the hydrogen bond numbers. A, D, G. Ligands-TNF-α complex. B, E, H. Ligands-IL-1β complex. C, F, I. Ligands-IL6 complex.\n\nThe fluctuations of the amino acid residues formed can also determine the stability of the ligand-receptor complex. The choline-TNF-α and inhibitor-TNF-α complexes are the most stable compared to other ligand-receptor complexes. This is indicated by fluctuations in amino acid residues that are not too high compared to the others. It is known that the more amino acid residues that experience fluctuation, the more unstable the ligand-receptor complex (Azminah et al., 2019). The number of hydrogen bonds in the choline-TNF-α and inhibitor-TNF-α complexes were the highest compared to the other complexes. A large number of hydrogen bonds makes the two ligand-receptor complexes the most stable compared to other ligand-receptor complexes.\n\nA toxicity test was conducted to evaluate the effect of the extract on the viability of TIG-1 cells. TIG-1 cell viability was determined by the MTT-assay method, using mitochondrial dehydrogenase activity in living cells against 3[4,5-dimethylthiazole-2-yl]-2,5-diphenyltertrazolium bromide (MTT) into insoluble purple formazan crystals. The MTT test requires the addition of a solvent (DMSO) to dissolve the formed insoluble formazan product.\n\nThe data obtained were the absorbance values of purple formazan crystals measured at a wavelength of 595 nm, which were then converted to determine the percentage of living cells. The number of viable cells was measured by colorimetry and the quantity of formazan product was directly proportional to the number of live cells in the culture (McCauley et al., 2013).\n\nThe results of the toxicity test (Figure 3) showed a decrease in the viability of TIG-1 cells in line with the increase in the concentration of the extract, but not significant. Exposing the lowest extract concentration of 12.5 μg/mL showed no decrease in TIG-1 cell viability compared to control cells, which was 96.1%. Meanwhile, exposing the highest extract concentration of 800 μg/mL showed a decrease in cell viability of 78.4%. TIG-1 cell viability in the range of extract concentrations of 25-400 μg/mL did not show a significant difference in the range of 84.5-92.5%. The results of the extract toxicity test on TIG-1 cells showed that the extract did not have a toxic effect on TIG-1 cells.\n\nReferring to international guidelines such as those of the International Organization for Standardization (ISO 10993-5:2009) it has been shown that if the viability is reduced to <70% of the control, then the test substance has the potential for cytotoxicity. Based on these guidelines, giving the extract to TIG-1 cells with the highest dose has a viability value above 70% which indicates that the extract has no cytotoxic potential on normal cells.\n\nAn in vitro cytotoxicity test was used to evaluate the toxicity of extracts following international guidelines such as those by the International Organization for Standardization (ISO 10993-5:2009). This test can be used to determine the structure–activity relationship of the extract used, as well as determine the initial concentration for further research. Inhibition of cell death, cell growth, or cell proliferation are important parameters used to evaluate the cytotoxicity of an extract (Aydin et al., 2016).\n\n\nConclusions\n\nBioactive constituents of Syringodium isoetifolium can be extracted efficiently using the UAE-probe method with 50% ethanol; in this study, it resulted in both the highest yields and total phenol, as well as the richest bioactive constituents. Moreover, choline was the most dominant compound in extracts yielded by different extraction techniques. Since choline was an abundant compound, the in silico assay was done using this compound, and resulted in choline having the ability to be anti-inflammatory. Furthermore, based on a cytotoxicity test, the S. isoetifolium extract by UAE-Probe with 50% ethanol did not have a toxic effect on TIG-1 cells. Hence, Syringodium isoetifolium extract had a high potential as a bioactive food ingredient.", "appendix": "Data availability\n\nFigshare: Experimental Data: Potency Development of Seagrass (Syringodium isoetifolium) as Food Bioactive Constituents by Different Extraction Techniques, https://doi.org/10.6084/m9.figshare.21780674.v7 (Susilo et al., 2022).\n\nThis project contains the following underlying data:\n\n- Experimental Data (yield, total phenolic, bioactive compounds & cytotoxicity).xlsx\n\n- RCSB and Protein DataBank accession numbers of molecular docking.docx\n\nFigshare: Experimental Data: Potency Development of Seagrass (Syringodium isoetifolium) as Food Bioactive Constituents by Different Extraction Techniques, https://doi.org/10.6084/m9.figshare.21780674.v7 (Susilo et al., 2022).\n\nThis project contains the following extended data:\n\n- Instruments.xlsx\n\n- Minitab Project - ANOVA OF YIELD DATA.MPJ.BAK\n\n- Minitab Project - ANOVA of Yield Data.MPJ\n\n- Minitab Project - ANOVA OF TOTAL PHENOLIC DATA.MPJ.BAK\n\n- Minitab Project - ANOVA of Total Phenolic Data.MPJ\n\n- GraphPad Prism - Graphic of TIG-1 cell viability profile.pzfx\n\n- Table. Molecular docking and amino acid residues on ligand-receptor interactions.docx\n\n- Figure. Molecular dynamic simulation.png\n\n- Figure. Ligand-receptor interaction and amino acid residues involved.png\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe authors are grateful to research center and community services (LPPM); central laboratory of life sciences (LSIH), Faculty of Agricultural Technology, Universitas Brawijaya, Malang-Indonesia for the funding and the instrumentation support.\n\n\nReferences\n\nAbd Aziz NA, Hasham R, Sarmidi MR, et al.: A review on extraction techniques and therapeutic value of polar bioactives from Asian medicinal herbs: Case study on Orthosiphon aristatus, Eurycoma longifolia and Andrographis paniculata. Saudi Pharm. J. 2021; 29(2): 143–165. 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PubMed Abstract \| Publisher Full Text\n\nPhuong NNM, Le TT, Dang MQ, et al.: Selection of extraction conditions of phenolic compounds from rambutan (Nephelium lappaceum L.) peel. Food Bioprod. Process. 2020; 122: 222–229. Publisher Full Text\n\nRengasamy KRR, Sadeer NB, Zengin G, et al.: Biopharmaceutical potential, chemical profile and in silico study of the seagrass– Syringodium isoetifolium (Asch.) Dandy. S. Afr. J. Bot. 2019; 127: 167–175. Publisher Full Text\n\nSusilo B, Midia Lestari WH, Rohim A: Impact of using low-cost packaging material of commercial herbal oil on its antibacterial compounds. All Life. 2020; 13(1): 516–523. Publisher Full Text\n\nSusilo B, Rohim A, Handayani MLW: Experimental Data: Potency Development of Seagrass (Syringodium isoetifolium) as Food Bioactive Constituents by Different Extraction Techniques. [Dataset]. figshare. 2022. Publisher Full Text\n\nvan Katwijk MM , van der Welle MEW , Lucassen ECHET, et al.: Early warning indicators for river nutrient and sediment loads in tropical seagrass beds: A benchmark from a near-pristine archipelago in Indonesia. Mar. Pollut. Bull. 2011; 62(7): 1512–1520. PubMed Abstract \| Publisher Full Text\n\nVarma AK, Patil R, Das S, et al.: Optimized Hydrophobic Interactions and Hydrogen Bonding at the Target-Ligand Interface Leads the Pathways of Drug-Designing. PLoS One. 2010; 5(8): e12029. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWargasetia TL, Ratnawati H, Widodo N, et al.: Bioinformatics Study of Sea Cucumber Peptides as Antibreast Cancer Through Inhibiting the Activity of Overexpressed Protein (EGFR, PI3K, AKT1, and CDK4). Cancer Informat. 2021; 20: 117693512110318. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu L, Li L, Chen S, et al.: Deep eutectic solvent-based ultrasonic-assisted extraction of phenolic compounds from Moringa oleifera L. leaves: Optimization, comparison and antioxidant activity. Sep. Purif. Technol. 2020; 247: 117014. Publisher Full Text\n\nXiong Y, Zhang P, Warner RD, et al.: Sorghum Grain: From Genotype, Nutrition, and Phenolic Profile to Its Health Benefits and Food Applications. Compr. Rev. Food Sci. Food Saf. 2019; 18(6): 2025–2046. PubMed Abstract \| Publisher Full Text\n\nYang Q-Q, Gan R-Y, Ge Y-Y, et al.: Polyphenols in Common Beans (Phaseolus vulgaris L.): Chemistry, Analysis, and Factors Affecting Composition. Compr. Rev. Food Sci. Food Saf. 2018; 17(6): 1518–1539. PubMed Abstract \| Publisher Full Text\n\nZeisel SH, da Costa KA : Choline: an essential nutrient for public health. Nutr. Rev. 2009; 67(11): 615–623. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "238828", "date": "07 Feb 2024", "name": "Carlos Eduardo Barão", "expertise": [ "Reviewer Expertise Emerging technologies Extraction Processes" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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\nThe article is interesting, but some points need to be improved Abstract: Name the bioactive compound of interest.\nKeywords: Use words other than the title.\nExtraction process: The conditions, even based on the work of Albuquerque (2017), seem random and do not offer similar conditions in each treatment to compare the results and define which would be better.\nIdentification of bioactive compounds by LC-HRMS: the type of solvent and concentration used in extraction can offer different profiles of phenolic compounds. It would be interesting to identify all extracts and not just the one that provides the highest yield.\nThe authors state that the extraction time influences the yield, so the same time should have been used in all conditions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11043", "date": "13 Apr 2024", "name": "Bambang Susilo", "role": "Author Response", "response": "Dear Reviewer, Thank you very much for your comments and suggestions. Here Author would like to respond to your comments. Abstract: Name the bioactive compound of interest. --> Choline and phenolic are the bioactive compounds of interest. Keywords: Use words other than the title. --> Thank you. Other relevant keywords i.e. extract yield, bioactive compounds, and solvents are added as replacements. Extraction process: The conditions, even based on the work of Albuquerque (2017), seem random and do not offer similar conditions in each treatment to compare the results and define which would be better. --> Our research does not compare Albuquerque's (2017) method with our method so we do not present data comparisons. However, we apply Albuquerque's (2017) method by making some modifications have need to be constructed, based on the conditions of the tools we used in the research laboratory. Identification of bioactive compounds by LC-HRMS: the type of solvent and concentration used in extraction can offer different profiles of phenolic compounds. It would be interesting to identify all extracts and not just the one that provides the highest yield. --> We do agree. However, based on the LC-HRMS results the dominant compounds detected are very good and relevant bioactive compounds as mentioned in Table 2. In addition, the highest yield extract is needed as the bioactive food ingredient, since even though it contains bioactive compounds, if the yield is low, it is less efficient as the bioactive food ingredient candidate. Accordingly, other extracts (by different solvents and concentrations) whose yield is not the highest do not need to be identified by LC-HRMS. The authors state that the extraction time influences the yield, so the same time should have been used in all conditions. --> Although the extraction time is different. However, all extraction techniques have used the optimum time based on citations from the previous research." } ] }, { "id": "261681", "date": "15 May 2024", "name": "Larry Chañi", "expertise": [ "Reviewer Expertise Extraction of bioactives by emerging green methods", "especially by supercritical fluids." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Potential transformation of seagrass (Syringodium isoetifolium) into a bioactive food ingredient using different extraction techniques\" presents interesting and updated scientific information about seagrass, it is a promising source of bioactive with potential application for the industry. The manuscript adequately describes the methods and results of the application of emerging technologies for the extraction of bioactive compounds from seagrass. It also details the quantification of total phenolic compounds and the identification of phenolics by LC-HRMS. The most abundant component was choline, this compound shows anti-inflammatory properties with potential for the pharmaceutical industry.\nMinor comments:\nThe manuscript requires minor corrections, which are detailed below:\nImprove the keywords, do not use very general words or words used in the title.\nI recommend changing “Extraction technique” and “Phytochemical” for the word’s “Green extraction methods” and “Marine plant phytochemicals”, respectively. Use the scientific name as a keyword as long as it is not used in the title. Other keyword alternatives are to include the name of the extraction methods.\n\nIs the work 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": "11585", "date": "29 Jun 2024", "name": "Bambang Susilo", "role": "Author Response", "response": "Dear Reviewer, Thank you kindly for your minor comments. Here with Author(s) response addressed for your comments and recommendations: 1. Keywords: The keywords are improved into Choline., Cytotoxicity., Marine plant., Microwave., Ultrasound. 2. Changing “Extraction technique” and “Phytochemical” words: Author(s) approve changing the “Extraction technique” and “Phytochemical” words into the “Green extraction methods” and “Marine plant phytochemicals” words, respectively, for changes the appropriate words in the entire manuscript." } ] } ]	1	https://f1000research.com/articles/12-1078
https://f1000research.com/articles/12-71/v1	18 Jan 23	{ "type": "Research Article", "title": "Anatomical variations of frontal sinus pneumatization: A computed tomography-based study", "authors": [ "Asma Sulaiman Al Hatmi", "Eiman Al Ajmi", "Halima Albalushi", "Meetham Al Lawati", "Srinivasa Rao Sirasanagandla", "Asma Sulaiman Al Hatmi", "Eiman Al Ajmi", "Halima Albalushi", "Meetham Al Lawati" ], "abstract": "Background: The pneumatization of the frontal sinus is variable between individuals, including monozygotic twins. The volumetric anatomic variants of the frontal sinus are classified into aplasia, hypoplasia, medium-sized, and hyperplasia. We aimed to study the frontal sinus morphology in Omani patients using computed tomography (CT) evaluations. Methods: Retrospectively, 1220 paranasal sinus CT scans from 610 patients investigated at Sultan Qaboos University Hospital, Oman, from January 2019 to December 2020 were reviewed. The frontal sinus morphology was classified according to the classification proposed by Guerram et al. The Chi-square test was used to determine the influence of sex. Results: With regard to the unilateral occurrence, the most prevalent frontal sinus category observed was medium-sized (13.3%), followed by hyperplasia (7.9%), hypoplasia (5.4%), and aplasia (2%) categories. Similarly, in bilateral occurrence, the most common frontal sinus category observed was medium-sized (53%), followed by hyperplasia (13.1%), hypoplasia (3.4%) and aplasia (2%) categories. Right and left frontal sinus aplasia were observed in 2.1% and 1.8% of cases, respectively. In terms of sex influence, the left unilateral (p<0.01) and the bilateral hypoplasia (p<0.05) were significantly higher in females. On the other hand, the left unilateral (p<0.01) and the bilateral hyperplasia (p<0.05) were higher in males. Conclusions: The baseline data of frontal sinus category frequencies reported in the present study is helpful in the diagnostic evaluation of sinusitis in the clinical setting. The preoperative recognition of frontal sinus types, particularly frontal sinus aplasia in multiplanar CT scans, is crucial to avoid unexpected complications while performing endoscopic sinus surgery.", "keywords": [ "Frontal sinus", "sinusitis", "hyperplasia", "aplasia", "surgery" ], "content": "Introduction\n\nFrontal sinuses are a pair of funnel-shaped pneumatic cavities situated in the squamous part of the temporal bone. Two frontal sinuses are separated by a bony septum, which is rarely located in the midline.1 Frontal sinuses emerge as an outgrowth in the region of the frontal recess of the nose in the fourth month of intrauterine life. At birth, they are rudimentary or absent. They begin to develop and become evident only after the second year of life. They grow at the age of seven or eight and mature after puberty.2,3 After 20 years of age, the size of the sinuses remains unchanged until the atrophic changes begin to appear due to advancing age.4 Frontal sinuses drain into the anterior part of the middle meatus of the nose through an ethmoid infundibulum or a frontonasal duct. The volume of the frontal sinus is highly variable between the two individuals. In adults, the mean size of the sinus is around ten cc and it may reach a maximum of 37 cc.5\n\nFrontal sinuses are rarely symmetrical as both sinuses develop individually.5 The pneumatization of the frontal sinus is known to be highly variable. The frontal sinus morphology varies from aplasia to hyperplasia within the same individual and even between monozygotic twins.6 The anatomical variations of the frontal sinus morphology have been reported in various populations worldwide.1,7–12 These studies have confirmed that climate and geography influence the frontal sinus morphology differences between the populations. In addition, few studies have demonstrated sexual dimorphism in frontal sinus anatomic variations.6,13–15 Frontal sinus anatomic variations are clinically important as they are closely associated with frontal sinusitis physiopathology, clinical presentation, development of complications and treatment.16–20 Furthermore, these variations and the unique morphology of the frontal sinus are helpful in the identification of subjects for forensics.14,21 Previously, few studies have proposed classifications for frontal sinus morphology based on two-dimensional and three-dimensional evaluations. Despite tremendous clinical significance, few recent papers have dealt with frontal sinus morphology, particularly in Middle Eastern populations. Hence, the objective of the present study was to assess the frontal sinus morphology in Omani patients using computed tomography (CT) according to the classification by Guerram et al.10\n\n\nMethods\n\nThe present study is a retrospective review of the electronic medical records database (TrakCare Unified Health Information System) at the Department of Radiology and Molecular Imaging, Sultan Qaboos University Hospital, Oman. All Omani patients aged ≥18 years referred for CT scan of the paranasal sinuses from January 2019 to December 2020 were included in the study. Patients with anterior skull base trauma, fibro-osseous lesions or significant motion artifacts that impaired the visualization of the frontal sinuses were excluded from the study. The present study obtained institutional ethical approval from the Medical Research Ethics Committee, Sultan Qaboos University.\n\nAll the scans were performed using a 64 multidetector CT scanner (Siemens Sensation 64) with the following parameters: 120 kVp, tube current modulation with reference mAs of 130 and 0.75-mm slice thickness. The Picture Archiving and Communication System (PACS) (Synapse PACS, FUJIFILM Worldwide, version 5.7.102) was used for screening the scans.\n\nWe evaluated right and left frontal sinuses from 610 patients’ CT scans, based on the standard method described by Guerram et al., to determine the prevalence of frontal sinus size categories.10 Using this method, frontal sinus size was categorized into four types, including aplasia, hypoplasia, medium-sized, and hyperplasia. To evaluate the frontal sinus categories, supraorbital and mid-orbital lines were generated on CT sections. The supraorbital line was a horizontal tangent connecting the superior margins of both orbits, while the mid-orbital line was a vertical line drawn at the midpoint of the distance between the medial and lateral borders of the orbit parallel to the mid-sagittal plane. Based on these lines, frontal sinus size categories were classified as follows: Aplasia: no pneumatization; Hypoplasia: minimal pneumatization under the supraorbital line; Medium-sized: pneumatization over the supraorbital line but medial to the mid-orbital line; Hyperplasia: lateral to the mid-orbital line (Figure 1). All the scans were reviewed by a single observer who is a board-certified radiologist. After screening, the data from each patient was recorded in the Microsoft Excel spreadsheet.\n\nSOL: supraorbital line. MOL: midorbital line. Image was prepared using biorender.com.\n\nThe statistical analysis was performed using SPSS software package (v.23) for Windows v24.0 (IBM Corp., Armonk, N.Y., USA). Descriptive statistics (e.g., frequency and percentage) were used to present the data. The sex difference was determined using the Chi-square test. A p-value <0.05 was considered statistically significant.\n\n\nResults\n\nIn the present study, the morphometry of 1220 frontal sinuses from 610 patients was recorded concerning the frequency of each type of frontal sinus. Among the study group, 314 were males (51.5%), and 296 (48.5%) were females, with a mean age of 43.1 ± 15.5 (SD) years. The unilateral and bilateral occurrence of each type of frontal sinus frequency was summarized in Tables 1 and 2. With regard to the unilateral occurrence, the most common frontal sinus category observed was medium-sized (13.3%), followed by hyperplasia (7.9%), hypoplasia (5.4%), and aplasia (2%). Similarly, in bilateral occurrence, the most common frontal sinus category observed was medium-sized (53%), followed by hyperplasia (13.1%), hypoplasia (3.4%) and aplasia (2%) categories, respectively. Right and left frontal sinus aplasia were observed in 2.1% and 1.8% of cases, respectively. The sex-wise distribution of frontal sinus categories was presented in Tables 1 and 2. There was a significant sex difference in the frequencies of left frontal sinus hypoplasia and hyperplasia categories (Table 1). Regarding the bilateral occurrence, a statistically significant sex difference was observed in the frequencies of hypoplasia and hyperplasia categories. The left unilateral (p<0.01) and the bilateral hypoplasia (p<0.05) were significantly higher in females than in males (Tables 1 and 2). On the other hand, the left unilateral (p<0.01) and the bilateral hyperplasia (p<0.05) were higher in males than in females (Tables 1 and 2). The representative images of CT scans showing frontal sinus categories are provided in Figure 2.\n\n# p<0.01.\n\n* p<0.05; Chi-square test. Values presented as number (%).\n\n# p<0.01.\n\n* p<0.05; Chi-square test. Values presented as number (%).\n\n\nDiscussion\n\nPreviously, to address the volumetric anatomic variants of the frontal sinus, three different studies have classified the frontal sinus morphology into four patterns, including aplasia, hypoplasia, medium-sized, and hyperplasia.10–12 These studies have used different parameters for classification. In the present study, we followed Guerram et al.’s classification to determine the morphology of the frontal sinuses.10 Similar to previous studies, in the present study, the medium-sized category of frontal sinus was the most common type of frontal sinus morphology.10,12,22 The values of medium-sized frontal sinus frequency observed in the present study are comparable with the frequency of 65.84% reported in a recent study by Ozdemir et al.22 In contrast, in a study by Yüksel Aslier et al., hyperplasia (44.5%) was the most common type, followed by medium-sized (37.2%), hypoplasia (14.2%) and aplasia (4.1%) categories.11 Similarly, in a study by Buller et al., following Guerram et al.’s morphologic classification, hyperplasia was found to be the most frequent sinus category (66%), followed by medium-sized (30.2%) and hypoplasia (3.8%) categories.23 However, no cases of aplasia were observed in this study.23 The study’s small sample size and inclusion criteria could possibly be the reason for these contrasting results. After aplasia, hypoplasia of the frontal sinuses is a rare morphology of the frontal sinuses. In studies by Yuksel Aslier et al. and Guerram et al., hypoplasia was observed in 14.2% and 9.5%, respectively.10,11 The frequency of hypoplasic frontal sinuses observed in the present study (8.7%) is comparable with Guerram et al. study findings.10 Regarding sex influence on frontal sinus categories, our study findings are comparable with the previous study by Guerram et al.10 In their study, hypoplasia was higher in females (13.7%) than males (5%). On the other hand, hyperplasia was higher in males (16.3%) than in females (7.5%).10\n\nAmong different frontal sinus morphology variations, the frontal aplasia type is well documented in different populations worldwide. In the existing literature, the reported frequency of bilateral frontal sinus aplasia greatly varies among populations worldwide. In a recent study on Saudi individuals, bilateral frontal sinus aplasia was found to be 3.3%.24 In Jordanian25 and Iranian26 individuals, prevalences of 4.2% and 8.3% were reported, respectively. A study from Turkey reported a low prevalence of 0.73%.7 Similarly, two studies on Indian subjects reported low frequencies of 2.05 and 2.5%, respectively.15,27 Contrary to these studies, high frequencies were reported in Northern Irish (10%) and Chinese individuals (16.6%).28,29 Surprisingly, unusually high frequencies of 43% and 40%, respectively, in Canadian Inuit males and females, and 25% and 36%, respectively, in Native Alaskans males and females, were observed.30,31 These highest frequencies were thought to be due to the influence of extremely cold climatic conditions.25 In our study, the frequency of bilateral frontal sinus aplasia was noted in 2% of cases. This frequency is close to that reported in the Indian population.15 Similar or comparable frequencies of frontal sinus aplasia in relative populations could be attributed to the ontogenic development of the frontal sinus.23 In most studies, bilateral frontal sinus aplasia is found to be more frequent in females than in males.1,7,32,33 In contrast, in Jordanian subjects, the frequency is higher in males than females.25 Similar to most of the studies, bilateral frontal aplasia is found to be more common in females than in males though it was not statistically significant.\n\nThe reported frequency of unilateral frontal sinus aplasia among different populations has varied between 0.8% and 12.7%. Higher frequencies of 12.7%, 10%, 6.5%, and 6.6% unilateral aplasia were reported in Chinese,29 Indian,15 Saudi,24 and Jordanian subjects,25 respectively. In contrast, low frequencies of 1.2%, 2%, and 2.5% were reported in Turkish,7 Northern Irish,28 and upper Rhine subjects,10 respectively. In Iranian subjects, unilateral aplasia was identified in 5.6% of cases.26 In Omani subjects, the recorded unilateral aplasia frequency was low and similar to Turkish subjects. Concerning the sex differences, in Indian,33 Saudi,24 and Turkish subjects,1 unilateral aplasia was more common in females. On the other hand, in Jordanian,25 Japanese13 and Iranian subjects,26 unilateral aplasia was more frequent in males. In the present study, unilateral aplasia was more frequent in females than in males though it was not statistically significant. With regard to laterality differences, most of the studies from Saudi Arabia,24 Japan,13 Turkey,7 Iran,26 and India33 have reported aplasia more frequently on the right side. In contrast, no laterality difference was observed in the present study.\n\nThe anatomy of the frontal sinus is the most complex compared to other paranasal sinuses. Due to its close relationship with the anterior cranial fossa and orbits, frontal sinusitis is considered a main source of orbital and cranial complications.34,35 Evidence from recent studies indicates that frontal sinus anatomical variations, particularly size and shape, are positively associated with the development of sinusitis.16–20 Another recent study reported a significant association between the frontal sinus type and frontal sinusitis frequency.22 In the same study, the prevalence of sinusitis in medium-sized and large sinuses was significantly higher than in small sinuses.22 Hence, the baseline data of frontal sinus types reported in the present study is helpful in the diagnostic evaluation of sinusitis in the clinical setting. The preoperative recognition of frontal sinus types, particularly frontal sinus aplasia in multiplanar CT scans, is crucial to avoid unexpected complications while performing endoscopic sinus surgery. For example, in endoscopic sinus surgery, opening a non-existent frontal sinus is a disastrous step.25 Furthermore, frontal sinus aplasia is known to increase the risk of having traumatic brain injuries.36 Hence, the frontal sinus morphology reported in the present study alert surgeons to rely on the preoperative radiological evaluation of the frontal sinus.\n\n\nConclusions\n\nIn our study, the most prevalent frontal sinus category was medium-sized, followed by hyperplasia, hypoplasia, and aplasia categories. The sex factor influenced the frequencies of hypoplasia and hyperplasia categories. The frequencies of unilateral and bilateral aplasia were low, and these values were comparable with values reported in Indian and Turkish populations. The baseline data of volumetric anatomic variations of frontal sinuses is crucial to minimize the risk factors associated with surgical procedures.", "appendix": "Data availability\n\nTo protect the patients’ privacy the present study data access was restricted. The anonymous raw data of the study showing the different types of frontal sinus morphology can be shared with readers and reviewers. To apply for access to the data, readers or reviewers can contact Dr. Srinivasa Rao Sirasanagandla (srinivasa@squ.edu.om). While applying for access, reader or reviewer should give a signed letter mentioning that they will not share the data with a third party and it will used only for academic purpose. The anonymous data will be provided in a password-protected file.\n\n\nReferences\n\nAydinlioglu A, Kavakli A, Erdem S: Absence of frontal sinus in Turkish individuals. Yonsei Med. J. 2003; 44: 215–218. 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PubMed Abstract \| Publisher Full Text\n\nVazquez A, Baredes S, Setzen M, et al.: Overview of frontal sinus pathology and management. Otolaryngol. Clin. N. Am. 2016; 49: 899–910. PubMed Abstract \| Publisher Full Text\n\nTezer MS, Tahamiler R, Canakcioglu S: Computed tomography findings in chronic rhinosinusitis patients with and without allergy. Asian Pac. J. Allergy Immunol. 2006; 24: 123–127. PubMed Abstract\n\nNatsis K, Karabatakis V, Tsikaras P, et al.: Frontal sinus anatomical variations with potential consequences for the orbit. Study on cadavers. Morphologie 2004; 88: 35–38. PubMed Abstract \| Publisher Full Text\n\nJohari HH, Mohamad I, Sachlin IS, et al.: A computed tomographic analysis of frontal recess cells in association with the development of frontal sinusitis. Auris Nasus Larynx 2018; 45: 1183–1190. PubMed Abstract \| Publisher Full Text\n\nAngélico FV Jr, Rapoport PB: Analysis of the agger nasi cell and frontal sinus ostium sizes using computed tomography of the paranasal sinuses. Braz. J. Otorhinolaryngol. 2013; 79: 285–292. PubMed Abstract \| Publisher Full Text\n\nKim DI, Lee UY, Park SO, et al.: Identification using frontal sinus by three-dimensional reconstruction from computed tomography. J. Forensic Sci. 2013; 58: 5–12. PubMed Abstract \| Publisher Full Text\n\nOzdemir M, Kavak RP, Öcal B, et al.: A novel anatomical classification of the frontal sinus: can it be useful in clinical approach to frontal sinusitis? Egypt. J. Otolaryngol. 2021; 37: 1–6. Publisher Full Text\n\nBuller J, Maus V, Grandoch A, et al.: Frontal Sinus Morphology: A Reliable Factor for Classification of Frontal Bone Fractures? J. Oral Maxillofac. Surg. 2018; 76: 2168.e1–2168.e7. PubMed Abstract \| Publisher Full Text\n\nAssiri KS, Alroqi AS: Frequency of the frontal sinus aplasia among Saudi Arabian population. A single-center retrospective case review. Saudi Med. J. 2021; 42(2): 228–231. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Balas HI, Nuseir A, Alzoubi F, et al.: Prevalence of Frontal Sinus Aplasia in Jordanian Individuals. J. Craniofac. Surg. 2020; 31(7): 2040–2042. PubMed Abstract \| Publisher Full Text\n\nDanesh-Sani SA, Bavandi R, Esmaili M: Frontal sinus agenesis using computed tomography. J. Craniofac. Surg. 2011; 22(6): e48–e51. Publisher Full Text\n\nJain A: Frontal sinus aplasia. Indian Streams Res. J. 2013; III: 1–8. Publisher Full Text\n\nCameriere R, Ferrante L, Molleson T, et al.: Frontal sinus accuracy in identification as measured by false positives in kin groups. J. Forensic Sci. 2008; 53: 1280–1282. PubMed Abstract \| Publisher Full Text\n\nTang JP, Hu DY, Jiang FH, et al.: Assessing forensic applications of the frontal sinus in a Chinese Han population. Forensic Sci. Int. 2009; 183: 104.e1–e3. PubMed Abstract \| Publisher Full Text\n\nKoertvelyessy T: Relationships between the frontal sinus and climatic conditions: a skeletal approach to cold adaptation. Am. J. Phys. Anthropol. 1972; 37: 161–172. PubMed Abstract \| Publisher Full Text\n\nHanson CL, Owsley DW: Frontal sinus size in Eskimo populations. Am. J. Phys. Anthropol. 1980; 53: 251–255. PubMed Abstract \| Publisher Full Text\n\nOzgursoy OB, Comert A, Yorulmaz I, et al.: Hidden unilateral agenesis of the frontal sinus: hu-man cadaver study of a potential surgical pitfall. Am. J. Otolaryngol. 2010; 31: 231–234. PubMed Abstract \| Publisher Full Text\n\nSheriff RM, Moideen CP: Incidence of frontal sinus aplasia in Indian population. Int. J. Otorhinolaryngol. Head Neck Surg. 2017; 3: 108.\n\nBetz CS, Issing W, Matschke J, et al.: Complications of acute frontal sinusitis: a retrospective study. Eur. Arch. Otorhinolaryngol. 2008; 265: 63–72. PubMed Abstract\n\nGoldberg AN, Oroszlan G, Anderson TD: Complications of frontal sinusitis and their management. Otolaryngol. Clin. N. Am. 2001; 34: 211–225. Publisher Full Text\n\nPajic SS, Antic S, Vukicevic AM, et al.: Trauma of the frontal region is influenced by the volume of frontal sinuses. A finite element study. Front. Physiol. 2017; 8: 493. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "160660", "date": "01 Mar 2023", "name": "Aspalilah Alias", "expertise": [ "Reviewer Expertise Anatomist", "geometric morphometric analysis", "shape analysis expert", "forensic anthropology", "forensic odontology and radiology research." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is accepted and ready for publication after the minor changes I stated below. Overall, this finding can help in clinical cases such as sinusitis and trauma and the forensic case for identification. I hope that more statistical analysis should be done in the future, for example, the traditional morphometric methods, such as measurement of length and the volume of the frontal sinus, and more advanced analysis of the shape, such as geometric morphometric methods and artificial intelligence research for the future. The parameter for the identification, for example, age determination, can also increase the manuscript's value. Please add the ethics approval number in the methodology part as you use the patient data for the research.\n\nThe comments for the manuscripts as below\n\nTitle - Suggested topic Anatomical variations of the frontal sinus: A computed tomography-based study.\n\nAbstract - Please change the paranasal sinus to the frontal sinus.\n\nIntroduction -The introduction is enough for introducing the paper. Initially, the authors explain the anatomy of the frontal sinus, link with the previous study, and relate with the clinical and forensic function of forensic sinuses.\n\nResult: Table 1 and Table 2 Gender association is significant on the left side for hypoplasia and hyperplasia.p<0.01.p<0.05\n\nPlease change gender to sex, while p<0.01 to p<0.01 and maintain p<0.05\n\nDiscussion - \"Two studies on Indian subjects reported low frequencies of 2.05 and 2.5%, respectively.\"\nPlease add percentage 2.05%\n\nDiscussion - Please add the forensic contribution for identification from the frontal sinus.\n\nConclusion - Please add also the forensic contribution for identification from frontal sinus.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10107", "date": "31 Aug 2023", "name": "Dr Srinivasa Rao Sirasanagandla", "role": "Author Response", "response": "Title - Suggested topic Anatomical variations of the frontal sinus: A computed tomography-based study. Response: The title of the study was changed as suggested by the reviewer. Abstract - Please change the paranasal sinus to the frontal sinus. Response: The correction was made in the abstract. Introduction -The introduction is enough for introducing the paper. Initially, the authors explain the anatomy of the frontal sinus, link with the previous study, and relate with the clinical and forensic function of forensic sinuses. Response: Thank you for your valuable comments Result: Table 1 and Table 2 Gender association is significant on the left side for hypoplasia and hyperplasia.p<0.01.p<0.05 Please change gender to sex, while p<0.01 to p<0.01 and maintain p<0.05 Response: The suggested changes were made in the results section and highlighted with track changes. Discussion - \"Two studies on Indian subjects reported low frequencies of 2.05 and 2.5%, respectively.\" Please add percentage 2.05% Response: The correction was made. Discussion - Please add the forensic contribution for identification from the frontal sinus. Response: The importance of frontal sinus for the forensic investigation was added in the discussion and highlighted with the track changes. Conclusion - Please add also the forensic contribution for identification from frontal sinus. Response: The importance of frontal sinus for the forensic investigation was added in the conclusion and highlighted with the track changes." } ] }, { "id": "172540", "date": "16 Aug 2023", "name": "Ka Suprasanna", "expertise": [ "Reviewer Expertise Neuroradiology", "Head and neck Radiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 an interesting study regarding variations in frontal sinus pneumatisations. These are the various observations made during the review\nMany authors have previously studied the pneumatisation of paranasal sinuses. What this study adds to the existing knowledge needs to be highlighted.\n\nThe authors have extensively discussed the variations in pneumatisations in various studies and in various ethnic groups. It would be more clear, if this data could be presented in a tabulated form.\n\nCrista galli pneumatisation extending from the frontal sinus has been previously studied. How many of the cases in this study had crista galli pneumatisation\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10108", "date": "31 Aug 2023", "name": "Dr Srinivasa Rao Sirasanagandla", "role": "Author Response", "response": "Many authors have previously studied the pneumatisation of paranasal sinuses. What this study adds to the existing knowledge needs to be highlighted. Response: The importance of the study was highlighted in the discussion section. The authors have extensively discussed the variations in pneumatisations in various studies and in various ethnic groups. It would be more clear, if this data could be presented in a tabulated form. Response: Table 3 summarizing the prevalence of frontal sinus variations was added in the discussion. Crista galli pneumatisation extending from the frontal sinus has been previously studied. How many of the cases in this study had crista galli pneumatization. Response: Our study aim was to record the volumetric anatomic variants of the frontal sinus according to the Guerram et al.’s classification. Hence, crista galli pneumatization was not evaluated in this study." } ] } ]	1	https://f1000research.com/articles/12-71
https://f1000research.com/articles/12-391/v1	13 Apr 23	{ "type": "Data Note", "title": "The identification of high-performing antibodies for Superoxide dismutase [Cu-Zn] 1 (SOD1) for use in Western blot, immunoprecipitation, and immunofluorescence", "authors": [ "Riham Ayoubi", "Walaa Alshafie", "Zhipeng You", "Kathleen Southern", "Peter S. McPherson", "Carl Laflamme", "Riham Ayoubi", "Walaa Alshafie", "Zhipeng You", "Kathleen Southern", "Peter S. McPherson" ], "abstract": "Superoxide dismutase [Cu-Zn] 1 (SOD1), is an antioxidant enzyme encoded by the gene SOD1, responsible for regulating oxidative stress levels by sequestering free radicals. Identified as the first gene with mutations in Amyotrophic lateral sclerosis (ALS), SOD1 is a determinant for studying diseases of aging and neurodegeneration. With guidance on well-characterized anti-SOD1 antibodies, the reproducibility of SOD1 research would be enhanced. In this study, we characterized eleven SOD1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.", "keywords": [ "Uniprot ID P00441", "SOD1", "Superoxide dismutase [Cu-Zn]", "Superoxide dismutase 1", "antibody characterization", "antibody validation", "Western blot", "immunoprecipitation", "immunofluorescence" ], "content": "Introduction\n\nSuperoxide dismutase [Cu/Zn] 1 (SOD1) is an essential enzyme that protects the body against oxidative stress by acting as the first line of defense against reactive oxidative species.1,2 Largely cytosolic but also found in the mitochondrial intermembrane space, SOD1 is a 153 amino acid protein functioning as a homodimer to bind copper and zinc in order to carry out its role in scavenging free radicals.3,4\n\nSOD1 was the first gene in which its mutations were identified in ALS over 30 years ago, predicting it to be a causative factor in motor neuron degeneration.5 A hallmark of SOD1-associated ALS is the misfolding and aggregation of SOD1 into neurotoxic species induced by gene mutations.6 The disease mechanism in which this occurs remains unknown.6 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.\n\nHere, we compared the performance of a range of commercially available antibodies for SOD1 and validated several antibodies for Western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of SOD1 properties and function.\n\n\nResults and discussion\n\nOur standard protocol involved comparing readouts from wild-type (WT) and knockout (KO) cells.7–9 To identify a cell line that expressed adequate levels of SOD1 protein to provide sufficient signal to noise, we examined public proteomics databases, namely PaxDB10 and DepMap.11 HeLa was identified as a suitable cell line and thus HeLa was modified with CRISPR/Cas9 to knockout the corresponding SOD1 gene (Table 1).\n\nFor Western blot experiments, we resolved proteins from WT and SOD1 KO cell extracts and probed them side-by-side with all antibodies in parallel8,9 (Figure 1). SOD1 is an common essential gene12 and the remaining SOD1 expression in the KO lysate could be detected with various antibodies.\n\nLysates of HeLa (WT and SOD1 KO) were prepared, and 20 μg of protein were processed for Western blot with the indicated SOD1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies ab51254 and 10269-1-AP, which were titrated to 1/15000 and 1/1000, respectively, as the signals were too weak when following the supplier’s recommendations. Antibody dilution used: ab252426 at 1/1000, ab51254 at 1/15000, ab79390 at 1/10000, MAB3418* at 1/1000, GTX100554 at 1/1000, GTX100659 at 1/1000, 10269-1-AP at 1/1000, 67480-1-Ig* at 1/10000, 702783 at 1/200, 711818 at 1/200, MA1-105* at 1/1000. Predicted band size: 16 kDa. = monoclonal antibody, = recombinant antibody.\n\nFor immunoprecipitation experiments, we used the antibodies to immunopurify SOD1 from HeLa cell extracts. The performance of each antibody was evaluated by detecting the SOD1 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates8,9 (Figure 2).\n\nHeLa lysates were prepared, and IP was performed using 2.0 μg of the indicated SOD1 antibodies pre-coupled to protein G or protein A Sepharose beads. Samples were washed and processed for Western blot with the indicated SOD1 antibody. For Western blot, MAB3418 was used at 1/1000, 67480-1-Ig* at 1/1000, 10269-1-AP at 1/2000, MA1-105* at 1/2000, ab79390** at 1/15000 and GTX100554 at 1/2000. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitate; = monoclonal antibody; = recombinant antibody.\n\nFor immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.13 In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).\n\nHeLa WT and SOD1 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated SOD1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody. Acquisition of the green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the red (grayscale) channel are shown. WT and KO cells are outlined with yellow and magenta dashed line, respectively. Antibody dilutions were chosen according to the recommendations of the antibody supplier. An exception was given for antibody 10269-1-AP, which was titrated to 1/500, as the signal was too weak when following the supplier’s recommendations. When the concentration was not indicated by the supplier, which was the case for ab252426, GTX100554, GTX100659, 702783, 711818* and MA1-105, we tested antibodies at 1/200, 1/500 or 1/1000. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab252426* at 1/500, ab51254 at 1/200, ab79390 at 1/200, MAB3418* at 1/500, GTX100554 at 1/200, GTX100659 at 1/500, 10269-1-AP at 1/500, 67480-1-Ig* at 1/500, 702783 at 1/500, 711818 at 1/500, MA1-105* at 1/1000. Bars = 10 μm. = monoclonal antibody; = recombinant antibody.\n\nIn conclusion, we have screened SOD1 commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions. The underlying data can be found on Zenodo.14,15\n\n\nMethods\n\nAll SOD1 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.16 Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat. number A21424 and A21429).\n\nWb=Western blot; IF= immunofluorescence; IP=immunoprecipitation;\n\n = monoclonal antibody;\n\n** = recombinant antibody.\n\nHeLa SOD1 KO clone was generated with low passage cells using an open-access protocol available on Zenodo. Two guide RNAs were used to introduce a STOP codon in the SOD1 gene (sequence guide 1: CCGTTGCAGTCCTCGGAACC, sequence guide 2: GCGCGGGGGGACGAGCGGGT).\n\nBoth HeLa WT and SOD1 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.17 Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065), 100 IU penicillin and 100 μg/mL streptomycin (Wisent cat. number 450201).\n\nWestern blots were performed as described in our standard operating procedure.18 HeLa WT and SOD1 KO were collected in RIPA buffer (50 mM Tris-HCl pH 8.0, 150mM NaCl, 1.0 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1x protease inhibitor cocktail mix (MilliporeSigma, cat. number 78429). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 x g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western blot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.\n\nWestern blots were performed with large 8-16% polyacrylamide gels and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. number BP103-10) which is scanned to show together with individual Western blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095) in TBS with 0,1% Tween 20 (TBST) (Cell Signaling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/mL in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL (Thermo Fisher Scientific, cat. number 32106) prior to detection with the HyBlot CL autoradiography films (Denville, cat. number 1159T41).\n\nImmunoprecipitation was performed as described in our standard operating procedure.19 Antibody-bead conjugates were prepared by 2.0 μg of antibody to 500 μL of phosphate-buffered saline (PBS) (Wisent, cat. number 311-010-CL) with 0,01% triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) in a 1.5 mL microcentrifuge tube, together with 30 μL of protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) Sepharose beads. Tubes were rocked overnight at 4°C followed by two washes to remove unbound antibodies.\n\nHeLa WT were collected in HEPES lysis buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor. Lysates were rocked 30 min at 4°C and spun at 110,000 x g for 15 min at 4°C. One mL aliquots at 0.5 mg/mL of lysate were incubated with an antibody-bead conjugate for ~2 hours at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of HEPES lysis buffer and processed for SDS-PAGE and Western blot on 8-16% polyacrylamide gels, as described above. Prot-A:HRP (MilliporeSigma, cat. number P8651) and VeriBlot for IP Detection Reagent HRP (Abcam, cat. number ab131366) were used as secondary detection systems for an experiment where a rabbit antibody was used for both immunoprecipitation and its corresponding Western blot. Similarly, anti- mouse IgG for IP HRP (Abcam, cat. number ab131368) was used for an experiment where a mouse antibody was used for immunoprecipitation and it’s corresponding Western blot.\n\nImmunofluorescence was performed as described in our standard operating procedure.8,9,13 HeLa WT and SOD1 KO were labelled with a green and a far-red fluorescence dye, respectively. The fluorescent dyes used are from Thermo Fisher Scientific (cat. number C2925 and C34565). WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator at 37 oC, 5% CO2. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10ml) in PBS for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0,1% Triton X-100 for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary SOD1 antibodies overnight at 4 °C. Cells were then washed 3 × 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 hr at room temperature. Cells were washed 3 × 10 min with IF buffer and once with PBS. Coverslips were mounted on a microscopic slide using fluorescence mounting media (DAKO).\n\nImaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apo 40x oil objective (NA = 1.40). Analysis was done using the Zen navigation software (Zeiss). All cell images represent a single focal plane. Figures were assembled with Adobe Photoshop (version 24.1.2) to adjust contrast then assembled with Adobe Illustrator (version 27.3.1).", "appendix": "Data availability\n\nZenodo: Antibody Characterization Report for Superoxide Dismutase [Cu-Zn] (SOD1), https://doi.org/10.5281/zenodo.5061103. 14\n\nZenodo: Dataset for the Superoxide Dismutase 1 Cu-Zn (SOD1) antibody screening study, https://doi.org/10.5281/zenodo.7709943. 15\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. Members of the group can be found below.\n\nNeuroSGC/YCharOS/EDDU collaborative group: Riham Ayoubi, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina, Kathleen Southern and Zhipeng You\n\nAn earlier version of this of this article can be found on Zenodo (doi:10.5281/zenodo.5061103).\n\n\nReferences\n\nKangralkar V, Patil SD, Bandivadekar R: Oxidative stress and diabetes: a review. Int. J. Pharm. Appl. 2010; 1(1): 38–45.\n\nLandis GN, Tower J: Superoxide dismutase evolution and life span regulation. Mech. Ageing Dev. 2005; 126(3): 365–379. PubMed Abstract \| Publisher Full Text\n\nKim J, Lee H, Lee JH, et al.: Dimerization, oligomerization, and aggregation of human amyotrophic lateral sclerosis copper/zinc superoxide dismutase 1 protein mutant forms in live cells. J. Biol. Chem. 2014; 289(21): 15094–15103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOkado-Matsumoto A, Fridovich I: Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria. J. Biol. Chem. 2001; 276(42): 38388–38393. PubMed Abstract \| Publisher Full Text\n\nRosen DR: Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993; 364(6435): 362. Publisher Full Text\n\nMcAlary L, Aquilina JA, Yerbury JJ: Susceptibility of Mutant SOD1 to Form a Destabilized Monomer Predicts Cellular Aggregation and Toxicity but Not in vitro Aggregation Propensity. Front. Neurosci. 2016; 10: 499. PubMed Abstract\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nAlshafie W, Ayoubi R, Fotouhi M, et al.: The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 12: 172. Publisher Full Text\n\nWang M, Herrmann CJ, Simonovic M, et al.: Version 4.0 of PaxDb: Protein abundance data, integrated across model organisms, tissues, and cell-lines. Proteomics. 2015; 15(18): 3163–3168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNusinow DP, Szpyt J, Ghandi M, et al.: Quantitative Proteomics of the Cancer Cell Line Encyclopedia. Cell. 2020; 180(2): 387–402.e16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDepMap, Broad: DepMap 19Q3 Public ed.2019.\n\nAlshafie W, McPherson P, Laflamme C: Antibody screening by Immunofluorescence.2021.\n\nAyoubi R, Alshafie W, You Z, et al.: Antibody Characterization Report for Superoxide dismutase [Cu/Zn] (SOD1).2021.\n\nLaflamme C: Dataset for Superoxide dismutase 1Cu/Zn (SOD1) antibody screening study. [Data set]. Zenodo. 2023.\n\nBandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51(D1): D358–D367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021.\n\nAyoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021." }	[ { "id": "186895", "date": "18 Aug 2023", "name": "Nishant N Vaikath", "expertise": [ "Reviewer Expertise Neuroscience", "Antibody development", "Antibody engineering", "Nanobody" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Ayoubi et al, have characterized commercially available SOD1 antibodies for use in biochemical techniques, including Western Blotting, Immunoprecipitation, and Immunofluorescence, utilizing lysates from knockout cell lines and isogenic parental controls. The aim was to identify high-performing antibodies suitable for specific applications. While the authors have screened and compared the antibodies using different applications, there are some areas that require improvement, as outlined below:\nQuantification of HeLa lysates: The authors need to provide information on how the HeLa lysates were quantified. Adding details about the quantification method will enhance the reroducibility and accuracy of the results.\n\nElaboration of the results section: The results section needs to be more detailed and informative. Currently, it lacks clarity regarding the outcomes for each technique and the identification of the best-suited antibodies for each application.\n\nTable of tested antibodies: To facilitate better understanding for readers, the authors should consider providing a table that lists the antibodies tested along with the techniques they were evaluated in. This table could highlight which antibodies performed best for each technique, making it easier for researchers to identify suitable antibodies for their experiments.\nBy addressing these minor comments and enhancing the results section, the study's findings will become more accessible and beneficial to the scientific community.\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": "10128", "date": "19 Oct 2023", "name": "Kathleen Southern", "role": "Author Response", "response": "Thank you to Nishant N Vaikath for reviewing this article and providing a peer-review report. To respond to your first suggestion, we have added a short description of the quantification method used. Please review the Western Blot methods section of the second version of the article we have uploaded. As for your second and third points, YCharOs does not engage in result analysis nor do we offer explicit antibody recommendations. The primary goal of our initiative is to deliver high-quality antibody validation data to the scientific community. We have found that, for the most part, scientists viewing our articles have the expertise to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Recognizing the potential ambiguity of this point within the article, we have taken proactive measures to rectify the situation. As such, a new paragraph has been added to the Results and Discussion section, explaining our reasonings as to why we do not recommend or score the antibodies tested. Thank you again for your suggestions, we always appreciate feedback on how to improve the reproducibility of our data. After reviewing the second version of the article, newly submitted, we hope that you chose to reconsider your approval status." } ] } ]	1	https://f1000research.com/articles/12-391
https://f1000research.com/articles/12-97/v1	26 Jan 23	{ "type": "Research Article", "title": "Islamic legal perspectives on digital currencies and how they apply to Jordanian legislation", "authors": [ "Nasir Albalawee", "Amjed S. Al Fahoum", "Amjed S. Al Fahoum" ], "abstract": "Background: The industrial transformation requires a speedy shift to financial digitization. One of the needs for financial digitalization in the study of Islamic contracts and Islamic business law is the use of digital platforms with digital currencies. Regarding the merits and downsides of its Sharia restrictions and its halal certification, which is currently under discussion, digital currencies and perks have generated controversy in Jordan and other Islamic countries. Methods: This study intends to analyze the legal foundations of digital currency from Jordanian and Islamic legal perspectives. The descriptive-qualitative research approach was utilized, and data collection processes included documentation and a literature review. All legal possibilities that may be drawn from Islamic law in order to investigate the legality of digital currencies are explored further and used to obtain the conclusions of this study. Results: A review of Sharia reasons and consideration for the wellbeing of the people suggests that digital currencies in their current form are incompatible with it and must adhere to the stipulations of Islamic finance. Therefore, digital currencies are unsuitable as a store of value or wealth due to their erratic swings, lack of purchasing power, and instant responsiveness to any technical problem, technical penetration, or official circumstance. Due to market instability, digital currencies can't be utilized to defer payments, settle debts, or repay loans. Conclusions: Digital currencies are speculative; not real money. Most of those who have this money are speculators seeking a quick payoff. Sharia views digital currency trading as gambling due to its high degree of volatility. Jordan's government should regulate digital currency use to meet demand. Digital currencies must be addressed under e-commerce laws.", "keywords": [ "Digital Currencies", "Bitcoin", "Digital Representation", "The Physical Presence of Currency", "Functions of Money", "Jordan Law." ], "content": "Introduction\n\nThe most important recent rapid development in the areas of communication, IT, banking, and business is the expansion of payment options and the rise of electronic transfer. The design of a new electronic payment system could have a high effect on privacy issues and criminal behavior related to money transfers. Therefore, a new payment system should ideally support these two conflicting objectives.1 In 1983, a paper called “Blind Signatures for Untraceable Payments” was published by David Chaum, a cryptographer who came up with the idea of encrypted payment system and started the company.1 In 1990, a company called DigiCash made eCash, which was the first cryptocurrency. Therefore, cryptocurrencies existed before Bitcoin, but most people only paid little attention to them a few years after 2009. Bitcoin, the well known digital currency, appeared in 2009 and marked the beginning of the electronic currency era.2 Unlike traditional currencies, Bitcoin has no physical form, is not issued by central banks, and is not regulated by any government agency. Despite the technical and legal challenges that surround electronic currencies and the legality or illegality of dealing with them, there is no doubt that the emergence of electronic currencies has received a great deal of interest.2 Even though it doesn't fundamentally address the demands of the time, its attributes have made it a huge hit with its distributors.3\n\nAccordingly, this paper will first introduce the notion of digital currencies, before moving on to examine the legal norms governing digital currencies from an Islamic legal perspective. Electronic currencies are quite popular, as demonstrated by their widespread adoption.2,3 Bitcoin is less expensive for retailers than credit cards, but these fees could increase with less storage capacity. Along with the technical difficulties, there are concerns about fraud and privacy. A cryptocurrency's purpose is to protect its users' privacy, but the Bitcoin identities created randomly need to be more secure.4 Bitcoin was the first widespread electronic currency where money could be made and traded without a third party. Due to the popularity of digital currencies, Jordanian law must deal with its most important problems. Second, the variety of geopolitical conditions worldwide increases the demand for it. Jordanian laws linked to this study motivate the adoption of new regulations that figure out the issues with digital currencies. Also, the results and suggestions proposed for the new Jordanian law and how it will deal with Sharia would increase trust and confidence in digital currency for Jordanians and Muslims everywhere.\n\nThe paper will examine the issue of the Jordanian legal system of digital currencies according to Islamic jurisprudence and law in two sections addressing, respectively, the notion of digital currencies and the legal regulations governing digital currencies. The risks of using digital currencies in violation of Jordanian law and the consequences of legislative gaps in the regulation of digital currency-specific rules are discussed in this article. Furthermore, the following matters will explain how it functions, its distinctive traits, and how the law interacts with digital money, shedding light and clarifying the repercussions of “digital currencies” when correctly described. Finally, the best ways to use digital currencies from an Islamic legal perspective will be discussed, as well as whether or not a new legal framework for digital money exists as a result of current legislation.\n\nTo examine the legalization methodology of digital currency, approaches such as explaining, analyzing, and comparing are employed. First, the research problem is defined, described, and discussed. Furthermore, jurisprudence opinions and judicial jurisprudence are used to assess the legal documents that govern the subject of the inquiry. In conclusion, the comparative method is applied to determine if digital currency can be legalized based on comparing national, Islamic, and international laws. The paper is structured as follows: first, digital currencies are introduced, ideas are explained, and their legal status is discussed. The second section explains the laws regulating digital currencies by comparing the views of national legislation and Islamic jurisprudence on dealing with digital currency. Finally, both results and conclusions will follow.\n\n\nMethods\n\nBitcoin has been described as a decentralized virtual currency. Virtual currencies, such as bitcoins, are both a form of money and a payment system. However, being a decentralized system, there is no central issuer, authority, or register-keeper.5 Bitcoin is unique, not because it is a virtual currency, but because it is a proof of concept for a decentralized, non-issued electronic currency.5 The regulation of virtual currencies is at a very early stage.5 Most regulatory systems need to be made to work well with this kind of payment system.5 Nevertheless, building and keeping trust is vital to regulating new payment services and getting people to use them.3–5 Most agree that proper regulation is critical for encouraging people to use new payment methods such as mobile banking and payments.5 Side by side, Jordanian law still needs to develop modern laws to deal with digital currencies because they are still relatively new and depend on advanced technologies, which makes it hard for lawmakers to understand them or figure out their secrets. Also, Jordan does not have laws that directly deal with digital currencies.6 The lack of laws means that a legal framework needs to be made to deal with these currencies in a way that goes within Islamic law and is in line with Jordan's laws. This research adds to the literature on digital currency by looking at how it works and giving a clear plan for how it should be regulated. Also, it will show how Islamic legal principles can be incorporated into future or current Jordanian regulations about cryptocurrencies.\n\nMoney is a cultural norm that helps sustain economic and social systems. Various exchange systems have emerged for centuries, including barter, precious metals, fiat currency, and, most recently, a distributed digital currency based on blockchain technology. As cryptocurrencies and stablecoins have become more popular, the world’s central banks have realized that they need to provide an alternative—or let the future of money pass them—by introducing virtual money backed and issued by a central bank called central bank digital currency (CBDC).7 Central banks worldwide would develop a global CBDC standard that is accepted everywhere. CBDC's potential implications on monetary policy and liquidity, as well as its technological and economic feasibility, have been the subject of much research.8,9 To do so, Omar, M. N applied the “maqsid al-shar'ah” paradigm to CBDC and uses it to explain a central Islamic principle and discuss its ethical implications.10 The research results produce an ethical ecosystem that can be used to evaluate the potential of CBDC with specific characteristics and features. Such a moral environment can also be realized by basing the digital currency system's compliance elements on the Islamic monetary framework. When used in the financial sector, technology can be used to create innovative new services, such as FinTech. There is much talk about financial technology these days. Muslim nations are fertile ground for developing Islamic banking and financial technology.11 FinTech is an industry buzzword that uses technology to improve financial services. Due to the widespread use of mobile and smartphone technology, Islamic banking and FinTech will do well in Muslim countries.12,13 However, these opportunities are currently limited because they need to be protected by a legal framework. Islamic FinTech startups struggle with regulation and Sharia FinTech research. Islamic FinTech must keep up with traditional financial developments, maintain stability, and prevent fraudulent trade.11–14 For electronic transactions to grow, many groups must work together, including the central bank, public policy, international and multilateral institutions, and Islamic banking itself (through the regulatory and supervision functions).\n\nDigital currency is defined as “currencies that do not have a tangible physical entity or physical presence and are traded via the Internet and are not subject to control or control by a central bank or because it operates outside the traditional monetary system, it is also referred to as virtual money”.15 It is a digital representation of monetary value issued by entities other than the central bank of Jordan and credit institutions, and its value is generated by the voluntary acceptance of this currency.16 Therefore, in Al-Bahouth, A. and later Al-Najjar, A., they characterized digital currency as a nonexistent currency that is essentially dependent on encryption. It is a decentralized currency that is not issued by a central bank, administered by any government, or tied to any local or global currency. It is electronically mined and manufactured by computers, and it is used and shared through the Internet.15,16\n\nBitcoin, a new monetary system for electronic payment, is without a doubt the nucleus of the emergence of digital currencies. This currency is based on encryption between the two parties and is built on an anonymous system of electronic transactions, with the aim of moving away from the centralization of major banks, as they are not monitored by different types of banks and bodies and are not subject to bank laws.17 The development of Bitcoin adds to our knowledge of monetary systems. The ongoing debate about how to pay for it demonstrates the value of this contribution. According to Bergstra, J.A. and Weijland, P., Bitcoin is categorized as a highly adaptable money-like informational commodity (MLIC), hence it is unnecessary to decide in advance whether or not it is a debt. By this definition, Bitcoin can start and finish its life as a non-money, with a period of “true” moneyness (as opposed to \"money-likeness\") in the middle.18 Innovations in microfinance distribution and repayment technology for the Islamic banking model have inspired research into a commercial bank-backed initiative to bring mobile banking to Malaysia's Islamic microfinance institutions.19 From the client's perspective, technological applications in Islamic microfinance organizations' payment systems and repayments present challenges.19\n\nTo investigate the relationship between financial inclusion and Islamic financial services in Muslim countries, Zulkhibri M., took a qualitative approach that has shown there have been improvements in the financial infrastructure of many Muslim countries over the past few decades, but this has not reached most of the population.20 Only 27% of people and businesses in Muslim countries have access to formal financial services, well below the average of 51% in emerging economies. Disadvantages include money, time, distance, paperwork, distrust, and religious beliefs.20 Although it represents only 0.5% of global microfinance, Islamic microfinance is small and insignificant since it does not use a cost-effective service paradigm. This research suggests that 40 million people currently excluded from the formal financial system due to their religion, can be included if Islamic wealth redistribution techniques such as awqaf, qard-al-hassan, sadaqa, and zakah are implemented.20 The Islamic financial services industry has a long way to go in many Muslim countries due to its small size and inadequate infrastructure.21 Crypto assets such as Bitcoin (digital money) are intriguing in Indonesia, because they have the potential to affect the global economy. Today, trade is conducted primarily via the Internet (digitalization).20 There needs to be regulatory certainty for crypto investments in Indonesia. In Jubaedah et al., they used Islamic law and creed philosophy principles to examine crypto assets held for trading purposes in Indonesia. Information on laws, government regulations, the Fatwa DSN MUI, and the Islamic tenets of creed, witness, and shahadah is culled from scholarly library collections for the study.21 This rule is only in effect for those who are open to and able to use cryptographic assets like Bitcoin.21 In Indonesia as well as Jordan, it is against the law to invest in bitcoin.6,21 The use of cryptocurrency as a medium of online exchange represents a novel and sustainable contribution to the growth of Indonesia's economy. Particular guidelines are required for the use of cryptography.21\n\nThere are about 1300 other cryptocurrencies on the digital currency market, such as Ethereum, Ripple, NEM, and LitCoin. On the other hand, Bitcoins are the most sought-after cryptocurrency on the market.22 The fact that it cannot be traded like other electronic currencies has made investors curious about it.16 In general, understanding money means understanding objects approved by the community as an intermediary tool for conducting exchanges or trade. What is meant by “approved” in this definition is that there is an agreement among community members to use one or several objects as an intermediary tool in exchange activities. Bitcoin, also known as “BTC,” is a digital currency not issued by any institutions, organizations, or governments. Bitcoin utilizes a peer-to-peer network as a distribution medium using advanced cryptographic protocols.23\n\nUnderstanding money's role is essential for establishing whether or not digital currency may be considered legal tender. In forms such as paper money, money is a means of trade and a measure of the values of products and services; therefore, it has buying power.24 Maurer delved into the burgeoning topic of “mobile money,” or value transfer and storage systems that are facilitated by mobile phones and are often hailed as a “signal intervention” to increase financial inclusion and bank the “unbanked” in developing countries. He discusses how economic techniques and social narratives about markets—specifically, narratives about the opportunities for profit and financial inclusion in the “payments space”—format a consumer market for mobile money and focuses on the stories that circulate in the emerging network of expertise that is calling “mobile money” into being. More importantly, he speculated on the possibility of a new form of money by asking if consumers' usage of mobile money and airtime as currency.25 In conclusion, services may be used as a kind of payment, a means of saving, and a means of protecting one's capital.\n\nBased on the precedent set by the Faqih (Sharia jurisprudence), it is clear that virtual currencies cannot be considered legal tender or perform the functions traditionally associated with money.26 The fact that digital currency only exists in cyberspace precludes it from being utilized as payment for physical goods and services.27,28 Money (also referred to as the “money supply”) is anything that is created and commonly accepted as payment for goods or debt repayment.29 Economists describe money as the frequently accepted (or generally accepted) medium of exchange.30 The argument that bitcoin is not money is predicated on the notion that money serves as a medium of trade, a store of value, and a unit of account. Bitcoin partially satisfies the first condition.31 Money is efficient and effective if it is acceptable, divisible, homogeneous in value, durable, transportable, uncommon, and stable.32 Acceptable might be interpreted to suggest that money must have intrinsic value and be desired for its own sake. Due to the lack of intrinsic value of fiat currency, acceptance standards remain difficult. The second need is that money must be easily divisible into small quantities, allowing individuals to acquire goods and services at any price. For money to be easily divided, it must be uniform or homogenous. Durability, the final condition, requires that currency be durable, not easily destroyed, and portable. “Must be scarce” signifies that money must be relatively difficult to acquire or scarce, and its value must be relatively steady through time.33 Virtual currencies have their own units of account, cannot be denominated in fiat currencies, and are convertible to variable degrees.34\n\nThe value of digital currencies is not an independent standard; it requires a fiat currency equivalent. Referring to Adam, cryptocurrency can still be used as a medium of exchange.35 Regarding legal tender, the government declares something to be a valid form of payment, and it must be issued by a central authority.36 Bitcoin is created by no commercial legal entity, limiting its use to people who make payments.37 To be referred to as “money,” an asset must fulfill the following functions: (a) average payment, (b) unit of account, and (c) store of value.38 Bitcoin is still a long way from supplanting fiat currency as the predominant form of legal cash. Bitcoin unit accounts are ineffective because their prices are too high for retail transactions and their splitting fees are excessive. Frequently, the bitcoin pricing of retail goods requires a significant number of left-sided zeros, making it impossible for consumers to compare prices across products and services.31 Regarding the function of a store of value, the extreme price volatility of bitcoin is also a barrier to the steady storage of wealth.39 In recent years, the price of Bitcoin, which can be compared to a fundamentally useless computer entry, has increased dramatically. So, cryptocurrencies are known for their high volatility, lack of buying power, and price changes in response to any technical or technological event. Moreover, they suffer from technical penetration or official legalization. Digital currencies are also unsuitable for delayed payments and cannot be used to pay off debts or loans because they are unstable and change with the market. As a result, they are unfit for use as a store of value or wealth.27,28 Therefore, researchers have found that digital currencies are not the same as conventional money and cannot even replace it. This is because of the aforementioned significant variances. Additionally, digital currencies are not commonly acknowledged or utilized. As a result of the negative publicity it has received, several nations have also rejected cryptocurrencies.40 For instance, the FBI closed the Silk Road case, which received considerable negative coverage.41 The Central Bank of China stated in December 2013 that virtual currency has no value and is not adequately protected by law.40 Cryptocurrency is not an exception to Indonesia's monetary policy, which prohibits using any medium of exchange other than fiat money authorized for trade and payment.42 Due to negative headlines, speculation, and other hazards, cryptocurrencies, and bitcoin, in particular, are viewed as investments rather than currency.43 Many countries do not use cryptocurrency because its value changes quickly; some have even made it illegal to trade it in their monetary systems and with their bankers.44\n\nLegislative provisions governing digital currencies\n\nAs we've previously mentioned, the majority of Islamic countries’ laws lack legal texts that address the subject of digital currencies.11–13 As a result, we will discuss the regulations that apply to digital currencies by explicating the stance of Jordanian laws on dealing with digital currencies and the position of Islamic law on digital currencies, each in accordance with its own need.\n\nThe Jordanian legislator is concerned about the financial policies regulating digital currencies' economic impact. Jordan seeks to safeguard Jordanian assets against the hazards associated with digital currency. Due to a technical breakthrough or a dramatic shift in their market value, cryptocurrencies are susceptible to significant price fluctuations and the loss of their entire worth. Also, since these losses are not covered by any public or private organization known locally or globally, the person using these currencies is legally responsible for everything that happens. There will be no accountability for risk or loss.45 As for national legislation, the Central Bank of Jordan responded to cryptocurrencies in Jordan beginning in 2014, when it issued its first circular prohibiting banks and all other financial institutions under its supervision, from engaging in any manner with cryptocurrencies, followed by the publication of two additional circulars in 2018 and 2019; to reinforce what was mentioned in the first circular.6 As such, the attitude of the Jordanian legislature is clear and unambiguous in its refusal to deal with any digital currencies.\n\nIn accordance with Egyptian law, the Egyptian Dar Al Iftaa (the House of Islamic Legislation in Egypt) has issued a religious edict, saying that the circulation of electronic currency is unlawful in Egypt.46 As was the situation in Algeria, where electronic money was forbidden under a law forbidding the acquisition, sale, use, and possession of the so-called electronic currency, and fines were levied for breaking these laws.46 Though some countries, like Egypt and Algeria, have openly banned digital currencies, others, like Germany, have opened the door to trading in them.46 Bitcoin is now officially accepted as legal tender in Germany.46 This means that the German government must impose a tax on the profits made by companies dealing with Bitcoin, while individual transactions are exempt from taxation. Ohio, in the United States, has officially acknowledged electronic money but has imposed limits on it, such as requiring sellers to register on an official website. Additionally, New York also created the first regulatory framework for regulating electronic money operations.47\n\nThe Islamic religion and the traditional economic system share the same vision of acceptable currency standards and their perceived economic functions. According to the two views, cryptocurrencies are illegal money even though they are an innovative way to pay for things and a tool for exchange and trading. This illegality is because they lack money's three most important characteristics: a medium of exchange and trade, a unit of account, and a store of value.48\n\nAlthough these three features are present in cryptocurrencies to some extent, they are not so obvious or inherent in them as to qualify them as valid currency. In the future, these currencies can develop and become globally recognized currencies and replace traditional currencies. However, this matter takes some time and requires appropriate governance measures to organize and subject them to supervision and control to avoid the significant risks and concerns associated with dealing in this currency.49\n\nIslamic and civil laws agree that Bitcoin is an unknown, unregulated digital currency not supported by any central bank and cannot be converted into physical commodities such as gold.29–44 It relies on decentralized communications and encryption to keep its data secure.39 Compared to traditional currencies and online payment services like PayPal, Bitcoin has a lot of people willing to buy and sell it, and it has low transaction costs. Also, Bitcoin could grow and become a legal currency used worldwide if it is accepted by governments everywhere and is subject to the proper rules and limits. In principle, there is no legitimate objection to creating new currencies, such as digital ones or cryptocurrencies, as long as they meet the legal requirements for legal currency.50\n\nReferring to the jurisprudential judgments on the legitimacy of transacting with digital currencies, most notably Bitcoin, we discover that Islamic Sharia scholars have divergent views on the actuality of encrypted currencies.49 Electronic currency must adhere to the standards and provisions outlined by Sharia law. The popularity of electronic money may also depend on how much people want it and how useful it is. Avoid using Sharia e-money for usury transactions: The exchange rate between cash and Sharia E-Money must be the same. The exchange of cash value for Sharia E-Money value must also occur in cash. Avoid spending too much money, and do not use it to buy things against Islamic law.51\n\nBuying Bitcoin, Dogecoin, and other digital currencies with fiat money is almost the same as buying currency with currency. There are two rules governing currency exchange: first, if it is the same type of item, the scale (quantity) must be the same, and payment must be given in cash. Digital money can be equated with money because it “has a price or value,” akin to gold, a valuable commodity whose price fluctuates, and the Jordanian dinar, which also has a value. However, the currency rate fluctuates as well. Then, buying and selling digital currency must follow the same rules, standards, and pillars as buying and selling physical currency.51,52\n\nTo be compliant with Islamic law, a financial system must have the following requirements: It should be interest-free (Interest prohibition (IP), which implies neither interest on debt shall be requested nor paid. It should be transparent and straightforward (no misrepresentation) so as not to deceive their trading partners. Trade partners have the right to know what they're purchasing. Additional trading partners must be free to make their own selections. The financial system should utilize legal entities. Thus, transactions using currency must involve existent commodities and services (real entities), and gambling is prohibited (GP). Lastly, Muslims are required to provide a decent portion of their income to those in need.53 Bitcoin is not recognized as a form of currency since it lacks a central issuing body, a legal framework, and a supervisory body. Furthermore, there are currently no guardian-type regulations in place in Jordan. If digital currencies fall under the supervision and control of the state, then Bitcoin would be deemed a kind of money, according to the most accurate interpretation of Sharia.\n\n\nResults and discussion\n\nThis study investigated the idea of digital currency as a tool for doing business in general and as a kind of electronic transaction in particular. The purpose of this study was to provide the reader with a better understanding of the notion of this currency and the opinion of Sharia and law in Jordan about its usage, as well as its characteristics and benefits.\n\nThe research results included the following elements: digital money is the consequence of technological and scientific progress, and it has evolved to keep pace with this technological era, which is the present and the future of the global economy. Electronic currency has grown extremely popular and stable among a huge number of people due to its convenience and speed in transactions, as well as its low costs because it is decentralized and does not require a significant number of formalities required by paper cash. Due to the obligation to encrypt them in order to transact with them, digital currencies offer a high level of privacy to its dealers. The majority of national laws do not establish a legal framework for digital currencies, despite their fast and massive rise. There is no legal existence for digital currencies, and the function of a currency does not apply to digital currencies; therefore, the definition of the legal status of digital currencies remains contested. Therefore, Sharia does not regard it as ordinary money that may be transacted immediately. On the other side, substantial changes in the global regime encourage the usage of digital money, primarily because individuals lack confidence in the present global financial system.\n\n\nConclusion\n\nIn this paper, we examined the relationship between Islamic law and cryptocurrency legislation. To present the most accurate descriptions of digital money, a number of credible sources were analyzed to establish a correct legal definition of digital currencies, types of digital currencies, and a discussion of digital currency legal status. Furthermore, the topic of debate and comparison was the legal system and Islamic law norms related to digital money was elaborated. Therefore, this study argues, digital currencies cannot be viewed as traditional money or even take its place, due to the vast differences that have been cited, and at the same time they do not enjoy widespread acceptance and popularity, especially considering that many countries have banned the trading of digital currencies within their monetary systems.\n\nIt can be concluded in the position of Sharia on encrypted digital currencies, according to what has been documented and deduced from researchers, that the concept of currency in Sharia is not limited to gold or silver but that Sharia approves any currency that people use as pricing tool. This idea means that, in theory, there is no legal problem with making new currencies, like digital or encrypted currency, as long as they meet the Islamic legal requirements of legal currency and are subject to the same legal rules in their circulation and transactions.\n\nFrom a legal point of view, a currency with a technical and immaterial value must be backed by assets with a real, tangible value or be supervised by a reputable financial institution. This requirement is to protect its dealers from fraud or considerable currency value changes.\n\nBitcoin's success is due to its use as a tool for price speculation rather than as a real currency. Most of those who bought this currency are speculators who aim to make a quick profit from speculation without taking on the risk of keeping it for a long time. For them, Bitcoin is an investment opportunity, not a currency that is dealt with like other currencies. As long as this is the case, people who want to make money quickly may stop using it and start using a newer speculative tool instead. This situation is especially true now that technology has made it easier to invest in ways that are a lot like gambling. As a result, trading in cryptocurrencies involves high risk due to their high instability, which makes investing in them more like gambling, which Sharia rejects. Therefore, it is advised that the Jordanian state should capitalize on digital currencies and utilize the huge demand for them from people by developing legal texts governing their use. There is also a need to alter e-commerce legislation by introducing new legal texts governing the problem of dealing with electronic currencies.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nAcknowledgments\n\nAuthors would like to thank Jadara and Yarmouk Universities for their support and facilitation.\n\n\nReferences\n\nChaum D:Blind Signatures for Untraceable Payments.Chaum D, Rivest RL, Sherman AT, editors. Advances in Cryptology. Boston, MA:Springer;1983. Publisher Full Text\n\nKethineni S, Cao Y: The rise in popularity of cryptocurrency and associated criminal activity. International Criminal Justice Review. 2020; 30(3): 325–344. Publisher Full Text\n\nYelowitz A, Wilson M: Characteristics of Bitcoin users: an analysis of Google search data. Applied Economics Letters. 2015; 22(13): 1030–1036. 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Publisher Full Text\n\nJubaedah D, Ahyani H, Putra HM, et al.: LEGAL ANALYSIS OF CRYPTO INVESTMENT IN ERA 4.0 VIEW FROM CREDO THEORY. Diponegoro Law Review. Oct. 2022; 7(2): 262–278. Publisher Full Text\n\nBasu S, Saha TR, Maity SK: Implications of cryptocurrency: a new business proposition of today’s entrepreneurial horizon. International Journal on Recent Trends in Business and Tourism (IJRTBT). 2018; 2(3): 64–70.\n\nChaira CN, Furqani H, Amanatillah D: Konsep Mata Uang Dalam Ekonomi Islam (Analisis Bitcoin Sebagai Mata Uang Virtual). EKOBIS SYARIAH. 2021; 3(2): 34–44.\n\nOdeh M: The functions of money and the extent of its realization in virtual currencies, the fifteenth international conference of the College of Sharia and Islamic Studies, University of Sharjah, United Arab Emirates.2021.\n\nMaurer B: Mobile money: Communication, consumption and change in the payments space. The Journal of Development Studies. 2012; 48(5): 589–604. Publisher Full Text\n\nAl-Jumaili O: Virtual currencies: the irreality, jurisprudential adaptation legal and ruling, the fifteenth international conference of the College of Sharia and Islamic Studies, University of Sharjah, United Arab Emirates.2019.\n\nSedrati A, Mezrioui A: A survey of security challenges in internet of things. Advances in Science, Technology and Engineering Systems. 2018; 3(1): 274–280. Publisher Full Text\n\nMeliza J, Sadalia I: CRYPTOCURRENCY. Journal of Trends Economics and Accounting Research. 2021; 1(3): 82–86.\n\nMishkin FS, Matthews K, Giuliodori M: The Economics of Money, Banking, and Financial Markets: European Edition.2013.Reference Source\n\nAndolfatto D, Berentsen A, Martin FM: Money, banking, and financial markets. The Review of Economic Studies. 2020; 87(5): 2049–2086. Publisher Full Text\n\nHazlett PK, Luther WJ: Is bitcoin money? And what that means. The Quarterly Review of Economics and Finance. 2020; 77: 144–149. Publisher Full Text\n\nMeera AKM: Cryptocurrencies From Islamic Perspectives: The Case Of Bitcoin. Buletin Ekonomi Moneter Dan Perbankan. 2018; 20(4): 443–460. Publisher Full Text\n\nSelcuk M, Kaya S: A Critical Analysis of Cryptocurrencies from an Islamic Jurisprudence Perspective. Turkish Journal of Islamic Economics. 2021; 8(1): 137–152. Publisher Full Text\n\nLikharev KK: On Money and Wealth. In Essential Quotes for Scientists and Engineers. 2021. Publisher Full Text\n\nAdam MF: Bitcoin: Shariah compliant. Amanah Finance Consultancy;2017.\n\nNair J, Motwani A: Crypto Currency: Bubble or Boom. International Journal of Advance Research in Computer Science and Management Studies. 2018; 327(1).\n\nGoanta C, Hopman M: Crypto communities as legal orders. Internet Policy Review. 2020; 9(2). Publisher Full Text\n\nSöderberg G: Are Bitcoin and other crypto assets money? Sveriges Riksbank: Economic Commentaries;2018; 5.\n\nYussof SA, Al-Harthy A: Cryptocurrency as an Alternative Currency in Malaysia: Issues and Challenges. Islam and Civilisational Renewal. 2018; 9(1): 48–65. Publisher Full Text\n\nChoudhury SR: Chinese ICOs: China bans fundraising through initial coin offerings, report says. CNBC;2017.\n\nGoanta C, Hopman M: Crypto communities as legal orders. Internet Policy Review. 2020; 9(2). Publisher Full Text\n\nSoehartono, Pati UK: The Regulation of Cryptocurrency Investation in Indonesia.2019. Publisher Full Text\n\nLham RN, Erlina, Fachrudin KA, et al.: Comparative of the supply chain and block chains to increase the country revenues via virtual tax transactions and replacing future of money. International Journal of Supply Chain Management. 2019; 8(5).\n\nKatsanov V: Cryptocurrencies, the way to an electronic concentration camp, 1st floor, Ugarit House for Printing, Publishing and Distribution, Syria.2021.\n\nAl-Tabbak K: “Trading Virtual Currency in Financing Terrorism and New Crimes”Confrontation, 1 Legal of, Mechanismsst EditionAl- Al-Nahda, DarArabiya for Publishing and Distribution, Cairo, Egypt.2020; Page 183.\n\nShdeifat O: Virtual Process Techniques: Bitcoinas a Model, Ph.D. Thesis, University of Islamic Sciences, Amman, Jordan.2018.\n\nAl-Hamidi H: Legalization of electronic currency: a comparative study in American and German legislation, Master's thesis, YarmoukUniversity, Irbid, Jordan.2019.\n\nAl-Saqaf R: Bitcoin and its monetary recipe: an inductive study in the light of Islamic jurisprudence. Al-Andalus Journal for Humanities and Social Sciences. 2021; 8(50).\n\nRabei A: The reality of Bitcoin and the ruling on dealing with it: A comparative jurisprudential study. Journal of the college of Islamic Studies, Aswan. 2020; (3): 2515.\n\nGrinberg R: Bitcoin: An Innovative Alternative Digital Currency. Hastings Science & Technology Law Journal. December 9, 2011; 4: 160. SSRN.Reference Source\n\nFageh A: Digital Currency under the Perspective of Islamic Law. Maliyah: Jurnal Hukum Bisnis Islam. 2021; 11(1): 110–128. Publisher Full Text\n\nMuhammad Ridwan Firdaus: E-money in the Perspective of Sharia Economic Law. Tahkim. June, 2018; 14(1): 152. Publisher Full Text\n\nOseni UA, Ali SN: Fintech in Islamic finance. Fintech In Islamic Finance. Routledge;2019; (pp. 3–14)." }	[ { "id": "161600", "date": "01 Feb 2023", "name": "Mohamed Aslam Akbar", "expertise": [ "Reviewer Expertise Shari'ah Sciences", "Islamic Economics and Finance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary\nIn this paper, the authors analyze the legal foundations of digital currencies from a Jordanian and Islamic legal perspective. The descriptive-qualitative research approach was used, and data was collected through documentation and literature review. The results show that digital currencies are incompatible with Shariah law and unsuitable for use as a store of value or wealth due to their instability. The paper concludes that digital currencies are speculative and considered as gambling in Shariah views, and that the Jordanian government should regulate their use. Overall, the work is properly constructed and well-articulated; however, there are some issues in the paper that can be rectified for the betterment of the argument.\nCritical Reflections\nIn the literature, the authors must emphasize that the functions and characteristics of money are distinct and not synonymous. Throughout history, money has undergone various transformations but has consistently fulfilled three main functions: serving as a store of value, a unit of account, and a medium of exchange. The specific characteristic of what constitutes money is contingent upon the complexity of society and can include factors such as portability, divisibility, and durability, etc. (Frasser & Guzmán, 2020)1. Digital money can be analyzed from both a functional and characteristic perspective, with a focus on the difficulties in implementing digital money within the framework of current Jordanian laws and regulations. For instance, there is a recent work that has been published on the characteristics of digital money from Maqasid al-Shari'ah perspective, where the paper analyzes the relationship between FIVE simultaneous characteristics of wealth and the cryptocurrency ecosystem, namely storable (iddikhār), desirable (marghūb), exchangeable (tadāwul), measurable (miqdār), and acquirable (muktasab). For more, you may refer to the Table 1 (Akbar, 2022)2.\nThe discussion may delve deeper into the feasibility of the Jordanian government's implementation of digital money in the country's legislation. It may explore the challenges and opportunities that come with the adoption of digital currencies and how they compare to traditional fiat money. One of the key points that need to be addressed is the difference between fiat money and crypto-commodities. Fiat money refers to the traditional physical currency issued and backed by the government, while crypto-commodities are digital currencies that use cryptography to secure and verify transactions. These digital currencies are decentralized, meaning they are not backed by any government or central authority.\nIt's important to differentiate these two types of currency before reaching a conclusion on the feasibility of implementing digital money in the Jordanian legislation. The discussion need to arrange and assess the various factors, such as security, stability, and adoption, that contribute to the success or failure of digital currencies. The discussion also need to evaluate the potential risks and benefits of using digital currencies in the country. For example, the use of digital currencies may increase the speed and efficiency of transactions and reduce the cost of transactions, but it may also bring about security risks such as hacking and fraud.\nConclusion\nUltimately, the paper must reach a conclusion on whether digital currencies can be considered as traditional money and whether the Jordanian government should implement the use of digital money in the country's legislation. The conclusion should be based on a thorough and balanced analysis of the differences between fiat money and crypto-commodities and a careful evaluation of the potential risks and benefits of digital currencies.\nAdditionally, the sub-level headings in a research article are not properly organized and this is causing some disorders. To clarify and make the content more understandable, the authors should arrange the sub-headings in a standard format such as Introduction, Literature Review, Methods, Discussions, Conclusion, etc. The Discussion section should be the longest part of the article as it highlights the main findings and results of the research. It is recommended that the paper follow a formal academic writing style to ensure that the work is of high quality and meets the standards of academic publishing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10083", "date": "31 Aug 2023", "name": "Nasir Albalawee", "role": "Author Response", "response": "We thank the reviewer for his insightful comments on our manuscript titled \"Islamic legal perspectives on digital currencies and how they apply to Jordanian Legislation.\" His feedback has been instrumental in refining our work and enhancing its overall quality. Below, we address each of the reviewer' comments and provide our responses: Reviewer 1 Comments Reviewer Comment: In the literature, the authors must emphasize that the functions and characteristics of money are distinct and not synonymous. Response: “Within the realm of scholarly discourse about monetary systems, it is crucial to emphasize the inherent lack of interchangeability in the functions and qualities of money. These two dimensions encompass distinct facets that, together, influence the function of money in an economy. Throughout human history, various types of currency have emerged, each with its own unique characteristics. However, despite these differences, the fundamental functions of money have remained relatively consistent. These functions include facilitating the exchange of goods and services, acting as a store of value through time, and serving as a standardized unit of measurement for financial transactions. The characteristics ascribed to money have a complex nature, frequently shaped by the level of sophistication of the cultural structure within which it functions. Within this particular setting, the examination of digital currency becomes increasingly significant as this discussion delves into the domains of both practical and inherent perspectives. The present investigation sheds light on the complex difficulties of integrating digital money into Jordan's legal system. This endeavor necessitates thoroughly assessing its compatibility with conventional monetary functions and characteristics (Fraser & Guzmán, 2020)1. Through further exploration, a comprehensive comprehension of the concurrent attributes of wealth, namely iddikhr, marghb, tadwul, miqdr, and equitability, illuminates the enigmatic intricacies of the cryptocurrency domain. Significantly, recent academic inquiries have delved into the attributes of digital currency from the perspective of Maqasid al-Shari'ah, mainly focusing on the idea of muktasab. This scholarly investigation skillfully examines the intricate relationship between the fundamental attributes of wealth and the constantly shifting landscape of cryptocurrencies. Table 1 presents a comprehensive overview of the synergies and differentiations discussed in the insights, offering a visual synthesis of the information (Akbar, 2022)2. Fundamentally, recognizing the nuanced distinction between the uses and properties of money is a fundamental aspect of the ongoing discourse. The transformation of currency over time is readily apparent. However, its fundamental roles of enabling transactions, maintaining value, and establishing a uniform measuring system continue to be the foundation of its nature. In the current financial environment, the emergence of digital currency has garnered significant attention and warrants careful analysis. This examination requires a comprehensive analysis that considers both the functional alignment and the symbiotic link between inspection and the complex aspects of wealth. The current discussion concerns the potential integration of digital currency into Jordan's legal system, highlighting the significant impact it may have on restructuring the country's economic landscape. ” Comment: The discussion may delve deeper into the feasibility of the Jordanian government's implementation of digital money in the country's legislation. It may explore the challenges and opportunities that come with the adoption of digital currencies and how they compare to traditional fiat money. One of the key points that need to be addressed is the difference between fiat money and crypto-commodities. Fiat money refers to the traditional physical currency issued and backed by the government, while crypto-commodities are digital currencies that use cryptography to secure and verify transactions. These digital currencies are decentralized, meaning they are not backed by any government or central authority. It's important to differentiate these two types of currency before reaching a conclusion on the feasibility of implementing digital money in the Jordanian legislation. The discussion need to arrange and assess the various factors, such as security, stability, and adoption, that contribute to the success or failure of digital currencies. The discussion also need to evaluate the potential risks and benefits of using digital currencies in the country. For example, the use of digital currencies may increase the speed and efficiency of transactions and reduce the cost of transactions, but it may also bring about security risks such as hacking and fraud. Response: Thank you for highlighting this point. We agree that a more comprehensive distinction between digital currencies and traditional fiat money is essential. In the revised manuscript, we have expanded the discussion section comparing these two forms of currency, elucidating their underlying characteristics, mechanisms, and regulatory implications. “The Feasibility of Implementing Digital Money in Jordanian Legislation This study thoroughly examined the viability of incorporating digital currency into the legal structure of Jordan. The present investigation examined the complex terrain of digital currency adoption, comparing and contrasting the obstacles and prospects concerning conventional fiat money. One crucial aspect of this study is distinguishing between fiat currency and crypto-commodities. Crypto-commodities refer to digital currencies that employ cryptographic techniques to ensure secure transactions, distinguishing them from fiat money, representing traditional physical cash regulated by governmental authorities. A notable distinction arises in the context of crypto-commodities, characterized by their intrinsic decentralization, which results in their lack of direct support from governmental or central authorities. The research rigorously examined multiple variables to evaluate the viability of integrating digital currency into the legal framework of Jordan. The abovementioned elements addressed in this study include security, stability, adoption rates, and broader societal ramifications. The study sought to clarify the potential for seamless integration of digital currencies within Jordan's economic and legal framework through a comprehensive examination of these components. A crucial aspect of this investigation entailed thoroughly assessing the potential advantages and disadvantages associated with using digital currency. Although digital currency has the potential to enhance transaction speed and reduce expenses, it is crucial to acknowledge the associated vulnerabilities it may create, specifically concerning security issues. The report acknowledges the potential dangers associated with cybersecurity threats, hacking incidents, and fraudulent activities that may develop due to utilizing digital currencies. Additionally, the study explored the possible ramifications of implementing digital currencies on Jordan's economic trajectory. Proponents said the digital currency could potentially augment financial accessibility and inclusivity, particularly among marginalized people. However, these perceived advantages were carefully evaluated in light of concerns regarding the potential disruption of the established financial system. The researchers actively and passionately delved into these complex aspects, highlighting the importance of conducting a comprehensive and nuanced assessment of the adoption of digital money in Jordan. The thorough comprehension of the complex dynamics that underlie digital currencies is crucial, as it highlights the investigation of difficulties, opportunities, and broader ramifications. Conducting a comprehensive analysis is crucial before formulating definitive judgments regarding incorporating digital currency into the legislative framework of Jordan.” Comment: Ultimately, the paper must reach a conclusion on whether digital currencies can be considered as traditional money and whether the Jordanian government should implement the use of digital money in the country's legislation. The conclusion should be based on a thorough and balanced analysis of the differences between fiat money and crypto-commodities and a careful evaluation of the potential risks and benefits of digital currencies. Response: “In conclusion, this paper's detailed research sheds light on the complex landscape pertaining to the incorporation of digital currencies inside the legislative framework of Jordan. A comprehensive understanding of the contrasting characteristics of fiat currency and crypto-commodities, along with a careful assessment of the associated advantages and disadvantages, leads to a well-rounded viewpoint. Digital currencies have some advantageous characteristics, including enhanced transaction efficiency and increased financial inclusion. However, it is important to acknowledge that they also possess inherent risks, particularly in the realm of security. The fundamental differentiation between these digital entities and conventional fiat money resides in their decentralized nature, which lacks direct official support. Upon thorough examination, it becomes apparent that digital currencies, despite their potential for transformation, exhibit notable distinctions from traditional monetary systems. The implementation of utilization strategies by the Jordanian government requires a careful and thoughtful approach that takes into account various complexities, while also being in line with the country's economic, social, and security priorities.” Comment: Additionally, the sub-level headings in a research article are not properly organized and this is causing some disorders. To clarify and make the content more understandable, the authors should arrange the sub-headings in a standard format such as Introduction, Literature Review, Methods, Discussions, Conclusion, etc. The Discussion section should be the longest part of the article as it highlights the main findings and results of the research. It is recommended that the paper follow a formal academic writing style to ensure that the work is of high quality and meets the standards of academic publishing. Response: “ The structure of the paper has been revised to align with the specifications outlined in the aforementioned feedback.” 1. Frasser C, Guzmán G: What do we call money? An appraisal of the money or non-money view. Journal of Institutional Economics. 2020; 16 (1): 25-40 2. Akbar DMA: Towards an interpretation of cryptocurrency as a commodity from a Maqasid al-Shari'ah perspective. International Journal of Islamic Economics and Finance Research. 2022. 99-112" } ] }, { "id": "169020", "date": "24 Apr 2023", "name": "Dmaithan Almajali", "expertise": [ "Reviewer Expertise ERP", "Technology Adoption", "Organizational Strategy", "IT-Business Alignments", "SCM", "and E-Commerce", "Cryptocurrency." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present many works already done and therefore they loose their goal's study since we notice too many repetitions in this paper.\nI will suggest to the researchers at the start of your literature review, please elaborate on the intention to use Cryptocurrency in Jordan. This is your focus on the study. You have missed the debate in the literature review section. Please use the recent citations in the literature review section.\nThe researchers have written the background of the study with a generic statement. Please try to link with your study objectives. Its shows no connection with your study. The researchers should provide the statistics related to Cryptocurrency used in Jordan.\nMention the implications and limitations of the study in the abstract.\nProvide the research gap in the introduction section which should derive the research questions and to fulfill the research questions, research objectives needs to be added.\nLiterature review need to be structured properly consisting the background of the study.\nProvide latest references.\nABSTRACT - the background of study and the problem being solved are not stated. They should be clearly stated at the beginning of the abstract. A good abstract should explain about the content of the entire paper, comprising the background of study, problem being solved, aim of paper, techniques/method, brief findings, and significance of the paper. Everything should be short and precise\nINTRODUCTION - The section discusses about the state of the art of the cryptocurrency. The domain of study is not discussed. As the background of the study, the section should discuss the domain of study first (to explain the extent people have studied), then follows with the context of study (current situation/the current practice). The problem being solved is also not clear. It should be on the domain knowledge. The section should also express the aim of paper, and ends with a paragraph that outlines the paper.\nMETHODOLOGY: Is the paper's argument built on an appropriate base of theory, concepts, or other ideas? Has the research or equivalent intellectual work on which the paper is based been well designed? Are the methods employed appropriate?:\nIn the method, the section 2 \"The idea behind digital currencies\", I am not sure about this paragraph. What actually does the idea behind digital currencies means to the reader.\nCONCLUSION - the conclusions do not adequately tie together the other elements of the paper\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "10084", "date": "31 Aug 2023", "name": "Nasir Albalawee", "role": "Author Response", "response": "We thank the reviewer for his insightful comments on our manuscript titled \"Islamic legal perspectives on digital currencies and how they apply to Jordanian Legislation.\" His feedback has been instrumental in refining our work and enhancing its overall quality. Below, we address each of the reviewers' comments and provide our responses: Reviewer Comment: Exploring potential economic and social impacts specific to Jordan would be beneficial. Response: We appreciate this suggestion. To address this, we have incorporated a dedicated section discussing the potential economic and social impacts of implementing digital currency within the unique context of Jordan. This addition provides a more localized perspective on the implications of our study. Reviewer Comment: Discuss the technological infrastructure required for digital currency adoption. Response: Your observation is valued. The revised manuscript includes a subsection discussing the necessary technological infrastructure for successful digital currency adoption. This edition offers insights into the technical considerations Jordan would need to address to ensure a smooth implementation. Reviewer Comment: at the start of your literature review, please elaborate on the intention to use Cryptocurrency in Jordan. Response: “The decision to integrate cryptocurrencies into the economic framework of Jordan is a deliberate and calculated response to the dynamic and changing global financial landscape. In light of the global phenomenon of digitization in financial transactions and the emergence of blockchain technology, the government of Jordan is contemplating the integration of cryptocurrency into its financial infrastructure as a strategy to enhance modernization efforts and leverage the potential advantages associated with this novel form of digital currency. Due to its decentralized nature, cryptographic protections, and ability to transform established financial frameworks, cryptocurrency, whose most well-known representative is Bitcoin, has drawn much attention. A desire for novelty does not merely drive the adoption of Bitcoin in Jordan, but rather, it is motivated by an acknowledgment of the potential transformative impact it might have. The primary driving force behind the desire to incorporate cryptocurrency stems from its capacity to augment financial inclusivity. In a country where certain population groups may face limited access to conventional banking services, digital currencies can function as a means to obtain financial services that surpass geographical limitations. In addition, the prompt and efficient cross-border transactions enabled by cryptocurrencies can enhance the efficiency of remittances and international trade, potentially strengthening Jordan's economic engagements at the international level. In addition, adopting Bitcoin is consistent with broader worldwide patterns that emphasize the shift towards digitalization and the prevalence of cashless societies. By adopting digital currencies, Jordan can establish itself as a leader in technical progress, which might attract international investments and stimulate innovation within its territory. Nevertheless, the endeavor necessitates a comprehensive comprehension of the complexities entailed. The utilization of cryptocurrencies in Jordan needs a thorough examination of legislative frameworks. It is imperative to balance the advantages of Bitcoin, such as expedient and cost-effective transactions, and the possible obstacles it presents, such as security risks and adherence to legal requirements. Achieving this equilibrium will be crucial in protecting various stakeholders' concerns, including individual users, financial institutions, and the government. Reviewer Comment: Please use the recent citations in the literature review section. Response: “ More Recent references were utilized in the literature review.” Reviewer Comment: The researchers should provide the statistics related to Cryptocurrency used in Jordan. Response: “Approximately 129,000 Jordanians, predominantly men, trade cryptocurrencies, according to a January report by the cryptocurrency payment company TripleA.3 In (AlMajali et. el, 2022) presented the inaugural empirical investigation of cryptocurrency in Jordan. Consequently, their model enhances theoretical understanding by providing the initial empirical evidence within the Jordanian setting. Their initiative aims to enhance understanding of cryptocurrency by offering valuable insights and recommendations to investors, government officials, and the wider public. The first figure in their research illustrates the demographic composition of cryptocurrency users in Jordan. 4 . “ 3. https://www.al-monitor.com/originals/2022/02/jordanians-turn-cryptocurrency-despite-ban#ixzz8AqNobzPc 4. Dmaithan Abdelkarim Almajali, Ra’Ed Masa’Deh & Zulkhairi M.d. Dahalin (2022) Factors influencing the adoption of Cryptocurrency in Jordan: An application of the extended TRA model, Cogent Social Sciences, 8:1, DOI: 10.1080/23311886.2022.2103901 Reviewer Comment : Mention the implications and limitations of the study in the abstract. Response: the abstract is updated to touch on the implications and limitations of the study.Reviewer Comment: Exploring potential economic and social impacts specific to Jordan would be beneficial. Response: We appreciate this suggestion. To address this, we have incorporated a dedicated section discussing the potential economic and social impacts of implementing digital currency within the unique context of Jordan. This addition provides a more localized perspective on the implications of our study. Reviewer Comment: Discuss the technological infrastructure required for digital currency adoption. Response: Your observation is valued. The revised manuscript includes a subsection discussing the necessary technological infrastructure for successful digital currency adoption. This edition offers insights into the technical considerations Jordan would need to address to ensure a smooth implementation. Reviewer Comment: at the start of your literature review, please elaborate on the intention to use Cryptocurrency in Jordan. Response: “The decision to integrate cryptocurrencies into the economic framework of Jordan is a deliberate and calculated response to the dynamic and changing global financial landscape. In light of the global phenomenon of digitization in financial transactions and the emergence of blockchain technology, the government of Jordan is contemplating the integration of cryptocurrency into its financial infrastructure as a strategy to enhance modernization efforts and leverage the potential advantages associated with this novel form of digital currency. Due to its decentralized nature, cryptographic protections, and ability to transform established financial frameworks, cryptocurrency, whose most well-known representative is Bitcoin, has drawn much attention. A desire for novelty does not merely drive the adoption of Bitcoin in Jordan, but rather, it is motivated by an acknowledgment of the potential transformative impact it might have. The primary driving force behind the desire to incorporate cryptocurrency stems from its capacity to augment financial inclusivity. In a country where certain population groups may face limited access to conventional banking services, digital currencies can function as a means to obtain financial services that surpass geographical limitations. In addition, the prompt and efficient cross-border transactions enabled by cryptocurrencies can enhance the efficiency of remittances and international trade, potentially strengthening Jordan's economic engagements at the international level. In addition, adopting Bitcoin is consistent with broader worldwide patterns that emphasize the shift towards digitalization and the prevalence of cashless societies. By adopting digital currencies, Jordan can establish itself as a leader in technical progress, which might attract international investments and stimulate innovation within its territory. Nevertheless, the endeavor necessitates a comprehensive comprehension of the complexities entailed. The utilization of cryptocurrencies in Jordan needs a thorough examination of legislative frameworks. It is imperative to balance the advantages of Bitcoin, such as expedient and cost-effective transactions, and the possible obstacles it presents, such as security risks and adherence to legal requirements. Achieving this equilibrium will be crucial in protecting various stakeholders' concerns, including individual users, financial institutions, and the government. Reviewer Comment: Please use the recent citations in the literature review section. Response: “ More Recent references were utilized in the literature review.” Reviewer Comment: The researchers should provide the statistics related to Cryptocurrency used in Jordan. Response: “Approximately 129,000 Jordanians, predominantly men, trade cryptocurrencies, according to a January report by the cryptocurrency payment company TripleA.3 In (AlMajali et. el, 2022) presented the inaugural empirical investigation of cryptocurrency in Jordan. Consequently, their model enhances theoretical understanding by providing the initial empirical evidence within the Jordanian setting. Their initiative aims to enhance understanding of cryptocurrency by offering valuable insights and recommendations to investors, government officials, and the wider public. The first figure in their research illustrates the demographic composition of cryptocurrency users in Jordan. 4 . “ 3. https://www.al-monitor.com/originals/2022/02/jordanians-turn-cryptocurrency-despite-ban#ixzz8AqNobzPc 4. Dmaithan Abdelkarim Almajali, Ra’Ed Masa’Deh & Zulkhairi M.d. Dahalin (2022) Factors influencing the adoption of Cryptocurrency in Jordan: An application of the extended TRA model, Cogent Social Sciences, 8:1, DOI: 10.1080/23311886.2022.2103901 Reviewer Comment: Mention the implications and limitations of the study in the abstract. Response: the abstract is updated to touch on the implications and limitations of the study “Islamic legal views on digital currency have major implications for Jordan's legal system. While the above approaches emphasize transparency and justice in transactions, concerns about speculative trading and financial instability arise. Jordan's legal system must be cautious when balancing Islamic principles and technological innovation.” Reviewer Comment: “Provide the research gap in the introduction section which should derive the research questions and to fulfill the research questions, research objectives needs to be added” Response: “ The subject under discussion is Islamic perspectives of digital currencies, with Bitcoin serving as a prominent example. It examines the concept of cash's digital representation and its implications for its physical presence. In addition, the discourse explores the primary functions of money in this context. Consequently, the following questions are implicit in the research problem: What are Digital currencies? What functions and characteristics define digital currencies? What is the status of digital currencies under the law? Is there a distinct legal framework for regulating digital currencies as a result of the enactment of beneficial laws? What is the position of Islamic law regarding the use of digital currencies in financial transactions? Reviewer Comment: ABSTRACT - the background of study and the problem being solved are not stated. They should be clearly stated at the beginning of the abstract. A good abstract should explain about the content of the entire paper, comprising the background of study, problem being solved, aim of paper, techniques/method, brief findings, and significance of the paper. Everything should be short and precise Response: “ The abstract now is structured as a background defining the problem being solved and the aim of the study. Also, it specifically presents in a systematic approach the background, methods, results, limitations, implications, and conclusions.” . Reviewer Comment: INTRODUCTION - The section discusses about the state of the art of the cryptocurrency. The domain of study is not discussed. As the background of the study, the section should discuss the domain of study first (to explain the extent people have studied), then follows with the context of study (current situation/the current practice). The problem being solved is also not clear. It should be on the domain knowledge. The section should also express the aim of paper and ends with a paragraph that outlines the paper. Response: “The introduction is updated to reflect on the aforementioned comment” Reviewer Comment: METHODOLOGY: Is the paper's argument built on an appropriate base of theory, concepts, or other ideas? Has the research or equivalent intellectual work on which the paper is based been well designed? Are the methods employed appropriate?: In the method, section 2 \"The idea behind digital currencies\", I am not sure about this paragraph. What actually does the idea behind digital currencies means to the reader. Response: “ The section is further elaborated and explained to make it clear to reader” Further the following additions were elaborated; Considering the legal nature of digital currencies, it is pertinent to include the following paragraph as an extension to the section mentioned above: The legal status of digital currencies varies across jurisdictions, with some countries recognizing them as legal tender. In contrast, others classify them as commodities or assets. For instance, digital currencies like Bitcoin are recognized as legal payment methods in Japan, subject to specific regulations. Conversely, digital currencies are not considered legal tender in countries like China, and their use is heavily restricted. Additionally, regulatory frameworks surrounding digital currencies are still evolving, with governments and financial institutions grappling with consumer protection, money laundering, and taxation issues. As a result, the legal landscape of digital currencies remains complex and subject to ongoing developments.5 5. Almajali, D., Hammouri, Q., Majali, T., Al-Gasawneh, J., & Dahalin, Z. (2021b). Antecedents of consumers’ adoption of electronic commerce in developing countries. International Journal of Data and Network Science, 5(4), 681–690. https://doi.org/10.5267/j.ijdns.2021.7.013 Response: to the aforementioned comment in the Discussion: “ In summary, integrating Bitcoin in Jordan reflects the country's desire to adopt digital transformation and foster innovation within its financial industry. The motivation behind this action stems from the aspiration to augment financial inclusivity, optimize cross-border transactions, and conform to the worldwide shift toward digitalization. Nevertheless, implementing cryptocurrency in Jordan's economic environment necessitates careful and thorough preparation, considering the intricate factors of regulation, economics, and technology, to guarantee a seamless and prosperous integration.” Reviewer Comment: Consider delving deeper into the legal challenges of aligning digital currency with Jordanian legislation. Response: Thank you for this suggestion. We have expanded the section on legal challenges to provide a more comprehensive exploration of potential legal hurdles that might arise when integrating digital currency into Jordanian legislation. This enhancement offers readers a more nuanced understanding of the complexities involved. In the revised version more recent references are cited in the literature review as recommended: Clark, E., Lahiani, A., & Mefteh-Wali, S. (2023). Cryptocurrency return predictability: What is the role of the environment?. Technological Forecasting and Social Change, 189, 122350. Darwish, D. (2023). Blockchain and Artificial Intelligence for Business Transformation Toward Sustainability. In Blockchain and its Applications in Industry 4.0 (pp. 211-255). Singapore: Springer Nature Singapore. Yeong, Y. C., Kalid, K. S., Savita, K. S., Ahmad, M. N., & Zaffar, M. (2022). Sustainable cryptocurrency adoption assessment among IT enthusiasts and cryptocurrency social communities. Sustainable energy technologies and assessments, 52, 102085. Jagrič, T., Fister, D., Amon, A., Jagrič, V., & Beloglavec, S. T. (2022). The Banking Industry in the Ecosystem of Digital Currencies and Digital Central Bank Currencies. In The New Digital Era: Digitalisation, Emerging Risks and Opportunities (Vol. 109, pp. 89-115). Emerald Publishing Limited. Rudd, M. A. (2022). 100 important questions about Bitcoin’s energy use and ESG impacts. Challenges, 14(1), 1. Dmaithan Abdelkarim Almajali, Firas Omar, Abdullah Alsokkar, Ala’a Saeb Alsherideh, Ra’Ed Masa’Deh & Zulkhairi Dahalin (2022) Enterprise resource planning success in Jordan from the perspective of IT-Business strategic alignment, Cogent Social Sciences, 8:1, DOI: 10.1080/23311886.2022.2062095" } ] } ]	1	https://f1000research.com/articles/12-97
https://f1000research.com/articles/12-1072/v1	31 Aug 23	{ "type": "Research Article", "title": "Antibiotic utilization and resistance in diarrheagenic Escherichia coli isolated from children under 5 Years in Nakuru County: a case-control study", "authors": [ "Titus Suge", "Dennis Magu", "Peter Wanzala", "Dennis Magu", "Peter Wanzala" ], "abstract": "Background: Children under the age of 5 years continue to suffer the ravaging effects of microbial resistance. Majority of the infections associated with this age are of bacterial and viral nature. Highest mortalities in this age group are those associated with enteric and diarrheal diseases. Diarrheagenic Escherichia coli (DEC) is among the leading causes of these diseases due to their ubiquitous nature.\nMethods: The study adopted a case-control design and aimed at investigating antibiotic utilization and resistance in DEC strains isolated from children under 5 years in Nakuru County. A total sample size of 384 children were considered. Stool samples from anal swabs were obtained and cultured on Eosin Methylene Blue (EMB). Antimicrobial susceptibility testing was done using the Kirby-Bauer disk diffusion method to segregate the resistant DEC isolates against amoxicillin, ampicillin, erythromycin, cefoxitin and nalidixic acid.\nResults: All the DEC (n=192, 100%) strains were resistant to amoxicillin, n=168, 87.5% were resistant to ampicillin, n=156, 81.3% to erythromycin n=72, 37.5% to cefoxitin and only n=64, 33.3% to nalidixic acid. Based on demographic factors, it was that observed self-medication leads among factors contributing to the observed trend in antimicrobial resistance (AMR). Other factors such the length of antibiotic use did not show any level of significance (p<0.05).\nConclusions: Thus, we conclude that a conglomerate of several factors is associated with the rising cases of AMR among the DEC strains. Notably, the use of first-line antibiotics especially the β-lactams poses a critical health concern being the most resisted class of antibiotics. Therefore, the current study unravels the need to remediate the effects of AMR among the DEC strains through proper formulation and implementation of guidelines on antibiotic usage.", "keywords": [ "Escherichia coli", "antibiotic resistance", "children" ], "content": "Introduction\n\nA significant portion of the global health burden is caused by enteric infections and diarrheal diseases (EIDD), which are frequently found in developing nations and are closely linked to inadequate water supply, sanitation, and hygiene conditions (Saka et al., 2019). The prevalence of EIDD is high throughout the world and is a major cause of morbidity and mortality in developing nations, with higher rates among children and the elderly. Notably, various microbial agents, such as viruses, bacteria, and parasites, are capable of causing diarrhea. Escherichia coli, belonging to the Enterobacteriaceae family (Wallace et al., 2020), is one of the most frequent bacterial causes of diarrhea among children under 5 years of age.\n\nUp to 40% of all diarrheagenic cases in developing nations are caused by strains of diarrhoeagenic E. coli (DEC), including enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and enterotoxigenic E. coli (ETEC), with an estimated 2.5 million child fatalities worldwide (Raghavan et al., 2017). Antibiotics have always been used to manage and treat enteric infections and diarrheal diseases (EIDD) majorly caused by DEC strains. Resistance patterns are currently on the rise among clinical cases of diarrhea in children under the age of five. These cases have been linked to the current emergence of new strains that are resistant to antibiotics. According to the World Health Organization [WHO] (2017), multi-drug resistance among important medical bacteria, including DEC, is causing global concern. Resistance to several antibiotics is largely caused by genetic mutations in bacteria allowing them to evade the lethal effects of these antibiotics (Verstraete et al., 2022). However, the acquisition of mobile genetic elements, such as gene cassettes, integrons, and mobile genetic elements, is connected to the use of third-generation cephalosporins and β-lactams (amoxicillin or ampicillin) (Rozwadowski & Gawel, 2022).\n\nCephalosporin resistance has been linked to extended-spectrum β-lactamases (ESBLs), enzymes involved in the degradation of β-lactams (Ali et al., 2018). Numerous asymptomatic DEC infections have been documented as a result of DEC’s widespread distribution, which is frequently characterized by the development of DEC-specific antibodies in young children (Buskirk et al., 2022). Children in DEC-prone and endemic areas, including urban slums, have been observed to experience this phenomenon. Kenya, one of Africa’s developing nations, deals with the threat of DEC antimicrobial resistance brought among children under the age of 5 years (Gitahi et al., 2018; Leting et al., 2022). Due to their multi-drug resistance, these resistant bacterial strains are proving challenging to control. In Kenya, just like other countries globally, drug-resistant pathogens are primarily caused by the improper and excessive use of antibiotics (Webale et al., 2020). This study, therefore, investigated the cases of multi-drug resistance in children under the age of five with prior use of the selected antibiotic agents in Nakuru County.\n\n\nMethods\n\nThis study was conducted at the Nakuru County General Hospital, specifically in the in-patient and out-patient departments. The geographical location of Nakuru County makes it a suitable location to investigate antibiotic utilization and resistance in diarrheagenic Escherichia coli, as it is an area with high rates of diarrheal diseases. Additionally, the high patient turnover at the hospital made it easier to obtain the required sample size for the study. The data were collected between October 2017 and May 2019.\n\nThe study received ethical approval from the KNH-UoN ERC (reference number KNH-ERC/A/216) on July 12, 2017, and research authorization from NACOSTI (reference number NACOSTI/P/22/20744) on 30th August 2017. Permission was also obtained from the County Medical Director and Public Health, as well as the Hospital Medical Superintendent of Nakuru Provincial General Hospital (Ref No. RII/Vol 1/08) on 24th October 2017. Caregivers provided informed consent for their children to participate by signing a consent form. Data collection was confined to the specified study period.\n\nTo investigate risk factors linked to antibiotic resistance, a case-control study design according to (Schlesselman, 1982) was used. Children who had developed an outcome (antimicrobial resistance) were identified in a case-control study, and their previous exposure to putative etiological factors was compared to that of controls or referents who did not have antimicrobial resistance. The starting point of a case-control study was the identification of cases.\n\nChildren under the age of five from Nakuru County and whose parents or legal caregivers signed assent forms constituted the study’s target group. Any subject who had diarrheal episodes 2 weeks preceding sample collection was excluded. To account for confounding variables, a matched design was utilized. Age, sex, diarrhea type, and residence were used to match participants.\n\nA web-based calculator was used to compute sample size (Philippe 2003). Random sampling technique was employed to select subjects. All children under the age of five who visited the Nakuru provincial general hospital with diarrheagenic Escherichia coli had their stool samples taken to identify cases and controls. A total sample size of 384 children were sampled. As a result, n equals 384 children, and the number of pairs equals 192.\n\nSamples were collected between July 12th 2017 to July 11th, 2018. Before the clients were assessed by the clinicians, the researcher assisted in identifying prospective eligible clients with the help of a trained clinic staff. Each child and his or her guardian were referred to the researchers, who explained to them about the study and requested them to sign a consent form. Participation in the study was voluntary.\n\nA pre-test was conducted at Kabarnet Hospital in Baringo County. Findings from the study were leveraged to standardize and validate the research instruments. For the computation of the instrument’s dependability, Cronbach’s Coefficient Alpha (α) was employed. A Cronbach’s Coefficient Alpha (α) of 0.75 was achieved which indicated that the instrument was reliable.\n\nThe anal swabs were collected under sterile conditions while the participants were lying on their backs. Each swab was assigned a unique participant number before being obtained. A sterile cotton-tipped swab was gently inserted into the anal canal, but not more than one inch, before being withdrawn. The anal swab was then placed into a plastic casing containing Amies transport media (Oxoid, Basingstoke, UK), which helps preserve the microorganisms in a viable but slow growth state during transportation to the laboratory. Subsequently, portions of the sample were transferred to a special container containing Cary-Blair transport medium (ThermoFisher Scientific), stored at 4°C, and sent to the laboratory for analysis.\n\nIsolated E. coli strains were transferred into sterile tubes containing buffered peptone water (P6682-500ML, Sigma Aldrich), a non-selective enrichment media. The swabs were rolled several times onto the sides of the tube to wash off all bacteria from the swab into the tube and the tube containing buffered peptone rinse water. The inoculated buffered peptone rinse solution was incubated at 37°C for 3–4 hours. Subsequently, 200 μl of peptone water cultures were streaked on Eosin Methylene Blue (EMB) agar (Oxoid, Basingstoke, UK) and incubated overnight at 37°C for 24 hours. The plates were removed from the incubator after 24 hours and visually examined to presumptively identify E. coli colonies. Gram staining was done by first heat fixing the bacterial smear on a microscope slide, followed by flooding the slide with crystal violet for one minute. The slide was then rinsed with water and flooded with iodine solution for one minute, followed by rinsing with water again. The slide was then decolorized with alcohol for a few seconds, rinsed with water, and counterstained with safranin for one minute. The slide was then rinsed, air-dried, and examined under oil immersion using a microscope (Smith & Hussey, 2005). To determine the morphology of the growing colonies, a pure culture was obtained by sub-culturing a single E. coli colony on another EMB agar plate. The plate was then incubated at 37°C for 24 hours. A single colony was then selected for confirmative identification of E. coli through a series of biochemical tests. IMViC biochemical tests were done by inoculating a pure culture of E. coli in the appropriate media for each test. Indole production was detected by adding Kovac’s reagent to a culture in Tryptone broth. Methyl red test was performed by adding Methyl red reagent to a culture in MR-VP broth. Voges-Proskauer test was performed by adding Barritt’s A and B reagents to a culture in MR-VP broth. Citrate utilization was determined by inoculating E. coli in Simmons citrate agar. Urease activity was determined by inoculating E. coli in urea agar. The tests were interpreted according to the results obtained by Cheesbrough (2006). Isolates that were positive to Indole and Methyl Red tests but negative to Voges Proskauer, Citrate and Urease tests were identified as E. coli. Escherichia coli ATCC 25922 and K. pneumonia were used as negative and positive controls respectively. Genetic characterization of diarrheagenic E. coli was done by amplification of target genes using polymerase chain reaction (PCR) as described by Shetty et al. (2012) and Fujioka et al. (2013). The target genes used for the identification of the different pathotypes of DEC were InvE for enteroinvasive E. coli (EIEC), st and lt for enterotoxigenic E. coli (ETEC), eaeA for enteropathogenic E. coli (EPEC), and astA for entero-aggregative E. coli (EAEC). PCR products were visualized on agarose gels and analyzed to determine the presence of target genes, which allowed for the identification of the different pathotypes of DEC.\n\nThe Kirby-Bauer disk diffusion method was used to test the susceptibility of the isolated E. coli to antibiotics commonly used in animal and human health as described by Hudzicki (2009). The Mueller-Hinton (MH) agar (Oxoid, UK) was utilized according to the recommendations provided by the Clinical Laboratory Standards Institute (CLSI) to perform antimicrobial sensitivity testing. The microorganisms were subjected to testing on disks containing frequently used antimicrobials to assess their susceptibility. Penicillin/Amoxicillin (Oxoid, UK) The inoculum of each E. coli isolate was adjusted to a concentration of about 106 CFU/mL by comparing its turbidity to that of McFarland standard 0.5. Adjusted bacterial inocula were inoculated on the whole surface of the media. The disks were then placed upside down on the inoculated culture plates and the inoculated agar plates were incubated at 37°C for 18 hours. The zones of complete inhibition, along with the disk diameter, were measured using Vanier calipers. The Petri dish was placed above a black, non-reflective surface that was illuminated by reflected light. The edge where no visible growth was observed was considered the zone boundary, which was measured to the nearest millimeter. Disk susceptibility results were interpreted using criteria established by the Clinical and Laboratory Standards Institute (CLSI, 2006).\n\nThe data obtained was analyzed using SPSS version 25 software. Descriptive statistics, including frequencies and percentages, were employed to summarize the patterns of antibiotic utilization and resistance, as well as the susceptibility of DEC to antibiotics. Odds ratios (OR) with corresponding 95% confidence intervals (CI) were calculated to measure the strength of association between the exposure of children to antibiotics within the past 12 months and antimicrobial resistance. A p-value of less than 0.05 was considered statistically significant.\n\n\nResults\n\nFigure 1 depicts a graphical distribution of resistant cases. All DEC samples were resistant to amoxicillin while only 12.5% of DEC samples were susceptible to ampicillin (Suge, 2023). Further, the DEC strains from the samples were 81.3%, 37.5% and 33.3% resistant to erythromycin, cefoxitin and nalidixic acid respectively (Table 1). Resistance was noted to decrease moving from β-lactam antibiotics through macrolides to cephalosporins and quinolones.\n\nThe analysis observed that the number of respondents whose children used antibiotics within the last 12 months was 384 (100%). This is an indication that all the children in Nakuru were frequent users or had been exposed to antibiotics within the study period (2017/2018). The study results also indicated that 148 (38.5%) children consumed antibiotics acquired over the counter and that 236 (61.5%) children in Nakuru county consumed prescription acquired antibiotics (see Table 2).\n\nFrom the analysis, it can be observed that the β-lactam antibiotics were the most used antibiotics in Nakuru (248, 64.6%). The results also indicate that sulfonamides were the least used antibiotics drug (16, 4.2%). The study further shows that most children used antibiotics for less than one week, with 288 (75%) of the total respondents. While 96 (25%) indicated usage of more than one week. Consequently, it can be observed that 156 (40.6%) of the children were on medication/self-medication with antibiotics when rarely sick. While 192 (50%) of the children were on medication/self-medication with antibiotics sometimes when they were sick. Finally, it can be observed that 36 (9.4%) of the children were on medication/self-medication with antibiotics always when sick. This show that there is a substantial proportion (90.6%) of the children who are on medication/self-medication with antibiotics (see Table 2).\n\nBased on the level of satisfaction, 140 (36.5%) parents indicated that they were not satisfied, while 244 (63.5%) of the respondents recorded that they were satisfied. Further, 148 (38.5%) of the children rarely finished the full prescription when using antibiotics. The study also indicates that 236 (61.5%) of the children within Nakuru always or sometimes finished the full prescription when using antibiotics (see Table 2).\n\nFurthermore, 140 (36.5%) of the respondents indicated that they always made an informed decision that an antibiotic was needed for the child’s illness. The results also indicated that 244 (63.5%) of the respondents sometimes decided on their own that an antibiotic was needed for the child’s illness (see Table 2).\n\nFinally, 200 (52.1%) of the respondents, based on experience, would make personal decisions on using antibiotics in the future. The results also indicate that 184 (47.9%) of the respondents do not foresee making a personal decision on using antibiotics on children in the future (see Table 2).\n\nThe analysis showed that the likelihood of a child occasionally completing the entire antibiotic prescription was 1.439 (0.605, 3.425), with a p-value of 0.000, which is significant at the 0.05 level. This odds ratio tells us that individuals who finished the full prescription of antibiotics were not a risk factor for antimicrobial resistance. The results also indicate that the odds of rarely a child finishing the full prescription was 6.75(2.754, 16.546), a p-value = 0.000< 0.05 significant level. This odds ratio tells us that individuals who rarely finished the full prescription of antibiotics have seven times the odds of antimicrobial resistance than individuals who always finished the full prescription (see Table 2).\n\nThe results also indicate that the odds of always making an informed decision on their own that antibiotics were needed for the child’s illness was 1.31(0.864, 1.988) with a p-value = 0.122 > 0.05. This odds ratio tells us that individuals who always made an informed decision on their own that antibiotics are needed for the child’s illness were not a risk factor for antimicrobial resistance (see Table 2).\n\nThe results showed that the odds to foresee making a personal decision on antibiotic use was 0.3 (0.197, 0.456) with a p-value = 0.000 < 0.05 significant level. Thus, the odds of antimicrobial resistance among individuals who foresee making a personal decision on antibiotic use was only 0.3 times the odds among individuals who did not foresee making a personal decision. This adjusted odds ratio indicates that individuals who foresee making a personal decision on antibiotic use seem to be protective against antimicrobial resistance (see Table 2).\n\nThe results show that the odds of how the antibiotic was acquired was 2.692 (1.759, 4.122) with a p-value = 0.000 < 0.05 significant level. These odds show that individuals who obtained antibiotics by walking into a pharmacy without a prescription have three times the odds of antimicrobial resistance than individuals who obtained antibiotics by walking into a pharmacy with a prescription. The results also indicate that the odds of β-lactam 6.167(4.076, 9.330) with a p-value = 0.000 < 0.05 significant level. This odds ratio tells us that individuals who used β-lactams antibiotics during medication/self-medication have six times the odds of antimicrobial resistance than individuals who didn’t use them. The results also indicate that the odds of macrolides 0.909 (0.581, 1.423) with a p-value = 0.422 > 0.05 significant level. This odds ratio tells us that individuals who used macrolides antibiotics during medication/self-medication were not a risk factor for antimicrobial resistance. The results also indicate that the odds of sulfonamide 0.35 (0.184, 0.667) a p-value = 0.000 < 0.05 significant level. Thus, the odds of antimicrobial resistance among individuals who used sulfonamide antibiotics during medication/self-medication were only 0.35 times the odds among individuals who didn’t use them. This adjusted odds ratio tells us that the use of sulfonamide antibiotics during medication/self-medication seems to be protective against antimicrobial resistance. The results also indicate that the odds of quinolones 0.818 (0.529, 1.265) a p-value = 0.25 > 0.05 significant level. This odds ratio tells us that individuals who used quinolones antibiotics during medication/self-medication were not a risk factor for antimicrobial resistance (see Table 3).\n\nThe results indicate that the odds on the length of usage of antibiotics by the child of less than a week was 0.8 (0.504, 1.272) a p-value = 0.205 > 0.05 significant level. This odds ratio tells us that the length of usage of antibiotics for a period of less than one week was not at risk factor for antimicrobial resistance (see Table 3).\n\nThe results indicate that the odds of sometimes a child being on medication/self-medication with antibiotics when sick was 0.228 (0.077, 0.672) a p-value = 0.000 < 0.05 significant level. The results also indicate that the odds of a child always being on medication/self-medication with antibiotics when sick was 0.038 (0.012, 0.113)), a p-value = 0.000 < 0.05 significant level. Thus, the odds of antimicrobial resistance among individuals who sometimes/always had their child being on medication/self-medication with antibiotics when sick was only 0.23 and 0.038 times, respectively, the odds among individuals who rarely had their child on medication/self-medication with antibiotics when sick. This adjusted odds ratio tells us that individuals who sometimes/always had their child being on medication/self-medication with antibiotics when sick seems to be protective against antimicrobial resistance (see Table 3).\n\nThe results indicate that the odds of unsatisfaction with experience of antibiotic medication was 4.705 (2.995, 7.391) a p-value = 0.000 < 0.05 significant level. This odds ratio tells us that individuals who were unsatisfied with the use of antibiotic medication have five times the odds of antimicrobial resistance than individuals who were satisfied with antibiotic medication (see Table 3).\n\n\nDiscussion\n\nParticularly in developing nations, DEC poses a concern to children’s health. In this study, 384 children under the age 5 years with acute diarrhea from Nakuru Provincial General Hospital (NPGH) were assessed for the incidence of DEC, epidemiological traits, and patterns of drug resistance. The DEC isolates showed the highest resistance to amoxicillin followed by ampicillin, erythromycin, cefoxitin and nalidixic acid.\n\nOur findings generally indicated that first-line antibiotics, such as ampicillin, were more resistant to DEC infection than second-line antibiotics, such as erythromycin, cefoxitin, and nalidixic acid. Notably, there exist a six-fold risk of developing antimicrobial resistance for individuals who used β-lactams antibiotics during medication/self-medication than those who did not use them. Both previous and current studies concur with these findings (Eltai et al., 2020; Cannatelli et al., 2016; Aslani et al., 2011; Jafari et al., 2009). Due to their affordability (mostly, because they are readily available over the counter) and accessibility first-line antibiotics are frequently, empirically and indiscriminately used to treat diarrhea in developing nations (Shah et al., 2016).\n\nInterestingly, at least all the children in this study had prior exposure to antibiotics within 12 months of birth. There are several studies that support this finding (Marra et al., 2009; Scott et al., 2016). This observation could be attributed to several factors including sensitivity of the immune system during childhood, teething and general propensity of children to harbor diverse pathogens.\n\nAdditionally, a large proportion of the children sampled obtained antibiotics directly from pharmacies without prescriptions. Seemingly, the policies governing the use of antibiotics are flawed. It is evident that most African countries, including Kenya, have non-stringent regulations regarding the use of antibiotics. Due to the need to maximize the profit margins and ensure constant flow of medicines, pharmacies tend to bend the regulations to meet such demands. Several studies have observed this tradition. Compounding this observation, most first line antibiotics, more specifically β-lactams (in case of the current study) are the mostly used. However, sulfonamides account for only a small fraction of the total medications used during treatment. The availability of and affordability of the first line antibiotics could be largely attributed to these observations. Several studies have been able to explain this trend in many populations. For example, a study by Erku et al. (2017) demonstrated that amoxicillin was the most commonly utilized antibiotic due to its easy acquisitions and affordability. Also, Saleem et al. (2019), while exploring the use of antibiotics inappropriately by Pakistani hospital patients, observed a significant use of cephalosporins and penicillin and thus their potential risk of developing resistance against them. A similar study by Okubo et al. (2018) also supports this observation. Also, Singh et al. (2018) showed the existence of a high prevalence of antibiotic resistance to widely used drugs like ampicillin, ceftazidime, cefoxitin, streptomycin, and tetracycline.\n\nNotably, the duration of use of antibiotics seems to be a factor contributing overall to the emergence of resistance among the sample population. Only a quarter of the population used medications past one week. In contrast, the majority of the sampled population used antibiotics for less than seven days. Most of the antibiotics that are correctly prescribed are more than a week. This observation indicates that the majority of the children could be withdrawn of their medications when their caregivers/parents observe their improved health status. Also, based on the data of how often a child finishes the entire prescription, it was noted only 7% finished their medications. As it is expected that the medications, especially antibiotics be used completely, this trend indicates a higher possibility of emergence of resistant DEC strains due to exposure to the sub-lethal doses of these antibiotics as compared to children completing their dosages. This study concurs with several studies. For instance, Atif et al. (2019) showed that majority of patients, including pediatric patients, stopped the antibiotic course when they felt better and did not finish. Thus the ‘feeling better’ contributes significantly on inconsistent use of antibiotics.\n\nInterestingly, it was observed that more than 90% of the children were under medications despite not having any symptoms of infections. This trend augments inappropriate use of antibiotics. It is expected that existence of active infections warrants the correct use of antibiotics. Cases of non-adherence to recommended antibiotic usage thus are the majority. As mentioned above, most antibiotics used in such medications are the β-lactams especially amoxicillin and ampicillin due to their affordability and availability. Thus, high number of usages of these antibiotics can be described by the need to ‘protect’ or ‘boost’ children’s immunity against the looming infections. The findings of the study however, further confirm the protective effect of using antibiotics only when sick against antimicrobial resistance. A report by Malik and Bhattacharyya (2019) indicates that combined effects of the economy, infections, and self-medication produce synergistic interactions through feedbacks on each other, portraying the evolution of drug resistance as a self-reinforcing loop in the population. Thus self-medication, compounded by other socio-economic factors, play a major role in the distribution of resistant strains. Other studies are further consistent with the current study (Ajibola et al., 2018; Gillani et al., 2017). There is however limited information on extent to which self-medication and without presentation of any symptoms among children.\n\nSatisfaction levels on the use of antibiotics showed that majority of the parents were satisfied with the extent to which antibiotics were efficacious and beneficial to their children. The findings further showed a five-fold protective effect against antimicrobial resistance of being satisfied with medication. This could be explained by the positive effect of satisfaction to adherence to prescribed antibiotics and hence reduction in the cases of antibiotic resistance. Conversely, unsatisfaction on the effectiveness of antibiotics is one of the major potential risks associated with antimicrobial resistance. Conversely, Cohen et al. (2017) presumes that there is little strong evidence that patient satisfaction will lead to better medical results. Thus, patient satisfaction levels are based on the progression of the disease and the inherently equivocal and unstable character of human reactions. This means therefore that if there have been situations of improved child’s health after medication, there are higher chances that the subsequent prescriptions will be adhered.\n\nWith regards to finishing antibiotics, majority of the children completed their prescriptions of antibiotics fully. Only 7% did not finish their doses. Evidently, finishing a full prescription of antibiotics is not a risk factor for antimicrobial resistance. However, the 7% are seven times more likely to develop antimicrobial resistance. Therefore, findings have indicated that there is a seven-fold likelihood of developing antimicrobial resistance when one does not complete their prescriptions. From this study we can establish that sub-lethal exposure of bacteria is a major factor contributing to antimicrobial resistance. Jamhour et al. (2017) demonstrated that in developing nations, knowledge of antibiotics and self-medication techniques is a power driver of antimicrobial resistance. Most of the populations including the caregiver prefer not to use antibiotics to treat a cold. The study further demonstrated that the majority of the people did not prefer completing doses, instead keeping them for future use. This situation exacerbates emergence of antimicrobial resistant strains.\n\nThe decision to use antibiotics is solely dependent on the caregiver. The majority of the parents/guardians who participated in the study reported that they always make decisions on their own regarding when and how their children should use antibiotics. Informed decision seems to be a protective factor in the development and emergence of antimicrobial resistance. These decisions could be made based on the experience with antibiotic use. Interestingly, there is a probability slightly above 50% that the caregiver will make an informed decision regarding the use of antibiotics by their children. Nonetheless there exists a protective effect of making personal informed decisions on antimicrobial resistance. Boiko et al. (2020) demonstrated that expectations and experiences on antibiotic use constitute many reasons for the choices and decisions on the use of antibiotics.\n\n\nConclusion\n\nThe current study established an interplay of various factors associated to antimicrobial resistance among the DEC isolates. The results are consistent with almost all the studies that have been conducted to assess resistance patterns of DEC isolates. That is, first-line antibiotics such as amoxicillin, ampicillin and erythromycin almost always present the highest proportions of resistance. Also, it is explicit that self-medication is significantly associated with the escalating rates of resistance among children under five years of age.\n\nThis study recommends appropriate measures be taken to mitigate this trend. This can be done through formulation and implementation of more stringent antibiotic utilization policies. Finally, more robust techniques can be employed to assess the extent to which resistance of antibiotics is associated to molecular characteristics of the DEC strains.", "appendix": "Data availability\n\nHarvard Dataverse: Antibiotic utilization and resistance in diarrheagenic escherichia coli isolated from children under 5 years in Nakuru County. https://doi.org/10.7910/DVN/IIGIAH (Suge, 2023).\n\nThis project contains the following underlying data:\n\n• RAW DATA 1.pdf (Data on the subject’s demographics, antibiotic consumption patterns, antibiotic resistance and genetic variants.)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAjibola O, Omisakin OA, Eze AA, et al.: Self-medication with antibiotics, attitude and knowledge of antibiotic resistance among community residents and undergraduate students in Northwest Nigeria. Diseases. 2018; 6(2): 32. Dantas Wallace. Publisher Full Text\n\nAli T, Ali I, Khan NA, et al.: The growing genetic and functional diversity of extended spectrum beta-lactamases. Biomed. Res. Int. 2018; 2018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAslani MM, Alikhani MY, Zavari A, et al.: Characterization of enteroaggregative Escherichia coli (EAEC) clinical isolates and their antibiotic resistance pattern. Int. J. Infect. Dis. 2011; 15(2): e136–e139. Publisher Full Text\n\nAtif M, Asghar S, Mushtaq I, et al.: What drives inappropriate use of antibiotics? A mixed methods study from Bahawalpur, Pakistan. Infect. Drug Resist. 2019; 12: 687–699. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoiko O, Gulliford MC, Burgess C: Revisiting patient expectations and experiences of antibiotics in an era of antimicrobial resistance: qualitative study. Health Expect. 2020; 23(5): 1250–1258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuskirk AD, Ndungo E, Shimanovich AA, et al.: Mucosal Immune Profiles Associated with Diarrheal Disease Severity in Shigella-and Enteropathogenic Escherichia coli-Infected Children Enrolled in the Global Enteric Multicenter Study. MBio. 2022; 13: e00538–e00522. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCannatelli A, Giani T, Antonelli A, et al.: First detection of the mcr-1 colistin resistance gene in Escherichia coli in Italy. Antimicrob. Agents Chemother. 2016; 60(5): 3257–3258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheesbrough M: District laboratory practice in tropical countries. Cambridge University Press; 2006. Publisher Full Text\n\nCohen JB, Myckatyn TM, Brandt K: The importance of patient satisfaction: a blessing, a curse, or simply irrelevant? Plast. Reconstr. Surg. 2017; 139(1): 257–261. Publisher Full Text\n\nEltai NO, Al Thani AA, Al Hadidi SH, et al.: Antibiotic resistance and virulence patterns of pathogenic Escherichia coli strains associated with acute gastroenteritis among children in Qatar. BMC Microbiol. 2020; 20(1): 54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErku DA, Mekuria AB, Belachew SA: Inappropriate use of antibiotics among communities of Gondar town, Ethiopia: a threat to the development of antimicrobial resistance. Antimicrob. Resist. Infect. Control. 2017; 6(1): 112–117. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFujioka M, Otomo Y, Ahsan CR: A novel single-step multiplex polymerase chain reaction assay for the detection of diarrheagenic Escherichia coli. J. Microbiol. Methods. 2013; 92(3): 289–292. PubMed Abstract \| Publisher Full Text\n\nGillani AH, Ji W, Hussain W, et al.: Antibiotic self-medication among non-medical university students in Punjab, Pakistan: a cross-sectional survey. Int. J. Environ. Res. Public Health. 2017; 14(10): 1152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGitahi N, Gathura P, Gicheru M, et al.: Multidrug resistant Escherichia coli isolated from asymptomatic school going children in Kibera slum, Kenya. African. J. Bacteriol. Res. 2018; 10(5): 70–77. Publisher Full Text\n\nHudzicki J: Kirby-Bauer disk diffusion susceptibility test protocol.2009. Publisher Full Text\n\nJafari F, Hamidian M, Rezadehbashi M, et al.: Prevalence and antimicrobial resistance of diarrheagenic Escherichia coli and Shigella species associated with acute diarrhea in Tehran, Iran. Can. J. Infect. Dis. Med. Microbiol. 2009; 20(3): e56–e62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJamhour A, El-Kheir A, Salameh P, et al.: Antibiotic knowledge and self-medication practices in a developing country: A cross-sectional study. Am. J. Infect. Control. 2017; 45(4): 384–388. PubMed Abstract \| Publisher Full Text\n\nLeting S, Musyoki S, Maiyoh G: Molecular characterization and drug susceptibility of diarrheagenic Escherichia coli isolates from children under five at a referral hospital in north-Western Kenya. East Afr. Med. J. 2022; 42(4): 4711–4720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalik B, Bhattacharyya S: Antibiotic drug-resistance as a complex system driven by socio-economic growth and antibiotic misuse. Sci. Rep. 2019; 9(1): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarra F, Marra CA, Richardson K, et al.: Antibiotic use in children is associated with increased risk of asthma. Pediatrics. 2009; 123(3): 1003–1010. Publisher Full Text\n\nNational Committee for Clinical Laboratory Standards: Performance standards for antimicrobial disk susceptibility tests: approved standards. National Committee for Clinical Laboratory Standards; 2006. Publisher Full Text\n\nOkubo Y, Michihata N, Morisaki N, et al.: Recent patterns in antibiotic use for children with group A streptococcal infection in Japan. J. Glob. Antimicrob. Resist. 2018; 13: 55–59. PubMed Abstract \| Publisher Full Text\n\nRaghavan PR, Roy S, Thamizhmani R, et al.: Diarrheagenic Escherichia coli infections among the children of Andaman Islands with special reference to pathotype distribution and clinical profile. J. Epidemiol. Glob. Health. 2017; 7(4): 305–308. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRozwadowski M, Gawel D: Molecular Factors and Mechanisms Driving Multidrug Resistance in Uropathogenic Escherichia coli—An Update. Genes. 2022; 13(8): 1397. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaka HK, Dabo NT, Muhammad B, et al.: Diarrheagenic Escherichia coli pathotypes from children younger than 5 years in Kano State, Nigeria. Front. Public Health. 2019; 7: 348. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaleem Z, Saeed H, Hassali MA, et al.: Pattern of inappropriate antibiotic use among hospitalized patients in Pakistan: a longitudinal surveillance and implications. Antimicrob. Resist. Infect. Control. 2019; 8(1): 187–188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchlesselman JJ: Case-control studies: design, conduct, analysis. Oxford University Press; Vol. 2. . 1982.\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\nShah M, Kathiiko C, Wada A, et al.: Prevalence, seasonal variation, and antibiotic resistance pattern of enteric bacterial pathogens among hospitalized diarrheic children in suburban regions of central Kenya. Trop. Med. Health. 2016; 44(1): 39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShetty VA, Kumar SH, Shetty AK, et al.: Prevalence and characterization of diarrheagenic Escherichia coli isolated from adults and children in Mangalore, India. J. Lab. Physicians. 2012; 4(1): 24–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh AK, Das S, Singh S, et al.: Prevalence of antibiotic resistance in commensal Escherichia coli among the children in rural hill communities of Northeast India. PLoS One. 2018; 13(6): e0199179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith AC, Hussey MA: Gram stain protocols. Am. Soc. Microbiol. 2005; 1: 14.\n\nSuge T: Antibiotic Utilization And Resistance In Diarrheagenic Escherichia Coli Isolated From Children Under 5 Years In Nakuru County.2023. Publisher Full Text\n\nVerstraete L, Van den Bergh B, Verstraeten N, et al.: Ecology and evolution of antibiotic persistence. Trends Microbiol. 2022; 30(5): 466–479. PubMed Abstract \| Publisher Full Text\n\nWallace M, Fishbein S, Dantas G: Antimicrobial resistance in enteric bacteria: current state and next-generation solutions. Gut Microbes. 2020; 12(1): 1799654. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWebale MK, Guyah B, Wanjala C, et al.: Phenotypic and genotypic antibiotic resistant diarrheagenic Escherichia coli pathotypes isolated from children with diarrhea in Nairobi City, Kenya. Ethiop. J. Health Sci. 2020; 30(6).\n\nWorld Health Organization [WHO]: Antimicrobial resistance.2017. Reference Source" }	[ { "id": "259234", "date": "11 Apr 2024", "name": "Benno H Ter Kuile", "expertise": [ "Reviewer Expertise My specialization is the molecular mechanisms of development and spreading of antimicrobial resistance" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe major problem with this study is that the analytical procedure is not sufficiently clear described. The authors state that: “Cronbach’s Coefficient Alpha (α) of 0.75 was achieved which indicated that the instrument was reliable”. The instrument itself is, however, not described. Several other points are unclear: 1)\n\nIn total 384 children were included in the study. A random sampling techniques was used to identify cases and controls. How many children were that were in principle eligible visited the hospital during the sampling period? Cases and controls were 1:1. How were the children assigned to either of two categories? What was the difference between case and control? 2)\n\nAll 384 children had “experiences with use of antibiotics within the past 12 months”. Still, in 44 cases no antibiotic was used.\n\n3)\n\nMost parents made “an informed decision that an antibiotic is needed for the child's illness” by themselves, but most antibiotics were prescribed. Who made the actual decision, the physician or the parent? 4)\n\nThe heading of table 3 indicates that the “Association between antimicrobial resistance and exposure of children to antibiotics within the past 12 months.” will be elucidated. This is not the case. There is barely a difference in antibiotic usage between the “cases” and the “controls”.\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "272516", "date": "29 May 2024", "name": "Niels Frimodt-Møller", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Suge et al. reports on an interesting subject of the relationship between antibiotic use and resistance in diarrheagenic E. coli in a district in Kenya. The study presented has, to be corrected or revised.\nThe main issues:\nThe study is supposed to be a case control study, but it is difficult to understand, how this was set up. How were controls defined? Were they patients with E. coli infections, diarrhea or any disease? The sample size calculation must inform which outcome defined the calculation? The antibiotic susceptibility testing should not include both ampicillin and amoxicillin; they are supposed to be completely equal in susceptibility studies. Usually ampicillin is used as representative of the aminopenicillins. Erythromycin is not active against E. coli, so the resistance rate should be 100%. It can be discussed whether azithromycin can be used, since this is active against Salmonella sp., but it is not used for E. coli infections. Why was cefoxitin included? This is not a commonly used drug for diarrhea, since it is only available for intravenous treatment – for diarrhea, one would usually only consider oral antibiotics. Nalidixic acid is not used any more for diarrhea, ciprofloxacin would be more appropriate. Nalidix acid could be used as a surrogate marker for fluoroquinolones, but it will miss the qnr-genes. The OR-calculations in Table 3 are difficult to understand; what was tested against what? E.g. an OR of 6.1 for beta-lactams is incommensurable with the data shown. Since all children received antibiotics it is a question whether this type of analysis can be done? If use was tested against resistance, this has to be revised, since resistance data are not correct.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-1072
https://f1000research.com/articles/12-1069/v1	31 Aug 23	{ "type": "Study Protocol", "title": "Comparative evaluation of the effectiveness of bioresorbable collagen membrane in combination with deproteinized bovine bone matrix xenograft (DBBM), and advanced platelet rich fibrin membrane in combination with deproteinized bovine bone matrix xenograft for periodontal regeneration", "authors": [ "Shweta Bhagat", "Priyanka Jaiswal", "Deepika Masurkar", "Priyanka Jaiswal", "Deepika Masurkar" ], "abstract": "Introduction: Periodontitis is a chronic, multifactorial, inflammatory disease that leads to gradual loss of bone and periodontal attachment. Thus, periodontal surgery is required according to severity of diseases. The goal of periodontal reconstructive surgery is to regenerate soft and hard tissues destroyed during periodontal disease. Aim: To compare the effectiveness of bioresorbable collagen membrane in combination with Deproteinized Bovine Bone Matrix xenograft (group A); and Advanced Plasma rich fibrin(A-PRF) membrane in combination with Deproteinized Bovine Bone Matrix xenograft (group B) in the treatment of human infrabony defects. Methods: This randomised controlled trial will be carried out at the periodontics outpatient department. We will choose 24 patients with moderate to advanced chronic periodontitis who are otherwise systemically healthy. Infrabony defects will be treated by open flap debridement using bovine-derived xenograft and bioresorbable collagen membrane (group A) and open flap debridement using bovine-derived xenograft and A-PRF after the completion of first therapy (group B). At baseline and six months after undergoing periodontal surgery, probing pocket depth, clinical attachment level, radiographic bone fill, gingival recession, oral hygiene status, and gingival health will be measured. Expected results: It is suggested that Bioresorbable Collagen Membrane in combination with Deproteinized Bovine Bone Matrix xenograft will show better results as compared to Advanced Platelet Rich Fibrin Membrane with Deproteinized Bovine Bone Matrix Xenograft. Clinical trial registry of India (CTRI): REF/2023/05/066732. Protocol version: v1 (12/05/23)", "keywords": [ "Periodontitis", "Infrabony defects", "Bioresorbable Collagen Membrane", "Xenograft", "Advanced platelet rich fibrin", "periodontal regeneration", "CBCT", "scaling and root planning." ], "content": "Introduction\n\nPeriodontitis is a chronic multifactorial inflammatory disease characterised by a dysbiotic dental biofilm that leads to gradual loss of periodontal attachment and bone which ends with a variety of negative consequences on speech, aesthetics, masticatory function, psychological well-being, as well as quality of life.1\n\nRegeneration of periodontium is defined as “the reproduction or reconstitution of a lost or injured part to restore the architecture and function of the periodontium. Regenerative periodontal therapies aim with the repairment of the periodontal structure and function.”2,3 With loss of attachment apparatus due to periodontal diseases, the main goal is to regenerate the attachment of periodontium to involve both development of newer cementum on the root of the tooth and newer periodontal attachment between recently formed bone and cementum.\n\nEven though autologous bone grafts are still the preferred method for bone augmentation surgeries, heterologous bone substitutes are successfully used to address the limited supply of autologous tissue, reducing morbidity of donor site, potential resorption, mismatch of size, insufficient graft material, operating time, and costs.4,5 Yet, to prevent site of a second surgical harvest and medical issues such as ankylosis and root resorption, people resort more to alternate choices for periodontal treatment.\n\nXenografts are bone grafts that are transplanted in humans from different species (usually porcine and bovine). Anorganic bovine bone graft (ABM) is deproteinized bovine bone mineral that is naturally generated. Hard tissue repairs intrabony defects in humans and clinical attachment level have been improved using anorganic bovine bone. The most prevalent source of xenografts in dentistry is deproteinized bovine bone and is utilised extensively for regeneration of alveolar bone and has a high rate of success, particularly in intraoral treatments.6\n\nAn open flap debridement alone is less effective than guided tissue regeneration (GTR). Based on biological basic principles, the membrane is positioned to prevent epithelial cells of gingiva from moving apically. Deproteinized bovine bone matrix xenograft (DBBM), a deproteinized form of bovine bone mineral, is routinely added to GTR.7,8 An X-ray beam with a conical form utilised in cone-beam computed tomography (CBCT) makes a 2-dimensional array of image detectors more sensitive. Studies that evaluated infrabony abnormalities using CBCT indicated greater precision and accuracy.9 Because CBCT readings are more accurate, it is possible to detect anomalies in buccal and lingual regions of the teeth.\n\nTo the best of our knowledge, this is the first clinical trial for infrabony defects that will compare collagen membrane combined with deproteinized bovine bone matrix and A-PRF combined with deproteinized bovine bone matrix.\n\nThis protocol is reported in line with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklist.16\n\n\nAim\n\nTo evaluate the effectiveness of biodegradable collagen membrane in combination with xenograft in the treatment of human infrabony defect.\n\n\nObjectives\n\nThe objectives of this study are:\n\n1. To evaluate the effectiveness of Collagen Membrane combined with Deproteinized Bovine Bone Matrix regarding increase in clinical attachment level, probing pocket depth reduction and radiographic bone fill in human infrabony defects at 6 months after surgery.\n\n2. To evaluate the effectiveness of A-PRF combined with Deproteinized Bovine Bone Matrix regarding increase in clinical attachment level, decrease in probing pocket depth and radiographic bone filled, 6 months after surgery.\n\n3. To compare the effectiveness of biodegradable Collagen membrane and A-PRF membrane in combination with Deproteinized Bovine Bone Matrix bone graft at 6 months after surgery.\n\n\nMethods\n\nCalculation of sample size was done with the use of previous study data by Gorkhali et al. in 2020.7 The result of the calculation is 12 in each group, thus around 24 samples will be undertaken for the entire study.\n\nRatio of sample size (Group 1/Group 2) = 1:1\n\nTotal sample size 24 with each 12 per group.\n\nThe outpatient department of periodontics, Sharad Pawar dental college, Sawangi, Wardha will choose 24 systemically healthy individuals with mild to advanced chronic periodontitis based on the following inclusion criteria:\n\nInclusion criteria\n\n1) Presence of at least 1 or 2 radiographically detectable interproximal infrabony osseous defect with probing pocket depth ≥5 mm and clinical attachment loss ≥5 mm following initial therapy (assessed radiographically by CBCT).\n\n2) Depth of intraosseous component of the defect ≥3 mm by clinical and radiographic means, which will be confirmed on intra-surgical measurement (assessed using UNC 15 probe).\n\n3) A radiographic base of the defect at least 3 mm coronal to the apex of the tooth (assessed radiographically by CBCT).\n\n4) Presence of at least 3 mm width of keratinized gingiva around test teeth to allow complete soft tissue coverage of the defect (assessed using UNC 15 probe).\n\nExclusion criteria\n\n1) Evidence of localized aggressive periodontitis.\n\n2) Patients with unacceptable oral hygiene (Plaque Index >1).10\n\n3) Smokers (recent history of smoking more than 10 cigarettes/day) or who used any of tobacco products.\n\n4) Tooth with inadequate endodontic/restorative treatments.\n\n5) Tooth with mobility exceeding grade II and exhibiting a class III or class IV furcation defect.\n\n6) History of periodontal surgical therapy of the selected quadrant.\n\n7) Women who are pregnant or lactating.\n\nIn a specially created chart, information about food status, oral hygiene practises, systemic history, gingival and also periodontal state, in addition to standard clinical information, will be documented.\n\nSupragingival and subgingival scaling and root planning will be done as a part of the initial therapy. If required, a coronoplasty will be carried out. Patient will receive repeated plaque control instructions until they have a plaque score of less than 1. Following the conclusion of the initial therapy, a re-evaluation will be conducted to do assessment of the patient towards therapy. If during re-evaluation test after 6 weeks, the periodontal pocket depth is more than 3 mm, plaque score is less than 1 then only we will take the patient for surgery.\n\nEach patient will be given an explanation of the study’s goal and design prior to getting started, and their informed consent will be obtained.\n\nThe probe angulations and position will be standardised using a specially designed occlusal acrylic stent. A cast model made from an alginate impression will be used to create an occlusal stent. The occlusal stent will be stretched to encompass coronal one-third of the teeth. A reference point or slot will be created on the deepest site of affected tooth. Linear, fixed reference point will be the apical border.\n\nAfter receiving initial therapy, a total of 24 systemically healthy individuals will be chosen for the study. A coin flip will be used to randomly distribute the participants, in keeping with a randomised parallel design, each of which will have 12 defects, prior to surgery (Figure 1).\n\nThe clinical parameters that will be documented like Plaque index, papillary bleeding index, probing pocket depth, relative clinical attachment level, and relative gingival marginal level on the day of surgery as baseline and then after six months. Moreover, additional data will be collected including intraoral periapical radiographs, intraoral clinical pictures, and periodontal charting on a specially prepared form.\n\nIndices\n\nPlaque index, a measurement of the buildup of full mouth supragingival plaque, will be used in order to assess the patient’s oral hygiene health. Papillary bleeding index will be used to evaluate inflammation of gingiva. These are:\n\n1. “Plaque Index (PI) (Turkesy-Gillmore-Glickman Modification of Quigley-Hein 1970).10\n\n2. Papillary Bleeding Index (Mühlemann H. R 1977).11\n\nThe best score will be less than 1. Worst score will be more than 1.\n\nProbing measurements\n\nA UNC-15 probe will be inserted into the slot of acrylic stent at the gingival margin level, with the tip positioned at that level. The measurement will be taken up to the bottom edge of the stent, will be recorded as the relative gingival margin level. A relative attachment level will then be determined. The depth of the probing pocket will be measured (PPD).\n\nWith a Periodontal Probe calibrated to UNC-15, width of keratinized gingiva will be determined. At baseline, three months, all probing measurements will be recorded.\n\nRadiographic analysis\n\nCBCT\n\nUsing CBCT, osseous defect sites will be examined at baseline and six months post-surgery. The lowest discontinuous point of the periodontal ligament will serve as landmark for the location of the base of defect. Root surface will be intersected by a line that runs perpendicular to the alveolar crest (AC). AC is the point of intersection along the root surface.\n\nRinsing the patients mouth using 0.2% chlorhexidine gluconate solution at the start of the surgical procedure is required for the patients. The condition of asepsis will be preserved during the entire process. The area undergoing for surgery will be anaesthetized using a local anaesthetic solution (2% w/v Lignocaine adrenaline solution (1:100,00) will be used. Maximum of 15 ml I.M. dosage will be given).\n\nFlap design (incisions)\n\nUsing BP blades number 12 or 15, intracrevicular (sulcular) incisions will be made to include periodontal access flap on the buccal and lingual sides. To retain the complete interdental papillae and accomplish primary wound closure, the incisions will be carried. The flap will include a tooth mesially and distally to the tooth and is connected with the defect. If additional access is needed, divergent vertical relief incisions will be made one tooth away from the problem.\n\nFlap reflection\n\nA periosteal elevator will be used for reflecting a full thickness mucoperiosteal flap to expose alveolar bone inside the osseous defect. By removing granulomatous tissue, extreme care will be taken to prevent perforation of the reflected periodontal flap.\n\nDebridement and root surface management\n\nHand tools (Hu-friedy Gracey curettes) and ultrasonic tools (EMS, piezo 150) will be used to remove granulation tissue from the osseous defect. It will be taken care of to ensure that the flap is not extremely thin. Plaque and calculus on the root surfaces will be removed. The root surfaces will be planed until they have smooth and firm surface.\n\nAt this point, UNC15 probe will directly assess the vertical bone defects and quantity of the bony walls present. If the vertical depth of the bone defect is less than 3 mm, the subject’s eligibility will be determined in the end. If the base of the defect doesn’t bleed enough, a half-round bur will be used to perform intra-marrow penetration. At this time, deproteinized bovine bone matrix graft will be used in combination with porcine collagen membrane to correct the test site defects.\n\nPreparation of APRF\n\nVenous blood (10 ml) will be drawn under aseptic conditions from antecubital fossa (10 ml venous blood will be withdrawn in 18 gauge needle) and moved to sterile test tubes before being centrifuged (REMI, (R-8C)) at 1500 rpm for the next 14 minutes to prepare A-PRF, according to Ghanaati et al.12 In the test sites, the infrabony defect will first get a Porcine Collagen Membrane with DBBM.\n\nProcedure for test group\n\nIt will be possible to completely isolate and hemostasize the defect. To enable quick flap closure after the implantation of the graft material, the flap will be presutured without making a knot. Deproteinized Bovine Bone Matrix bone graft will be inserted inside the osseous defect in the test site until maximal coronal level is reached of the osseous wall by raising the flap, on which Collagen membrane will be placed over the defect. Membrane will be applied so that it extends 3 mm or more. The flap will then be coronally repositioned and then sutured accordingly.\n\nNow the reflected flap will be held in place combining vertical mattress sutures and interproximal sutures. After two minutes, saline-soaked gauze will be used to close any gaps that could allow a clot to develop and prevent reattachment. Periodontal dressing will be applied.\n\nProcedure for control group\n\nThe surgical process on selected control site and the test site will be the same, with the exception that the osseous deficiencies at control sites will be filled with Deproteinized Bovine Bone Matrix graft and then APRF membrane will be applied on the top of the filled bone graft.\n\nAntibiotics including Amoxicillin 500 mg thrice a day and analgesics with the combination of Ibuprofen 325 mg and Paracetamol 400 mg thrice a day will be prescribed post-surgical for 5 days. Patients will be instructed to rinse daily with 0.2% chlorhexidine gluconate twice for 6 weeks. Periodontal dressing and sutures will be removed after 8-10 days post-surgery. Patients will be instructed to clean the treated site with cotton pellet saturated with 0.12% chlorhexidine gluconate for additional 2-3 weeks in an apico-coronal direction and later using a soft toothbrush. After this period, patients will be again instructed to resume mechanical oral hygiene measures, use of interdental cleaning aids such as interdental brush and to discontinue chlorhexidine.\n\nMonitoring\n\nThe study will be monitored till completion by data monitoring committee (DMC) which includes PG Guide, head of the department, research convener and chief advisor of university research cell.\n\nThe principal investigator (PI) will have access to these interim results and make the final decision to terminate the trial.\n\nData will be collected, assessed, and spontaneously reported during adverse events and other unintended effects of trial interventions or trial conduct.\n\nThe project management group meet will review the trial conducted every month. The trial steering group and the independent data monitoring and ethics committee meet will review and conduct the trial period till the trial is complete.\n\nPatients will be recalled back at the first, third, and sixth months after surgery. All patients will be given oral hygiene instructions together with oral prophylaxis (supragingival scaling). In the first six months following surgery, neither probing nor subgingival instrumentation will be done.\n\nAt six months, during follow-up visit a thorough post-operative evaluation will be carried out. Plaque index, gingival index, probing pocket depth, relative clinical attachment level, and relative gingival marginal level are the main clinical parameters that will be recorded. Radiographs and CBCT will also be required.\n\nAfter 6 months, we are expecting increase in clinical attachment level and decrease in periodontal pocket depth with radiographic bone fill in the test group.\n\nData analysis\n\nThe data collected from patients will be entered on Microsoft excel by PI ensuring the quality checks. Software for statistical analysis will be SPSS version 15.0, SPSS, Chicago, USA). Student’s unpaired t-test will be done.\n\n\nConclusion\n\nWe expect both surgeries to lead to significant reductions in probing pocket depth and clinical attachment gains, and treatment with open flap debridement with bovine-derived xenografts and collagen membrane will lead to significantly higher probing pocket depth reduction and clinical attachment gain than treatment with open flap debridement alone.\n\nThe results will be published in an indexed journal.\n\nNot yet started.\n\n\nDiscussion\n\nThe reconstruction of periodontal osseous defects is a continuous challenge with periodontal therapy. The periodontal wound healing after periodontal therapy occurs by periodontal repair. In recent years, efforts have been concentrated towards periodontal regenerative procedures aimed at restoring the vanished periodontal support. The present investigation aims to evaluate the effectiveness of bioresorbable collagen membrane together with bovine xenograft for periodontal regeneration in human infrabony defects. The current discussion is based on effectiveness of combination of xenograft with bioresorbable collagen membrane in comparison to combination of xenograft with A-PRF for periodontal regeneration in human infrabony defects (Periodontal defects).\n\nXenograft has been shown to exhibit good biocompatibility and osteoconductivity in both animal and human study. In addition it has been found that resorption rate of xenograft corresponds to the formation of newer bone.7 According to the findings of the study by Gorkhali et al.,7 patients with chronic periodontitis who underwent surgical therapy with open flap debridement alone or open flap debridement (OFD) with bovine-derived xenograft and collagen membrane experienced a clinically significant PPD reduction and CAL gain, but only in the case of CAL at six months across both groups. Various investigations have been done for the evaluation of bone grafts and collagen membrane in adjunct to surgical therapy for regenerative procedure.\n\nSculean et al.13 had a similar study where they compared the treatment of deep intrabony defects with bovine-derived xenograft and a bioresorbable collagen membrane to open flap debridement. Bio-oss showed better regenerative properties in infrabony defects than open flap debridement.14,16 According to Luepke et al.,15 resorbable barrier membrane combined with DFDBA produced superior results than resorbable barrier membrane used alone.\n\nProcedures will be performed in keeping with the ethical standards of the institutional. This protocol was permitted by the Institutional Ethics Committee of Datta Meghe Institute of Higher education and Research, Sawangi, Meghe, Wardha (approval number DMIHER (DU)/IEC/2023/576) on 6th February 2023. This trial has been registered at the Clinical trial registry of India (CTRI) (REF/2023/05/066732; registration pending).\n\nCase histories will be taken for each patient. A unique I’d number will be provided to each case histories which will be stored in password protected folder in desktop and while sharing it will be ensured that identifying information such as age, address, IPD number and OPD number will be deleted. PI and institutional authorities will be accessible to the final trial dataset.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: SPIRIT checklist for ‘Comparative Evaluation of the Effectiveness of Bioresorbable Collagen Membrane in Combination with Deproteinized Bovine Bone Matrix Xenograft and Advanced Platelet Rich Fibrin Membrane in Combination with Deproteinized Bovine Bone Matrix Xenograft for periodontal regeneration in Human Infrabony Defects: A Clinical and Radiographic Study’ https://zenodo.org/record/8022776. 16\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nI acknowledge the help from my university, college, professors, department, statistician and resources which provided the necessary data and their inputs for this given clinical study.\n\n\nReferences\n\nOrlandi M, Muñoz Aguilera E, Marletta D, et al.: Impact of the treatment of periodontitis on systemic health and quality of life: A systematic review. J. Clin. Periodontol. 2022; 49(Suppl.24): 314–327. PubMed Abstract \| Publisher Full Text\n\nNibali L, Sultan D, Arena C, et al.: Periodontal infrabony defects: Systematic review of healing by defect morphology following regenerative surgery. J. Clin. Periodontol. 2021 Jan; 48(1): 101–114. Epub 2020 Nov 11. PubMed Abstract \| Publisher Full Text\n\nBhedasgaonkar SY, Kapadia J, Patil NA: Treatment of infrabony defects with platelet-rich fi brin along with bone graft: Case report. J. Int. Clin. Dent. Res. Organ. January-June 2015.\n\nDhonge RP, Vishwakarma A: Successfulmanagement of three-wall periodontal intrabony defect using Novabone Dental Putty®. Indian J. Dent. Sci. 2018; 10: 172–175. Publisher Full Text\n\nZhou S, Sun C, Huang S, et al.: Efficacy of Adjunctive Bioactive Materials in the Treatment of Periodontal Intrabony Defects: A Systematic Review and Meta-Analysis. Biomed. Res. Int. 2018; 2018: 1–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeenu G, Thomas George V, Manohar R, et al.: Applications of xenografts in periodontal regeneration. IP Int. J. Periodontol. Implantol. 2021; 6(4): 184–191. Publisher Full Text\n\nGorkhali RS, Pradhan S, Shrestha R, et al.: Evaluation of Bovine-derived Xenograft Combined with Bioresorbable Collagen Membrane in Treatment of Intrabony Defects. J. Nepal Soc. Perio. Oral Implantol. 2020 Jul-Dec; 4(8): 61–67. Publisher Full Text\n\nGhanaati S, Booms P, Orlowska A, et al.: Advanced platelet-rich fibrin: a new concept for cell-based tissue engineering by means of inflammatory cells. J. Oral Implantol. 2014 Dec; 40(6): 679–689. PubMed Abstract \| Publisher Full Text\n\nChoi IGG, Cortes ARG, Arita ES, et al.: Comparison of conventional imaging techniques and CBCT for periodontal evaluation: A systematic review. Imaging Science in Dentistry. 2018; 48(2): 79–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTuresky S, Gilmore ND, Glickman I: Reduced plaque formation by the chloromethyl analogue of victamine C. J. Periodontol. 1970 Jan; 41(1): 41–43. PubMed Abstract \| Publisher Full Text\n\nMühlemann HR: Psychological and chemical mediators of gingival health. J. Prev. Dent. 1977 Jul-Aug; 4(4): 6–17. PubMed Abstract\n\nMelcher AH: On the repair potential of periodontal tissues. J. Periodontol. 1976 May; 47(5): 256–260. PubMed Abstract \| Publisher Full Text\n\nSculean A, Nikolidakis D, Schwarz F: Regeneration of periodontal tissues: combinations of barrier membranes and grafting materials - biological foundation and preclinical evidence: a systematic review. J. Clin. Periodontol. 2008 Sep; 35(8 Suppl): 106–116. PubMed Abstract \| Publisher Full Text\n\nStavropoulos A, Karring T: Guided tissue regeneration combined with a deproteinized bovine bone mineral (Bio-Osss) in the treatment of intrabony periodontal defects: 6- year results from a randomized-controlled clinical trial. J. Clin. Periodontol. 2010; 37: 200–210. PubMed Abstract \| Publisher Full Text\n\nLuepke PG, Mellonig JT, Brunsvold MA: A clinical evaluation of a bioresorbable barrier with and without decalcified freeze-dried bone allograft in the treatment of molar furcations. J. Clin. Periodontol. 1997 Jun; 24(6): 440–446. Publisher Full Text\n\nBhagat S: SPIRIT Checklist- “Comparative Evaluation of the Effectiveness of Bioresorbable Collagen Membrane in Combination with Deproteinized Bovine Bone Matrix Xenograft and Advanced Platelet Rich Fibrin Membrane in Combination with Deproteinized Bovine Bone Matrix Xenograft for periodontal regeneration in Human Infrabony Defects: A Clinical and Radiographic Study.” (Version v1). Zenodo. 2023. Publisher Full Text" }	[ { "id": "308991", "date": "19 Aug 2024", "name": "Carlos M. Ardila", "expertise": [ "Reviewer Expertise Periodontology", "artificial intelligence", "clinical trials", "clinical epdemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 offers an interesting proposal, but various aspects require further elaboration throughout its content, as outlined below:\nTITLE: - Consider adding \"A Randomized Controlled Trial\" to the title for clarity. - Specify the type of periodontal defects (infrabony) in the title.\nABSTRACT: - Provide a brief background on the importance of periodontal regeneration. - Clearly state the null and alternative hypotheses. - Mention the primary outcome measures.\nIntroduction: - Expand on the pathogenesis of periodontitis and its consequences. - Discuss the limitations of current periodontal regenerative therapies. - Clearly justify the choice of materials (collagen membrane and A-PRF) and xenograft.\nMethods: - Specify the randomization method and allocation concealment. - Describe the surgical procedures in detail, including defect classification. - Clarify the timing of A-PRF preparation and application. - Define the outcome measures and their assessment methods. - The diagnosis of chronic periodontitis is outdated. Revise.\nExpected Results:\n- Rephrase as \"Hypothesized Outcomes\" or \"Expected Differences.\" - Provide a clear rationale for the expected superiority of Group A.\nAdditional Recommendations:\n- Describe the blinding procedure. - Ensure adherence to CONSORT guidelines for randomized controlled trials. - Provide a description of the xenograft material and its characteristics. - Clarify the follow-up period and any additional treatments.\nKeywords: Make sure they correspond to MeSH terms.\nINTRODUCTION - Reorganize the introduction to flow logically:\nDefine periodontitis and its consequences. Discuss the importance of periodontal regeneration. Introduce xenografts and DBBM as treatment options.\n- Clarify the rationale for combining DBBM with collagen membrane and A-PRF. - Specify the type of infrabony defects being targeted. - Mention the clinical significance of improving attachment level, reducing pocket depth, and enhancing radiographic bone fill. - Avoid abrupt transitions between topics (e.g., CBCT discussion feels disconnected). - Remove redundant phrases (e.g., \"to the best of our knowledge\" is already implied). - Ensure consistent terminology (e.g., use \"deproteinized bovine bone matrix\" consistently instead of \"anorganic bovine bone mineral\" or \"ABM\").\nAim and Objectives:\n- Rephrase the aim to focus on comparing the two treatment combinations. - Clarify the primary outcome measures in the objectives. - Consider adding a timeline for the 6-month evaluation.\nMETHODS - Ensure adherence to CONSORT guidelines for randomized controlled trials. - The calculation of sample sizes must accurately present the data considered, averages or proportions, standard deviations, significance value, power, etc. - - The diagnosis of aggressive periodontitis is outdated. Revise. - Clarify the randomization process (e.g., block randomization, stratification). The one presented can generate inequitable groups. - Specify the blinding procedure. - Provide more details on the data monitoring committee (DMC) and trial steering group. - Nothing is indicated about the clinicians who will participate, their level of training, the agreement and calibration exercises (how they will be measured). Similarly, information must be presented about the clinicians who will perform the clinical evaluations. - Clarify the statistical analysis plan, including:\nPrimary and secondary outcome measures. Comparison between groups (e.g., collagen membrane vs. A-PRF). Adjustment for confounding variables. The type of analysis to be performed: by protocol or intention-to-treat analysis.\n- Consider adding a section on adverse event reporting and management. - Clarify the maintenance care and re-examination procedures. - Provide more details on the expected outcomes and hypotheses. - The statistical analysis of the data must be more detailed, starting with the determination of the normal distribution that will establish the statistical tests to be used and more complex tests that could control confounding variables. Evidence must be presented that will analyze the quantitative and qualitative descriptive data, etc. - Ensure consistency in terminology and formatting throughout the methodology section.\nCONCLUSIONS:\nConsider adding a statement on the potential clinical implications of the study findings.\nDISCUSSION: - Clarify how the current study builds upon existing research. - Provide more details on the mechanisms by which xenografts and collagen membranes promote periodontal regeneration. - Discuss potential limitations and future research directions.\nAdditional Suggestions:\nConsider adding a section on limitations, addressing potential study limitations and biases. Provide a clear statement on the dissemination plan, including publication and presentation intentions. Ensure registration with the Clinical Trial Registry of India is completed and provide the registration number. Consider adding a statement on the availability of data and materials.\nLanguage and Grammar:\nUse precise and concise language throughout the manuscript. Avoid repetitive phrases and sentences. Ensure consistent terminology and formatting. Proofread for grammatical errors, punctuation, and spelling mistakes, for example “planning”. Reorganize sections for logical flow and clarity. Use transitional phrases to connect ideas between sentences and paragraphs. Clearly define key terms and concepts. Use active voice to convey a sense of agency.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1069
https://f1000research.com/articles/12-1068/v1	31 Aug 23	{ "type": "Case Study", "title": "Performance evaluation and comparative study of three 52-kW PV plants in India: a case study", "authors": [ "Divya Navamani Jayachandran", "Boopathi Kadhirvel", "Lavanya Anbazhagan", "Geetha Anbazhagan", "Pradeep Vishnuram", "Reddy Prasad", "Boopathi Kadhirvel", "Lavanya Anbazhagan", "Geetha Anbazhagan", "Pradeep Vishnuram", "Reddy Prasad" ], "abstract": "Developing countries like India are rapidly transitioning from traditional energy sources to sustainable energy sources, due to the increase in demand and the depletion of fossil fuels. Grid-connected photovoltaic (PV) systems attract many investors, organizations, and institutions for deployment. This article studies and compares the performance evaluations of three 52-kW PV plants installed at an educational institution, SRMIST (SRM Institute of Science and Technology), in Tamil Nadu, India. This site receives an annual average temperature of 28.5°C and an average global horizontal irradiation of 160 kWh/m2/m. The prediction model for the 52-kW power plant is obtained using solar radiation, temperature, and wind speed. Linear regression model-based prediction equations are derived using the Minitab 16.2.1 software, and the results are compared with the real-time AC energy yield acquired from the three 52-kW plants for the year 2020. Furthermore, this 52-kW plant is designed using PVsyst V7.1.8 version software. The simulation results are compared with the energy yield from the plants in 2020 to identify the shortfall in the plant performance. The loss analysis for the plant is performed by obtaining the loss diagram from the PVsyst software. This study also proposes a methodology to study the commissioned PV plant's performance and determine the interaction between variables such as direct and diffused solar radiations, air temperature, and wind speed for forecasting hourly produced power. This article will motivate researchers to analyze installed power plants using modern technical tools.", "keywords": [ "52-kW PV plant", "energy yield", "regression", "prediction", "Solar Energy." ], "content": "Introduction\n\nSolar energy has grown to be among the most popular sources of clean energy in recent years across many industries, and numerous studies are being conducted to improve its application and benefits. A continent like Asia has a higher potential for power generation from solar energy, as depicted in Figure 1(a). In this continent, developing country like India has vast potential, and their demand also increases with a population of nearly 140 crores. This power demand must be met through renewable power sources due to fossil fuel depletion. Since the country has higher global solar radiation, depicted in Figure 1(b), the country has set a target of 300 GW for solar energy by 2030. However, the country has already reached its installed capacity of 63 GW in March 2023, and the total installed capacity of renewable sources is depicted in Figure 1(c). Hence it is necessary to concentrate on this to reach the set target. Recently, many researchers have been concentrating on research based on several configurations in which solar photovoltaic (PV) systems can be installed, grid-connected PV and standalone PV, which may be designated as off-grid systems.1 However, the installation capacity of both differs significantly as, through the years, it has been observed that a grid-connected PV system is much more developed than an off-grid system. There is another topology which is known as a Hybrid solar energy system. It can charge the system from the grid and solar PV directly, but these are expensive and are not usually preferred. Several studies are being conducted to improve its application in daily life and determine how its potential applications might be broadened. It has been observed that solar thermal collectors are utilized to turn energy into heat while also generating electricity with panels.2 Some problems include variations in output energy due to changes in irradiance level.3 As we know, PV modules are made from silicon cells, thus limiting their efficiency to significantly less. Therefore, it is essential to increase their efficiency so that more people can be ready to invest in this.4 Solar insolation determines the sustainability and dependability of PV-based power generation systems; hence optimization is crucial to satisfy load demand.5 These factors are crucial during system installation because the performance is also influenced by the environment, location, and plant varieties.6\n\nAccording to the IRENA report, even though many technologies exist, most governments concentrate on solar and wind, and major investments were made in PV and wind technology.7 The government has recently initiated a series of efforts to deploy roof panels over various offices and organizations, which can assist in addressing the situation.8,9 Sometimes, PV degradation may also lead to variation in series, shunt resistances, and decreased output power.10 It can also diminish the impact of greenhouse gases brought by fossil fuels. PV energy is more affordable than any other sustainable energy source, and research has shown that it is incredibly profitable in rural regions.11,12 At every stage of the global solar PV supply chain, China is currently by far the largest supplier; with a manufacturing capacity for PV modules of around 340 GW/year, it has more than twice the installed PV modules worldwide, with a manufacturing capacity utilization rate for solar components of between 40 and 50% in 2021. China directly supplies all markets, except North America, where import taxes on Chinese solar PV components have been imposed. However, Chinese businesses have been actively investing in production capacity in Southeast Asia to supply the area and export to the United States.13 Many studies suggest the appropriate areas for implementing PV systems, but they could be more extensive. The utilized parameters and sites discussed in this research are identified as an outcome of the literature review, and their applicability is noted in Table 1.\n\nOne of the first R analysis types thoroughly explored and applied realistically in many situations is linear regression. Creating a mathematical model that may be used to forecast one variable, known as the dependent variable, can be characterized using another variable, the independent variable. The degree of the linear relationship between two variables is measured using correlation analysis. This is because models with a linear dependence on their unknown parameters are more readily fitting than models with non-linear dependence because it is simpler to identify the data samples of the resulting estimators.24\n\nOn the other hand, a significant amount of data must be managed, so the regression model is useful.25 It is frequently used to predict time-series and regression models using conventional estimate approaches, which involve consideration of the predictor variables, the target variable, and their relationship.26 This study compares the two models’ abilities to accurately forecast PV module performance: linear and non-linear regression models. A logarithmic linearized equivalent model serves as the mathematical representation of the non-linear model. In this paper, the site which has been selected is based on SRM Institute of Science and Technology in Kattankulathur, Chennai City, in Tamil Nadu, India. Many studies were conducted in our literature to investigate the behavior during one year, from January 2020 to December 2020. Performance parameters like global horizontal irradiation, energy yield, and capacity factor have been calculated. The power plants are installed on the rooftop of the Mechanical C Block, Civil Engineering Block, and Science & Humanities Block of SRM Institute of Science and Technology (SRMIST), Kattankulathur, 603203. In the paper, the description of installed PV systems and site details are discussed.\n\nFurther, the simulation of the grid and the calculated results are shown through tables and graphs. Towards the end of the paper, the economic factor and environmental impacts are discussed. Global solar radiation (GHI) of Asia and global solar radiation (GHI) of India are illustrated in Figure 1. These global solar radiation maps are downloaded from Solargis, where several collections of solar resource maps are available for research purposes.27 Various case studies on the PV performance are accomplished by researchers by considering certain locations,28,29 based on techno-economic assessment,30,31 feasibility studies,32,33 comparative studies,34 optimizing the performance,35 solar insolation studies and estimations36–38 and several other factors.39–46\n\n\nMethods\n\nThis section discusses the methodology followed to make this case study. Initially, the linear regression model is obtained using solar radiation, temperature, and wind speed data from NREL (National Renewable Energy Laboratory). Then, regression equations are obtained from this prediction model for further study. This statistical analysis will give the correlation among the control factors and its significance. The results from the prediction model will be compared with the running 52-kW plant installed in the institution. Finally, a complete description of all three 52-kW grid-connected PV systems is presented with the photographs, satellite map, and the specification of BoS (Balance of Solar PV system).\n\nAfter that, a comparative study is performed on all three 52-kW grid-connected PV systems with respect to energy yield, performance ratio, capacity utilization factor, CO2, and diesel saved. The procedure followed for this comparative analysis is presented in detail in a separate section. This 52-kW plant is simulated in PVsyst V7.1.8 simulation software and the results obtained are compared with the real-time data for 2020. This comparison will help us observe the performance of all three 52-kW PV systems. Finally, the inferences from the study are observed and listed for the conclusion. The flowchart of the methodology followed for the study is shown in Figure 2.\n\n\nDescription of three 52-kW grid-connected PV system\n\nThe major components of the grid-connected PV system are a solar array, inverter with maximum power point tracking (MPPT), AC and DC disconnect, and other protective and connective equipment to the grid. It is more effective than a standalone PV system because it eliminates the losses incurred in energy storage. Another significant advantage of the grid-connected system is the eradication of the problem incurred due to the presence of batteries, i.e., cost and replacement. The general schematic diagram of all three 52-kW PV systems is represented in Figure 3.\n\nAll three 52-kW solar power plants are located at SRMIST with latitude and longitude of 12.8231° N, 80.0442° E, and elevation above the sea level of 51 m. Since the generated PV power significantly depends on the sun’s position and its radiation intensity, the institute studied solar radiation for one year and opted for these three locations on the campus.\n\nFigure 4(a)-(c) presents the description of all the sites taken for the study. In addition, satellite map images and photographs of the Mechanical ‘C’ block, Science and Humanities, and Chemical Engineering block are depicted in Figure 4(a), (b), and (c), respectively.\n\nThree 52-kW PV plants occupy a rooftop area of 304 square meters. The plant is divided into ten strings with 16 panels in series. Each string has the capacity to generate 5.2 kW of power and the ten strings are combined to generate the power of 52 kW. All ten strings are connected to the main string combined box, which is connected to Delta RPI M50 A commercial inverter. All three 52-kW plants are installed with the structure as mentioned above. All these plants work with a central inverter system. The output of the plant is connected to the grid. The generated power is used for the lighting and other appliances in the institute.\n\nTypically, in many solar plants, the tilt angle of the PV panels is made equal to the latitude of the geographical location of the PV plant. All three plants have fixed tilt angles, and the institute does not plan for any modern techniques to tilt the panel to produce efficient output. Since the latitude of the Kattankulathur location is 12.83°, the tilt angle of the three 52-kW solar PV plants is 13.3°.\n\nThe rating of PV panels in the 52-kW plant is 325 W polycrystalline. It is a fixed type with a weight of 21.5 kg. The efficiency of the panel is 16.72%. The number of cells in this panel is 72 cells. This 325 W panel has a maximum voltage of 37.88 V and an open circuit voltage of 45.86 V. This polycrystalline PV panel’s maximum and short circuit current are 8.59 A and 9.06 A, respectively. To maintain the efficiency of the PV modules, the maintenance team regularly cleans the panels.\n\nA 50 kVA inverter converts the DC power to AC power. The range of DC and AC voltage of the inverter are 200-1000 V and 320-480 V, respectively. The inverter’s efficiency is 98.60%, and the total input current is 100 A. The total harmonic distortion is less than 3%, with a 45-55 Hz frequency range. It has an inbuilt disconnect switch.\n\nTo observe the DC and AC voltage, current, and power of the plant, a few graphs are presented in Figures 5 and 6. Figure 5(a)-(f) presents the AC and DC voltage and current of all the 52-kW power plants. This observation is drawn from June 15th, 2020. Similarly, the AC power output of three solar PV plants was observed on October 22nd, 2020; these graphs are presented in Figure 6. Figure 3(a)-(c) presents the AC power output of the 52-kW plant of the Faculty of Science and Humanities, Chemical Engineering lab, and mechanical ‘C’ block, respectively.\n\n\nRegression analysis for the selected site\n\nThe chosen site data was gathered on an hourly basis. The selected location receives 9 hours of solar radiation every day on average. For prediction, the AC hourly produced energy, direct beam and diffused radiation, ambient temperature, and wind velocity of the chosen site were considered. The regression model for the creation of AC energy outputs was developed using average hourly data at the location SRMIST, Kattankulathur, Tamil Nadu, India. From the prediction model, the regression equation was derived. Equation ((1), which has a linear relationship with beam radiation, diffused radiation, temperature, and wind speed, is used to estimate AC power from PV panels.\n\nA regression model is a statistical method for determining the connection between the control variables. It is essential to check for residual plots before developing a regression equation to ensure linear regression. The statistical analysis and generation of the regression model for the system under consideration were carried out using Minitab software version 16.2.1.\n\nWhere ρ - AC energy output (kWh), α - beam irradiance (W/m2), β - diffuse irradiance (W/m2), γ - ambient temperature (C), and μ - wind speed (m/s).\n\nA graphical method of analyzing residuals is essential to assess for a ‘good fit’ regression model the best fit of a set of data in a regression line. The residual plots reveal the degree of correlation between the variables and the projected results. The figure shows the residual plots of AC energy yield. As demonstrated in the figure, the points are spaced linearly straight. It indicates that the projected and actual values are more closely related, which is denoted as a normal probability plot.\n\nFigure 7 shows a comparison between residual and anticipated values. Both appear to be the most similar to one another, so there is very little difference between them. The histogram plot of AC energy is shown in Figure 7. In the histogram graph, clear data regarding the residuals are shown. The figure demonstrates the residuals vs. trial run order. Both positive and negative residual values are present, indicating the existence of certain relationships. The models show promise for adequacy due to the thorough study of AC residual plots.\n\nThe R2 (Coefficient Determination) value of the generated regression model is higher, indicating appropriate accuracy. For AC energy, the R2 value achieved is 96.39 percent. The corrected R2 (Radj) value is 96.95 percent, indicating that the generated regression model is very significant. In addition, the R2 (Rpred) value obtained is 93.54 percent. Figure 8 depicts the influence of irradiance and temperature on the AC energy produced. At maximum beam irradiance and temperature median, the maximum array energy production is seen in Figure 8.\n\nIncreased temperature may result in a drop in production. The figure shows a high array output at the median of diffused radiation and temperature. It has been discovered that for the installed plant to produce more power, the temperature must be between low and high. The figure depicts the influence of irradiance and wind speed on the AC energy produced. The illustration depicts the effects of wind speed and beam irradiance on AC output shown in the image. For a high Voltage AC output, a full beam irradiance and a medium wind speed are required.\n\nIn the illustration, the median of the graph yields the highest output. As a result, it is found that maximum beam irradiance, medium dispersed radiation, temperature, and wind speed are the finest examples of high production yields.\n\n\nProcedure taken for the analysis\n\nThese 52-kW plants are analyzed, and their performance is studied by dividing the study into three stages.\n\nFirst stage: Retrieving data from the online (DelREMO) monitoring system of all three plants. The plant location and its structure are also studied thoroughly.\n\nSecond stage: The key metrics like yield ratio, performance ratio, and capacity utilization factor of the plants are analyzed and compared.\n\nThird stage: Finally, the energy yield of the plants is compared with the result obtained from the modelling software PVsyst 7.1.8. The loss diagram of the plant is obtained and discussed.\n\nThere are specific performance parameters like reference yield, array yield, final yield, performance ratio and capacity utilization factor to determine the overall system’s performance. International Energy Agency has developed certain performance parameters for evaluating and analyzing the performance of grid-connected PV systems.9 DelREMO online monitoring system is shown in Figure 9. Comparison of all three 52-kW PV plant is illustrated in Figure 10. Performance parameters of the 52-kW power plant at Mechanical ‘C’ block, chemical engineering block, Faculty of Science and Humanities is listed in Tables 2, 3 and 4, respectively. A comparison of key highlights of 52-kW power plants at the institute is given in Table 5.\n\n\nSimulation using PVsyst\n\nThe maximum energy generated is in the month of March (6627 kWh), and the minimum is generated during July (4428kWh). The total energy produced during that year was 64606 kWh.\n\nAs shown in Table 6, the annual global irradiation is 1913.8 kWh/m2. The total energy obtained is 66903 kW/h. The average ambient temperature is 28.17°C and obtained annual average performance ratio obtained is 88.1%. A comparison of monitored results with the results acquitted from PVsyst V7.1.8 is listed in Table 7.\n\nFigure 11 shows the Lc value recorded as 0.45kWh/kWp/day and the La value as 0.16kW/kWp/day. Similarly, YF is recorded as 4.55kWh/kWp/day.\n\nThe global horizontal irradiance is 1927 kWh/m2/y, as shown in Figure 12. The effective irradiation on the collector plane is 1842 kWh/m2/y. After the PV conversion, the nominal array energy is 5559 kWh. The efficiency of the PV array is 15.46% at STC, while the virtual energy is 4726 kWh. The energy at the output after having the inverter losses comes out to be 4524 kWh.\n\nThe results obtained from the online monitoring system (DelREMO) is compared with the data acquired from the linear regression model and PVsyst software. From Table 5, the following observations are made:\n\n• The actual performance 52-kW plant in Science and Humanities closely matches the results obtained from the PVsyst.\n\n• The energy yield of the 52-kW plant in Chemical Engineering is slightly higher compared with the results obtained from the PVsyst.\n\n• The 52-kW plant in the Mechanical ‘C’ block operates with underperformance compared with the results obtained from the PVsyst and the other two PV plants.\n\n• The energy yield of the 52-kW plant in Chemical Engineering is high from March to July.\n\n• The energy yield of the 52-kW plant in Science and Humanities is high from August to December.\n\n• The Science and Humanities building is located in a place without any hindrance caused by tall buildings and trees, whereas the plant on the Mechanical ‘C’ block is surrounded by many tall buildings adjacent to the location.\n\n\nConclusions\n\nA performance study of three 52-kWgrid connected solar photovoltaic power plants installed on the Mechanical Engineering Block, Chemical Engineering Block, and Science & Humanities Block of rooftop SRMIST Kattankulathur was evaluated on an annual basis. The observations drawn from this case study are:\n\n• The maximum energy yield was observed in February (6828 kWh), whereas the lowest was recorded in May (81 kWh) from the Mechanical ‘C’ block.\n\n• The maximum energy yield was observed in March (7273 kWh), whereas the lowest was recorded in January (3543 kWh) from the Chemical Engineering block.\n\n• The maximum energy yield was observed in January (6989 kWh), whereas the lowest was recorded in February (2925 kWh) from Science and Humanities block.\n\n• By comparing the energy yield of the three 52-kW power plants, it is noted that the month of the maximum and minimum of the three plants are not similar. However, the energy yield value is nearer for the solar plants except for the Mechanical ‘C’ block.\n\n• The lowest energy yield in May in the Mechanical ‘C’ block is due to the plant’s shutdown during the lockdown during the pandemic covid situation.\n\n• The comparison of measured energy yield with PVsyst divulges that the 52-kW solar plant is functioning closer to the forecasted generation of energy yield from the PVsyst 7.1.8.\n\n• This case study gives an insight into identifying the location for large-scale implementation of PV plants across India. Also, this study helps calculate and evaluate other operational data based on net energy output. The obtained data on the PV system can also be helpful in large-scale applications.\n\n• All the operating plants have a good PR ratio. Also, the plant has been operating and feeding energy to the grid at a good percentage.\n\n• A precise forecast of solar energy generated is critical in today’s scenario for a specific site. This article may help researchers to have an insight understanding of forecasting AC power produced by the installed power plants.", "appendix": "Data availability\n\nOSF: Solar data, https://doi.org/10.17605/OSF.IO/NCRDU. 47\n\nThis project contains the following underlying data:\n\n• AC power 1st May 2020.xlsx2023-06-14 11:38 AM\n\n• AC voltage and current 1st May 2020.xlsx2023-06-14 11:38 AM\n\n• Solar On Grid power plant monthly Generation_2020-Specific data.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nDondariya C, Porwal D, Awasthi A, et al.: Amit Bhimte, Performance simulation of grid-connected rooftop solar PV system for small households: A case study of Ujjain, India. Energy Rep. 2018; 4: 546–553. 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Publisher Full Text\n\nMansour RB, Mateen Khan MA, Alsulaiman FA, et al.: Optimizing the Solar PV Tilt Angle to Maximize the Power Output: A Case Study for Saudi Arabia. IEEE Access. 2021; 9: 15914–15928. Publisher Full Text\n\nQureshi S, Phan-Van L, Nguyen LD, et al.: Rooftop solar policies feasibility assessment model: Vietnam case study. Energy Policy. 2023; 177: 113577. Publisher Full Text\n\nAn Y, Chen T, Shi L, et al.: Solar energy potential using GIS-based urban residential environmental data: A case study of Shenzhen, China. Sustain. Cities Soc. 2023; 93: 104547. Publisher Full Text\n\nJayaswal K, Sharma GS, Bhaskar MS, et al.: Global Solar Insolation Estimation and Investigation: A Case Study of Various Nations and Cities. IEEE Access. 2021; 9: 88069–88084. Publisher Full Text\n\nNguyen TBP, Wu Y-K, Pham M-H: A Novel Data-Driven Method to Estimate Invisible Solar Power Generation: A Case Study in Taiwan. IEEE Trans. Ind. Appl. Nov.-Dec. 2022; 58(6): 7057–7067. Publisher Full Text\n\nNguyen TBP, Wu Y-K, Pham M-H: A Novel Data-Driven Method to Estimate Invisible Solar Power Generation: A Case Study in Taiwan. IEEE Trans. Ind. Appl. Nov.-Dec. 2022; 58(6): 7057–7067. Publisher Full Text\n\nMontorfano M, Sbarbaro D, Morán L: Economic and Technical Evaluation of Solar-Assisted Water Pump Stations for Mining Applications: A Case of Study. IEEE Trans. Ind. Appl. Sept.-Oct. 2016; 52(5): 4454–4459. Publisher Full Text\n\nDi Grazia S, Tina GM: Optimal site selection for floating photovoltaic systems based on Geographic Information Systems (GIS) and Multi-Criteria Decision Analysis (MCDA): a case study. Int. J. Sustain. Energy. 2023; 1–23. Latest article. Publisher Full Text\n\nArtaş SB, Kocaman E, Bilgiç HH, et al.: Why PV panels must be recycled at the end of their economic life span? A case study on recycling together with the global situation. Process Saf. Environ. Prot. 2023; 174: 63–78. Publisher Full Text\n\nAbraim M, Salihi M, El Alani O, et al.: Techno-economic assessment of soiling losses in CSP and PV solar power plants: A case study for the semi-arid climate of Morocco. Energy Convers. Manag. 2022; 270: 116285. Publisher Full Text\n\nAbdel-Basset M, Mohamed R, Sharawi M, et al.: A comparative study of optimization algorithms for parameter estimation of PV solar cells and modules: Analysis and case studies. Energy Rep. 2022; 8: 13047–13065. Publisher Full Text\n\nHuppmann D, Browell J, Nastasi B, et al.: A research agenda for open energy science: Opportunities and perspectives of the F1000Research Energy Gateway [version 1; peer review: not peer reviewed]. F1000Res. 2022; 11: 896. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAljuboury AS, Al-Azzawi WK, Shakier LM, et al.: Analysis of a self-sufficient photovoltaic system for a remote, off-grid community [version 1; peer review: awaiting peer review]. F1000Res. 2022; 11: 1540. Publisher Full Text\n\nDivya NJ:Solar data. [Dataset]. OSF. 2023. Publisher Full Text" }	[ { "id": "202437", "date": "24 Nov 2023", "name": "Arvind R. Singh", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. What insights or suggestions may be obtained from the loss analysis conducted using PVsyst software to identify the elements contributing to the performance gap in the 52-kW photovoltaic (PV) plants? Furthermore, how can these observations be utilised to develop methods aimed at optimising the effectiveness and productivity of grid-connected photovoltaic (PV) systems operating in comparable environmental circumstances?\n2. What specific challenges or issues are associated with this rapid growth, and how does the research aim to address them in the context of performance evaluations of the 52-kW PV plants?\n3. This study offers a comprehensive analysis of the advantages and limits associated with different models. By examining these factors, we may get valuable insights into how the selection of a certain model type may influence the accuracy of photovoltaic (PV) performance forecasts.\n4. Can you elaborate on the practical applications and implications of the insights gained from this case study in terms of identifying suitable locations for large-scale PV plant implementations and the forecasting of solar energy generation in India, especially in the context of sustainable energy planning and grid integration?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Partly", "responses": [ { "c_id": "10652", "date": "29 Nov 2023", "name": "Divya Navamani J", "role": "Author Response", "response": "Rebuttal letter 1. What insights or suggestions may be obtained from the loss analysis conducted using PVsyst software to identify the elements contributing to the performance gap in the 52-kW photovoltaic (PV) plants? Furthermore, how can these observations be utilised to develop methods aimed at optimising the effectiveness and productivity of grid-connected photovoltaic (PV) systems operating in comparable environmental circumstances? The losses can be obtained for the whole year or month-wise from PVsyst software. This loss diagram gives an insight into the seasonal effect and is used to evaluate the impact of different losses. Furthermore, the loss diagram, which shows the primary causes of losses, offers a quick and insightful look at the quality of a PV system design. In the presented article, we have given the losses incurred in the plant. As a future scope, we have planned to suggest an optimizing methodology to work on the productivity of grid-connected photovoltaic (PV) systems based on the losses incurred. In addition, further studies can be made to increase the efficiency of PV systems by reducing the loss. 2. What specific challenges or issues are associated with this rapid growth, and how does the research aim to address them in the context of performance evaluations of the 52-kW PV plants? This article compares the performance evaluation of three PV plants with a 52-kW rating. The two plants are close to each other. The mechanical ‘C’ block plant is 1.3 km from the other two plants. The PR ratio of the Mechanical ‘C’ block plant is 60 %, whereas the other two plants are 68-69 %. All three plants were commissioned in 2019. The observations are made and informed the maintenance engineer after this case study. 3. This study offers a comprehensive analysis of the advantages and limits associated with different models. By examining these factors, we may get valuable insights into how the selection of a certain model type may influence the accuracy of photovoltaic (PV) performance forecasts. Yes. This case study is carried out to perform a comprehensive analysis of three 52-kW plants by theoretical study and using PVsyst software. Inference from the survey is drawn and presented in the manuscript. 4. Can you elaborate on the practical applications and implications of the insights gained from this case study in terms of identifying suitable locations for large-scale PV plant implementations and the forecasting of solar energy generation in India, especially in the context of sustainable energy planning and grid integration? This study helps to calculate and evaluate other operational data based on net energy output. The obtained data on the PV system can also be helpful in large-scale applications. The global horizontal solar irradiation of the location is presented with PR and CUF calculations. This will help us go for a large-scale solar PV plant on a site with a similar GHI." }, { "c_id": "11429", "date": "21 Jun 2024", "name": "Dr. Arvind R. Singh", "role": "Reviewer Response", "response": "The revision is satisfactory" }, { "c_id": "11883", "date": "29 Jun 2024", "name": "Divya Navamani J", "role": "Author Response", "response": "Thank you for the response" } ] }, { "id": "202434", "date": "12 Dec 2023", "name": "Deepa Kaliyaperumal", "expertise": [ "Reviewer Expertise Renewable Energy technologies", "Power electronics converters for application in Renewable", "Electric Vehicle", "Power Quality ...etc", "Machine learniong and deep learning applications in all fields" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWork has been reported professionally with good technical analysis. the analysis is excellent. 1)data availability to the user has to be mentioned in the content. 2)the conclusion of before pandemic, during the pandemic and after the pandemic can be listed in a table 3) The conclusion for summer and winter seasons can be listed\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [ { "c_id": "10761", "date": "13 Apr 2024", "name": "Divya Navamani J", "role": "Author Response", "response": "1)data availability to the user has to be mentioned in the content. Data availability-Underlying data OSF: Solar data, https://doi.org/10.17605/OSF.IO/NCRDU. 47 It is mentioned in the article. 2)the conclusion of before pandemic, during the pandemic and after the pandemic can be listed in a table. The plants were commissioned by Nov 2019. So, before pandemic, we took data from Jan 2020 -March 2020 and analysed in the article. 3) The conclusion for summer and winter seasons can be listed Entire analysis is carried out for the year 2020 (Jan-Dec) for both summer and winter season, The PR for both the seasons are presented." } ] }, { "id": "202435", "date": "25 Jun 2024", "name": "Zahira R.", "expertise": [ "Reviewer Expertise microgrid", "power quality", "renewable energy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 content is very well written. The prediction model for the 52-kW power plant is analyzed using PV syst software and the data's are compared well with the different scenario in institution buildings. In addition the model-based prediction equations are derived using the Minitab 16.2.1 software, and the results are compared with the real-time AC energy yield acquired from the three 52-kW plants for the year 2020.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1068
https://f1000research.com/articles/12-1067/v1	31 Aug 23	{ "type": "Policy Brief", "title": "Implementation of the digital government public policy in Colombia: Lessons learned from public entities", "authors": [ "Maribel Puentes-Poloche", "Alfredo Guzmán Rincón", "Favio Cala-Vitery", "Favio Cala-Vitery" ], "abstract": "The digital government policy aims to improve the efficiency, transparency, and quality of the services provided by the state. Research on this policy has been conducted from the perspective of the interaction between the state and each of the actors in society, neglecting the decisive role of public entities in its proper implementation. This policy brief focuses on the Colombian case and aimed to provide evidence to decision-makers and public entities on which variables should intervene to effectively implement the digital government policy. The recommendations given arise from a regression model, which identified that variables of web accessibility and usability, IPV6 adoption, the strategic component of Information and Communications Technology (ICT) use, the use of emerging technologies, transparency, and access to public information should be intervened.", "keywords": [ "digital government", "public policy", "public entities", "policy brief", "Colombia" ], "content": "Introduction\n\nThe digital government policy is conceptualized as “the use of digital technologies as an integral part of government modernization strategies to create public value”.1 The goal of this policy is to improve the efficiency, transparency, and quality of services offered by the State through the public entities that comprise it.2–5 There has been a growing interest in the study of this public policy from social actors and the academic community (see Refs. 6–8). Thus, most of the research that has been carried out analyses digital government public policy from the perspective of the interaction between the state and each of the actors in society, especially citizens (G2C), companies (G2B) and the state itself (G2G). In the context of these interactions, there has been a tendency to study which variables explain the implementation of the digital government policy from the perspectives of citizen value perceptions (e.g., Refs. 9, 10) and business owners (e.g., Ref. 11).\n\nThis policy brief approaches digital government policy from a perspective centred on the use of technologies, considering both the interaction of the state with society’s actors and the central role of public entities in the implementation of this policy.12 While previous research has examined governmental actions for the implementation of the digital government policy (e.g., Refs. 13–15), the approach adopted here highlights the importance of understanding the strategies and efforts implemented by public entities to achieve effective implementation of the digital government policy. This allows decision-makers and public entities to have stronger evidence and recommendations on the variables they should intervene in to achieve successful implementation of this policy.\n\nDue to the heterogeneity of digital government policy worldwide, this policy brief is framed within the Colombian public policy. The selection of the country is based on its performance in this policy compared to other OECD countries. According to the OECD (2019), Colombia had a 75% progress in the implementation of this policy, being surpassed only by Korea and the United Kingdom with 76% and 78%, respectively, and surpassing countries such as Denmark, Japan, Canada, Spain, and Israel, as well as the OECD average, which was 50%. Therefore, the study on public entities in Colombia provides feedback on the variables that public entities should intervene to achieve the implementation of the digital government policy, especially in developing countries.\n\n\nContextualization of the digital government policy in Colombia\n\nIn 2009, the Colombian government enacted the “Gobierno en Línea” (Online Government) as a policy to adopt the use of Information and Communication Technologies (ICTs) in public entities, with the aim of providing efficient services to different interest groups of the state (citizens, businesses, employees, and public entities). Subsequently, Law 1712 of 2014 regulated the application of the online government policy in all sectors.16 In 2015, “Gobierno en Línea” became the digital government policy through Decree 1078, becoming one of the policies for institutional planning and performance of public entities. Decree 1078 of 2015 regulated the digital government policy, where governance is one of its elements based on the relationship between national and territorial entities, central and decentralized level, under the leadership of the Ministry of Information and Communications Technology.17 Currently, Decree 767 of 2022 established guidelines articulated with Decree 1263 of 2022, orienting them towards the integration of the digital government policy with digital transformation.\n\nRegarding the evaluation of the implementation of the digital government policy, the state developed the “Formulario Único de Reporte de Avances de la Gestión” (Single Management Progress Report Form – FURAG in Spanish), it consists of 54 questions, of which 27 are single selection (50%). In these questions, entities are asked about their level of progress in the development of documents such as the Information Technology Strategic Plan (PETI in Spanish), the implementation of topics such as privacy and information security, information systems, data analytics, among others. Similarly, of the 20 multiple-choice questions (37%), entities can select one or several options associated with web access or usability, information technology topics such as management performed, PETI content, projects, purchases, IPV6 adoption, use and appropriation, process automation, implementation of emerging technologies, and information contained in the Electronic Headquarters for transparency and access to public information. Finally, the remaining 17 questions (13%) are numerical selection and are associated with amounts of open data, services on the interoperability platform, exchange of information, and the status of IT projects in each entity.\n\nThe measurement methodology, as well as its periodicity and other guidelines for the application of FURAG, are carried out by the Administrative Department of Public Function in coordination with the Council for Institutional Management and Performance. The latter is composed of the leading policy entities, including the Ministry of Information and Communication Technologies. In the first measurement, a baseline was established for each entity to report its progress in the implementation and development of the digital government policy. Based on the annual results, a self-diagnosis of strengths and weaknesses is conducted, along with the proposal of an action plan that establishes concrete actions to improve performance in areas where lower scores were obtained.\n\nFinally, the calculation of the level of implementation of the public digital government policy is the result of a polytomous model that describes the probability that an entity i with a performance θi responds to an item j in category k or any higher category, compared to responding in a category lower than k. The probability Pxj(θi) is calculated using the following formula:\n\nWhere:\n\n• θi represents the performance of entity i in relation to the evaluated item or set of items.\n\n• xj represents the number of categories k for item j, i.e., the number of response options or levels available for that item.\n\n• αj is the item discrimination parameter, which determines the sensitivity of the item to differentiate between different levels of performance.\n\n• δj is the parameter that defines the item difficulty, indicating at what performance level individuals are expected to have a 50% probability of responding in the category or higher.\n\n\nMethods\n\nTo fulfill the objective of the policy brief, which is to provide evidence to decision-makers and public entities on which variables should intervene to effectively implement the digital government policy, a model based on multiple linear regression was proposed using data obtained from the FURAG of the 147 national public entities that make up the executive branch of the State. The multiple linear regression model is mathematically defined as:\n\nIn the case of the policy brief, the dependent variable (y) was the level of implementation of the digital government policy, which is evaluated on a scale of 0 to 100. For each of the independent variables, they were quantified as the total number of requirements that each entity complies with regarding the FURAG evaluation model. The independent variables are described in Table 1.\n\nThe operationalization of the model was performed in version 29 of the SPSS software18 using the multiple linear regression technique INTRO, For the replicability of the results in case they do not have SPSS, they can use R Studio.19 To evaluate the quality of the model, various statistics were used, including R2, adjusted R2, ANOVA test, and VIF collinearity test. The R2 coefficient represents the proportion of the total variability in the dependent variable explained by the multiple linear regression model, where values close to 1 indicate that the model explains the variability in the data well. The adjusted R2 coefficient, which takes into account the number of predictor variables in the model, represents a better fit of the model when values close to 1 are obtained. The ANOVA test evaluates the overall significance of the multiple linear regression model, with a p-value less than 0.05 indicating that the model is significant and at least one of the predictor variables is important in explaining the variability in the dependent variable.\n\nThe Variance Inflation Factor (VIF) collinearity test measures how much the variance of the regression coefficient of a predictor variable is inflated due to the presence of other predictor variables in the model. In this case, the presence of collinearity problems in the model was discarded if the VIF result for each variable was less than 4. Finally, the statistical significance of the predictive variables in the final model was analyzed, with those having a p-value less than 0.05 considered statistically significant.\n\n\nPerformance of public entities in the implementation of digital government public policy in Colombia\n\nIn Colombia, there are 147 national-level public entities distributed across 24 sectors, where 17.7% are affiliated with the Ministry of Finance and Public Credit, 11.6% with the Ministry of Defense, 7.5% with the Ministry of Health and Social Protection, 6.1% with the Ministry of Education, and 57.1% with other administrative departments. Based on the results obtained from the FURAG for the year 2021, last measurement, the average level of implementation of the digital government policy was 85.67% with a deviation of 9.95%. As shown in Figure 1, the results obtained by the “Corporación De Alta Tecnología Para La Defensa” (Defence High Technology Corporation) with an implementation level of 44.02%, “Sociedad De Televisión De Las Islas LTDA” (Society Of Television Of The Islands LTDA) with 52.81%, “Centro De Diagnostico Automotor De Caldas LTDA” (Centre For Automotive Diagnostics Of Caldas LTDA) with 58.96%, and “La Imprenta Nacional De Colombia” (The National Printing Office of Colombia) with 59.22% are considered outliers.\n\n\nVariables that affect the implementation of digital government policy in Colombia\n\nBased on the results of the FURAG for the 2021 period and using a linear regression model, it was possible to establish which variables influence the level of implementation of the digital government policy in Colombia. The developed model showed an R2 of 0.819 and an adjusted R2 of 0.79. Table 2 shows that the regression model was statistically significant overall (F = 29.20, p-value < 0.001), indicating that the predictors were collectively related to the dependent variable.\n\nWith that being said, the model did not present any problems of collinearity (see Table 3) since the VIF statistic values were less than 4. Thus, it was considered that the variables that affect the implementation of the digital government policy were PAW, PUW, PAIPV, PEAUTIC, PTE, and PTAIP (See the meaning of the acronyms in Table 3), as they presented p-values less than 0.05. Finally, Figure 2 presents the model’s predictions contrasted with the actual data obtained in FURAG, where the dependent variable was the level of implementation of the digital government policy.\n\n\nActionable recommendations\n\nBased on the level of implementation of digital government policy in Colombia, there are several actionable recommendations that can be given to decision makers and government entities to achieve the implementation of this policy in various contexts. In relation to web accessibility (PAW), the State should establish standardized criteria so that the portals of public entities allow all people, including those with disabilities or limitations, to access the information and services available to them. This is materialized in the case of Colombia in Resolution 1519 of 2020 from the Ministry of Information and Communication Technologies, where 23 criteria that public entities must comply with in relation to web accessibility are established.20\n\nFor web usability (PUW), public entities must design a clear and organized structure of content so that portal users can find the information they are looking for. Similarly, usability testing should be performed to confirm the clarity and organization of the contents. Furthermore, it is necessary for web pages and platforms used for service provision to be compatible with various devices. The processes related to the services provided by the entity should also be simplified so that all stakeholder groups can access them from the same application.\n\nFurthermore, the adoption of IPV6 (PAIPV) is essential for the sustainability of digital government and its implications, since the IPV4 protocol has reached the limit of the number of IP addresses it can support. Therefore, if public entities do not migrate, they may have difficulties in expanding their networks and providing services. This results in the limitation of users’ ability to access certain resources of the public entity. Given the relevance of the adoption of IPV6, the transition should be planned carefully, considering the difficulties in accessing information and service provision that may arise from not executing it correctly. In addition, the variety of platforms to be linked within and outside the public entity should be recognized, and the transition should have sufficient budget and time for its implementation.\n\nDue to the role of ICTs in digital government policy, a strategic component for their use and appropriation should be developed (PEAUTIC). In this sense, public entities should plan for the long term, train employees and stakeholders, promote collaboration and citizen participation, ensure the security and privacy of information, ensure universal access and digital inclusion, and evaluate and measure the impact of the strategy.\n\nOn the other hand, as public entities become familiar with emerging technologies (PTE) (e.g., blockchain, big data, artificial intelligence, robotics, etc.), it is important that they are prepared to integrate them effectively into their operations, especially in the provision of services. In this context, the implementation and use of emerging technologies by entities should be based on an analysis of how they can be used. Thus, the use of partnerships with the private sector and academia to take advantage of specialized knowledge and technical experience, especially in cases where public entities have no experience in the implementation and use of such ICTs, should be evaluated. Finally, it is not enough to have emerging technologies; the entity’s staff, as well as the social actors who will use them, must be trained.\n\nFinally, transparency and access to public information (PTAIP) are fundamental in digital government policy, as they allow stakeholders to be informed and participate actively in decision-making. In this sense, the use of digital technologies facilitates the dissemination of public information and promotes accountability by public entities. Thus, entities should implement tables of retention of documentary evidence, complaint and claim forms, access to reports and open data, public transition reports, directories of information on public servers and employees, and budget execution, among others.\n\n\nConclusions\n\nThe implementation of digital government policy in countries such as Colombia is a crucial step towards the transformation of the state, allowing for the improvement of efficiency, transparency, and quality of services. In this context, it is recognized that the implementation of digital government policy is not an easy task. Therefore, from the particular case of Colombia, public entities require the adoption of standardized criteria regarding web accessibility and usability, the transition to IPV6, and the integration of emerging technologies, as well as a clear and effective strategy for the appropriation of ICTs by employees and citizens. Additionally, it is important to highlight the relevance of transparency and access to public information as a fundamental tool for accountability and the participation of stakeholder groups in the entities.\n\nIn this regard, it is important to reflect on the challenges and opportunities presented by the implementation of digital government policy in developing countries. First, it is necessary to consider the importance of investment in technology and training of employees and stakeholders for the success of these policies. Similarly, the need for adequate strategic planning that allows public entities to effectively adapt to changes and technological advances in the long term is highlighted. Second, the need for effective collaboration between the public sector, the private sector, and academia to leverage specialized knowledge and technical expertise in the implementation and use of ICTs should be considered. In this sense, the construction of strategic alliances can be a useful tool for the success of digital government policy. Finally, it is important to highlight the importance of the participation of stakeholder groups in the implementation and evaluation of these policies. Inclusion and universal access to digital information and services are essential to ensure a more just and equitable society.\n\nWith the actionable recommendations outlined in this guidance document, as well as the documentation of the Colombian case through modelling, new tools are provided for decision-makers and public entities to implement digital government policy. Furthermore, this policy brief strengthens the literature on the implementation of digital government policies from the perspectives of public entities.", "appendix": "Data availability\n\nData used in this study are from the FURAG dataset of the Colombia 2023 self report, available from the Función Pública [Public Function] website https://www.funcionpublica.gov.co/web/mipg/resultados-medicion . A guide for how to apply for dataset access is available at: https://www.funcionpublica.gov.co/web/mipg/resultados-medicion .\n\n\nReferences\n\nOECD: Government at a Glance 2019. OECD; 2019 [cited 2023 Feb 20]. Reference Source\n\nZein RM, Twinomurinzi H: Towards Blockchain Technology to Support Digital Government.Kő A, Francesconi E, Anderst-Kotsis G, et al., editors. Electronic Government and the Information Systems Perspective. Cham: Springer International Publishing; 2019 [cited 2023 Apr 14]; pp. 207–220. Publisher Full Text\n\nZeng Y, Zhang Q, Zhao Q, et al.: Doing more among institutional boundaries: Platform-enabled government in China. Rev. Policy Res. 2022 [cited 2023 Feb 20]; 40: 458–478. Publisher Full Text\n\nRico-Pinto JS, Sánchez-Torre J: A review of studies about factors in G2G interoperability. Proceedings of the European Conference on e-Government, ECEG. Santiago de Compostela: Academic Conferences Limited. 2018; p. 188.\n\nMohamed MA: Towards Adoption of Government Enterprise Architecture: The Cases of Egypt and Syria. Proceedings of the 13th European Conference on eGovernment: ECEG 2013. 2013; p. 345.\n\nTwizeyimana JD, Andersson A: The public value of E-Government – A literature review. Gov. Inf. Q. 2019 [cited 2023 Apr 21]; 36: 167–178. Publisher Full Text Reference Source\n\nBaheer BA, Lamas D, Sousa S: A Systematic Literature Review on Existing Digital Government Architectures: State-of-the-Art, Challenges, and Prospects. Administrative Sciences. 2020 [cited 2023 Apr 21]; 10: 25. Publisher Full Text Reference Source\n\nLiva G, Codagnone C, Misuraca G, et al.: Exploring digital government transformation: a literature review. Proceedings of the 13th International Conference on Theory and Practice of Electronic Governance. Athens Greece: ACM. 2020 [cited 2023 Apr 21]; pp. 502–509. Publisher Full Text\n\nHu G, Chohan SR, Liu J: Does IoT service orchestration in public services enrich the citizens’ perceived value of digital society? Asian J. Technol. Innov. 2022 [cited 2023 Apr 21]; 30: 217–243. Publisher Full Text\n\nLi Y, Shang H: Service quality, perceived value, and citizens’ continuous-use intention regarding e-government: Empirical evidence from China. Inf. Manag. 2020 [cited 2023 Apr 21]; 57: 103197. Publisher Full Text Reference Source\n\nKankanhalli A, Charalabidis Y, Mellouli S: IoT and AI for Smart Government: A Research Agenda. Gov. Inf. Q. 2019 [cited 2023 Apr 21]; 36: 304–309. Publisher Full Text Reference Source\n\nSandoval-Almazán R, Luna-Reyes LF, Luna-Reyes DE, et al.: Building Digital Government Strategies. Cham: Springer International Publishing; 2017 [cited 2023 Apr 21]. Publisher Full Text\n\nAl-khafaji NJ, Shittu AJK, Osman WRS: G2G interaction among local agencies in developing countries based on diffusion of innovations theory. 2014 Fourth International Conference on Digital Information and Communication Technology and its Applications (DICTAP). 2014; pp. 125–131.\n\nOuma FK: Impediments to interagency statistical information sharing amongst government agencies in Uganda: A G2G adoption. 2014 IST-Africa Conference Proceedings. Le Meridien Ile Maurice, Pointe Aux Piments, Mauritius: IEEE. 2014 [cited 2023 Jan 26]; pp. 1–11. Reference Source\n\nVan Cauter L, Snoeck M, Crompvoets J: Removing the Blinkers: What a Process View Learns About G2G Information Systems in Flanders.Tambouris E, Janssen M, Scholl HJ, et al., editors. Electronic Government. Cham: Springer International Publishing; 2015 [cited 2023 Mar 3]; pp. 209–221. Publisher Full Text\n\nCongress of the Republic of Colombia. Law 1712 de 2014. 1712 2014. http\n\nCongress of the Republic of Colombia. Decree1078 de 2015. 1078 2015.\n\nSPSS Statistics - Visión general: [cited 2023 Apr 25]. Reference Source\n\nR Studio: [cited 2023 Apr 25]. Reference Source\n\nMinTIC: Colombia in Resolution 1519 of 2020." }	[ { "id": "202563", "date": "13 Sep 2023", "name": "Diana Teresa Parra-Sánchez", "expertise": [ "Reviewer Expertise Digital transformation", "IoT adoption." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe policy brief focuses on the Colombian case to provide evidence to decision-makers and public entities on which variables should intervene to implement the digital government policy effectively.\nIntroduction: It is recommended to present the article's structure in sections.\nThere is a need to delve deeper into the digital government policy implemented in Colombia.\nThe presentation of the equation to calculate the level of implementation of public policy is suggested to be presented in the method section.\nThe document does not have a Discussion section. The implications of the study carried out are not appreciated. Likewise, there is a lack of greater depth in the recommendations. It is recommended that the authors of the document present the method in greater detail. In this way, readers can understand and replicate what is presented in the document.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Partly\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? No\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "240803", "date": "29 Mar 2024", "name": "Giancarlo Vecchi", "expertise": [ "Reviewer Expertise Digitalisation policies - Public Sector modernization" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 suggest authors to: a) to provide comments to the Figure 2, underlining the programs/actions that are in a good or bad trend\nb) See the sentence p. 3: “the approach adopted here highlights the importance of understanding the strategies and efforts implemented by public entities to achieve effective implementation of the digital government policy” Here change as: “…the importance of understanding the strategies and efforts used/adopted by public entities.”\nc) Variables p. 5: I cant understand if the ‘internal and external interoperability among system’ is present or covered by the different variables\nd) Page 8: See the sentence: “For web usability (PUW), public entities must design a clear and organized structure of content so that portal users can find the information they are looking for. Similarly, usability testing should be performed to confirm the clarity and organization of the contents.” You can underline the use of co-design processes with different categories of citizens and internal users. See: https://services.blog.gov.uk/2021/03/23/from-user-centred-design-to-co-design/ https:/www3.weforum.org/docs/WEF_Co_designing_Tech_with_Civil_Society_2021.pdf\n\"A government strategy in the field of digitalization policies, could promote - as in some experiences (e.g. the task forces on national digitalization in UK, Estonia, Italy, etc. (see Mergel I. 2019. “Digital service teams in government.” Government Information Quarterly vol. 36, n.4) - a community of developers, based on the involvement of private and third sectors expert, able to test the digital solution, to collaborate in the development of apps, etc.\"\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Partly\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Yes\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1067
https://f1000research.com/articles/12-1065/v1	31 Aug 23	{ "type": "Study Protocol", "title": "Assessing the impact of hospital marketing in attracting the patient for service consumption in tertiary care hospital in central India.", "authors": [ "Yohana Sheikh", "Deepika Kanyal", "Deepika Kanyal" ], "abstract": "Background: Hospital service is a field that is constantly changing, with the availability of creativity, opportunities motivating passion, and allowing experts to thrive. Healthcare marketing is a scientific discipline since it uses concepts, plans, and methods that are exclusive to both conventional and social marketing. The importance of this study is to build up the bridge and relationship between the hospital and the patient. To introduce the healthcare services to the patient to let them know what services the hospital provides and what advantages they will receive for their health betterment. With the help of this study, we can track the flow of the patient from which median they are coming, for example, traditional marketing or digital marketing. This study will promote and analyse where the marketing strategies are lacking and where they need to be improved with the help of recommendations of this study. The objectives of this study to bridge the gap by providing the corrective information and developing the relationship between patient and hospital, to create a leadership position and differentiate from competitors. Methods: A retrospective study approach will be adopted in the study. It includes a collection of data and information direct from the marketing department. Study will be conducted in the tertiary care hospital of central India. Secondary type of data will be collected from the Hospital Management Information system. Expected Result: The expected result of the study will be to bridge the gap by providing the corrective information and developing the relationship between patient and hospital, to create a leadership position and differentiate from competitors to know where the institute is standing in the market.", "keywords": [ "Healthcare", "Marketing", "Patient", "Services", "Quality", "Hospital", "Customers", "Branding." ], "content": "Introduction\n\nHospital service is a field that is constantly updating, with the availability of creativity, chances motivating passion, and allowing experts to thrive. Hospital marketing is a scientific discipline since it uses concepts, plans, and methods that are exclusive to both conventional and social marketing. There are marketplaces and services, but there are no monetary equivalents in healthcare marketing, which makes it unique. The depiction of a healthy community, the identification of a group of chronically ill individuals, the certainty that sick individuals receive adequate medical care by undergoing the entire rehabilitation program, the professional and social reintegration of patients, etc., are examples of how its application is practical. The need for marketing in the healthcare industry originated from worries about society's health. An effective marketing plan includes a detailed analysis of the patient's needs, the identification of latent expectations, and introducing novel healthcare services that sick people have not directly requested. Healthcare professionals may help to develop their target market, engage with it, and provide value to it. New marketers begin with the client rather than the goods or facilities. They focus more on creating a long-lasting connection than completing a single transaction. They aim for excellent customer satisfaction because they want customers to continue doing business with the same provider. Marketers have traditionally employed various techniques, such as promotional products, market analysis, pricing, product creation, advertising, and distribution.1\n\nMarketing tools are an essential consideration in this situation. The effectiveness of the healthcare system as a whole is significantly impacted by marketing. For instance, branding a hospital as a supplier of specialized services signals a change in focus for customers. Still, it also impacts the executives, doctors, and staff that make the hospital run and its role in the community. Promotional initiatives also impact healthcare systems by affecting how the general public views healthcare issues and objectives. Marketing may have a significant effect on the medical system by serving as a crucial conduit linking customers and healthcare providers and encouraging the dissemination of health information. Healthcare marketing really affects costumer’s behaviour and significantly affects how well the healthcare system runs.2\n\nQuality of service in healthcare is characterized as a discrepancy between patient views and standards. The definition of service quality is the customer's total perception or appraisal of the business and its services relative inadequacy or superiority. It may be calculated by comparing consumers' hopes with their views of the real performance of the service. Before using the services, customers build their expectations. Individuals form impressions during the service delivery process, and when assessing the conclusion of the service encounter, they contrast their views with their expectations. Service quality implies that the customer's needs and expectations should be met during service delivery and suggests that patients judge a hospital's service quality based on its service output, service procedure, and physical surroundings. This is because service quality is thought to satisfy patients' demands.3\n\nNovel marketing tactics used by private hospitals to promote their facilities are explored, as are sample hospitals managed by various corporate managements, using the 7Ps of marketing, which stand for place, product, promotion, people, physical evidence, pricing, and process.\n\n1) Product: Inpatient services, outpatient, and healthcare promotion comprise the three product lines that comprise the length of the healthcare product mix.\n\n2) Pricing: The term ‘pricing’ is most of the time used to refer to the actual price that an institute makes. Pricing for healthcare services is often done while considering factors such as bed occupancy, service quality, the value of infrastructure, overhead, wages of the medical staff, nursing staff, administrative personnel, and others.\n\n3) Place: A crucial component of a hospital is its location. The hospital should be conveniently located for patients and have proper pollution protection. Each hospital will work to make services accessible and convenient for its intended clients.\n\n4) Promotion: Promoting the goods and facilities as a marketing strategy facilitates communication between the dealer and client. The vendor hopes to convince and influence the people to buy their goods or facilities by doing this.\n\n5) People mix: Hospitals are a knowledge-based sector; thus, people are essential. Patients and employees are included in this population.\n\n6) Physical evidence: proof of physical is the setting where the facilities are provided using intangible or physical goods and where the business and the client interact.\n\n7) Process: In the services marketing sector, it is called ‘interactive marketing’. One of the critical seven aspects of services marketing. According to one definition, customer engagement is the “performance of the implemented through which a sequence of connections between vendors of services and consumers are designed to facilitate efficient involvement in both service manufacturing and consumption that meets the needs and desires that consumers have and generates positive perceived quality”.4\n\nBy creating guiding principles and moral standards, bioethics aims to define the therapeutic activity and any other associated activity required to keep a healthcare institution operating. The field of bioethics is quite broad and has a history encompassing several academic fields, including sociology, theology, law, philosophy, and medicine. The approach to build and sustain connections with the target customer includes advertising and marketing (patients). It was required to create ethical guidelines for healthcare marketing in order to control this activity. Promotional messaging needs to be truthful and avoid setting up false expectations. The hospital or the doctor must be able to provide the services listed in the advertisement. While marketing information aims to draw attention to more intriguing topics, it should still be more grounded in reality. In this context, it is important to identify the groups and subcategories that are more receptive to different aspects of the advertising message. A client who is in severe pain will be easily swayed and have a great motive to get better. The information presented must not misrepresent reality or offer patients false hope to be ethical.5 New, creative applications are giving branding a newfound prominence. Even though there have been times when branding hasn't worked, marketers are starting to identify the right situations where it operates. The choice of a brand name is one of today's branding strategy issues and concerns. The effectiveness of a branding plan will be impacted by this underlying problem.6 Since these communities may aid in understanding customer requirements and in fostering brand loyalty and involvement, they have developed into a potent tool for marketers. This paper attempts to investigate how consumers can be persuaded to participate in online communities in this regard by analysing the impact of involvement in virtual communities on consumer commitment.7 Branding is the process by which a firm or organization becomes unique in the eyes of its customers. The achievement of any business depends on its ability to create and manage a brand, encompassing its name, reputation, and identity. A firm's branding and the degree to which customers connect to its brand dictate how, where, and when it engages with its customers. The company's most robust differentiation in the market now is its branding.8 There is no evidence indicating that using advertising campaigns to influence the worldwide public's views of an entire city, region, or country is anything more than a vain and irrational waste of taxpayers' money. Marketing communications are acceptable when the task is simply one of selling a product, and the product can just as easily be the vacation resorts or potential investment possibilities of a nation as the goods of a corporation.9\n\nThe importance of the study ‘Impact of hospital marketing in attracting the patient for service consumptions in tertiary care hospital in central India’ is to build up the bridge and relation between the healthcare industry and the patient. To introduce the healthcare services to the patient to let them know what services the hospital is providing and what advantages they are going to receive for their health betterment in an easy manner. With the help of this study, we can track the flow of the patient from which median they are coming, for example tradition marketing or digital marketing. The patient is getting information from the radio, social media, pamphlet, or other sources (ATL-above the line, BTL-below the line). delivering exceptional consumer experiences and creating a strong, prosperous, and dominant reputation on markets for healthcare services. To understand how the patient’s, feel about the effectiveness and consequences of their interactions with the medical institution. We can track which median of marketing is affecting more in the mind of customers and which are the target population, and on which source the hospital has to work more for analyzing the gap between the patient’s expectations and the services the hospital is providing. This study may help the hospital to improve or enhance its services or the marketing strategies by analysing the data.\n\n\n\n1) To bridge the gap by providing the corrective information and developing the relationship between patient and hospital.\n\n2) To create a leadership position and differentiate from competitors.\n\n\nMethods\n\nThis will be a quantitative cross sectional retrospective study. It includes a collection of secondary data and information direct from the marketing department and software of marketing department. This study will be conducted in the tertiary care hospital of central India. Duration of this study will be three months.\n\nAs all the data of this study is going to be collected directly from the marketing department’s software so sample size and sample techniques will be not applicable in this study.\n\nThe inclusion of this study is the software of the marketing department. This study will be based on secondary data and will be collected form the marketing department under the guidance of marketing administrative officer, head of the department and the manager of that department from the software of hospital that is HMIS and secondary data. Exclusion of this study will be the hospital staff, healthcare workers and patient and patient’s relatives\n\nThe procedure of the data collection will be conducted in the tertiary care hospital of central India which is highly reputed hospital. Type of the data of this study is secondary data which will be directly collected from the software. Primarily data of this study will be collected from the Hospital Management Information system (HMIS) software version 2.08 (version 2 and patch.08) which was installed on 10th March 2022.\n\nThe study will be published in an institutional indexed journal and the data will be publicly available after publication.\n\n\nResults\n\nThe expected result of the study will be to bridge the gap by providing the corrective information and developing the relationship between patient and hospital, to create a leadership position and differentiate from competitors.\n\n\nDiscussion\n\nIn their study, Mark J. Kay in the USA, stated that to compare and contrasts the customer (or patient) viewpoint with the organizational perspective to generate a view on what is ‘salient’ or vital to the healthcare marketing’s sector.2 The structural issues in healthcare, such as social service systems, technological advancements in medicine, and rising healthcare expenses, aggravate this ‘salience dilemma’. The report reviews a few studies and examines how customers face more challenging health decisions.2\n\nAccording to the Chao-Chan Wu study, their findings show that patient loyalty is influenced by hospital brand image both directly and indirectly.3 This indicates that a strong hospital brand not only fosters customers’ loyalty directly but also boosts patient happiness by elevating the perceived quality of service, encouraging patients to repeat. Moreover, a hospital's brand image is a leading determinant in patient experience, improving service quality, and patient loyalty. The results also point to the importance of the link between service quality and patient satisfaction in influencing patient loyalty to healthcare institutions. This study suggests that to improve loyalty, service quality and patient happiness, hospital management should work to establish and maintain a positive brand image of the healthcare institute. This study also makes some recommendations about the development and upkeep of a positive hospital brand image.3\n\nAccording to Garnick et al., the study stated that an example can be used to show the varied definitions of hospital market areas.10 As a single-county, Fresno County, where Fresno Community Hospital is located, has 12 additional hospitals inside its borders. Seven hospitals can be found within a 15-mile range of Fresno Community Hospital. Five healthcare institutions admit at least 5% of their patients from the census tracts that make up to 60% of admissions to the Fresno Community. All five hospitals fall within a 15-mile radius. The area of that organization, the 15-mile group, and the able to-share origin cluster each has 12–14 hospitals from a commercial standpoint.10\n\nAccording to the Wani et al., study, despite efforts to improve the healthcare of the Indian populace, the circumstances wasn’t same as before.11 It is ironic that while spending a disproportionately significant portion of its gross domestic product (GDP) on health, India's results are average. The Declaration and Fundamental Principles of the Indian Constitution constantly stress the need for an ‘equal society’, which includes the government's obligation to provide basic healthcare.11\n\nThe synopsis for this study has been sent to the Internal Ethical Committee in the Datta Meghe Institute of Higher Education and Research for ethical approval and is currently under consideration.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgments\n\nWe appreciate the support of the faculty of MHA, School of allied health sciences, Datta Meghe Institute of Higher Education and Research.\n\n\nReferences\n\nPurcarea EV: The impact of marketing strategies in healthcare systems. J. Med. Life. 2019 Apr; 12(2): 93–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKay MJ: Healthcare marketing: what is salient? Int. J. Pharm. Healthc. Mark. 2007 Sep 11; 1(3): 247–263. Publisher Full Text\n\nWu CC: The impact of hospital brand image on service quality, patient satisfaction and loyalty. Afr. J. Bus. Manag. 2011 Jun 18; 5(12): 4873.\n\nSreenivas T, Srinivasarao B, Srinivasa Rao U, et al.: An analysis of marketing mix in hospitals. Int. J. Adv. Res. Manag. Soc. Sci. 2013 Feb; 2(4): 187–207.\n\nSolomon M, Radu G, Hostiuc M, et al.: Ethical issues in advertising and promotion of medical units. Rom J. Ophthalmol. 2016 Oct; 60(4): 216–218. PubMed Abstract\n\nRooney JA: Branding: a trend for today and tomorrow. J. Prod. Brand. Manag. 1995 Oct 1; 4(4): 48–55. Publisher Full Text\n\nCasaló LV, Flavián C, Guinalíu M: Promoting consumer's participation in virtual brand communities: A new paradigm in branding strategy. J. Mark. Commun. 2008 Feb 1; 14(1): 19–36. Publisher Full Text\n\nAl-Zyoud MF: Social media marketing, functional branding strategy, and intentional branding. Probl. Perspect. Manag. 2018; 16(3): 102–116. Publisher Full Text\n\nPralea AR: Branding in health marketing. Bulletin of the Transilvania University of Brasov. Econ. Sci. 2011 Jul 1; 4(2): 65.\n\nGarnick DW, Luft HS, Robinson JC, et al.: Appropriate measures of hospital market areas. Health Serv. Res. 1987 Apr; 22(1): 69–89. PubMed Abstract\n\nWani NU, Taneja K, Adlakha N: Health System in India: Opportunities and Challenges for Enhancements. IOSR Journal of Business and Management (IOSR-JBM). 2013 Mar. e-ISSN." }	[ { "id": "202722", "date": "05 Oct 2023", "name": "Manoja Das", "expertise": [ "Reviewer Expertise Health systems research", "Implementation research", "Mixed methods 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\nThe study protocol aims to document the effect of the business promotion efforts at a tertiary care private hospital. There are some critical observations.\nRationale\nThe rationale and objectives need to be aligned. The research components are to be clearly mentioned and the study design, data collection and analysis must be aligned with the same.\nThe objective is to document impact on the patient turnover/attendance due to marketing. The marketing efforts are not clearly mentioned, and what was done new or differently, which is under study for the impact documentation.\n\nMethods\n\nA cross-section study can not be retrospective in nature, unless secondary data is used for analysis. Please check the study design.\n\nThe retrospective data from the marketing team shall be a trend analysis of repeated data collected over time.\n\nIs the data to be collected over three months or retrospective data for three month shall be collected?\nThe objective and data collection planned do not match.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "227882", "date": "20 Dec 2023", "name": "Babatunde Abiodun Balogun", "expertise": [ "Reviewer Expertise Healthcare marketing", "health communication", "consumer behaviour", "customer engagement", "health service management" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. Abstract The 267-word abstract of the protocol provides a broad overview on hospital service and healthcare marketing, the research design of the study and the resultant potential outcome. Clarity is needed on the two major concepts on which the study is based – hospital service and healthcare marketing. The reader ought to know the focal points of the study. The method should state how the data collected will be analysed and interpreted. The expected result should indicate the presumption of the hypothesis being tested.\n2. Introduction: The introduction is an assemblage of many concepts, but none stands out for its depth. The literature review is disjointed, and does not discuss, analyse, and critique the current state of research in the field of hospital service or healthcare marketing. The authors have mentioned some novel marketing tactics used by private hospitals, but this claim is unfounded. Numerous published studies have documented the use of such tactics. The term ‘bioethics’ is misplaced. It is unclear why the author has referred to the term for this study.\n3. Rationale The study aims to uncover how a tertiary private hospital can advance its services or marketing strategies by evaluating two aspects of the interaction between the hospital and its patients, namely, effectiveness and consequences. This rationale is vague. The authors may consider focusing on the hospital’s service or marketing strategies, but not the two in one study. Each concept has its variables that could be tested by this study. What do you mean by \"patients' feel\"? What are the parameters to measure? The potential benefit of this study should be elaborated. Does the study intend to contribute to theory? In which field will this contribution be? What are the existing gaps?\n4. Methods The authors have provided inadequate details about the data to be collected and analysed, and how. It is difficult to ascertain the relevance and appropriateness of the research design. Has the research design been tested or used previously by other researchers? Conducting quantitative analysis requires a considerable sample size, which the authors should clarify. Please remove the ambiguity about the duration of the study or reword it.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-1065
https://f1000research.com/articles/12-1064/v1	31 Aug 23	{ "type": "Genome Note", "title": "Complete mitochondrial genome and the phylogenetic position of a new Annelida species belonging to the genus Syllis", "authors": [ "Jun Young Chae", "JinHo Kim", "Tae-Wook Kang", "Jae il Lee", "Hyung-Ho Lee", "Moo-Sang Kim", "Jun Young Chae", "JinHo Kim", "Tae-Wook Kang", "Jae il Lee", "Hyung-Ho Lee" ], "abstract": "The family Syllidae is the most taxonomically complex of the phylum Annelida. Although the gene order in the phylum Annelida's mitochondrial DNA (mtDNA) is well conserved, several exceptional cases have been reported. In this study, we describe the mitochondrial genome of a Syllis sp. that is 17,092 bp in length and contains 13 mitochondrial protein-coding genes (PCGs), 23 transfer RNA (tRNA) genes, including two tRNA-M, two ribosomal RNA (rRNA) genes, and a putative control region between tRNA-W and tRNA-G distinguished by a single short noncoding region. However, the gene order is not similar to those of other species in the family Syllidae. Phylogenetic analyses of 18S rRNA and 13 PCGs sequences demonstrated that this worm was clustered with other Syllis species in the family Syllidae. This is the first study to reveal the complete mitochondrial genome sequence of a previously unidentified Syllis sp. improving our understanding of the molecular biological characteristics of the poorly known genus Syllis.", "keywords": [ "Annelida", "Syllidae", "Syllis", "Mitochondrial genome" ], "content": "Introduction\n\nMitochondria are cellular organelles that function as an energy factory in animals. They are inherited from the maternal lineage and can be used to trace phylogenetical relationships. The mitochondrial genome sequence thus provides very valuable data for genetic or taxological research. In addition, the sequence and order of genes in the mitochondrial DNA (mtDNA) can be used to uncover phylogenetic relationships between evolutionarily close or distant species (Vallès and Boore 2006). Owing to the startling progress of sequencing technology leading to next-generation sequencing (NGS), many mitogenome sequences from diverse species have accumulated in public databases in recent years. However, obtaining complete mitogenome data remains difficult because of the diversity of mitogenome structure, gene arrangement, and transfer RNA (tRNA) structure, according to species (Bernt et al. 2013).\n\nMore than 700 species of the family Syllidae have been classified into 74 genera (Aguado and Martín 2009). Despite the number of species, the taxonomy of the family Syllidae remains incomplete with many unclarified correlations. Few studies describing the mtDNA sequence of members of the phylum Annelida are available from the first decade of the 2000s (Boore 2004; Jennings and Halanych 2005; Bleidorn et al. 2006; Zhong et al. 2008; Mwinyi et al. 2009; Shen et al. 2009). However, some mtDNA sequences from the phylum Annelida were reported using NGS (Aguado et al. 2016), which was revealed that the gene order in the mtDNA sequence in this phylum is well conserved, although Weigert et al. (2016) reported that differences in gene order occur more frequently than expected. Despite this controversy, information on the mtDNA sequences of the phylum Annelida remains insufficient. In this study, we obtained the complete mitochondrial genome sequence of a novel species belonging to the genus Syllis. This mitogenome contributes to our understanding of the conservation of mitochondrial gene order in the phylum Annelida.\n\n\nMethods\n\nThe molecular biology experiments were conducted at Pukyong National University. The specimen was obtained (36°12′N, 129°25′E) from a small worm attached to Farrer’s scallop (Chlamys farreri K. H. Jones & Preston, 1904) and this small worm had a lots of legs on the body. Genomic DNA (gDNA) was extracted from the whole body of the worm using the Bead™ Genomic DNA Prep Kit For Animal Tissue (Biofact, Republic of Korea). We deposited the gDNA at Pukyong National University (Voucher no. PKNU_2021_002: Jun Young Chae, jychae@pukyong.ac.kr) because the whole worm was used for extraction gDNA owing to its very small size. The cox1 gene was amplified using an invertebrate universal primer set (LCO1490, 5′-GGTCAACAAATCATAAAGATATTGG-3′; HCO2198, 5′-TAAACTTCA-GGGTGACCAAAAAATCA-3′; Folmer et al. 1994) by PCR. PCR was conducted using HelixAmp™ Taq-Plus Polymerase (NANOHELIX, Republic of Korea) and SimpliAmp™ Thermal Cycler (RRID:SCR_023004). The PCR condition was pre-denaturation at 95°C for 2 minutes; 30 cycles at 95°C for 20 seconds, 40°C for 40 seconds, and 72°C for 45 seconds; final extension at 72°C for 5 minutes. The amplicon was sequenced, and the cox1 sequence was compared to identify this species using the NCBI Basic Local Alignment Search Tool (BLAST) (RRID:SCR_004870) (Johnson et al. 2008).\n\nThe bioinformatics experiments were conducted at theMOAGEN. gDNA (1 μg) was sheared using the S220 Ultra sonicator (Covaris, USA). MGIEasy DNA library prep kit (MGI, China) was used for library preparation according to the manufacturer’s instructions. Briefly, fragmented gDNA was selected based on its size using AMPure XP magnetic beads and the fragmented gDNA was end-repaired and a-tailed at 37°C for 30 minutes, and 65°C for 15 minutes. Indexing adapter was ligated to the ends of the DNA fragments at 23°C for 60 minutes. PCR was performed to enrich those DNA fragments that have adapter molecules after purifying the adapter-ligated DNA. Thermocycler conditions were as follows: 95°C for 3 minutes, 7 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes. The double stranded library is quantified using QauntiFluor ONE dsDNA System (Promega, USA). The library is circularized at 37 °C for 30 minutes, and then digested at 37°C for 30 minutes, followed by cleanup of circularization product. The library is incubated at 30°C for 25 minutes using DNB enzyme for making DNA nanoball. Finally, the library was quantified by QauntiFluor ssDNA System (Promega, USA). NGS was conducted on the MGISEQ-2000 (MGI, China) platform with 150 bp paired-end reads. The raw reads were screened using the cutadapt tool (RRID:SCR_011841) (Martin 2011), and all clean sequences were used for de novo assembly using the assembler of the CLC Genomics Workbench (RRID:SCR_011853) ver. 20.04 (Qiagen). The circular form of the mitogenome was confirmed using the “Map to Reference” tool of Geneious (RRID:SCR_010519) software ver. 2021.2.2 by remapping the filtered data into the contig sequence from the de novo assembly. Annotation of the complete mtDNA sequence was performed using the MITOS WebServer and manually corrected using SnapGene (RRID:SCR_015052) software ver. 5.3.2 based on previously released mtDNA annotation information (Aguado et al. 2016). The mitogenome map was prepared using ORDRWA (Greiner et al. 2019). We also obtained a contig that included the 18S rRNA sequence from the de novo assembly of CLC Genomics Workbench, registered at GenBank (accession no. OP341337) (Chae and Kim 2022b), and used this sequence to construct a phylogenetic tree for a more detailed taxonomic classification of this worm.\n\nBayesian inference (BI) with MrBayes (RRID:SCR_012067) 3.2.6 (Huelsenbeck and Ronquist 2001), was used to perform phylogenetic analysis based on nucleotide sequences of 13 protein-coding genes (PCGs) of 16 available mitochondrial genomes in the class Polychaeta, and Orbinia latreilliid (accession no. NC_007933) (Bleidorn et al. 2006) was set as an outgroup. Additional phylogenetic analysis was conducted based on the nucleotide sequences of 18S rRNA of 41 species belonging to the family Syllidae.\n\n\nResults\n\nBLASTN analysis showed that the partial sequence of cox1 from the worm had the highest similarity to that of Syllis pigmentata (accession no. EF123774.1), at 87.76%, which is a relatively low identity score. The sequence was also similar to that of S. ehlersioides (accession no. EF123773.1), S. alternat (accession no. HQ932467.1), S. albae (accession no. KX792209.1), Typosyllis antoni (accession no. KX752426.1), and T. augeneri (accession no. JF903788.1) in decreasing order at 87.62%, 83.07%, 82.76%, 81.90%, and 77.70%, respectively. All these species belong to the genus Syllis, suggesting that our worm may be a new species belonging to this genus.\n\nThe raw data output of NGS was deposited in the Sequence Read Archive (SRA) database (accession no. SRR18465399) (theMOAGEN 2022b). The length of the complete mtDNA sequence was 17,092 bp, and the sequence was registered in GenBank (accession no. ON312495) (Chae and Kim 2022a). A total of 38 genes were predicted in this mitochondrial genome, including 13 PCGs, 23 tRNA genes, and two rRNA genes, and all genes are encoded on the positive strand. The gene composition of this mtDNA is similar to that of T. antoni (Aguado et al. 2016), with two tRNA-M being present and consistent with previously reported data (Kurabayashi et al. 2006; Zhang et al. 2009).\n\nAlmost all PCGs start with an ATG codon (atp6, cox3, nad2, cytb, atp8, cob, nad3, nad1, cox2, nad4, and nad4l), whereas nad6 and nad5 have ATA as a start codon and cox1 uniquely starts with an AAC codon found in insects (Kim et al. 2016). Nad2 and nad1 terminate with truncated T- codons. The remaining 11 PCGs have a TAA stop codon, except for nad4 and nad4l, which have TAG stop codons. The mtDNA of T. antoni contains cox3 as the first gene and tRNA-P as the last gene. Although the worm mtDNA examined in this study begins with tRNA-G and ends with tRNA-W, the gene order is the same from nad6 to cox2 (nad6, tRNA-F, tRNA-D, tRNA-T, tRNA-S2, tRNA-K, tRNA-Y, Large rRNA, tRNA-R, tRNA-S1, tRNA-E, tRNA-V, tRNA-I, atp8, cob, nad3, tRNA-N, tRNA-M, tRNA-M, nad5, nad1, and cox2) in the two species (Figure 1). By contrast, the mitogenome sequences of Ramisyllis multicaudata and R. kingghidorahi are entirely dissimilar from that of the worm described in this study. Their gene order is the same because these two species belong to the same genus. Similarly, the differences in gene order between the mitogenomes of Syllis sp. and T. antoni result from these species being from different genera. These results are consistent with gene order in mitogenomes being more diverse than expected and accord with previous reports of different gene orders in the Phylum Annelida (Aguado et al. 2016; Weigert et al. 2016). We identified 23 tRNA genes, including two tRNA-L, two tRNA-S, and two tRNA-M. The standard cloverleaf structure was observed in the predicted secondary structure of 15 tRNAs, except for tRNA-R and tRNA-S2, which lack a D-arm, and five tRNAs (tRNA-D, tRNA-F, tRNA-G, tRNA-M, and tRNA-Y), which lack a loop structure in the T-arm. Small rRNA with 880 bp was located between tRNA-L1 and tRNA-A. Large rRNA was observed between tRNA-Y and nad2, and its length was 1016 bp. The length of the putative control region was 1,291 bp and was located between tRNA-W and tRNA-G. The gene order is different from that of mitogenomes of other species in the family Syllidae (Figure 1).\n\nThe mitogenome gene order of the Syllis sp. described in this study was compared with that of other species belonging to the family Syllidae. The circular mitogenomes in these mtDNA maps are shown linearly to more easily compare the gene orders. The tRNAs, ATP synthase F0 subunits, cytochrome c oxidase subunits, NADH dehydrogenase subunits, ribosomal RNAs, and cytochrome b are marked blue, green, pink, yellow, red, and purple, respectively. The gene name is indicated at the top of each box. mtDNA, mitochondrial DNA; tRNA, transfer RNA.\n\nPhylogenetic analysis showed that the Syllis sp. was clustered with T. Antoni, and the clade had a close relationship with the family Syllidae members belonging to the order Phyllodocida (Figure 2), suggesting that this worm is a previously unclassified species. Additional phylogenetic analysis was performed to confirm the genus of the worm based on the 18S rRNA sequence. The results show that it was grouped with S. busseltonensis, and this node was assembled with various Syllis spp. rather than with the genus Haplosyllis or Branchiosyllis (Figure 3). These results are consistent with the examined worm being a novel Syllis sp. that has not yet been described.\n\nA phylogenetic tree was constructed using the nucleotide sequence of PCGs of the Syllis sp. described in this study and 16 species obtained from GenBank with Orbinia latreilliid (NC_007933) as an outgroup. GenBank accession numbers are given with species names. Posterior probabilities of the Bayesian inference are indicated as node numbers. Class, order, and family taxonomic ranks are shown adjacent to vertical black bars. The Syllis sp. analyzed in this study is indicated by an arrowhead. PCG, protein-coding genes.\n\nA phylogenetic tree was constructed based on the nucleotide sequence of the 18S rRNA gene from the Syllis sp. described in this study and the nucleotide sequences of 40 species from the family Syllidae in GenBank. GenBank accession numbers accompany species names. Node numbers indicate the posterior probabilities of the Bayesian inference. The Syllis sp. described in this study is indicated by the arrowhead. rDNA, ribosomal DNA.\n\n\nEthics and consent\n\nThere is no human or animal involvement in the study. Since the sample is an insect, there is no need for ethical approval or permission to collect the sample.", "appendix": "Data availability\n\nGenBank: Syllis sp. JYC-2022 mitochondrion, complete genome. Accession number ON312495; https://identifiers.org/ncbi/insdc:ON312495 (Chae and Kim 2022a).\n\nGenBank: Syllis sp. JYC-2022 small subunit ribosomal RNA gene, partial sequence. Accession number OP341337; https://identifiers.org/ncbi/insdc:OP341337 (Chae and Kim 2022b).\n\nBioProject: Syllis sp. JYC-2022. Accession number PRJNA818342; https://identifiers.org/NCBI/bioproject:PRJNA818342 (theMOAGEN 2022a).\n\nSRA: Syllis sp. JYC-2022. Accession number SRR18465399; https://identifiers.org/insdc.sra:SRR18465399 (theMOAGEN 2022b).\n\nBioSample: Syllis sp. JYC-2022. Accession number SAMN26856052; https://identifiers.org/biosample:SAMN26856052 (theMOAGEN 2022c).\n\n\nReferences\n\nAguado MT, Martín GS: Phylogeny of Syllidae (Polychaeta) based on morphological data. Zool. Scr. 2009; 38: 379–402. Publisher Full Text\n\nAguado MT, Richter S, Sontowski R, et al.: Syllidae mitochondrial gene order is unusually variable for Annelida. Gene. 2016; 594: 89–96. PubMed Abstract \| Publisher Full Text\n\nBernt M, Braband A, Schierwater B, et al.: Genetic aspects of mitochondrial genome evolution. Mol. Phylogenet. Evol. 2013; 69(2): 328–338. Publisher Full Text\n\nBleidorn C, Podsiadlowski L, Bartolomaeus T: The complete mitochondrial genome of the orbiniid Polychaete Orbinia Latreillii (Annelida, Orbiniidae) – A novel gene order for Annelida and implications for Annelid phylogeny. Gene. 2006; 370: 96–103. PubMed Abstract \| Publisher Full Text\n\nBoore JL: Complete mitochondrial genome sequence of Urechis Caupo, a representative of the phylum Echiura mtDNA. BMC Genomics. 2004; 5: 1–8. Publisher Full Text\n\nChae JY, Kim MS: Syllis sp. JYC-2022 mitochondrion, complete genome. [Dataset]. GenBank. 2022a. Reference Source\n\nChae JY, Kim MS: Syllis sp. JYC-2022 small subunit ribosomal RNA gene, partial sequence. [Dataset]. GenBank. 2022b. Reference Source\n\nFolmer O, Black M, Hoeh W, et al.: DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 1994; 3: 294–299. PubMed Abstract\n\nGreiner S, Lehwark P, Bock R: OrganellarGenomeDRAW (OGDRAW) version 1.3. 1: expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Res. 2019; 47(W1): W59–W64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuelsenbeck JP, Ronquist F: MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001; 17(8): 754–755. Publisher Full Text\n\nJennings RM, Halanych KM: Mitochondrial Genomes of Clymenella Torquata (Maldanidae) and Riftia Pachyptila (Siboglinidae): Evidence for Conserved Gene Order in Annelida. Mol. Biol. Evol. 2005; 22: 210–222. PubMed Abstract \| Publisher Full Text\n\nJohnson M, Zaretskaya I, Raytselis I, et al.: NCBI BLAST: a better web interface. Nucleic Acids Res. 2008; 36(Web Server issue): W5–W9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim MJ, Jeong SY, Jeong JC, et al.: Complete mitochondrial genome of the endangered flower chafer Osmoderma Opicum (Coleoptera: Scarabaeidae). Mitochondrial DNA B Resour. 2016; 1: 148–149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurabayashi A, Usuki C, Mikami N, et al.: Complete nucleotide sequence of the mitochondrial genome of a Malagasy poison frog Mantella madagascariensis: evolutionary implications on mitochondrial genomes of higher anuran groups. Mol. Phylogenet. Evol. 2006; 39: 223–236. PubMed Abstract \| Publisher Full Text\n\nMartin M: Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. Journal. 2011; 17(1): 10–12. Publisher Full Text\n\nMwinyi A, Meyer A, Bleidorn C, et al.: Mitochondrial genome sequence and gene order of Sipunculus Nudus give additional support for an inclusion of Sipuncula into Annelida. BMC Genomics. 2009; 10: 1–16. Publisher Full Text\n\nShen X, Ma X, Ren J, et al.: A close phylogenetic relationship between Sipuncula and Annelida evidenced from the complete mitochondrial genome sequence of Phascolosoma esculenta. BMC Genomics. 2009; 10: 111–136. Publisher Full Text\n\ntheMOAGEN: Syllis sp. JYC-2022. [Dataset]. BioProject. 2022a. Reference Source\n\ntheMOAGEN: Syllis sp. JYC-2022. [Dataset]. SRA 2022b. Reference Source\n\ntheMOAGEN: Syllis sp. JYC-2022. [Dataset]. BioSample. 2022c. Reference Source\n\nVallès Y, Boore JL: Lophotrochozoan mitochondrial genomes. Integr. Comp. Biol. 2006; 46: 544–557. PubMed Abstract \| Publisher Full Text\n\nWeigert A, Golombek A, Gerth M, et al.: Evolution of mitochondrial gene order in Annelida. Mol. Phylogenet. Evol. 2016; 94: 196–206. PubMed Abstract \| Publisher Full Text\n\nZhang JF, Nie LW, Wang Y, et al.: The complete mitochondrial genome of the large-headed frog, Limnonectes bannaensis (Amphibia: Anura), and a novel gene organization in the vertebrate mtDNA. Gene. 2009; 442: 119–127. PubMed Abstract \| Publisher Full Text\n\nZhong M, Struck TH, Halanych KM: Phylogenetic information from three mitochondrial genomes of Terebelliformia (Annelida) worms and duplication of the methionine tRNA. Gene. 2008; 416: 11–21. PubMed Abstract \| Publisher Full Text" }	[ { "id": "223333", "date": "14 Dec 2023", "name": "Yingdong Li", "expertise": [ "Reviewer Expertise Hydrobiology" ], "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 presented the complete mitogenome of a potentially new Annelida species and explored its gene order and phylogenetic relationship with other Polychaeta species. I strongly recommend that the authors address the following issues and resubmit the manuscript.\nFirstly, the authors should provide additional evidence, such as morphological characteristics to confirm the classification of this as a new species. Secondly, the authors only compared the gene orders among four species. If the reasons for the limited comparison are not outlined in the Methods section, providing this clarification would enhance the manuscript. Otherwise, expanding the comparison would strengthen the study.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? No\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Partly", "responses": [] }, { "id": "278083", "date": "01 Jul 2024", "name": "Zhi Wang", "expertise": [ "Reviewer Expertise Taxonomy and phylogeny of Annelida", "Biodiversity and Ecology of marine benthos" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article focuses on the Syllidae family, which is one of the most taxonomically complex families in Annelida. It describes the mitochondrial genome of a speciesÃÂ Syllis sp., which is 17,092 bp long and contains 13 PCGs, 23 tRNA genes, 2 rRNA genes, and a putative control region. The gene order is different from other species in the family. Phylogenetic analyses show that this worm clusters with other Syllis species. This is the first study to reveal the complete mitochondrial genome sequence of a previously unstudiedÃÂ Syllis sp., enhancing our understanding of the genus.\nDetailed comments:\n1. Introduction-paragraph 2: The first sentence should introduce the number of valid syllid species described in the world, and the updated data should cite the WoRMS (2024).\n2. Methods-paragraph 1: \"this small worm had a lots of legs on the body\". \"legs\" should be replaced by the specific term \"parapodia\".\n3. Results-paragraph 4: \"suggesting that this worm is a previously unclassified species\". I don't think this conclusion isÃÂ rigorously accurate, since the database currently only include a small part of the described annelid species. I suggest to revise this sentence to \"suggesting that this worm may be a previously unclassified species\"\n4. I suggest to provide the morphological characterisitics or high solution photos of this Syllis sp., which would be helpful for taxonomists to do further studies on this group of Polychaetes.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1064
https://f1000research.com/articles/12-1063/v1	31 Aug 23	{ "type": "Research Article", "title": "Complementary and alternative medicine use by Bangladeshi adult patients with diabetes and hypertension: A multicenter study", "authors": [ "Faroque Md. Mohsin", "Sudipta Das Gupta", "Shahriar Hasan", "Sarah Tahera Mahmud", "Irin Yasmin", "Md. Anwar Hossain", "Md. Mahbub Alam", "Md. Nazrul Islam", "Sudipta Das Gupta", "Shahriar Hasan", "Sarah Tahera Mahmud", "Irin Yasmin", "Md. Anwar Hossain", "Md. Mahbub Alam", "Md. Nazrul Islam" ], "abstract": "Background: The use of complementary and alternative medicine (CAM) in Bangladesh is widespread, but scarce information exists about its use in treating hypertension and diabetes in adults. The study aimed to understand the distribution and determinants of CAM use among Bangladeshi adult patients with hypertension and diabetes. Methods: This multicenter cross-sectional study was conducted among 400 adult patients with hypertension (n=200) and diabetes (n=200). Data were collected from two specialised diabetes and hypertension treatment centers. Interviews were used to gather socio-demographic data, while medical records were used to obtain information on hypertension and diabetes. A multivariate logistic regression model was used to identify the most influential predictors of CAM usage. Results: Overall, 29% (n=116) of patients used CAM for diabetes and hypertension, with 37.5% (n=41) for hypertension and 37.5% (n=75) for diabetes. Older patients (aged >59) were 7.5 times more likely to use CAM (OR=7.527; 95% CI: 3.224 to 17.574, p<0.001) than young adults (aged <35 years). The currently employed patients were less likely to be CAM users (OR=0.429; 95% CI: 0.264 to 0.697, p<0.001) than the unemployed patients. It was less common for patients from nuclear families to use CAM (OR=0.178; 95% CI: 0.111 to 0.286, p<0.001) than those from extended families. Patients with hypertension were less likely (OR=0.430; 95% CI: 0.275 to 0.672, p<0.001) to be CAM users than patients with diabetes. Most of the CAM users were influenced by media and family members. The most common reasons for CAM use were its perceived effectiveness, fewer side effects, and low cost. Conclusions: The study observed a high prevalence of CAM users among patients with hypertension and diabetes. The most common CAM modalities were homeopathy, ayurveda and unani medicine. The findings suggest future directions for research and have practical implications for chronic disease treatment.", "keywords": [ "Complementary and alternative medicine", "Hypertension", "Diabetes", "Chronic diseases", "Bangladesh" ], "content": "Introduction\n\nNon-communicable diseases (NCDs), namely hypertension and diabetes, have emerged as a global pandemic in the form of higher morbidity and mortality rate in the last few years (Terzic & Waldman, 2011; Mendis et al., 2014). The World Health Organization (WHO) predicts NCDs will rise globally in the next decade. About 74% of all deaths worldwide are attributed to NCDs, which kill 41 million people annually.\n\nThe prevalence of NCDs is significantly higher in developing nations (Terzic & Waldman, 2011). Notably, the number of fatalities from NCDs has already surpassed the total number of deaths from communicable, maternal, perinatal, and nutritional diseases on every continent except Africa (Wang & Wang, 2020). Hypertension is the reason for half of coronary heart disease and around two-thirds of the burden of cerebrovascular disease globally (Benjamin et al., 2019; Stanaway et al., 2018) and also enhances the risk of stroke, kidney failure, disability, and premature death (Rapsomaniki et al., 2014; Wei et al., 2017).\n\nIn Bangladesh, hypertension is the major preventable cause of a wide range of health issues and the resulting financial strain (Chowdhury et al., 2022; Hossain et al., 2022a). Approximately 68% of fatalities in Bangladesh are attributable to NCDs, and hypertension is responsible for 15–20% (El-Saharty et al., 2013). There is a wide range of variation in the prevalence of hypertension reported by several studies ranging from 11 to 44% in Bangladesh (Hossain et al., 2022b; Khanam et al., 2015). It’s estimated that the number of people living with diabetes worldwide is expected to climb to 783 million by 2045 (Saeedi et al., 2019). According to the International Diabetes Federation’s (IDF) latest report, 90 million people are living with diabetes in South East Asia. The report highlighted that diabetes is expected to reach 113 million adults by 2030 and 151 million by 2045 in South East Asian countries. The latest IDF report also showed that over 3 in 4 adults with diabetes live in low- and middle-income countries like Bangladesh. The IDF also reported that 13.1 million cases of diabetes were diagnosed in 2021 and will increase to 22.3 million cases by 2045 in Bangladesh. Because there are only a handful of studies related to the prevalence of hypertension and diabetes in Bangladesh, the findings are not consistent yet. Although, there is a dearth of research that properly combines systematic reviews and meta-analyses to compile the current collection of knowledge on the incidence of hypertension.\n\nIn the current health system, Bangladesh lacks a population-based surveillance system to monitor the prevalence and inadequate treatment and control mechanisms for hypertension and diabetes (Chowdhury et al., 2018). Despite the progress of modern medicine, complementary and alternative medicine (CAM) such as homeopathic medicine, Ayurveda, Unani, acupuncture, acupressure, yoga and other CAM therapies have traditionally been accepted as necessary treatments and found to be more prevalent among Asians (Calcagni et al., 2019; Peltzer & Pengpid, 2015), including Bangladeshi (Shahjalal et al., 2022a). A study suggested that CAM may help to promote an integrative, participatory model of diabetes care (DiNardo et al., 2012). Several studies showed that 2 to 36 million diabetic patients used CAM in the USA (Dham et al., 2006; Egede et al., 2002; Eisenberg et al., 1998). A large number of studies showed that 39.3% of diabetic patients used CAM in the UAE (Radwan et al., 2020), 36.7% of diabetic patients used CAM in Turkey (Yıldırım & Marakoğlu, 2018), 46.3% in Australia (Manya et al., 2012), 53.5% in Pakistan (Jawed et al., 2019), and 67.7% in India (Kumar et al., 2006). A systemic review reported that the prevalence of CAM use for diabetes ranged from 17 to 72.8% (Chang et al., 2007).\n\nIn the USA, CAM used for hypertension was higher among those without diagnosed hypertension than those with diagnosed hypertension, and only 7.8% of CAM users reported using CAM to treat hypertension (Bell et al., 2006). About 29% of hypertensive patients use CAM in Nigeria (Osamor & Owumi, 2010), 63.5% in Germany (Jeschke et al., 2009), and 63.9% in India (Shafiq et al., 2003).\n\nSustainable Development Goal (SDG) 3 has set target 3.4 of reducing premature mortality from NCDs through prevention and treatment, and target 3.8 of achieving universal health coverage through the access to quality essential health-care services and safe, effective, quality and affordable essential medicines and vaccines for all. In that case, CAM can play a significant role in achieving these two targets because in recent studies of Bangladesh, 33% of patients utilized CAM exclusively for treatment purposes (Shahjalal et al., 2022b) while 32.8% NCD patients used CAM for the treatment of chronic illness (Shahjalal et al., 2022a). The abovementioned findings of Bangladesh-based studies indicated that even though conventional medicine has come a long way, the number of people using CAM keeps growing across the country.\n\nDespite the widespread acceptance of CAM alongside conventional treatment, there is still a lack of data on the prevalence, utilization, and associated factors of using CAM for chronic illness among NCD patients in Bangladesh, as the studies conducted in Bangladesh did not address the current situation of CAM usage for diabetes and hypertension exclusively. As one of the studies conducted in three tertiary care hospitals in Dhaka explored the CAM usage among the chronic illness patients only (Shahjalal et al., 2022a); another study was conducted on CAM usage for diabetes patients but only in a specific region. These studies did not explore the nationwide perspective of CAM usage for diabetes and hypertension only, although despite the widespread acceptance of CAM alongside modern treatment, there is still a lack of data on the prevalence, utilization, and associated factors of using CAM among diabetes and hypertension patients in Bangladesh. For that reason, this study aimed to assess the prevalence of using CAM for diabetes and hypertension, along with exploring the factors associated with both CAM usage.\n\n\nMethods\n\nThis cross-sectional study was conducted at the National Healthcare Network (NHN), Adabor Center, Dhaka and Hypertension and Research Center, Rangpur, Bangladesh. The two health care centers were chosen purposively and the survey took place March to May 2021. When patients arrived at the specified hospitals’ outpatient departments, they were assessed and asked about their diagnosis of diabetes and hypertension as well as duration of suffering for diseases. Then, the selected patients were approached for enrollment in our study. Patients were included in the study based on the following inclusion criteria: (i) age of equal or more than 18 years, (ii) having confirmed diabetes and hypertension, and (iii) were able to follow common instructions from the interviewer. Patients who had any psychological disorders and those who refused to give the consent were excluded from the study.\n\nThe data collection was done by the research team. Patients who sought treatment at the study health facilities during the data collection window were eligible to participate. Every eligible patient was selected using a purposive sampling technique at the health care centre. Upon screening the inclusion criteria and obtaining informed consent from all eligible patients, a face-to-face interview was conducted in the outpatient department of the survey health facilities using a structured questionnaire. A pilot survey was conducted among 24 patients. It was intended to investigate the capacity to comprehend the relevant techniques and trouble-some situations while interviewing. Following piloting, we made the necessary corrections in the questionnaire. In order to avoid repatriation and potential skewing of the results, we used the patients’ IDs to ensure they did not repeat the questionnaire. The required minimum sample size was 288 at 90% power, 95% CI of 0.05 to 1.96, with 29 % of a patient used CAM for NCDs in Bangladesh (Shahjalal et al., 2022a), and the margin of error was 5%. Finally, we collected 400 samples (response rate was 94%), including 200 diabetes and 200 hypertensive patients from the outpatient departments between March and May 2021. The process of data collection, identification and inclusion flow diagram is presented in Figure 1. A detailed description of the study variables has been discussed in Supplementary File 1 (Hasan et al., 2023).\n\nOutcome variables\n\nThe outcome variable was the use of CAM for diabetes and hypertension. The sample was classified according to CAM usage such as (i) CAM user (code, 1) and (ii) CAM non-user (code, 0). The outcome was determined using one question. “Did you ever use CAM for diabetes or hypertension?”\n\nIndependent variables\n\nIndependent variables included patients’ sociodemographic, clinical and healthcare data on diabetes and hypertension. Participants’ gender, age, marital status, education, religion, family type, employment status, monthly household income and smoking history were included as the socio-demographic variables.\n\nStatistical analyses were carried out using SPSS (IBM version 22.0). The qualitative variables were described in terms of frequencies and percentages, and continuous variables in terms of means and standard deviations. The association between socio-demographic and clinical variables with the CAM user determined. Multivariate logistic regression was used to identify the most influential predictors of CAM usage. Multivariate logistic regression adjusted for socio-demographic and clinical characteristics were used to find out the effect of CAM usage.\n\nParticipation in this study was entirely voluntary. Informed written consent was obtained from study participants following explanation of the study aims and objectives. The study was approved by the Ethics Board of Government Unani & Ayurvedic Medical College (GUAMC), Dhaka, Bangladesh (Ref-2021/OR-GUAMC/IRB-No.101). Ethics approval was obtained in February 2021 before the study commenced. All procedures were performed in accordance with relevant guidelines and regulations.\n\n\nResults\n\nA total of 400 patients participated in the study. The frequency distribution of their sociodemographic characteristics is presented in Table 1. The age of the respondents ranged from 21 to 81 years with a mean of (47.04±11.7) years. The majority of the patients’ ages were between 35 to 59 years (n=268, 67.0%) and most participants were female (n=230, 57.6%). The majority of the respondents were Muslim (n=347, 86.8%) and had tertiary-level education (n=170, 42.5%). Most respondents were married (n=392, 98%) and lived in a nuclear family (n=236, 59%.). More than half of the respondents were unemployed (n=251, 62.8%) and household income was 11,000 to 30,000 BDT (n=213, 53.3%). Most participants were non-smokers (n=328, 82.0%).\n\nIn total, 29% (n=116) of patients utilised CAM for hypertension and diabetes, where 20.5% (n=41) of hypertension and 37.5% (n=75) of diabetes patients used CAM (Table 2). Among CAM users, we found three types of CAM that the patients use for the treatment purpose of diabetes and hypertension. Homeopathic medicine, ayurvedic medicine and Unani medicine were the most common CAM modalities (Figure 2). About 50.86% (n=68) used homeopathic medicine, while 31.80% (n=37) used Ayurvedic medicine and 15.51% (n=18) of patients used Unani medicine to treat diabetes and hypertension.\n\nTable 2 describes the relationship between the socio-demographic and clinical characteristics with the CAM use. In order to predict the use of CAM on socio-demographic and clinical characteristics, logistic regression was employed.\n\nFor respondents who were aged more than 59 years, they were 7.5 times more likely to use CAM (OR=7.527; 95% CI: 3.224 to 17.574, p<0.001), and the respondents who were aged between 35 to 59 years old were 1.7 times more likely to use CAM (OR=1.779; 95% CI: 0.830 to 3.814, p=0.139) than the respondents who were aged less than 35 years old, respectively. It was observed that male respondents were less likely to use CAM (OR=0.848; 95% CI: 0546 to 1.316, p=0.462) than the female respondents.\n\nOn the other hand, Muslims were 1.6 times more likely to use CAM (OR=1.657; 95% CI: 0.821 to 3.344, p=0.159) than the other religious participants. The respondents who had no education were 1.4 times more likely to use CAM (OR=1.495; 95% CI: 0.784 to 2.851, p=0.222) than the higher secondary level educated participants; additionally, primary educated level respondents were also 1.09 times more likely to be a user of CAM (OR=1.094; 95% CI: 0.639 to 1.879, p=0.742). However, the secondary level educated respondent were less likely (OR=0.772; 95% CI: 0.408 to 1.459, p=0.425) to use CAM than tertiary level educated respondents.\n\nThe never married respondents were less likely (OR=0.813; 95% CI: 0.162 to 4.087, p=0.801) to be users of CAM rather than married respondents, and the respondents who lived in a nuclear family were less likely to use CAM (OR=0.178; 95% CI: 0.111 to 0.286, p<0.001) than the respondents who lived in an extended family. The respondents who were currently employed were less likely to be CAM users (OR=0.429; 95% CI: 0.264 to 0.697, p<0.001) than the unemployed respondents.\n\nTable 2 also describes that the respondents who earned less than or equal to 10,000 BDT (OR=0.339; 95% CI: 0.134 to 0.862, p=0.023) and the respondents whose income was between 11,000 to 30,000 BDT (OR=0.794; 95% CI: 0.504 to 1.251, p=0.321) were less likely to be a user of CAM than the respondents whose income was between 31,000 to 50,000 BDT, respectively.\n\nThe current smoker respondents were 1.2 times more likely (OR=1.283; 95% CI: 0.743 to 2.214, p=0.372) to use CAM rather the non-smoker respondents. The respondents who had hypertension were less likely (OR=0.430; 95% CI: 0.275 to 0.672, p<0.001) to use CAM user rather the respondents who had diabetes mellitus.\n\nThe reasons for choosing CAM are shown in Table 3. The patients reported several reasons for utilising CAM. Mainly, external influences and internal influences played a significant role in utilising CAM. In external influences, media influence was the most common (n=43, 37%), followed by patients’ family members (n=33, 28.4%) and friends (n=25, 21.5%).\n\nSimilarly, in the case of internal influences, the patients reported several reasons for utilising CAM. The most popular internal influence was the fewer side effects of CAM (n=63, 54.3%). The other common influences were: patients believed that CAM works better (n=57, 49.5%), experienced poor results from modern medicine (n=53, 46%), CAM has a lower cost (n=38, 33.3%), and the advice from others influenced of patients (n=45, 38.5%).\n\n\nDiscussion\n\nThis study investigated the prevalence of using CAM for diabetes and hypertension among Bangladeshi adults, and the factors associated with CAM use. In this study, a majority of the diabetes patients used CAM rather than hypertension patients. Among older aged patients, CAM usage had the highest preferences in Bangladesh. This result is concurrent with the previously reported findings among NCD patients (61.7%) (Shahjalal et al., 2022a) in Bangladesh. A study undertaken in India revealed that 63% of diabetic patients utilize CAM (Bhalerao et al., 2013). Another investigation carried out in Sydney determined that 28% of diabetic patients utilized CAM (Manya et al., 2012). On the other hand, the patients with chronic illness had a lower percentage of CAM use in Singapore (22.7%) and South Africa (27.2%) (Hughes et al., 2015; Lee et al., 2004). The findings of these studies suggested that CAM usage is shared among diabetic and hypertension patients globally.\n\nThe female patients of this study were more likely to use CAM than the male ones. In India, 30% of male patients of age 51-60 years old used CAM, whereas 57% female participants used CAM for treatment (Roy et al., 2015). On the other hand, other studies reported that younger adults had a higher prevalence of using CAM, and that women mostly used CAM. A previous Bangladeshi study revealed that 60.6% of patients of age 26 to 45 years old used CAM, which was the highest figure of all age groups, and 39.8% of female participants used CAM for treatment (Shahjalal et al., 2022b). Another study revealed that 15.6% of patients aged between 20-39 years old and 40-59 years old used CAM to treat chronic illness, whereas 16.7% of female patients used CAM (Shahjalal et al., 2022a). In Pakistan, 63% of patients of age less than 45 years old and 71% of female patients used CAM for diabetes treatment (Jawed et al., 2019).\n\nAccording to the findings of this study, socio-demographic characteristics such as educational status, employment status and smoking behaviour were significantly related to CAM use among the diabetes and hypertension patients in Bangladesh, which reflects the similar findings of earlier studies from Malaysia, Nepal and Pakistan (Hasan et al., 2009; Kadayat et al., 2012; Shaikh & Hatcher, 2005). These findings indicated that patients with no formal education, unemployed patients and patients with smoking habits were more likely to use CAM for treating diabetes and hypertension. These findings support the findings of previous studies of Bangladesh and Pakistan, where 17.5% of patients who had schooling less than five years of and diabetic patients with less than ten years of education, respectively, used CAM for chronic illness treatment purpose (Jawed et al., 2019; Shahjalal et al., 2022a). This study revealed that married patients and members of joint families are more likely users of CAM than single ones. In a previous study in Bangladesh, 27.7% of married patients utilized CAM for chronic illness treatment (Shahjalal et al., 2022a).\n\nOur results depicted that older aged patients use higher amounts of CAM to treat diabetes and hypertension. This result supports the findings of other studies in South-East Asian countries like India, Malaysia, and China (Hasan et al., 2009; Hughes et al., 2015; Xin et al., 2020). The main reason could be the tendency of seeking traditional medical aid for severe complications. Considering the growing popularity among NCD patients, especially diabetes and hypertension patients, the government should promote CAM along with conventional or modern medicine. This will help to lessen the pressure on the existing healthcare system and will ensure proper treatment for the patients.\n\nHomeopathic medicine was the most used type of CAM by hypertension and diabetic patients in this study. A previous study in Bangladesh reported that 52.2% of patients used homeopathy treatment for treating chronic illnesses (Shahjalal et al., 2022a). 36% of patients in India used Ayurveda, followed by homeopathy for their treatment purpose (27%) (Roy et al., 2015).\n\nExternal and internal influences were prominent among the patients for utilizing CAM. Media influence and fewer side effects were the most influential factors among the patients. Along with these, media influence, better performance from CAM, and poor results from modern medicine were also common influential factors in using CAM. The study in Bangladesh reported that 55.5% of patients used CAM because of its less adverse effects and 41.6% of patients utilised it for its effective disease management (Shahjalal et al., 2022b). In India, patients used CAM because of its effectiveness (50%), advice from family (43%), and on-time usage on illness (51%) (Roy et al., 2015). Another study in India reported that the effectiveness (70.4%), own will (38.5%) and immediate usage of CAM (65.7%) were significant factors in using CAM among elderly patients (Sharma et al., 2017).\n\nThis research has several positive aspects. First, we gathered data by physicians from the surveyed health care centers. By involving physicians as the data collectors from the study hospitals, we reduced possible information bias. Secondly, we collected data from two specialised health care centres that provide only hypertension and diabetes services. There are also some limitations to the study. The study was cross-sectional, and it isn’t easy to determine causality. Further, using cardinal-based items may reduce the response validity of the variables studied.\n\n\nConclusion\n\nThough patients with hypertension and diabetes use a high percentage of modern medicine, CAM is also widely used in Bangladeshi adults. Our results show a relationship between CAM usage and age, gender, employment history, economic status and family size. Homeopathy, Ayurvedic medicine and Unani medicine were the most common CAM practices in Bangladeshi adults with hypertension and diabetes. Most CAM users were influenced by the media, family members and friends. CAM use was most common for perceived effectiveness, fewer side effects, and low cost. The findings suggest future directions for research and have practical implications for diabetes and hypertension.", "appendix": "Data availability\n\nMendeley: Complementary and alternative medicine use by Bangladeshi adult patients with diabetes and hypertension: A multicenter study, https://doi.org/10.17632/8dtp5twpds.2 (Hasan et al., 2023).\n\nThis project contains the following underlying data:\n\n- data.xlsx (complete survey responses).\n\nMendeley: Complementary and alternative medicine use by Bangladeshi adult patients with diabetes and hypertension: A multicenter study, https://doi.org/10.17632/8dtp5twpds.2 (Hasan et al., 2023).\n\nThis project contains the following extended data:\n\n- A blank copy of the questionnaire\n\n- Data key for the xlsx file.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nBell RA, Suerken CK, Grzywacz JG, et al.: CAM use among older adults age 65 or older with hypertension in the United States: General use and disease treatment. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "223669", "date": "22 Nov 2023", "name": "Jun Jie Benjamin Seng", "expertise": [ "Reviewer Expertise Complementary and alternative medicine" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the kind invitation to review this manuscript.\nAbstract\nSuggest to add in the types of CAM that is mostly commonly used.\n\nAdd in section regarding the variables adjusted for in the multivariate analyses\nIntroduction\nBriefly an short paragraph on CAM should be described in the introduction before talking about the prevalence of its usage. The broad categories that are well described include mind and manipulative methods, alternative medical systems which has been studied in patients with NCD. -> Relevant citations: https://pubmed.ncbi.nlm.nih.gov/36694496/; https://pubmed.ncbi.nlm.nih.gov/35439704/; https://pubmed.ncbi.nlm.nih.gov/33373760/\n\nThe relevance of sustainable development goal and interpretation of the target numbers need to be briefly described.\n\nWhat is the main reason for focusing on specifically HTN and T2DM\nMethods\nuse STROBE checklist\n\nSuggest to provide a copy of the questionnaire used\nResults\nSuggest to stratify results in Table 1 by CAM use and perform appropriate univariate analyses\n\nSuggest to report odds ratio in 2 decimal places\nDiscussion\nWhat is the implications of the study\n\nAre there any unique findings in the study\n\nWhat are the safety concerns about CAM usage in the population\n\nBriefly suggest to discuss the CAM landscape in Bangladesh - whether practitioner are formally trained, and any regulation in tandem with the study results\n\nWhat are the limitations of the 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "274117", "date": "29 May 2024", "name": "Afisulahi Abiodun Maiyegun", "expertise": [ "Reviewer Expertise Family medicine", "complementary and alternative medicine" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research objectives were to determine the prevalence of complementary and alternative medicine (CAM) use among adult Bangladeshi adult patients with diabetes and hypertension and to determine factors associated with CAM use. The combined prevalence for CAM use among the study population was 29%, with women, the non-educated and the elderly among those more likely to use CAM. There is an error in the abstract: the prevalence of both hypertension and diabetes were reported as 37.5% each, contrary to the results presented in the article. The introduction need to be more concise. Specifically, the last two sentences of the first paragraph of the introduction need referencing. The study on CAM and diabetes (mentioned in the last paragraph of the introduction after Shahjalal et al) needs to be referenced as well. The last two sentences of the third paragraph and the third sentence of the fourth paragraph (on prevalence) can be deleted: they are more like deviations from the central theme of the introduction. The penultimate sentence of the introduction contains a repetition of ideas already mentioned at the beginning of that paragraph (on the inadequacy of research into utilization of CAM). This should be re-framed. The study design was cross-sectional, which was appropriate. However, the method was inadequate for the following reasons:\nfor a national study, only two study sites were used. It is difficult to determine how representative of the national Bangladeshi population these two centres are. It is even possible the two centres are located in the same region of the country, which would further limit the generalisability of the study to the whole country as projected by the authors. participants were selected \"purposively\", meaning there was no random sampling. Results drawn from such a selection may not be appropriate to determine prevalence of the use of CAM because the method is prone to bias. the meaning of \"purposively\" was not explained in the study. The author need to state exactly why and how the sampling was done \"duration of suffering for diseases\" should be replaced with less graphic and more scientific expressions like \"duration since diagnosis\". the pilot testing of the questionnaire should be discussed first mentioning how the questionnaire was administered Which sample size calculator was used to arrive at the sample size? How many patients with diabetes and hypertension were recruited from each of the hospitals.\nThe results were presented as if they were all statistically significant, contrary to the fact that many of the findings had P value greater than 0.05. Also, income of the study participants was reported in \"BDT\". This should be spelt out and preferably converted to the dollar equivalent at the time of the study to enable non-Bangladeshi readers appreciate the levels of participants' income and for comparison with other similar studies reporting patient income. The discussion section mainly comparison of the study's findings with other studies'. More effort should be made to explain the findings in the study, on one hand, and the agreement or disparity of this study and the other referenced studies, on the other hand. In addition, this section has many problems with the use of English. For instance, it says \" a majority of the diabetes patients used CAM rather than hypertensive patients...\", \"this result is concurrent with the previously reported findings...\" Results \"agree with\", but is not \"concurrent\" as used here. (There are many instances of linguistic problems elsewhere in the article). In the penultimate paragraph of the discussion section, phrases like \"own will\" and \"immediate usage of CAM\" used in the referenced article need to be explained here to improve readers' comprehension. The \"future directions for research\" mentioned in the study were not specified. The authors should highlight a few of such directions that arise from this study. To improve this article, these points should be addressed. Furthermore, employing the services of expert English medical writers is recommended to improve the quality of the article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1063
https://f1000research.com/articles/12-1062/v1	31 Aug 23	{ "type": "Systematic Review", "title": "Mental health problems of entrepreneurs during the COVID-19 health crisis: Fear, anxiety, and stress. A systematic review", "authors": [ "Víctor Hugo Fernández-Bedoya", "Monica Elisa Meneses-La-Riva", "Josefina Amanda Suyo-Vega", "Johanna de Jesús Stephanie Gago-Chávez", "Monica Elisa Meneses-La-Riva", "Josefina Amanda Suyo-Vega", "Johanna de Jesús Stephanie Gago-Chávez" ], "abstract": "Background: The COVID-19 pandemic has had a global impact, affecting millions of people and causing various symptoms, leading to social distancing, and quarantine measures. This has impacted the global economy, employment, and education, resulting in widespread school and business closures. Entrepreneurs have had to face the difficult decision of whether to continue or pause their operations during the pandemic, which could result in mental health problems such as fear, anxiety, or stress. The primary objective of this research is to identify mental health issues faced by entrepreneurs during the pandemic, including the countries of origin, conclusions, and lessons learned. Methods: A systematic review was conducted in the SCOPUS database, providing 93 initial results that were later filtered, resulting in 18 final records. These records allowed for the identification of scientific evidence detailing the mental health problems that entrepreneurs faced during the COVID-19 pandemic. Results: We found that the COVID-19 pandemic has presented challenges for entrepreneurs, leading to increased stress and burnout. However, some have coped by seeking support, using digital technology, and adopting innovative business models. Entrepreneurship can positively impact well-being by allowing individuals to pursue meaningful work aligned with their values. Effective communication with customers is essential for maintaining businesses and building loyalty. The pandemic has highlighted the need for specialized psychological support tailored to entrepreneurs' unique challenges. Finally, entrepreneurship can address societal challenges with a focus on social impact and sustainability. Conclusion: The COVID-19 pandemic has brought about significant challenges for entrepreneurs, resulting in heightened stress and burnout. Despite this, many entrepreneurs have adapted and coped by seeking support, leveraging digital technology, and embracing innovative business models.", "keywords": [ "Mental health", "entrepreneurship", "COVID-19", "business ventures", "fear", "anxiety", "stress" ], "content": "Introduction\n\nCOVID-19 is an infectious disease caused by the SARS-CoV-2 virus, which was first detected in Wuhan, China in December 2019 (Yu et al., 2021). Since then, it has become a global pandemic that has affected millions of people worldwide (Wang et al., 2020a; Wu et al., 2020).\n\nSymptoms of COVID-19 can range from mild to severe and include fever, cough, difficulty breathing, headache, fatigue, and loss of smell or taste (Çalıca Utku et al., 2020). The virus primarily spreads through respiratory droplets when an infected person coughs, sneezes, or talks (Esakandari et al., 2020).\n\nThe pandemic has had a significant impact on the global economy, causing many businesses to close temporarily or permanently due to decreased demand and supply chain disruptions (Alekseev et al., 2023; Fairlie et al., 2022; Fernández-Bedoya et al., 2021). It has also had a negative impact on employment, with many people losing their jobs or being required to work from home (Galanti et al., 2021; Kramer & Kramer, 2020; Russo et al., 2023; Suhariadi et al., 2023).\n\nIn addition, the pandemic has led to widespread school and university closures, resulting in an educational crisis (Gaikwad & Kulkarni, 2021; Ober & Kochmańska, 2022; Pazos et al., 2020; Suyo-Vega, Meneses-La-Riva, Fernández-Bedoya, Alarcón-Martínez, et al., 2022a). It has also had a negative impact on the mental health of many individuals due to social isolation, financial stress, or fear of contracting the virus (Becerra-Medina et al., 2022; Martínez Rodríguez et al., 2020; Meneses-La-Riva et al., 2023; Son et al., 2022; Valero Cedeño et al., 2020).\n\nTo prevent further spread of the virus, many countries have implemented social distancing and quarantine measures, such as border closures (De Beukelaer, 2021; Emeto et al., 2021; Grimée et al., 2022; Spennemann, 2021), the shutdown of non-essential businesses (Berg et al., 2021; Murillo VIllanueva et al., 2021; Song et al., 2021), and limitations on social gatherings (Ahmed & Memish, 2020; Guay et al., 2021; Moberly, 2020). Vaccination has also been a crucial tool in the fight against the pandemic (Dagan et al., 2021; Mathieu et al., 2021; Wang et al., 2020b).\n\nEntrepreneurs, as widely recognized, are known for starting with an initial idea and taking the initiative and decisive action to pursue a business project that enables them to enter the market by manufacturing a product or providing a service (Campana Añasco & Chamorro Bacilio, 2022; Fernández-Bedoya, Meneses-La-Riva, Suyo-Vega, & Gago-Chávez, 2023c; Nicolás Martínez & Rubio Bañón, 2023; Rasvanis & Tselios, 2023; Salas Ruiz, 2019; Satar et al., 2023). They have faced the difficult decision of whether to continue, pause, or carry on with their operations throughout the pandemic (Anggadwita et al., 2023; Fernández-Bedoya et al., 2021; Maldonado-Cueva et al., 2023; Ngcongo & Ramraj, 2022; Sahi et al., 2023). These conditions, combined with the uncertain and challenging circumstances, could result in mental health problems, such as fear, anxiety or stress, for example (Khan et al., 2022).\n\nFear is a universal emotion that arises instinctively and serves a fundamental role in human survival (Ekman, 1992; LeDoux, 2014). This emotional response is triggered by the perception of danger, threat, or uncertainty, and can be evoked by a variety of stimuli, including unfamiliar situations, traumatic events, or physical threats (Lang, 1995). For instance, an entrepreneur may experience fear of failure or contracting COVID-19. This emotion is adaptive, as it facilitates individuals to avoid dangerous situations and take self-protective measures. Nonetheless, excessive fear or fear that occurs in the absence of real threat can become an emotional problem that impairs a person’s daily life (Shin & Liberzon, 2010).\n\nAnxiety is a natural emotional response to perceived threatening or stressful situations (McHugh, 2019; Reddy & Tekulapally, 2022). It is commonly expressed as a feeling of worry, fear, apprehension, or restlessness, and is frequently accompanied by physical symptoms such as palpitations, sweating, tremors, and muscle tension (Stein & Sareen, 2015; Taylor et al., 1991). Anxiety can be evoked by various circumstances, including stressful events, uncertainty, fear of the future, or daily concerns (Blair et al., 2008; Shostak & Peterson, 1990). In contrast to fear, anxiety typically involves a more protracted emotional response over time, whereas fear is usually a more brief and intense reaction (Davis et al., 2011). Anxiety may present as a persistent sense of worry or restlessness, whereas fear is characterized by an intense emotional response that subsides once the danger is eliminated (Shostak & Peterson, 1990).\n\nStress is also a physiological and emotional response of the body to a situation perceived as a threat or challenge (Chan et al., 2022; Rijal et al., 2023; Suyo-Vega, Meneses-La-Riva, Fernández-Bedoya, Polonia, et al., 2022b). It can be triggered by various factors, such as work pressure (Lovallo et al., 1986; Smith et al., 2019), tight deadlines (Bowen et al., 2013, 2014; Herrero et al., 2012), financial uncertainty (Apouey et al., 2020; Caggiano et al., 2021), market competition (Amable, 2004; Pandey & Sharma, 2016), among others, which are exacerbated during times of COVID-19. In the case of an entrepreneur, stress can result from multiple tasks and responsibilities, such as making important decisions, managing limited resources, and the need to innovate and adapt to rapid and constant changes in the business environment (Fernández-Bedoya, Meneses-La-Riva, Suyo-Vega, Grijalva-Salazar, et al., 2023b; Mark et al., 2008; Vasilescu et al., 2016). Stress in an entrepreneur can develop gradually as pressure increases and responsibilities become more demanding (Cardon & Patel, 2015). The entrepreneur may experience physical and emotional symptoms, such as fatigue, insomnia, irritability, difficulties in concentrating, and an increase in alcohol or tobacco consumption (Palmer et al., 2021). These symptoms can have a negative impact on their cognitive abilities, interpersonal relationships, and ability to perform everyday tasks effectively (Mäkiniemi et al., 2021).\n\nThe primary objective of this research was to identify studies that delineate the mental health issues that entrepreneurs faced during the COVID-19 era. The specific objectives were to determine the countries from which these investigations originated, to elaborate on their conclusions and extract the lessons learned in each case.\n\n\nMethods\n\nIn order to achieve the proposed objectives, we conducted a systematic review (Moher et al., 2009). We searched for all scientific research that details the mental health problems that entrepreneurs may have experienced during the health crisis caused by the COVID-19 virus.\n\nThe search was conducted on the Scopus database, which is considered to be of high quality and contains information from around the world (Baas et al., 2020). The search was carried out on March 15, 2023, approximately three years after the alert issued by the World Health Organization, which led to the suspension of non-essential businesses worldwide. It is important to note that access to the Scopus database was granted through our credentials provided by Universidad César Vallejo.\n\nIn particular, we conducted four search equations, which are detailed in Table 1.\n\nSearch Equation 1: TITLE-ABS-KEY (entrepreneur AND “mental health” AND COVID-19). This search resulted in 20 initial records, of which 11 were downloaded directly from Scopus, while 3 had to be downloaded from the journal’s website. 6 records could not be accessed due to various restrictions.\n\nSearch Equation 2: TITLE-ABS-KEY (entrepreneur AND “fear” AND COVID-19). The initial results were 23, of which 9 were downloaded directly and 3 through the journal’s website. 11 records could not be accessed due to various restrictions.\n\nSearch Equation 3: TITLE-ABS-KEY (entrepreneur AND “anxiety” AND COVID-19). The initial results were 17 documents, 7 of which were downloaded directly from Scopus, and 4 through the journal’s website. 6 records could not be accessed due to various restrictions.\n\nSearch Equation 4: TITLE-ABS-KEY (entrepreneur AND “stress” AND COVID-19). This search resulted in 33 initial records, of which 10 were downloaded from Scopus and 4 from the original source. 19 studies could not be downloaded due to lack of access.\n\nSubsequently, some inclusion and exclusion criteria were applied. This allowed us to filter out the records that we intended to evaluate. Regarding the type of scientific evidence, we decided to use only original research, excluding editorials, protocols, and even other review articles. In terms of language, we worked with scientific evidence in English. Specifically, we searched for records that detail mental health problems in entrepreneurs worldwide, so articles that did not meet this condition were excluded. The applied filters resulted in a preliminary result of 26 records. After removing duplicate records, the final result was 18 records.\n\nFour experienced reviewers formed the team responsible for the inclusion criteria screening process in this systematic review. Initially, the team performed a comprehensive assessment of titles and abstracts from all identified records obtained through selected databases and other sources. Subsequently, they obtained full-text reports of potentially relevant records identified during the initial screening. The reviewers meticulously analysed these full-text reports to determine whether they aligned with the predetermined inclusion criteria. To enhance the accuracy and consistency of the screening process, a random sample of records screened by each reviewer underwent verification by another reviewer. This quality assurance measure aimed to identify and address any discrepancies or inconsistencies. The entire screening process, including the total number of records identified, screened, and ultimately included in the review, was thoroughly documented. For clarity and transparency, this information was presented in a PRISMA flow diagram—a visual representation illustrating the systematic progression from record identification to the final inclusion of relevant studies in the review (see Figure 1).\n\n\nResults\n\nTable 2 displays the 18 identified records, which are indicated by their respective internal codes, citations, original titles, and the scientific journals of publication.\n\nAdditionally, in order to meet the research objectives, it was identified which country each of the experiences of mental health problems in entrepreneurs during the COVID-19 pandemic belong to, as exposed in the 18 scientific articles. These results are found in Table 3 and Figure 2.\n\nFinally, Table 4 presents the conclusions of each article, accompanied by the lessons learned that can be drawn from such research.\n\n\nDiscussion and conclusion\n\nAs a global pandemic, COVID-19 has affected millions of people worldwide, causing a range of symptoms from mild to severe. The virus primarily spreads through respiratory droplets, leading many countries to implement social distancing and quarantine measures. The pandemic has had a significant impact on the global economy, employment, and education, leading to widespread school and university closures, as well as business shutdowns.\n\nEntrepreneurs have faced the difficult decision of whether to continue, pause, or carry on with their operations throughout the pandemic. These conditions, combined with the uncertain and challenging circumstances, could result in mental health problems, such as fear, anxiety, or stress. Fear is a universal emotion that arises instinctively and serves a fundamental role in human survival. Anxiety and stress are also natural emotional responses of the body to a situation perceived as a threat or challenge.\n\nThe primary objective of the research is to identify studies that delineate the mental health issues that entrepreneurs faced during the COVID-19 era. The specific objectives were to determine the countries from which these investigations originated, to elaborate on their conclusions, and extract the lessons learned in each case.\n\nThe results of the systematic review allowed for the identification of 18 scientific articles that detailed the mental health issues faced by entrepreneurs during the COVID-19 pandemic, as well as how they demonstrated resilience in the face of this situation.\n\nAn interesting finding was that the scientific journal where the most articles related to this topic were published was Sustainability, with 3 records. On the other hand, the rest of the publications were found in various journals, with topics related to business, psychology, and public health.\n\nBased on the 18 results provided, the main themes related to entrepreneurship during the COVID-19 pandemic are:\n\n• The pandemic has presented significant challenges for entrepreneurs, including financial difficulties, reduced demand for products and services, supply chain disruptions, and social isolation. These challenges have made it difficult for many entrepreneurs to maintain their businesses and achieve their goals.\n\n• The pandemic has also had a significant impact on the mental health of entrepreneurs, with many experiencing increased levels of stress, anxiety, and burnout. The pressures of running a business during a pandemic have been particularly challenging, and it is important for entrepreneurs to prioritize their mental health and seek support when needed.\n\n• Despite these challenges, many entrepreneurs have found ways to cope with the pandemic, such as seeking social support, using digital technology, and adopting innovative business models. These coping strategies and mechanisms have helped entrepreneurs to adapt and overcome the challenges presented by the pandemic.\n\n• The pandemic has highlighted the potential for entrepreneurship to positively impact eudaimonic well-being, emphasizing personal growth and fulfillment. Entrepreneurship allows individuals to pursue their passions and create meaningful work that aligns with their values, which can lead to a greater sense of purpose and well-being.\n\n• Effective communication with customers has been crucial during the pandemic, particularly regarding safety measures and changes in operating procedures. Entrepreneurs who have been able to effectively communicate with their customers have been better able to maintain their businesses and build customer loyalty.\n\n• The pandemic has highlighted the need for specialized psychological support and interventions tailored to the needs of entrepreneurs in times of crisis. Entrepreneurs face unique challenges and stresses, and it is important for them to have access to resources that can help them manage their mental health and well-being.\n\n• Finally, the pandemic has highlighted the potential for entrepreneurship to address societal challenges, with a focus on social impact and sustainability over profit maximization. Entrepreneurs who are able to adapt their businesses to address the challenges presented by the pandemic can make a positive impact on their communities and contribute to a more sustainable future.\n\nAccording to the given data, it is clear that the mental health and overall well-being of entrepreneurs have been significantly affected by the COVID-19 pandemic. Table 5 provides a concise overview as a general interpretation of the results in the context of other evidence.\n\nThese themes highlight the multifaceted nature of the challenges faced by entrepreneurs during the COVID-19 pandemic and the need for tailored support measures to address them effectively.\n\nIn conclusion, the COVID-19 pandemic has posed considerable difficulties for entrepreneurs, leading to increased stress and exhaustion. Nevertheless, numerous entrepreneurs have managed to overcome these challenges by seeking assistance, utilizing digital technology, and adopting creative business approaches. Entrepreneurship can have a positive effect on individuals’ well-being, as it allows them to pursue fulfilling work that aligns with their values. Maintaining effective communication with customers is crucial for ensuring business sustainability and building loyalty. The pandemic has emphasized the necessity for specialized psychological support tailored to address the distinctive challenges that entrepreneurs face. Furthermore, entrepreneurship has the potential to tackle societal issues with a strong emphasis on social impact and sustainability.\n\nA probable limitation of this systematic review could be the regional confinement of the results, rendering them incapable of generalization to other regions. For instance, certain studies concentrate on entrepreneurs from India, France, China, and Thailand, implying that the conclusions may not be transferable to entrepreneurs from different parts of the world. Additionally, a possibility of publication bias exists, whereby studies exhibiting noteworthy or affirmative outcomes may be more prone to publication. Hence, the reader should consider this factor while evaluating the overall robustness of the evidence.\n\nAs for future work, some ideas include:\n\n• Further research can be conducted to explore the specific challenges faced by entrepreneurs during the pandemic and how they have adapted to overcome them. This can help to inform policies and interventions that support entrepreneurship during times of crisis.\n\n• More research is needed to understand the impact of the pandemic on the mental health of entrepreneurs and the most effective ways to support their well-being. This may involve developing tailored psychological support and interventions that meet the unique needs of entrepreneurs.\n\n• Future studies could investigate the potential for entrepreneurship to promote eudaimonic well-being and personal growth, particularly in times of crisis. This can help to identify the conditions under which entrepreneurship can contribute to a more fulfilling and purposeful life.\n\n• More research is needed to understand how effective communication with customers can help entrepreneurs maintain their businesses during a crisis. This may involve exploring different communication strategies and their effectiveness in different contexts.\n\nFurther work is needed to explore the potential for entrepreneurship to address societal challenges and promote sustainability. This can involve developing innovative business models and strategies that prioritize social impact and environmental sustainability over profit maximization.", "appendix": "Data availability\n\nZenodo: Data for “Mental health problems of entrepreneurs during the COVID-19 health crisis: Fear, anxiety, and stress”, https://doi.org/10.5281/zenodo.8127850 (Fernández-Bedoya et al., 2023a).\n\nThis project contains the following underlying data:\n\n- Data for “Mental health problems of entrepreneurs during the COVID-19 health crisis: Fear, anxiety, and stress” (Data for “Mental health problems of entrepreneurs during the COVID-19 health crisis: Fear, anxiety, and stress”).\n\nZenodo: PRISMA 2020 checklist for “Mental health problems of entrepreneurs during the COVID-19 health crisis: Fear, anxiety, and stress”, https://doi.org/10.5281/zenodo.8127809 (Fernández-Bedoya et al., 2023d).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAhmed QA, Memish ZA: The cancellation of mass gatherings (MGs)? 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "205345", "date": "27 Sep 2023", "name": "Andrew Kweku Conduah", "expertise": [ "Reviewer Expertise Population Estimates and Projections", "Population Health and Mortality- Health Emergencies & Preparedness", "Ageing & Intergeneration Relations and Non- Communicable disease & Health Projection (Physical Activity", "Obesity", "Tobacco Control etc.)", "Business Demography", "Business Policy and Strategy", "Business Management", "Public Administration", "Entrepreneurial Innovation" ], "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 article describes Mental health problems of entrepreneurs during the COVID- 19 health crisis: Fear, anxiety, and stress. A systematic review. However, the study design used is not clear and there are major methodological flaws in the review and much crucial information missing. Without a clear study design and methods information, it has not been possible to assess the validity and trustworthiness of the results in this paper.\nREQUESTED REVISIONS:\nAbstract - please revise and follow the PRISMA for Abstracts reporting guideline (if this is deemed a SR, see comment later). There are many essential elements of the method and results missing.\n\nInclude a statement of originality / value in the abstract after the conclusion and before keywords.\n\nPlease refer to the current PRISMA guideline - which is PRISMA 2020. There are many items on PRISMA2020 that are not reported in this review.\n\nWhy were other data bases like PubMed/Medline, Web of Science, etc not searched. PsyINFO would have been a more appropriate choice for this topic.\nI am concerned that the search strategy is insufficient. For example, no MeSH terms are used, no truncation or synonyms, etc. Was a librarian with SR search expertise involved with developing the search? Was a forward and backward citation search done?\n\nPlease describe the process of screening the search results, deciding on eligibility and data extraction. For example, was this process done by 2 researchers independently? If so, state this and describe how consensus was achieved.\n\nPlease list what data were extracted, how this was determined and include the data extraction form as an appendix.\n\nPg 10 - in a SR, the methodological quality (or risk of bias) of included studies needs to be assessed and reported. I find no mention of this in the paper.\n\nAgain, data analysis section is required\n\nThe 'coding process' that was followed needs to be described in the methods section. Also, were only qualitative data extracted? Or quantitative data as well? If so, how were both studies handled? Were they only eligible if they contained some narrative or qual data? This all needs to be clearly explained in the methods section. How about the quantitative data as well?\nThe introduction was skewed to certain areas in Europe, Latin America and Asia without any mentioning of sub- Saharan Africa. How do the authors justify that? Authors should tell us the situation in North America, Europe, Asia, Latin America and sub- Saharan Africa.\nMethods: State the Inclusion and exclusion criteria very clearly as this seems quite blurred in your paper.\nThe choice of databases is limited, and one would expect MEDLINE or PubMed at a minimum to appear given the topic. Furthermore, the inclusion criteria should be more explicit based on a PICO, PCC, or other such review guidance criteria. On reading this, I get the impression this work is simply a literature review.\nA description of the type of data that was extracted is missing, and who conducted this (i.e., the normal process in a systematic review is for two reviewers to undertake this independently).\nThe findings could lend itself to a thematic analysis but this is missing in the analysis section. This further confuses the review methodological approach taken by the reviewers, as there are distinct methods available for qualitative evidence syntheses.\nOverall, based on the methodological concerns outlined above, the work presented in this manuscript, in my opinion, needs redoing. The authors need to adopt a discrete methodology and adhere to the methodological guidance of whichever approach is ultimately chosen for the manuscript to reach a publishable standard and provide confidence in the scientific soundness of the review.\nPlease find a word document with further comments here.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1062
https://f1000research.com/articles/12-1061/v1	31 Aug 23	{ "type": "Systematic Review", "title": "Exploring the Landscape of Latent Autoimmune Diabetes and Maturity Onset Diabetes of the Young in Africa: A Scoping Review", "authors": [ "Davis Ombui", "Ahmed Murtaz Khalid", "Ahmed Murtaz Khalid" ], "abstract": "Background: Latent autoimmune diabetes in adults (LADA) and maturity onset diabetes of the young (MODY) are two forms of diabetes with varied disease symptoms. The prevalence of LADA is higher in adults than MODY. Both diseases contribute to the general burden of diabetes globally. While LADA is a sporadic autoimmune disorder, MODY is a heritable genetic disorder. The prevalence of LADA and MODY has not been fully documented in Africa due to the lack of robust diagnostic tools and the exorbitantly high cost of the available diagnostic tools. Methods: To understand the prevalence landscape of LADA and MODY in Africa, we conducted an extensive scoping review and mapped the various studies performed in Africa. We adopted the Joanna Briggs Institute literature review framework to conduct the scoping review of literature. Results: Research articles were included in the review analysis following exhaustive inclusion criteria to ensure that only qualified articles were included in the final analysis. Overall, 16 research articles met the inclusion criteria and were critically analyzed. An in-house data extraction sheet was used for data extraction from all the shortlisted articles. Information about the sample size, inclusion criteria, age, gender, and study design extracted from all the articles and analyzed. Majority of the studies adopted cross-sectional study design. In terms of sample sizes, the studies used relatively smaller sample sizes due to the high cost of the diagnosis and nature of the diseases. The prevalence rates of LADA and MODY varied in various countries ranging from 1.8 to 18%. Conclusion: African countries are significantly under-represented. The scarcity of research on LADA and MODY research in Africa is evidence of the urgent need to invest more resources in this area. This would guide future research and shape the road towards understanding diabetes in Africa.", "keywords": [ "Latent Autoimmune Diabetes in Adults", "Maturity onset Diabetes of the young", "Prevalence", "scoping review", "diabetes", "Africa" ], "content": "Introduction\n\nLatent autoimmune diabetes of the adults (LADA) is the most common form of autoimmune diabetes diagnosed in adults accounting for approximately 2-12% of all the diabetes cases1 while maturity onset diabetes of the young (MODY) is a common form of monogenic diabetes accounting for 2% of diabetes worldwide.2 LADA is more commonly diagnosed in patients under 35 years, has intermediate levels of beta cell dysfunction characterized by normal to low levels of c-peptide. Positive diagnosis is confirmed by reactivity to at least one of autoantibodies against glutamic acid decarboxylase-65 (GAD-65), islet antigen 2 (IA-2), insulin , zinc transporter 8 (ZnT8) and islet-cell (ICA).1 On the other hand, MODY is a rare heritable disorder transmitted in an autosomal dominant fashion. The disease is mostly asymptomatic and is often misdiagnosed as either type 1 or type 2 diabetes.3\n\nFor effective management of LADA and MODY, accurate and early diagnosis is paramount. However, the current diagnosis approaches heavily depend on clinical symptoms, which overlap with other forms of diabetes and become visible in advanced stages of the disease. Thus, the need for more sensitive and accurate diagnostic tools is more urgent than ever. Unlike in the developed countries, the true prevalence, morbidity, and mortality of these conditions has not been fully defined in Africa. The international diabetes foundation estimated overall prevalence of diabetes in Africa to be 4.5% and it is projected to rise to 5.2% by 2045.4 Despite the efforts made by individual African countries to establish country-specific prevalence rates, this data is missing for most African countries making it difficult to establish the overall prevalence rate. Therefore, to resolve this conundrum, it is imperative to explore research studies in Africa to estimate an overall prevalence rate for the continent.\n\nThe population of Africa is skewed towards the young people, with a median age of 19.5 years.5 LADA and MODY are often diagnosed in this age category implying that the African population structure provides the right population structure for perpetuation of the diseases. A high prevalence of LADA and MODY in Africa would negatively impact the socio-economic productivity of the continent considering the high proportion of the young people in Africa. There is thus, a compelling need to accurately estimate the prevalence of these conditions in this age category constituting the majority in African population. However, efforts to establish the prevalence of LADA and MODY are subdued by the lack of accurate and timely diagnostic approaches. The currently used tools rely on the identification of specific biomarkers which are costly and out of reach in Africa.6 Moreover, the lack of universal consensus guidelines on case definition and diagnosis criteria compounds the problem of diagnosis.7\n\nAlthough there exists literature on different forms of diabetes, Africa is largely underrepresented in published research and review articles. This study sought to consolidate published literature on LADA and MODY to provide a comprehensive pool of African research studies in a single document. Through pooling the available research, this review provides a snapshot of LADA and MODY landscape in Africa. Therefore, this is a baseline evaluation of status of LADA and MODY in Africa that will inform subsequent research focus to better manage these diseases.\n\n\nMethods\n\nThe literature review framework proposed by Joanna Briggs Institute8 was adopted for this scoping review. The framework uses the Preferred Reporting Items for Systematic Review and Meta-Analysis Extension for Scoping Review Protocols (PRISMA-ScR) guidelines to ensure rigor and reduce bias in reporting the methodology.9\n\nStudies on LADA and/or MODY patients in African countries were considered for inclusion. Studies with prospective, cross-sectional, retrospective, interventional designs or dissertations and grey literature published in English or French were included. Systematic and narrative reviews on LADA and/or MODY were excluded, however, there reference lists were hand-searched to identify original research articles. Exclusion criteria was studies conducted among non-Africans and studies whose data sets could not be extracted.\n\nThe search was conducted using broad search terms to increase number of articles retrieved. Search terms used were LADA, MODY, autoimmune diabetes, prevalence, morbidity, mortality, characteristics, latent autoimmune diabetes in adults, maturity onset diabetes in adults, Africa, African countries either singly or in combination using Boolean connectors (“AND”, “OR”, “AND OR”). Articles were searched across various databases including EMBASE, MEDLINE, PubMed, Scopus, Google Scholar, Web of Science, and African Journal Online (AJOL).\n\nMicrosoft excel and Endnote X8 were used to manage the search results. The first screening step involved assessing the articles titles for conformity with inclusion criteria. Abstracts of shortlisted articles were screened to select articles with relevant information and full text of relevant articles downloaded. An in-house data extraction sheet was used to chart shortlisted articles. Information extracted included first author, year of publication, country of study, study design, source population, age range, gender, risk factors/inclusion criteria, clinical parameters, biochemical parameters, prevalence, and main findings. Details of extracted information can be accessed through Extended data table S1 and S2: DOI: 10.6084/m9.figshare.22592893.\n\n\nResults and discussion\n\nA total of 40 articles (26 on LADA and 14 on MODY) were retrieved. After further screening, 17 papers on LADA and seven papers on MODY were excluded from final analysis for various reasons (Figures 1a and 1b) respectively. Sixteen papers (nine on LADA and seven on MODY) satisfied the inclusion criteria. The summary of final analysis of the 16 papers is presented in Extended data tables S1 and S2 DOI: 10.6084/m9.figshare.22592893. The papers were published between 2000 and 2022.\n\nThe screening steps included identification, screening, and selection of eligible articles for inclusion.\n\nNigeria was the most represented country in terms of LADA research, accounting for three of the identified research articles10–12 the rest of the countries contributed one research paper each; South Africa,13 Tanzania14 Ghana,15 Tunisia.16 Sudan17 and Kenya.18 For MODY, four papers were identified from Tunisia19–22 and one each from Morocco,23 South Africa24 and Sudan25 that had evaluated the prevalence of MODY in those countries (Figure 2).\n\nNine papers were analyzed for LADA and seven for MODY.\n\nThe map representation of the studies based on the countries from which they were conducted has been shown in Figures 3a and 3b.\n\nCross sectional study design was adopted by all the nine papers on LADA and six out of the seven papers on MODY. One paper on MODY23 adopted a mixed study design approach with a prospective participant recruitment with a cross-sectional analysis approach. The widespread use of cross-sectional study design is informed by the relatively lower cost and shorter time involved. Thus, the cost and duration of data collection appeared to be important determinants of the choice of a study design.26 The downside of this approach, however, is the high risk of bias since the data give a snapshot of event at a particular point in time, without considering time dependent factors that could confound the study outcomes.27 hence findings from such studies must be interpreted with caution.28\n\nParticipants included in the LADA studies were enrolled in hospitals, with most studies adopting a purposive sample collection strategy. The lowest sample size was 47 participants17 while the highest sample size was 235.10 In MODY studies, the sample sizes ranged between 1120 to 1643.24 The low sample sizes in these studies could be partly due to the high cost of diagnosis. Participants were sampled from patients who were undergoing diabetes type II management and screened for LADA.1 The purposive sampling approach applied in these is mainly employed when screening general population would be costly.29 Due to the smaller sample sizes used in the LADA studies, statistical power of the reported result may be put to question. Consequently, the interpretation of findings of such studies should be made with caution. MODY on the other hand is a genetic disease and familial studies recruiting patients linked to confirmed cases are preferred. MODY is an autosomal dominant disorder inherited disorder, justifying the need to identify individuals related to confirmed cases for enrollment. The south African study,24 however screened the general population to identify MODY cases. Taken together, it can be said that both the inclusion criteria and sample sizes recorded in the various studies in this analysis were based on convenience and cost minimization, the danger of which is reduced statistical power of the study findings.\n\nAge and sex are independent variables that can potentially confound dependent variables. Thus, age or sex stratification of data during analysis is important in age or sex dependent diseases. For instance, LADA is most common among adults suggesting that the researchers would be more interested in enrolling adult participants. Indeed, LADA studies recruited participants who are 30 years to 85 years. On the contrary, the onset of MODY can be as early as <5 years, explaining why studies in MODY recruited participants from five years.3,23 Both LADA and MODY are not sex-linked disorders, hence, the sex of participants was not considered as an inclusion criterion. However, patterns of disease in male and female participants could provide some insights into association of the disease with a particular sex and shape the design of interventions.\n\nA questionnaire was used to collect clinical information. Clinical parameters such as duration of diabetes, weight, and height were collected alongside patient’s demographic data including age and sex. BMI, used as a surrogate marker for obesity, was calculated to determine the prevalence of obesity among the participants. In addition, some studies collected data on diabetes related complications.10,14 The MODY studies collected information about age, sex, and BMI. The current diabetes treatment was also recorded for some of the studies.22–24 Clinical parameters can be used as prognostic markers for disease outcome. Thus, analysis of these parameters is important for understanding disease trajectory. Moreover, these markers can be used to answer questions such as the link between LADA and BMI and establish the cutoff age for LADA diagnosis.\n\nA similar approach as that used in LADA studies to collect clinical parameters was adopted for MODY, underscoring the importance of clinical data in prevalence studies. In addition to regular clinical data, information about current treatment is also key for MODY as these would not only influence the clinical presentation of the disease but also help to evaluate the performance of management strategies. It is also worth noting that some types of MODY patients may develop diabetes related complications. Hence subsequent studies should screen for such complications during participants enrollment.\n\nMeasurement of biochemical laboratory parameters was made in all the 16 studies reviewed. The HBA1C and fasting blood glucose levels were measured for all participants and four studies10,12,13,18 measured the C-peptide levels for diagnosis of diabetes. The pancreatic antibody, anti-GAD65 was used as a diagnostic marker for LADA diagnosis.\n\nBaseline measurement of HBA1C was performed prior to patient enrollment into the MODY studies. Negative pancreatic antibody was screened prior to participant enrollment in six studies apart from one.21 Normally, the diagnosis of LADA is based on several biochemical markers including at least a positive pancreatic antibody and low to normal c-peptide levels.1 The reactivity of GAD-65 antibody has been established to indicate LADA positivity.30 However, this antibody is not specific to LADA, its presence may also indicate type I diabetes. There is therefore need for subsequent studies to uncover more specific markers for LADA diagnosis.\n\nThe prevalence of LADA in selected African countries where the studies were performed ranged from 1.8 to 18%. The Nigerian study reported a prevalence of 14% of LADA among patients who were on treatment for type II diabetes.10 The mean BMI of LADA positive participant was 27.6, which exceeded the normal BMI range of 18.5 to 24.9. However, the studies could not find a link between the elevated BMI levels to LADA due to the possible confounding effect of type II diabetes patients.1 Contrary to the high BMI levels reported here, another study conducted in northern Nigeria,12 where LADA prevalence was found to be 10.5%, participants did not attain high BMI levels. Other confounding factors that were not accounted for by study design or adjusted for during data analysis might influence the variation in BMI levels. Another study from Nigeria reported a prevalence of 11.9%10 based on anti-GAD65 seroprevalence. Compared to the Ghana study, which reported a prevalence of 11.7%,15 there was no significant difference in prevalence rate. The Tanzanian study14 reported a 5.1% prevalence of LADA among individuals type II diabetes. An evaluation of the association of LADA and diabetes related complications was conducted and found no significant association. A longitudinal study design to follow the participants over time would have more suited to explore the associations.\n\nThe study conducted in Kenya reported a moderately lower prevalence rate of 5.7% (n=124).18 The study recruited patients with clinically defined type 2 diabetes. Similarly, the south African study reported a low prevalence rate of 2.5% (n=80).13 LADA in Sudan was the lowest at 1.8% (n=15) among all studies analyzed.17\n\nFor MODY studies, prevalence was assessed based on presence of mutations associated with the diseases. For instance, study conducted in Tunisia21 used molecular tools to screen mutations in HNF1A, GCK, HNF4A and INS genes. Mutations in GCK and HNF4A genes were found among 13.05% (n=23) of study participants. Another study in Tunisia by Ref. 20 performed sequencing of 27 genes previously linked with presence of MODY and found two mutation in HNF1A and two others in GCK. However, the presence of these mutations does not imply the presence of MODY, rather, they predispose individuals to MODY. The prevalence data must therefore be interpreted carefully to show the risk of developing the disease but not necessarily the presence of the disease.\n\nIt is therefore important to carefully interpret this data. Recently, Ref. 22 screened for GCK and HNF1A genes and reported 1 (n=17) variant in GCK associated with MODY pathogenesis. It is important to note that the studies conducted in Tunisia did not identify genes associated with MODY in most of the confirmed MODY cases. Thus, the involvement of other novel genes not screened in the Tunisia study.19 The Moroccan study23 reported 10% (n=22) prevalence of GCK and HNF1A mutations, while the rest of the patients did not carry mutations in any of the genes screened despite the high level of HBA1C. Community screening for MODY in the South African study24 found a 5.9% (n=1643) prevalence of HNF1A mutations and only two participants had GCK mutations.\n\n\nConclusion\n\nA considerable number of research articles have been published in African countries, with majority of the papers being published in west and northern part of Africa. A significantly smaller number of research articles were retrieved from the eastern and southern Africa countries. The countries in the central part of Africa were not represented in our analysis, underscoring the need to encourage research on LADA and MODY in this part of Africa. Albeit the small sample sizes, the studies addressed important questions, while shedding light on the estimates of LADA and MODY prevalence, which is missing in most African countries. This scoping review reveals the lack of deliberate effort towards these two conditions in Africa. This disparate situation may be exacerbated by the high burden of infectious diseases in Africa hence resources and attention are directed to them due to their apparently high mortality than LADA and MODY.", "appendix": "Data availability\n\nFigshare: Exploring the Landscape of Latent Autoimmune Diabetes and Maturity Onset Diabetes of the Young in Africa: A Scoping Review. DOI: https://doi.org/10.6084/m9.figshare.22592893.v1. 31\n\nThis project contains the following data:\n\n- Extended data.docx: The data contains summary tables of all the research articles analyzed in this scoping review.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY4.0).\n\nOpen Science Framework. PRISMA checklist, DOI: https://doi.org/10.17605/OSF.IO/X8P6E.\n\n\nAcknowledgement\n\nThe authors acknowledge the immense intellectual input and guidance offered by Edwin Magomere of University of Ghana. Dr. Kevin Omondi Ndede and Dr. Yubrine Gachemba Moraa have been of great support during the process of writing this report.\n\n\nReferences\n\nLiu B, Xiang Y, Liu Z, et al.: Past, present and future of latent autoimmune diabetes in adults. Diabetes Metab. Res. Rev. 2020; 36: e3205. Publisher Full Text\n\nUrakami T: Maturity-onset diabetes of the young (MODY): current perspectives on diagnosis and treatment. Diabetes MetabSyndrObes. 2019; 2019(12): 1047–1056.\n\nPeixoto-Barbosa R, Reis AF, Giuffrida F: Update on clinical screening of maturity-onset diabetes of the young (MODY). Diabetol. Metab. Syndr. 2020; 12: 1–14. Publisher Full Text\n\nIDF: Diabetes Atlas Tenth edition 2021.2021.\n\nRoser M, Ritchie H, Ortiz-Ospina E, et al.: World population growth. Our world in data.2013.\n\nMutyambizi C, Pavlova M, Chola L, et al.: Cost of diabetes mellitus in Africa: A systematic review of existing literature. Global. Health. 2018; 14(1): 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUrbanová J, Brunerová L, Brož J: Hidden MODY-Looking for a needle in a Haystack. Front. Endocrinol (Lausanne). 2018; 9: 355. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBriggs-Institute: The Joanna Briggs Institute Reviewers’ Manual 2015: Methodology for JBI scoping reviews.2015.\n\nTricco A, Lillie E, Zarin W, et al.: PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation.2018; 467–473.\n\nAdeleye OO, Ogbera AO, Fasanmade O, et al.: Latent Autoimmune Diabetes Mellitus in Adults (LADA) and it’s characteristics in a subset of Nigerians initially managed for type 2 diabetes. Int. Arch. Med. 2012; 5: 23–28. Publisher Full Text\n\nIpadeola A, Adeleye JO, Akinlade KS: Latent autoimmune diabetes amongst adults with type 2 diabetes in a Nigerian tertiary hospital. Prim. Care Diabetes. 2015; 9: 231–236. Publisher Full Text\n\nMuazu SB, Okpe I, Anumah F: The prevalence and characteristics of latent autoimmune diabetes in adults subset among type two diabetes mellitus patients in Northern Nigeria. Ann. Afr. Med. 2016; 15: 163–170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPanz V, Kalk W, Zouvanis M, et al.: Distribution of autoantibodies to glutamic acid decarboxylase across the spectrum of diabetes mellitus seen in South Africa. Diabet. Med. 2000; 17: 524–527. PubMed Abstract \| Publisher Full Text\n\nManisha AM, Shangali AR, Mfinanga SG, et al.: Prevalence and factors associated with latent autoimmune diabetes in adults (LADA): a cross-sectional study. BMC Endocr. Disord. 2022; 22: 1–9. Publisher Full Text\n\nAgyei-Frempong M, Titty F, Owiredu W, et al.: The prevalence of autoimmune diabetes among diabetes mellitus patients in Kumasi, Ghana. Pak. J. Biol. Sci. 2008; 11: 2320–2325. PubMed Abstract \| Publisher Full Text\n\nAmrouche C, Kamoun HJ, Trabelsi N, et al.: Latent autoimmune diabetes in Tunisian adults (LADA): identification of autoimmune markers. Tunis. Med. 2008; 86: 316–318. PubMed Abstract\n\nBolad A, Abdelmageed R, Elnimeiri MK: Diagnostic value of Autoantibodies to GAD65 and IA-2 in Patients with Latent Autoimmune Diabetes in Adult (LADA). Sudan J. Med. Sci. 2011; 6.\n\nOtieno C, Huho A, Omonge EO, et al.: Type 2 diabetes mellitus: clinical and aetiologic types, therapy and quality of glycaemic control of ambulatory patients. East Afr. Med. J. 2008; 85: 24–29. PubMed Abstract \| Publisher Full Text\n\nAmara A, Chadli-Chaieb M, Ghezaiel H, et al.: Familial early-onset diabetes is not a typical MODY in several Tunisian patients. Tunis. Med. 2012; 90: 882–887. PubMed Abstract\n\nDallali H, Pezzilli S, Hechmi M, et al.: Genetic characterization of suspected MODY patients in Tunisia by targeted next-generation sequencing. Acta Diabetol. 2019; 56: 515–523. Publisher Full Text\n\nKhelifa SB, Martinez R, Dandana A, et al.: Maturity Onset Diabetes of the Young (MODY) in Tunisia: Low frequencies of GCK and HNF1A mutations. Gene. 2018; 651: 44–48. PubMed Abstract \| Publisher Full Text\n\nMoalla M, Safi W, Babiker Mansour M, et al.: Tunisian maturity-onset diabetes of the young: a short review and a new molecular and clinical investigation. Front. Endocrinol. 2021; 12: 684018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrhanint S, Bouguenouch L, Abourazzak S, et al.: Molecular and clinical assessment of maturity-onset diabetes of the young revealed low mutational rate in Moroccan families. Int. J. Pediatr. Adolesc. Med. 2022; 9(2): 98–103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatsha TE, Raghubeer S, Tshivhase AM, et al.: Incidence of hnf1a and gck mody variants in a South African population. Appl. Clin. Genet. 2020; 13: 209–219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdalazeem Osman A, Nouraldayem Altayeb H, MohammedAbdel Hamid M: Molecular Detection of Glucokinase (GCK) Gene Mutation in Maturity-onset Diabetes of the Young (MODY2) Among Sudanese Families With Type 2 Diabetes Mellitus.2018.\n\nWang X, Cheng Z: Cross-Sectional Studies: Strengths, Weaknesses, and Recommendations. Chest. 2020; 158: S65–S71. Publisher Full Text\n\nSedgwick P: Bias in observational study designs: cross sectional studies. BMJ. 2015; 350. Publisher Full Text\n\nWang X, Cheng Z: Cross-sectional studies: strengths, weaknesses, and recommendations. Chest. 2020; 158: S65–S71. Publisher Full Text\n\nRobinson RS: Purposive Sampling. Encyclopedia of Quality of Life and Well-Being Research. Michalos AC, editor. Springer Netherlands; 2014; pp. 5243–5245. Publisher Full Text\n\nJones AG, McDonald TJ, Shields BM, et al.: Latent autoimmune diabetes of adults (LADA) is likely to represent a mixed population of autoimmune (type 1) and nonautoimmune (type 2) diabetes. Diabetes Care. 2021; 44: 1243–1251. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOmbui D: Exploring the Landscape of Latent Autoimmune Diabetes and Maturity Onset Diabetes of the Young in Africa: A Scoping Review. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "244072", "date": "01 Mar 2024", "name": "Christine Bellanné-Chantelot", "expertise": [ "Reviewer Expertise Genetics of diabetes" ], "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\nMajor points ·\n\nThe authors restricted their analysis to articles reporting specific country-specific data. Several other papers analyzing cohorts from North Africa or sub-Saharan Africa are available; It would be interesting for the authors to include these data in their discussion. For example[1],[2]. ·\n\nThe analysis of clinical-biological data is very limited. It would be desirable for the authors to present a minimum of clinical and biological characteristics of the cohorts by country; the minimum set could include age at diabetes discovery, prevalence of obesity, treatment (insulin or not), GAD antibodies for LADA, and the prevalence of the 3 main etiologies GCK or HNF1A/4A for MODY. Minor points ·\n\nIntroduction: 1st paragraph. It cannot be said that MODY is asymptomatic. They are, as indicated by the authors, often mislabeled as type 1 or type 2 diabetes or diagnosed late, on average 10 years after the onset of diabetes. ·\n\nTwo observations are missing in the introduction: (i) MODY identification criteria were defined in European populations. Studies need to be conducted in non-Caucasian populations to define diagnostic criteria adapted to these populations, and (ii) the prevalence of type 2 diabetes is higher in Africa with an earlier onset age overlapping with MODY and LADA (PMID: 35900910), leading to a more complicated identification of LADA and MODY diabetes. ·\n\nThe following sentence is incorrect: \"However, the presence of these mutations does not imply the presence of MODY, rather, they predispose individuals to MODY.\" The subjects in the analyzed cohorts are diabetic or have hyperglycemia; they have MODY diabetes associated with clinical variability.\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? No", "responses": [] }, { "id": "272666", "date": "22 May 2024", "name": "Yang Xiao", "expertise": [ "Reviewer Expertise Diabetes", "Immunometabolism", "Endocrinology and Metabolism" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary of the Article: The article is a scoping review exploring the landscape of Latent Autoimmune Diabetes in Adults (LADA) and Maturity Onset Diabetes of the Young (MODY) in Africa. The study aims to understand the prevalence of these diseases in Africa by analyzing research articles from various African countries. The authors conducted an extensive review of the literature, identifying 16 research articles that met their inclusion criteria. The study designs were predominantly cross-sectional, and the sample sizes varied, with costs and convenience influencing these choices. The prevalence rates for LADA and MODY were found to range from 1.8% to 18% across different African countries. The authors concluded that there is a significant under-representation of African countries in research on LADA and MODY, highlighting an urgent need for more investment in this area.\nConstructive Feedback and Recommendations: 1. Rationale and Objectives: Based on the information provided in the article, the review partly meets the criteria for clarity in rationale and objectives. a. The rationale is stated in the first paragraph: \"Although there exists literature on different forms of diabetes, Africa is largely underrepresented in published research and review articles. This study sought to consolidate published literature on LADA and MODY to provide a comprehensive pool of African research studies in a single document.\" b. The objectives are also clearly stated: \"Through pooling the available research, this review provides a snapshot of LADA and MODY landscape in Africa. Therefore, this is a baseline evaluation of status of LADA and MODY in Africa that will inform subsequent research focus to better manage these diseases.\" The authors aim to consolidate the available literature on LADA (Latent Autoimmune Diabetes in Adults) and MODY (Maturity Onset Diabetes of the Young) in Africa, in order to provide a comprehensive overview of the current landscape and inform future research in this area. However, the rationale for conducting the study and the specific motivation behind it are not clearly articulated. To improve the scientific soundness of the article, the authors should provide a more detailed explanation of why this research is important and necessary.\n2. Methods and Analysis: Based on the information provided in the article, the details of the methods and analysis are not sufficiently provided to allow replication by others. The article states that the Joanna Briggs Institute framework and PRISMA-ScR guidelines were used, but does not provide enough specifics on the search strategy, databases searched, inclusion/exclusion criteria, and data extraction process. The article mentions that an \"in-house data extraction sheet\" was used, but do not specify the data points that were extracted from the included studies. A more detailed description of the data extraction process would be helpful. Apart from that, the authors should provide a detailed description of their search strategy, including the databases searched, the full search strings used, and the process of study selection. Additionally, they should describe any data extraction tools or protocols used. Overall, the methods section lacks the level of detail that would be required for another researcher to be able to reproduce this systematic review. To enhance the replicability of the study, the authors should provide more detailed information on these aspects, including the specific search terms used, screening process, and data extraction sheet.\n3. Statistical Analysis: Not applicable, as the review is primarily a narrative synthesis of the included studies, without any quantitative meta-analysis. The article states: \"Due to the smaller sample sizes used in the LADA studies, statistical power of the reported result may be put to question. Consequently, the interpretation of findings of such studies should be made with caution.\" This suggests the authors are aware of the limitations in interpreting the statistical findings given the small sample sizes in the primary studies. However, the article does not go into any further detail on the statistical methods or interpretation. Given the nature of a scoping review, the lack of in-depth statistical analysis is understandable. The authors appropriately acknowledge the limitations of the primary study designs and sample sizes, and caution against over-interpretation of the prevalence estimates reported. However, a more comprehensive discussion of the statistical considerations would strengthen the review.\n4. Support for Conclusions: While the article presents some research results on LADA and MODY prevalence in African countries, the connection between these results and the conclusions drawn is not adequately explained. To strengthen the article, the authors should provide a more thorough discussion of how the results support the conclusions and clarify the relationship between the findings and the conclusions. They should discuss the implications of the findings in the context of the broader African population and the specific challenges faced in diagnosing and managing LADA and MODY in Africa. The article provides a high-level summary of the study designs, sample sizes, and prevalence estimates reported, but does not delve into a critical analysis of the limitations, biases, and generalizability of the existing evidence. Without a more thorough evaluation of the current literature, the conclusions drawn seem to go beyond what can be reasonably inferred from the results presented. Overall, the conclusions appear to be somewhat overstated given the scope and depth of the analysis provided in the article. A more cautious interpretation of the available evidence would be warranted to align the conclusions with the results reported.\n5. Living Systematic Review Methodology: The review is not a living systematic review; hence this criterion is not applicable. However, if the authors intend to update the review periodically, they should define the process and schedule for updating the search and analysis.\n6. Specific Criticisms and How to Address Them:\nA. Inclusion of Additional Data: The reviewer suggests including data from other papers analyzing North African or sub-Saharan African cohorts. The authors should consider expanding their search to include these cohorts to provide a more comprehensive overview of the landscape. B. Clinical-Biological Data Analysis: The authors are advised to present clinical and biological characteristics of the cohorts by country. This could include age at diabetes discovery, prevalence of obesity, treatment modalities, and specific biomarker prevalence for LADA and MODY. C. Correct Interpretation of Genetic Data: The authors should clarify the relationship between the presence of mutations and the diagnosis of MODY, ensuring that the discussion reflects the clinical context of the subjects in the analyzed cohorts. D. No competing interests were disclosed, which is appropriate for the transparency of the review. E. Quality assessment: The article does not mention any formal quality assessment or risk of bias evaluation of the included studies. This is an important step in a systematic review to critically appraise the methodological quality of the primary studies.\nRecommendations for Improvement: To make the article scientifically sound, the authors should address the following key points:\n1. Provide a more detailed and comprehensive description of the methods, including the search strategy, inclusion/exclusion criteria, and data extraction process. 2. Incorporate a formal quality assessment or risk of bias evaluation of the included studies to critically appraise the methodological rigor of the primary evidence. 3. Expand the analysis and interpretation of the findings, including a more in-depth discussion of the limitations, biases, and generalizability of the existing literature. 4. Align the conclusions and recommendations more closely with the results presented in the review, avoiding overstated or unsupported claims.\nBy addressing these areas, the authors can strengthen the scientific rigor and validity of the scoping review, providing a more robust and reliable assessment of the current state of LADA and MODY research in Africa.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) No", "responses": [] } ]	1	https://f1000research.com/articles/12-1061
https://f1000research.com/articles/12-1059/v1	31 Aug 23	{ "type": "Genome Note", "title": "The complete chloroplast genome of Phyllanthus acidus (L.) Skeels (Phyllanthaceae)", "authors": [ "Hoang Danh Nguyen", "Thi Diem Quynh Nguyen", "Minh Thiet Vu", "Hoang Dang Khoa Do", "Hoang Danh Nguyen", "Thi Diem Quynh Nguyen", "Minh Thiet Vu" ], "abstract": "Phyllanthus acidus (L.) Skeels (Phyllanthaceae) is a potential medicinal plant recognized for its sour and tart tasted fruits. In this study, the chloroplast genome of P. acidus was sequenced, assembled, and characterized. The chloroplast genome size was 156,331 bp and the overall GC content was 36.9%. Additionally, the chloroplast genome had a quadripartite structure consisting of a large single copy (LSC; 85,807 bp in length; GC content: 34.6%), a small single copy (SSC; 19,262 bp in length; GC content: 30.6%), and two inverted repeat regions (IR; 25,631 bp in length; GC content: 43.1%). A total of 113 unique genes were annotated in the chloroplast genome, comprising 79 protein-coding genes, 30 tRNAs, and four rRNAs. The phylogenetic analysis based on 79 protein-coding genes revealed the paraphyly of the Phyllanthus genus. These findings provided additional genetic information for further research on P. acidus and the cp genome in the Phyllanthaceae family.", "keywords": [ "Malpighiales", "Phyllanthaceae", "phylogenetic relationships", "plastome", "star gooseberry" ], "content": "Introduction\n\nPhyllanthus acidus (L.) Skeels, also known as star gooseberry, belongs to the Phyllanthaceae family and commonly distributes in the wet tropical regions, including South/ Southeast Asia, Central Africa, the Caribbean region, and Central/South America (POWO 2022). P. acidus has been traditionally used to treat various diseases, including inflammation, gastrointestinal problems, rheumatism, bronchitis, Alzheimer, and hepatic diseases (Jain et al. 2011; Chakraborty et al. 2012; Srirama et al. 2012; Uddin et al. 2016). The leaves and roots of P. acidus also possess antidotal properties against viper venom (Jayvir 1998). Moreover, P. acidus exhibited the potential for alleviating hypertension (Leeya et al. 2010).\n\nThe chloroplast (cp) genome is extremely effective at inferring phylogeny since it is predominantly maternally inherited, has a conversed structure and gene content, and has a slow mutation frequency (Palmer et al. 1988). Additionally, the cp genomes provide essential data for conducting studies on population genetics, molecular identification, and genetic engineering (Powell et al. 1995; Daniell et al. 2016; Cao et al. 2022). In the current study, the characteristics of the P. acidus cp genome and its phylogenetic implication were explored to gain more information about the evolution and phylogenetic relationships within the Phyllanthaceae family and closely related taxa.\n\n\nMethods\n\nThe P. acidus sample (young branches with leaves) was collected from Can Tho, Viet Nam (9°56′55.7″N, 105°30′16.0″E) and labeled with voucher number: NTT-2022.12.CR (contact person: Dr. Do Hoang Dang Khoa, dhdkhoa@ntt.edu.vn). It was deposited at the NTT Hi-tech Institute, Nguyen Tat Thanh University. No specific permit was required to collect and study the species in Vietnam. The leaf sample was dried with silica gel and stored in a -80°C freezer until conducting DNA extraction.\n\nThe total genomic DNA extraction from the dried leaves was carried out using the Cetyltrimethylammonium bromide (CTAB) protocol (Doyle & Doyle 1987). The quality of genomic DNA samples was checked using gel electrophoresis and NanoDrop OneC Spectrophotometer. The DNA samples that showed a clear band on agarose gel and had a 260/280 ratio between 1,8-2 and a 260/230 ratio between 2.0-2.2 were selected for conducting the next-generation sequencing step. Subsequently, the library was prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, USA). The library was sequenced using Illumina Miseq platform to generate paired-end reads of 150 bp (Ktest Science Co. Ltd., Vietnam). The raw reads were qualified and filtered low-quality reads (Q score < 20 and length < 100 bp) and reads containing primers or adapters using FastQC v0.12.1 and Trimmomatic v0.39 programs (Andrews 2010; Bolger et al. 2014). For the assembly of cp genome, the NOVOPlasty v4.3.1 program was used (Dierckxsens et al. 2017). Preliminary annotation was conducted by Geseq with default parameters (Tillich et al. 2017). The complete annotation genome was illustrated using OrganellarGenomeDRAW v1.3.1 (Greiner et al. 2019). All 79 protein-coding regions in the cp genomes of P. acidus and 16 related taxa from the Phyllanthaceae were extracted and aligned for phylogenetic analysis using MUSCLE v5 program (Edgar 2004). The chloroplast genome of Acalypha hispida (Euphorbiaceae; Genbank accession no. NC_070339) was selected as an outgroup. A maximum likelihood phylogenetic tree was reconstructed using IQTREE with 1000 bootstrap replicates and GTRGAMMA substitution model (Nguyen et al. 2015).\n\n\nResults\n\nApproximately 349.8 MB of clean reads were obtained and used for completing the cp genome of P. acidus. The assembly process utilized 1,166,034 paired-end reads, resulting in an average coverage depth of 2,234.3X (Nguyen, Nguyen, Do, and Vu, 2023; Nguyen, Do, and Vu, 2023) The quadripartite cp genome of P. acidus (GenBank accession number OR050568) had a length of 156,331 bp and consisted of an LSC region of 85,807 bp, a SSC region of 19,262 bp, and a pair of IR regions of 25,631 bp (Figure 1). The overall GC content of the genome was 36.9%, and the GC content of the LSC, SSC, and IR regions were 34.6%, 30.6%, and 43.1%, respectively. The cp genome of P. acidus contained a total of 130 genes, including 85 protein-coding regions, 37 tRNA genes, and eight rRNA genes (Table 1). Among 85 protein-coding genes, 17 genes contained intron, of which ycf3 and clpP contained two introns. In IR regions, a total of 19 genes were duplicated, including eight protein-coding regions (i.e., rps19, rpl2, rpl23, ycf1, ycf2, ndhB, rps12, and rps7), seven tRNAs (trnI_CAU, trnL_CAA, trnV_GAC, trnI_GAU, trnA_UGC, trnR_ACG, and trnN_GUU), and four rRNAs (rrn16S, rrn23S, rrn4.5S, and rrn5S). Notably, rps19 and ycf1 duplications were incomplete. The phylogenetic analysis revealed a paraphyly of Phyllanthus species, in which Breynia futicosa and Glochidion chodoense formed a clade with Phyllanthus amarus (Figure 2). Therefore, more genomic data and samples of Phyllanthaceae species are required for further phylogenetic studies.\n\nGenes located inside the circle are transcribed in a clockwise direction, while genes outside the circle are transcribed counterclockwise. The inner circle depicted in dark gray that represents the GC content, while the light-gray circle represents the AT content of the genome. LSC: large single copy; SSC: small single copy; IRA/IRB: inverted repeat regions.\n\n* Gene with introns; 2x – duplicated gene in IR region; 2xa – incomplete duplicated gene in IR region.\n\nThe asterisk indicates P. acidus sequenced in this study. The numbers next to each node are bootstrap values.", "appendix": "Data availability\n\nNCBI Short Read Archive (SRA): DNA-seq of Phyllanthus acidus. Accession number SRR24772537; https://www.ncbi.nlm.nih.gov/sra/SRR24772537 (Nguyen, Nguyen, Do, and Vu, 2023).\n\nNCBI Assembly database: Phyllanthus acidus chloroplast, complete genome. Accession number OR050568; https://www.ncbi.nlm.nih.gov/nuccore/OR050568 (Nguyen, Do, and Vu, 2023).\n\n\nAcknowledgements\n\nThe authors thank to anonymous reviewers for their helpful comments to improve the quality of this manuscript.\n\n\nReferences\n\nAndrews S: FastQC: A Quality Control Tool for High Throughput Sequence Data.2010.\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCao D-L, et al.: Application of chloroplast genome in the identification of Traditional Chinese Medicine Viola philippica. BMC Genomics. 2022; 23(1): 540. Publisher Full Text\n\nChakraborty R, et al.: Antiinflammatory, antinociceptive and antioxidant activities of Phyllanthus acidus L. extracts. Asian Pac. J. Trop. Biomed. 2012; 2(2): S953–S961. Publisher Full Text\n\nDaniell H, et al.: Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol. 2016; 17(1): 134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDierckxsens N, Mardulyn P, Smits G: NOVOPlasty: De novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 2017; 45(4): gkw955. Publisher Full Text\n\nDoyle JJ, Doyle JL: A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 1987; 19: 11–15.\n\nEdgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32(5): 1792–1797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreiner S, Lehwark P, Bock R: OrganellarGenomeDRAW (OGDRAW) version 1.3.1: expanded toolkit for the graphical visualization of organellar genomes. Nucleic Acids Res. 2019; 47(W1): W59–W64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJain NK, et al.: Effects of Phyllanthus acidus (L.) Skeels fruit on carbon tetrachloride-induced acute oxidative damage in livers of rats and mice. Zhong Xi Yi Jie He Xue Bao = Journal of Chinese Integrative Medicine. 2011; 9(1): 49–56. PubMed Abstract \| Publisher Full Text\n\nJayvir A: Nature heals, a glossary of selected indigenous medicinal plants of India. Indian J. Pharm. 1998; 30(2): 126.\n\nLeeya Y, et al.: Hypotensive activity of an n-butanol extract and their purified compounds from leaves of Phyllanthus acidus (L.) Skeels in rats. Eur. J. Pharmacol. 2010; 649(1–3): 301–313. PubMed Abstract \| Publisher Full Text\n\nNguyen L-T, et al.: IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 2015; 32(1): 268–274. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNguyen DH, Nguyen TDQ, Do KHD, et al.: DNA-seq of Phyllanthus acidus. NCBI Short Read Archive. [Dataset]. 2023. Reference Source\n\nNguyen DH, Do KHD, Vu TM: Phyllanthus acidus chloroplast, complete genome. [Dataset]. GenBank. 2023. Reference Source\n\nPalmer JD, et al.: Chloroplast DNA variation and plant phylogeny. Ann. Mo. Bot. Gard. 1988; 75: 1180–1206. Publisher Full Text\n\nPowell W, et al.: Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines. Proc. Natl. Acad. Sci. 1995; 92(17): 7759–7763. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPOWO: Plants of the World Online. Facilitated by the Royal Botanic Gardens, Kew.2022.\n\nSrirama R, et al.: Hepatoprotective activity of Indian phyllanthus. Pharm. Biol. 2012; 50(8): 948–953. Publisher Full Text\n\nTillich M, et al.: GeSeq – versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 2017; 45(W1): W6–W11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUddin MS, et al.: Neuroprotective Effect of Phyllanthus acidus L. on Learning and Memory Impairment in Scopolamine-Induced Animal Model of Dementia and Oxidative Stress: Natural Wonder for Regulating the Development and Progression of Alz. Adv. Alzheimer's Dis. 2016; 05(02): 53–72. Publisher Full Text" }	[ { "id": "202362", "date": "22 Jan 2024", "name": "Jessica D. Rey", "expertise": [ "Reviewer Expertise Molecular characterization of different plant species i.e. Philippine endemic medicinal plants", "strawberry varieties planted in the Philippines", "genetics of disease resistance cultivated rice", "soilless culture of leafy and fruiting vegetables" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good genome note on the sequencing and assembly of the plastome of Phyllanthus acidus. However, the paper lacks enough details on the reasons behind investigating the species (i.e., limited genetic and genomic resources? problems with identification?).\nThe protocol on sequencing and assembly is appropriate and is clearly described in the paper. To improve data transparency, I suggest including supplementary data such as depth of coverage/read mapping depth and the list of genes which are hard to annotate (i.e., cis-splicing/trans-splicing genes). In addition, I would also like to suggest the use of at least two different methods of phylogenetic analyses (e.g., Maximum likelihood and Bayesian inference) and to indicate the statistical support values [of the two methods] in the tree.\nLastly, to improve readability of the paper I would like to recommend checking for grammatical errors.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1059
https://f1000research.com/articles/9-1196/v1	05 Oct 20	{ "type": "Study Protocol", "title": "UNICORNS: Uveitis in childhood prospective national cohort study protocol", "authors": [ "Salomey Kellett", "Jugnoo S Rahi", "Andrew D. Dick", "Rachel Knowles", "Valerija Tadić", "Ameenat Lola Solebo", "Salomey Kellett", "Jugnoo S Rahi", "Andrew D. Dick", "Rachel Knowles", "Valerija Tadić" ], "abstract": "Background: Childhood uveitis is a rare inflammatory eye disease which is typically chronic, relapsing-remitting in nature, with an uncertain aetiology (idiopathic). Visual loss occurs due to structural damage caused by uncontrolled inflammation. Understanding of the determinants of long term outcome is lacking, including the predictors of therapeutic response or how to define disease control. Aims: To describe disease natural history and outcomes amongst a nationally representative group of children with non-infectious uveitis, describe the impact of disease course on quality of life for both child and family, and identify determinants of adverse visual, structural and developmental outcomes. Methods: UNICORNS is a prospective longitudinal multicentre cohort study of children newly diagnosed with uveitis about whom a core minimum clinical dataset will be collected systematically. Participants and their families will also complete patient-reported outcome measures annually from recruitment. The association of patient (child- and treatment- dependent) characteristics with outcome will be investigated using logistic and ordinal regression models which incorporate adjustment for within-child correspondence between eyes for those with bilateral disease and repeated outcomes measurement. Discussion: Through this population based, prospective longitudinal study of childhood uveitis, we will describe the characteristics of childhood onset disease. Early (1-2 years following diagnosis) outcomes will be described in the first instance, and through the creation of a national inception cohort, longer term studies will be enabled of outcome for affected children and families.", "keywords": [ "Uveitis", "Child", "Prospective Cohort", "Vision", "Quality of Life" ], "content": "Background\n\nUveitis is a collective term for a varied group of rare conditions characterised by intraocular inflammation, in which uncontrolled disease can lead to irreversible ocular damage and visual loss1–3. It is less commonly seen in children than adults, with an estimated incidence of 5/100,000 children per annum2,4,5. Disease of childhood onset is typically chronic and relapsing-remitting in nature, and often complicated by the co-occurrence of systemic inflammatory disease2,6,7. The primary intraocular location of the inflammation is used to classify uveitis type into anterior, intermediate, posterior and pan-(global)-uveitis8. The majority of affected children (between 40–90% dependent on the studied population) have chronic anterior disease2,9,10. Whilst up to half of childhood uveitis occurs as isolated ocular disease, uveitis can also be classified by the presence of an associated disorder8, the most common being juvenile idiopathic arthritis (JIA, a group of diseases with an estimated pooled prevalence of 30 per 100,000)11. Infectious causes are also well recognised but uncommon2.\n\nAffected children are managed with topical corticosteroids and systemic immunotherapies. Disease control is achieved in between 30–70% by one year after diagnosis1–3,6,7,9,12. There are uncertainties around the long-term outcomes for children with uveitis, and the determinants of those outcomes. Several relatively large but methodologically heterogenous retrospective studies have reported conflicting findings on postulated predictors of ocular outcome or therapeutic response, including gender12–15, age at onset, disease duration, or serological markers such as anti-nuclear antibody (ANA)1,16–20. There are conflicting reports with regards to the beneficial or harmful role of topical steroids20,21, and the prognostic significance of low grade inflammation22,23. Consequently, there exists significant diversity of practice and absence of consensus on the management of disease24.\n\nIn order to improve outcomes for children with non-infectious uveitis, we need to describe long term outcomes and their predictors. Existing work on the predictors of arthropathy outcomes for children with JIA suggests that these predictors may be clinical, demographic, environmental, psychological and or social25–29. We shall undertake a multi-centre, prospective inception cohort study, which aims to (1) describe disease natural history and outcomes amongst a nationally representative group of children with all-cause non-infectious uveitis, (2) describe the impact of disease course on quality of life for both child and family, and (3) identify determinants of therapeutic response, adverse visual, structural and developmental outcomes, and quality of life outcomes. We present the protocol for this study, the ‘Uveitis in Childhood Prospective National Cohort Study’, or ‘UNICORNS’.\n\n\nMethods\n\nUNICORNS is a multicentre study which includes 26 secondary or tertiary care centres in the United Kingdom (UK) (see extended data - S130), although it will also be open to any hospital based paediatric ophthalmology service delivering care to children newly diagnosed with uveitis. Whilst participant identification and data sharing will occur across all sites, the primary research centre for the study will be the UCL Great Ormond Street Institute of Child Health.\n\nEligible children will be those aged under 18 years old (at diagnosis) who are newly diagnosed with uveitis. Exclusion criteria for the study includes (1) uveitis due to malignancy, ocular trauma (including iatrogenic, ie intraocular surgery), or due to confirmed ocular infection.\n\nThe families of eligible participants will be approached by their clinical team during the delivery of their routine care and informed of the study, and given contact details for the research team. Families will then contact the research team by email or by telephone to confirm their interest. A study video and study website have been established to support informed recruitment. Families will also contact researchers in person at sites where the research team are also members of clinical team. Families may also contact the research team electronically following notification of study via existing patient support groups which have agreed to support recruitment for this study (Olivia’s Vision, the largest uveitis patient group internationally). Following contact of the research team by the family, a study pack will be sent for completion. The study pack contains a participant information sheet, consent form, assent for young people, and a family background questionnaire which collects data on self-described ethnic background and the socioeconomic markers such as home ownership and parental educational attainment (see extended data - S230). On receipt of a completed consent form, the child’s clinical team and GP will be notified of the child’s recruitment.\n\nPatient reported outcome measures (PROMs) will be collected from families at study entry and then annually over the duration of the study. The PROMs to be completed at baseline comprise:\n\n- Strengths and Difficulties Questionnaire (SDQ): This is to screen the emotional and behavioural aspects of the participants lives. It comprises of 25 items split across 5 scales assessing emotional symptoms, conduct problems, hyperactivity-inattention, peer problems and prosocial behaviour31. It takes approximately 5–10 mins to complete, and will be completed by children aged over 10 years, and by parents for children aged over 2 years.\n\n- Child Health Utility 9D (CHU9D): This is a brief (<5 mins to complete) generic preference based measure of health related quality of life, specifically developed with and for young people32. It is to be completed by children aged over 7 years and parents for those aged over 2 years.\n\n- Children's Sleep Habits Questionnaire (CSHQ): This evaluates the incidence of behaviours linked with typical paediatric sleep difficulties33, and takes an estimated 5 to 10 mins to complete. It will be completed by children aged over 10 years, and by parents for children aged over 2 years.\n\n- 100-mm general evaluation (GE): This visual analogue score (a horizontal line 100mm long) will be used to capture overall perception of the burden of the child’s uveitis. It will be completed by children aged over 5 years, and by parents for children aged 2–17.\n\nAt one year after diagnosis, and annually, children and their families will be invited to complete and return the following PROMS:\n\n- 100-mm general evaluation (GE)\n\n- Child Health Utility 9D (CHU9D)\n\n- Paediatric Quality of life Score (PedsQL): This is used to evaluate health related quality of life in children. It is a brief questionnaire (typically 5 mins to complete) to be completed by children aged over 5 years, and parents of children aged over 2 years.\n\n- Vision related quality of life metric and Functional Vision Questionnaires for Children and Young People (VQoL_CYP & FVQ_ CYP): This pair of instruments captures the functional and broader impacts of living with a visual disability24,34. They are self-completed for children over 8 years, and take 15 minutes to complete.\n\nInterim analyses will be undertaken to understand the correlation between these PROMs, and in the event of strong correlation indicative of instrument redundancy, the use of the PROMS will be rationalised during the study in order to limit the time spent by the families in completing them.\n\nAt baseline all families will also be asked to complete a family background form (approx. time for completion 5 minutes). Parents of all children aged under 10 years will be asked (over the phone, or in person, approx. time for completion 5 – 10 minutes) for information available in pages of their child’s personal child health record (PCHR, or red book, current version in use since 2009). This information comprises perinatal adverse events, birth weight, vaccination history and any developmental concerns. Consent will also be sought from participating families and young people for future linkage of cohort data to routine health, social care and education databases for collection of long term broad developmental and health outcomes.\n\nAll participating centres have agreed in principle to collect, for all children presenting with uveitis, and then at every clinic appointment, a standardised clinical dataset comprising the core clinical and outcome variables (CCOV) (see extended data - S330). These comprise ocular and systemic clinical findings at presentation, serological and imaging investigations undertaken, treatments prescribed to the children and degree of therapeutic response, and clinical outcomes such as visual acuity of the development of a sight threatening complication. This will enable exploration of confounders of disease outcome such as severity of disease at onset, age at onset, disease duration, and use of immunosuppression. Harmonisation work has been undertaken across sites ahead of study, to reach consensus on CCOV and the clinical definitions in use. The CCOV will be incorporated into routine clinical notes in either paper or electronic format, thus avoiding additional administrative burden to the collaborating clinician. Data are to be returned via postal and/or electronic transfer, or direct from clinical records by the research team. Data will be pseudonymised (through use of an alphanumeric link code) prior to return. Acquired images of the affected eyes will also, where possible, be returned to the research team via secure NHS to NHS DICOM® (Digital Imaging and Communications in Medicine) image sharing networks. Annual case note review by researchers at large volume centres (>5 children recruited per year) will occur to ensure completeness of data capture.\n\nOur aim is to study all eligible children diagnosed across this multicentre network, over a period of 3 years, with an expectant consent rate of 60% and expectant attrition rate of 20%. This should result in a minimum of 250 children recruited over a three year period. This anticipated sample will allow us to reliably detect clinically important associations: at α=0.05, this sample size should >80% power to compare differences in proportions of at least 20% between groups. This assumes that the smaller group has at least 100 subjects35.\n\nThese endpoints will be used, as measured every year following diagnosis:\n\nPrimary endpoints:\n\n- New incidence of sight threatening ocular complications, including glaucoma, cataract, and macular oedema\n\n- Quality of life\n\nSecondary endpoints:\n\n- Total prescribed topical and systemic corticosteroid burden\n\n- Attainment of disease control (absolute control defined as absence of inflammation, relative control defined as the absence of inflammation greater than 0.5, the lowest grade of inflammation, with the use of less than one drop of topical corticosteroid)\n\nThe timeline for when these will be collected is provided in Table 1.\n\nSDQ: strength and difficulties questionnaire; CHU9D: Child Health Utility 9D; CSHQ: Children's Sleep Habits Questionnaire; GE: 100-mm General Evaluation; PedsQL: Paediatric Quality of life Score; VQoL_CYP: Vision related quality of life metric – Children and Young People.\n\n(x): in the event of strong correlation indicative of instrument redundancy, the use of these PROMS will be rationalised during the study in order to limit the time spent by the families in completing them.\n\nPatient demographic, socioeconomic and clinic characteristics, and outcomes, will be described, as will the use of and timing of the different topical and systemic agents. Continuous variables will be reported as means with standard deviations or medians with interquartile range. Categorical variables will be reported as proportions, with 95% confidence intervals. Outcomes will also be stratified by uveitis type (anterior versus other uveitis, and JIA associated versus other uveitis), by age of onset, and by use of systemic therapy within the first six months following diagnosis.\n\nThe association of patient (child- and treatment- dependent) characteristics with outcome for the largest population (those with chronic anterior uveitis, expected to be 80% of the total group), will be investigated using logistic and ordinal regression models (STATA / R software) which incorporate adjustment for time since disease onset. Multi-level modelling will be used for adjustment for within child correspondence between eyes for those with bilateral disease, and the clustered nature of repeated measures for this chronic disease.\n\nFunctional principal component analysis (FPCA) for sparse longitudinal data will be undertaken to investigate the different trajectories of ocular inflammation amongst children with chronic anterior uveitis. The covariates used in this investigation will be those identified as potential mediators through the regression analyses. Where children have bilateral disease, the most severely affected eye will be selected for use in modelling. For those children with symmetrical bilateral disease, one eye will be chosen at random for inclusion.\n\nWe shall also investigate the clustering of clinical findings within subtypes of all forms of uveitis disease using latent cluster analysis of demographic variables and clinical variables.\n\nThe study has been approved by the Health Research Authority Research Ethics Committee (IRAS 258638, REC 20/LO/0661). This study will be registered with ClinicalTrials.org.\n\nThe results from this study will be published on the institutional website and published in peer reviewed journals. Study reports will be in accordance with the Strengthening the\n\nReporting of Observational Studies in Epidemiology Statement Guidelines (STROBE). Study newsletters will be made available online for participants and distributed through patient support groups. A study ‘Open Day’ will be held at the end of year 3 to inform participants on study progress and invite input on planned dissemination processes, with an online link to videos created during the day to involve those unable to attend on the day.\n\nThe study aims were informed by the priorities identified by stakeholders (patients and professional groups) who participated in the 2013 James Lind Alliance Priority Setting Partnership (JLA PSP)36. Amongst the top research topic priorities for those affected with inflammatory eye disease were the effectiveness of treatments, the ability to predict disease severity, the development of early detection methods, and the safety of current monitoring for ongoing chronic uveitis.\n\nThis study is supported by the Childhood Uveitis Studies Steering group (SSG), which was formed in April 2019 in order to provide ‘stakeholder’ guidance for study aims, methodology, and dissemination plans. The group consists of three young people directly affected by childhood uveitis, and three parents of affected children. This group have been supported and trained in research methods through written materials / presentations. At least two SSG meetings have been held each year since group formation with email communications between meetings. This group has co-developed the cohort study methods – e.g. selection of patient reported outcomes and study participant literature. This study has also benefitted from regular communication with the UK based patient led support groups for childhood uveitis (Olivia’s Vision) and childhood arthritis (‘JIA matters / Versus Arthritis’).\n\nData are to be entered into study specific databases and manage by the research team (SK and ALS). All information collected during the course of the study will be kept strictly confidential. Forms will be anonymised and coded using a unique patient identifier assigned at the notification stage. Stored patient information will be kept on NHS and University computers, so as to be able to track the patient in the study. All patient information will be managed according to Data Protection Act 2018 requirements.\n\nThe datasets generated and/or analysed during the current study will be made available (following anonymization) by the corresponding author on reasonable request. Authorised collaborators will be granted access to aggregated anonymised data from participants who have consented to this level of sharing, following review of their study protocol. Requests will be reviewed by the research team and a patient representative. Data and material transfer agreements will be required to be completed, in order to ensure regulatory compliance and that the interests of the participants are upheld and respected throughout.\n\nResources created through study processes will be shared upon reasonable request.\n\nThe study is currently open to recruitment.\n\n\nDiscussion\n\nUveitis carries the risk of blindness and can result in life long burden of avoidable disability and the attendant high financial and social costs of medical care, education, rehabilitation and support needed for visually impaired children and the adults they become. Improved understanding of the factors associated with favourable and adverse outcomes for affected children are necessary for planning care and service provision. This multicentre study will result in a nationally representative cohort of children with non-infectious uveitis, providing externally valid findings on the determinants of outcome. The prospective collection of patient reported outcome alongside an agreed minimal core clinical dataset will enable robust evaluation of the role of postulated risk factors with adjustment for identified confounding. It will also provide valuable information on the lived experience of these children, through the use of a range of patient reported measures. This allows UNICORNS to capture a more complete spectrum of patient centred outcomes than have been reported by previous studies. This study also offers a timely opportunity to investigate the outcomes of childhood uveitis in the United Kingdom at a time when more targeted, biologic agents such as adalimumab and tocilizumab have either been commissioned, or recommended for use in children with refractory disease37,38. Within trial populations, up to a quarter of children continue to have uncontrolled disease despite the use of these agents37. UNICORNS will provide information on the feasibility of undertaking future ‘Trial within cohorts’ or other pragmatic interventional trial designs, and provide a data sharing infrastructure to support ‘classic’ randomised controlled trials of emerging novel interventions. Greater understanding of this population of children affected by a rare, chronic, inflammatory, idiopathic disease will also allow investigators to develop future nested trials of complex interventions which are targeted at patient (eg, packages of support around the time of transition and transfer to adult care), clinician (eg, decision support models for rare disease) or organisation level (eg, information sharing platform across primary and secondary care for rare disease). Thus, the findings of this cohort study will be of direct importance to clinical practice and future research within this disease area and beyond.\n\nThis study design involves additional burden on the participants and their family through the completion of multiple carefully chosen and validated patient report metrics. These metrics characterise the family experience, global and vision related quality of life and health related utility. This allows for data collection on outcomes that our PPI work has shown to be of crucial importance to the affected families.\n\nObservational studies such as UNICORNS are open to confounding, preventing causal inference when associations are reported. Our prospective design and use of a core minimal dataset should enable careful consideration of identified potential confounders. In order to strengthen study findings, all study reports will be reported in accordance with STROBE guidelines.\n\nAttrition is always a concern with regards to longitudinal studies. This study is supported by a national clinical network, the Paediatric Ocular Inflammation Group24,39, which will support follow up should children transfer from one clinical unit to another. The involvement of the child’s primary care giver, and the support of patient groups and their continued publication of study newsletters, is also expected to reduce attrition rates.\n\nChildhood onset uveitis confers particular burden due to chronicity, the association with systemic inflammatory disease, and the frequent requirement for systemic immunosuppression. There remain unanswered questions around disease phenotypes, long-term eye and wider developmental outcomes, and the determinants of those outcomes, with resultant limitations in the evidence base used to counsel affected families, balance treatment decisions, or plan further research. We propose a population based, prospective longitudinal study of childhood uveitis, in order to describe the characteristics of childhood onset disease. We will describe outcome and investigate socio-demographic, clinical, biological and treatment related determinants of outcome. Early (1–2 years following diagnosis) outcomes will be described in the first instance, and through the creation of a national inception cohort, we shall enable longer term studies of outcome for affected children and families.\n\nThis study is approved by the UK Health Research Authority. Patients or their legal representative will have to sign an informed consent form before study entry. This study is in accordance with the Declaration of Helsinki and in accordance with the Medical Research Involving Human Subjects Act (WMO). Risks for participating in this study are expected to be very small to negligible and all patients will receive standard care. Objection or incapacitation of a patient or their representative will lead to exclusion from the study and analysis.\n\n\nData availability\n\nNo data are associated with this article.\n\nOpen Science Framework: UNICORNS extended data: https://doi.org/10.17605/OSF.IO/EUBMA30\n\nThis project contains the following extended data:\n\n- S1.pdf (List of collaborating centres)\n\n- S2.pdf (Study information pack)\n\n- S3.pdf (Minimal uveitis core dataset)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors would like to thank the clinical teams at the collaborating unites for their contributions to the study. We should also like to thank Phillippa Cumberland, Ulverscroft Senior Biostatician previously based at UCL GOS ICH, for her guidance with statistical design.\n\n\nReferences\n\nGregory AC, Kempen JH, Daniel E, et al.: Risk factors for loss of visual acuity among patients with uveitis associated with juvenile idiopathic arthritis: the Systemic Immunosuppressive Therapy for Eye Diseases Study. Ophthalmology. 2013; 120(1): 186–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdelsten C, Reddy MA, Stanford MR, et al.: Visual loss associated with pediatric uveitis in english primary and referral centers. Am J Ophthalmol. 2003; 135(5): 676–80. PubMed Abstract \| Publisher Full Text\n\nde Boer J, Wulffraat N, Rothova A: Visual loss in uveitis of childhood. Br J Ophthalmol. 2003; 87(7): 879–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAcharya NR, Tham VM, Esterberg E, et al.: Incidence and prevalence of uveitis: results from the Pacific Ocular Inflammation Study. JAMA Ophthalmol. 2013; 131(11): 1405–12. PubMed Abstract \| Publisher Full Text\n\nGritz DC, Wong IG: Incidence and prevalence of uveitis in Northern California; the Northern California Epidemiology of Uveitis Study. Ophthalmology. 2004; 111(3): 491–500; discussion 500. PubMed Abstract \| Publisher Full Text\n\nMorelle G, Gueudry J, Uettwiller F, et al.: Chronic and recurrent non-infectious paediatric-onset uveitis: a French cohort. RMD open. 2019; 5(2): e000933. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerrara M, Eggenschwiler L, Stephenson A, et al.: The Challenge of Pediatric Uveitis: Tertiary Referral Center Experience in the United States. Ocul Immunol Inflamm. 2019; 27(3): 410–17. PubMed Abstract \| Publisher Full Text\n\nJabs DA, Nussenblatt RB, Rosenbaum JT: Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop. Am J Ophthalmol. 2005; 140(3): 509–16. PubMed Abstract \| Publisher Full Text\n\nPaivonsalo-Hietanen T, Tuominen J, Saari KM: Uveitis in children: population-based study in Finland. Acta Ophthalmol Scand. 2000; 78(1): 84–8. PubMed Abstract \| Publisher Full Text\n\nClarke LA, Guex-Crosier Y, Hofer M: Epidemiology of uveitis in children over a 10-year period. Clin Exp Rheumatol. 2013; 31(4): 633–7. PubMed Abstract\n\nThierry S, Fautrel B, Lemelle I, et al.: Prevalence and incidence of juvenile idiopathic arthritis: a systematic review. Joint Bone Spine. 2014; 81(2): 112–7. PubMed Abstract \| Publisher Full Text\n\nKalinina Ayuso V, ten Cate HA, van den Does P, et al.: Young age as a risk factor for complicated course and visual outcome in intermediate uveitis in children. Br J Ophthalmol. 2011; 95(5): 646–51. PubMed Abstract \| Publisher Full Text\n\nKalinina Ayuso V, Ten Cate HA, van der Does P, et al.: Male gender as a risk factor for complications in uveitis associated with juvenile idiopathic arthritis. Am J Ophthalmol. 2010; 149(6): 994–99.e5. PubMed Abstract \| Publisher Full Text\n\nCouto C, Frick MM, LaMattina K, et al.: Chronic Anterior Uveitis in Children. Ocul Immunol Inflamm. 2016; 24(4): 392–6. PubMed Abstract \| Publisher Full Text\n\nDana MR, Merayo-Lloves J, Schaumberg DA, et al.: Visual outcomes prognosticators in juvenile rheumatoid arthritis-associated uveitis. Ophthalmology. 1997; 104(2): 236–44. PubMed Abstract \| Publisher Full Text\n\nKalinina Ayuso V, Ten Cate HA, van der Does P, et al.: Male gender and poor visual outcome in uveitis associated with juvenile idiopathic arthritis. Am J Ophthalmol. 2010; 149(6): 987–93. PubMed Abstract \| Publisher Full Text\n\nSaboo US, Metzinger JL, Radwan A, et al.: Risk factors associated with the relapse of uveitis in patients with juvenile idiopathic arthritis: a preliminary report. J AAPOS. 2013; 17(5): 460–4. PubMed Abstract \| Publisher Full Text\n\nParoli MP, Speranza S, Marino M, et al.: Prognosis of juvenile rheumatoid arthritis-associated uveitis. Eur J Ophthalmol. 2003; 13(7): 616–21. PubMed Abstract \| Publisher Full Text\n\nSabri K, Saurenmann RK, Silverman ED, et al.: Course, complications, and outcome of juvenile arthritis-related uveitis. J AAPOS. 2008; 12(6): 539–45. PubMed Abstract \| Publisher Full Text\n\nBlum-Hareuveni T, Seguin-Greenstein S, Kramer M, et al.: Risk Factors for the Development of Cataract in Children with Uveitis. Am J Ophthalmol. 2017; 177: 139–43. PubMed Abstract \| Publisher Full Text\n\nThorne JE, Woreta FA, Dunn JP, et al.: Risk of cataract development among children with juvenile idiopathic arthritis-related uveitis treated with topical corticosteroids. Ophthalmology. 2010; 117(7): 1436–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAngeles-Han ST, Ringold S, Beukelman T, et al.: 2019 American College of Rheumatology/Arthritis Foundation Guideline for the Screening, Monitoring, and Treatment of Juvenile Idiopathic Arthritis-Associated Uveitis. Arthritis Care Res (Hoboken). 2019; 71(6): 864–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConstantin T, Foeldvari I, Anton J, et al.: Consensus-based recommendations for the management of uveitis associated with juvenile idiopathic arthritis: the SHARE initiative. Ann Rheum Dis. 2018; 77(8): 1107–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolebo AL, Rahi JS, Dick AD, et al.: Areas of agreement in the management of childhood non-infectious chronic anterior uveitis in the UK. Br J Ophthalmol. 2019; 104(1): 11–16. PubMed Abstract \| Publisher Full Text\n\nHanns L, Cordingley L, Galloway J, et al.: Depressive symptoms, pain and disability for adolescent patients with juvenile idiopathic arthritis: results from the Childhood Arthritis Prospective Study. Rheumatology (Oxford). 2018; 57(8): 1381–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcErlane F, Carrasco R, Kearsley-Fleet L, et al.: Growth patterns in early juvenile idiopathic arthritis: Results from the Childhood Arthritis Prospective Study (CAPS). Semin Arthritis Rheum. 2018; 48(1): 53–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShoop-Worrall SJW, Verstappen SMM, Baildam E, et al.: How common is clinically inactive disease in a prospective cohort of patients with juvenile idiopathic arthritis? The importance of definition. Ann Rheum Dis. 2017; 76(8): 1381–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHyrich KL, Baildam E, Pickford H, et al.: Influence of past breast feeding on pattern and severity of presentation of juvenile idiopathic arthritis. Arch Dis Child. 2016; 101(4): 348–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcErlane F, Foster HE, Carrasco R, et al.: Trends in paediatric rheumatology referral times and disease activity indices over a ten-year period among children and young people with Juvenile Idiopathic Arthritis: results from the childhood arthritis prospective Study. Rheumatology (Oxford). 2016; 55(7): 1225–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKellett S, Rahi J, Dick A, et al.: UNICORNS extended data. 2020.\n\nStone LL, Janssens JM, Vermulst AA, et al.: The Strengths and Difficulties Questionnaire: psychometric properties of the parent and teacher version in children aged 4-7. BMC Psychol. 2015; 3(1): 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRatcliffe J, Couzner L, Flynn T, et al.: Valuing Child Health Utility 9D health states with a young adolescent sample: a feasibility study to compare best-worst scaling discrete-choice experiment, standard gamble and time trade-off methods. Appl Health Econ Health Policy. 2011; 9(1): 15–27. PubMed Abstract \| Publisher Full Text\n\nOwens JA, Spirito A, McGuinn M: The Children's Sleep Habits Questionnaire (CSHQ): psychometric properties of a survey instrument for school-aged children. Sleep. 2000; 23(8): 1043–51. PubMed Abstract\n\nRobertson AO, Tadić V, Cortina-Borja M, et al.: A patient-reported outcome measure of functional vision for children and young people aged 8 to 18 years with visual impairment. Am J Ophthalmol. 2020; 219: 141–153. PubMed Abstract \| Publisher Full Text\n\nSolebo AL, Cumberland P, Rahi JS: British Isles Congenital Cataract Interest Group. 5-year outcomes after primary intraocular lens implantation in children aged 2 years or younger with congenital or infantile cataract: findings from the IoLunder2 prospective inception cohort study. Lancet Child Adolesc Health. 2018; 2(12): 863–871. PubMed Abstract \| Publisher Full Text\n\nRowe F, Wormald R, Cable R, et al.: The Sight Loss and Vision Priority Setting Partnership (SLV-PSP): overview and results of the research prioritisation survey process. BMJ Open. 2014; 4(7): e004905. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamanan AV, Dick AD, Jones AP, et al.: Adalimumab plus Methotrexate for Uveitis in Juvenile Idiopathic Arthritis. N Engl J Med. 2017; 376(17): 1637–46. PubMed Abstract \| Publisher Full Text\n\nTappeiner C, Mesquida M, Adan A, et al.: Evidence for Tocilizumab as a Treatment Option in Refractory Uveitis Associated with Juvenile Idiopathic Arthritis. J Rheumatol. 2016; 43(12): 2183–88. PubMed Abstract \| Publisher Full Text\n\nSolebo AL, Rahi JS, Edelsten C, et al.: Management of paediatric ocular inflammatory disease in the UK:national survey of practice. Eye (Lond). 2020; 34(3): 591–92. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "72432", "date": "23 Oct 2020", "name": "Joke de Boer", "expertise": [ "Reviewer Expertise My area of research is uveitis in children and adults." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important research project that will provide new information to improve treatment strategies in children with non-infectious uveitis. This project will provide an unique cohort of children with uveitis that longitudinally will be followed. The questionnaires will provide important information regarding the impact on patients suffering from childhood uveitis and their families. The outcome of QqL might also influence the care of these patients. The study is very well designed and patients are involved in the design of the project. The inclusion of 250 patients in 26 centers in 3 years is highly feasible. The topics that need further clarification are the serology tests that are performed and how infectious causes are excluded.\n\nHowever, several questions remain regarding the secondary endpoint for attainment of disease control. The authors state that absolute disease control is defined as absence of inflammation and relative disease control as absence of inflammation greater than 0,5, the lowest grade of inflammation. These definitions should be more specified because they might be different for the different anatomic subtypes of uveitis. For instance, is flare a grade of inflammation? Flare might permanently be raised after inflammation in anterior uveitis without cells in anterior chamber. Another example is intermediate uveitis where cells might be absent in anterior chamber in active diseases, however, cell in vitreous might persist for a long period without signs of active inflammation on fluorescein angiography. So disease control should be more specified for different anatomic subtypes of uveitis. Also the inflammatory parameters in the different anatomic subtypes that will be assessed should be mentioned.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "75910", "date": "04 Jan 2021", "name": "Ivan Foeldvari", "expertise": [ "Reviewer Expertise Pediatric Rheumatology", "outcome research", "juvenile idiopathic arthritis associated uveitis", "juvenile localized scleroderma", "juvenile systemic sclerosis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 unique project to learn more about childhood uveitis. Patients are assessed in a standardized manner regarding uveitis visual outcome and PROMs.\n\nI am missing the application for EYEQL ( 10.1002/acr.205241).\n\nIt would be a great chance to apply the MIWGUC outcome measures prospectively, which offer a definition for inactive disease and response to treatment (10.1186/s12969-019-0345-22).\n\nI would suggest an assessment of the patients every 6 months, the proposed annual follow up interval is to rare.\n\nI missed the collection of data of activity of the associated condition, like JIA, sarcoidosis...\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "6599", "date": "22 Apr 2021", "name": "Ameenat Lola Solebo", "role": "Author Response", "response": "We thank Prof Foeldvari very much for his comments. Responses to his suggestions for amendments to study methods are below: I am missing the application for EYEQL ( 10.1002/acr.205241). UNICORNS will only be using metrics which have been validated for use in a UK cohort, and we look forward to using EYEQL once it has not been validated for use in a UK cohort I would suggest an assessment of the patients every 6 months, the proposed annual follow up interval is to rare. Children will continue to receive routine clinical care, which involves follow up at intervals of between 1 week and 4 months. The annual interval refers only to the collection of the specific patient reported outcome metrics. I missed the collection of data of activity of the associated condition, like JIA, sarcoidosis. Data on general health status is routinely collected at clinical follow up (as indicated in the dataset documents) and will be extracted for use in UNICORNS analyses." } ] } ]	1	https://f1000research.com/articles/9-1196
https://f1000research.com/articles/12-1056/v1	30 Aug 23	{ "type": "Case Report", "title": "Case Report: Pleural effusion in Wilms tumor – always malignant?", "authors": [ "Keta Vagha", "Patel Zeeshan Jameel", "Jayant Vagha", "Ashish R. Varma", "Rupesh Rao", "Patel Zeeshan Jameel", "Jayant Vagha", "Ashish R. Varma", "Rupesh Rao" ], "abstract": "Wilms tumor (WT) is the most common renal malignancy seen in pediatric patients. Although lungs are the most common site of metastasis in Wilms tumor, non-malignant pleural effusion has been infrequently reported. Here, we report a case of an eleven-year-old female who presented with an abdominal mass and progressive breathlessness. On further evaluation, she was found to have a right-sided Wilms tumor with ipsilateral massive pleural effusion. The effusion resolved almost completely after four weeks of chemotherapy. We conclude that patients suffering from Wilms tumor presenting with pleural effusion need not be synonymous with metastatic disease and can have a favorable prognosis.", "keywords": [ "Wilms tumor", "pleural effusion", "pulmonary metastasis" ], "content": "Introduction\n\nWilms tumors are responsible for approximately 6% of all malignancies and more than 95% of renal malignancies in the pediatric age group.1 Early diagnosis, risk stratification, stage-based management and improved neo-adjuvant therapies have greatly improved the overall five-year survival up to >90%.2 Wilms tumor is most often diagnosed clinically as an incidental discovery of an asymptomatic abdominal mass by parents or attending pediatrician. Other common symptoms include abdominal pain, gross painless hematuria, constitutional symptoms, and hypertension. Rarely, fatal pulmonary embolism, hematological abnormalities and pleural effusion have been reported in children with Wilms tumor.3 Common sites for metastasis in advanced cases include abdominal lymph nodes, lungs and less often, liver and bone. Here, we report a rare case of Wilms tumor presenting clinically with a massive pleural effusion.\n\n\nCase presentation\n\nAn eleven-year-old, previously healthy adolescent girl from central India presented with a one-week history of abdominal distension with abdominal pain and a five-day history of progressive breathlessness.\n\nUpon initial physical examination, she had tachycardia (heart rate [HR]-130/min), tachypnea (respiratory rate [RR]-40/min), a normal blood pressure (110/80 mmHg) and was maintaining SpO2 on room air. Respiratory system examination showed tracheal deviation to the left, stony dull percussive note and absent breath sounds on the right side suggestive of right-sided pleural effusion. Examination of the abdomen showed a well-defined, firm, mildly tender mass (12×14 cm) palpable in right lumbar, hypochondrium, epigastric and umbilical regions. The upper border of the mass was distinctly palpable from the liver. There were no associated congenital malformations.\n\nHer hematological parameters were within limits, except thrombocytosis. Serum biochemistry was normal. Liver and kidney function tests were within limits. Urine analysis was also normal. Therapeutic thoracocentesis was done and around 750 mL of pleural fluid was aspirated gradually over the course of 48 hours, following which she improved symptomatically. Pleural fluid analysis revealed a blood-stained, sterile fluid, with protein content of 4.4 gm/dL, glucose of 91 mg/dL, and LDH of 1043 IU/L. Fluid cytology revealed markedly increased lymphoid cell with plenty of red blood cells. No malignant cells were visualized. Chest radiograph was suggestive of a massive right sided pleural effusion (Figure 1).\n\nContrast enhanced computed tomography (CECT) of abdomen showed a large, heterogeneously enhancing mass (22×16×14 cm) with multiple necrotic areas arising from the mid and upper pole of the right kidney (Figure 2).\n\nA right-sided massive pleural effusion with adjacent passive atelectasis can be seen.\n\nOn the basis of the above findings, a diagnosis of Wilms tumor with right-sided pleural effusion was made.\n\nAs per the International Society of Pediatric Oncology (SIOP), her management plan included preoperative chemotherapy followed by radical nephrectomy and post-operative chemotherapy. She received six cycles of chemotherapy prior to surgery comprising of vincristine (1.5 mg/m2), actinomycin D (45 mcg/kg) and adriamycin (50 mg/m2). Her pleural effusion completely resolved after four weeks of chemotherapy without the need for further thoracocentesis (Figure 3). She then underwent right radical nephrectomy. However, during surgery the mass was found to be densely adherent to the inferior vena cava (IVC) across its length as well as to posterior aspect of liver and diaphragm. Some residual mass adherent to IVC was left behind. Histopathological examination of the specimen was suggestive of Wilms tumor (SIOP stage III) with no lymph nodal metastasis. In view of the residual disease, she received post-operative radiotherapy with a total dose of 10.8 grays to the abdomen.\n\nFurther, as planned, she was started on weekly chemotherapy with vincristine, actinomycin D and adriamycin for 24 cycles. At time of writing, she has completed all her cycles with no further complications. Further management plan includes surveillance ultrasonography for abdominal recurrence or development of a second primary tumor in the contralateral kidney and chest CT for pulmonary metastasis after three months.\n\nInformed written consent was obtained from the patient and her parents for publication of this case.\n\n\nDiscussion\n\nThe current case is interesting because of the unusual clinical presentation of pleural effusion. Since pleural effusion shows the involvement of an organ system distant from the primary tumor site, there is a tendency to think of metastatic disease in such cases. However, there were no signs of primary pulmonary metastasis in this case. Therefore, the present case highlights that those patients suffering from Wilms tumor presenting with pleural effusion need not be synonymous with metastatic disease and can have a favorable prognosis.\n\nThe most frequent site for metastasis in Wilms tumor is the lung, occurring in up to >90% of patients with metastatic disease. Rarely, pleural metastasis has also been documented. Pleural effusion is a rarely presenting feature in children with Wilms tumor. The incidence of pleural effusion has been reported to be 4.3%.4 Different mechanisms implicated in the causation of pleural effusion are pleural metastasis, hypoproteinemia secondary to either chemotherapy or radiation-induced transient liver injury, or unrelated causes such as chylous exudate due to post-surgical lymphatic damage with associated infection.5 Sympathetic effusion due to proximity of tumor to diaphragm or damage to diaphragm due to adhesion may be the cause in our case. Even though pleural effusion is seen in patients with Wilms tumor, massive effusions are rarely seen so as to cause respiratory distress as in our case. We were able to find at least six publications (17 children) of Wilms tumor with pleural effusion with pulmonary metastasis being reported in four children which are summarised in Table 1.4–9 The significance of pleural effusion in these groups of patients is the fact that it dramatically upgrades the staging of tumor and therefore, changes the management of the patient. In a study by Wong et al.,10 the malignant positivity of pleural effusion in WT with pleural effusion was found to be 35%. In stark contrast, a retrospective analysis done at St. Jude Children’s Research Hospital, Memphis, Tennessee, USA over 16-year period detected that there were no signs of metastasis in children with WT presenting with pleural effusion.4 The treatment modality for WT with pulmonary metastasis includes chemotherapy with vincristine, actinomycin D and Adriamycin along with lung radiation therapy. The inappropriate upstaging of WT leads to over-treatment with consequent treatment-related toxicities. Pulmonary fibrosis and diffuse interstitial pneumonitis are complications secondary to lung radiation therapy for metastatic WT. Dilated cardiomyopathy is a potentially life-threatening complication due to Adriamycin by virtue of its ability to cause myocardial injury; it may also act as a radiosensitiser which further increases the potential for myocardial damage leading to reduced overall survival. Hence, appropriate staging as well as management are of utmost importance.\n\nThere is no consensus on the treatment of pleural effusion in WT. Canopolat et al. have documented the efficacy of chemotherapy alone in considerably resolving pleural effusion and noted a decrease in tumour size as well. Radiation therapy has also been documented to resolve pleural effusion.5 In our patient, although a therapeutic thoracocentesis was performed to reduce the acute symptoms, the pleural effusion resolved completely by chemotherapy alone. Moreover, CT thorax and pleural fluid cytology did not show evidence of any metastatic disease, hence, radiation therapy to lungs was not implemented.\n\n\nConclusions\n\nAlthough pleural effusion is a rare occurrence in cases of WT, it need not be synonymous with metastatic disease and can be treated effectively with a good outcome. We recommend a careful strategy in cases presenting with pleural effusion, so as to avoid chemotherapy and radiation therapy-related morbidities. The lack of consensus on management of these groups of patients necessitates further studies in determining risk factors as well as management strategies.\n\n\nPatient consent\n\nWritten informed consent was obtained from the patient and their parents for their anonymized information to be published in this article.\n\n\nAuthor contributions\n\nPZJ, KV was a major contributor for writing this manuscript and patient care. AD was majorly involved in the chemotherapy management. AA, ARR were overlooking the patient’s management and corrected the final manuscript. JV critically reviewed the abstract section as well as the final manuscript. All the authors have read and approved of the final manuscript.", "appendix": "Data availability\n\nAll data are available as part of the article and no additional data sources are required.\n\n\nReferences\n\nPastore G, Znaor A, Spreafico F, et al.: Malignant renal tumours incidence and survival in European children (1978–1997): report from the Automated Childhood Cancer Information System project. Eur. J. Cancer. 2006 Sep 1; 42(13): 2103–2114. PubMed Abstract \| Publisher Full Text\n\nDoganis D, Zborovskaya A, Trojanowski M, et al.: Wilms tumour event-free and overall survival in Southern and Eastern Europe: Pooled analyses of clinical data from four childhood cancer registries (1999–2017). Eur. J. Cancer. 2019 Jul 1; 115: 37–46. PubMed Abstract \| Publisher Full Text\n\nFriedman AD: Wilms tumor. Pediatr. Rev. 2013 Jul 1; 34(7): 328–330. Publisher Full Text\n\nCorey B, Yang CH, Wilimas JA, et al.: Significance of pleural effusion at diagnosis of Wilms tumor. Pediatr. Blood Cancer. 2004 Feb; 42(2): 145–148. PubMed Abstract \| Publisher Full Text\n\nBetkerur U, Lanzkowsky P: Pleural effusion in Wilms’ tumor. J. Pediatr. Surg. 1977 Aug 1; 12(4): 523–525. Publisher Full Text\n\nKüpeli S, Varan A, Akyüz C, et al.: Pleural effusion in wilms tumor after tru-cut biopsy. Pediatr. Hematol. Oncol. 2007 Jan 1; 24(7): 555–558. PubMed Abstract \| Publisher Full Text\n\nAl-Hadidi A, Lapkus M, Novotny NM, et al.: Wilms Tumor with Pleural Metastasis. Global. Pediatr. Health. 2020 Aug; 7: 2333794X2095229. Publisher Full Text\n\nCanpolat C, Jaffe N: Wilms’ tumor: cure of malignant pleural effusion exclusively with chemotherapy. Med. Pediatr. Oncol. 1995 Apr; 24(4): 274–277. PubMed Abstract \| Publisher Full Text\n\nSchinstine M, Abati A, Tsokos M, et al.: Cytological identification of metastatic epithelial nephroblastoma in pleural fluid: report of a case and review of literature. Diagn. Cytopathol. 2006 Sep; 34(9): 621–625. Publisher Full Text\n\nWong JW, Pitlik D, Abdul-Karim FW: Cytology of pleural, peritoneal and pericardial fluids in children. A 40-year summary. Acta Cytol. 1997 Mar 1; 41(2): 467–473. PubMed Abstract \| Publisher Full Text" }	[ { "id": "246039", "date": "07 Mar 2024", "name": "Marcello Della Corte", "expertise": [ "Reviewer Expertise Urology", "Uro-oncology", "Andrology", "Pediatric Urology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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\"Dear Authors, Thank you for submitting this manuscript. Your case report offers a valuable overview on Wilms tumor, focusing on an infrequently reported clinical finding: non-malignant pleural effusion. This article highlights that pleural effusion presenting in patients with Wilms tumor needs not to be considered a metastatic disease and can actually have a good prognosis. It represents a new finding in current literature and will certainly be useful in clinical practice. I read it with interest and I sincerely cannot find \"major points\" to be addressed, since your work is already valuable and well structured. I just would add few minor points as suggestions in order to further improve its quality.\nMinor points:\nThe introduction is complete, clear and well understandable. I just would suggest to add \"case report\" to the key words, as stated in CARE Case Report Guidelines (https://www.care-statement.org/checklist). The \"Case presentation\" section already is really comprehensive, I just have some suggestions in order to further improve its quality. \"Cinical findings\" paragraph: - Please specify the SPO2 on room air value, I think it is the only missing information in this detailed paragraph. \"Therapeutic intervention\": - I think the first sentence would be clearer if the Authors replaced the first \"her\" with \"the patient's\". \"Discussion\": - What do the Authors mean by \"primary metastasis\"? Did they mean \"first\"?\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": "11221", "date": "04 Apr 2024", "name": "Keta Vagha", "role": "Author Response", "response": "I am extremely grateful for your positive comments. I shall make the corrections advised by you and resubmit the manuscript." } ] }, { "id": "202399", "date": "22 Jul 2024", "name": "Keerti Swarnakar", "expertise": [ "Reviewer Expertise Pediatrics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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,\n\nThank you for submitting \"Pleural Effusion in Wilms Tumor – Always Malignant? A Case Report.\" In cases of Wilms tumor (WT) presence of pleural effusion either at the presentation or developing during the course of disease, it’s always important to decide whether it's malignant or non-malignant. In your case report detailed account of the eleven-year-old patient is insightful, highlighting the non-metastatic nature of pleural effusion in Wilms tumor. The comprehensive clinical presentation, effective management with chemotherapy, and thoughtful follow-up plan provide valuable guidance for clinicians. Your comparison with other cases and discussion on the etiology and prognosis enrich the medical literature. This case report is an excellent educational resource, offering practical insights into managing similar presentations.\n\nBrief Description of the case-\n\nThis case report details an eleven-year-old girl with Wilms tumor presenting with a right-sided abdominal mass and massive pleural effusion. After four weeks of chemotherapy, the pleural effusion resolved almost completely. The case highlights that pleural effusion in Wilms tumor patients does not necessarily indicate metastatic disease and can have a favorable prognosis with appropriate treatment.\n\nMinor Corrections-\n\n1. Kindly add 'Case Report' in the keywords as it is required under the Care Guidelines.\n\n2. In the case presentation- Specify the exact duration between the onset of symptoms and the diagnosis. This will provide a clearer understanding of the disease progression and the promptness of the medical intervention.\n\n3. In the case presentation - Mention the full form of SpO2 and also its value for better understanding.\n\n4. Mention whether Immunohistochemistry and immunofluorescence done or not.\n\n5. Please comment whether tuberculosis was ruled out or not.\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/12-1056
https://f1000research.com/articles/12-1055/v1	30 Aug 23	{ "type": "Case Report", "title": "Case Report: Synchronous primary location of gastrointestinal stromal tumors (GIST) and adenocarcinoma of the colon: an unusual occurrence", "authors": [ "Asma Sghaier", "Amine El Ghali", "Khalil Fradi", "Dorra Chiba", "Fehmi Hamila", "Sabri Youssef", "Amine El Ghali", "Khalil Fradi", "Dorra Chiba", "Fehmi Hamila", "Sabri Youssef" ], "abstract": "Background: We have little knowledge about the synchronous occurrence of gastrointestinal stromal tumors (GISTs) and other types of histologic tumors. This association is very rare. Case presentation: We describe a case of synchronous stromal tumor and adenocarcinoma of the left side colonic localization. Immunohistochemistry identified c-Kit expression. The discovery of colonic adenocarcinoma was on operative specimen after histologic examination. The patient underwent left carcinologic colectomy with stoma. Follow-up at one year postoperatively did not detect tumor recurrence. Discussion: Clinical implications of the association between these two neoplasms are not clearly described. Treatment depends on the dominance of one histologic type. Knowledge of the genetic data of this association offers opportunity of treatment with the new targeted-therapy molecules. Surgical resection, may remain the curative treatment. Conclusions: Synchronous adenocarcinoma and GIST has been more commonly described in the stomach. The pathogeneses of tumorigenesis may not be the same for the two tumors. More studies seem be necessary to clarify a potential role of different genes in the development of adenocarcinomas. And therefore, above all their therapeutic implications", "keywords": [ "Colon- adenocarcinoma-GIST- C-Kit-synchronous" ], "content": "Introduction\n\nStromal tumors are the most common mesenchymal tumors of the gastrointestinal tract. They derive from the interstitial cells of Kajal.1 The coexistence of gastrointestinal stromal tumors (GIST) and colorectal adenocarcinomas is unusual. This association has been rather more described in the stomach. Most of them were discovered during surgical intervention for primary gastrointestinal adenocarcinoma.1,2 The synchronous occurrence of primary colonic adenocarcinoma and stromal tumors brings us to think about the possibility of similar origin and carcinogenetic process, and the possibility of similar systemic drugs specially target therapy. Furthermore, the association of specific tumors often leads to the discovery of novel genetic pathways to carcinogenesis that could be important for the development of oncologic therapeutics protocols.\n\n\nCase presentation\n\nA 79-year-old White retired school-teacher male was admitted complaining of asthenia and diffuse abdominal pain. The patient had no notable pathological history and had never been operated on. The patient also had no familial pathologic history notably no cancer history. The physical examination revealed a large, solid pelvic mass extending to the epigastrium, which was responsible for abdominal pain and a feeling of tightness (Figure 1).\n\nA colonoscopy was performed but was incomplete due to the presence of an impassable stenosis at the sigmoid, which seemed to be extrinsic. An abdominal-pelvic CT scan was performed and described a large abdominal-pelvic mass of 25 cm in length, which was enhanced after injection of contrast product and seemed to have a digestive origin (Figure 2).\n\nLaparotomy confirmed the presence of a voluminous mass of the sigmoid adhering and invading the bladder dome extended to the upper rectum. This mass was friable, necrotic in places and centered by a liquefied hematoma (Figure 3).\n\nThere was no evidence of metastatic disease. The patient had undergone extensive carcinologic resection and the bladder dome was partially resected. In fact, a bladder bezel that was adhering to the tumor was removed. Given the hemorrhagic nature and the precarious nutritional state of the patient. We decided to postpone an anastomosis and perform a Hartmann stoma. Surgical follow-up was favorable, and the patient was discharged on the eighth day of the post-operative period.\n\nThe surgical resection piece was sent to the department of Pathology, macroscopically, the specimen corresponded to the left colon extended to the rectum measuring 20 cm in length, 3.5 cm at the colonic border and 2.5 cm at the rectal border. The wall was the site of a shredded tumor lesion extending 15 cm in height (Figure 4).\n\nOn opening, the colonic lumen was partially obstructed by a 4 cm high protrusion of the colonic mucosa, under which there was a whitish tumor proliferation with two macroscopic aspects, whitish fasciculated in the submucosa ulcerating the mucosa (Figure 4).\n\nThis aspect is partially separated by the muscularis propria from the other aspect of the tumor, which shows necrotic and hemorrhagic remodeling, and extends towards the serosa, where there is a capsular rupture.\n\nAt 1.5 cm from this tumor there was an intraluminal polypoid lesion measuring 1.5 cm long.\n\nRegarding histology, the main tumor was a mixed gastrointestinal stromal tumor (GIST), with spindle cells in the submucosa and epithelial cells in the outer layers of the colonic wall, with a high risk of recurrence due to the innumerable mitoses, which exceeded 100 mitoses per 50 fields at high magnification, and the capsular rupture, according to the Miettinen and Joensuu classificationv (Figure 5) and was classified pT4 according to TNM 2017.3\n\nThe polypoid lesion was an adenocarcinoma NOS type well differentiated developed on degenerated adenoma stadified pT1N0 (Figure 6).\n\nThe patient medical file was discussed with the multidisciplinary consensus staff indicated treatment with imatinib-based targeted therapies. Follow-up at one year postoperatively did not detect tumor recurrence.\n\n\nDiscussion\n\nStromal tumors are the most common mesenchymal tumors of the digestive tract. Yearly incidence rates range between 4.3 to 22 per million in the world, which is due to variability, the improving diagnostic criteria and a lack of GIST registries.4 Simultaneous presence of Colonic adenocarcinoma and stromal tumor is an uncommon occurrence. Because of the high incidence of adenocarcinoma histological type and the frequency of gastrointestinal stromal tumors (GIST), a fortuitous relationship based on the available data cannot be ruled out. The Genetic pathways of tumorigenesis are different for the two histologic types; c-Kit appears to be occasionally expressed in adenocarcinoma, and there is no evidence if the protein is indeed in the carcinogenetic process; this report is not available for stromal tumors. A review concluded that STI571 blocks the growth of colonic carcinoma cell lines.5 These results justified by preclinical investigations of c-Kit expression in colonic cancers had as objective to evaluate the use of tyrosine kinase inhibitors in the treatment of colorectal carcinomas.\n\nWe have presented a case of synchronous invasive colonic GIST with adenocarcinoma. Despite the relative common occurrence of GISTs, reports of synchronous adenocarcinoma and GISTs are quite rare. According to Kover et al., 7 of 43 patients with histologically evidence of GISTs were found with second histological type; three of these GISTs were colorectal adenocarcinomas.5 A second study realized by Au et al., found that nearly 41% of the stromal tumors were synchronous association with second malignant tumor, and 38% of these second malignancies were intestinal.6\n\nColonic adenocarcinoma and GIST present evidence of familial predisposition, except hereditary cancers. In another case, the patient did not have a family history of gastrointestinal or other malignancies.7 The genetic polymorphism of these two histologic types has been particularly investigated. Through progression from normal colonic epithelium to adenoma and adenocarcinoma, various genetics cancers can occur.8\n\nMechanisms have been clarified in sporadic colorectal cancer: chromosomal instability is responsible for 85% of the whole cases, and microsatellite instability, in the rest 15%.8 Unusually, none of the most commonly involved genes in colorectal carcinogenesis (APC, DCC, p53, K-ras, DNA mismatch repair genes) have been identified to be associated in the pathway of stromal tumors. Nevertheless, the GISTs seem to be related with the proto-oncogene mutation c-Kit, a tyrosine kinase receptor during embryonic growth and on postnatal. Activation of c-Kit by its ligand, SCF, may generates a cascade of cellular process involving transformation, differentiation, cell proliferation, adhesion, and chemotaxis.9\n\nWhen it is possible, surgery is the ideal therapeutic alternative with curative intention for non-metastatic stromal and adenocarcinoma at the same time. The operative strategies are in most situations wide and extensive.10 Since nodal involvement is rare, lymph-node clearance is not recommended.11 The prognosis of stromal tumors depends on tumor localization, its size, and the mitotic activity.12,13 The stage of synchronous malignancies is crucial because the dominant one is responsible of the outcome and survival.13 Imatinib provide special focus in the treatment of stromal tumors; particularly, for neoadjuvant process. The benefits of this target therapy are well established to downstage inoperable cases especially by decreasing size. As a result, safe resection margins and therefore an R0 resection are recommended.14\n\nGIST presents a high rate of recurrence (40% within 2 years).15 Just such colonic carcinoma, GISTs usually metastasize to the liver.16 Overall survival after complete resection of stromal tumors ranges from 47% to 66% at 5 years, and seems to be longer in patients with low-grade tumors: 100% at 10 years for tumors with 0–1 mitosis/30 hpf. High-grade lesions:>10 mitosis/10 hpf, have the worst outcome: 0% survival at 10 years. Nevertheless, the absence of a high mitotic index does not guarantee a better outcome.17 Overall, a 5-year survival for colonic adenocarcinoma correlates with the preoperative staging, and ranges from 3–8% for stage IV to 90% for stage I.18\n\nFor our case the predominant histologic type was the stromal one (GIST), with a high risk of recurrence due to the innumerable mitoses, which exceeded 100 mitoses per 50 fields at high magnification, and the capsular rupture, according to the Miettinen and Joensuu classification and was classified pT4 according to TNM 2017.\n\nThe adenocarcinoma type was well differentiated developed on degenerated adenoma stadified pT1N0. The patient was treated with imatinib-based targeted therapies.\n\nEticulous immunohistochemical and molecular biology study of all resection specimens are highly recommended whenever the combination of two histological types is found in the primary anatomopathological study.\n\nThese in-depth and ideally exhaustive studies guarantee the development of new targeted therapies and immunotherapies that would provide these patients with the opportunity of complete remission.\n\nNonetheless, all such cases must be discussed at a multidisciplinary concertation involving all the medical staff.\n\nFinally, this case certainly illustrates a rare association of two histological entities. There are few cases described in the literature, which limits the possibility of reaching well-codified conclusions regarding management.\n\nHowever, we believe that this case highlights the necessity for more thorough immunohistochemical and molecular biology studies.\n\nThe aim is to draw up recommendations with a high level of scientific evidence.\n\n\nConclusion\n\nSynchronous tumors rare cancer of the colonic with the co-existence of two histologically different neoplasms occurring in the same site without contact between them. This condition is rarely proven in preoperative investigations. Perfect histopathologic examination with multiple biopsies and pathologic examination of resection specimens is required to detect synchronous tumors. Those with advanced or aggressive behavior has pejorative prognostic significance and should receive adjuvant therapy.\n\nPathogeneses of such association are still not yet well identified. More studies are required to understand this incident to provide optimal curative management for patients. Sophisticated molecular biology studies are the bridge to innovative, more effective and targeted therapies.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for publication of this case report and accompanying images.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nWronski M, Ziarkiewicz-Wroblewska B, Gornicka B, et al.: Synchronous occurrence of gastrointestinal tumors and other primary gastrointestinal neoplasms. World J. Gastroenterol. 2006; 12: 5360–5362. Publisher Full Text\n\nMelis M, Choi EA, Anders R, et al.: Synchronous colorectal adenocarcinoma and gastrointestinal tumor (GIST). Int. J. Color. Dis. 2007; 22: 109–114.\n\nAmin MB, Edge S, Green F, et al.: AJCC Cancer Staging Manual (ed 8th Edition). New York: Springer; 2017. Publisher Full Text\n\nWaidhauser J, Bornemann A, Trepel M, et al.: Frequency, localization, and types of gastrointestinal stromal tumor-associated neoplasia. World J. Gastroenterol. 2019; 25(30): 4261–4277. Publisher Full Text\n\nGonçalves R, Linhares E, Albagli R, et al.: Occurrence of other tumors in patients with GIST. Surg. Oncol. 2010; 19(4): e140–e143. Publisher Full Text\n\nAu WY, Ho KM, Shek TW: Papillary renal cell carcinoma and gastrointestinal stromal tumor: a uniqueassociation. Ann. Oncol. 2004; 15: 843–844. Publisher Full Text\n\nMarcovalerio M, Eugene AC, Robert A, et al.: Synchronous colorectal adenocarcinoma and gastrointestinal stromal tumor (GIST). Int. J. Color. Dis. 2007; 22: 109–114. Publisher Full Text\n\nHahn M, Koufaki ON, Schackert HK: Molecular biology of colorectal cancer and clinical consequences for colorectal cancer syndromes. Langenbeck’s Arch. Surg. 1998; 383: 389–396. PubMed Abstract \| Publisher Full Text\n\nLinnekin D: Early signaling pathways activated by c-Kit in hematopoietic cells. Int. J. Biochem. Cell Biol. 1999; 31: 1053–1074. PubMed Abstract \| Publisher Full Text\n\nDeMatteo RP, Lewis JJ, Leung D, et al.: Two hundred gastrointestinal stromal tumors: recurrence patterns and prognostic factors for survival. Ann. Surg. 2000; 231: 51–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPidhorecky I, Cheney RT, Kraybill WG, et al.: Gastrointestinal stromal tumors: current diagnosis, biologic behavior, and management. Ann. Surg. Oncol. 2000; 7: 705–712. PubMed Abstract \| Publisher Full Text\n\nSchneider-Stock R, Boltze C, Lasota J, et al.: Loss of p16 protein defines high-risk patients with gastrointestinal stromal tumors: a tissue microarray study. Clin. Cancer Res. 2005; 11: 638–645. PubMed Abstract \| Publisher Full Text\n\nMiettinen M, El-Rifai W, Sobin LHL, et al.: Evaluation of malignancy and prognosis of gastrointestinal stromal tumors: a review. Hum. Pathol. 2002; 33: 478–483. Publisher Full Text\n\nVassos N, Agaimy A, Hohenberger W, et al.: Coexistence of gastrointestinal stromal tumors (GIST) and malignant neoplasms of different origin: Prognostic implications. Int. J. Surg. 2014; 12(5): 371–377. PubMed Abstract \| Publisher Full Text\n\nDeMatteo RP, Lewis JJ, Leung D, et al.: Two hundred gastrointestinal stromal tumors: recurrence patterns and prognostic factors for survival. Ann. Surg. 2000; 231: 51–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMudan SS, Conlon KC, Woodruff JM, et al.: Salvage surgery for patients with recurrent gastrointestinal sarcoma: prognostic factors to guide patient selection. Cancer. 2000; 88: 66–74. PubMed Abstract \| Publisher Full Text\n\nNowain A, Bhakta H, Pais S, et al.: Gastrointestinal stromal tumors: clinical profile, pathogenesis, treatment strategies and prognosis. J. Gastroenterol. Hepatol. 2005; 20: 818–824. PubMed Abstract \| Publisher Full Text\n\nEfron J, Wexner SD: Rectal cancer.Cameron JL, editor. Current surgical therapy. 7th edn.Philadelphia: Elsevier Mosby; 2001; pp. 235–245." }	[ { "id": "215037", "date": "20 Oct 2023", "name": "Shamus R. Carr", "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 case report highlights a unique finding of a patient with synchronous GIST tumor of the colon and colonic adenocarcinoma. There are areas where some editing would enhance the clarity of the text. The authors should consider offering some thought or recommendations on how they might further delve into the question of common carcinogenic pathways.\nPoints:\nIn the Abstract, the work “carcinologic” is used. The root of this work “carcinology” means “study of crustaceans”. This word is therefore incorrect and either should be deleted or changed.\n\nPlease add a “period” at the end of the last sentence in the Conclusions of the Abstract.\n\nThe term “dominance of one histologic type” is unclear as it is known that GIST arise from the stroma and adenocarcinoma of the colon arise from the mucosal lining. This also has implications on distant spread. Could the authors rewrite this sentence? I think they mean “the more aggressive or higher stage”, but I may be wrong.\n\nGIST tumors are thought to arise from the interstitial cells of Cajal (not Kajal). Named after the Spanish neuropathologist and Nobel laureate Ramón y Cajal.\n\nWas a PET scan done prior to surgery to evaluate for distant metastatic disease? If the case was deemed emergent, and therefore a PET would not have been able to be performed, please note that about the operation.\n\nWas the GIST tumor checked to see if it was SDHA deficient?\n\n“Colonic” does not need to be capitalized unless it is the first word of a sentence.\n\nThe last sentence of the third paragraph of in the Conclusions section is unclear. Can this be rewritten for clarity?\n\nIn the 5th paragraph of the Conclusions section, the authors imply that lymphadenectomy is not required for colon cancer. This is clearly not what the authors intend. Please review and rewrite to clarify.\n\nIn the 6th paragraph of the Conclusions section, the second sentence needs a small edit. It should read “Just like…” and not as written, “Just such…”.\n\nWhen mentioning the TNM staging system update from 2017 for GIST tumors, it is probably better to state the edition of the AJCC staging (which I believe was the 7th AJCC edition, but I would double check).\nWould the authors consider rewriting the last 7 paragraph of the Conclusions section into one or two paragraphs?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1055
https://f1000research.com/articles/12-1051/v1	29 Aug 23	{ "type": "Study Protocol", "title": "A comparative study on clinical outcome of conventional trabeculectomy and glaucoma drainage device in a tertiary health care center", "authors": [ "Ayushi Tayal", "Sachin Daigavane", "Sachin Daigavane" ], "abstract": "BACKGROUND: Glaucoma is becoming a more common cause of blindness as the world's population ages. Glaucoma, the second largest cause of blindness worldwide, accounts for approximately 15% of all blindness. However, it poses an even more significant public health concern than cataracts because the blindness it produces is irreversible. This study aims to compare the clinical outcomes of traditional trabeculectomy and glaucoma drainage devices. METHODOLOGY: This is a hospital-based study of 30 patients in which we compare clinical results after conventional trabeculectomy and implantation of a glaucoma drainage device. Intraocular pressure will be measured by an Applanation tonometer and best-corrected visual acuity testing will be performed in all patients. The data will be compared, and statistical analysis will be done. EXPECTED RESULTS: Patients with Glaucoma drainage implants will have better IOP control.", "keywords": [ "Aurolab aqueous drainage implant", "glaucoma surgery", "refractory glaucoma", "trabeculectomy", "intraocular pressure." ], "content": "Introduction\n\nHigh intraocular pressure (IOP), optic nerve deterioration, and visual impairments are the hallmarks of glaucoma, a condition that is a primary cause of blindness.1 Glaucoma is a multifactorial optic neuropathy that manifests as classical optic nerve head characteristics and corresponding modifications in the visual field and accelerated ganglion cell ageing in the retina that in the presence or absence of any angle abnormalities, may or may not be connected to underlying disease cause.2 Neovascular glaucoma, the secondary glaucoma brought on by uveitis, and angle recession are a few kinds of glaucoma that typically have poor responses to standard medical treatments and surgical methods.3,4 Intraocular pressure (IOP) is the main focus of currently available standard treatments because it has been demonstrated that increased IOP causes the death of brain cells in glaucoma patients.5 Additionally, IOP is now thought to be the only risk factor for glaucoma that is practically adjustable and has benefits for maintaining the visual field (VF).\n\nUveitis is an inflammation and swelling of uvea, the central layer of the eye. In Europe and the United States, it causes 5% to 20% of blindness cases.6 Because it could be challenging to achieve a suitable IOP with medical interventions and procedures, uveitic glaucoma is challenging to control. Uveitic glaucoma is treated using a variety of methods. Trabeculectomy is one of the most frequently performed surgeries. The primary difficulty with this is fibrosis of bleb.7,8 Although anti-fibrotic medications like mitomycin C have been applied during surgery, but the filtering surgery’s rate of success is still questionable.9\n\nCompared to other refractory glaucoma, filtration surgery for uveitic glaucoma has lesser success rates due to the repeated inflammatory response of uveitis that occurs among patients.10 Aqueous drainage devices have become popular in recent years as glaucoma treatments. A preference for tube shunts that is increasing and trabeculectomy that is losing favour is also seen in surveys conducted by the American Glaucoma Society.11\n\nThe increased usage of tube shunt surgery as a substitute to trabeculectomy is likely a result of concern over bleb-related problems like bleb leaks and bleb infections. The tube shunts when first developed, were often only used for glaucomas that had not responded to filtering surgery and were refractory. However, implantation of these devices in glaucomas with improved surgical prognosis has been motivated by a rising body of favourable experience with them. Refractory glaucoma is defined as uncontrolled intraocular pressure with evidence of deterioration in the optic nerve or visual field despite maximally tolerable anti-glaucoma medications (topical and/or systemic), previously unsuccessful non-seton surgical treatment, a combination of surgery and medications, or a high risk of failure for trabeculectomy.12\n\nWith untreated primary and refractory glaucomas, the current study compares the clinical outcomes of glaucoma drainage devices and traditional trabeculectomy in patients.\n\n\nAim and objectives\n\nTo research the effectiveness of traditional trabeculectomy and glaucoma drainage devices.\n\n\n\n1) To assess the clinical results of traditional trabeculectomy in glaucoma patients with uncontrolled primary glaucoma.\n\n2) To assess the clinical results of traditional trabeculectomy in glaucoma patients who have refractory glaucoma.\n\n3) To evaluate the clinical results of drainage devices in patients with uncontrolled primary glaucoma.\n\n4) To evaluate the effectiveness of glaucoma drainage devices in people with refractory glaucoma.\n\n5) To compare the treatment results of traditional trabeculectomy with glaucoma drainage devices in patients with uncontrolled primary glaucoma.\n\n6) To compare the therapeutic results of traditional trabeculectomy and glaucoma drainage devices in glaucoma patients who have not responded to other treatments.\n\nTrial design - Single Centric, hospital based cross sectional study.\n\nThis is a hospital-based study in which subjects would be recruited from the OPD of ophthalmology at Acharya Vinobha Bhave Rural Hospital Sawangi, Meghe, Wardha, Maharashtra, with clearance from the institutional ethical committee of Datta Meghe institute of higher education and research and will adhere to the principles of the Helsinki Declaration. The specifics of the surgery will be properly explained to each patient. The study will gradually recruit the patients who meet the inclusion criteria. This study will be carried out under standard preoperative, intraoperative and post operative conditions by a single experienced surgeon. Study participants will be divided into two groups. Group-A (Trabeculectomy group) and Group-B (Glaucoma Drainage Device group) taking into consideration factors such as staphyloma, scleral thinning and high myopia, participants with this pathology will be undergoing glaucoma drainage device implantation. Subjects will be invited & screened for inclusion and exclusion criteria for selection. All patients in both groups will be followed up on a monthly basis for the next two years. At each follow-up, we evaluate IOP, visual acuity and need of anti-glaucoma medications in post-operative patients, and both groups are compared.\n\n\n\n1) Patients with uncontrolled primary glaucoma.\n\n2) Patients who will be diagnosed as secondary glaucoma.\n\n3) Patients who will be diagnosed as refractory glaucoma.\n\n4) Patients consenting to study/giving valid consent.\n\n\n\n1) Patients with any ocular infection.\n\n2) Patients having corneal decompensation.\n\n3) Patients with neovascular glaucoma.\n\n4) Patients who did not successfully complete a minimum 1-year follow-up period were not included in this study.\n\nThere will be a clinical history gathered and best corrected visual acuity (Snellen’s chart), slit lamp examination, Intra Ocular Pressure measurement (applanation tonometer), fundoscopy (slit lamp biomicroscopy with 90 D, direct, and indirect ophthalmoscopy with 20 D) and gonioscopy are all included in a thorough ophthalmic checkup. OCT-RNFL and Perimetry will be done. Patients who underwent trabeculectomy and glaucoma drainage device surgery (Aurolab aqueous drainage implant) will be included in the study.\n\nA conjunctival flap precisely fornix based will be made, and the corneal traction suture will be positioned at the 12 o’clock position. Gentle diathermy will be used to cauterise the blood vessels. A rectangular scleral flap of 4×3 mm with partial thickness will be made and a trab of 0.8×2.0 mm will be achieved followed by a Peripheral Iridectomy. A single 10-0 nylon suture will be given to secure the scleral flap, with possible extra sutures in between the sutures and at the flap’s corners. The conjunctiva at the limbus will be stitched up with a 10-0 nylon suture.\n\nTenon’s tissue will be dissected after a conjunctival flap (fornix based). The operating surgeon will choose the implant insertion site depending on the amount of conjunctival scarring, if any, and whether it will be supero-temporal or infero-nasal. Using muscle hooks, adjoining recti muscles in the desired quadrant will be isolated. After that, the AADI implant will be prepped to look for possible manufacturing flaws. The end plate will then be positioned so that the front edge of the plate will be roughly 8 mm away from the limbus, in between the neighbouring recti muscles. After that, 9-0 Nylon sutures will be used to secure the plate to the underlying sclera by passing them through the implant’s fixation holes. The tube’s connection to the sclera will be stabilised using a non-compressing mattress suture. By tying it with a 6-0 vicryl suture, temporary tube occlusion will be accomplished. A 23-gauge needle will be used to produce a scleral fistula at a distance of roughly 3 mm from the limbus. In order for the tube to lie 1-2 mm past the surgical limbus, it will then be anteriorly bevelled up and introduced into the AC through the scleral tract. The operating surgeon will directly observe the tube to ensure its place. Fenestrations in the tube will be constructed, necessitating short-term IOP management in the immediate postoperative period until the vicryl suture lyses. After that, 8-0 Vicryl sutures will be used to close the conjunctiva and tenon’s capsule using both interrupted and running procedures.\n\nPrimary outcome measures\n\n1) Visual acuity testing by Snellen’s chart:\n\nIt is used to assess distant visual acuity. The patient is seated 6m away from the Snellen’s chart and is instructed to read the chart with both eyes, one after the other. When the patient can read up to a 6 m line, his visual acuity is reported as 6/6, and depending on the smallest line that the patient can read from a 6 m distance, his vision is recorded as 6/9, 6/12, 6/18, 6/24, 6/36, and 6/60.\n\n2) Intra Ocular Pressure measurement by applanation tonometry:\n\nThe Goldmann Applanation Tonometer is commonly recognized as the gold standard in intraocular pressure (IOP) measurement. A topical anesthetic is instilled into the conjunctival sac, followed by a fluorescein stain. The cobalt blue filter reveals a pattern of two green semicircular mires, with the inner borders of semicircles aligned. The IOP is calculated by multiplying the dial value by ten in mmHg.\n\nSecondary outcome measures\n\n1) Moorfields bleb grading (Figure 1):\n\nThe bleb is assessed and characterized with respect to:\n\n• Diffusion area\n\n• Height\n\n• Vascularity in central, peripheral bleb area and non-bleb area.\n\n2) Need of anti-glaucoma medication:\n\nIf the post-operative IOP measurement indicates greater values, we will administer anti-glaucoma drugs later.\n\n3) OCT-RNFL and Humphrey visual field analysis.\n\nUsing sample size formula with desired error of margin\n\nWhere Zα/2 is the level of significance at 5% i.e.:\n\n99.5% confidence interval = 2.8\n\nP = Prevalence of secondary glaucoma = 0.50% i.e. 0.005\n\nd = Desired error of margin = 5% = 0.05\n\nn = 14.8\n\nn = 15 patient needed in each group.\n\nTotal sample size = 30\n\nStudy reference: Arun Narayanaswamy et al.13\n\nFormula reference: Daniel et al.14\n\nSample size: After meeting the inclusion and exclusion requirements, a total of 30 SUBJECTS will be registered in this interventional cross-sectional hospital-based study.\n\nThis will be done with the aid of descriptive and inferential statistics, chi-square tests, odds ratios, and software analysis with the SPSS-24.0 system and Graph Pad prism 7.0 version and p < 0.05 is considered as level of significance.\n\nWe plan to publish in an indexed journal and present the study in national conference proceedings.\n\nThe recruitment of participants has been started.\n\n\nDiscussion\n\nIn this study, we compared the clinical outcomes of glaucoma drainage tube implantation to traditional trabeculectomy. The most frequent etiology of vision loss and blindness is glaucoma. In India, it is the second most frequent reason for blindness, and the majority of people are unaware of the cure. There has been very little study in this field to evaluate the efficiency of glaucoma drainage devices and routine trabeculectomy. As a result, we intend to compare the therapeutic effects of glaucoma drainage devices and conventional trabeculectomy in AVBRH patients with uncontrolled primary and refractory glaucoma.\n\nA detailed analysis of GDDs revealed that the Baerveldt and Ahmed implants’ respective IOP changes were roughly 54 and 51%.15 After a year, Harbick et al. discovered a considerable net decline in visual acuity, which they attributed to concurrent pre-existing retinal and corneal problems.16 Patients who underwent tube shunt surgery versus those who underwent trabeculectomy with MMC during the first three years of follow-up were more likely to maintain IOP control and avoid persistent hypotonia, or reoperation for glaucoma.17\n\nAccording to Joshi et al., glaucoma drainage implants have become an essential surgical option for treating secondary glaucoma. Although these implants were previously utilised solely after a failed trabeculectomy, they are now increasingly being employed as a main surgical technique in cases of uveitic glaucoma.18\n\nIn 10 patient’s 14 eyes with uveitic glaucoma associated with Behcet’s disease, Satana et al. reported that AGV implantation led to 90.9% success rates after 18 months and 72.7% success rates after 2 years.19 Retrospective investigation of uveitic glaucoma patients 60 eyes who underwent AGV implantation indicated success rates of 77.0% after one year and 50.0% after four years (IOP range of 5 to 21 mmHg and a 25.0% decrease in IOP from before surgery.20 Preoperative corticosteroid usage may increase AGV surgical outcome in uveitic glaucoma. Preoperative prednisone at 1 mg/kg/day was indicated by Mata et al. until the inflammation was controlled. Oral corticosteroids are decreased during a four-week period following surgery.21\n\nIn eyes with chronic inflammatory glaucoma, N. Bhardwaj, S. M. Iverson, W. Shi, and colleagues found that implantation of the Baerveldt Glaucoma Drainage Device was more likely to maintain IOP control and prevent glaucoma reoperation than trabeculectomy with antifibrotic therapy.22\n\nInstitutional Ethical Clearance has been obtained on 29/8/2022\n\nIEC No – DMIMS (DU)/IEC/2022/198\n\nCTRI Registration was done on 26/07/2023\n\nCTRI Number – REF/2023/07/070943", "appendix": "Data availability\n\nNo data as this is a study protocol.\n\n\nAcknowledgements\n\nI would like to acknowledge Mr Laxmikant Umate Sir, who has helped me in sample size calculation and data analysis planning.\n\n\nReferences\n\nBaneke AJ, Lim KS, Stanford M: The pathogenesis of raised intraocular pressure in uveitis. Curr. Eye Res. 2016; 41(2): 137–149. Publisher Full Text\n\nBurr J, Azuara-Blanco A, Avenell A, et al.: Medical versus surgical interventions for open angle glaucoma. Cochrane Database Syst. Rev. 2012; 9: CD004399. Publisher Full Text\n\nWiese C, Heiligenhaus A, Heinz C: Changes in inflammatory activity after glaucoma filtration surgery in children with chronic anterior uveitis. Ocul. Immunol. Inflamm. 2016; 24(4): 397–401. PubMed Abstract \| Publisher Full Text\n\nEksioglu U, Yakin M, Sungur G, et al.: Short- to long-term results of Ahmed glaucoma valve in the management of elevated intraocular pressure in patients with pediatric uveitis. Can. J. Ophthalmol. 2017; 52(3): 295–301. PubMed Abstract \| Publisher Full Text\n\nDreyer EB, Lipton SA: New perspectives on glaucoma. JAMA. 1999; 281(4): 306–308. Comment in: JAMA. 1999;281(23):2186-7. Publisher Full Text\n\nAmoozgar B, Lin SC, Han Y, et al.: A role for antimetabolites in glaucoma tube surgery: current evidence and future directions. Curr. Opin. Ophthalmol. 2016; 27(2): 164–169. PubMed Abstract \| Publisher Full Text\n\nSchoenberg ED, Blake DA, Swann FB, et al.: Effect of two novel sustained-release drug delivery systems on bleb fibrosis: an in vivo glaucoma drainage device study in a rabbit model. Transl. Vis. Sci. Technol. 2015; 4(3): 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMedeiros Pinto J, Pinto Ferreira N, Abegao PL: Ahmed valve upstream obstruction caused by fibrous ingrowth: surgical approach. J. Glaucoma. 2017; 26(1): e236–e238. PubMed Abstract \| Publisher Full Text\n\nYazdani S, Mahboobipour H, Pakravan M, et al.: Adjunctive mitomycin C or amniotic membrane transplantation for Ahmed glaucoma valve implantation: a randomized clinical trial. J. Glaucoma. 2016; 25(5): 415–421. Publisher Full Text\n\nKalinina Ayuso V, Scheerlinck LM, de Boer JH : The effect of an Ahmed glaucoma valve implant on corneal endothelial cell density in children with glaucoma secondary to uveitis. Am J. Ophthalmol. 2013; 155(3): 530–535. PubMed Abstract \| Publisher Full Text\n\nJoshi AB, Parrish RK, Feuer WF: 2002 Survey of the American Glaucoma Society. Practice preferences for glaucoma surgery and antifibrotic use. J. Glaucoma. 2005; 14: 172–174. PubMed Abstract \| Publisher Full Text\n\nNassiri N, Kamali G, Rahnavardi M, et al.: Ahmed glaucoma valve and single-plate Molteno implants in treatment of refractory glaucoma: a comparative study. Am J. Ophthalmol. 2010; 149(6): 893–902. PubMed Abstract \| Publisher Full Text\n\nNarayanaswamy A, Baskaran M, Zheng Y, et al.: The prevalence and types of glaucoma in an urban Indian population: the Singapore Indian Eye Study. Invest. Ophthalmol. Vis. Sci. 2013; 54(7): 4621–4627. Publisher Full Text\n\nDaniel WW: Biostatistics: A Foundation for Analysis in the Health Sciences. 7th ed.New York: John Wiley & Sons; 1999.\n\nHong CH, Arosemena A, Zurakowski D, et al.: Glaucoma drainage devices: A systematic literature review and current controversies. Surv. Ophthalmol. 2005; 50: 48–60. PubMed Abstract \| Publisher Full Text\n\nHarbick KH, Sidoti PA, Budenz DL, et al.: Outcomes of inferonasal Baerveldt glaucoma drainage implant surgery. J. Glaucoma. 2006; 15: 7–12. PubMed Abstract \| Publisher Full Text\n\nGedde SJ, Schiffman JC, Feuer WJ, et al.: The Tube Versus Trabeculectomy Study was followed up on after three years. Am J. Ophthalmol. 2009 Aug 10; 148: 670–684. PubMed Abstract \| Publisher Full Text\n\nJoshi AB, Parrish RK 2nd, Feuer WF: 2002 survey of the American Glaucoma Society: practice preferences for glaucoma surgery and antifibrotic use. J. Glaucoma. 2005; 14: 172–174. PubMed Abstract \| Publisher Full Text\n\nSatana B, Yalvac IS, Sungur G, et al.: Ahmed glaucoma valve implantation for uveitic glaucoma secondary to Behcet disease. J. Glaucoma. 2015; 24(8): 607–612. PubMed Abstract \| Publisher Full Text\n\nCosta VP, Azuara-Blanco A, Netland PA, et al.: Efficacy and safety of adjunctive mitomycin C during Ahmed glaucoma valve implantation: a prospective randomized clinical trial. Ophthalmology. 2004; 111(6): 1071–1076. PubMed Abstract \| Publisher Full Text\n\nMata AD, Burk SE, Netland PA, et al.: Management of uveitic glaucoma with Ahmed glaucoma valve implantation. Ophthalmology. 1999; 106(11): 2168–2172. PubMed Abstract \| Publisher Full Text\n\nIverson SM, Bhardwaj N, Shi W, et al.: Surgical outcomes of inflammatory glaucoma: a comparison of trabeculectomy and glaucoma-drainage-device implantation. Jpn. J. Ophthalmol. 2015; 59(3): 179–186. Publisher Full Text" }	[ { "id": "220712", "date": "14 Nov 2023", "name": "Nader Bayoumi", "expertise": [ "Reviewer Expertise Glaucoma" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 needs thorough English language revision.\nIntroduction\nHigh intraocular pressure (IOP), optic nerve deterioration, and visual impairments are the hallmarks of glaucoma” The hallmark of glaucoma is optic nerve affection with subsequent visual field deterioration. IOP is an important factor, but not the first of the hallmarks of glaucoma. Please rephrase to reflect this concept.\n\nIncreased IOP causes the death of brain cells in glaucoma patients” Please modify to “death of retinal ganglion cells”.\n\nA preference for tube shunts that is increasing and trabeculectomy that is losing favour” Please specify to the reader whether the authors are referring to “primary” or “repeat / subsequent” interventions for glaucoma.\nAim and Objectives\nPlease unify the terminology used for the study participants (“patients, people”)\n\nPlease specify the objectives of “clinical results” (do the authors refer to efficacy, or safety?).\n\nPlease present the objectives in concise statements avoiding undue repetition. All 6 objectives proposed apparently fit into “comparison of trabeculectomy to GDDs in primary and refractory glaucoma”.\nProtocol\nPlease mention the Country of the study setting(s).\n\nGradually recruit” Do the patients refer to “sequential recruitment”? Please rephrase.\n\nPlease mention the method of randomisation if this study is randomised.\n\nTaking into consideration factors such as staphyloma, scleral thinning and high myopia,” Do the authors imply that eyes with these conditions will undergo GDDs without randomisation? This would represent a significant sampling bias to the study and would confound the results.\n\nPlease mention clearly whether the study eyes (especially in the GDDs group) will be treatment-naïve or would have been subjected to prior glaucoma surgery.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-1051
https://f1000research.com/articles/12-793/v1	06 Jul 23	{ "type": "Research Article", "title": "Immunological changes in a cohort of COVID-19 survivors: Mansoura University experience", "authors": [ "Tamer Elhadidy", "Heba Wagih Abdelwahab", "Doaa Shahin", "Asem Hewidy", "Eman Khashaba", "Rehab Ahmad Elmorsey", "Nermin Abo El Kheir", "Elsayed A. Eid", "Ahmed El-Mesery", "Marwa O. Elmaria", "Tamer Elhadidy", "Heba Wagih Abdelwahab", "Doaa Shahin", "Asem Hewidy", "Rehab Ahmad Elmorsey", "Nermin Abo El Kheir", "Elsayed A. Eid", "Ahmed El-Mesery", "Marwa O. Elmaria" ], "abstract": "Background: COVID-19 is a global pandemic that has affected millions of people all over the world since 2019. Infection with COVID-19 initiates a humoral immune response that produces antibodies against specific viral antigens, which in turn is supposed to provide immunity against reinfection for a period of time. The aim of this research was to study the kinetics of IgM and IgG antibodies against SARS-CoV-2.\nMethods: One hundred and seventeen post-COVID-19 participants were enrolled in the study. Qualitative assessment of IgM and IgG antibodies over six months (three visits) post recovery was conducted. Results: The current study revealed a significant reduction in IgM and IgG titers between the first and second visits (p <0.001). After six months, the antibody titer had declined by 78.8% from the first visit for IgM and by 49.2% for IgG antibodies. Regarding younger age and male sex, statistically significant persistence of IgM antibodies was noticed at the six months follow up. Also, statistically significant persistent IgG immunity was found in male patients and diabetics by the end of the six months follow up. Conclusions: We observed a significant waning of IgM and IgG titers over a period of six months follow up.. The persistence of positive IgM and IgG antibodies by the end of six months was variable due to differences in age, gender and presence of diabetes mellitus.", "keywords": [ "Post COVID immunity", "COVID-19", "IgM", "IgG", "antibodies", "diabetes", "follow up study" ], "content": "Introduction\n\nCOVID-19 is considered the most catastrophic pandemic in the 21st century until now. Globally, as of 3 June 2022, 5:37pm CEST, there have been 528,816,317 confirmed cases of COVID-19, including 6,294,969 deaths, reported to WHO (WHO, 2022).\n\nInfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, initiates a humoral immune response that produces antibodies against specific viral antigens. These include anti-S protein antibodies that target the virus spike S1 protein subunit and receptor binding domain (RBD) (CDC, 2022).\n\nAntibodies are detected in the blood of people who have been previously infected with a virus that causes a disease; they show the body’s efforts to fight off a specific virus. Antibodies may protect people from reinfection or becoming severely ill for some time afterward (Qu et al., 2020). However, antibodies wane over time and how quickly antibodies wane is different for each disease and each person.\n\nThe aim of this study was to identify the kinetics of IgM and IgG antibodies in a cohort of COVID-19 survivors over six months’ follow up and evaluate the longevity of immunity provided by COVID-19 infection.\n\n\nMethods\n\nThis longitudinal follow up study was conducted between May 2020 to April 2021 at post-COVID-19 outpatient clinics at several Mansoura University Main Hospital. Ethical approval was obtained from the Mansoura University Institutional Research Board (IRB) (approval number: RP.20.05.67). Written informed consent was taken from all participants and all study participants were assured of the confidentiality and anonymity of the data.\n\nA convenience sample of 123 post-COVID-19 patients who attended the outpatient’s clinic were recruited. The follow up schedule was explained to them and they were free to leave the study at any time. Six participants were excluded because they refused follow up. Consequently, 117 post-COVID-19 participants were studied. Follow up was conducted at one month (first visit), three months (second visit) and six months (third visit) after recovery from COVID-19. Of 117 survivors who attended the first visit, only 98 attended the second visit and 76 completed the third visit (Figure 1). Demographics, comorbidities, severity of previous COVID-19 and laboratory data of the participants were recorded. Severity of previous COVID-19 infection was classified according to WHO severity definitions (WHO, 2021).\n\nA clinical sheet was used including demographic data such as age, gender, marital status, smoking status, history relevant to clinical presentation and medical comorbidity, pulmonary function testing, electrocardiogram, necessary laboratory analysis such as complete blood picture (CBC), COVID-19 IgG and IgM antibodies titer and computed tomography (CT) chest (see Extended data (Khashaba, 2023)).\n\nSerum samples using the Abbott SARS-CoV-2 IgG and IgM assays following the manufacturer’s instructions (SARS-CoV-2 IgG (https://www.fda.gov/media/137383/download); SARS-CoV-2 IgM (https://www.fda.gov/media/142940/download) (Abbott Laboratories, Abbott Park, IL, USA (reference 06R8620 and 06R8720; Abbott ARCHITECT, 2020; Abbott AdviseDx, 2020)). The assay is a chemiluminescent microparticle immunoassay for qualitative detection of IgG and IgM in human serum or plasma against the SARS-CoV-2 nucleoprotein. The amount of IgG and IgM antibodies to SARS-CoV-2 in each sample is determined by comparing its chemiluminescent relative light unit (RLU) to the calibrator RLU (index S/C). Using an index S/C threshold of 1·4 (for IgG) and 1 (for IgM) (Bryan et al. 2020).\n\nThe collected data was revised, coded, tabulated and introduced to a PC using Statistical Package for Social Science software (IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp). Descriptive statistics in the form of means and standard deviation (±SD) was used for normally distributed numerical data, while median and range was used for non-normally distributed numerical data. The frequency and percentage of qualitative data was recorded. The Wilcoxon signed rank test was used to compare quantitative data at different times, which was non-normally distributed. A Chi square test was used to compare qualitative data in two groups. The McNemar test was used to compare immune status at different points of time. Logistic regression analysis was used for predictors of positive immunity among the studied group using a forward Wald model. Adjusted odds ratios and their 95% CIs were calculated. A p-value less than 0.05 was considered as statistically significant.\n\n\nResults\n\nBaseline characteristics of the cohort (117 post-COVID-19 participants) were as follow: mean age was 54 years old, about 54.7% of the participants were male, diabetes mellitus was detected in 38.5% while hypertension was detected in 34.5%. In addition, 46.2% and 47.9% of participants previously had non-severe and severe COVID-19 infection respectively (Underlying data (Khashaba, 2023)).\n\nOf 117 participants, only 98 were enrolled in the second visit (three months post COVID-19) of which one IgM result was not recorded. Only 76 participants completed the third visit (six months post COVID-19) (Table 1).\n\nRegarding the COVID-19 IgM antibody titer, there was significant reduction in IgM titer between the first and second visits (percentage change 68.8%; p<0.001). Moreover, a reduction in the IgM titer was observed between the second and third visit (percentage change 32.3%). After 6 months follow up (third visit) COVID-19 IgM titer declined by 78.8% from the first visit (Underlying data (Khashaba, 2023)).\n\nA total of 88% (103 out of 117) of participants had a positive COVID-19 IgM in the first visit and after six months follow up 53.9% (41 out of 76) participants had a positive COVID-19 IgM and the difference was statistically significant (p<0.05) (Table 1).\n\nFor the COVID-19 IgG antibody titer, there was a significant reduction in IgG titer between the first and second visits (p<0.001). Also, a significant reduction in IgG titer was observed between the first and third visit. After six months follow up, at the third visit, the COVID-19 IgG titer had declined by 49.2% from the first visit (Table 1).\n\nA total of 93.2% (109 out of 117) participants had positive COVID-19 IgG at the first visit and after six months follow up 60.5% (46 out of 76) of participants had positive COVID-19 IgG (Table 1).\n\nThe existence of a positive IgM test at the third visit was significantly associated with the age of the studied participants (mean age was 52 in participants with a positive test compared to 57.4 in participants with a negative test (p=0.04). Also, a positive test was more frequent among males than females (p=0.04). Moreover, positivity was significant among healthcare workers compared to non-healthcare workers (p=0.04). Although a non-significant association was detected between a positive IgM test and diabetes mellitus, a positive IgM test was frequent among non-diabetic participants (60.9% versus 43.3%). In addition, a positive IgM test was frequent among non-smokers and participants who had previously had severe and critical COVID-19 despite no statistical significance (Table 2).\n\nOn studying persistent IgG positivity at the third visit, it was slightly associated with older age of studied participants (mean age was 56.7 in participants with a positive test compared to 51.4 in participants with a negative test (p=0.05)). Also, a positive test was more frequent among males than females (p=0.008). A significant association was detected between a positive IgG test and diabetes mellitus as it was more frequent among diabetic than non-diabetic participants (83.3% versus 45.7%). Finally, a positive IgG test was more frequent among non-smokers, and participants previously having had severe and critical COVID-19 despite the absence of statistical significance (Table 3).\n\nBased on logistic regression analysis, age, sex and occupation are significant predictors for positive IgM immunity; older age is associated with a decreased chance of positive IgM [OR (95%CI) 0.9 (0.8–0.9)]. However, males and health care workers had an increased chance of a positive IgM [OR (95%CI) 5.06 (1.6–16.2), 6.2 (1.0–35.5) respectively]. Regarding IgG immunity, males and diabetic participants had an increased chance of a positive IgG [OR (95%CI) [4.2 (1.4–12.3), 7.3 (2.2–24.3) respectively] (Table 4).\n\nThere was an overall reduction in the level of IgM from 10.9 units at one month to 3.4 units after three months and 2.3 units at six months (Figure 2). There was an overall reduction in the level of IgG from 6.7 units at one month to 4.3 units after three months and 3.4 units at six months (Figure 3).\n\n\nDiscussion\n\nUnderstanding the persistence of neutralising antibodies in COVID-19 survivors is essential for developing effective vaccination programmes and COVID-19 pandemic control measures. Here, we looked into COVID-19 survivors’ natural neutralising antibody survival rates (Greaney et al., 2021; Sette and Crotty, 2021). We studied the changes of IgM and IgG to the spike protein of SARS-CoV-2 in 117 post COVID-19 participants. There was significant reduction in IgM and IgG antibody titer over time. By the end of six months follow up, IgM and IgG titer declined by 70.5% and 46.8% from the first visit respectively. Forty-one out of 76 participants remained IgM positive by the end of the sixth month. When relating this positivity with different factors, there was significant relation to mean age of 52 years old and male sex. Although not statistically significant, there was persistently more positive IgM in non-smokers, non-diabetics, non-hypertensive and in more severe and critical cases. Forty-six out of 76 cases remained IgG positive by the end of the sixth month. Positive IgG immunity is found more frequently and statistically significant in older participants, male sex and diabetics. Although not statistically significant, there was persistently more positive IgG in non-smokers, hypertensive, and in more severe and critical cases.\n\nThe persistence of IgG antibodies in diabetics compared to non-diabetic participants may be explained by more severe disease in diabetic patients. This is supported by several studies which stated that ICU patients showed a faster and increased neutralising antibody response compared to non-ICU patients (Lau et al., 2021; Liu et al., 2020; Qu et al., 2020; Williamson et al., 2020). The association between serum IgA, IgG, and IgM levels and glycated haemoglobin, an indicator of long-term diabetes control, fructosamine, and 111 healthy non-diabetic people was examined in 110 diabetic patients. While the concentration of IgM was considerably lower (by 46.7%, p<0.001) in diabetic patients compared to non-diabetic participants, there were significant increases in serum IgA (by 82.7%, p 0.001) and IgG (by 35.2%, p 0.001) concentrations (Ardawi et al., 1994).\n\nThe anti-SARS-CoV-2 antibody titers were statistically higher in older compared to younger participants (Lau et al., 2021; Ozgocer et al., 2022) and in men compared to women (Jin et al., 2020; Klein et al., 2020; Ozgocer et al., 2022). Indeed, clinical outcomes showed that males experience both a higher severity and fatality for COVID-19 infection than females (Mukherjee and Pahan, 2021; Peckham et al., 2020; Qu et al., 2020).\n\nDuring the period of follow up, none of the studied participants had documented reinfection with COVID-19, which may be explained by protection provided by neutralizing antibodies. Although the onset of detection and duration of persistence of neutralizing IgM and IgG antibodies differ among studies, they were detectable in sera of most COVID-19 survivors (Alzaabi et al., 2021; Hou et al., 2020; Iyer et al., 2020; Lee et al., 2010; Li et al., 2003; Ogega et al., 2021; Petersen et al., 2021; Qu et al., 2020; Sariol and Perlman, 2020).\n\nWhat amount of the neutralising antibody response could provide protection against infection or re-infection to properly time the vaccination strategy to sustain antibody-mediated protection against SARS-CoV-2 is an important question that must be answered by future studies. Although COVID-19 convalescents or vaccination recipients experienced re-infection, symptomatic re-infections and severe illnesses happened less frequently than in initial infections (Butt et al., 2021; Hacisuleyman et al., 2021; Leidi et al., 2022) and this is the rationale for studying antibody response against SARS-CoV-2 and implementation of vaccination strategies.\n\nOur study was limited by a small number of participants, and we cannot detect a cutoff value for protecting antibody level. Further studies on larger numbers of participants are required to detect the level of the neutralizing antibody needed to protect against re-infection to correctly schedule the vaccination plan.\n\n\nConclusions\n\nWe concluded that the titer of neutralizing IgM and IgG antibodies against COVID-19 virus waned over time until they became negative. In our study, we found a significant reduction in IgM and IgG titers over a period of six months follow up. Regarding younger age, male sex and health care workers, statistically significant persistence of IgM titer was noticed at six months follow up. Also, statistically significant persistent of IgG immunity was found in males and diabetic participants by the end of the six months follow up.\n\n\nConsent\n\nWritten informed consent for publication of the participants’ details was obtained from the participants.", "appendix": "Data availability\n\nHarvard Dataverse: Underlying data for ‘Immunological changes in a cohort of COVID-19 survivors: Mansoura University experience’, https://www.doi.org/10.7910/DVN/O3QGSS (Khashaba, 2023).\n\nThis project contains the following underlying data:\n\n- Post Covid project.sav\n\n- Post Covid project.xlsx\n\nHarvard Dataverse: Extended data for ‘Immunological changes in a cohort of COVID-19 survivors: Mansoura University experience’, https://www.doi.org/10.7910/DVN/O3QGSS (Khashaba, 2023).\n\nThis project contains the following extended data:\n\n- COVID 19 sheet final (MUHs)-1.pdf\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgements\n\nWe would like to thank Marwa Ghanem, Mohamed Hamoda, Ahmed Abd-Elgawad and Mohamed Abd-Elmoneim who are Assistant Lecturers of Chest Medicine at Mansoura University.\n\n\nReferences\n\nAlzaabi AH, Ahmed LA, Rabooy AE, et al.: Longitudinal changes in IgG levels among COVID-19 recovered patients: A prospective cohort study. 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World Health Organization; 2021." }	[ { "id": "187529", "date": "26 Jul 2023", "name": "Zhiwei Chen", "expertise": [ "Reviewer Expertise Immunity" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, Tamer Elhadidy et al. reported the anti-NP IgM and IgG titer in individuals at 1-, 3- and 6-months post SARS-CoV-2 infection and further explain the correlation between antibody and disease severity, gender and diabetes. There are several major issues that need to be addressed.\n\nThe authors measured IgG and IgM titer against SARS-CoV-2 NP, not the spike. However, neither anti-NP IgM nor IgG have neutralizing activity. Although the results provide some information on humoral responses, the value for vaccine-mediated protection is limited. The authors should discuss this limitation of the study.\n\nIn method of the Chemiluminescent immunoassay, the authors mention that they used human serum or plasma for IgG and IgM measurements. It should be noted that plasma was isolated from the vacutainer contained EDTA or heparin. There might be a difference in dilution factor depending on the isolation methods used. It is not clear if such dilution factor would affect the results with 2 times difference between IgG_1m and IgG_6m (Figure 3). The authors should clarify this.\n\nThere were no standard assays used in this study for comparison with other studies. WHO already established the International Standard for anti-SARS-CoV-2 immunoglobulin (https://cdn.who.int/media/docs/default-source/biologicals/bs-documents-(ecbs)/2022-documents/new-2022-document-susan/bs-2022.2427_mattiuzzo-g._sars-cov-2_ab_2ndisandrpfor-voc_final.pdf?sfvrsn=90585abb_1&download=true). The authors may test some samples using a standard assay to increase the value of this study.\n\nWhat’s the detection limit of the IgM and IgG assay or how to define the positive and negative of the test? In Table 2 and Table 3, the authors separated the positive test and negative test for comparison. Can the authors explain?\n\nHuman serum or plasma samples isolated from individuals without infection history should be included as cutoff values. Since NP is quite conserve between common cold coronavirus and SARS-CoV-2, the issue of antibody cross reactivity should be addressed.\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? Yes", "responses": [ { "c_id": "10089", "date": "29 Aug 2023", "name": "Eman Khashaba", "role": "Author Response", "response": "Comment 1: The authors measured IgG and IgM titer against SARS-CoV-2 NP, not the spike. However, neither anti-NP IgM nor IgG have neutralizing activity. Although the results provide some information on humoral responses, the value for vaccine-mediated protection is limited. The authors should discuss this limitation of the study. Reply: Vaccine mediated immunity was not studied in current research as all study subjects were post-Covid and none of them received vaccination. This note will be added to limitations of study after discussion. Comment 2: in method of chemiluminescent immunoassay, the authors mentioned that they used human serum or plasma IgG and IgM. It should be noted that plasma was isolated from vacutainer contained EDTA or heparin there might be difference in dilution factor There might be a difference in dilution factor depending on the isolation methods used. It is not clear if such dilution factor would affect the results with 2 times difference between IgG_1m and IgG_6m (Figure 3). The authors should clarify this.……. Reply: All samples included in the study were serum samples, the chemiluminescent microparticle immunoassay can detect IgG and IgM in human serum or plasma but only serum samples were included in this study as stated on page 3 so there was no need for a dilution factor ……. Comment 3: There were no standard assays used in this study for comparison with other studies. WHO already established the international standard for anti-SARS-CoV-2 immunoglobulin….. (https://cdn.who.int/media/docs/default-source/biologicals/bs-documents- (ecbs)/2022-documents/new-2022-document-susan/bs-2022.2427_mattiuzzo-g._sars-cov-2_ab_2ndisandrpfor-voc_final.pdf?sfvrsn=90585abb_1&download=true). The authors may test some samples using a standard assay to increase the value of this study. Reply: We have used FDA approved standardized commercial kits from Abbott laboratories with provided calibrators used in calculation of antibody levels Comment 4: What is the detection limit of IgM and IgG assay or how to define the positive or negative …… In Table 2 and Table 3, the authors separated the positive test and negative test for comparison. Can the authors explain? Reply: The amount of IgG and IgM antibodies to SARS-CoV-2 in each sample is determined by its chemiluminescent relative light unit (RLU) to the calibrator RLU using an index S/C threshold of 1.4 for IgG and 1.0 for IgM as stated on page 3 Comment 5: Human serum or plasma samples isolated from individuals without infection history should be included as cutoff values. Since NP is quite conserve between common cold coronavirus and SARS-CoV-2, the issue of antibody cross reactivity should be addressed. Reply: We used FDA approved standardized commercial kits from Abbott laboratories with provided calibrators used in calculation of antibody levels. Cross reactivity could not be addressed in this study. This comment was added to limitations of study" } ] }, { "id": "191284", "date": "07 Aug 2023", "name": "Ritthideach Yorsaeng", "expertise": [ "Reviewer Expertise Clinical Trials", "Vaccine Trials", "Immunology", "Infectious Diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor concerns.\nHave all study participants not received any COVID-19 vaccination before the study termination? Vaccination during the study followed-up may alter the immunologic outcome and distort the results.\n\nHave all study participants got re-infection before the study termination? Infection during the study followed-up may alter the immunologic outcome and distort the results.\nMinor concerns.\n\"The assay is a chemiluminescent microparticle immunoassay for qualitative detection of IgG and IgM in human serum or plasma against the SARS-CoV-2 nucleoprotein.\" Are you sure that the IgM assessment in this study targeted anti-nucleocapsid?\n\nThere was a lacks of information about the immunity trend (immunity waning).\nComments.\nYou can add a further keyword, such as; anti-nucleocapsid.\n\n\"Using an index S/C threshold of 1·4 (for IgG) and 1 (for IgM).\" Suggest revising a decimal separator to \" .\".\n\nSuggest using one style of English. I found this manuscript contained both UK and US styles. For example, neutralising and neutralizing.\n\nThe age group can be stratified (adolescent, young adult, middle-aged and elderly) to see more immunity waning trend in all Tables and Figures. Because a young age has higher immunity than an elder. If it works.\n\nIs the work 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": "10090", "date": "29 Aug 2023", "name": "Eman Khashaba", "role": "Author Response", "response": "Major concerns. Have all study participants not received any COVID-19 vaccination before the study termination? Vaccination during the study followed-up may alter the immunologic outcome and distort the results. Reply: None of the participants received COVID-19 vaccination before the study termination as our study was conducted post COVID-19 infection and the participants were not convinced to receive any vaccination before at least one-year post infection. This was proved by history taking during follow up. Have all study participants got re-infection before the study termination? Infection during the study followed-up may alter the immunologic outcome and distort the results. Reply: Re-infection was excluded based on review of clinical symptoms during follow up visits. This was mentioned in discussion. Minor concerns. \"The assay is a chemiluminescent microparticle immunoassay for qualitative detection of IgG and IgM in human serum or plasma against the SARS-CoV-2 nucleoprotein.\" Reply: The amount of IgG and IgM antibodies to SARS-CoV-2 in each sample is determined by its chemiluminescent relative light unit (RLU) to the calibrator RLU using an index S/C threshold of 1.4 for IgG and 1.0 for IgM as stated on page 3 Are you sure that the IgM assessment in this study targeted anti-nucleocapsid? Reply: Yes, the chemiluminescent microparticle assay detects the IgM targeted against the SARS-CoV-2 nucleocapsid protein There was a lacks of information about the immunity trend (immunity waning). Reply: The authors illustrated in figures the immunity waning according to measured levels of I g M and IgG at 1,3,6 months (Table1, Figure 2 &3) in results section. Comments. You can add a further keyword, such as; anti-nucleocapsid. Ok. The keyword is added \"Using an index S/C threshold of 1·4 (for IgG) and 1 (for IgM).\" Suggest revising a decimal separator to \" .\". Ok. Done Suggest using one style of English. I found this manuscript contained both UK and US styles. For example, neutralising and neutralizing. Ok done UK style The age group can be stratified (adolescent, young adult, middle-aged and elderly) to see more immunity waning trend in all Tables and Figures. Because a young age has higher immunity than an elder. If it works. Reply: our study results revealed that there was statistically significant difference according to mean age between positive and negative groups (Table 2). Based on these results ,we did logistic regression analysis which revealed that increased age (continuous variable) is associated with decreased chance of positive IgM ONLY (Table 4) . These results agree with the reviewer' s comments that young age is associated with better immunity (necessary changes will be made in discussion)." } ] } ]	1	https://f1000research.com/articles/12-793
https://f1000research.com/articles/12-1050/v1	29 Aug 23	{ "type": "Research Article", "title": "The total factor productivity growth of health systems in African least developed countries", "authors": [ "Edward Musoke", "Bruno Lule Yawe", "John Ddumba Ssentamu" ], "abstract": "Background: Given the several health policy reforms in various countries in the Sustainable Development Goals (SDGs) era, the need for efficient and productive health systems has become of great concern. This is even more urgent in African Least Developed Countries (African LDCs) that need to improve the health of their populations. Thus, the objective of this study is to assess the total factor productivity growth of health systems of 29 African Least Developed Countries for the 2008-2018 period. Methods: The study uses data from the World Bank and the World Health Organization. Using the Data Envelopment Analysis (DEA) Malmquist index, the inputs that were used in the study included domestic general government health expenditure, domestic private health expenditure, external health expenditure and out of pocket health expenditure while the outputs were life expectancy at birth, maternal mortality rate, under five mortality rate, and infant mortality rate. Results: Sixteen African LDCs registered progress in the total factor productivity growth of their health systems while thirteen registered a decline the total factor productivity growth of their health systems. Overall, there was 0.3% average increase in total factor productivity growth of health systems in African LDCs. This was attributed to a 1.2% increase in technical efficiency change and a 0.9% average decrease in the technical change of health systems in African LDCs. Conclusions: African LDCs with less productive health systems are advised to bench mark the policies of African LDCs with productive health systems.", "keywords": [ "Total factor productivity growth", "technical efficiency", "Data Envelopment Analysis Malmquist Index", "Health Systems", "African Least Developed Countries" ], "content": "Introduction\n\nDeveloped and Least Developed Countries (LDCs) have a clear, pressing, and ongoing need for a healthy population and productive health system (Mohamadi et al., 2020). This according to Kim et al. (2016) has prompted health sector reforms to raise health systems performance in these countries. Key amongst these reforms indicating a country’s commitment to the health of its citizens is health expenditure (Kim et al., 2016). According to Anaemene (2018), unlike developed countries that have high levels of health expenditure, least developed countries and in particular African LDCs represent 1% of global health spending and are responsible for 25% of the world’s disease burden which accounts for 60% of their deaths (United Nations, 2017). In light of this Ojwang’Oyieke and Karamagi (2023) state that without efficient and productive use of scarce health resources, these health challenges could potentially overwhelm the health systems of African LDCs. Thus estimating the total factor productivity growth of health systems of African LDCs which is the maximization of outputs for a given level of scarce inputs, is critical. Thus, the general objective of this study is to assess the total factor productivity growth of health systems of 29 African LDCs for the 2008-2018 period. The findings in this paper are particularly important for a number of reasons. First, African LDCs with less productive health systems can benchmark practices of African LDCs with productive health systems. Second, it adds to the small but growing body of knowledge about the total factor productivity growth of health systems in resource-constrained settings. Future researchers might find use for such important literature.\n\nThe rest of this paper is organized as follows: the literature review is presented in the next section followed by the methodology and discussion of findings. After the discussion of findings, the conclusions and policy recommendations are presented followed by acknowledgements, statement on data availability, declaration of competing interests, grant information and the acknowledgement.\n\n\nLiterature review\n\nTo comprehend the total factor productivity growth of health systems in African LDCs, the theory of constraints by Goldratt and Cox (1984) is adopted. According to Ochiel (2019), the premise of this theory is that the transformation of inputs into outputs is through the production process is faced with constraints. Thus the elimination of these constraints is the ultimate goal of each health systems to witness progress in their total factor productivity growth (Aguilar-Escobar & Garrido-Vega, 2016; Ochiel, 2019). This is why Ochiel (2019) suggests that several steps like investing in technology can be adopted to counteract the negative effects of the constraints in health systems and witness progress in the total factor productivity growth of these health systems.\n\nSeveral studies have assessed the total factor productivity growth of health systems comparing several countries from different regions of the world like Organization for Economic Co-operation and Development (OECD) countries (Adang & Borm, 2007; Kim et al., 2016), European and Central Asian countries (Hsu, 2014), Visegrád group countries (Grausová et al., 2014), Continental African Countries (Kirigia et al., 2007), World Health Organization countries from the Eastern Mediterranean region (Masri & Asbu, 2018), Upper Middle Income Countries with focus on Iran (Mohamadi et al., 2020), countries from the Association of South East Asian Nations (Singh et al., 2021) and developed countries (Almessabi, 2020). They used several inputs like number of medical personnel (Adang & Borm, 2007; Almessabi, 2020), health expenditure (Almessabi, 2020; Kim et al., 2016; Masri & Asbu, 2018), number of hospital beds (Almessabi, 2020; Grausová et al., 2014), education (Kim et al., 2016; Kirigia et al., 2007). Several outputs like infant mortality rate (Almessabi, 2020; Kim et al., 2016; Masri & Asbu, 2018), under five mortality rate (Almessabi, 2020), life expectancy (Adang & Borm, 2007; Grausová et al., 2014; Hsu, 2014) and maternal mortality ratio (Ibrahim et al., 2019) have been used as well.\n\nAll these studies employed the Data Envelopment Analysis (DEA) based Malmquist index and established variations in the total factor productivity growth of their health systems, with health systems of some countries experiencing a regress in productivity (Almessabi, 2020; Hsu, 2014; Masri & Asbu, 2018) while others demonstrating progress in productivity (Adang & Borm, 2007; Kim et al., 2016; Kirigia et al., 2007). The regress in productivity was attributed increase or decrease in efficiency (Almessabi, 2020; Hsu, 2014; Masri & Asbu, 2018) and an increase or decrease in technology (Almessabi, 2020; Hsu, 2014; Masri & Asbu, 2018). Similarly, the progress in productivity was attributed to an increase or decrease in efficiency (Adang & Borm, 2007; Kim et al., 2016; Kirigia et al., 2007) and an increase or decrease in technology (Adang & Borm, 2007; Kim et al., 2016; Kirigia et al., 2007).\n\nThe contribution and originality of this paper is based on Sajadi et al. (2020) suggestion of selecting the best input and output combinations which is so crucial in the estimation of the total factor productivity growth of health systems. According to Wagner and Shimshak (2007), most of the studies assessing the total factor productivity growth of health systems consider the input and output combinations as simply “givens” based on literature and do not focus on the choice of the best input and output combinations. Yet if the choice of input and output combinations is not given the attention it deserves, results of total factor productivity growth are biased and inconsistent. Adang and Borm (2007) further note that much of the critique of the 2000 World Health Report from the World Health Organization (2000) had to do with completeness of the production function and the choice of the inputs and output combinations. This study addresses this gap by using correlational analysis as used by Rooijakkers (2018); Kizza (2012); Hisali and Yawe (2011) and Yawe (2006) to select the best input and output combinations for the estimation of the total factor productivity growth of health systems.\n\n\nMethods\n\nFollowing Kizza (2012) and Yawe (2006) each African LDCs is considered to be a Decision Making Unit (DMU) or unit of analysis. Twenty-nine African LDCs are considered for this study based on the availability of data (see Table 1). According to Table 1, of the twenty-nine African LDCs, twelve are found in west Africa, eight are found in east Africa, six are found in south Africa and three in central Africa. According to Wale-Oshinowo et al. (2022), the geographic configurations resulting from the colonial and post-colonial delineation of these regions of Africa are to blame for the high proportion of African LDCs in West and East Africa.\n\nBased on studies like: Hadad et al. (2013); Çelik et al. (2017): Ibrahim et al. (2019); Masri and Asbu (2018); Behr and Theune (2017); Retzlaff-Roberts et al. (2004) and Mohamadi et al. (2020), four inputs and outputs are considered for this study. Since the production of health at a macro level is complicated, health outcomes are used as health outputs (Çelik et al., 2017; Ng, 2008; Peacock et al., 2001). The input, output data and their definitions based on the World Bank (2021) and World Health Organization (2019) are shown in Table 2.\n\nThe estimation of the total factor productivity growth of health systems require the use of output variables that capture good health outcomes. To conform to isotonicity and devise output variables that capture good health outcomes in infants, mothers and children under five years (Ibrahim et al., 2019; Zhou et al., 2020). The Infant Mortality Rate (IMR); Maternal Mortality Ratio (MMR) and under-five mortality rate (U5MR) values are converted to infant survival rate (ISR) (ISR=1,000−IMR/IMR), maternal survival ratio (MSR) (MSR=100,000−MMR/MMR) and under five survival rate (U5SR) (U5SR=1,000−U5SR/U5SR).\n\nCorrelation analysis recommended by Cetin and Bahce (2016); Yawe (2006) and Kizza (2012) is used to select the best input and output combinations. According to Cetin and Bahce (2016), input and output combinations that are highly correlated and significant are redundant and dropped from the further analysis of the total factor productivity growth of health systems. Furthermore, following Kizza (2012) and Yawe (2006) input and output combinations that provide the highest average total factor productivity growth are chosen for the DEA based Malmquist model.\n\nAccording to Santín and Sicilia (2017) and Cordero et al. (2013) endogeneity occurs when the technical efficiency scores are strongly correlated with any one input. Correlation analysis suggested by Dhaoui (2019) is used to test for potential endogeneity in the assessment of the total factor productivity growth of health systems in African LDCs.\n\nThe theoretical framework for estimating the total factor productivity growth of health systems in African LDCs is based on Solow (1957) model which is summarized as:\n\nWhere Qt is the output and At is the total factor productivity, which measures the shift in the production function at given the X, Y inputs and technology set. This total factor productivity (At) is measured using a non-parametric index number (Hulten, 1986). Following Ojwang’Oyieke and Karamagi (2023), since this approach does not impose a specific form on the production function, equation (1) is converted to a (logarithmic) differential of the production function as:\n\nAccording to Hulten (1986), the growth rate of real output can be factored out into the growth rate of X and Y inputs weighted by their output elasticities and the growth rate of the hicksian efficiency index.\n\nBy total differentiation of equation (2), Solow (1957) showed that the hicksian efficiency index is a residual growth rate of output that is not accounted for by the growth in inputs which is given as:\n\nWhere St=xtyt:xtcanproduceyt Is the feasible technology set which contains a combination of xtinputs and xtoutputs. Thus the solow residual Rt= the hicksian index. Solow concluded that, theoretically, this growth rate was equal to the growth rate of the hicksian efficiency parameter At∗At (Ojwang’Oyieke & Karamagi, 2023). Abramovitz (1956) called this residual as a measure of the degree of our ‘ignorance.’ “This ignorance could be wanted (technical, scale and technological innovation) or unwanted like (measurement errors, omitted variables, aggregation bias, and model misspecification)” (Ojwang’Oyieke & Karamagi, 2023). For the case of African LDCs, assuming that the unwanted ignorance is minimal, and hence attribute the solow residual to technical, scale and technological innovation in the decomposition of total factor productivity growth (Zofio, 2007).\n\nFollowing Masri and Asbu (2018), the output-oriented Variable Returns to Scale Data Envelopment Analysis (VRS-DEA) based Malmquist total factor productivity index is adopted for estimating the total factor productivity growth of health systems in African LDCs. The output-oriented VRS malmquist total factor productivity index is chosen over the input-oriented Constant Returns to Scale (CRS) approach because it is better suited for least developed countries while the input oriented is better suited for developed countries that have better health outputs (Dhaoui, 2019; Dingake, 2017).\n\nIf countries produce multiple outputs yt using multiple inputs xt, productivity change is measured using total factor productivity index also called multifactor productivity index. The output distance function each country over a given period of time is given as\n\nThe first distance function, in equation (5), measures the maximum proportional change in outputs required to make xt+1yt+1 feasible in relation to the technology at the previous period t. Similarly, the second mixed-period distance function, equation (6), measures the maximum proportional change in output required to make xtytfeasible in relation to the technology at t+1 which we call D0t+1xtyt.\n\nThe Malmquist total factor productivity index (MTFP) measures total factor productivity (TFP) change between two time points in terms of ratios of distance functions. The MTFP between two time periods (t and t+1) using periodtand period t+1 technologies respectively is given as for period t\n\nFor period t+1\n\nWhere;\n\nM0t and M0t+1denote the MTFP in period tand t+1 respectively;\n\nD0txt+1yt+1 refers to the output distance function which evaluates period t+1 data relative to the technology in period t;\n\nD0txtyt is output distance function evaluating period t data relative to technology in periodt;\n\nD0t+1xt+1yt+1is the output distance function evaluating period t + 1 data relative to technology in period t+1;\n\nD0t+1xtytis the output distance function evaluating period t data relative to technology in period t+1;\n\nUsing periodt and t+1 technologies, the MTFP is defined as the geometric mean if the equations as follows;\n\nThe MTFP in equation (9) is further decomposed into efficiency change EFFCH and technical/technological TECHCH change as follows\n\nThat is: M0t = TEFFCH×TECHCH. The MTFP index value greater than 1 indicates growth in productivity, whereas a value less than 1 indicates a decline in productivity between periods t and t+1. A value of 1 denotes stagnation in productivity (Kirigia et al., 2007). Likewise for efficiency change and technical change, if TEFFCH>< and TECHCH>< then there is an increase (decrease) in efficiency and technical progress (regress).\n\nThe DEA based Malmquist model is estimated using DEAP version 2.1 a free DEA Program developed by Coelli (1996). STATA version 15 by Stata Corp (2015) is used for the pre estimation techniques (choice of the best input/output combinations and checking for endogeneity issues regarding the total factor productivity growth of health systems). R, a free software environment for statistical computing and graphics, can be used for this analysis as well. Please see Underlying data (Musoke et al., 2023) for access to the specific datasets used in the study.\n\n\nResults and discussion\n\nThere is variation among the chosen inputs and outputs for various Africa LDCs (see Table 3).\n\nThe minimum and maximum amounts for domestic general government health spending were 0.927 and 89.097 million US dollars, respectively, while the minimum and maximum amounts for external health spending were 1.121 and 74.705 million US dollars. The difference between domestic private health spending and out-of-pocket medical expenses is even greater, with minimum and maximum values of 2.182 and 1.825 million US dollars and 139.601 and 147.569 million US dollars, respectively. For the health outputs, the average life expectancy at birth is 59.056 years, with a range of 43.384 to 68.7 years. With minimum values of 0.005, -0.405, and 7.734 and maximum values of 0.046, 8.259, and 35.63, respectively, the average under-five survival rate, maternal survival ratio, and infant survival rate are 0.013, 1.438, and 17.453, respectively.\n\nTo determine the interrelationships between various input and output variables, the Pearson’s correlation matrix for the input and output variables in Table 4 is calculated.\n\n* p < 0.05.\n\n p < 0.01.\n\n* p < 0.001 indicates 5%. 1% and 0.1% significance level.\n\nSeveral input/output combinations for three (3) DEA model specifications based on the output orientation and Variable Returns to Scale (VRS) assumption are presented in Table 5. The results in Table 5 are in light of the results of the Pearson’s correlation matrix in Table 4. Only two outputs and all inputs are included in the DEA Model 1. Under five survival rate and maternal survival rate are dropped from DEA Model 1 as outputs because they have a strong significant positive correlation r=0.837>0.5p<0.001.\n\nDEA malmquist model 2 has two outputs and all inputs. DEA malmquist model 2’s outputs life expectancy at birth and infant survival rate are dropped due to their strong significant positive correlation r=0.775>0.5p<0.001. DEA malmquist model 3 only has two inputs and four outputs. Due to their significant positive correlation r=0.980>0.5p<0.001, domestic private health expenditure and out-of-pocket health expenditure inputs were dropped for DEA malmquist model 3.\n\nResults of the total factor productivity growth of the three estimated DEA Malmquist models based on the several input and output combinations for DEA malmquist model specifications in Table 5 are presented in Table 6. Over the 2008-2018 period, the average total factor productivity changes in DEA malmquist model 1 is 0.990 indicating a 1% regress in productivity. Similarly, the average total factor productivity changes in DEA malmquist model 3 is 0.983 indicating a 1.7% regress in productivity. The average total factor productivity change in DEA malmquist model 2 is 1.003 which indicates a productivity progress of 0.3%.\n\nA comparison of all the three (3) DEA Malmquist models in Table 6 indicated that model 2 is the most preferred model with an average total factor productivity growth of 1.003 and 16 of 29 African LDCs on the frontier.\n\nTest for endogeneity for the most preferred DEA malmquist model 2\n\nResults of the pearsons correlation between inputs and technical efficiency scores based on VRS for the most popular DEA Malmquist model 2, are presented in Table 7.\n\nSince endogeneity typically denotes a strong correlation between inputs and the technical efficiency scores based on the VRS (Orme & Smith, 1996). According to Table 7, there isn’t much of a correlation between the input variables and technical efficiency scores. As a result, the DEA Malmquist Model 2 does not have an endogeneity issue and can be adopted for analysis.\n\nResults of the Malmquist index summary of annual means for DEA Malmquist Model 2 of the African LDCs from 2008 to 2018 are presented in Table 8. According to Kizza (2012) and Yawe (2006), the annual means of the Malmquist Index are geometric in nature and represent the efficiency change, technical change and total factor productivity change.\n\n* Note that 2010 refers to the change between 2008 and 2010.\n\nThe findings in Table 8 demonstrate that over time, the average technical efficiency change of health systems in African LDCs improved by 1.2%, the average pure efficiency change of African LDCs’ health systems improved by 0.9%. The average scale efficiency change and total factor productivity change improved by 0.3%. The mean pure efficiency change and mean scale efficiency change were responsible for the 1.2% increase in the average technical efficiency change of health of health systems. The highest progress in technical efficiency change of 11.9% was registered in the year 2013 while the highest regress of 12.1% was registered in the year 2017.\n\nThe findings in Table 8 also indicate a 0.9% regress in the technological change of health systems in African LDCs. The highest progress and regress of 18.4% and 11.4% of technological change were registered in the years 2017 and 2018 respectively. Furthermore, the average total factor productivity change for health systems in African LDCs in Table 8 was 1.003, representing a 0.3% increase in total factor productivity. The highest progress in total factor productivity change of 13.4% was in the year 2016 while the lowest of 0.1 was during the 2008-2010 period. The highest regress in total factor productivity change of 5.3% was in the year 2012 while the lowest regress in total factor productivity change was in the year 2014. The growth in total factor productivity over the years was largely from the technical efficiency change than the technical change.\n\nAccording to Kizza (2012), group averages like the malmquist index summary of annual means for the best DEA Malmquist Model 2 in Table 8 hide individual results. As a result, it is crucial to run estimates for summary means for each African LDC for the 2008-2018 period (see Table 9). Results in Table 9 indicate a 1.2% progress in the technical efficiency of health systems for African LDCs over the 2008-2018 period. since technical efficiency = pure efficiency change × scale efficiency change, the mean technical efficiency change of 1.012 was as a result of 1.009 and 1.003 progress in pure efficiency change and scale efficiency change respectively.\n\nSeventeen (Angola: 2.8%, Benin: 2%, Burundi: 3.8%, Central African Republic: 2.2%, Eritrea: 4.5%, Gambia: 5.6%, Guinea: 6.4%, Guinea Bissau: 7.3%, Liberia: 0.4%, Madagascar: 2.4%, Malawi: 2.7%, Mauritania: 0.1%, Mali: 5%, Niger: 3.4%, Uganda: 8.4%, Tanzania: 3.9%, Zambia: 5.5%) African LDCs had progress in technical efficiency change meaning that they moved towards the frontier. Five (Burkina Faso, Democratic Republic of Congo, Mozambique, Rwanda and Sierra Leone) neither registered regress or progress in the technical efficiency change. Seven (Chad: 0.8%, Djibouti: 4.7%, Ethiopia: 0.6%, Lesotho: 3.8%, Senegal: 4.5%, Sudan: 11.4% and Togo: 4%) African LDCs registered regress in the technical efficiency change. These results are consistent with those of studies like Kim et al. (2016) and Hsu (2014) who also reported a progress in the technical efficiency change. However, they are in disagreement with those of Singh et al. (2021) and Kirigia et al. (2007) who reported a decline in the technical efficiency change. A possible explanation for this is the efficient use of resources in countries that demonstrated a progress and inefficient use of resources in countries that demonstrated regress.\n\nAll African LDCs experienced a 0.9% mean reduction or regress in technology indicating that technical change for the African LDCs was less than one (<1). This meant that the technology (production) frontier shifted downwards. Sixteen African LDCs (Central African Republic, Democratic Republic of Congo, Eritrea, Ethiopia, Gambia, Guinea, Guinea Bissau, Liberia, Malawi, Mali, Mozambique, Rwanda, Sierra Leone, Tanzania, Togo and Uganda) had a regress in technical change while twelve African LDCs (Angola, Benin, Burkina Faso, Burundi, Chad, Djibouti, Lesotho, Madagascar, Niger, Senegal, Sudan and Zambia) had progress or improvement in technical change over the 2008-2018 period. Mauritania is the only African LDC that had stagnation in technical Change. The regress in technical change experienced by the African LDCs during the 2008-2018 period is attributed to low adoption of technologies and to the use of outdated technologies. Similar results are reported by (Hsu, 2014; Masri & Asbu, 2018; Singh et al., 2021).\n\nOver period 2008 to 2018 period, there was a 0.3% progress in the total factor productivity change of health systems in African LDCs. This progress was due to 1.2% progress in technical efficiency change and 0.9% regress in technical change. Sixteen African LDCs (Angola = 6.2%, Benin = 2.2%, Burkina Faso = 0.5%, Burundi = 7.7%, Central African Republic = 0.7%, Chad = 0.3%, Eritrea = 1.8%, Gambia = 4.9%, Madagascar = 8.4%, Malawi = 1.3%, Mali = 2.4%, Mauritania = 0.1%, Niger = 6.2%, Uganda = 7.5%, Tanzania = 3.7% and Zambia = 7.1%) registered progress in the total factor productivity change of health systems in African LDCs. Thirteen African LDCs (Democratic Republic of Congo, Djibouti, Ethiopia, Guinea, Guinea Bissau, Lesotho, Liberia, Mozambique, Rwanda, Senegal, Sierra Leone, Sudan and Togo) registered regress in the total factor productivity change of health systems in African LDCs. These findings are in agreement with those of Ibrahim et al. (2019), Kim et al. (2016) and Hsu (2014) and in disagreement with those of Kirigia et al. (2007). A possible explanation for this according to Kim et al. (2016) and Cashin and Dossou (2021) are the several health policy reforms such as (easy access to primary care, better treatment procedures implementation of information technology and payment systems) amongst the African LDCs.\n\n\nConclusion and policy recommendations\n\nResults of the total factor productivity growth of health systems in African LDCs from 2008 to 2018 indicated a 0.3% progress in the total factor productivity change. Sixteen African LDCs registered a decline in the total factor productivity growth while thirteen witnessed progress in the total factor productivity growth of their health systems. The variations in the total factor productivity growth of health systems of African LDCs are attributed to a 1.2% progress in technical efficiency change and a 0.9% regress in technical change. Less productive African LDCs are advised to bench mark the policies of productive African LDCs.", "appendix": "Data availability\n\nData for each of 29 African LDCs on life expectancy at birth, maternal mortality ratio, under five mortality rate and infant mortality rate for the 2008-2018 period used in this study were sourced from the World Bank: https://databank.worldbank.org/source/world-development-indicators.\n\nThe domestic general government health expenditure, out of pocket health expenditure, domestic private health expenditure and external health expenditure data used in this study were sourced from the World Health Organization health financing indicators section: https://www.who.int/data/gho/data/indicators/indicators-index . To access the data for each of the 29 African LDCs for the 2008-2018 period, each of the indicators is searched for from the list of indicators which are arranged in alphabetical order.\n\nAccess to the source data is free of charge subject to the terms and conditions set by the World Bank (https://data.worldbank.org/summary-terms-of-use) and the World Health Organization (https://www.who.int/about/policies/publishing/data-policy/terms-and-conditions). The input and output data has been compiled and is provided on Zenodo below.\n\nZenodo: Input and output data. https://doi.org/10.5281/zenodo.8007631 (Musoke et al., 2023).\n\nThis project contains the following underlying data:\n\n- Input and Output Data.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank Makerere University, through its staff welfare and development that enabled the researcher to undertake this study. The entire team at the Makerere University school of economics that organized work in progress presentations and gave valuable contributions to the betterment of this work are also acknowledged.\n\n\nReferences\n\nAbramovitz M: Resource and output trends in the United States since 1870. Resource and output trends in the United States since 1870. NBER; 1956; (pp. 1–23).\n\nAdang EM, Borm GF: Is there an association between economic performance and public satisfaction in health care? Eur. J. Health Econ. 2007; 8: 279–285. PubMed Abstract \| Publisher Full Text\n\nAguilar-Escobar V-G, Garrido-Vega P: Applying the theory of constraints to the logistics service of medical records of a hospital. Eur. Res. Manag. Bus. Econ. 2016; 22(3): 139–146. Publisher Full Text\n\nAlmessabi BN: A NEXUS BETWEEN COST EFFICIENCY AND COST PRODUCTIVITY: A CASE STUDY OF HEALTH SYSTEMS OF DEVELOPED COUNTRIES. PalArch's J. Archaeol. 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Publisher Full Text\n\nMohamadi E, Manesh AO, Takian A, et al.; Technical efficiency in health production: A comparison between Iran and other upper middle-income countries. Health Policy Technol. 2020; 9(3): 335–347. Publisher Full Text\n\nMusoke E, Yawe LB, Ddumba SJ: Input and output data. [Data set]. Zenodo. 2023. Publisher Full Text\n\nNg YC: The productive efficiency of the health care sector of China. Rev. Reg. Stud. 2008; 38(3): 381–393. Publisher Full Text\n\nOchiel JW: Technical and Scale Efficiency of Hospitals in Kisumu County Kenya. University of Nairobi; 2019.\n\nOjwang’Oyieke S, Karamagi IJ: Decomposition of Total Factor Productivity Growth in Referral Hospitals in Kenya: 2012-2016. Tanzanian Economic Review. 2023; 12(2).\n\nOrme C, Smith P: The potential for endogeneity bias in data envelopment analysis. J. Oper. Res. Soc. 1996; 47(1): 73–83. Publisher Full Text\n\nPeacock S, Chan C, Mangolini M, et al.: Techniques for measuring efficiency in health services. Productivity Commission: Staff Working Paper. 2001.\n\nRetzlaff-Roberts D, Chang CF, Rubin RM: Technical efficiency in the use of health care resources: a comparison of OECD countries. Health Policy. 2004; 69(1): 55–72. PubMed Abstract \| Publisher Full Text\n\nRooijakkers P: Data Envelopment Analysis in determining factors that influence technical efficiency levels on Dutch dairy farms.2018.\n\nSajadi HS, Goodarzi Z, Takian A, et al.: Assessing the efficiency of Iran health system in making progress towards universal health coverage: a comparative panel data analysis. Cost Eff. Resour. Alloc. 2020; 18: 1–11. Publisher Full Text\n\nSantín D, Sicilia G: Dealing with endogeneity in data envelopment analysis applications. Expert Syst. Appl. 2017; 68: 173–184. Publisher Full Text\n\nSingh S, Bala MM, Kumar N, et al.: Application of DEA-Based malmquist productivity index on health care system efficiency of ASEAN countries. Int. J. Health Plann. Manag. 2021; 36(4): 1236–1250. PubMed Abstract \| Publisher Full Text\n\nSolow RM: Technical change and the aggregate production function. Rev. Econ. Stat. 1957; 39: 312–320. Publisher Full Text\n\nStataCorp, L: StataCorp stata statistical software: Release 14. College Station, TX, USA: StataCorp LP; 2015.\n\nUnited Nations: Health care systems: time for a rethink report 2017-Pain from crash in commodity prices.2017.\n\nWagner JM, Shimshak DG: Stepwise selection of variables in data envelopment analysis: Procedures and managerial perspectives. Eur. J. Oper. Res. 2007; 180(1): 57–67. Publisher Full Text\n\nWale-Oshinowo B, Omobowale A, Adeyeye MM, et al.: Least developed countries in Africa. Palgrave Macmillan; 2022.\n\nWorld Bank: The World Bank. DataBank. World Development Indicators. Washington, DC, USA: World Bank; 2021.\n\nWorld Health Organization: The world health report 2000: health systems: improving performance. World Health Organization; 2000.\n\nWorld Health Organization: Global Health Observatory (GHO) data. Geneva: 2019.\n\nYawe BL: Technical efficiency and total factor productivity growth in Uganda’s district referral hospitals. University of Dar es Salaam; 2006.\n\nZhou L, Ampon-Wireko S, Dauda L, et al.: Empirical analysis of factors influencing healthcare efficiency among emerging countries. Healthcare; 2020.\n\nZofio JL: Malmquist productivity index decompositions: a unifying framework. Appl. Econ. 2007; 39(18): 2371–2387. Publisher Full Text" }	[ { "id": "231745", "date": "25 May 2024", "name": "Kwadwo Arhin", "expertise": [ "Reviewer Expertise Health Economics", "Healthcare Financing", "Health Systems Efficiency and Productivity Analysis", "and Health Econometrics." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper examines the productivity of healthcare resources in 29 least developed countries in Africa. Though the authors have made great efforts to contribute to the literature on health system efficiency and productivity, I think incorporating the following suggestions would go a long way to improve the quality of the paper.\n1. One relevant recent literature on the productivity of health systems in Africa was left out in the literature review Arhin et al.,(2023) 1 2. How were the least developed countries (LDCs) defined? Is it based on the World Bank's definition for LDCs? What range of per capita GDP defines LDCs? The authors must clearly define LDCs. 3. To ensure that input variables used in the analysis are comparable across countries, the authors must use the variables measured in purchasing power parity (PPP) rate per capita instead of the current US$ per capita used in the analysis. The World Health Organization's Global Health Expenditure Database has all the input variables measured in PPP. 4. Out of Pocket Health Expenditure (OOP) is a subset of Domestic Private Health Expenditure (DPE) as correctly defined in Table 2. The addition of OOP to DPE as in Models 1 and 2 (see Table 5) adds little to no information to the data. The authors must consider including one of these two input variables in the estimation of models 1 and 2. 5. The policy recommendations section of the paper is a major weakness. There are no clear-cut policies emanating from the results and discussion of the paper. The authors must clearly explain at least three policy implications of the results of the paper and which agencies must implement those policy recommendations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1050
https://f1000research.com/articles/12-1049/v1	29 Aug 23	{ "type": "Research Article", "title": "Comparison of root end sealing ability of three retrograde filling materials in teeth with root apices resected at 900 using dye penetration method under fluorescent microscope", "authors": [ "Dr. Payal Chaudhari", "Manoj Chandak", "Dr. Aditya Patel", "Manoj Chandak", "Dr. Aditya Patel" ], "abstract": "Background: The main factor contributing to surgical endodontic dissatisfaction is an inadequate seal at the apex. The material used for retrograde filling should prevent contaminants from penetrating periradicular tissue. So the objective of this study was to evaluate and compare the ability of Mineral Trioxide Aggregate (MTA), Zirconomer and bioactive bone cement to seal the root end as retrograde filling material with apices resected at 900 angles using dye penetration method under fluorescent microscope. Methods: 36 extracted teeth samples were cut at Cementoenamel junction horizontally. Following the customary protocols of, cleaning, shaping and obturation with gutta percha and AH Plus sealer, resection of samples was done 3mm at the apical end at 90 degree angle along the long axis of the tooth with the help of diamond disc. A depth of 3mm of root end cavity was made with diamond coated ultrasonic surgical tip. The samples were randomly allocated in 3 groups: Group I: MTA ; Group II : Zirconomer; Group III: Bioactive Bone Cement . Coating of nail varnish was done with the roots except at the tip. Each material was compacted in the retrocavity using small pluggers. All the retrofilled samples were kept in acridine orange for 24 hours, following which cleaning & bucco-lingual sectioning (vertical) was carried out. Confocal laser scanning fluorescent microscope was used for checking the dye penetration in these samples thereby evaluating sealing ability. Results: Group III (Bioactive Bone Cement) had the highest root end sealing ability with mean (1.4 ± 0.51) followed by Group I (MTA) (2.3 ± 0.65) and lastly Group II (Zirconomer) (4.1 ± 0.57) as root end filling material with apices resected at 900 angle using dye penetration method under fluorescent microscopy which were statistically significant. Conclusions: Bioactive Bone cement can be recommended as an acceptable retrograde filling material.", "keywords": [ "Endodontic surgery", "Retrograde filling material", "Sealing ability", "Retrograde Endodontics" ], "content": "Introduction\n\nA successful endodontic therapy is determined by accurate diagnosis, efficient chemo-mechanical preparation of the root canal (RC), and creation of a hermetic barrier which blocks every path of communication between the canal space as well as periapical tissues.1 The reaction of the host to pathogenic bacteria inhabiting the RC system is called apical periodontitis. The essential objective of traditional endodontic therapy is preventing and/or eliminating the state of apical periodontitis.2,3 The success rate of endodontic therapy, which is widely used for treating irreversible pulpitis or necrosis of the RC content, is currently between 85% and 95%.4 But chances of successful treatment are quite slim when infection permeates the periapical tissues.2\n\nDisinfecting a root canal thoroughly is challenging due to its complexity, predominantly in the apical area. The apical third of the root constitutes of the lateral & accessory canals, apical delta, isthmus, ramifications. The lateral and accessory canals, which are situated a few millimeters within the root apex, create an apical delta, which forms channels that allow communication between the canal and the periodontal apparatus.5 Despite endodontic therapy, nutrient supply to bacteria in such ramifications as well as apical deltas will be unchanged, resulting in a persistent presence of bacteria along with remnants of necrotic tissue in the apical end.5 Apical periodontitis and periapical lesions are caused by these root canal irritants egressing into the periradicular tissues.6\n\nThe biofilm-resistant microorganisms, poor medicament/irrigant penetration, low irrigant concentration, short exposure period & low volume of the irrigant, and poor irrigant exchange in the apical part continue to pose challenges in nonsurgical endodontic therapy. Additionally, extraradicular biofilms that are connected to the apical root surface, are thought to be a potential reason of post-treatment apical periodontitis, leading to root canal therapy failure.7\n\nWhen nonsurgical retreatment fails, is impractical, or is unlikely to improve the initial endodontic therapy, endodontic microsurgery is frequently the last resort.8 The primary indications for periradicular surgery include failed non-surgical root canal therapy, the need for surgical drainage of periodontal/periapical abscess, calcific metamorphosis of the pulpal space, procedural errors, anatomic variations, biopsy, corrective surgery, and replacement surgery.8 Particularly, cases with a persistent lesion whose origin is connected to complex canal anatomy, extra-radicular infection, foreign body reacting material, and/or cystic tissue may only be resolved through surgical intervention.4\n\nHarty et al. (1970)9 quantified that most of the non-surgical root canal therapies which failed were due to inadequate apical seal. In order to ensure sufficient apical sealing, periapical surgery facilitates thorough root canal debridement & the introduction of a retrograde filling. For accomplishing the aforementioned objectives, the surgical technique entails a series of consecutive procedures: The RC system is sealed by performing (a) apicoectomy, (b) root-end cavity preparation, & (c) root-end filling with a bioactive & biocompatible material.8,10\n\nSince 98% of apical ramifications, lateral canals, & accessory canals are located in the apical region, the surgical treatment entails resection of 3mm of root.5 This is then followed by preparation of the retrograde cavity & restoration using root-end filling material. The apical resection should be performed in a plane perpendicular to the long axis of the tooth, resulting in a shallow bevel angle (0°–10°), which decreases the exposure of the dentinal tubules thereby reducing microleakage.11 The purpose of endodontic surgery is to provide a sufficient apical seal while also seemingly eliminating the persisting microbes in the apical third by restricting them source of nutrients.12\n\nThe retrograde filling material provides an apical seal to an otherwise unobturated root canal and perhaps even enhances the seal of already existing root canal filling material & is biocompatible with periapical tissues because majority of endodontic failures occurs from the outflow of irritants from pathologically involved canals.13 For periapical surgery to be successful, the quality of the apical seal established by the retrograde filling material is reflected to be vital.14\n\nThere have been numerous attempts to determine the best methods, tools, and equipment for apical resection and cavity preparation. Numerous investigations examined the possibility of crack formation affecting the apical seal when root end resections were carried out using a variety of equipments, including diamond burs, Lindemann burs, multipurpose burs, Erbium doped yttrium-aluminium-garnet (Er: YAG) laser, carbide fissure tungsten burs, and diamond-coated ultrasonic tips.15–17 Traditionally, high speed micro handpieces have been used for preparing the root end cavity using micro, rounded, and inverted conical burs. This method may result in a number of issues, including lingual perforation of the root and nonparallel cavity walls.18\n\nIn comparison to traditional rotary burs, ultrasonic tips offer a better solution and exhibit numerous benefits when used for retro cavity preparation. Because of the availability of tips in variety of shapes along with angulations that are carefully chosen in accordance with the root features and positioning, the introduction of ultrasonic tips significantly improved retrograde preparation.19 Additionally, usage of ultrasonic tips has added benefits, such as the ability to create conservative osteotomy site and to obtain root-end resection with negligibly small or non-existent bevel angles,20 thereby minimizing the number of exposed dentinal tubules and mitigating the risk of microleakage.10 Additionally, these techniques allow in removing isthmus tissue that is situated in between two canals in the same root9 and has a minimal potential for injuring the neighbouring soft tissues during surgery.11 The ultimate effects of ultrasonic preparation include smaller, cleaner, and more retentive root-end cavities that are also more centrally positioned and aligned along with the usual root canal’s orientation.21 Moreover, the possibility of microcracks formation apically after the retrograde cavity preparation using ultrasonic tips has been noted which could have an impact on the apical seal.22 Recently, some efforts have been undertaken to enhance the performance and usability of ultrasonic equipment. The development of new zirconium- and diamond-coated retro preparation tips signify a key subject in this arena.23\n\nThe advancement of dental materials is fundamental for raising diagnostic evidence in endodontic surgery. The incorporation of these new materials into clinical settings, as well as developments in equipment technology, procedures, and therapeutic expertise, can, in the author’s opinion, offer treatment possibilities which would have been otherwise unfathomable to foresee. The ideal retrograde filling material should be biocompatible, insoluble in oral tissue fluids, dimensionally stable, and unaffected by moisture. It should also adhere to and adapt to the dentinal walls of the retrograde cavity preparation, preventing the leakage of microbes & their byproducts into the periapical tissues.24,25 With the optimal healing response from periradicular tissues, Mineral Trioxide Aggregate (MTA) is an ideal retrograde filling material for periapical surgery.26 As a result of its capacity to facilitate the biomineralization process, MTA has been demonstrated to be bioactive.27\n\nBut nevertheless, MTA has received criticism for failing to meet the necessities of the ideal retrograde filling material in two areas: handling challenges and a delayed setting reaction,28 which may be a factor in leakage,29 surface disintegration, marginal adaptation loss, and continuity of the material, among other issues.30–32\n\nThe chemical constitution of MTA (Angelus), according to the manufacturer, includes tricalcium silicate, dicalcium silicate, tricalcium aluminate, calcium oxide, calcium tungstate and certain insoluble residues. According to Duarte et al. (2003),33 Pro-Root MTA is an analogous product to MTA-Angelus, which was developed in Brazil. MTA Angelus offers the same desired qualities as regular MTA but has a shorter setting time & is provided in containers that allow for added precise dispensing.\n\nBone cement made of polymethylmethacrylate (PMMA), one of the novel materials, may possess the qualities needed for a root-end filling. Orthopedic surgery has frequently used bone cement.34 In the 1970s, the US Food and Drug Administration authorized the usage of bone cement for prosthetic fixation in the hip and knee.34 Since then, bone cement has been used extensively to secure prostheses to living bone, but usage trends have varied.34 Bone cement offers a number of qualities that could make them an excellent choice for a repair material for a number of endodontic procedures. Good handling and working characteristics, superior strength and load bearing capacity,34,35 quicker setting times of about 15 minutes,36,37 and good operating properties. It possesses high tolerance to moisture in the environment and strong marginal adaptability to function as a retrograde filling material,36 and also has low cytotoxicity that is comparable to MTA.37 Furthermore, bone cement lacks bioactivity, a crucial characteristic of reparative materials. Extensive research has demonstrated that incorporating a bioactive substance into bone cement, such as amorphous calcium phosphate, hydroxyapatite, tetra calcium phosphate, or bioactive glass, can aid in the induction of bioactivity.38 The polymer/powder component of bone cement has been modified with MTA in this in vitro study in an attempt to create a bioactive bone cement while retaining all of the material’s beneficial characteristics and avoiding any potential pitfalls of MTA.\n\nIn the 1970s, glass ionomers cement was introduced. These cements have superior adhesive qualities because they chemically interact with dentin and are based on the reaction of ion-leachable, acid-soluble calcium fluoro aluminosilicate glass particles with polyalkenoic acid.39 They cause a severe inflammatory reaction, which subsides and is further replaced by bone.39 Novel formulations of Glass Ionomer Cement (GIC) have been introduced, which are original materials, such as “Zirconomer and zirconomer improved”40 that comprises of ceramic & zirconia reinforced GIC and helped combatting the drawbacks of silver amalgam along with tooth-coloured restorative materials exhibiting adequate antibacterial effect. The greater strength of amalgam is demonstrated with Zirconomer, and it preserves the ability of GIC to release fluoride as well as chemical adherence to the surrounding dentine, which can be investigated as well for retrograde filling material for its sealing ability.\n\nSealability of retrograde filling materials has been gauged via various approaches including dye penetration,41 bacterial leakage,42 fluid filtration,43 capillary flow porometery44 and glucose leakage.45 Optical magnification with or without the use of dyes,46 histological sections,46 stereomicroscopy,47 scanning electron microscopy (SEM),48 & fluorescence confocal microscopy49 are frequently used techniques for analysing the sealability of root-end materials of which the fluorescence confocal microscopy is the most reliable.\n\nThis is attributed to the fact that clear images with appropriate shapes of samples can be achieved by exclusion of the light which is not produced by focal plane of microscope. Additionally, images that are more contrasted and less hazy than those produced by conventional microscopes can be obtained.\n\nThis study aimed to compare the sealing ability of MTA, Bioactive bone cement, and Zirconomer as root-end filling materials by assessing the degree of microleakage through fluorescence confocal microscopy.\n\n\nMethods\n\nThe protocol of this research has been published and the same methodology is being followed in this study.50\n\nThe research was performed in the Department of Conservative Dentistry and Endodontics at Sharad Pawar Dental College Sawangi, Wardha.\n\nFor better understanding of the study, materials and methods have been discussed under the following headings: Study design, ethical approval, materials, and assessment of outcome parameters.\n\nThe study was in vitro experimental study. The present study was performed within a span of two (2020-2022) years.\n\nEthical approval was obtained from Institutional Ethical Committee DMIMS (deemed to be university) (Ref No – DMIMS (DU)/IEC/2020-21/9386). Written consent was taken from the participants for using the extracted teeth samples for the study. The teeth extracted were due to periodontal and orthodontic reasons.\n\nSource of specimens\n\nThirty-six extracted single-rooted maxillary incisors and canines were collected from the Department of Oral and Maxillofacial Surgery, Sharad Pawar Dental College, DMIMS, Wardha.\n\nThe recommendations and guidelines of the Occupational Safety and Health Administration (OSHA) and the Center for Disease Control and Prevention (CDC) were followed for collection, storage, sterilization, and handling of extracted teeth. Gloves, a mask, and safety glasses were always worn when handling teeth. Any visible blood and gross debris were removed from teeth samples. Wide opening plastic jars were utilised for the initial collection of teeth in distilled water. After being submerged in 10% formalin for 7 days, the teeth were removed from the solution & placed in separate jars with distilled water. The first collection jars, their lids, and the used gloves were disposed of in biohazard disposal containers. With cotton pliers as needed, the teeth were taken out of the jars and cleaned under running water.\n\nTeeth with fully developed and anatomically sound roots, devoid of caries & root canal fillings and with a Single patent canal.\n\nTeeth with fractured roots, open apices, calcified root canals, internal and external resorption, cracks/fractures on examination and multirooted teeth.\n\nFormula Using Mean difference\n\nPrimary Variable:- Sealing ability (Dye penetration)\n\nMean Difference between for sealing ability between Resin modified glass Ionomer and MTA: 1.60-0.40=1.20 (as per reference article48)\n\n(Estimated difference between two groups)\n\nPooled Standard Deviation Estimated= (0.516 + 0.966)/2 = 0.741\n\n(As per reference article)\n\nTotal samples required = 9 per group.\n\nTo remove the residual debris and tissues, teeth samples were submerged in 2.5% NaOCl solution for 10 minutes. Using hand scalers, mechanical removal of calculus from the surface of the root was done. Fresh distilled water was used to store the teeth until usage.\n\nFor this research, 36 maxillary anterior teeth were selected for sample preparation. Crowns of the samples were resected, and length of the teeth was standardized to 16 mm (from apex of root to coronal reference point) by sectioning with a double faced diamond disc perpendicular to the long axis of the root. Access opening was done with high-speed diamond burs along with copious water cooling. A 10 K file was inserted into the root canal until it reached the apical foramen. This measurement was then subtracted by 1 mm to get the working length, which was then radiographically confirmed. A reproducible glide path was established using a 15 K file. Biomechanical preparation of the canals was done with ProTaper Universal file system until F3 size using X-Smart Endomotor and handpiece. For maintaining the apical patency between rotary file insertions, size 10 K files were used. After change of each file, irrigation of root canals with 2 ml of 1% hypochlorite was carried out during instrumentation. For removal of the smear layer, root canals were dried & irrigated with 17% ethylenediaminetetraacetic acid with pH 7.2 for a duration of three minutes.\n\nThe canals were dried with paper points, and then sealed with gutta-percha using the single cone obturation technique. The prepared root canals of each tooth sample were fitted with F3 gutta-percha points and sealer (AH Plus) to provide sufficient tug back and successful obturation. The excess gutta-percha was sheared off. Temporary cement was used to seal the access cavities.\n\nThe specimens were then stored at 37° Celsius in 100% humidity for 48 hours thus allowing the sealer to completely set.\n\nIn all samples, root resection was performed by resecting 3 mm from the apical third of root with a “diamond disc” under continuous irrigation with normal saline solution at 90° angle with respect to the long axis of tooth.\n\nWith the usage of ultrasonic Satelac Retrotips (S12 90ND) in a Satelac NSK ultrasonic unit, preparation of retrograde cavities was carried out in all teeth samples measuring 3 mm in depth and 1mm in width. This class I cavity was made parallel to the long axis of the tooth using ultrasonic retro tip with light pressure in a brushing motion. A graduated periodontal probe (API) was utilized for gauging the size of the root end cavity.\n\nExcept for the resected apical portion, all specimens were painted with two layers of clear nail varnish for sealing all possible portals of communication with the root canals.\n\n36 teeth specimens were allotted in three groups (n = 12) at random & retrograde filling was carried out as follows (Table 1).\n\nGroup I: The retrocavities were filled with MTA\n\nTo create a thick putty-like consistency, MTA powder was mixed with liquid provided by the manufacturer in a “3:1 powder to liquid ratio”. MTA carrier was used to transfer MTA in order to fill the retro gaps. Following which it was condensed using “hand pluggers & burnished with a ball burnisher” to remove any material in excess thereby improving marginal adaptation.\n\nGroup II: The retrocavities were filled with Zirconomer (Conventional GIC, SHOFU, Japan)\n\nMixing of this material was done manually as per the manufacturer’s instructions on a paper pad at a “powder to liquid ratio of 2:1” before placing it into the retrograde cavity & is further condensed with micro hand pluggers.\n\nGroup III: The retrocavities were filled with Bioactive bone cement (Surgical Simplex P, Stryker)\n\nPreparation of Bioactive bone cement: “In preliminary investigations, the optimal amount of MTA and silane coupling agent that is needed to modify bone cement without altering its handling properties is identified”.51\n\nPowder modification: “Until all of the MTA particles were miscible in the polymer powder, 0.4 mg MTA was mixed with 0.6 mg bone cement”.\n\nLiquid modification: “One mL of monomer liquid was combined with one drop of the silane coupling agent (Monobond S). The generated powder and liquid of the modified bone cement were combined in a 2:1 ratio under ambient conditions at room temperature. Material was compressed into the specified cavity using micro hand pluggers.\n\nFollowing this the specimens were kept in incubator at 37° Celsius at 100% humidity for 24 hours.\n\nPreparation of reagent\n\nIn 10 ml of distilled water, 50 mg of acridine orange was made as a stock solution and stored in a fridge. 50 mL of distilled water was added to “1 mL of acridine orange stock solution and 0.5 mL of glacial acetic acid” to formulate a working solution.\n\nPrinciple of the fluorescent dye\n\nAcridine orange (AO), a metachromatic stain, intercalates with nucleic acid, altering the dye’s optical properties and causing it to fluoresce bright orange red under ultraviolet light. All cells containing nucleic acids will fluoresce orange. Against a green-fluorescing or dark background, the empty voids and spaces will fluoresce bright orange. It is an intercalating dye capable of penetrating both living and dead cells. To generate green fluorescence, AO will stain all nucleated cells.\n\nIts maximum excitation/emission pair is 502 nm/525 nm in the DNA bound state & 460 nm/650 nm in the RNA bound state. It can also cross acidic compartments such as lysosomes, where the cationic dye is protonated. Acridine Orange is excited by light in the blue spectrum in this acidic environment, whereas emission is strongest in the orange region.\n\nThe samples were submerged in Acridine orange dye 24 hours. Following this, the samples were retrieved from the dye reservoir and rinsed using running water for 15 minutes to remove any excess dye.\n\nEach root was sectioned longitudinally into two halves with the usage of a diamond disc. Using a Confocal Fluorescent microscope (ZEISS LSM 800) at 10x magnification and a green filter, each sliced specimen was examined for marginal adaptation of the retrograde filling material to the walls of the prepared cavity, the presence and absence of gaps/voids, & the extent of penetration of the dye (wavelength 546 nm). Digimizer Image Analysis Software 6.3.0 was employed in assessing the amount of penetration of dye in three groups.\n\nScoring criteria for dye penetration for checking sealing ability.49\n\n0-No dye penetration; 1 - Dye penetration into apical one third of retrograde filling material; 2 - Dye penetration into apical middle third of retrograde filling material; 3 - Dye penetration into full length of retrograde filling material; 4 - Dye penetration beyond retrograde filling material”.\n\nFor this study, descriptive and analytical statistics were used. The Shapiro-Wilk test was used to determine whether the data were normal.52 The data were analysed using parametric tests because they had a normal distribution.\n\nThe mean differences between the groups were examined using the one-way analysis of variance (ANOVA) test. Tukey’s HSD test was used in the post hoc analysis.\n\nThe software used was SPSS (Statistical Package for Social Sciences) Version 22.0 (IBM Corporation, Chicago, USA).\n\n\nResults\n\nGroup II (Zirconomer group) had the maximum mean (4.1 ± 0.57) followed by Group I (MTA group) (1.74 ± 0.65) and lastly Group III (Bioactive Bone Cement group) (1.4 ± 0.51) demonstrating highest microleakage due to increased dye penetration in Group II Zirconomer and least microleakage in Group III bioactive bone cement, thus indicative of highest sealing ability of Bioactive bone cement and least sealing ability with Zirconomer (Figures 1-3). There was statistically discernible dissimilarity in the amount of sealing ability admist the three groups of retrograde filling material with the p value of <0.001 (Table 2).\n\nOn intergroup comparison (Table 3), it was seen that the mean difference observed between Group I (MTA) and Group II (Zirconomer) of 1.8 is a statistically significant difference (i.e. p = 0.02) followed by the mean difference observed between Group I (MTA) and Group III (Bioactive Bone cement) of 1.1 which is a statistically significant difference (i.e. p = 0.03). Although, the Mean difference observed between Group III (Bioactive Bone cement) and Group II (Zirocnomer) of 3.3 is a highly statistically significant difference (i.e. p = 0.001).\n\n\nDiscussion\n\nMost endodontic tribulations can be resolved by utilising a standard conventional therapeutic approach.1 The fundamental goal of an endodontic therapy is to establish a three-dimensional hermetic seal between the periodontium and the root canal. For prevention of leakage and the entry of oral fluids along with organisms that could contribute to reinfection of the tooth, this seal should be established apically as well as coronally.53 It is essential to reduce the microbial load in these conditions to address the main etiological cause.54 Another cause of endodontic failure is complicated root morphology with an apical delta and lateral canals. Most of the endodontic failures arise as a consequence of irritants and bacteria seeping into the periapical region from infected root canals.1 According to dental estimates, 10 to 38% of root canal reintervention attempts have been shown to fail.55 The presence of microorganisms aggregated as biofilm in the root canal is a primary factor cited in those failed cases.54 Reduced microbial load is achieved in part by mechanically debriding out the length and diameter of the infected root canal.56 The retrograde filling serves as a second line of defense against persistent bacteria entering periapical tissues, hence the chosen material’s ability for sealing is crucial. Additionally, under specific circumstances, such as those that involve significant periapical lesions, instruments that are separated in the canals, apical variations, inadequate obturations, canal calcification, and dilacerated roots, etc., necessitates surgical intermediation as cleaning and shaping of the apical third becomes very challenging.57–59\n\nIntroducing the paradigm shift to endodontic surgery which is an approach employed in cases of periapical pathology that remains persistent or resistant to healing following nonsurgical retreatment60 and is cantered on the usage of equipment, materials, and techniques that integrate biological principles with tested clinical outcomes for healing of endodontic lesions.59\n\nResection of the root end, retrograde cavity preparation, and root-end filling are the measures taken to accomplish the goal of securing the root canal at the cut root end against microbes.10 Periradicular surgery followed by retrograde filling not only removes the infected periapical tissues & root apex, but also reseals the root canal. Creating a hermetic seal at the root apex with a root-end filling will inhibit bacteria and their byproducts to enter or exit the canal.1\n\nA strong apical seal established with a retrograde filling material that is properly adapted is essential for the success of periradicular surgery. Improper marginal seal of the root ends, characterised by insufficient contact between the restorative material & the tooth surface, is typically the cause of apical surgery failure.61 Interstitial fluid may drive bacteria into the root canal, therefore apical closure should limit such leakage into the canal. Retrograde preparations and apicectomy increases the likelihood of leakage from the residual root, which emphasizes the need for retrofilling.62 So, a good apical seal must be provided by a retrograde filling substance that adheres well to the canal wall. Additionally, it should be biocompatible as well as capable of exhibiting osteoinductive/osteoconductive properties that will hasten healing in the periapical area and lower the risk of failures. The purpose of introduction of a retrograde filling is to create a fluid-tight apical seal, which prevents the leaking of residual irritants from the canal into the perirapical tissue & vice versa.63 Therefore, in the present study, the apical sealing ability of three root end filling materials to the root dentine was assessed.\n\nGilheany et al. (1994)11 stated that root resection at angles other than 90 degree and <3 mm from the apex exposes a greater number of dentinal tubules and leads to ineffective removal of root ramifications. Therefore, in the present investigation, root end resections were performed perpendicular to the long axis of the tooth and 3 mm from the apex. According to a research by Mjör et al. (2001),64 reduction in 98% of apical ramifications and 93% of lateral canals while decreasing the amount of exposed dentinal tubules was achieved by removing at least 3 mm of the root end.\n\nRoot-end resection can be carried out in three different planes to the tooth’s long axis: 30°, 45°, and 90°. Large number of exposed dentinal tubules, increased mechanical loads, and loss of dentine-cementum bone that arise from 30° & 45° resection angles impede healing after periapical surgery.65 The most preferred of them is 90° because it has the least impact on adaptability of retrograde material and reduces the possibility of leakage via the dentinal tubules that have been severed.\n\nThe performance of retrograde filling material and the outcome of surgical endodontic therapy are both influenced by a diverse range of variables. The strategy employed to prepare the root end cavity is one of them. Burs were traditionally employed as the tools to prepare the root end cavities. In comparison to cavities made with diamond-coated ultrasonic tips, it has been found that cavities prepared utilizing rotary instruments resulted in increased production of debris and smear layer.66 These remnants are permeable67 and permit microleakage, which prevents filling material from making complete contact with cavity walls.\n\nErgo, in the present era of dentistry, piezoelectric devices demonstrate a revolutionary method to accomplish the same goal while conserving soft tissue in the surgical field and selectively cutting mineralized tissue.68 With walls that are “parallel to & coincide with the anatomic outline of the root canal space”, the optimal root-end preparation should be at least 3mm into the root dentine.59 The apical cavities conform considerably more easily, safely, and precisely; they are smaller and more centrally positioned, which lowers the possibility of root perforation; and finally, improved cavity disinfection, which lowers the amount of dentinal remnants.69–71 Diamond-coated tips aid in preventing the propagation of dentinal cracks.72 Therefore, in the current investigation, S12 90ND Retrotips (NSK, Satelac) with diamond coating were used to prepare retrograde cavities at the optimal depth of 3 mm.\n\nDifferent techniques have been devised to evaluate the sealing ability of root canal filling materials. These techniques are typically based on the same concept, which is to analyse the tracer’s capacity to penetrate along the obturated canal of an extracted tooth. For assessment of microleakage and subsequently the sealing ability, number of tracers including dye, radioisotope, bacteria and their products have been employed.73 Dye penetration technique is most often utilised for microleakage research since the dyes are affordable, secure, widely accessible, reasonably simple to store & use, and most significantly, their penetration can be analysed quantitatively.74\n\nIndicators of potential leakage, such as dye penetration, should be taken into consideration. This is due to the fact that a filler material that can withstand the entry of small compounds like dyes may also be able to withstand the penetration of larger bacteria and their byproducts, according to Torabinejad et al. (1994).75 As a result, the dye penetration method was used in this investigation since it provides reliable information.\n\nFor dye leakage experiments, several different dyes are employed, such as India Ink, erythrosine B solution, aqueous fuchsin solution, fluorescent solution, silver nitrate, Methylene blue, rhodamine B, acridine orange, and others.76 Dye leakage studies depend on a number of factors, including the length of time the specimens are immersed in the marker, the time of immersion, whether negative pressure (vacuum) is used or not to release air trapped within filling gaps, whether the specimens are immersed completely in the dye, or only partially, the type of seal used, the quantity of specimens, the size of the marker, the position of the specimens during immersion, and, most importantly, the kind of material used.77 Methylene blue is frequently utilised in various studies of dye leakage. However, it has drawbacks, such as discoloration caused when methylene blue comes in proximity with an alkaline filling material. This occurs as a consequence of the ‘hydrolysis of methylene blue’, which produces a clear molecule named thioxine.78 MTA exhibits a high pH (12.5) & contains calcium oxide, which when in contact with water forms calcium hydroxide and is stained by methylene blue. Therefore, dye solutions that do not adversely affect the alkalinity of their marking capability are employed in evaluating the sealing capability of MTA.78\n\nIn the present investigation, acridine orange dye was utilised in place of methylene blue since it has additional benefits, such as smaller particle size, greater penetration, water solubility, diffusability, and hard tissue non-reactivity. The water-soluble fluorescent dye acridine orange, is a metachromatic vital stain, is not harmful to the substrate or material in touch, is readily identifiable even at low concentrations, flows freely along the interface, has low toxicity, and is stable in an aqueous environment. It diffuses more effectively on human dentin in comparison to methylene blue. When excited by blue light, the fluorescent dye helps in differential staining where the voids or spaces fluoresce bright orange against a green-fluorescing dark background.\n\nA linear or spectrophotometric measurement of dye penetration is achievable. However, in a study comparing longitudinal splitting, cross sectioning, and cleaning of the specimens, it was shown that longitudinally split samples exhibited improved dye penetration.77 Hence, after longitudinally splitting the samples with a diamond disc, the linear measurement of dye penetration is assessed in the current investigation. This study employed Confocal Fluorescent Microscopy (ZEISS LSM 800) at 10× magnification in conjunction with a green filter (wavelength 546 nm) to assess the sealing ability. This method provides advantages over other traditional techniques such as “scanning electron microscopy or transmission electron microscopy. The advantages of this technique include the elimination of out-of-focus blur in images as well as the ability to create three-dimensional images that disclose more precise information than two-dimensional images.79 The confocal fluorescent microscope does not require a specific sectioning method, hence the incidence of artifacts is lower when compared to a scanning electron microscope.80\n\nThe retrograde filling material groups were as follows: Group I (MTA), Group II (Zirconomer) and Group III (Bioactive Bone cement). The sealing ability values were as given below: Group I (MTA): 2.3 ± 0.65; Group II (Zirconomer): 4.1 ± 0.57 and Group III (Bioactive Bone cement): 1.4 ± 0.51. Group II (Zirconomer group) had the highest mean (4.1 ± 0.57) followed by Group I (MTA group) (2.3 ± 0.65) and lastly Group III (Bioactive Bone cement group) (1.4 ± 0.51).\n\nWhen Group I (MTA) (2.3 ± 0.65) was compared with Group II (Zirconomer) (4.1 ± 0.57), the difference of 1.8 ± 0.80 was found. There was statistically discernible dissimilarity found amid Group I and II with the p value <0.001.\n\nThe study’s findings revealed that every material examined demonstrated microleakage. The highest microleakage values were produced by Group II Zirconomer which is attributed to the high solubility possessed by this material. Compromised sealing produced by Zirconomer could be described by the presence of zirconia ceramic filler particles that make up Zirconomer’s chemical makeup. It is probable that the zirconia fillers would interfere with the chelating process between the calcium ions (Ca2+) in tooth apatite and the carboxylic group (-COOH) of polyacrylic acid.81 This was in congruence with a study done by Asafarlal S et al. (2017)82 which showed inferior adaptation of Zirconomer at the tooth restoration interface owing to larger size of zirconia filler particles.\n\nMTA showed better sealing ability when compared to Group II zirconomer. Good sealing ability & marginal adaptation of MTA is caused by the deposition of hydroxyapatite crystals & the formation of a hybrid layer between dentine and calcium silicate–based materials. The formed hydroxyapatite crystals gradually grow and nucleate in order to fill the microscopic gaps between calcium silicate based cements & the dentinal wall.83 The sealing ability of MTA may have improved as a result of Ca ions bioavailability inside these materials which stimulates the production of bone-associated proteins through Ca channels and large amounts of Ca ions could eventually activate adenosine triphosphate, which is important in the mineralization process.84 Additionally, the good sealing ability of MTA may be due to dihydrogen monoxide absorption during the powder’s hydration, which causes expansion during setting. The positioning of the cementum and periodontal ligament strands adjacent to MTA is another intriguing feature.85\n\nDue to the MTA Angelus’s reduced particle size and higher specific surface area of its particles, the wetting volume, water binding capacity, and hydration rate are all increased. Additionally, because MTA is hydrophilic and expands during setting expansion when cured in a moist environment, moisture does not alter its setting or its qualities during periapical surgery.86 This finding is reinforced by a study by Xavier et al. (2005),41 which found that MTA Angelus had better marginal adaptability and sealing capability than super EBA and Vitremer.\n\nConversely, studies by Ahirwar et al. (2020),87 Bolbolian et al. (2020)88 and Fatinder Jeet Singh et al. (2022)89 were in contradiction to this present study as they concluded that the production of tag-like structures made of crystalline deposits rich in calcium or phosphate at the junction of tooth and retrograde filling materials and the shorter setting period, i.e., 12 minutes would be the reason for the greatest sealing ability of Biodentine over MTA.\n\nAn invitro study by Dhivya et al. (2022)90 was in contradiction to the present study as it concluded that Zirconomer had enhanced sealing ability due to “incorporation of zirconia fillers which is an uneven compound and hence deviations in its phase from monoclinic to tetragonal and then to cubic and vice versa lead to increase in volume thereby counteracting the volumetric shrinkage expressed during polymerization”.91\n\nWhen Group I (MTA) (2.3 ± 0.65) was compared with Group III (Bioactive Bone cement group) (1.4 ± 0.51), the difference of 1.1 ± 0.14 was found. There was statistically discernible dissimilarity seen amid Group I and III with the p value <0.001.\n\nThe current study revealed that Group I MTA had inferior sealing ability when compared to Bioactive bone cement. This can be attributed to MTA’s extended maturation phase, which is linked to the creation of a passivating trisulfate coating over hydrated crystals, simulating a terrible clinical condition” in congruence to a study conducted by Chaudhari et al. (2022).92\n\nIn comparison to MTA & Zirconomer, the acquired results showed that group III bioactive bone cement had superior adaptation to the root dentine in the longitudinal sections. These findings are consistent with those of the earlier dye diffusion investigation conducted by High and Russell et al. (1989),93 who demonstrated a successful marginal seal when bone cements (CMW and Palacos bone cement with gentamicin) were used as retrograde filling materials. Additionally, Holt and Dumsha’s (2000)94 leakage research demonstrated that Simplex P bone cement offered a reliable retrofill seal. The results of earlier studies by Charnley et al. (1970),95 who used a fluid displacement model and observed that the volume of cement increases to a maximum during polymerization before shrinking slightly, but not to its initial volume, may help to explain how bone cement is able to adhere to the dentin wall properly despite the well-known polymerization shrinkage of acrylics.\n\nAccording to Miyazaki et al.96 (2003), Si-OH groups produced on the material’s surface can act as a catalyst to promote the formation of apatite by releasing Ca2 + ions into the body fluid. The addition of silane to the liquid component results in the formation of a Si-OH group due to the hydrolysis of alkoxysilane following exposure to the body environment, which causes heterogeneous HA nucleation. In contrast, the addition of MTA powder to bone cement results in the formation of Ca ions. The addition of silane to bone cement improves its mechanical properties because chemical bonding can be achieved with PMMA. Additionally, any PMMA polymerization shrinkage is offset by the slight MTA expansion that occurs during setting, increasing the sealing and strength of modified bone cement. This could account for the better apical sealing capabilities of Bioactive bone cement.\n\nAdditionally, the exothermic reaction released during the setting of bone cement did not seem to have any negative effects due to very small amount needed in root end fillings as opposed to quantities required in orthopaedics.97 Less quantities needed would result in a considerably lower exothermic reaction & a reduced quantity of free monomer as stated by Badr et al (2010).25 Bone cement could thus be used in both dentistry and medicinal applications. There are other bone cements available with antibiotics incorporated.\n\nAn in vitro study performed by Chordiya et al. (2022)98 & Akbulut et al. (2018)99 are in contradiction to the present study. They claimed that the cell reduction caused by the bone cement was comparable to that caused by the Micro-Mega mineral trioxide aggregate (MM-MTA), Smart Dentin Replacement (SDR) and ProRoot MTA (PMTA). These results are in accordance with those of Badr et al. (2010),25 who observed that PMMA bone cement & MTA have similar cytotoxic effects on fibroblasts. Although, the poor biocompatibility of PMMA bone cement may be caused by residual methylmethacrylate monomer.100\n\nWhen Group II (Zirconomer) (4.1 ± 0.57) was compared with Group III (Bioactive Bone cement group) (1.4 ± 0.51), the difference of 3.3 ± 0.06 was found. There was statistically discernible dissimilarity found amid Group II and III with the p value <0.001.\n\nThis study proved bioactive bone cement to have highest sealing ability. This can be attributed to its excellent interlocking property. With superior handling capabilities that make it possible to mould it into a dough form and place it in the root end cavity area, the bone cement could also endure a damp environment and is unaffected by blood contamination. This is in congruence to a study performed by Ann Saji et al. (2022).101\n\n\nConclusions\n\nWithin the limitations of this investigation, it can be concluded that the seal created by bioactive bone cement in retrograde filling was superior to Ziconomer and comparable to MTA. It offered more potent operating qualities that could outweigh any prospective MTA drawbacks. In light of this, bioactive bone cement appears to be an advantageous and promising material for retrograde filling. It also has the potential to be employed as a repair material in a variety of other clinical settings, including furcation repair and resorptive defects.\n\nAdvancements in dental materials will help broaden the perspective to have a holistic approach with better marginal adaptability and sealing ability in retrograde preparations preventing microleakage. More recent materials for root-end filling can be evaluated and compared. The teeth with multiple canals can be evaluated that might change the outcome of the study. Further in vivo studies should be carried out to provide more accurate results.", "appendix": "Data availability\n\nZenodo: Comparison of Root End Sealing Ability of three Retrograde Filling Materials in Teeth with Root Apices Resected at 900 using dye penetration method under fluorescent microscope. https://doi.org/10.5281/zenodo.7857676.\n\nThis project contains the following underlying data:\n\n- master chartt csv.csv\n\nZenodo: Strobe checklist for the study. https://doi.org/10.5281/zenodo.7861151.\n\nThis project contains the following extended data:\n\n- STROBE-cross-sectional_checklist.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nChaurasiya S, Yadav G, Tripathi AM, et al.: Endodontic failures and its management: a review. Int. J. Oral Health Med. Res. 2016 Feb; 2(5): 144–148.\n\nChugal N, Mallya SM, Kahler B, et al.: Endodontic Treatment Outcomes. Dent. Clin. N. 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Publisher Full Text" }	[ { "id": "235065", "date": "25 Jan 2024", "name": "Amjad Abu Hasna", "expertise": [ "Reviewer Expertise Endodontics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, I express my gratitude for the opportunity to provide feedback on your work and commend your dedication to advancing the field. The introduction commendably delves into the challenges of endodontic therapy, the necessity for surgical intervention, and the considerations in choosing retrograde filling materials. It effectively sets the groundwork for the subsequent exploration of the comparative study involving MTA, Bioactive bone cement, and Zirconomer as root-end filling materials. However, there appears to be a confusion between pro-root MTA (Dentsply) and MTA (Angelus) by the author, warranting closer attention. The methods section is well-structured, offering a clear delineation of each step in the experimental process, laying a solid foundation for the comparative study of retrograde filling materials. Nonetheless, my major concern centers around Group 3, where retrocavities were filled with Bioactive bone cement (Surgical Simplex P, Stryker), featuring both powder and liquid modifications. Disagreeing with the sole classification of this group as the bioactive bone cement group, I note that it comprises a mixture of bioactive bone cement, MTA, and monomer liquid. I advocate for increased attention to this particular aspect of the study. In my view, the study should have encompassed the following groups:\nMTA Glass ionomer cement (GIC) Bioactive bone cement (BBC) MTA + GIC MTA + BBC GIC + BBC MTA + GIC + BBC\nAdditionally, I would appreciate clarification on the main differences between the current study and the previously published one at Chaudari et al.,(2021)[Ref-1] Finally, the conclusion should mention the mixture of bioactive bone cement, MTA, and monomer liquid as a unique group (group 3), not solely the bioactive bone cement Sincerely,\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?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "248290", "date": "16 May 2024", "name": "Muhammad Amber Fareed", "expertise": [ "Reviewer Expertise Dental biomaterials", "restorative dentistry", "prosthodontics", "dental education", "mechanical properties" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI've reviewed this research article by Dr. Payal Chaudhari et al which aims to evaluate and compare the sealing ability of Mineral Trioxide Aggregate (MTA), Zirconomer, and bioactive bone cement using dye penetration method under a fluorescent microscope. Please find comments bellow: 1. The clarification on the unique composition of Group 3, comprising a mixture of bioactive bone cement, MTA, and monomer liquid, is crucial for the accurate interpretation of results and should be highlighted in the conclusions. 2. Please explain and discuss on how does the use of a Confocal Fluorescent microscope enhance the visualization of fluorescently labeled samples compared to conventional microscopy techniques? Moreover, how does the exclusion of out-of-focus light in Confocal Fluorescent microscopy contribute to the accuracy and reliability of assessing the sealing ability of retrograde filling materials? 3. Figures 1-3 presented in the study provide valuable visual representations of the dye penetration and sealing ability of the retrograde filling materials. However, a critical comment would be that the figures could be enhanced by including scale bars for reference, labeling specific regions of interest, and providing clear legends to aid in the interpretation of the results. 4. It would be beneficial to discuss how the results contribute to the selection of optimal materials for endodontic procedures and highlight any potential limitations or areas for further research. Providing a more robust discussion on the practical implications of the study results would enhance the overall impact and relevance of the conclusion. 5. Regarding the conclusion, while the study effectively compares the sealing ability of the retrograde filling materials using the dye penetration method and Confocal Fluorescent microscopy, the conclusion could be strengthened by emphasizing the clinical implications of the findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1049
https://f1000research.com/articles/12-326/v1	23 Mar 23	{ "type": "Research Article", "title": "Optimal control model of human-to-human transmission of monkeypox virus", "authors": [ "Joseph Ackora-Prah", "Samuel Okyere", "Ebenezer Bonyah", "Atinuke Olusola Adebanji", "Yaw Boateng", "Joseph Ackora-Prah", "Ebenezer Bonyah", "Atinuke Olusola Adebanji", "Yaw Boateng" ], "abstract": "Background: The number of monkeypox cases is rising globally, but it’s unclear how many instances there will be in the near future. The disease has been one of the major problems for sub-Saharan Africans in the past few years. Methods: A deterministic mathematical model incorporating optimal controls has been developed in this research to investigate the transmission of the monkeypox virus. The model’s fundamental properties such as positivity and boundedness of solution, and basic reproduction number have been examined. In order to assess the efficacy of two preventative control strategies—public education and vaccination—optimal controls were included in the model and Pontragyin’s maximum principle used to characterized the model. Results: Public education was found to have less of an effect on those who were vulnerable than vaccine control. However, both approaches were successful in reducing the number of people who were exposed to the illness and reducing the number of fatalities. Additionally, vaccination increases a person’s immunity, which speeds up their recovery. Conclusions: A deterministic classical model incorporating optimal controls was proposed to study the monkeypox virus dynamics in a population. The disease is not endemic, which is explained by the model’s basic reproduction number, which was less than unity. Based on the findings of this study, we advise vaccine control plan as the most effective preventative measure.", "keywords": [ "Monkeypox", "Equilibrium points", "Sensitivity analysis", "basic reproduction number", "optimal control." ], "content": "Introduction\n\nThe rare disease monkeypox is brought on by the monkeypox virus according to the Centers for Disease Control and Prevention (CDC). The variola virus, which causes smallpox, is related to the virus that causes monkeypox CDC. Although less severe, monkeypox symptoms are similar to those of smallpox, but it seldom causes fatalities as detailed by the World Health Organization (WHO). Despite being known as “monkeypox,” the disease’s genesis remains a mystery according to the CDC. However, the virus might spread from non-human animals like monkeys to humans.\n\nA human case of monkeypox was reported by the WHO for the first time in 1970. In 1970, while smallpox eradication initiatives were intensifying, a nine-month-old infant in the Democratic Republic of the Congo became the first human to be identified as having monkeypox. In a number of central and western African countries, incidences of monkeypox were known from 1970 before the outbreak in 2022 as reported by WHO. Until recently, almost all cases of monkeypox in people living outside of Africa were attributed to either imported animals or international travel to places where the disease regularly occurs (CDC). These occurrences have been recorded over numerous continents (CDC). Direct contact with animals or other people who have the monkeypox virus can cause human infection.1\n\nThe number of cases of monkeypox is rising globally as reported by (WHO), but it’s unclear how many instances there will be in the near future. At least 110 countries around the globe have already been identified to have the virus (CDC). Earlier in 2022, the disease started spreading in non-endemic regions at an unprecedented rate.2 The Centers for Disease Control and Prevention (CDC) data reveals that more than 84,716 cases have been documented globally in the 110 countries, including 83,511 in 103 nations that have never before reported instances of monkeypox (CDC). The US, 29,980 cases is the greatest number of confirmed cases followed by Brazil with 10,625.3 As of January 16th, 2023, the Ghana Health Service (GHS) had identified 121 cases of the disease with 4 recorded deaths (CDC).\n\nHistorically, the disease has received little attention, which has made it difficult to understand how it spreads. Mathematical models has been used to study the dynamics of diseases in the past.4–8 The transmission of the Cholera-COVID-19 co-infection in Yemen was studied using an ordinary differential equation model, according to Hezam et al.4 To reduce the number of cases in the community, they built the model with the best control strategies, including social isolation, lockdown, the number of tests, and the quantity of chlorine water tablets. To analyze the transmission of cholera, Tessema et al.5 devised a mathematical model that included drug resistance and immunization of newborns. The model included the best control strategies, including prevention, instruction, and treatment of sick people. In order to better understand the COVID-19 disease’s transmission dynamics in Senegal, Perkins and España7 proposed a mathematical model that included both of the disease’s primary therapeutic approaches: immunization of susceptible individuals and recovery/treatment of infected individuals. A model was proposed by Deressa and Duressa6 to investigate COVID-19 transmission in Ethiopia. The model was altered to include the best preventative methods, including treating hospitalized cases and promoting public health awareness. The authors in Ref. 8 proposed a model to study the co-dynamics of diabetes and COVID-19 in Ghana.\n\nA few researchers including Okyere and Ackora-Prah9 have tried to analyze the monkeypox virus mathematically.9–14 A mathematical model was developed by Somma et al.10 to investigate the spread of monkeypox throughout the human and rodent populations. According to the model proposed in Ref. 10, the human population is provided with a quarantine class and a public awareness campaign to prevent the spread of the disease. In order to restrict the spread of the monkeypox virus infection among humans and rodents, Usman and Adamu11 devised a mathematical model that incorporates both vaccination and treatment interventions for the human population. Peter et al.12 created and investigated a classical model for the spread of the monkeypox virus from people to rodents in Nigeria. Peter et al.13 developed both traditional and Caputo-Fabrizio fractional-order derivative models to investigate the disease’s spread in Nigeria. To examine the transmission of the monkeypox and smallpox viruses in the Democratic Republic of the Congo, Grant et al.14 devised a model. In our previous work in Ref. 9, a fractional-order model was formulated to study the disease dynamics in Ghana, taking into consideration individuals immune to the virus, however the model proposed in Ref. 9 fails to suggest preventive mechanisms to curtail the spread of the disease.\n\nAfter looking over a number of academic publications, we discovered that very few research on the monkeypox virus and its mechanisms of transmission considered the virus’s capacity to propagate from person to person and that no previous research has taken into account the implications of individuals who have been deceased. Although there is evidence of virus transmission from human to human,9,15,16 which is the main mechanism of illness transmission in Ghana, current models of the disease concentrate on rodent-to-human transmission. This study extends our previous work done by Okyere and Ackora-Prah in Ref. 9 by introducing a compartment for the deceased individuals and incorporating optimal preventive methods in the proposed model.\n\nThe remainder of the paper is divided into the following sections: We extend a previous model proposed in Ref. 9 by two authors of this study and investigate the classical model in the methods section. We identify the model’s qualitative characteristics such as the positivity and boundedness of solutions, equilibrium points and basic reproduction number. We finally include optimal controls in the new model that has been developed in the optimal control section. We numerically solve the optimal control model.\n\n\nMethods\n\nWe propose a deterministic model to study the viral transmittal of monkeypox by examining our previous fractional monkeypox model proposed in Ref. 9. Our previous model fails to suggest preventive mechanisms for curtailing the spread of the disease. In order to avoid the spread of the disease, this conventional model instead offers preventative control measures thereby excluding the immune compartment proposed in the previous model and introducing a compartment for individuals deceased of monkeypox virus. It was relevant to exclude the immune compartment as the recovered compartment serves it purposes in this new model. Only human-to-human virus transmission was taken into account in Ref. 9 and in this current model. The population is segmented into six compartments, including: Susceptible HS, exposed HE, infected HI, hospitalised HQ, recovered HR and individuals deceased from the disease D. We let N denotes the total population. Therefore, Nt=HS+HE+HI+HQ+HR+D.\n\nThe rate of new recruits into the vulnerable class is ζ, whereas the rate of natural mortality is ζ1. Those who are vulnerable have a ζ2 risk of contracting the sickness from those in HI. The recovery rates of a hospitalized person and infected patients are shown by the values ζ6 and ζ3, respectively. The infectious rate and disease-induced death rate are denoted by ε and ζ5, respectively. ζ4 gives the rate of progression from the infected segment to the hospitalized segment. Figure 1 depicts the model’s flowchart.\n\nOur modified model is described by the following ordinary differential equations.\n\nWith initial conditions HS0=HSo,HE0=HEo,HI0=HIo,HQ0=HQo,HR0=HRo,D0=Do\n\nIn order to prove that system (1) is mathematically and epidemiologically well-posed in a workable region Φ, we present the results shown below.\n\nThere is a domain 0 ≤ Φ < ∞ that contains and bounds the solution set (HS, HE, HI, HQ, HR, D) of the system (1).8\n\nConsidering the solution set HSHEHIHQHRD with positive initial conditions\n\nWe let, Nt=HSt+HEt+HIt+HQt+HRt+Dt, then\n\nThe results of solving the differential inequalities are\n\nTaking the limits as t→∞ gives N′t≤ζζ1\n\nTo put it another way, every solution is constrained to the Φ zone of feasibility. The system (1) solutions are now shown to be nonnegative in Φ.\n\nFor any t>0 in the domain ϕ, the initial states must not be negative in order to remain such.8\n\nWe construct our argument based on the concepts from.13 Clearly, it is simple to understand that HS>0 if not for the condition that t∗>0 such that HSt∗>0 and HS′ ≤0 for all 0<t≤t∗\n\nBy integrating from 0 to t∗, we get the following:\n\nThe result is obtained by multiplying through by e−ζ2HIN+ζ1t,\n\nHSt∗=HS0e−ζ2HIN+ζ1t+e−ζ2HIN+ζ1t∫0t∗ζeζ2HIN+τdτ>0, which contradicts HSt∗=0.\n\nThe following conclusions can be drawn from the remaining three (3) equations in system (1).\n\nWhich goes against the logic HEt∗=HIt∗=HQt∗=HRt∗)=0. This concludes the proof.\n\nThe monkeypox-free and endemic equilibrium point given by the model in Ref. 9 are\n\nThe endemic equilibrium point, ee=HS∗HE∗HI∗HQ∗HR∗ is\n\nThe basic reproduction number R0 is the key element that determines whether or not a disease will proliferate throughout a community. The R0 as computed in Ref. 9, is\n\n\nOptimal control\n\nIn this section, we introduce the control ut into the system (1), where control u1t stands for public education on monkeypox and control u2t stands for vaccination/immunization of those who are vulnerable. By incorporating time-dependent controls onto system (1), we are able to produce\n\nPontragyin’s maximal principles are used to examine the behaviour of the system (6). The objective function for a fixed time tf is given by\n\nwhere tf is the control’s final time and the parameters b1, b2, b3, b4, and c1 indicate the balancing cost factors for the two controls, respectively. Therefore, we investigate to find the best controls u1∗,u2∗ so that\n\nThe Pontryagin maximum concept, which is described in Ref. 18, was utilized to determine the prerequisite for the ideal control. This idea transforms the (6), (7) and (8) into a problem of minimizing a Hamiltonian, H, which is described by\n\nwhere the adjoint or costate variables are represented by ΛHS,ΛHE,ΛHI,ΛHQ and ΛHR represents the adjoint variables or costate variables. By considering the correct partial derivatives of system (9) with regard to the related state variables, the system of equations is generated.\n\nGiven an optimal control u1∗u2∗ and corresponding solution HS∗,HE∗,HI∗,HQ∗,HR∗ that minimizes Ju1u2 over U, there exist adjoint variables ΛHS,ΛHE,ΛHI,ΛHQ,ΛHR, satisfying\n\nThe right hand side differentiation of the system (9) calculated at the optimal control is taken into consideration to produce the differential equation described by the adjoint variables. The obtained adjoint equations are presented as\n\nObtaining the solution for u1∗ and u2∗ subject to the constraints gives,\n\nThis gives\n\nIn this part, we numerically analyze the behavior of the (6) optimum control model and display the effects of varying the controls u1 and u2. In the first instance, using the parameter values listed in Table 1. The outcome of using the parameter values from Table 1 to evaluate the R0 is R0=0.1940, proving that the illness is not endemic to the country. We begin our simulation on May 24, 2022, the day the first five cases of monkeypox are confirmed by the Ghana Health Service. According to the Ghana Statistical Service, in the 2021 population and housing census, there are 30.8 million people living in Ghana. Our model is consistent and includes the entire population under study (N = 30.8 million). We begin with HS=30799995,HE=0,HI=5,HQ=0,and HR=0,D=0. The outcomes of the MATLAB (version R2016a) (RRID:SCR_001622)19–21 (Python (RRID:SCR_008394) could potentially be used as an open alternative using similar methods described in this manuscript) ODE45 using the parameter values and the initial conditions are shown in Figures 2–19.\n\nSetting control u2=0 and varying control u1, the results obtained are displayed in Figures 2–7. This strategy was ineffective on the susceptible, infected and hospitalised compartments (see Figures 2, 4 and 5 respectively). However, the strategy effectively reduced the number of individuals exposed to the virus (see Figure 3). There were a marginal reduction in the number of recoveries and individuals deceased at the end of the period (see Figures 6 and 7).\n\nThe results obtained by setting control u1=0 and varying control u2 are shown in Figures 8-13. Figure 8 and Figure 9 depicts a decline in the number of susceptible and exposed individuals respectively. The vaccination control led to a marginal decrease in the number of infected, hospitalized and deceased individuals (see Figures 10, 11 and 13). Recoveries increases as people get permanent immunity through vaccination (see Figure 12).\n\nWe set u1≠0 and u2≠0 and compare the effect of the two control measures on the system (1). The results obtained are given as Figures 14–19. Vaccination strategy was the only effective control measure in curbing vulnerability of the population (see Figure 14). The two stategies were effective in bring down the number of exposed individuals (see Figure 15). However, they were ineffective in the infected, hospitalised and deceased classes (see Figures 16, 17 and 19). Greater number of recoveries with vaccination strategy unlike the public education (see Figure 18).\n\n\nConclusion\n\nTo better understand how the monkeypox virus spreads, a deterministic model has been put forth. The disease is not endemic, which is explained by the model’s basic reproduction number, which was less than unity. In order to assess the efficacy of two preventative control strategies–public education and vaccination–optimal controls were included in the model. Public education was found to have less of an effect on those who were vulnerable than vaccine control. However, both approaches were successful in reducing the number of people who were exposed to the illness. Vaccination reduced number by 32.35% and public education by 50% at the peak of the exposed phase. Additionally, vaccination increases a person’s immunity, which speeds up their recovery. Both strategies had a minor impact on the number of fatalities during the course of the time period. Based on the findings of this study, we advise a vaccine control plan as the most effective preventative measure.", "appendix": "Data availability\n\nOSF: Raw_data_monkeypox. https://doi.org/10.17605/OSF.IO/69RYP. 19\n\nThis project contains the following underlying data:\n\nRaw data.docx (data input into matlab simulations)\n\nRaw data.pdf (data input into matlab simulations)\n\nOSF: Monkeypox_data. https://doi.org/10.17605/OSF.IO/ZQ65J. 20\n\nThis project contains the following extended data:\n\nMATLAB Command Window.pdf (Matlab output data that accompanied the numerical simulation figures)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nHammarlund E, Lewis MW, Carter SV, et al.: Multiple diagnostic techniques identify previously vaccinated individuals with protective immunity against monkeypox. Nat. Med. 2005; 11(9): 1005–1011. PubMed Abstract \| Publisher Full Text\n\nKalyar F, Chen X, Kunasekaran M, et al.: An unprecedented global resurgence of monkeypox. Global Biosecurity. 2022; 4. Publisher Full Text\n\nAlah MA, Abdeen S, Tayar E, et al.: The story behind the first few cases of monkeypox infection in non-endemic countries. J. Infect. Public Health. 2022; Volume 15(Issue 9): Pages 970–974. 1876-0341.\n\nHezam IM, Foul A, Alrasheedi A: A dynamic optimal control model for COVID-19 and cholera co-infection in Yemen. Adv. Differ. Equ. 2021; 2021: 108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTessema FS, Koya PR, Bole BK: Optimal Control and Cost-Effectiveness Analysis of Cholera with Vaccination. J. Math. 2022; 2022: 1–16. Publisher Full Text\n\nDeressa CT, Duressa GF: Modeling and optimal control analysis of transmission dynamics of COVID-19: the case of Ethiopia. Alex. Eng. J. 2021; 60(1): 719–732. Publisher Full Text\n\nPerkins A, España G: Optimal control of the COVID-19 pandemic with nonpharmaceutical interventions. Bull. Math. Biol. 2020; 82(9): 118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOkyere S, Ackora-Prah J: A mathematical model of transmission dynamics of SARS-CoV-2 (COVID-19) with an underlying condition of diabetes. Int. J. Math. Math. Sci. 2022; 2022: 1–15. Publisher Full Text\n\nOkyere S, Ackora-Prah J: Modelling and analysis of monkeypox disease using fractional derivatives. Results Eng. 2023; Vol. 17: 100786. 2590–1230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSomma SA, Akinwade NI, Chado UD: A mathematical model of monkey pox virus transmission dynamics. Ife Journal of Science, AJOA. 2019; 21(1). elSSN: 0794-4896.\n\nUsman S, Adamu I: Modeling the Transmission Dynamics of the Monkeypox Virus Infection with Treatment and Vaccination Interventions. J. Appl. Math. Phys. 2017; 5(12). Reference Source\n\nPeter OJ, Kumar S, Kumari N, et al.: Transmission dynamics of Monkeypox virus: a mathematical modelling approach. Model. Earth Syst. Environ. 2021; 8: 3423–3434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeter OJ, Oguntolu FA, Ojo MM, et al.: Fractional order mathematical model of monkeypox transmission dynamics. Phys. Scr. 2022; 97(8): 084005. Publisher Full Text\n\nGrant R, Nguyen LL, Breban R: Modelling human-to-human transmission of monkeypox. Bull. World Health Organ. 2020; 98(9): 638–640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVaughan A, Aarons E, Astbury J, et al.: Human-to-Human Transmission of Monkeypox Virus, United Kingdom, October 2018. Emerg. Infect. Dis. 2020; 26(4): 782–785. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThornhill JP, Barkati S, Walmsley S, et al.: Monkeypox virus infection in humans across 16 countries – April–June 2022. N. Engl. J. Med. 2022; 387: 679–691. PubMed Abstract \| Publisher Full Text\n\nCastillo-Chavez C, Blower S, van den Driessche P , et al.: Mathematical approach for emerging and reemerging infectious diseases. New York: Springer Verlag.\n\nPontryagin LS, Boltyanskii VG, Gamkrelidze RV, et al.: The mathematical theory of optimal processes. New York/London: Wiley and sons; 1963; vol. VIII +360S. . Publisher Full Text\n\nOkyere S: Raw_data_monkeypox. [Data]. OSF. 2023 February 14. Publisher Full Text\n\nOkyere S: Monkeypox_data. [Data]. OSF. 2023 February 14. Publisher Full Text\n\nokyere2015: okyere2015/Matlab_codes: Matlab codes for monkeypox deterministic models (v1.0.0). [Code] Zenodo. 2023. Publisher Full Text" }	[ { "id": "168328", "date": "20 Apr 2023", "name": "Olumuyiwa James Peter", "expertise": [ "Reviewer Expertise Mathematical Biology", "Infectious Disease Modelling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 investigates an optimal control model of human-to-human transmission of monkeypox virus. This work has potential and my comments are as follows:\nPlease add the main findings and objective of the current study in the abstract.\n\nWhat are the benchmark cases in your study?\n\nWhat are the special cases of your study?\n\nTable needs to be referenced. Main equations and propositions need to be referenced. Punctuation is missing after some equations.\n\nTry to show more of the physical situation in the results and discussion to reflect the proposed notion of the whole study.\n\nFor enhancing the introduction section please include the following recent studies on monkeypox models: Peter et al. (20231) and Peter et al. (20222).\n\nIs the work 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": "10062", "date": "29 Aug 2023", "name": "Samuel Okyere", "role": "Author Response", "response": "1. Please add the main findings and objective of the current study in the abstract The objective has been specified in the background of the abstract. The main findings have been included in the results of the abstract. 2. What are the benchmark cases in your study? The study extends and analyses the monkeypox model proposed in Ref. 9, which also takes inspiration from the study in Ref. 12 and 13 and suggests intervention strategies in curbing the disease in Ghana and preventing future occurrences. 3. What are the special cases of your study? The study reveals an insignificant number of monkeypox-related deaths in Ghana. It also reveals the disease getting extinct in the first two weeks of the outbreak. The study reveals the effectiveness of the two control measures with each strategy leading to a greater percentage reduction in the number of exposed individuals. 4. Table needs to be referenced. Main equations and propositions need to be referenced. Punctuation is missing after some equations. All equations and theorems have been referenced with the exception of the model formulated by the authors. Punctuations and corrections have been done. 5. Try to show more of the physical situation in the results and discussion to reflect the proposed notion of the whole study. This has been done with the introduction of even new figures to give more insight into the model." } ] }, { "id": "193971", "date": "08 Aug 2023", "name": "Stephen Lasong", "expertise": [ "Reviewer Expertise Mathematical Modeling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is good. The findings are new, accurate, and comprehensive. The procedures are clearly laid out. The findings fully support the discussion and conclusions. The writing is decent. There are corrections that need to be done:\n\nnot\n\nThe same applies to t approaches infinity.\n\nCheck equation 6:\n\nnot\n\nCheck equation 11:\n\nnot\n\nIs the work 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": "10063", "date": "29 Aug 2023", "name": "Samuel Okyere", "role": "Author Response", "response": "Corrections have been rectified in the revised version, however, the first equation is still valid and not the one suggested by the Reviewer." } ] } ]	1	https://f1000research.com/articles/12-326
https://f1000research.com/articles/12-1048/v1	29 Aug 23	{ "type": "Case Report", "title": "Case Report: Idiopathic pleuroparenchymal fibroelastosis", "authors": [ "Selsabil Daboussi", "Ben Hmida Lenda", "Samira Mhamedi", "Boubaker Nouha", "Chiraz Aichaouia", "Aida Ayadi", "Zied Moatemri", "Ben Hmida Lenda", "Samira Mhamedi", "Boubaker Nouha", "Chiraz Aichaouia", "Aida Ayadi", "Zied Moatemri" ], "abstract": "Background: Idiopathic pleuroparenchymal fibroelastosis (IPPFE) is a very rare and a slowly conspicuous progressing chronic lung disease, which usually involves the upper lobes of the lung. This unusual disease, first recognized as a rare idiopathic interstitial pneumonia in 2013, is characterized by dense fibrosis of the visceral pleura and the subjacent lung parenchyma accompanied by elastosis predominating in the subpleural alveolar walls. In the interest of improving our understanding of this uncommon disease, we report a case of IPPFE established by pathology results. Case report: A 73-year-old male patient, smoker, with a medical history of chronic obstructive pulmonary disease, presented since January 2022 with a gradual worsening of dyspnea on exertion and productive cough with weight loss. The chest X-ray detected a thoracic distention. The chest high resolution computed tomography revealed biapical subpleural parenchymatous condensations with tractive bronchiectasis and pleural retraction in the right upper lobe and diffuse bilateral cento-lobular emphysema. A scan-guided trans-parietal lung biopsy showed lung parenchyma tattooed with anthracosic deposits, largely remodeled by fibrous tissue, intermingled with numerous wavy and refractive dyselastotic structures in polarized light. The orcein staining confirmed the presence of excess elastosic fibers within these lesions. All etiological investigations were negative. His lung function studies revealed a reversible obstructive ventilatory disorder. Following a multidisciplinary discussion, the diagnosis of IPPFE was confirmed on the basis of the distribution in the upper lungs on chest computed tomography combined with pathology pattern. Conclusions: This case emphasizes the atypical misleading radiological presentation of IPPFE and the key role of pathological results in establishing the diagnosis. Hence, further studies are needed to improve our understanding of this uncommon disease and to establish clear-cut guidelines for IPPFE diagnosis and management.", "keywords": [ "elastofibrosis", "idiopathic", "interstitial lung disease", "lung function", "pleural thickening", "pleuropulmonary elastosis" ], "content": "Introduction\n\nIdiopathic pleuroparenchymal fibroelastosis (IPPFE) is a very rare chronic lung disease, that usually involves the upper lobes of the lungs.1 This unusual disease was first recognized as a rare idiopathic interstitial pneumonia (IIP) in 2013.1 It is characterized by dense fibrosis of the visceral pleura and the subjacent lung parenchyma accompanied by elastosis predominating in the subpleural alveolar walls, with perilobular or bronchocentric distribution.2 It is a slowly conspicuous progressing entity, striking firstly the upper lobes close to the lung apices, producing lung volume loss, leading to platythorax and ultimately an irreversible respiratory failure and early death.1 IPPFE usually presents in adults without gender predilection.2 Although a number of disease associations have been described, its exact cause is still unknown.1 Pleuroparenchymal fibroelastosis (PPFE) is schematically separated into IPPFE and PPFE secondary to a number of conditions.3 Patients often have a history of recurrent pulmonary infections, shortness of breath, and dry cough.2 The diagnosis is based on chest high resolution computed tomography (HRCT), requiring in some cases a pathological confirmation.1 Except for lung transplantation, to this day, there is no specific treatment.4 In the interest of improving our understanding of this uncommon disease, we report a case of IPPFE established by pathology results.\n\n\nCase presentation\n\nWe represent the case of a 73-year-old male patient, admitted to the Pneumology Department of the Military Hospital of Tunis, Tunisia. He had a medical history of chronic obstructive pulmonary disease (COPD) group E treated with a long-acting beta-agonist (LABA) and long-acting muscarinic antagonist (LAMA). His tobacco consumption amounted to 100 packets per year with no particular exposition to toxics. He had a complete immunization schedule with no history of vaccine reaction or infectious disease. He had no family history of interstitial pneumonia or cancer. In January 2022, he had a gradual worsening of respiratory symptoms, which consisted of productive cough with white sputum and dyspnea on exertion. Recent weight loss was reported. The physical examination had revealed sibilant rales and the saturation at room air was 88%. Cyanosis or digital clubbing were not found. General examination was unremarkable. The laboratory findings were as follows: white blood cells, 10600/L; neutrophils, 5900/L; eosinophil count, 900; hemoglobin, 13.9 g/L; and platelets, 216,000/L. Liver and kidney function was normal. The C-reactive protein was 8 mg/L. Sputum acid-fast bacillus smear, sputum culture and sputum fungal culture test results were all negative. Aspergillus antigenemia and serology were also negative. The chest X-ray detected a thoracic distention. He was treated, symptomatically, as an acute exacerbation of COPD.\n\nFive months after the acute event, a chest HRCT revealed biapical subpleural parenchymatous condensations; 1025 mm in the right upper lobe (Figure 1) and 1811 mm in the left upper lobe (Figure 2). There was tractive bronchiectasis and pleural retraction in the right upper lobe and diffuse bilateral cento-lobular emphysema.\n\nA bronchoscopy was performed with unremarkable results. A scan-guided trans-parietal lung biopsy showed lung parenchyma tattooed with anthracotic deposits, largely remodeled by fibrous tissue, intermingled with numerous wavy and refractive dyselastotic structures in polarized light (Figure 3). The orcein staining confirmed the presence of excess elastotic fibers within these lesions (Figure 4). Following a multidisciplinary discussion, the diagnosis of IPPFE was confirmed on the basis of the distribution in the upper lungs on chest CT combined with pathology pattern.\n\nHis lung function studies revealed a reversible obstructive ventilatory disorder. The results are as follows: forced vital capacity (FVC), 74%; forced expiratory volume in the first second (FEV1), 48%; FEV1/FVC, 50.2% and total lung capacity in a single breath (TLC-SB), 119%. He presented with oxygen desaturation at a level of 90% after walking 350 meters during a six-minute walk test.\n\nSerum levels of antinuclear antibodies, rheumatoid factor, anti-cyclic citrulline peptides, antineutrophil cytoplasmic antibodies, extractable nuclear antigen antibodies and dot myositis were all negative.\n\nWe recently began treatment with oral corticosteroids, specifically prednisone at a dosage of 40 mg per day, taken once daily.\n\n\nDiscussion\n\nIPPFE is a rare interstitial pneumonia, characterized by its unique radiological and pathological pattern.3 Historically, IPPFE was acknowledged for at least 20 years as a case of pulmonary upper lobe fibrosis (PULF).1 Only in 2004 was it formally labeled as a PPFE in a mini-series of five patients sharing a distinct radiological and pathological pattern of chronic interstitial and pleural fibrosis of the upper lungs that did not fit within other categories of IIP.1 Most cases were reported in Japan.1 In 2013, IPPFE was first introduced into the classification of IIP as a new category along with idiopathic lymphocytic interstitial pneumonia.3 The true prevalence of IPPFE is still unknown due to the absence of set criteria for its diagnosis.5 Shioya et al. detected 29 cases (7.7%) of IPPFE out of 375 cases of IPP over a 10-year period.1\n\nAccording to a review of 78 cases published up to 2013, IPPFE had a bimodal age distribution ranging between 13 and 85 years of age, with a mean of 49 years; as was reported in our case.5 Although gender predilection is still a controversial topic, a female predominance was found in non-smoking, younger and lightweight patients.1 Unlike our case, indeed, IPPFE mostly occurs in nonsmokers.2\n\nCommonly, clinical presentation is typically characterized by exertional dyspnea, dry cough, weight loss, chest discomfort and recurrent respiratory infections.5 Pneumothorax and pneumomediastinum are less common.5 Our patient, considering his COPD background, had productive cough with white sputum. Clinical exam features include inspiratory crackles on auscultation, platythorax and deepened suprasternal notch; which is contrasting with our case.5\n\nThe typic imaging features of PPFE are highly suggestive showing symmetric, bilateral, apical and irregular pleural thickening with few calcifications and bronchiectasis in some cases.4 Middle and lower lungs may be affected in some patients.4 For our patient, chest HRCT showed biapical subpleural parenchymatous condensations, tractive bronchiectasis and pleural retraction in the right upper lobe. In this regard, our case is noteworthy by its atypical radiological presentation suggesting a neoplastic pulmonary disease, considering our patient background.\n\nThe main pathological features of PPFE are upper zone pleural fibrosis with subjacent intra-alveolar dense fibrosis and elastosis, keenly dissociated from the adjacent normal parenchyma, with mild focal lymphocyte and plasma cell infiltration on the periphery of the fibrosis.4\n\nIn our case, the diagnosis of IPPFE was clearly confirmed based on typical pathology results of a scan-guided trans-parietal lung biopsy.\n\nFor a positive diagnosis of PPFE, an agreed consensus statement has yet to be defined.2 Thus, a list of criteria has been proposed in the literature and adopted in clinical practice.3 Reddy et al. suggested both radiological and pathological criteria including confidence levels: “definite”, “consistent with” and “inconsistent with” PPFE.3 Considering the unavailability of pathology and the unfavorable risk-effectiveness profile of invasive procedures, Enomoto et al. proposed modified criteria requiring progression of disease on imaging rather than pathologic confirmation.3 In all cases, a multidisciplinary discussion is still mandatory for a conclusive diagnosis of IPPFE, as it was reported in our patient.2\n\nPPFE, to this day, has no clear pathogenetic explanation.1 The pathogenesis is thought to involve acute or subacute lung injury, including diffuse alveolar damage with aberrant tissue repair, leading to exuberant interstitial inflammation and subsequent fibrosis.1,5 The triggering stimuli remains unknown.1\n\nPPFE is schematically separated into IPPFE and PPFE secondary to a number of conditions.3 Given that a clear causative relationship has yet to be established, these conditions are considered more like disease-associated factors or inciting triggers rather than etiologies.2,4 So far, the strongest association seems to be with a previous organ transplant [lung, bone marrow, hematopoietic stem cell and liver transplantation].1,2 The other associated factors are fibrotic interstitial lung disease, recurrent pulmonary infection, autoimmune disease, radiation therapy, chemotherapy, alkylating drugs, environmental exposures to asbestos, silica or aluminum and familial or genetic telomeropathy.3,4 In our case, our patient was diagnosed with IPPFE after ruling out all these conditions.\n\nThis disease prognosis is poor, and most of the patients show disease progression after diagnosis.4 To date, there is no specific medical effective treatment for PPFE, and lung transplantation remains the only therapeutic option.4\n\n\nConclusions\n\nIn conclusion, this case emphasizes the importance of improving our understanding of this uncommon disease, presenting an atypical radiological pattern, thus requiring pathology results for establishing the IPPFE diagnosis. Further experiences and studies are needed to better understand the pathogenesis and to establish clear-cut guidelines for PPFE diagnosis and management.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nChua F, Desai SR, Nicholson AG, et al.: Pleuroparenchymal Fibroelastosis. A Review of Clinical, Radiological, and Pathological Characteristics. Ann. Am. Thorac. Soc. nov 2019; 16(11): 1351–1359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBonifazi M, Montero MA, Renzoni EA: Idiopathic Pleuroparenchymal Fibroelastosis. Curr. Pulmonol. Rep. 2017; 6(1): 9–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCottin V, Si-Mohamed S, Diesler R, et al.: Pleuroparenchymal fibroelastosis. Curr. Opin. Pulm. Med. 1 sept 2022; 28(5): 432–440. Publisher Full Text\n\nCao M, Li H, Cai HR: Idiopathic pleuroparenchymal fibroelastosis confirmed by pathology: a case report. J. Int. Med. Res. févr 2021; 49(2): 300060521992217. PubMed Abstract \| Publisher Full Text\n\nTavakolian K, Udongwo N, Douedi S, et al.: Idiopathic Pleuroparenchymal Fibroelastosis. J. Med. Cases. mai 2022; 13(5): 235–239. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "286245", "date": "01 Jul 2024", "name": "Dongmei Zhou", "expertise": [ "Reviewer Expertise immunogenetics", "endocrine system" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 provides a comprehensive overview of the diagnostic process involved in managing a rare disease characterized by its challenging diagnosis and treatment. Insufficient awareness among clinicians regarding this particular condition necessitates the dissemination of this case report, which aims to enhance their understanding and recognition of the disease.\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": "291326", "date": "17 Jul 2024", "name": "Claudia Valenzuela", "expertise": [ "Reviewer Expertise Interstitial lung 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\nThis case report describes FEPP, emphasizing its complexity, clinical, radiological and pathological characteristics to establish the diagnosis and the need for specific treatment. It comprehensively reviews the main characteristics of the disease, which is informative for clinical practice. Perhaps in the treatment part the authors could detail more the treatments used in clinical practice and the level of evidence for it.\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/12-1048
https://f1000research.com/articles/12-804/v1	10 Jul 23	{ "type": "Research Article", "title": "Symptoms trend and challenges in dental practice during delta variance COVID-19 pandemic in Indonesia: Google Trends Analysis", "authors": [ "Faizul Hasan", "Noor Rohmah Mayasari", "Eisner Salamanca", "Odontuya Dorj", "Rahmat Dani Satria", "Kamaluddin Latief", "Mokh. Sujarwadi", "Hendrik Setia Budi", "Noor Rohmah Mayasari", "Eisner Salamanca", "Odontuya Dorj", "Rahmat Dani Satria", "Kamaluddin Latief", "Mokh. Sujarwadi" ], "abstract": "Background: The COVID-19 pandemic has grown to be a serious issue on a global scale. Dental care is one of the industries affected by COVID-19. The surveillance utilizing lifetime data, however, is still not clear. The purpose of this study was to use Google Trends (GT) analysis to examine symptom trends and challenges during the COVID-19 outbreak in Indonesia. Methods: Covid-19 cases retrieve from Our World in Data. The cases were collected between 1 April 2021-30 September 2021. The GT was used to discover Indonesian relative search volume (RSVs) covering the timeframe of the first outbreak covid-19 pandemic in Indonesia on 1 March 2020 until 13 February 2022. The duration of the search was chosen to reflect the relative popularity of the keywords \"symptoms and dentistry practice challenge-related terms\" and \"coronavirus\". Results: We observed that there was a significant and positive correlation between the COVID-19 daily case using GT RSV data and the COVID-19 case from Our World in Data. The COVID-19 daily case had a strong correlation with search terms related to symptoms (such as fever, sore throat, flu, toothache, and cough), drugs (such as ibuprofen, paracetamol, demacolin, bodrex, and antibiotic), and health management (such as self-isolation and telemedicine). Conclusion: Using GT may be helpful to observe the current symptoms trends as well as its challenge tendencies as a surveillance tool for a continuing pandemic like COVID-19. GT should be considered and used as it has the potential to be a powerful digital epidemiology tool that can provide more insight into disease dynamics.", "keywords": [ "COVID-19", "google trends", "Dental care", "symptoms" ], "content": "Introduction\n\nThe coronavirus disease 2019 (COVID-19) was firstly found in China,1 was a virus that have been infected many people and caused many deaths worldwide.2 Unlike a severe acute respiratory syndrome (SARS)3–5 the COVID-19 incubation period was longer (4–12 days) than that of SARS (2–7 days).6 In addition, this virus, caused pandemic, has a rapid transmission speed and need special treatment approach.7 People suffering from COVID-19 can developed several symptoms such as fever, headache, and dry cough.8,9 Finding the best symptom management are clinically important.\n\nIn the early phases of the COVID-19 pandemic, dental healthcare professionals - including dentists, dental assistants, dental hygienists, and nurse practitioners - were aware of the significant risk of exposure.10,11 It is documented that dentists have a significant risk of transmitting COVID-19 from their patients because of the transmission through respiratory droplets, the use of dental handpieces that produces aerosols, and their near physical closeness with patients.11–13 Many dental clinics had not identified the spread of SARS-CoV-2 as a significant threat to their patients or themselves. The most recent research demonstrated that SARS-CoV-2 is not only present in saliva but also in the salivary glands, due to the salivary glands and tongue epithelium’s angiotensin-converting enzyme 2 (ACE2) high expression.14\n\nThe Centers for Disease Control and Prevention (CDC) advises people to cover their faces when in dental facilities and to take them off only when receiving treatment. It is nonetheless recommended that patients keep their distance from one another in order to reduce the risk of the virus spreading among potentially asymptomatic individuals.15 By integrating information technology, telemedicine or telehealth provides online medical care to patients who are dispersed out across different locations.16 Dental professionals can reduce patient interaction before acting by using teledentistry.17 The dentist who is concerned about patient who have COVID-19, can find employing remote evaluation helpful. Subdivisions of teledentistry with significant roles in dental practice include teleconsultation, tele-diagnosis, teletriage, and telemonitoring.18\n\nInternet search has become the major source of information, including medical and dental terms. In recent years, the internet has established itself as a major resource of information.19 Through keyword-driven internet searches, people have quick access to a vast amount of information.19,20 It was estimated that about eighty percent of internet users have looked for health information through online platforms.21 Big data such as Google Trends (GT) has become the largest potential major source of data for medical and dental studies that need to be properly analyzed and interpreted.22 The GT service assesses the popularity of internet search queries and can be used as a monitoring tool in a variety of languages and locations around the world.23 However, no study has investigated the search for symptoms trends and challenges in dental practice particularly during COVID-19 pandemic in Indonesian populations.\n\nProviding life data trend using GT may provide important and updated information for clinical practitioners and health policy makers. The aim of this study was to analyze symptoms trend and challenges in dental practice during the COVID-19 pandemic in Indonesia using the Google trends analysis.\n\n\nMethods\n\nCOVID-19 cases retrieve from https://github.com/owid/covid-19-data/tree/master/public/data/ maintained by Our World in Data. The cases collected between 1 April 2021-30 September 2021, this time periods were the occurrence of delta variance in Indonesia.\n\nGoogle trends is available at https://trends.google.com.tw/trends/?geo=TW. Google trends was used to discover Indonesian relative search volume (RSVs) covering timeframe of the first outbreak COVID-19 pandemic in Indonesia on 1 March 2020 until 13 February 2022. The search period was used to reflect the relative interest in “coronavirus” and “symptoms and dental practice challenge-related keywords”. RSVs ranges from 0-100, with 100 being the highest relative search term activity for specifies search keyword in the time frame period of interest. Google trends only allows a maximum of five terms search, which is enabled to be compared.\n\nBahasa was used to identified search term. Prior to identifying included search terms in each category, we search the potential keyword to understand the Indonesian search interest related symptoms and dental practice challenge. We identified search terms in to three categories: (1) symptoms (e.g., demam, sakit tenggorokan, flu, sakit gigi, batuk); (2) drugs (e.g., ibuprofen, paracetamol, ponstan, demacolin, bodrex, antibiotik), health management (e.g., isolasi mandiri, isoman, telemedicine, teledentistry).\n\nTo perform all statistical analyses, we used SPSS software version 23.0 (IBM, Armonk, NY, USA). Descriptive analysis using RSVs related keyword was used to identify the trend of symptoms and dental practice challenge during COVID-19 pandemic in Indonesia. A Kolmogorov-Smirnov test was used to test the normality data. The relationship between RSVs and coronavirus (1 April 2021-30 September 2021) was tested using Spearman’s rank-order correlation coefficient. The timeframe was chosen to emphasize the highest peak of interest search term during covid-19 pandemic in Indonesia. The correlation was interpreted use following category: r = 0.1-0.2 is very weak, r = 0.3-0.5 is fair, r = 0.6-0.7 is moderate, r = 0.8-0.9 is very strong and r = 1 is perfect.24\n\n\nResults\n\nThe trend of the COVID-19 case based on “Our Worlds Data” was seen in Figure 1A. The pick of COVID-19 cases sharply increased during June 2021 and was seen to have a downtrend after August 2021. In terms of COVID-19 cases using GT RSVs, we used four keywords of “Covid”, “Covid 19”, “Covid-19”, and “Coronavirus disease 2019” used to search the coronavirus-related terms (Figure 1B). We found a similar trend in that the COVID-19 case increased after June 2021 and decreased after August 2021.\n\n(A) COVID-19 case based on our Worlds Data. (B) COVID-19 based on Google Trends RSV.\n\nRSV = relative search volumes.\n\nThe symptoms-related search terms including fever (In: demam), sore throat (In: sakit tenggorokan), flu (In: flu), toothache (In: sakit gigi), and cough (In: batuk) were seen in Figure 2A. The RSVs curves showed increased trends during the COVID-19 uptrend period between June, 2021 and August, 2021. The top five interest by sub region of those symptoms mentioned above were depicted in Figure 3A-E.\n\n(A) Symptoms-related search terms; fever (In: demam), sore throat (In: sakit tenggorokan), flu (In: flu), toothache (In: sakit gigi), cough (In: batuk). (B) Drugs-related search terms; ibuprofen, paracetamol, ponstan, demacolin, bodrex, antibiotic (In: antibiotik). (C) Health managements-related search terms; self-isolation (In; isolasi mandiri or isoman), telemedicine, teledentistry. RSV = relative search volumes.\n\nWe examined the search interest related to drugs used during the COVID-19 pandemic. As seen in Figure 2B, there were six drugs that were commonly used during the COVID-19 pandemic (ibuprofen, paracetamol, ponstan, demacolin, bodrex, and antibiotic). Interestingly, those six drugs have shown to have a similar trend that increased during June 2021 and was down after August 2021. Figure 4A-F showed the top five sub-regions related to the drugs-search terms.\n\nDuring the COVID-19 lockdown, RSVs curves revealed a rise in interest in terms associated to health management, including telemedicine, teledentistry, and self-isolation (In: isolasi mandiri or isoman) (Figure 2C). Figure 4A–D listed the highest five searches by sub-region. The heat map in Figure 5D, however, could not be created due to little of data.\n\nAs seen in Table 1, the daily confirmed COVID-19 cases were having a strong and significant positive correlation with COVID-19 using GT (P < 0.01). The symptoms-related search terms (fever, sore throat, flu, toothache, and cough) were seen to have positive correlation with COVID-19 case (all P < 0.01). Similarly, drugs-related search term also had significant correlation with COVID-19 case (the P-values of ibuprofen, paracetamol, demacolin, and antibiotic were all P < 0.01, and ponstan was P < 0.05). For health management, the self-isolation, “isoman”, telemedicine but not teledentistry were significantly have correlation with COVID-19 case (all P < 0.01).\n\n* P < 0.05.\n\n** P < 0.01.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first study investigating the symptoms trend, drugs, and health managements, during COVID-19 pandemic in Indonesia. We observed that there was a significant and positive correlation between the COVID-19 daily case using GT RSVs data and the COVID-19 case from Our World in Data. The COVID-19 daily case had a strong correlation with search terms related to symptoms (such as fever, sore throat, flu, toothache, and cough), drugs (such as ibuprofen, paracetamol, demacolin, bodrex, and antibiotic), and health management (such as self-isolation and telemedicine).\n\nWe found that symptoms-related terms such as fever and cough were significantly related to COVID-19 cases. In accordance with evidence that the health experts alerted the public to several primary characteristics of COVID-19, such as fever, persistent coughing, and an absence of taste and smell.25 In contrast, toothache, the second higher symptom (see, Figure 2A), did not show any statistically significant result. However, with the highly infectious Delta variance, various oral symptoms might be emerging including dysgeusia, ageusia, a burning sensation in the mouth, a dry mouth, hyposmia, and severe halitosis26,27 that may lead to toothache. Further investigations are warned.\n\nTelemedicine is strongly correlated with COVID-19 based on this present study. The characteristic of the SARS-CoV-2 Delta variant implies that Delta may replicate more quickly and be more aggressive in the early stages of illness.28 Compared to the Delta variation, the Omicron form is less likely to experience loss of or changes in smell, whereas wheezing of voice and sore throat are much more common. Acute symptom duration was greater for people with the Delta variant than for people with the Omicron variant.29,30 Patients should be always encouraged to visit a doctor if they have worrying symptoms that are becoming worse. However, due to social distancing and lockdown, hence many patients were searching for terms related to telemedicine. In order to avoid infection transmission, a modification of inpatient treatment, such as using telemedicine, during the COVID-19 pandemic should be promoted.\n\nCreate awareness about the use of teledentistry should be emphasized to become a tool for patient’s oral health evaluation and a safe approach to start new treatments during COVID-19 pandemic, and for other diseases with similar pathway of transmission. However, from our findings, teledentistry has an insignificant correlation with COVID-19. These may be due to less knowledge of the patients or dentist to use teledentistry methods, that could lead to a low search level in internet.31,32 Teledentistry is an effective method for screening the patient’s health condition previous to an in person dental consultation.16,17 Using teledentistry for consultations before patients reach dental facilities in a pandemic situation should be incorporated for both, dentists and patients’ safety.\n\nWe acknowledge several limitations in this study. First, the timeframe was only focused on the specific time of April 2021 until September 2021. The application of the finding in another timeframe during the pandemic may be underestimated. However, that time frame was the important time to see the sever symptom and mortality rate due to COVID-19 in Indonesia. Second, the language-related search terms were used in the national language called Bahasa. As Indonesia has hundred local languages, those terms may be not adopted in this current study. However, since Bahasa become the only national language and is mostly considered the first language to share information on the internet, this current study was still rigorous. Lastly, GT did not measure symptoms directly but used the search term trends. The real symptom’s characteristics should be validated further.\n\n\nConclusion\n\nUsing GT may be helpful to observe the current symptoms trends as well as its challenge tendencies as a surveillance tool for a continuing pandemic like COVID-19, particularly in the countries consisted of many islands and supported with proper internet accessed. GT should be considered and used as it has the potential to be a powerful digital epidemiology tool that can provide more insight into disease dynamics. Future improvements can be made, such as merging other digital data types such as Twitter and Facebook, in an attempt to increase the model’s capacity for prediction.", "appendix": "Data availability\n\nCOVID-19 case data available from: https://github.com/owid/covid-19-data/tree/master/public/data/\n\nGoogle Trend data available from: at https://trends.google.com.tw/trends/?geo=TW. Search terms and other parameters are provided in the text.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nChina Novel Coronavirus I RT: A Novel Coronavirus from Patients with Pneumonia in China. N. Engl. J. Med. 2019; 2020.\n\nOrganization WH: Novel Coronavirus (2019-nCoV): situation report, 3.2020.\n\nHuang Y: The SARS epidemic and its aftermath in China: a political perspective. 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Oral Dis. 2020.\n\nBrandão T, Gueiros L, Melo T, et al.: Oral lesions in SARS-COV-2 infected patients: could the oral cavity be a target organ. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2020; 131: e45–e51. Publisher Full Text\n\nGlavina A, Biočina-Lukenda D, Mravak-Stipetić M, et al.: Oral symptoms and lesions in SARS-CoV-2 positive patient. Oral Dis. 2020; 28: 979–980. Publisher Full Text\n\nHart WS, Miller E, Andrews NJ, et al.: Generation time of the alpha and delta SARS-CoV-2 variants: an epidemiological analysis. Lancet Infect. Dis. 2022; 22(5): 603–610. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWise J: Covid-19: Symptomatic infection with omicron variant is milder and shorter than with delta, study reports. BMJ: British Medical Journal. 2022; 377. Publisher Full Text\n\nMenni C, Valdes AM, Polidori L, et al.: Symptom prevalence, duration, and risk of hospital admission in individuals infected with SARS-CoV-2 during periods of omicron and delta variant dominance: a prospective observational study from the ZOE COVID Study. Lancet. 2022; 399(10335): 1618–1624. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaucci-Neto W, de Souza PM , Cerqueira NM, et al.: Knowledge, Perception, and Experience of Dentists About Teledentistry. Int. Dent. J. 2021; 72: 456–462. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAboalshamat KT: Awareness of, beliefs about, practices of, and barriers to teledentistry among dental students and the implications for Saudi Arabia Vision 2030 and coronavirus pandemic. J. Int. Soc. Prev. Community Dent. 2020; 10(4): 431–437. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "186204", "date": "19 Jul 2023", "name": "Sri Susanty", "expertise": [ "Reviewer Expertise Gerontologies", "healthy ageing", "community health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe purpose of this manuscript is to help improve oral health status. However, I would like to point out some issues, as below:\nWhat are the restrictions on dental care practice (dental issues) in relation to the “bahasa” search terms used by the authors? Please be descriptive (are they in the mouth, throat, teeth, or gums).\n\nPlease have a look at the list of medications that dentists frequently use based on the issue. You can utilize diclofenac, paracetamol, ibuprofen, and mefenamic acid if the issue is pain. This is significant because these medications cannot be used for other dental conditions.\n\nIn terms of the drug terminology used in your search terms, the drugs you list are still very general (there are symptomatic drugs to reduce pain symptoms, and there are causative bacteria). I suggest the authors group them.\n\nWhat was the reason the authors used \"demacolin and bodrex,\" which contain antihistamines while having a dental issue?\n\nWhy would the authors choose to use painkillers (ibuprofen, paracetamol, ponstan) or antibiotics for tooth infections? I suggest the authors write it clearly.\n\nWhy do the authors only take terminology from the patent name (brand) of a drug? And why were these brands chosen? Did the authors consider the price of branded drugs which are expensive and not necessarily used in dental health facilities?\n\nTreatment of dental problems does not only use pharmacological therapy. However, Indonesia also uses traditional medicine and non-pharmacological therapy. Why don't the authors consider this?\n\nManagement of dental problems such as plaque does not always use oral medication, but also gargling with an antiseptic. Why did the authors not include mouthwash terminology? Why are the authors only considering oral medication? What about using albothyl and betadine? I know this is viral in Indonesia.\n\nWhat about other dental problems such as canker sore? Generally, people use vitamin C before going to the doctor. Why did the authors not consider including the keyword vitamin C?\n\nI failed to understand the use of antibiotics in the search item. What antibiotics did the authors use? Amoxicillin? Cotrimoxazole? Or penicillin? I suggest the authors consider this to avoid bias.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "10014", "date": "29 Aug 2023", "name": "Faizul Hasan", "role": "Author Response", "response": "Reviewer 1 (Dr. Sri Susanty): The purpose of this manuscript is to help improve oral health status. However, I would like to point out some issues, as below: Response: Thank you for your thorough review. We carefully considered your suggestions and comments and revised our manuscript accordingly. The following are our specific responses to each of the comments. We hope the revisions are satisfactory and that our responses adequately address the comments. What are the restrictions on dental care practice (dental issues) in relation to the “bahasa” search terms used by the authors? Please be descriptive (are they in the mouth, throat, teeth, or gums). Response: The study primarily focuses on highlighting the symptoms, trends, and challenges in dental practice during the COVID-19 pandemic in Indonesia. The search term \"sakit gigi\" (translated to \"toothache\" in English) was used to emphasize major dental problems and related issues. However, regarding specific restrictions on dental care practice in relation to the \"bahasa\" search terms, the study does not provide explicit information on that matter. Instead, it aims to shed light on the impact of the pandemic on dental health and practices in Indonesia. Please have a look at the list of medications that dentists frequently use based on the issue. You can utilize diclofenac, paracetamol, ibuprofen, and mefenamic acid if the issue is pain. This is significant because these medications cannot be used for other dental conditions. Response: We agree with you. Hence, in this study, we only focus on the frequent drug used to counter dental pain such as “ibuprofen” and “paracetamol”. In terms of the drug terminology used in your search terms, the drugs you list are still very general (there are symptomatic drugs to reduce pain symptoms, and there are causative bacteria). I suggest the authors group them. Response: Thank you for your suggestion. In the first stage, we list several drugs and see the trend. However, some of the terms did not provide a good trend. Therefore, in the final results, we only include the term that showed a significant trend in association with COVID-19. What was the reason the authors used \"demacolin and bodrex,\" which contain antihistamines while having a dental issue? Response: We found that “demacolin” and “bodrex” have increased trends during the delta variance periods (see Figure 2B). As we know these two medications are generally and regularly used by Indonesian people if they feel unwell as the first self-medication before going to healthcare providers. Because the symptoms of COVID-19 include fever and flu, so the search terms for administration of these medications were high. In addition, some users considered taking medication (paracetamol, an ingredient of bodrex) before or after vaccination (see Figure 4B). Why would the authors choose to use painkillers (ibuprofen, paracetamol, ponstan) or antibiotics for tooth infections? I suggest the authors write it clearly. Response: As mentioned in the response to question number 3, painkillers (ibuprofen, paracetamol, ponstan) or antibiotics provide increasing trends during the delta variance periods (Figure 2B). However, we are not sure whether this medication is used for tooth infection because it is out of our study aim. Why do the authors only take terminology from the patent name (brand) of a drug? And why were these brands chosen? Did the authors consider the price of branded drugs which are expensive and not necessarily used in dental health facilities? Response: As we explained in the previous response, firstly we include all related terms including generic and patent names of the drug, however, some of them did not provide good trends. As a note, Google trends provided data based on the users' search on Google machine but not the real symptom, so the final term included in this study actually the real-time search engine data. We already acknowledge this in the limitation section. Treatment of dental problems does not only use pharmacological therapy. However, Indonesia also uses traditional medicine and non-pharmacological therapy. Why don't the authors consider this? Response: We also used the term for traditional medicine such as “jamu” or non-pharmacological therapy such as “pijat”. However, we did not find trends during the intended periods. Management of dental problems such as plaque does not always use oral medication, but also gargling with an antiseptic. Why did the authors not include mouthwash terminology? Why are the authors only considering oral medication? What about using albothyl and betadine? I know this is viral in Indonesia. Response: We mainly focus on dental pain as it is considered the main problem. The other problem such as cleaning plaque was not our study aim. In addition, the term albothyl or betadine also did not provide a good trend during the study periods. What about other dental problems such as canker sore? Generally, people use vitamin C before going to the doctor. Why did the authors not consider including the keyword vitamin C? Response: We did use the term “Vitamin C”, however, it did not show a significant trend. I failed to understand the use of antibiotics in the search item. What antibiotics did the authors use? Amoxicillin? Cotrimoxazole? Or penicillin? I suggest the authors consider this to avoid bias. Response: The detail of the antibiotic was provided in Figure 4F. It included amoxicillin, oseltamivir, “obat antibiotic covid”." } ] } ]	1	https://f1000research.com/articles/12-804
https://f1000research.com/articles/12-431/v1	21 Apr 23	{ "type": "Systematic Review", "title": "A framework for doctoral education in developing students’ mental well-being by integrating the demand and resources of the program: An integrative review", "authors": [ "Vrinda Acharya", "Ambigai Rajendran", "Sandeep Shenoy", "Sandeep Shenoy" ], "abstract": "Background: Research on doctoral students’ mental well-being has gained significant importance in recent years. The findings of such studies were uncertain about the critical demands and resources of a doctoral program that substantially influence the students’ mental health. This review aims to integrate the current evidence in bringing out the nature and significance of differentiated demands, contextual and personal resources, and their influence on the well-being of the students. Methods: An integrative literature review was conducted based on the five-stage framework of Whittemore and Knafl. The study identified 45 articles published from 2000 onwards following the Joanna Briggs Institute quality evaluation criteria and PRISMA reporting guidelines for selecting eligible articles. Results: The integrative review findings divulge that differentiated demands of doctoral programs were categorized into challenge-hindrance demands. The differentiated demands experienced by doctoral students were grouped as ambiguity in doctoral program structure, resource inadequacy, workload, complexity, and responsibility. Additionally, institutional support, research supervisory support, and intrinsic motivation were treated as essential resource in mitigating the effects of the differentiated demands of the doctoral program. Conclusions: An integrated conceptual model was built exclusively for doctoral programs and suggests that the universities and supervisors design and structure healthy, constructive doctoral programs. As an outcome of the review, theoretical underpinnings of demands-resources and mental well-being are reported. The current review is an initial attempt to synthesize challenge-hindrance demands and contextual-personal resources in determining the mental well-being of doctoral students.", "keywords": [ "Challenge-hindrance demands", "well-being", "doctoral students", "Job Demands-Resources model", "doctoral education" ], "content": "1. Introduction\n\nGrowing mental health problems are the major contributor to the global health burden; hence there is a need to address this concern in various settings. Mental health refers to individuals’ social, psychological, and emotional well-being. Ensuring the good psychological health of an individual improves their potential to work and productivity. Several studies witnessed the mental health problems of doctoral students and are at high risk compared to the general population, i.e., 32% of the students experience psychological disorders because of stress, depression, and anxiety (Darley, 2021; Herrmann & Wichmann-Hansen, 2017; Levecque et al., 2017). Psychological distress among doctoral students results in program dropout, reduced quality research outputs, academic disengagement, detachment from learning and development activities, and work-family conflict.\n\nEvidence has confirmed the rise in mental health problems among doctoral students were viz: rigorous research policies, dynamic work conditions with increased program demands, a growing number of publication targets, financial commitments to manage the research and family, conflicting inputs from the expert panel, resulted in unpleasant experience in maintaining work-life balance of the doctoral students (Levecque et al., 2017; Pappa et al., 2020; Skopek et al., 2020). These existing studies have highlighted the various stressors of doctoral students subjectively instead of demarcating the intensity of the stressors as challenge-hindrance demands. A recent study by McCauley and Hinojosa (2020) conceptualized doctoral demands as challenge-hindrance stressors that influence the learning experience of doctoral students. The study claims that hindrance demands strain doctoral students and challenge demands motivate them in professional development. However, the effect of these demands on mental health of the student in the presence of resources remains unanswered. Therefore, studies that examine the degree of challenge-hindrance demands in the context of doctoral students are missing.\n\nJob Demands-Resources (JD-R) model postulates that high demands and inadequate resources result in growing academic strain (Pappa et al., 2020) and distress among doctoral students (Sufyan & Ali Ghouri, 2020). Mental disorder caused by the lack of resources that includes: less scope for receiving research grants (Maloshonok & Terentev, 2019), inadequate training from institutions and supervisor, lack of research infrastructure at institutions, limited emotional-technical support and guidance from the supervisor, insufficient access to peer and family support and financial responsibilities (Curtin et al., 2016; Haven et al., 2019; McCauley & Hinojosa, 2020). The resources from institutions, supervisors, family, and peers are likely to moderate doctoral students’ stress and are considered contextual resources (Cornwall et al., 2019; Lynch et al., 2018). At the same time, studies investigated the explicit requirement of intrinsic motivation rather than any other personal resources (Matheka et al., 2020; Ryan et al., 2021; Sverdlik & Hall, 2019). However, the moderating role of contextual and personal resources in the presence of high demands has not been applied to doctoral education in JD-R model.\n\nPrior studies on doctoral program challenge and their impact on students’ well-being are often inconclusive to build a comprehensive model. Thus, we propose a framework, by identifying demands-resources of doctoral education, using what we learn from academic literature. Existing literature offers rich sources of input in developing a current integrated framework and offers a quality insight for doctoral educators, policymakers, institutions, and supervisors.\n\n\n2. Theoretical background\n\nStudies have adopted Bronfenbrenner’s ecological framework by considering the personal, supervisor, university, family, and community as multi-faceted factors influencing doctoral students’ mental health status (Beasy et al., 2019; Usher & McCormack, 2021). As anticipated by Bronfenbrenner’s ecological framework, also grounded on the JD-R model, this section explores the antecedents of mental well-being at supervisor, university, and doctoral students’ levels.\n\nJD-R model is a leading stress-coping model that explains the well-being of an employee (Demerouti et al., 2001). This model highlights that the demands and resources stem from the job, and its imbalance causes burnout and ill-being. Although the JD-R model is designed to study employees’ well-being in the work environment, it has also been applied to the learning environment. In the doctoral education context, job demands refer to the demands appraised by the researchers in their doctoral studies and roles that may influence their mental health (Levecque et al., 2017). Job resources refer to “support from the institution, supervisors, peers, and family that may foster students’ motivation and reduce health impairment by buffering against the negative impact of job demands”. Doctoral programs’ demands and resources are originated from various sources during the program journey, that influence the students’ well-being (Pyhältö et al., 2012). These research demands and resources are appraised differently by doctoral researchers, institutions, and supervisors (Ellis et al., 2015). Prior studies used the JD-R model to explain the intensified strain in the doctoral journey is due to high program demands and limited resources. However, these studies fail to predict its impact on mental health of the students (Haven et al., 2019; Ryan et al., 2021; Sufyan & Ali Ghouri, 2020). Therefore, we reviewed all the prior studies to understand the doctoral programs’ demands and resources that influence doctoral students’ well-being at three levels (i.e., institutions, supervisor and personal).\n\nIndividuals appraise a stressful situation or work environment either as a challenge or hindrance demands. These demands are conceptualized as “two factor stress model” or ‘differentiated demands model’ (Cavanaugh et al., 2000; LePine et al., 2005). Challenge demands promote the desirable outcomes such as learning, job satisfaction, and organizational commitment. In contrast, hindrance demands lead to destructive outcomes, such as withdrawal behavior, burnout, and turnover intentions (LePine et al., 2005). Individuals experience burnout due to hindrance demands, whereas challenge demands can be overcome by acquiring an essential resource (Ellis et al., 2015). Hindrance demands include role conflict, organizational politics, role ambiguity, administrative hassles, and interpersonal conflict. Challenge demands include time pressure, workload, and responsibility (Lepine et al., 2005). This dual-process framework of job demands has been examined in the working environment, but limited studies are reported in the doctoral education environment (McCauley & Hinojosa, 2020). In summary, it is vital to examine the influence of dual role of doctoral program demands of the doctoral journey that impacts the mental health of doctoral students.\n\nJob resources positively affect a worker’s accomplishment, learning, growth, and psychological and physical well-being (Demerouti et al., 2001). The Conservation of Resources (COR) theory separates job resources into contextual and personal resources. Contextual resources are those located outside the individual and are related to work or social environment (Hobfoll, 2001). In doctoral education, contextual resources include supervisory support, the social environment in which the doctoral student is situated, and the institution’s research training (Sufyan & Ali Ghouri, 2020). During doctoral studies, contextual resources may be obtained from outside (e.g., from family or friends) or within the institution (e.g., from the institution, supervisor, or peers) (Skopek et al., 2020; Waight & Giordano, 2018). A study by Dericks et al. (2019) empirically analyzed the combination of peer, supervisor, and department support as a contextual resource in predicting doctoral students’ satisfaction. Critical resources from the institutions and transparency in the program requirements enhanced the Ph.D. completion rates (Skopek et al., 2020). Despite the moderating role of contextual resources in the stressful environment, there is a dearth of studies that underpin its influence on doctoral students’ mental health.\n\nPersonal resources refer to individual characteristics that contribute to goal achievements by lessening the negative influence of job demands and positively influencing their well-being (Ellis et al., 2015; Hobfoll, 2001). Significant personal resources include self-efficacy, optimism, intrinsic motivation, and resilience (Schaufeli & Taris, 2014). Personal resources of the doctoral student are more critical than contextual resources in fostering program completion (Pyhältö et al., 2012). Many theories reflect the robustness of personal resources in the doctoral journey. One such theory is Self-Determination Theory (SDT), conceptualized based on intrinsic motivation, which is critical personal resource of the doctoral students. Intrinsic motivation refers to doctoral students’ innate interest, enjoyment, and excitement in pursuing a Ph.D. (Litalien & Guay, 2015). A student with high intrinsic motivation devotes time for research, and contributing to knowledge (Shin et al., 2018). Existing studies have reported a strong link between intrinsic motivation and doctoral students’ well-being (De Clercq et al., 2021). However, there is lag of studies on doctoral programs that insight the moderating role of intrinsic motivation between the mental health well-being and demands.\n\nThe well-being of a doctoral student refers to the state of mind of the researcher that is primarily influenced by the demands of their role and the support provided by the program (Juniper et al., 2012). The empirical findings confirm a positive association between employees’ mental well-being and favorable individual and organizational-level outcomes (Sverdlik & Hall, 2019). The previous studies have examined various antecedents of the well-being of doctoral students, including domain-specific expertise, inadequate social support, unclear expectations from supervisors, access to financial resources, relationships mismatch with supervisors, and the scholarly community networks (Barry et al., 2018; Juniper et al., 2012; Marais et al., 2018).\n\nThe previous literature has enumerated different factors of mental well-being of doctoral students, but there is limited evidence that theorized the distinct nature of dual role of demands and contextual-personal resources. There is a lack of clarity in the past studies on how the demands of doctoral programs are categorised in determining its positive and negative impact on well-being. Also there exists a paucity of the literature that examines the multiple and inter-connected resources that enhance the well-being of the students. The doctoral education is inextricably associated with a stressful journey, and we suggest that the intrinsic motivation of the student moderates the program’s high demands on the mental health of the students. However, little is known about the significance of intrinsic motivation in the framework of JD-R model.\n\nTo address the above-mentioned gap in the literature, the present study adopted the integrative review method. We adopted Bronfenbrenner’s ecological framework, which recognizes that multiple and inter-connected demands and resources impact students’ well-being status. Thus, this study classifies the cause of the mental health problems among doctoral students as either challenge or hindrance demands, and we illustrate the utility of the contextual-personal resources in shaping the well-being of the doctoral students. The current review offers opportunities for university advisors and institutions to comprehend and design the policy structure of doctoral education fully. We propose theory-driven recommendations for doctoral educators to maximize the challenge demands of the program rather than a hindrance and offer supportive contextual resources in the program journey.\n\n\n3. Methodology\n\nAccording to Whittemore and Knafl (2005) and Hopia et al. (2016), the “integrative review method helps the reviewer to assess the methodological clarity, compare the data, analyze and generate the patterns within the selected articles” (pp. 550–551). Integrative reviews are performed to synthesize the previously published theoretical and empirical research as policy initiation. This methodology focuses on understanding the broad constructs, relationships, and theory-driven reasoning to design a framework for the proposed study (Torraco, 2005). The review has adopted five steps of integrative review methodology suggested by Whittemore and Knafl (2005): (1) problem formulation and setting of the broad purpose and review questions, (2) literature search, (3) quality and relevance appraisal of selected literature, (4) data analysis through data abstraction, comparison, and synthesis of the selected article, and (5) the presentation of results. The reviewers have been involved in the Joanna Briggs Institute (JBI) standard for quality evaluation and interpretation of the selected articles (The Joanna Briggs Institute, 2016). After conducting the preliminary search, the reviewers performed title and abstract screening to check the articles as per the eligibility criteria by the first two authors to exclude irrelevant reports. Articles rejected by both authors were not included in the review during the title and abstract screening process. In abstract screening, approved abstracts by either author are included in the review. Then the final search of full-text screening is performed for reports accepted by both authors. Suppose any difference of opinion among authors on the inclusion criteria was resolved by discussion in the presence of a third author and reached a consensus. The included studies were saved in M.S. excel, and duplicates were removed. Subsequently, the selected studies were classified based on the methodology of the studies.\n\nCompared to the general population, doctoral students are at increased risk of experiencing stress and mental health problems, including anxiety and depression (Hazell et al., 2020; Levecque et al., 2017). The existing studies on doctoral education recommended the integration of JD-R and differentiated demands for assessing the mental well-being of doctoral students. Three review questions were framed by integrating the JD-R and differentiated demands. (1) What are the challenge-hindrance demands of the doctoral program that impact the mental health of doctoral students? (2) What contextual and personal resources moderate doctoral students’ stress? (3) What relevant guiding theories strengthen doctoral students’ well-being? These questions aided in synthesizing our research results, which enabled us to review the prior work and formulate an integrated framework for doctoral education. The proposed framework can be validated further by a quantitative approach.\n\nThe literature search was conducted using two databases, namely Scopus, and Web of Science, using the Boolean operators as keywords. The following were the keywords used as search strategy: “doctoral program” OR “doctoral education” OR “doctoral scholar” OR “doctoral student” AND “differentiated demands” AND “challenge and hindrance”, AND “JD-R” AND “motivation” AND “well-being” AND “mental health” AND “stressors”. A similar Boolean operation was done for the “doctoral candidates”, “Ph.D. scholar” and “Ph.D. students”. The author performed the manual search by reviewing the cited articles from the selected list to include the relevant articles.\n\n3.2.1 Inclusion and exclusion criteria\n\nStudies undertaken in an “academic setting” were considered for inclusion, which had been determined by the criteria that include studies on the JD-R model, motivation studies, differentiated job demands, and well-being among doctoral students. Also, quantitative, qualitative, conceptual, and mixed methods studies published in the English language from 2000 onwards were included because of the significant transformation in structured doctoral education across different countries starting from the 20th century onward (Kot & Hendel, 2012). The reviewers excluded the studies beyond the scope of the above inclusion search criteria. The articles focusing on institutional or individual outcomes were excluded from the study, for example, program satisfaction, Ph.D. dropout rate, and students’ performance. Based on the quality scores allotted by the reviewers as per JBI, the articles not meeting the quality requirement were excluded. The articles were chosen based on whether the selected final articles had been empirically tested, and theoretically supported. Finally, 45 studies were included in the review (see Figure 1). We followed the PRISMA integrative review checklist to identify, select, appraise, and synthesise studies.\n\nQuality appraisal of the selected articles was performed using the JBI quality appraisal checklist. This tool estimates the article’s methodological quality, the possibility of bias in the design, analysis, reliability, and validity measure used in the selected article. Two review authors independently assessed the selected articles’ quality assessment. The third author helped to resolve discrepancies that arose during the process. This method helped to overcome the bias during the assessment phase. In Annexures A to E in the extended data (Acharya et al., 2023a), a collated quality assessment checklist is given for reference. Based on JBI critical appraisal checklist, we have exacted a total of 26 cross-sectional design studies: two articles on the randomized experiment, one observational study, four conceptual papers, and 12 qualitative studies for the data analysis.\n\nThe selected articles were screened based on methodological process, antecedents, consequences, theoretical articulation, and facilitators of mental well-being of doctoral students. From the final 45 articles, the associated variables were identified, iteratively compared, coded, categorized, and summarized for an integrated conclusion (see Table 1).\n\n3.4.1.1 Differentiated demands and doctoral students’ well-being\n\nDoctoral students are required to work for long hours, present the findings of their work regularly, learn rigorous methodologies and analysis skills, obtain tangible research outcomes in terms of high-quality publications, and balance the student-supervisor relationship (Litalien & Guay, 2015; Sufyan & Ali Ghouri, 2020). To meet these bounded requirements, complexity, study responsibility and workload of the doctoral program have increased and are considered as challenge demands (Pervez et al., 2021). The doctoral program also confined with a lack of transparency in the program, communication gap between the supervisor-student, low quality mandatory coursework, lack of infrastructures, which hinder the progress of doctoral studies. (Levecque et al., 2017; Sin et al., 2021). These hindrance demands summarize the ambiguity in the doctoral program, poor relationship with the supervisor, family, and advisory members, and resource inadequacy that threatens the students’ mental health (McCauley & Hinojosa, 2020). The students appraise challenge demands of the doctoral program as an opportunity to advance and hindrance demands as a threat to their learning (McCauley & Hinojosa, 2020). In summary, challenge demands positively influence the well-being of the student, and hindrance demands adversely influence.\n\n3.4.1.2 Contextual resources of doctoral programs\n\nJob resources support doctoral students in achieving their goals, research growth, and development. A recent integrative review of the JD-R model postulated the significance of resources at three levels: the institution level, the team level (supervisor style and co-workers), and the individual level (personal resources) (Kwon & Kim, 2020). Similarly, empirical works on doctoral programs examined the primary resources from the institution, supervisor, and personnel is essential in maintaining doctoral students’ well-being (Ryan et al., 2021; Waight & Giordano, 2018). Access to research learning infrastructure, financial assistance in the form of scholarship, transparency in the policy structure, and valuable guidance from the scholarly community are referred to as contextual resources provided by the institutions that enhance the intrinsic motivation of doctoral students (Janssen et al., 2021). Emotional support from family, online mentoring groups, face-to-face support from peers and faculty members, are the social resources that assist the doctoral student in dealing with stress (Boone et al., 2020). Supervisors’ open and honest communication, emotional and technical support, flexible supervision, patience, insightfulness, and the propensity to honour the research students’ self-reflection and listening skills are significant supervisor resources that facilitate the students’ well-being (Dericks et al., 2019). Contextual resources such as supervisors and social support buffer the negative relationship between hindrance demands and work engagement. Job resources also boost the positive association of challenge demands and work engagement (Tadić et al., 2015).\n\n3.4.1.3 Intrinsic motivation as a personal resource in the doctoral study\n\nUnlike other personal resources, the intrinsic motivation is a significant internal resource of doctoral students that has been witnessed in literature. Intrinsic motivation is the internal desire to study, that is critical in achieving program completion (Dericks et al., 2019; Matheka et al., 2020). Intrinsic motivation of the doctoral student is often associated with their positive personal experience and its influence on improving their performance and academic engagement (Litalien & Guay, 2015). Intrinsic motivation limits the influence of hindrance demands on the strain and, in turn, improves the mental health of the doctoral student (McCauley & Hinojosa, 2020). Intrinsically motivated doctoral students undertake doctoral education due to their inherent interest in acquiring knowledge in their domain area and are capable of withstanding hindrance demands (Matheka et al., 2020). Intrinsic motivation with contextual resources supports doctoral learning outcomes and reduces the effect of burnout (van den Broeck et al., 2011). Empirical studies have highlighted the moderating role of intrinsic motivation in strengthening job resources and basic psychological needs (van den Broeck et al., 2011).\n\n3.4.1.4 Significance of well-being in JD-R model\n\nExisting evidence recommend that adoption of JD-R model in doctoral education setting would allow to examine the influence of challenges on two different levels of outcomes i.e., organisation and individual level. (McAlpine et al., 2022). Also, studies to date have focused mainly on mental health problems or mental distress rather than the positive side of the construct. Therefore, the current study sheds light on the mental well-being of doctoral students by considering both positive and negative perspectives of doctoral program demands and resources.\n\n3.4.2 Guiding and contributing theories\n\nIn explaining the challenge-hindrance demands, the current review evolved from Folkman and Lazarus’s (1985) Transactional Model of Stress and Coping (TMSC). The theory argues that doctoral student appraises the program demands depending on their subjective environment as challenge or hindrance demands. The theory also contends that hindrance demands can be a source of stress, and challenge demands support the personal resources to lessen the individual strain (Pervez et al., 2021). A limited number of studies analyzed the challenge-hindrance demands of a doctoral program that are theoretically articulated based on the TMSC model (McCauley & Hinojosa, 2020). Nevertheless, this study did not report the influence of differentiated program demands on the well-being of students. In this connection, one may concur that there is a need for an empirical study on the magnitude of doctoral students’ appraisal of their program demands.\n\nUsing insights from the Conservation of Resources (COR) Theory, we explain the doctoral program is bound with resources along with the demands. Indeed, from the COR theory, doctoral students are always required and willing to develop the resources to manage stressful demands. Absence of these resources creates a stressful situation for the student and results in burnout. Adequate contextual and personal resources help students to mitigate work demands and maintain individual well-being. Under the COR theory, institution, supervisor, and social support are viewed as contextual resources, and intrinsic motivation is a personal resource that improves the mental health of doctoral students (Sufyan & Ali Ghouri, 2020). Drawing on COR theory, resources gained from peer support, supervisor mentoring, learning support from institutions, and emotional support from the family will enhance an individual’s intrinsic motivation by protecting against resource loss (Boone et al., 2020). Sufyan and Ali Ghouri (2020) adopted the COR theory and JD-R model to study doctoral students’ stress. The study results conveyed that high demand and lack of resources will impact their academic journey. Thus, their findings suggest that the scope for inclusion of COR and JD-R has a significant role in determining the mental health of students.\n\nIntrinsic motivation derives its theoretical underpinning from Causality Orientations Theory (COT), a sub-theory under SDT, stating how individuals acquaint themselves with autonomous, controlled, and impersonal external environments. COT theory drives that doctoral students to act out of their desire during the autonomous orientation. In contrast, in controlled orientation, students focuse on their gain and rewards, and they experience anxiety in impersonal orientation (Deci et al., 2001). COT suggests that the higher the degree of self-determination and self-esteem among doctoral students, the greater the autonomous orientation and the intrinsic motivation that reduce burnout levels, helping them to achieve a higher level of engagement (Lynch et al., 2018). The contrary is expected when controlled, and impersonal orientations are more predominant among doctoral students (Litalien & Guay, 2015). The intrinsic motivation fostered by the intervention or motivational program that facilitates the researcher’s autonomous orientation instead of promoting the controlled orientation, such as reward policy, schemes, and programs.\n\nThe mental well-being of the present framework is underpinned by Basic Psychological Need Theory (BPNT), a mini theory under SDT that makes it novel in doctoral program research. BPNT suggests that individuals experience mental health and higher quality behavior when their social environment supports (contextual resources) their basic psychological needs (Deci et al., 2001). The social settings that boost individual well-being are termed autonomy-supportive resources (Ryan & Deci, 2000). Autonomy-supportive resources include supervisors, faculty members, and peers that increase the doctoral students’ basic psychological needs (Devos et al., 2015; Sverdlik & Hall, 2019). In line with the BPNT, it is believed that the autonomy-supportive doctoral program setting strengthens doctoral students’ autonomy orientation (Van Rooij et al., 2021). Hence, doctoral students’ work autonomy and challenge demands induce the mental health of the researcher during the doctoral program journey. The findings of differentiated demands, resources, and mental well-being are presented in Figure 2.\n\nTMSC=Transactional model of stress and coping; COR=Conservation of Resources; COT=Causality orientations theory; BPNT=Basic Psychological Needs Theory.\n\n\n4. Discussion\n\nMuch of the existing literature on doctoral education addresses students’ stress and mental health problems (Barry et al., 2018; Darley, 2021; Levecque et al., 2017; Marais et al., 2018). It is evident from the review that the existing studies need a precise classification of the doctoral program demands, resources and their interaction effect on the students’ well-being. This section discusses possible research opportunities under the theoretical purview and presents propositions that may encourage future research.\n\nConcept 1\n\nAs highlighted, doctoral students undergo a tremendous amount of stress due to intrapersonal regulation, lack of supervisors’ support, completion timeline (Cornwall et al., 2019), publication targets (Haven et al., 2019), and work-life conflict (Sufyan & Ali Ghouri, 2020). Although a significant number of articles iterates that job demands are the major stress-causing factors, surprisingly, only a few studies found the differentiating nature of job demands in the doctoral program setting (McCauley & Hinojosa, 2020). Thus, based on the transactional theory of stress, the current review has clarified doctoral students’ challenge-hindrance demands that impact their mental health through the job strain as a mediator.\n\nHence operationalization of challenge and hindrance demands is proposed as follows: 1) challenge demands of doctoral programs, motivate, and inspire doctoral students to engage in research to produce high-quality research output. Moreover, 2) hindrance demands act as a constraint and impede the quality of research output. As per the proposed definition, there are two sub-categories of challenge demands: doctoral research complexity and workload demands. The categories of hindrance demands are research ambiguity demands and resource inadequacies demands. The operationalization of the challenge and hindrance demands in doctoral education are postulate as below:\n\nHigher ambiguity in the doctoral program structure and insufficient resources in the program journey situate more stressful for the doctoral students, which hinders their well-being.\n\nGreater complexity and responsibility in the doctoral program foster the study engagement of the doctoral students that internally lessens the burnout.\n\nConcept 2\n\nThe current review considered Bronfenbrenner’s ecological framework to acknowledge the multi-faceted resources required for doctoral programs. With this framework, the current reviewers proposed that contextual (supervisor, institution, family) and personal (intrinsic motivation) resources, boost the doctoral students’ well-being. Waight & Giordano (2018) has highlighted the external resources in the non-academic context as online support, family, and mentor. Pervez et al. (2021) reported that support from supervisor and peers are essential in diminishing the impact of depression and anxiety among students. The study by Cassens et al. (2014) reported that support from supervisors, institutions, and co-workers mediate the relationship between perceived stress and engagement. It is surprising to see existing literature on doctoral programs independently emphasizing contextual and personal resources. Thus, the current review attempts to understand the combined effect of contextual and personal resources that enhance students’ well-being. The study has reviewed the role of supportive resources as a moderator between high demands and well-being of the student. With this, we propose the following research proposition by considering the contextual resource of doctoral programs:\n\nSufficient supervisor, institutional and social resources during the doctoral journey diminish the stressful demands by protecting against future resource loss and enhancing the doctoral students’ well-being.\n\nConcept 3\n\nMuch of the existing literature on doctoral programs covered the positive influence of intrinsic motivation on students’ mental health, satisfaction, and productivity (Kemp et al., 2014; De Clercq et al., 2021). Based on SDT articulation, Litalien & Guay (2015) empirically validated that autonomous orientation among doctoral students positively predict the program satisfaction, well-being, performance and is negatively associated with anxiety, turnover intention, and health impairment. Despite the 11 studies borrowed from the SDT to understand the significance of intrinsic motivation, there are only two studies have witnessed the autonomous orientation as an independent variable in predicting the well-being of doctoral students (Litalien & Guay; Litalien et al., 2015). However, the literature ignored the moderating effect of intrinsic motivation in the presence of high doctoral demands. Thus, future research can also explore how the autonomous orientation moderates the influence of stress on doctoral students’ well-being. Hence, we posit the following third proposition with the significance of intrinsic motivation:\n\nA sense of higher autonomy emphasizes greater intrinsic motivation and lessens burnout among doctoral students. A greater level of doctoral students’ self-determination helps to achieve higher engagement that foster their well-being.\n\nDuring the integrative review, we understood that most ofthe studies were conducted on doctoral education in the UK (56%), followed by the USA (34%), and only 10% are from Asia. Since the significant studies are from USA and UK, the context-specific factors could be changed. Levecque et al. (2017) have reported significant overlap in doctoral education characteristics in Asian countries, the UK, the USA, and the universities across the globe, such as scholarship, enrolment protocol for the doctoral program, intensive course work, supervisors’ roles, and time-to-degree, with the notable difference in terms of fees and publication requirements. The review results align with the results that it is vital to design standard instruments to validate the differentiated demands and motivational interventions to support the mental health of doctoral students is vital (Sufyan & Ghori, 2020). The theoretical underpinning of each construct has been reported in Table 2.\n\nPrior studies on JD-R model have limited their analysis to explain the influence of demands and resources on organization outcomes such as service quality and organizational commitment. Also, its impact on individual outcomes includes in-role and extra-role behaviours, creativity, and job satisfaction. There are limited studies that examined the effect of demands-resources on well-being of the individuals using JD-R model. However, future research could empirically test the influence of demands-resources on the mental health of the students in a learning context. Second, future studies can explore the effect of the demands and resources on physical health in a highly demanding environment as it reported sleep disturbance, gastrointestinal problems, frequent headaches, and eyestrain among individuals. Third, future studies can develop the framework for doctoral students’ well-being by considering the job crafting technique (Demerouti, 2014). Here researchers are “proactive crafters” of their studies by initiating the changes in demands and resources so that they can engage themselves in their study. In line with job crafting, researchers can improve their interpersonal relationships with supervisors, peers, and scholarly communities, and learn various methodologies and academic writing skills through online collaborations.\n\nFourth, the mental health of doctoral researchers has considered only two factors of Ryff’s six-factor psychological well-being model, i.e., autonomy and positive relationships (Ryff, 1989). Researchers are mandated with a high degree of autonomy, aware of the core domain knowledge, and develop a research identity. Thus, future studies might consider adopting a holistic definition of Ryff’s model to explain the mental health of the researchers.\n\nThe proposed framework on doctoral students’ mental health serves as an implication for institutions, doctoral educators, and practitioners by incorporating the challenge demands in their doctoral education. Institutions are suggested to frame the workload of the doctoral program at the beginning of the journey that a student needs to complete and inform the students about the rationale behind the rigorous journey. Here institutions are expected to be reasonable towards students while allocating the teaching workload, providing research training, engaging administrative work, number and quality of journal publications. Doctoral programs are bound with many responsibilities that ensure the students to learn varied competencies and skills requirements throughout the journey. This research complexity is addressed by providing professional development training such as advanced statistics workshops, sessions on writing for high-impact journal publications, substantial research methodologies, and dealing with the cumbersome publication process. At the beginning of the doctoral journey the institutions need to communicate precisely with the students about the accomplishment of program responsibilities that include presenting research output at the conference, managing the research fund, and personal expenses, developing required research competencies, learning, and adopting new methodologies of the research.\n\nDoctoral educators could assist the students in delivering the resources at three levels: institutions, supervisors, and peers. Institutions could provide funding for doctoral students to participate in professional conferences, doctoral research colloquiums, and statistical analysis workshops. Offering a research infrastructure such as separate workspaces, essential research tools, software, and databases boosts the students’ morale. Interventions such as mindfulness, stress and time management workshops, and workshops that are designed to build research resilience would assist students’ emotional and psychological development. Supervisors should assist in accumulating the supporting resources by scheduling a weekly meeting with students to discuss their research progress, providing extensive, specific, and constructive feedback, and collaborating with experts in the student’s domain. Supervisors also can act as mentors by providing the student with emotional support, respecting the student’s both personal and academic commitments such as time for family, household chores, and exercise. Supervisors also can provide authorship credits to the students by involving them in other research projects, and being friendly with students helps to preserve and protect resources for the future. Sharing research ideas with peers, networking with peers from other institutions in the same domain, participating in various research groups, and socio-emotional support from the peers, motivates the student.\n\nInstitutions and supervisors are urged to promote doctoral students’ autonomous motivation to reduce mental distress. They recommend designing an intervention that creates and promotes interest for the doctoral students in their study topic. Supervisors could be trained to encourage the students’ psychological needs, which goes beyond the research project supervision.\n\n\nConclusion\n\nThe present integrative review developed a holistic framework of doctoral students’ mental well-being by integrating the insights from literature. From a theoretical perspective, our review has revisited the JD-R model by conceptualizing the nomological framework of demands and resources of doctoral students at the individual, supervisor, and institution levels. Due to a significant increase in mental distress among doctoral students, the study has synthesized the four critical predictors: challenges, hindrances, demands, and contextual-personal resources. Finally, the findings highlighted contributing variables for improving doctoral students’ mental health and implications for practice. Future researchers shall develop new empirical insights and apply suggested theories to understand how demands and resources interact in predicting the mental health of doctoral students.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nFigshare: Quality assessment checklist by Joanna Briggs Institute (JBI), for “A framework for doctoral education in developing students’ mental well-being by integrating the demand and resources of the program: An integrative review”, https://doi.org/10.6084/m9.figshare.22298995 (Acharya et al., 2023a).\n\nThis project contains the following extended data:\n\n- Quality assessment checklist by Joanna Briggs Institute (JBI)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 Public domain).\n\nFigshare: PRISMA checklist for “A framework for doctoral education in developing students’ mental well-being by integrating the demand and resources of the program: An integrative review”, https://doi.org/10.6084/m9.figshare.22300792 (Acharya et al., 2023b).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 Public domain).\n\n\nReferences\n\nAcharya V, Rajedran A, Shenoy S: Quality assessment checklist by Joanna Briggs Institute (JBI). 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Publisher Full Text" }	[ { "id": "184790", "date": "20 Jul 2023", "name": "Rebekah Layton", "expertise": [ "Reviewer Expertise Biomedical graduate education training and education", "training environment", "professional development resources/accessibility", "and mental health and well-being in graduate populations", "intersectional approached to equitable environments in academic and research culture", "doctoral and postdoctoral (PhD) career development" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall: This is a well-designed systematic review on a timely and important topic, examining mental health and well-being of trainees in doctoral education - a topic which was previously under-studied but is beginning to accrue a robust literature to allow for more complex multi-study examinations such as the current work. The authors embed their review in sound theoretical frameworks, taking a holistic view of both mental health (negative) and well-being (positive). The authors effectively enumerate factors in doctoral training which can contribute to mental health and well-being outcomes that have been studied. Another strength of this paper is the consideration and inclusion of the work of international scholars broadly and across academic settings. Important themes, implications, and recommendations are provided. While this work has excellent merit, the writing has consistent errors that obscure the important content the authors are trying to convey. Examples and suggestions for improving readability are included below. This is the main area of improvement needed to elevate this manuscript to a high-quality publication. I hope the authors choose to revise this manuscript to increase the readability and writing quality, which is the primary barrier to it currently being indexable work.\nAs this is a systematic review article, statistical analyses are not applicable; it is difficult to evaluate whether conclusions are adequately supported in some cases due to the errors in word choice and sentence structure - hence, if this can be revised to be more readable - it is likely this issue would be resolved. This has the potential to provide a valuable contribution to the literature with some additional revisions and careful editing.\nExample Edits/Wording Needed: 1. The word \"significant\" is (over) used in a number of places that are confusing to the reader - especially since the authors are not referring to statistical analyses - alternative word choices should be used whenever possible to avoid creating the impression that a statistical result is being described. The word significant/significance should be replaced throughout, to the greatest extent possible. For example:\nAbstract - \"has gained significant importance in recent years\"\n\n\"little is known about the significance of intrinsic motivation...\"\n\n-Although a significant number of articles iterates...\"\n2. A number of verbs are used out of context to the extent that the sentence no longer makes sense; in some cases, I could guess at the meaning I thought the authors intended, but in other cases the meaning was unclear.\nAbstract - \"findings divulge\"\n\n\"studies witnessed the the mental health problems of doctoral students..\"\n\n\"Although a significant number of articles iterates...\" - maybe consider: \"a number of articles demonstrate that...\" etc.\n\n\"studies witnessed...\"\n\n\"grounded on the JD-R model\"\n\n\"however there is lag of studies that insight the moderating role...\" both lag and insight are problematic in this sentence\n\n\"has been witnessed in the literature\"\n\nGenerally, other terms/phrases to consider could include: \"evidence suggests that...\" \"has been demonstrated by X studies\" \"has been shown to X\"\n\n\"The theory argues that doctoral student appraise the program demands depending on their subjective environment as challenge or hinderance demands\" - this is confusing and needs to be reworded - perhaps making doctoral student[s] plural, perhaps adding on how they view their subjective environment, perhaps adding \"the\" ahead of the word challenge - or perhaps changing to \"doctoral student appraisal of program demands depends on...demands change\"?\n\n\"in this connection, one may concur...\" - surmise, perhaps? (in this context \"connection\" does not make sense)\n\n\"....(COR), we explain the doctoral program is bound with resources along with the demands\" - unclear - maybe add \"explain that...\" and \"bound\" does not make sense - unsure what word is intended\n\n\"...the program journey situate...\" - replace situate\n\n\"foster the study engagement...\" - replace foster\n\n\"based on SDT articulation...\" - replace articulate\n\n\"despite the 11 studies borrowed from the SDT to understand the significance of intrinsic motivation, there are only two studies have witnessed...\" - \"borrowed\" does not make sense, \"witness\" does not make sense, there are/have witnessed grammar/tense do not match... revise sentence\n\nProposition 3 needs to be reworded - \"higher autonomy\" does not make sense - \"greater autonomy,\" perhaps? - emphasizes does not make sense - \"amplifies\" or \"increases,\" perhaps? - \"a greater level of ...\" does not make sense, \"more autonomous feelings...\" or \"greater perception of self-determination by graduate students...\" perhaps? - \"foster\" in the current context should be plural - and both sentences need to be revised, currently its guesswork to try to figure out what the authors mean here.\n\n3. There are a number of consistent grammatical errors, such as errors in plural versus singular nouns/verbs and missing or incorrect prepositions. These make it very hard to follow the flow and meaning of sentences in some cases and need to be revised. Examples are included below, but the manuscript should be checked carefully for grammar, sentence structure, and wording choice throughout.\nAbstract - \"theoretical underpinnings of demands-resources and mental well-being are reported\" - is there a word missing here - do the authors mean the demands-resources framework/model?\n\nsimilarly, \"jobs demands and resources\" throughout - add model/framework or rework the sentence to use the singular form if generally referring to the construct and not the model\n\n\"viz\" - unsure if the authors mean \"vis-à-vis,\" perhaps?\n\n\"resulted in unpleasant experience in...\" - unpleasant does not make sense in this context\n\n\"these demands are conceptualized as 'two-factor stress model' or 'differentiated demands model'\" - unclear if words are missing, perhaps \"the\" ahead of each model - or perhaps \"as part of\" - or maybe just awkward wording?\n\n\"lack of infrastructures\" should be singular not plural\n\n\"unlike other resources, the intrinsic motivation is a significant...\" - \"the\" should be removed along with \"significant\" (see earlier comment above)\n\n\"in turn, improves the health of the doctoral students...\" should remove second \"the\"\n\n\"through the job strain as mediator\" - remove \"the\"\n\n\"Greater complexity and responsibility in the doctoral program foster the study engagement of the doctoral students that internally lessens the burnout\" - unclear what is meant by \"the study engagement\" - do the authors mean engagement is improved by autonomous motivation to engage in the students' own research? Clarify/rephrase\n\n\"Thus future research can explore how the autonomous orientation motivates....\" - remove \"the\"\n\n\"There are limited studies that examined the effect of demands-resources on wellbeing...\" grammatically confusing, revise - \"have examined the interaction of demand and resources,\" perhaps? - or \"have examined the demands-resources model in the context of...\"?\n\n\"Researchers are mandated with a high degree of autonomy...\" - \"mandated\" does not make sense, reword - \"tend to display a high degree,\" perhaps?\n\n\"The proposed framework... serves as an implication...by incorporating challenge-demands in their doctoral education\" - has implications perhaps? - incorporating the challenge-demands framework perhaps? - very confusing sentence as currently written.\n\n\"Institutions are suggested to frame...\" - different word needed - \"Recommendations for institutions include reframing the workload...\" perhaps?\n\nOther wording suggestions:\n\n\"Scope for future\" more commonly referred to as \"Future Directions\" as the header title\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": [ { "c_id": "9941", "date": "24 Jul 2023", "name": "Vrinda Acharya", "role": "Author Response", "response": "Title of the article \"A Framework for doctoral education in developing students’ mental well-being by Integrating the Demands and Resources of the Program: An integrative review\" Appreciate your constructive feedback and detailed report on the article's improvement. The author team will definitely work on your suggestions and resend the revised article at the earliest. We will work rigorously on grammar, sentence structure, and wording choice of the article. Thank you once again for the recommendations and suggestions. Regards Vrinda" } ] } ]	1	https://f1000research.com/articles/12-431
https://f1000research.com/articles/11-740/v1	04 Jul 22	{ "type": "Research Article", "title": "Methylene blue sentinel lymph node biopsy for breast cancer learning curve in the COVID-19 era: How many cases are enough?", "authors": [ "Yohana Azhar", "Birgitta M. Dewayani", "Kiki Lukman", "Birgitta M. Dewayani", "Kiki Lukman" ], "abstract": "Background: Sentinel lymph node biopsy (SLNB) is now the gold standard procedure for early breast cancer with clinically negative lymph nodes (N0). According to the Indonesian Board-Certified oncologist surgeon, the learning curve for evaluating fellow breast surgeons to achieve this competency could have been shorter due to the COVID-19 pandemic. This study aims to see if the learning curve for sentinel lymph node (SLN) identification can be shortened and if imprint cytology (IC) can replace frozen sections (FS) for intraoperative analysis. Methods: Fellow breast surgeons were taught to perform SLNB on breast cancer patients. Intraoperative assessment and completion of axillary lymph node dissection (ALND) were performed in the first setting for standardization with the attending surgeon. Sentinel lymph node (SLN) identification was plotted on cumulative sum chart (CUSUM) limitations for evaluating the variability competency between attending surgeon and fellow surgeon based on a target identification rate of 85%. In addition, the accuracy of imprint cytology versus frozen section for identifying lymph node metastases was compared. Results: Consecutive 50 SLNBs were conducted during this period with attending and trainees split into two groups. After 13 consecutive tests, the CUSUM plot positively identified SLN as a significant achievement level of competency. Imprint cytology was shown to be inferior to frozen section cytology. The accuracy of imprint cytology is 91.8%, while the accuracy of frozen sections is 95.9%. Conclusion: According to a CUSUM chart based on a reasonable set of parameters, the learning curve for SLNB using methylene blue dye is reached after 13 consecutive positively detected SLN. Meanwhile, the frozen section is still the gold standard for determining the disorder of axillary lymph nodes, but the accuracy between the two methods can be comparable.", "keywords": [ "Breast Cancer", "Frozen Section", "Methylene Blue", "Selective Lymph Node Biopsy" ], "content": "Introduction\n\nSentinel lymph node (SLN) biopsy has replaced axillary lymph node dissection (ALND) as the standard minimally invasive staging procedure in patients with clinically node-negative disease.1–3 Dual tracers, such as blue dye and radiotracer mapping, are recommended in the Asia Pacific and Europe to achieve a higher SLN identification rate than blue dye alone. However, because radiotracer mapping is more expensive and has several disadvantages, methylene blue dye as a single agent is well-tailored to use in developing countries without significantly compromising test quality.4–6\n\nBoth the Indonesian Board-Certified oncologist surgeon and the American Board-Certified surgeon recommend that at least 20 SLNB procedures be performed under the supervision of an attending physician to achieve competency, with a false-negative rate of no less than 5% and an SLN identification rate of more than 85%. Given that the SLN identification rate is more susceptible to technical failure, it is now a more reliable estimate for learning curve analysis. The Indonesian Board-Certified oncologist surgeon admitted that the learning curve used to assess the fellow surgeon objectively might be shorter, especially during the COVID-19 pandemic, when surgery volume and timeframe should be reduced.6 A method for plotting learning curves that can be used to check and predict the performance of others.7,8\n\nIt is necessary to develop a method for plotting learning curves to test and predict individual performance concerning a standardized degree of proficiency. Cumulative sum chart (CUSUM) plots are an excellent method for determining learning curves for any technique with specific and difficult output variables. They will be able to customize qualifications, knowledge, and skill certification criteria and deal with training issues throughout the COVID-19 pandemic.9\n\nThe presence of nodal metastasis during the SLNB procedure can be determined by frozen section and imprint cytology. Although a few studies have shown that a frozen section is more accurate in determining lymph node metastasis during SLNB, many have claimed the advantages of imprint cytology in this setting.10–13 In this study, we compare the usefulness of imprint cytology and frozen section in the intra-operative diagnosis of sentinel node metastasis.\n\nCUSUM analysis was used on the SLNB learning curve as a retrospective quality control tool, but it is not suitable for prospective learning curve analysis. For SLNB, other learning curve methods were often used. In this study, a CUSUM control chart is used to prospectively compare the learning curve of a fellow surgeon for SLNB and check it as an accomplishment level of qualifications and relate it with an attending surgeon using methylene blue dye as the visualization agent.9\n\nFrozen section (FS) analysis has become more prominent for intraoperative assessment; however, it takes more time, tissue loss, and expense than imprint cytology. The notified accuracy rates for each method in the literature are roughly equivalent.13,14 This research focuses on determining whether a SLN identification learning curve could be shortened, especially during the COVID-19 pandemic, and whether imprint cytology can substitute frozen sections for intraoperative evaluation. We conducted this study in accordance with The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.\n\n\nMethods\n\nAfter receiving approval from Hasan Sadikin General Hospital's Ethics Committee (no. LB.02.01/X.7.4/272/2021). Written informed consent forms were obtained from the patients.\n\nWe conducted a prospective, cross-sectional study between January and July 2021 at Hasan Sadikin General Hospital, Bandung, West Java, Indonesia. Consecutive 50 SLNB (operable primary tumor less than 5 cm and clinically negative ipsilateral axilla) was conducted by attending and fellow surgeons during the research period. As the International Board stated that competency could be achieved after 20 cases, the fellow was given 25 cases to be evaluated. We compared the result with the attending surgeon, who had already experienced this procedure for ten years.\n\nFollowing anesthesia induction, five milliliters (ml) of 0.1% methylene blue (Methylene blue S.A.L.F 1% ® for injection) were infiltrated into the subareolar tissue, a significantly higher identification rate than the subareolar tissue the average in the other sites. The breast was massaged for about five minutes, and the surgical sites were prepared. In the breast-conserving surgery procedure, the surgent went to the axilla to make an axillary incision and performed SLNB. The SLNB would be done after creating a superior flap when the procedure is mastectomy. The definitive procedure will begin 10-15 minutes after the massage. All blue nodes were removed, and any node received a blue lymphatic channel. After removing the blue nodes, the surrounding tissue was checked, and any remaining hard or large nodes were included in the specimen.\n\nIn all cases, the specimen was cut in the cryostat and subsequently stained with hematoxylin and eosin (H&E) for frozen section (FS) analysis and assessed by two cytopathologists. The rest of the tissue, as well as the FS blocks, were embedded in paraffin. Three slides were obtained from each block, and two or three sections were stained with H&E. The results of both imprint cytology (IC) and FS examinations of the SLNB(s) were sent to the surgical team during surgery. The result of the H&E examination was also sent to the surgery department after the operation.\n\nA total of 50 cases with an established diagnosis of breast carcinoma through core biopsy, a Tis, T1, or T2 primary breast tumor, and clinically no ipsilateral axilla lymph nodes were prospectively selected between January 2021 and July 2021.\n\nALND was completed on a total of 50 cases. Since this is the first study in Indonesia, the authors believe that definitive treatment of the axilla at this stage should not vary from the established standard treatment, as approved by Hospital Ethics Committee Board. Following that, patients were to have direct ALND only if the SLN was positive on intraoperative assessment or could not be identified or if an initially negative SLN turned out to be positive on the paraffin slice. The fellows who had established their SLN mapping technique by participating a training course for one week were included in this research.\n\nWe compared the ability of the fellow to make an identification of blue nodes after SLNB was done in 15 minutes as per Indonesia Board Certified Breast Surgeon guidelines.6\n\nThe following characteristics were then listed in the special form: surgeons (fellow and attending surgeon), mastectomy versus breast-conserving surgery, site of injection, identified SLN, number of SLNs, Berg’s level at which SLN was found, and a number of non-SLNs removed. The unfixed nodes were sent to the pathologist. The operation, which included ALND, was then completed. The same pathologist did all pathological analyses.\n\nNodes were sliced at 3mm intervals. Frozen section and imprint cytology were performed on more than 1cm in size nodes. For nodes smaller than 1cm, only imprint cytology was done. All the nodes were routinely processed for permanent paraffin sections, and immunohistochemistry was performed if the paraffin block examination was inconclusive.\n\nThe pathologist documented the following variables on the hospital form: SLN cytology (positive or negative), SLN frozen section, SLN paraffin sections, and ALND paraffin block sections.\n\nA CUSUM analysis was completed for the ability to identify the blue node during SLNM, and duration times were needed to identify the blue node. The results were presented in the CUSUM chart, a graphical presentation of a series of consecutive procedures performed by attending and fellow. The CUSUM plot shows randomly at or above the horizontal line at an acceptable level of performance. Nevertheless, the slope will be upward and cross the decision interval when the operation is performed at unacceptable level. The sloping plot represents the surgeon’s process of mastering a new skill.\n\nThe sensitivity, specificity, negative predictive value, positive predictive value, and accuracy were determined for the frozen section analysis alone, and imprint cytology alone compared with final pathology results from the paraffin block as the gold standard. Sensitivity was defined as the positivity rate by the given assessment and the final pathological diagnosis. Specificity was defined as the rate of negativity by the given assessment and final pathological diagnosis. The negative predictive value was defined as the rate of negativity by the given assessment and the final pathological diagnosis.\n\n\nResults\n\n50 SLNB procedures were performed during the study interval. Two fellows performed 25 SLNB during this research and three attending performed 25 cases. Quality indicators for successfull SLN mapping include identification rates (ID), false negative rates (FNR), the time need to identification, and complication rates. We compared the pattern of attending and fellow while doing SLNB (Figure 1).\n\nAS – attending surgeon; FS – fellow surgeon; LCL – lower cut limit; UCL – upper cut limit.\n\nThe blue line represents the attending surgeon, and the orange line represents the fellow surgeon. The yellow lines show the upper decision boundary H1 (if the learning curve crosses H1 from below, it means that the failure is more remarkable than expected and that the process is out of control, and associations must be found) while the blue lines show the lower decision boundary H0 (if the learning curve crosses H0 from above, it means that the measured percentage failure does not differ from the acceptable failure rate with a type1 error equal to, and a type 2 error equal to). When the student crosses the H0 boundary, it is considered that the proficiency level has been achieved for the assessed procedure. As we can see after the initial 13 cases, the trainee's performance to assess the blue node in the SLNB procedure is relatively flat, and competency was achieved. There was a significant difference (p<0.05) in how the attending and fellow surgeons completed that SLNB but, as we can see on the graph, the blue and orange lines are still acceptable because they never crossed the boundaries.9,15\n\nThe results of the frozen section examination show sensitivity 97.5%, specificity 11.1%, positive predictive value (PPV) 97.5%, negative predictive value (NPV) 88.9%, and accuracy value 95.9% (Table 1). This means that the examination of the number of lymph nodes using the frozen section is accurate enough to be able to predict the patient's lymph nodes. The imprint examination results are shown with sensitivity 97.4%, specificity 27.3%, PPV 92.5%, NPV 88.9%, and accuracy value 91.8%.\n\n\nDiscussion\n\nThe SLNB is one of the essential procedures in breast cancer surgery that oncologist surgeons should master during their training. Many criteria influence whether a surgeon can do a particular operation, including their medical knowledge, specific training, and level of expertise. Under the supervision of a supervisor, the skill to perform a surgical technique is usually acquired through a process of observation, learning, and repetition.\n\nIn general, a combination of informal assessment and peer review and more formal accreditation, credentialing, or privilege can be used to ensure the quality of medical practice. The assessment, review, or credentialing process is frequently subjective and lacks specific standards of practice. It has been suggested that comparative treatment result data on specific physician performance benchmarks are required to establish a single process's credibility.8\n\nThe CUSUM chart can be used as one tool to assess the level of competence and has been widely used to evaluate the achievement of the learning curve for some new procedures in surgical fields. The CUSUM curve is a control chart that can monitor shifts in the process mean. The target is the plot should not be widely variable from the reference value (attending performance).\n\nIn our result, we can see on Figure 1 that there wasn’t wide variability between the attending and fellow surgeons while doing this procedure. The required level of competence of fellow breast surgeons in Indonesia can be achieved in a shorter time. The fellow surgeon in this study comes from the general surgeon who has experience and is familiar with mastectomy procedures, including axillary clearance as a part competency that should be achieved to be a general surgeon in rural areas. Successful SLN biopsy depends primarily on accurate identification of the metatstatic route. Knowledge of the anatomy of the lymphatics is important. This accuisision part can be easily attained by fellow surgeons.\n\nHowever, there are several problems with using CUSUM analysis to assess performance in procedural skills. First, there are no nationally agreed definitions for success or failure at any given procedure, and those used in the literature vary greatly.\n\nThere is currently no consensus on where the acceptable and unacceptable boundaries should be set or to what degree alpha and beta errors should be tolerated. Tight boundaries are essential for quality control and for assessing trained individuals, but should these boundaries be much more comprehensive for the novice trainee to allow for their learning curve and to provide encouragement and a sense of achievement? The number of competent doctors produced can increase dramatically simply by altering the boundaries. Therefore, if procedural competency is to be defined by CUSUM, it would be necessary to establish national rates. These would need to be tailored to the trainee's experience.8,15\n\nSecond, in this research, we have not included the characteristic of patients in the analysis; the size of the tumor, location of the tumor, and type of surgery probably have influenced the achievement of the procedure.15 This assumption was based on our judgment that we only included breast cancer which restricted criteria to take part in this research.\n\nIntraoperatively, FS and IC can be used to evaluate SLNs. Although cytologic procedures are faster than FS and may not result in significant nodal tissue loss, they may be difficult to confirm because they rely on cytology material. Requiring FS would further limit the transferability of SLNB to tertiary hospitals and lengthen the duration of the procedure and the length of stay of the surgeon in the operating room. All surgery should be done in a quick in and out setting in the COVID-19 era. Imprint cytology has recently been shown to be equally effective as FS, as shown in our result. It turns out more specific than FS in detecting SLN metastatic activity when performed by skilled pathologists, particularly those with cytology competence. There were mixed results when it came to using IC instead of FS to detect lymph node metastases in SLNB.\n\nThe frozen section may provide information on metastasis size, but it results in tissue loss for permanent sections; it is a time-consuming and expensive technique that necessitates a cryostat and skilled professionals. Touch imprint cytology requires less work, is faster, and saves tissue for permanent sections; however, pathologists must be trained in cytology sample reporting. Intraoperative cytology yields rapid results with minimal artifacts. In cytology samples, however, the number of cells analyzed is less.14,16 Several studies comparing FS and IC in the intraoperative examination of sentinel lymph nodes found a substantial difference in sensitivity between 44% and 100% for FS and 34% to 95% for IC.17 However, the variations in methods used in intraoperative and permanent section histopathologic evaluations make it impossible to compare research with reliability. Tew et al., reviewed 31 papers that compared touch imprint cytology (TIC) and FS in the literature and found that touch imprint cytology had a sensitivity of 63% overall, with a pooled sensitivity of 81% for macrometastases and 22% for micrometastases.17 A similar meta-analysis of frozen section examination reported a sensitivity of 78% overall, with 94% for macrometastases and 40% for micrometastases. In most investigations that compared IC and FS, there was no statistically significant difference between the two methods despite FS having higher sensitivity.18–20 Inadequate sampling causes decreased TIC sensitivity, which can be solved by increasing the number of slides used in touch imprint cytology. This can improve the method's sensitivity without losing tissue for permanent histological examination. The high specificity of both the frozen section and intraoperative cytology approaches revealed that both procedures have low false-positive rates. Low nuclear grade metastatic tumors, particularly lobular carcinomas, have higher false-negative rates for both modalities of intraoperative SLN examination because the tumor cells are small and poorly cohesive. The authors compared the accuracy of the two approaches and discovered that imprint appears to be inferior for identifying the metastatic process in SLNB. By using CUSUM analysis we are able to analyze learning curves and threshold criteria for relevant individual achievement to mastering SLNB using methylen blue. Importantly, CUSUM analysis can be applied to assess these end points. A widely accepted methological approach in SLNB. Future studies are needed to validate the CUSUM analysis as a potential tool for defining milestones and competence benchmark that can be used to credentialing certification.\n\n\nConclusion\n\nUsing the CUSUM chart, a reasonable choice of other parameters shows that experienced breast surgeons have completed the SLNB learning curve after 14 successful methylene blue attempts. In the presence of attending, this form of learning curve analysis can be applied to fellow surgeons by utilizing a proxy measure for failure, such as failure to identify the SLN.\n\nIn terms of detecting SLN metastasis, IC appeared to be more specific than FS. Although IC is a simple and accurate method for screening SLN in breast cancer patients during surgery with high accuracy, FS remains the gold standard for detecting sentinel lymph node metastases.\n\n\nData availability\n\nZenodo: Underlying data for ‘Methylene Blue Sentinel Lymph Node Biopsy for Breast Cancer Learning Curve in Covid-19 era: How many cases are enough?.’ https://doi.org/10.5281/zenodo.6442807.21\n\nThis project contains the following underlying data:\n\n- Manuscript analysis.xlsx (underlying dataset for 50 procedures and analysis)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReporting guidelines\n\nZenodo: STROBE checklist for ‘Underlying data for ‘Methylene Blue Sentinel Lymph Node Biopsy for Breast Cancer Learning Curve in Covid-19 era: How many cases are enough?.’ https://doi.org/10.5281/zenodo.6442807.21\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 Dr. Ricarhdo Valentino Hanafi for arranging this manuscript. We are keen to make our data, analytic methods, and study materials available to other researchers. The data are available in the Data availability section.\n\n\nReferences\n\nClarke D, Khonji NI, Mansel RE: Sentinel node biopsy in breast cancer: ALMANAC trial. World J. Surg. 2001; 25(6): 819–822. PubMed Abstract \| Publisher Full Text\n\nHunt KK, Ballman KV, McCall LM, et al.: Factors associated with local-regional recurrence after a negative sentinel node dissection: results of the ACOSOG Z0010 trial. Ann. Surg. 2012; 256(3): 428–436. PubMed Abstract \| Publisher Full Text\n\nKrag DN, Anderson SJ, Julian TB, et al.: Sentinel-lymph-node resection compared with conventional axillary-lymph-node dissection in clinically node-negative patients with breast cancer: overall survival findings from the NSABP B-32 randomized phase 3 trial. Lancet Oncol. 2010; 11(10): 927–933. PubMed Abstract \| Publisher Full Text\n\nLi J, Chen X, Qi M, et al.: Sentinel lymph node biopsy mapped with methylene blue dye alone in patients with breast cancer: A systematic review and meta-analysis. PLoS One. 2018 Sep 20; 13(9): e0204364. PubMed Abstract \| Publisher Full Text\n\nBrahma B, Putri RI, Karsono R, et al.: The predictive value of methylene blue dye as a single technique in breast cancer sentinel node biopsy: a study from Dharmais Cancer Hospital. World J. Surg. Oncol. 2017 Feb 7; 15(1): 41. PubMed Abstract \| Publisher Full Text\n\nKonsil Kedokteran Indonesia: Peraturan Konsil Kedokteran Indonesia Nomor 78 Tahun 2020 tentang Standar Pendidikan profesi Dokter Subspesialis Bedah. Konsil Kedokteran Indonesia;2020.Reference Source\n\nClough KB, Nasr R, Nos C, et al.: New anatomical classification of the axilla with implications for sentinel node biopsy. Br. J. Surg. 2010; 97(11): 1659–1665. PubMed Abstract \| Publisher Full Text\n\nMaguire T, Mayne CJ, Terry T, et al.: Analysis of the surgical learning curve using the cumulative sum (CUSUM) method. Neurourol. Urodyn. 2013; 32(7): 964–967. Publisher Full Text\n\nPetropoulou T, Kapoula A, Mastoraki A, et al.: Imprint cytology versus frozen section analysis for intraoperative assessment of sentinel lymph node in breast cancer. Breast Cancer Targets Ther. 2017; 9: 325–330. PubMed Abstract \| Publisher Full Text\n\nLiang R, Craik J, Juhasz ES, et al.: Imprint cytology versus frozen section: intraoperative analysis of sentinel lymph nodes in breast cancer. ANZ J. Surg. 2003; 73(8): 597–599. Publisher Full Text\n\nHoda SA, Brogi E, Koerner FC, Peter P: Rosen’s Breast Pathology.2017; Vol. 4.\n\nTurner RR, Hansen NM, Stern SL, et al.: Intraoperative examination of the sentinel lymph node for breast carcinoma staging. Am. J. Clin. Pathol. 1999; 112(5): 627–634. Publisher Full Text\n\nVan Diest PJ, Torrenga H, Borgstein PJ, et al.: Reliability of intraoperative frozen section and imprint cytological investigation of sentinel lymph nodes in breast cancer. Histopathology. 1999; 35(1): 14–18. PubMed Abstract \| Publisher Full Text\n\nHoda SA, Resetkova E:Pathologic Examination of Breast and Lymph Node Specimens, Including Sentinel Lymph Nodes. Rosen’s breast pathology. 4th ed.2017; p. 1263–336.\n\nWoodall WH, Rakovich G, Steiner SH: An overview and critique of the use of cumulative sum methods with surgical learning curve data. Stat. Med. 2021; 40: 1400–1413. PubMed Abstract \| Publisher Full Text\n\nOmranipour R: Intraoperative Evaluation of Sentinel Lymph Nodes by Touch Imprint Cytology Technique in Breast Cancer Patients. Annu. Res. Rev. Biol. 2014; 4(24): 3751–3757. Publisher Full Text\n\nTew K, Irwig L, Matthews CP, et al.: Meta-analysis of sentinel node imprint cytology in breast cancer. Br. J. Surg. 2005; 92(9): 1068–1080. PubMed Abstract \| Publisher Full Text\n\nCreager AJ, Geisinger KR, Perrier ND, et al.: Intraoperative Imprint Cytologic Evaluation of Sentinel Lymph Nodes for Lobular Carcinoma of the Breast. Ann. Surg. 2004; 239(1): 61–66. PubMed Abstract \| Publisher Full Text\n\nFrancz M, Egervari K, Szollosi Z: Intraoperative evaluation of sentinel lymph nodes in breast cancer: Comparison of frozen sections, imprint cytology, and immunocytochemistry. Cytopathology. 2011; 22(1): 36–42. PubMed Abstract \| Publisher Full Text\n\nPerhavec A, Besić N, Hocevar M, et al.: Touch imprint cytology of the sentinel lymph nodes might not be indicated in early breast cancer patients with ultrasonically uninvolved axillary lymph nodes. Ann. Surg. Oncol. 2008; 15(8): 2257–2262. Publisher Full Text\n\nAzhar Y, Dewayani BM, Lukman K: Methylene Blue Sentinel Lymph Node Biopsy for Breast Cancer Learning Curve in Covid-19 era: How many cases are enough? [Data set]. Zenodo. 2022. Publisher Full Text" }	[ { "id": "148146", "date": "08 Sep 2022", "name": "Dedy Hermansyah", "expertise": [ "Reviewer Expertise Advanced oncologic care", "breast cancer", "general surgery", "immuno-oncology science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI acknowledge that the design and aim of this study are interesting to be presented in a global oncologic forum since the accelerated learning curve of oncologist trainees should provide better care to society. However, I also believe the design of this study creates multi-focus research, due to both the SNLB's procedure competency by CUSUM chart learning curve and diagnostic accuracies of imprint cytology (IC) being analyzed. Hence, it is recommended to draw a fine-connecting line between those dots, whether is there any direct correlation between CUSUM results and ICs results? Is it by the usage of methylene blue only as a single-tracer?\nUtilization of methylene blue in SNLB biopsy to accelerate the learning curve among fellow surgeons in this biopsy is evidently favorable. Thus the need to apply dual tracers analysis in this study or fellow training can be abolished, especially in the settings of limited resources. However, the authors may need to explain more the methylene blue's role in both diagnostics tools, since this study concluded that FS is statistically better than ICs.\nThe idea of applying CUSUM to accelerate the learning curve had been numerously implemented in several centers, e.g., by Park et al.,1 in the video-assisted mini-laparotomy partial nephrectomy skill learning or Ospina et al.,2 in anesthetic skills training. Similar accelerated outcomes were also reported, with fewer case-by-case learning rates necessary to reach specific competencies; even by the nationally established standards for breast surgeon fellows3,4.\nNevertheless, applying the CUSUM analysis method may provide a prospective comparison of learning curve improvement, especially in the surgical- or procedure-related learning processes. Still, the fact that the authors decided to imply additional study on the comparison of IC and frozen-section (FS) unquestionably raises a question; where does the correlation lie between those analyses?\n\nThe first analysis of the CUSUM chart did provide substantial results to be analyzed. However, I believe the authors should elaborate on the figure in more depth since I did not find any acceptable condition of why a p-value of <.05 is considered meaningful. What are the meaningful outcomes here? Is it the difference between attending and fellow surgeons? Thus if the differences were substantial, does it delineate that there was a \"substantial\" difference as well between both arm's skills? or it aimed to depict a significant result by reducing the need for SNLB procedure from 20 (national standard) to 13, especially during special conditions such as the COVID-19 pandemic? Please kindly re-elaborate it in the result section.\n\nThe authors also need to fix grammatical and rephrasing issues thoroughly, since I believe it is uneasy to understand the work at the first glance.\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": "9530", "date": "29 Aug 2023", "name": "Yohana Azhar", "role": "Author Response", "response": "Thank you for your valuable input. After discussing with our team, we decided not to combine the two studies but only focus on CUSUM analysis research to assess how many cases the minimum trainee must achieve in mastering the Sentinel Lymph Node Biopsy technique using a single tracer Methylen Blue by increasing the number of samples and re-analyzing. Please find the new version of analysis on this site." } ] }, { "id": "155578", "date": "28 Nov 2022", "name": "Tony Mallett", "expertise": [ "Reviewer Expertise Breast surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAttaining technical proficiency in sentinel lymph node biopsy with patent blue alone is clearly highly relevant to the region of practice. As mentioned in the authors discussion section, determining measures of success and failure of this technique is challenging. We have included some issues below that should be clarified.\nIt would be valuable to have more data included such as the number of nodes removed for each case, how many were sentinel & non-sentinel, distribution of tumour involvement by sentinel & non-sentinel nodes. This would show how the sensitivity/NPV for blue dye alone were calculated for the whole series. It would also be useful to compare these results for fellow and attending cases to assess for any differences in accuracy, which is the end goal of sentinel lymph node biopsy rather than simply number of nodes removed.\n\nWith regards to CUSUM reasoning for the upper and lower cut limits should be included. At -1 lymph nodes it is clear that the lower limit could never be crossed. Is zero nodes deemed a failure? The upper cut limit of 4 does correspond to the generally accepted maximum number of sentinel nodes to be removed, is that how it was selected? This should be made clear in methodology.\n\nA comment on any complications for the fellow & attending series of cases should also be included.\nWith the addition of the above attaining proficiency may be shown in terms of accuracy of results & complication rate in addition to the efficiency of finding blue sentinel lymph nodes within the 15 minute time frame.\nIt may be useful to review the SNAC trial1 to see how the authors presented their methodology & 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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9531", "date": "29 Aug 2023", "name": "Yohana Azhar", "role": "Author Response", "response": "Thank you for your valuable insight. After long discussion with the team in this research we focus on CUSUM analysis to get new number regarding the achievement of trainee learning SLNB using methylene blue only. In this regard To avoid bias we only included trainee had already obtained internal validation in performing SLNB, including doing 20 SLNB procedures with identification rates above 95% and false negative rates below 5%. We submit new version how CUSUM analysis be concluded in this site in detail. No systemic anaphylactic reactions and postoperative complications related to the subdermal injection of methylene blue, such as allergic reactions and skin and parenchymal necrosis, occurred throughout the study. Patients who underwent BCS exhibited blue skin staining in the skin around the injection site, which remained for approximately 1–2 months after the procedure. We didn’t mention this because we thought this topic is not the aim of study." } ] } ]	1	https://f1000research.com/articles/11-740
https://f1000research.com/articles/10-274/v1	06 Apr 21	{ "type": "Research Article", "title": "Nonlocal contrast calculated by the second order visual mechanisms and its significance in identifying facial emotions", "authors": [ "Vitaly V. Babenko", "Denis V. Yavna", "Pavel N. Ermakov", "Polina V. Anokhina", "Vitaly V. Babenko", "Pavel N. Ermakov", "Polina V. Anokhina" ], "abstract": "Background: Previously obtained results indicate that faces are preattentively detected in the visual scene, and information on facial expression is rapidly extracted at the lower levels of the visual system. At the same time different facial attributes make different contributions in facial expression recognition. However, it is known, among the preattentive mechanisms there are none that would be selective for certain facial features, such as eyes or mouth. The aim of our study was to identify a candidate for the role of such a mechanism. Our assumption was that the most informative areas of the image are those characterized by spatial heterogeneity, particularly with nonlocal contrast changes. These areas may be identified in the human visual system by the second-order visual mechanisms selective to contrast modulations of brightness gradients. Methods: We developed a software program imitating the operation of these mechanisms and finding areas of contrast heterogeneity in the image. Using this program, we extracted areas with maximum, minimum and medium contrast modulation amplitudes from the initial face images, then we used these to make three variants of one and the same face. The faces were demonstrated to the observers along with other objects synthesized the same way. The participants had to identify faces and define facial emotional expressions. Results: It was found that the greater is the contrast modulation amplitude of the areas shaping the face, the more precisely the emotion is identified. Conclusions: The results suggest that areas with a greater increase in nonlocal contrast are more informative in facial images, and the second-order visual mechanisms can claim the role of filters that detect areas of interest, attract visual attention and are windows through which subsequent levels of visual processing receive valuable information.", "keywords": [ "face", "emotion", "saliency", "spatial heterogeneity", "nonlocal contrast", "second-order visual mechanisms" ], "content": "Introduction\n\nExperiments involving a saccadic task (Crouzet et al., 2010; Kirchner & Thorpe, 2006) and registration of event-related potentials (Cauchoix et al., 2014; Dering et al., 2011; Herrmann et al., 2005; Liu et al., 2002; Liu et al., 2009; Pitcher et al., 2007) showed that face identification and processing are so fast that we can most probably speak here of a feedforward processing directed by preattentive mechanisms (Crouzet & Thorpe, 2011; Reddy et al., 2004; Reddy et al., 2006; VanRullen, 2006; Vuilleumier, 2000; Vuilleumier, 2002; Vuilleumier et al., 2001), that is without any involvement of attention (Allen et al., 2017; Eimer & Holmes, 2002; Liu et al., 2002). This may mean that low level information is used to distinguish a face from the background and to define its characteristics.\n\nMany researchers believe that faces are holistically coded within the low-frequency range, and this description is sufficient not just to detect the face but also to determine its emotional expression (Calder et al., 2000; Schyns & Oliva, 1999; Tanaka et al., 2012; White, 2000). Meanwhile the classical work by A.L. Yarbus (1967) clearly demonstrated that while viewing a face we fix our eyes at quite definite details. Further eye tracking experiments and experiments with the “bubbles” method showed that not all areas of the face are equally useful for emotion recognition (Blais et al., 2017; Duncan et al., 2017). Different facial features are significant for the discrimination of different emotions (Atkinson & Smithson, 2020; Calvo et al., 2014; Eisenbarth & Alpers, 2011; Fiset et al., 2017; Jack et al., 2014; Smith & Merlusca, 2014; Smith & Schyns, 2009; Smith et al., 2005; Wang et al., 2011), these emotions being probably processed at different rates too (Ruiz-Soler & Beltran, 2012).\n\nThe problem is that the lower levels of the human visual system lack neurons which would be selective to certain facial features. Nevertheless, there should exist a mechanism permitting the detection of faces automatically and to extract significant information preattentively. The aim of this investigation was to identify the possible candidate for the above mechanism.\n\nRealization of the importance of defining those areas of interest in the images that attract visual attention, was the impetus for those research trends aimed at finding the algorithm of formation of saliency maps (Borji et al., 2013; Judd et al., 2012; Rahman et al., 2014). At the same time, the choice of the attention goal should be based on the principle of information maximization (Bruce & Tsotsos, 2005).\n\nIn respect of the human visual system, one can only speak of the preattentive mechanisms actualized within low-level vision and able of “bottom-up” attention control. It is clear that the evenly lit areas are of no interest to the visual system. Of interest is something changeable, hence we may speak of changes in brightness. Indeed, there are specialized mechanisms for finding brightness gradients in the visual system, and these are striate neurons (Hubel & Wiesel, 1962; Hubel & Wiesel, 1968). However, these can only find local heterogeneities. To find areas of interest, there should exist mechanisms beyond local operations. Yet we first have to answer the question about the characteristics of these nonlocal areas of interest. In recent years, there appeared a viewpoint stating that the image areas whose information content differs from the surroundings are of the greatest interest for the visual system (Baldi & Itti, 2010; Hou et al., 2013; Itti & Baldi, 2009). This refers to difference in low-level feature distribution in the field of view (Itti et al., 1998), while salience in this case is determined by the degree of total difference of features within the analyzed area from features in the surrounding area (Bruce & Tsotsos, 2009; Gao & Vasconcelos, 2007; Perazzi et al., 2012).\n\nImportant is that the visual system really has mechanisms able to find space heterogeneities of brightness gradients: these are the so-called second-order visual mechanisms (see review Graham, 2011). The latter combine the outputs of striate neurons, and their receptive fields are organized in such a way that they do not respond to homogeneous textures, but are activated when the texture has modulations of contrast, orientation, or spatial frequency of brightness gradients.\n\nSo far these mechanisms have been predominantly studied and considered as an instrument of segmentation of textures (e.g. Graham & Sutter, 2000; Graham & Wolfson, 2004; Kingdom et al., 2003; Schofield & Yates, 2005). We are pioneers in raising the question whether the second-order visual filters can be of use in segmenting natural images and finding in them those saliency areas that are used for categorization. Our expectation was to obtain the answer through the task of detecting faces in a series of successively presented objects and determining their emotional expression.\n\nIt was shown earlier that the second-order mechanisms are specific to the modulated visual feature, i.e. whether it is contrast, orientation or spatial frequency of brightness gradients (Babenko & Ermakov, 2015; Kingdom et al., 2003). Then it was revealed that modulations of contrast take priority in competition for attention (Babenko et al., 2020). All this enabled us to work out a hypothesis stating that areas of maximum modulation of nonlocal contrast contain information helpful in identifying emotional facial expressions. To test this hypothesis, we developed a software program (gradient operator of nonlocal contrast) imitating operation of the second-order visual filters and calculating the space distribution of contrast modulation amplitude in the input image.\n\n\nMethods\n\nA total of 38 students between the ages of 19 and 21 took part in this investigation. All the participants had normal or corrected to normal vision and reported no history of neurological or psychiatric disorders. All the research participants were informed about the purpose and procedures of the experiment; they all signed a consent form that outlined the risks and benefits of participating in the study and indicated that they believed in the safety of the investigation. The study was conducted in accordance with the ethical standards consistent with The Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved by the local ethics committee. The design of the experiment, the methodological approach, the conditions of confidentiality and use of the consent of participants were 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 25 March, 2020.\n\nInitial digitized photos of faces and objects brought to a single size (8 ang.deg.), medium brightness (35 cd/m2) and RMS contrast (0.45), were processed by the nonlocal contrast gradient operator. A total energy of the image filtered at a frequency of 4 cycles per a diameter of this central area with a 1 octave bandwidth, was calculated in the center of the operator’s concentric area. In the peripheral part of the operator (a ring whose width equaled the central area diameter), the spectral power of the entire range of spatial frequencies perceived by a person was calculated, per 1 octave on average.\n\nThe contrast modulation amplitude amounted to the difference of values of the power spectrum obtained in the operator’s central and periphery areas. Operators of various diameters were used, and for each operator we defined those areas where the total contrast was maximum different from the surroundings, i.e. had the highest modulation amplitude.\n\nThe algorithm of stimuli formation is shown in Figure 1. An initial image example can be seen on the left. Then there are spatial frequencies in cycles per image (cpi) for which space distribution of the total nonlocal contrast was defined. On the right, one may see 3D maps of space distribution of contrast modulation amplitude when using operators of various diameters. The next column demonstrates the same maps in a 2D format. Red dots on them show local maximum apexes. While processing the image with the gradient operator of the largest size with its central area diameter making one half of the image size, we selected 2 maximums, after which, in the course of operator diameter two-fold reduction, selected were 4, 8 and 16 maximums correspondingly. A round aperture with a Gaussian transfer function transmitting four image cycles (hereinafter this aperture is referred to as a “window”) was placed within positions found this way. Areas of maximum contrast modulation amplitude were combined in a new image (the right column). The total diameter of the areas found at different spatial frequencies equaled the diameter of a conventional circle with the initial image fit to it. Stimuli were the faces synthesized from the areas extracted at one spatial frequency (examples can be seen in the right column of Figure 1), as well as those resulting from the combination of these images within one aggregate image (i.e. containing all the previously used spatial frequency ranges).\n\nTo create stimuli, we used 40 initial images of faces and 240 initial images of natural objects. Photos of faces were taken from FERET Database collected under the FERET program, sponsored by the DOD Counterdrug Technology Development Program Office (Phillips et al., 1998; Phillips et al., 2000). This database was created with the consent of participants and contains photographs of men and women of different races with different emotional facial expressions. We used part of the images from the database provided to us in full accordance with Color FERET Database Release Agreement.\n\nIn fact, we used the “bubbles” method (Gosselin & Schyns, 2001), yet unlike the traditional approach with the aperture located at random, the aperture of our research was placed in definite, previously pre-estimated positions which corresponded to the areas with a definite modulation value of the total nonlocal contrast.\n\nThen the same way we formed stimuli consisting of areas with the minimum contrast modulation amplitude, as well as images consisting of areas with a modulation having the medium amplitude between the closest minimums and maximums.\n\nWe employed a one-way design for independent samples having a three-level factor “Amplitude of modulation” (min, med, max). The percentage of correct identification of facial expressions was the dependent variable. The sample size was determined based on Anova's power = 0.8 and expected Cohen's f > 0.5 effect size. The minimum expected effect size was determined based on the results of the preview of the prepared images performed by the researchers themselves.\n\nThe observers were demonstrated synthesized images of Caucasian and Asian faces in frontal view (male and female) with neutral and happy facial expressions. These randomly alternated with synthesized images of objects of different categories, the probability of faces within the chains of consequent stimuli making 33%. The observer had to inform about the appearance of a face and possibly define its expression (the answer “I don’t know” was allowed). Exposure time was not limited. The percentage of correct recognitions of facial expressions for the images formed from the areas of different contrast modulation amplitudes, was calculated.\n\nIn order to anonymize the identity of the observers, all names were encrypted by md5 algorithm and raw data files were saved on the local disk storage with limited access.\n\n\nResults\n\nFirst, we compared task solution effectiveness where the face images had been formed from maximum nonlocal contrast areas belonging to the narrow spatial frequency range. It is worth reminding that the lesser the diameter of the areas, the higher the spatial frequency (cpi) contained in them and the greater the general number of the areas found. Where synthesized face images contained space frequencies of just one range of 1 octave, the general result of facial expression recognition was low (Figure 2). The performance was higher for the stimuli formed from the areas with the maximum increase in contrast having the central spatial frequency of 16 cpi. Somewhat lower were the values of 32 cpi frequency, and much lower these were for the lowest and the highest frequency ranges. The obtained distribution generally agrees with the data suggesting that the medium spatial frequency range expressed in cycles per face is more important in face recognition (Boutet et al., 2003; Collin et al., 2006; Näsänen, 1999; Parker & Costen, 1999; Tanskanen et al., 2005; see also review Ruiz-Soler & Beltran, 2006).\n\nAxis X shows the central spatial frequency of the areas from which the face stimulus was synthesized.\n\nHowever, our main purpose was to test the hypothesis stating that the most informative image areas are those with the greatest increase in nonlocal contrast using the example of faces of different emotional expressions.\n\nTo answer this question, we compared the effectiveness of task solution for the faces formed from the areas of different contrast modulation amplitudes: maximum, minimum and medium (Figure 3). The stimuli were combined from the areas found in all the applied spatial frequency ranges.\n\nIt was found that in the task of identifying the facial emotional expression the result approximately improves from 5% to 61% with the increase in the modulation amplitude of the total contrast in those fragments from which the stimulus is formed (see Figure 4).\n\nThe abscissa shows the modulation amplitude (see the text explanations).\n\nUsing ANOVA (JASP software, RRID:SCR_015823) has proved the statistical significance of the dependence obtained (see Table 1). The Levene's test calculation showed a need to use homogeneity corrections.\n\nNote. Type III Sum of Squares\n\nThe obtained effect is very high (Cohen’s f = 1.3). Post Hoc analysis with the application of Tukey’s test with Bonferroni and Holm’s corrections (see Table 2) also showed that accuracy with which the observers recognize emotions in the faces formed from the areas of different contrast modulation amplitudes, significantly grows with the amplitude increase.\n\nNote. P-value adjusted for comparing a family of 3\n\nThus the obtained results have verified our hypothesis stating that the face image areas of the greatest increase of total nonlocal contrast contain information which can be used by the visual system in recognizing emotional expressions.\n\n\nDiscussion\n\nWe used the task of recognizing face emotional expressions in order to demonstrate that the areas of the greatest nonlocal contrast modulation amplitude might possibly be the most informative ones, hence they may be used in categorizing face expressions. Meanwhile the same areas may be revealed by the second-order visual mechanisms.\n\nIt should be noted that in recent years there have been publications of a number of model studies where the assessment of the image area aggregate energy is making the basis of the algorithm of segmenting the scenes and selecting objects from the background (Cheng et al., 2011; Fang et al., 2012; Perazzi et al., 2012). These calculation operations demonstrate really good effectiveness, yet they generally have little in common with the true-life mechanisms of the human visual system.\n\nIn our study we too proceeded from the assumption that space heterogeneities of the image energy might contain helpful information. Yet the most important item of our work is that we propose a definite physiological mechanism able of detecting these areas of interest in the image. The developed gradient operator calculating the nonlocal contrast modulation amplitude imitates the functioning of the second-order visual filters with different spatial-frequency tunings. Moreover, we tried to maximally approximate these operators’ parameters to the well-known characteristics of the second-order filters. Thus, for example the spatial frequency (in cycles per “window”) passed from the extracted areas is constant for the “windows” of all the used sizes. This emphasizes the presence of a fixed ratio of the frequency tunings of the first- and second-order filters (Dakin & Mareschal, 2000; Sutter et al., 1995) and thus ensures the invariance of the description when changing the scale. We have also used a “window” resizing step which provides a change step in the spatial frequency passed by the “windows”, this step equaling 1 octave, which roughly corresponds to the step in the change of the spatial-frequency tuning of pathways in the human visual system (Wilson & Gelb, 1984). The bandpass of the second-order mechanisms also corresponds to the given bandwidth of our operator and is equal to 1 octave (Landy & Henry, 2007). We have used the Gaussian envelope in passing the extracted image area, thus imitating the spatial characteristics of the filters at the human visual system input. We have defined that a “window” transmits namely four cycles of the input image. This value is also based on the previously obtained results (Babenko et al., 2010).\n\nAt the same time there were parameters whose optimality remains doubtful to us. So, for example, the number of identified areas grows exponentially in cases where the operator’s size reduces, this chain starting from two “windows”. We have proceeded from the requirement that the total diameter of the identified areas should be equal to the diameter of the whole image. In this case the spatial frequency of the synthesized face may be easily calculated in cycles per image. However, in reality there might be some other number of areas identified at each frequency that is optimal. No doubt, increase in their number will lead to an improved result. Neither did we introduce eccentricity correction since we assumed that in natural conditions saliency maps may also be formed by the human visual system with the use of eye movements. However, the data concerning the time of facial expression perception might indicate that one fixation is sufficient for this (Liu & Ioannides, 2010; Pourtois et al., 2010; see also reviews George, 2013; Vuilleumier & Pourtois, 2007), although another opinion also exists (Duncan et al., 2019; Eimer & Holmes, 2007; Eimer et al., 2003; Erthal et al., 2005; Okon-Singer et al., 2007; Pessoa et al., 2002).\n\nNevertheless, it is impossible to take into account every parameter of the mechanisms providing search for areas of interest in the image and can hardly put into question the conclusion that the information content of the facial image reflecting its emotional expression increases with the growth of the nonlocal contrast amplitude of areas which form this image.\n\nIt is also noteworthy that the areas of a maximum nonlocal contrast amplitude can generally be found specifically around the eyes and the mouth (see Figure 1 and Figure 3), i.e. those parts of the face that are considered to be most informative in conveying emotional signals (Bombari et al., 2013; Eisenbarth & Alpers, 2011; Yu et al., 2018).\n\n\nConclusions\n\nThe obtained experimental results have confirmed the hypothesis stating that the image areas of the greatest increase in the nonlocal contrast contain information that contributes to the identification of emotional facial expressions. The second-order visual filters are those mechanisms able to find such information.\n\nWe also suppose that the second-order visual filters that highlight the image areas with the highest modulation amplitude of nonlocal contrast are able to attract visual spatial attention; these filters are the windows through which subsequent processing levels receive significant information.\n\n\nData availability\n\nOpen Science Framework: Nonlocal contrast calculated by the second order visual mechanisms and its significance in identifying facial emotions, https://doi.org/10.17605/OSF.IO/5YZGW (Yavna, 2021).\n\nThis project contains the following underlying data:\n\nemotions.csv –data,\n\nemotions.jasp – statistics\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nSource code available from: https://github.com/dvyavna/2ord_contrast\n\nArchived source code as at time of publication: https://doi.org/10.17605/OSF.IO/5YZGW (Yavna, 2021).\n\nLicense: MIT", "appendix": "References\n\nAllen PA, Lien MC, Jardin E: Age-Related Emotional Bias in Processing Two Emotionally Valenced Tasks. 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PubMed Abstract \| Publisher Full Text\n\nVuilleumier P: Faces Call for Attention: Evidence from Patients with Visual Extinction. Neuropsychologia. 2000; 38(5): 693–700. PubMed Abstract \| Publisher Full Text\n\nVuilleumier P: Facial Expression and Selective Attention. Curr Opin Psychiatry. 2002; 15(3): 291–300. Reference Source\n\nVuilleumier P, Armony JL, Driver J, et al.: Effects of Attention and Emotion on Face Processing in the Human Brain: An Event-Related FMRI Study. Neuron. 2001; 30(3): 829–41. PubMed Abstract \| Publisher Full Text\n\nVuilleumier P, Pourtois G: Distributed and Interactive Brain Mechanisms during Emotion Face Perception: Evidence from Functional Neuroimaging. Neuropsychologia. 2007; 45(1): 174–94. PubMed Abstract \| Publisher Full Text\n\nWang HF, Friel N, Gosselin F, et al.: Efficient Bubbles for Visual Categorization Tasks. Vision Res. 2011; 51(12): 1318–23. PubMed Abstract \| Publisher Full Text\n\nWhite M: Parts and Wholes in Expression Recognition. Cogn Emot. 2000; 14(1): 39–60. 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): 124–31. PubMed Abstract \| Publisher Full Text\n\nYarbus AL: Eye Movements and Vision. New York: Plenum Press. 1967. Reference Source\n\nYavna D: Nonlocal contrast calculated by the second order visual mechanisms and its significance in identifying facial emotions. 2021. http://www.doi.org/10.17605/OSF.IO/5YZGW\n\nYu D, Chai A, Chung STL: Orientation Information in Encoding Facial Expressions. Vision Res. 2018; 150: 29–37. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "82785", "date": "06 May 2021", "name": "Yuri E. Shelepin", "expertise": [ "Reviewer Expertise Vision perception and pattern recognition" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article solves the problem of the most informative areas of the face images. Authors argue that these areas are in the greatest increase of non-local contrast. A model of the second-order visual mechanism is used, with the help of which the image areas with the highest, lowest and intermediate amplitude of the total contrast modulation are extracted. The answers of the observers in the task of distinguishing the emotional expressions in face images created from areas with different modulation amplitudes are analyzed.\nEvidence is given that the second-order visual filters can play the role of preattentive operators, highlighting the most informative image areas. Overall, the finding of the current study is important and interesting, and analysis is reliable. However, I would like to highlight two problems:\nThe second-order visual mechanisms should be described in the Introduction in more detail, since the journal has a broader audience.\n\nThe authors used the task of distinguishing facial expressions and obtained a result confirming the informational significance of the image areas with the highest total brightness contrast. However, different information is useful for different visual tasks. At the same time, are the preattentive mechanisms specific to the visual task? Is the proposed algorithm for finding the interest areas effective in the task of discriminating emotions and will it be equally effective in other visual tasks? It would be useful to address this issue in the 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?\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": "6664", "date": "12 May 2021", "name": "Denis Yavna", "role": "Author Response", "response": "Thank you very much for your attention to our study and its positive assessment. We will add necessary information to the Introduction and Discussion." } ] }, { "id": "179353", "date": "20 Jun 2023", "name": "Tina Tong Liu", "expertise": [ "Reviewer Expertise Vision", "face recognition", "emotional processing", "primary visual cortex", "neuroplasticity" ], "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, the authors aim to identify a mechanism in human visual perception that would be selective for facial features/attributes, which are known to contribute to facial expression recognition. The authors hypothesized that the most informative areas of an image are those characterized by spatial heterogeneity, particularly with nonlocal contrast changes. To test this hypothesis, the authors first developed a software program to create three variants of a single face, each with maximum, minimum, and medium contrast modulation amplitudes. Then, the authors applied the \"bubbles\" method and ask the participant to judge the emotional expressions of faces. The study found that the greater the contrast modulation amplitude of the areas shaping the face, the more accurately participants were able to identify the emotion, revealing that nonlocal contrast can be a diagnostic feature for facial emotion recognition.\n\nWhile this manuscript addresses a very interesting question, it entails a number of noticeable limitations regarding the 6 review criteria:\n1. Is the work clearly and accurately presented and does it cite the current literature?\n1.1 I would like to suggest that the authors pay closer attention to citation accuracy.\nFor instance, the sentence below could be revised to reflect the appropriate techniques used in the cited studies:\n\"Experiments involving a saccadic task (Crouzet et al., 2010; Kirchner & Thorpe, 2006) and registration of event-related potentials (Cauchoix et al., 2014; Dering et al., 2011; Herrmann et al., 2005; Liu et al., 2002; Liu et al., 2009; Pitcher et al., 2007)...\"\nInstead of measuring ERP, Liu et al. (2002) used MEG, Pitcher et al. (2007) used TMS, and Liu et al. (2009) specifically measured intracranial field potentials, not event-related potentials.\n\nAnother example:\n\"Indeed, there are specialized mechanisms for finding brightness gradients in the visual system, and these are striate neurons (Hubel & Wiesel, 1962; Hubel & Wiesel, 1968). \"\nThese two seminal papers focused on the preferences of V1 neurons for orientation, direction of movement, and spatial frequency, not brightness gradients. It is important to note that V1 neurons do not \"find\" sinusoidal gratings in t he visual scene. The central concept revolves around receptive fields—specific regions in visual space that have an impact on the activity of V1 neurons.\n\n1.2 The authors may need to exercise caution when making negative claims that can be readily disproven.\n\nFor example, \"The problem is that the lower levels of the human visual system lack neurons which would be selective to certain facial features\" may not hold true based on recent evidence. Recent evidence show that human primary visual cortex is sensitive to emotional expressions of faces (Bo et al., 20211, Liu et al., 20222).\n\nAlso, \"It is clear that the evenly lit areas are of no interest to the visual system.\" The authors should be aware of the existence of multiple ON and OFF regions within receptive fields, before making this claim.\n\n1.3 I would encourage the authors to provide a more balanced view of the literature.\n\nThe claim that face processing is pre-attentive is not universally accepted. It is crucial for the authors to acknowledge the contradictory nature of prior studies regarding the topic of fast face detection and pre-attentive processing. Please include citation of Pessoa et al. (2002)3 for a balanced view of the literature.\n\n1.4 The authors should aim to provide a clearer description of the second-order visual processes as proposed by Graham (2011), rather than simply referring to it as a \"mechanism.\" \"Mechanism\" is a very strong word, meaning the underlying processes, principles, or components that are responsible for this phenomenon. Instead of using the term \"second-order visual mechanism,\" it would be more appropriate to adhere to Graham's (2011) terminology and refer to it as \"second-order processes\" or \"higher-order processes.\"\n\n2. Is the study design appropriate and does the work have academic merit?\nBased on the information provided, it seems that the stimuli used in the study consisted of 40 initial images of faces and 240 initial images of natural objects. However, it is unclear whether the same task of judging facial expressions was applied to both types of stimuli (faces and natural objects).\n\nThe authors may need to address the potential limitations regarding the limited number of emotional expressions used in the study. With only two expressions (neutral and happy) presented throughout the experiment, there is a possibility that participants may have identified the diagnostic features associated with these expressions.\nThe authors described all the faces have a frontal view, but the sample face using the bubbles procedure in Figure 3 does not have a frontal view.\n\n3. Are sufficient details of methods and analysis provided to allow replication by others?\nThe authors may wish to consider breaking down the Method section into subsections to enhance the readability and organization of the manuscript, such as Participants, Study Design, Stimuli, Procedure, etc.\n4. If applicable, is the statistical analysis and its interpretation appropriate?\nWithout detailed information about the specific experimental design, it is challenging to evaluate the appropriateness of using a one-way ANOVA versus a repeated-measures ANOVA.\nThere is no standard deviation or error in Figure 2. Simply presenting the mean is not acceptable.\n5. Are all the source data underlying the results available to ensure full reproducibility?\nI appreciate the authors' efforts to promote openness and transparency, However, to ensure a comprehensive replication of the study, it is important for the authors to provide access to all the processed stimuli, rather than just a limited number of sample images on https://osf.io/5yzgw/files/osfstorage. The emotions.csv file provided in the OSF repository is not sufficient or helpful for replication purposes either. It is recommended that the authors provide PII-stripped (Personally Identifiable Information), anonymized raw data in the OSF registry.\n6. Are the conclusions drawn adequately supported by the results?\nIt is misleading to say \"confirmed the hypothesis\" or \"proved the statistical significance\". In the context of null hypothesis testing framework, the stats only allow the authors to reject the null hypothesis.\n\nI am not sure if I understand the scope of the conclusions. Is the importance of nonlocal contrast is limited only to facial emotion recognition or if it extends to other aspects such as general face recognition or object recognition in a broader sense?\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?\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": "9984", "date": "29 Aug 2023", "name": "Denis Yavna", "role": "Author Response", "response": "Dear Reviewer We sincerely thank you for your attention to our manuscript and your valuable comments. We tried to take into account all of them and made changes to the text. Let me also make some comments. 1.1 I would like to suggest that the authors pay closer attention to citation accuracy. Thank you, your comments have been taken into account. The only thing we have left unchanged is the reference (Hubel & Wiesel, 1962). You are correct that \"These two seminal papers focused on the preferences of V1 neurons for orientation, direction of movement, and spatial frequency...\" However, it is in the article by Hubel & Wiesel (1962) that the authors first state: “The most effective stimulus configurations, dictated by the spatial arrangements of excitatory and inhibitory regions, were long narrow rectangles of light (slits), straight-line borders between areas of different brightness (edges), and dark rectangular bars against a light background\". This is a direct indication that striate neurons respond most strongly to brightness gradients (bands or edges). 1.2 The authors may need to exercise caution when making negative claims that can be readily disproven. We have included citations of relevant papers in the text. However, we are not sure that we have the right to insert citations of papers published after our manuscript was submitted. We also clarified what we mean by \"lower levels of the human visual system\" in the text. As for the On- and Off-reactions of the visual neurons, of course they take place. However, in the context of the problem of saliency, these reactions are not considered. At the same time, turning on or off the light stimulus can also attract the observer's attention. Therefore, we have made an appropriate addition to the text. 1.3 I would encourage the authors to provide a more balanced view of the literature. Indeed, \"The claim that face processing is pre-attentive is not universally accepted.\" We have added links. 1.4 The authors should aim to provide a clearer description of the second-order visual processes We have included in the text a more detailed description of the second-order filter model. As for the term \"mechanism\", you correct: \"Mechanism\" is a very strong word.\" Where possible, we have replaced the word “mechanism”. 2. Is the study design appropriate and does the work have academic merit? А) it is unclear whether the same task of judging facial expressions was applied to both types of stimuli (faces and natural objects). We have included in the text an explanation of how the subjects' task was formulated. B) there is a possibility that participants may have identified the diagnostic features associated with these expressions. We agree with you that such a possibility existed. Therefore, we tried to minimize it. To do this, the subjects had to categorize all presented stimuli (not just faces). Faces appeared much less frequently than non-faces and differed, in addition to expression, in sex, race, and frequency content. The subjects were not warned in advance which facial expressions would be used. They themselves had to determine the facial expression if they detected a face in a series of other stimuli. C) The authors described all the faces have a frontal view, but the sample face using the bubbles procedure in Figure 3 does not have a frontal view. We used face images from a database that labeled them as frontal. However, there were some variations of the angle. We considered this to be a positive factor, since it introduced additional variability into the incentives. 3. Are sufficient details of methods and analysis provided to allow replication by others? Based on your recommendation, we have structured the \"Method\" section into subsections. 4. If applicable, is the statistical analysis and its interpretation appropriate? We have made appropriate corrections to the text. 5. Are all the source data underlying the results available to ensure full reproducibility? Based on your recommendation, we have provided access to all source data. 6. Are the conclusions drawn adequately supported by the results? We have made corrections in accordance with your comments. Once again, we sincerely thank you for your attention to our manuscript and for your valuable comments." } ] } ]	1	https://f1000research.com/articles/10-274
https://f1000research.com/articles/12-1039/v1	25 Aug 23	{ "type": "Case Report", "title": "Case Report: Giant cell arteritis presenting as pyrexia of unknown origin", "authors": [ "Satish Mahajan", "Shailee Chandak", "Anamika Giri", "Kashish Khurana", "Satish Mahajan", "Shailee Chandak", "Anamika Giri" ], "abstract": "Giant cell arteritis, also known as temporal arteritis or cranial arteritis, is a type of systemic vasculitis that mainly affects large and medium-sized arteries in the head, neck, and upper extremities. It is characterized by inflammation of the walls of arteries, which can lead to narrowing or blockage of the affected vessels, and can result in serious complications such as vision loss, stroke and aortic aneurysm. The exact cause of giant cell arteritis is not fully understood, but it is believed to be an autoimmune disorder, where the immune system mistakenly attacks healthy cells and tissues in the body. Genetic, environmental and infectious factors may also play a role in its development. The disease is more common in people over 50 years of age and occurs more frequently in women than in men. Diagnosing this disease is challenging and requires high degree of clinical suspicion and use of advanced modalities for confirming the diagnosis. Here we report a case of a 70-year-old female with pyrexia of unknown of origin who was ultimately diagnosed as a case of giant cell arteritis by PET-CT.", "keywords": [ "Giant cell arteritis", "systemic vasculitis", "autoimmune disorder", "pyrexia of unknown origin", "stroke" ], "content": "Introduction\n\nGiant cell arteritis (GCA) is a systemic vasculitis that primarily affects large and medium-sized arteries, including the aorta. It can lead to serious complications such as aortic aneurysm and dissection. The presentation of GCA includes constitutional symptoms such as fatigue, malaise and fever, as well as headache, scalp tenderness and jaw claudication (pain with chewing). Vision changes, such as transient or permanent visual loss, double vision or diplopia can also occur due to involvement of the ophthalmic artery. Other symptoms may include weight loss, joint pain and proximal muscle weakness. In this case report, we discuss a 70-year-old female patient with a 2-month history of intermittent fever associated with temporal headache, which was relieved on taking over the counter medications. She was investigated thoroughly, but due to a lack of any significant findings, a positron emission tomography CT (PET-CT) scan was done to rule out any malignancy which guided us to the path of our diagnosis of giant cell arteritis.\n\nThe diagnostic criteria of GCA are based on a combination of clinical features, laboratory findings and histopathological findings. Table 1 shows the commonly used diagnostic criteria of GCA.\n\n\n\n1. Age > 50 years old\n\n2. New onset or recent exacerbation of headache\n\n3. Scalp tenderness or jaw claudication\n\n4. Elevated erythrocyte sedimentation rate (ESR) > 50 mm/hour\n\n5. Biopsy-proven temporal artery inflammation or skip lesions.\n\nThe presence of three or more of these criteria has a high specificity and sensitivity for the diagnosis of GCA. Clinical judgement is necessary when making the diagnosis. Additionally, the absence of these criteria does not rule out the possibility of GCA and further testing may be required. Early diagnosis and prompt treatment with high-dose steroids can prevent serious complications, such as vision loss, associated with this condition.\n\nDiagnosis of giant cell arteritis typically involves a combination of clinical evaluation, blood tests, imaging studies and biopsy of affected tissue. A biopsy of the temporal artery is the gold standard for diagnosis, as it can show characteristic inflammatory changes in the arterial wall. Blood tests may also reveal elevated levels of inflammatory markers such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). A definitive diagnosis of GCA is made through biopsy of the affected artery, typically the temporal artery.1\n\nTreatment for giant cell arteritis involves the use of corticosteroids, such as prednisone, to reduce inflammation and prevent further damage to affected arteries. The dosage and duration of treatment may vary depending on the severity of the disease and the individual patient’s response. In some cases, immunosuppressive drugs may also be used to help control the autoimmune response.\n\nPyrexia of unknown origin (PUO) is a condition characterized by persistent fever of at least 38.3°C for more than three weeks, with no apparent cause despite extensive diagnostic workup. The etiology of PUO is varied, with infectious diseases accounting for approximately one-third of cases, followed by inflammatory and malignant conditions.2 PUO can be challenging to diagnose and manage, as it requires a systematic approach in ruling out potential causes. The diagnosis involves a thorough medical history, physical examinationand laboratory tests, including blood cultures, serological testing, imaging studies and biopsy, if necessary. Despite its rarity, PUO poses a significant diagnostic challenge. Its early recognition and prompt intervention can prevent significant morbidity and mortality.2\n\n\nCase report\n\nA 70-year-old female patient, a teacher by profession and currently retired, presented to the outpatient department with chief complaints of fever on and off for two months along with headache, loss of appetite and weight loss. Patient was apparently alright two months ago when she started experiencing fever which was insidious in onset, high grade intermittent, associated with chills and rigour. Fever was relieved on taking anti-pyretics. Patient complained of headache which was localised to the temporal region, squeezing type, not associated with photophobia, phonophobia, nausea or vomiting. There were no aggravating factors for headache and it was relived on taking over the counter medications. Patient also complained of generalised weakness and loss of appetite for the last 2 months. Patient gives history of weight loss of around 12 kilograms which accounts to significant weight loss.\n\nPatient was diagnosed with hypothyroidism 10 years ago, taking Tabthyroxine 100mcg once daily. Patient also had a history of tubercular lymphadenitis 50 years ago and had taken the full course of anti-tubercular therapy, however no documents were available. Apart from these, the patient had no other complaints. Patient had no history of any constitutional symptoms prior to two months ago, no history skin rash, oral ulcers, joint pain or swelling.\n\nOn examination patient was vitally stable, there were no mucocutaneous lesions, and all peripheral pulses were well felt. General examination also did not reveal any abnormalities.\n\nPatient was investigated on the line of pyrexia of unknown origin as she was symptomatic for more than two months. All routine investigations, as mentioned in Table 2, were normal, fever profile was normal. ESR and CRP were on the higher side. Autoimmune profile including anti-nuclear antibodies, anti-dsDNA antibodies, anti-Smith antibodies, anti-Ro and anti-La were all negative. Chest X-ray and chest CT also did not reveal any abnormalities. Bone marrow examination was also normal. Table 1 shows various diagnostic modalities used to screen for PUO.\n\nIn view of no significant findings in examination and investigations, 3 days after admission to the hospital, it was decided to do a positron emission tomography CT (PET-CT) scan to rule out any malignancy. Due to the hospital not having a FDG-PET scan available, the patient was referred to another hospital for this procedure and then returned to our hospital with the results of the PET-CT. The results showed a small uptake in aorta and a differential of giant cell arteritis was considered. A CT aortogram and contrast enhanced CT abdomen were done, which revealed circumferential thickening of distal descending part of aorta as shown in Figures 1 and 2 respectively. Temporal artery biopsy was done, and the biopsy report showed giant cell arteritis. Patient was started on high dose steroids (oral prednisone at 1mg/kg/day) and was started on supportive management. Serial monitoring of ESR and CRP showed the values to be decreased 3-4 days after the initiation of steroids and the patient improved drastically. Patient was eventually discharged with follow up every two months.\n\n\nDiscussion\n\nGiant cell arteritis is a systemic inflammatory disorder that predominantly affects medium to large-sized arteries, commonly the temporal arteries. GCA usually presents with headache, scalp tenderness, and jaw claudication, but it can also present as pyrexia of unknown origin in some cases.\n\nWhen GCA presents as PUO, the patient has a persistent fever of at least 38.3°C for more than three weeks, with no apparent cause despite extensive diagnostic workup. In such cases, the patient may have constitutional symptoms such as fatigue, malaise, and weight loss. The diagnosis of GCA should be considered in patients over the age of 50 with PUO, especially in the presence of other symptoms of GCA such as headache, scalp tenderness, and jaw claudication.\n\nLaboratory tests may reveal elevated inflammatory markers such as erythrocyte sedimentation rate and C-reactive protein. Biopsy of the temporal artery can confirm the diagnosis of GCA, showing characteristic findings such as granulomatous inflammation and giant cells.3\n\nPET imaging with 2-fluoro-2-deoxy-D-glucose (FDG) allows for non-invasive visualization and quantification of metabolic activity in tissues, which can be used to differentiate between normal and inflamed or malignant tissues. In GCA, FDG-PET imaging can detect increased metabolic activity in affected arteries, including the aorta, and can provide valuable information about the extent and severity of disease involvement.4\n\nDiagnosis of GCA can be challenging, as the disease can present with nonspecific symptoms and physical examination findings, and laboratory tests may be inconclusive. PET imaging with FDG has emerged as a valuable tool in the diagnosis and management of GCA. Several studies have demonstrated the utility of FDG-PET imaging in the diagnosis and management of GCA. In a study by Blockmans et al.,5 FDG-PET imaging had a sensitivity of 87% and a specificity of 100% in the diagnosis of GCA, and was able to detect early disease involvement in patients with negative temporal artery biopsy results. In another study by Nuenninghoff et al., FDG-PET imaging had a sensitivity of 78% and a specificity of 91% in the diagnosis of GCA, and was able to detect aortic involvement in patients with clinically suspected but biopsy-negative disease.\n\nIn our case, PET scan came out to be the major modality which helped us in investigating a diagnosis of GCA which was later confirmed by biopsy and CT aortogram.\n\nAortic involvement in GCA is an important predictor of disease severity and risk of complications, including aortic aneurysm, dissection, and rupture. However, diagnosis of aortic involvement can be challenging, as the aorta is a deep-seated structure that is not easily accessible to physical examination or biopsy.\n\nFDG-PET imaging can be a valuable tool in the diagnosis and management of aortic involvement in GCA. In a study by Meller et al.,6 FDG-PET imaging had a sensitivity of 83% and a specificity of 90% in the detection of aortic involvement in patients with GCA, and was able to differentiate between active and inactive disease in the aorta. In another study by Salvarani et al., FDG-PET imaging was able to detect aortic involvement in 67% of patients with clinically suspected GCA and negative temporal artery biopsy results.\n\n\nConclusion\n\nA high index of suspicion for GCA should be maintained in patients presenting with PUO, especially in those over the age of 50 with additional symptoms of GCA. Prompt diagnosis and treatment with high-dose steroids are crucial to prevent irreversible complications associated with GCA, such as vision loss.\n\nFDG-PET imaging is emerging as a valuable tool in the diagnosis and management of GCA, particularly in patients with suspected aortic involvement. FDG-PET imaging can detect early disease involvement, differentiate between active and inactive disease, and provide valuable information about disease extent and severity. However, FDG-PET imaging should be used in conjunction with other diagnostic modalities, such as clinical evaluation, laboratory testing, and imaging studies, to confirm the diagnosis of GCA and guide appropriate treatment.\n\n\nConsent\n\nWritten informed consent for the publication of the case report and associated images was sought from the patient prior to submission.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nLaBuzetta JN, Conklin J, Hilliard J, et al.: Pyrexia of Unknown Origin: A Case of Giant Cell Arteritis. J. Am. Board Fam. Med. 2018; 31(6): 976–979. Publisher Full Text\n\nKumar N, Nasim A, Helfgott SM: Fever of Unknown Origin in a 74-Year-Old Woman. JAMA Intern. Med. 2018; 178(5): 712–713. Publisher Full Text\n\nKumar S, Bhandari S, Gupta S, et al.: Giant Cell Arteritis Presenting as Pyrexia of Unknown Origin: A Rare Case Report. J. Clin. Diagn. Res. 2015; 9(5): OD01–OD02. Publisher Full Text\n\nSundaram A, Tazelaar HD, Ryu JH: A 63-year-old woman with persistent fever and cough. Chest. 2004; 126(4): 1298–1301. Publisher Full Text\n\nBlockmans D, de Ceuninck L , Vanderschueren S, et al.: Repetitive 18F-fluorodeoxyglucose positron emission tomography in giant cell arteritis: a prospective study of 35 patients. Arthritis Rheum. 2006 Feb 15; 55(1): 131–137. PubMed Abstract \| Publisher Full Text\n\nMeller J, Sahlmann CO, Gürocak O, et al.: FDG-PET in patients with fever of unknown origin: the importance of diagnosing large vessel vasculitis. Q. J. Nucl. Med. Mol. Imaging. 2009 Feb; 53(1): 51–63. PubMed Abstract" }	[ { "id": "215844", "date": "14 May 2024", "name": "Andreas Wiggers Nielsen", "expertise": [ "Reviewer Expertise FDG-PET/CT imaging of polymyalgia rheumatica and giant cell arteritis" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is a case report of a patient presenting with fever of unknown origin. Due to prolonged symptoms with pyrexia and headache, the patient was referred for FDG-PET/CT evaluation. This imaging modality demonstrated a vascular uptake in aorta prompting a diagnosis of GCA.\nTitle:\nI find the title misleading. It should be stated that the patient also presented with other symptoms including temporal headache as well as other constitutional symptoms.\n\nIntroduction:\nIn my opinion, the structure of the introduction is confusing. I recommend to restructure this section and highlight the themes of this article:\n1. Diagnosing GCA can be challenging in some cases due to an unspecific symptomology. 2. FDG-PET/CT may aid the clinicians in diagnosing GCA.\n\nThe introduction should not contain description of the case patient. Remember to cite relevant reference - E.g.: “Table 1 shows commonly used diagnostic criteria of GCA” (reference) Temporal artery biopsy used to be the “gold standard” for diagnosing GCA. However, a biopsy of the temporal artery may not exclude extra-cranial vasculitis. Therefore, EULAR guidelines now recommend imaging for diagnosing GCA.\nCase report: Patients:\nThe description of the symptomology should be concise and precise. Avoid repetitions and rephrasing the same statements. Include relevant examinations for GCA like temporal artery palpation. Did the patient exhibit symptoms of polymyalgia rheumatica? Table 2 should contain normal range for each lab test. Can you include follow-up data in the table? Please state if prednisone resolved the symptoms of the patient. Figures: Include the FDG-PET/CT with FDG uptake of aorta instead. The CT image demonstrates a thickening of the abdominal aorta but not inflammation. Did the FDG-PET/CT show uptake in the temporal artery that subsequently underwent biopsy?\n\nDiscussion:\nThis section needs to be revised. Currently, this section mainly discusses the diagnostic performance of FDG-PET/CT for GCA. I cannot see how this relates to a case report. Discuss if this was an atypical case of GCA. As I understand it, this case patient had classic signs of GCA with temporal headache, constitutional symptoms and elevated acute phase reactants. Describe the knowledge gap in the introduction and how does this case report provide new or supportive evidence.\n\nConclusion:\nThe authors conclude that FDG-PET/CT can detect early disease involvement, differentiate between active and inactive disease, and provide valuable information about disease extent and severity. This statement are not supported from the evidence presented in this case report. Consequently, it is recommended that the authors reconsider their conclusion and instead emphasize the clinical implications implied by the findings of this case report.\nReference:\n\nThe authors should cite cohort studies instead of case reports to support their statements.\n\nAbstract:\nShould be changed to accommodate the comments to the main body of the paper.\n\nWriting: The manuscript is generally written in an easily understandable language.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "311661", "date": "23 Aug 2024", "name": "Silvia Grazzini", "expertise": [ "Reviewer Expertise Vasculitis Clinic (GCA fast track)", "Rheumatology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors described a case of large vessel GCA presenting as FUO and temporal headache, in which diagnosis was possible thanks to PET-CT. The theme is interesting and I appreciate the effort to describe a rare presentation of this fascinating disease, but the paper is not well written for many reasons: first of all, English should be improved; then, some sentences are not accurate (GCA subtypes, acute vs chronic complications, classification vs diagnostic criteria) but, most important, authors didn't deepen certain aspects which, in my opinion, are mandatory to mention in 2024, such as the role of less invasive imaging modalities - in this regard, it would be more than appropriate to cite ultrasound results - or treatment strategies besides prednisone. Moreover, I found some errors: - classification criteria should be mentioned as references, and at least citation 1 should be reset; - Table 2 is not well written: some measure units are missing, as well as normal values; - Table 1 is about diagnostic criteria for GCA, not diagnostic modalities used to screen FUO. I think the case report could be considered for indexing, but major revisions are required.\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? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/12-1039
https://f1000research.com/articles/12-1038/v1	25 Aug 23	{ "type": "Research Article", "title": "Mixed confine fusion experiment, evidence of feasibility of confinement", "authors": [ "Xijie Wei" ], "abstract": "Background: In 2022 3rd International Conference on Materials, Physics and Computers (MPC 2022), a promising fusion theory called Mixed confine fusion (MCF) was proposed. The limitation of that work was that no experiment was carried out to prove the feasibility. Methods: In order to provide more powerful evidence of feasibility of MCF, in this work, an experiment comparing properties of plasma under and without a MCF confinement provided by two groups of electrical magnets generating 30mT magnetic field is presented. Results: The result shows that the product of ion density and temperature under Lawson criterion increased 46 times, from 2.30811014m-3 and 39.3496eV to 3.98031015m-3 and 105.1625eV. Conclusions: The results suggest that hotter and dense plasma exists in the center of the chamber, which means all particles received a force towards the center of the chamber. This result provides confirming evidence that MCF can provide a confinement to plasma.", "keywords": [ "nuclear fusion", "plasma physics", "mixed confine fusion" ], "content": "Introduction\n\nNuclear fusion occurs with a huge amount of energy released. In order to use fusion energy as an energy supplier, thermonuclear fusion is used, which requires devices confining hot plasma. It seems that limitations exist in all kinds of fusion devices. For example, pressure produced on the coils is observed in almost all kinds of fusion devices using magnetic field as a driver, such as Tokamak, which was proposed by USSR scientist G.G.Dolgov-Saveliev.1,2 A much higher standard of material for the wall exists in other devices using inertia as the driver in inertial confinement fusion (ICF), which uses the method of very rapidly heating and compressing small pellets of suitable fuel.3 Lasers are used as drivers in ICF. This method causes the first wall to be exposed to more X-ray and debris meaning the wall must be made using a higher standard of material.4 Other fusion device using both magnetic field and inertia as the drivers of confinement like Magneto-internal fusion (MIF), have been designed using magnetic field to confine a low density plasma and then lasers are used, compressing the hot plasma until fusion occurs.5 Laser is used as the main driver to achieve fusion. Magnetized laser fusion, a liner which is a tube magnetically imploded is contained and used to heat and compress to achieve the condition of fusion,6 suffer both of the limitations mentioned in tokamak and ICF.\n\nMixed confine fusion (MCF), is a promising method of confining plasma proposed in the 2022 3rd International Conference on Materials, Physics and Computers (MPC 2022), is mainly about confining fusion plasma with a high speed change of three perpendicular magnetic field, using both magnetic field and inertia as drivers, in which some limitations might be avoided. Different from MIF, MCF uses magnetic field in the final stage to achieve fusion. As a consequence of this method not only does no extra laser need to be used, but in addition, the magnets are all used periodically, which causes a lower average pressure per time of the magnets as an interval of rest is produced. This allows for a much lower standard of electric magnet is required. Different from other devices using both magnetic field and inertia as drivers, in MCF, these two drivers are used equally, while in MIF magnetized laser fusion laser is the main driver to achieve fusion and magnetic field is used for initial confinement.5 MCF is considered as having some advantages comparing with Tokamak. It is shown with a simulated model (used in a previous study by the author) that there is no force exist in the center of the model, leading to a suitable distribution of energy for plasma confinement.7 At the same time, the limitation of the MCF method is that the ability of confinement of plasma is much weaker when compared with Tokamak is shown as the overall energy difference between the edge of the model and the center, which is considered as the cause of force of confinement, is much smaller than Tokamak in similar conditions.7\n\nHowever, in my last work,7 the feasibility of MCF was only proved with a simulation. No experiment was conducted. In this work, an experiment using a device based on the MCF theory is going to be introduced with the relationship between confinement ability and two variables, frequency of changing field and magnetic field strength.\n\n\nMethods\n\nAn experiment was conducted to determine the correlations between magnetic field intensity, frequency, and confinement ability. As depicted in Figure 1, the device may be broken down into three basic components: a plasma generator, a MCF confinement system, and a single Langmuir probe.\n\nA hand-made circle-shaped tungsten cathode using 0.3 mm diameter tungsten stick with 6mm diameter is connected to a 100 kV high-power voltage supply brought from e-commerce platform Taobao which produced a 100 kV potential difference between the cathode and annular brass anode with a 6mm nuzzle. The current in this circuit can be ignored since it is smaller than 1mA, maximal power of the voltage supplier is 16W. Ambient air is used in the experiment, so it is expected that the plasma is mainly made by nitrogen. The experiment’s gas pressure is 13.3 ± 0.2 pa, measured by MKS 901P, with the usage of Edwards Rv3 vacuum pump. The pressure is controlled by adjusting a valve so that the rate of air entering the chamber is equal to the rate of air pumped out of the chamber.\n\nWater-cooling coils are used to generate magnetic field needed in this experiment. Every coil has about 2000 turns and is powered by a 30 V laboratory power supply, MP6020D from MAISHENG, with the usage of a hall probe, approximately 30mT magnetic field is measured to be generated. The current in coil is about 10A as supplied by the power supply.\n\nThe 4mm of the Langmuir probe’s tungsten stick that is exposed to the plasma has a radius of 0.125 mm and is coated in a shell made of 3D-printed photosensitive resin, which is used to ensure the area the probe exposed to the plasma is constant all the time, this shell can be replaced by any insulated shell, the replacement would not be considered as an influential factor of the result measured, since the shell is so thin that its influence can be ignored. The accuracy of the ammeter brought from e-commerce platform Taobao used to measure current in the probe circuit is 10 nA. Every reading that is recorded. The voltage supplied across the probe and chamber are at a range of 0-60 V by MS-602DS from MAISHENG.\n\nA camera brought from e-commerce platform Taobao was used to find out the external physical characters of the plasma. A pancratic lens with the focal length changing between 12 mm to 120 mm is used. The photo was taken only when a 60 V potential difference is provided across the Langmuir probe, as plasma would be in that state of electron saturation, when plasma would be brighter so that it is much easier to distinguish the edge of the plasma.\n\nThe gas pressure is adjusted by adjusting the valve controlling rate of gas entering into the chamber, after that, high voltage supplier is turned on so that plasma is generated by the plasma source. The voltage across Langmuir probe and chamber is adjusted with different values between 0 V and 60 V. Current readings with a voltage interval of 2.5 V were recorded when the voltage is between 0 V and 10 V, voltage intervals were about 5 V when voltage was between 10 V and 60 V. The interval is not strictly equal to 5 V as a result of that the probe voltage output by the supplier is adjusted by human, considering the method of analysis using, this is not considered to cause an influence of the results. This process is repeated measuring the properties of plasma with no MCF confinement and under a MCF confinement. Pressure is kept constant so that the plasma generated by the source has its property constant all the time. Reading of accurate ampere meter is recorded.\n\n\nResult and discussion\n\nReadings of Langmuir is record and a graph of probe current and voltage is plotted, shown as Figure 2A and Figure 2B.10\n\nSecond derivative of probe current against probe voltage is shown as Figure 3A and Figure 3B.10\n\nProf. Azooz’s MATLAB program8 is used to analysis the data collected authors may be able to reproduce this analysis using open-source alternatives like Python or GNU Octave. With the usage of Prof. Azooz’s MATLAB program,8 with the output of ion density and electron temperature generated, the property of plasma in two situations is shown, it is found that plasma with no MCF confinement has ion density and electron temperature of 2.30811014 m-3 and 39.3496 eV respectively. When plasma is under an MCF confinement, its ion density and electron temperature increased to 3.98031015 m-3 and 105.1625 eV. This software is chosen because the input it requires, i.e. current and voltage readings across the Langmuir probe is identical to what is recorded in this experiment. After the data is inputted into the program, it would find out the best parameters from a1 to a4, that make the equation, equation 1.8 fit the data.\n\nThen the result is generated within the usage of this fitted equation.\n\nThe ion saturation current is calculated with equation 2.8\n\nWhere H is the maxima number of I in the data inputted, V min is the minima number of V inputted.\n\nPlasma potential can be calculated with equation 3.8\n\nVector VV in increments of 3 is created with equation 4.8\n\nwhere Vmax is the maxima number of voltage input.\n\nVector t in increments of 3 is created with equation 5.8\n\nElectron temperature T can be calculated with equation 6.8\n\nWhere trapz(X,Y) integrates Y with respect to the coordinates or scalar spacing specified by X, E =t-Vp.\n\nIon density can be calculated with equation 7.8\n\nWhere Mi is the mass of ions in kg, in this work, it is assumed only Nitrogen ion is considered, so Mi is chosen as 2.32489449*10-26 kg.\n\nIn order to find out the properties of plasma under a MCF confinement, Lawson’s criterion of product of ion density, ion temperature and energy confinement time is considered in this work.9 In order to find out the promotion, some assumptions are made. First, it is assumed the energy confinement time of plasma in all conditions are the same, as a consequence of usage of same plasma generator. Influence on energy confinement time caused by magnetic field confinement is ignored as it is too small and hard to measure. Second, it is also assumed that the plasma is under thermionic mode, i.e. ion has same temperature electrons, this assumption is made as a result of only electron temperature can be measured with Prof. Azooz’s program. After making these assumptions, the property of promotion of fusion efficiency can be written as equation 8.\n\nWhere nic and Tec are ion density and electron temperature under MCF confinement, ninc and Tenc are ion density and electron temperature with no MCF confinement.\n\nIn this work, it is found that p = 46.0880. Lawson’s criterion is used to evaluate fusion rate in fusion devices, p >1 means with the MCF magnetic field, a higher fusion rate existed, which provides evidence that MCF magnetic field provides a positive influence on fusion rate, i.e. evidence that the feasibility of using MCF as a fusion device. More dense and hotter plasma is found in the center of the chamber, as a consequence of that all particles in the plasma experienced a force towards the center of the chamber, due to MCF confinement. This is considered evidence of feasibility of confinement of MCF theory.\n\nMore evidence is found through the image shot with the camera, shown as Figure 4.\n\nIn Figure 4, it can be seen that a plasma sphere is confined by magnetic field. The sphere-shaped plasma fits the simulation of magnetic field in my previous work, i.e. the magnetic field energy is expected to be independent to direction,7 which produces a centripetal force on the particles, so that the plasma is expected to have a spheroidal shape.\n\n\nConclusions\n\nIn this work, an experiment it made to prove the feasibility of plasma confinement of MCF theory. It is found that the property of plasma under Lawson’s criterion is increased 46 times. This produces compelling evidence that MCF magnetic field can provide a confinement to plasma. Drawbacks of this study included the limited magnetic field and chamber size existed. It is suggested in further study, a larger chamber and a greater magnetic field should be used.", "appendix": "Data availability\n\nFigshare: The data of paper ‘Mixed Confine Fusion experiment, evidence of feasibility of confinement’ https://doi.org/10.6084/m9.figshare.23283920.v1. 10\n\nThis project contains the following underlying data:\n\n‐ Data of figure 4. 20230331_225913.bmp\n\n‐ 20230331.xlsx (raw data collected from physical experiment)\n\nFigshare: The data of paper ‘Mixed Confine Fusion experiment, evidence of feasibility of confinement’ https://doi.org/10.6084/m9.figshare.23283920.v1. 10\n\nThis project contains the following extended data:\n\n‐ Data of Figure 2.a I-Vgraph confinement.fig\n\n‐ D2I-Vgraphnoconfinement.svg\n\n‐ Data of Figure 2.b I-Vgraph noconfinement.fig\n\n‐ D2I-Vgraph confinement.svg\n\n‐ Data of Figure 3.a D2I-Vgraph confinement.fig\n\n‐ I-Vgraph confinement.svg\n\n‐ Data of Figure 3.b D2I-Vgarph noconfinement.fig\n\n‐ I-Vgraph noconfinement.svg\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nReferences\n\nManheimer W: Challenges facing high-field tokamaks. Phys. Today. 2019; 72(4): 12. Publisher Full Text\n\nDolgov-Saveliev GG: Investigations of the Stability and Heating of Plasmas in Toroidal Chambers.\n\nYamanaka: Twenty years of “NUCLEAR FUSION”: an overview of highlights in the evolution of controlled nuclear fusion research Inertial confinement. Nucl. Fusion. 1980; 20: 1084–1085. Publisher Full Text\n\nAIP Conference Proceedings.1996; 369: 200. Publisher Full Text\n\nWurden GA, Hsu SC, Intrator TP, et al.: Magneto-Inertial Fusion. United States.Publisher Full Text\n\nRoth JR: Industrial plasma engineering. Vol.1 principles. IOP Publishing Ltd; 1995.\n\nWei X: Simulation of Special Distribution of energy of plasma in a High-speed Changing Magnetic Field. J. Phys. Conf. Ser. 2022; 2371: 012002. Publisher Full Text\n\nAzooz A: Langmuir probe data analysis code. MATLAB Central File Exchange.2023. Retrieved April 2, 2023. Reference Source\n\nLawson JD: Some criteria for a useful thermonuclear reactor (Technical report). Harwell, Berkshire, U. K.: Atomic Energy Research Establishment; December 1955.\n\nWei: The data of paper ‘Mixed Confine Fusion experiment, evidence of feasibility of confinement. figshare. Figure. 2023. Publisher Full Text" }	[ { "id": "201774", "date": "16 May 2024", "name": "Marin Marin", "expertise": [ "Reviewer Expertise Applied Mathematics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic of this manuscript can be of interest for the readers having the speciality in this domain. There seems to be no other approach for this mixed confine fusion experiment, evidence of feasibility of confinement. I've rebuilt most of the calculations and seem to be correct.\n\nGenerally speaking, the manuscript is well written and organized.\nFor these reasons I can recommend the acceptance of this paper.\nHowever, I suggest that the author must takes into account the following corrections:\n\nI think the title needs to be reformulated to become more “friendly”. The words “confine” and “confinement” can be considered a repetition.\n\nThe meaning of I and V in relation (1) is not specified.\n\nOrigin of relation (2) is not specified.\n\nIt is not clear how relation (8) was obtained.\n\nThe results of this manuscript are theoretical and abstract. It would be in the best interest of the manuscript if their minimal utility were presented.\n\nFrom where were the data taken used in the graphic representations?\n\nSome editing \"glitches\" need to be corrected.\n\nPunctuations are used randomly. Insert comma or full stop after each and every equation accordingly.\n\nMany notions and results are \"borrowed\" from different already published paper. As such, I think the authors need to emphasize more clearly the contribution of the manuscript from a scientific point of view.\n\nReferences are not uniformly written. See, for instance, [2], [5].\n\nAlso, I think, the author must strengthen the References section with some articles that use some similar techniques, to make the techniques used more plausible, for instance: (Marin,1996) (Ref-1) ,(Marin,1994)(Ref-2)\n\nIf the author takes into account all these corrections, then this manuscript deserves to be indexed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "239926", "date": "22 May 2024", "name": "Oriza Kamboj", "expertise": [ "Reviewer Expertise Laser Plasma Interaction" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study provides an overview of nuclear fusion, specifically focusing on the Mixed Confinement Fusion (MCF) method Here's a breakdown of the review for improvement:\n1. Lack of Citations: The text mentions a simulated model used in a previous study by the author but lacks proper citations for readers to refer to. Including references to these studies would enhance the credibility of the information. Kindly add the following for stimulated scattering: Strengthens the Context: By referencing existing research on SRS suppression, the current paper demonstrates a broader understanding of the challenges faced in fusion research.(Ref-1) (Kamboj et al,2022) Encourages Further Exploration: These references highlight potential areas for future investigation within the MCF framework, particularly regarding the combined effects of MCF and other SRS suppression techniques.(Ref-2) (Kamboj et al,2023) These citations are crucial for a well-rounded discussion of the feasibility of MCF. They offer valuable context and encourage further exploration of the topic (Ref-3) (Kamboj et al, 2023)\n2. Grammar and Syntax: There are instances of awkward phrasing and grammar issues, which can hinder the flow of the text. A thorough proofreading to address these concerns would enhance readability.\nThe text provides a comprehensive overview of fusion methods, focusing on MCF. Improvements in citations, simplifying technical language and addressing grammar issues would enhance the overall quality of the review.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1038
https://f1000research.com/articles/12-1037/v1	24 Aug 23	{ "type": "Research Article", "title": "Multi-centric Phase III, single-arm, open-label clinical study to assess clinical safety, tolerability, and efficacy of intravenous lacosamide in focal onset seizures", "authors": [ "Ashutosh Kakkad", "B S Keshava", "Bashir Ahmadi", "GRK Sarma", "Praveen Gupta", "Rajaram Agarwal", "Rajendra Dugani", "Rajnish Kumar", "Ravindra Lodha", "Sanjay Varade", "Hitesh Raval", "Dhaval Rathod", "Shohini Ghosh", "Ram Gupta", "Krishnaprasad K", "B S Keshava", "Bashir Ahmadi", "GRK Sarma", "Praveen Gupta", "Rajaram Agarwal", "Rajendra Dugani", "Rajnish Kumar", "Ravindra Lodha", "Sanjay Varade", "Hitesh Raval", "Dhaval Rathod", "Shohini Ghosh", "Ram Gupta", "Krishnaprasad K" ], "abstract": "Background: In hospitalized seizure patients and during clinical seizure emergencies, parenteral administration of antiepileptic therapy (AED) is primary treatment modality. Appropriate selection and administration of AED is essential for immediate seizure control. The purpose of this study was to evaluate the safety, tolerability, and efficacy of lacosamide injection in adult patients with focal onset seizures (FOS) with or without secondary generalized tonic-clonic seizures or focal to bilateral tonic-clonic seizures. Methods: In this Phase 3 study, we enrolled 60 patients (≥18 years) with FOS and maintained on stable doses of oral lacosamide. Patients were switched to intravenous (i.v.) lacosamide (10 mg/ml); daily i.v. dosage and frequency were kept equivalent to oral lacosamide per patient. Lacosamide was infused intravenously over for 30 to 60 minutes at 12 hourly intervals for five consecutive days. Primary outcome was evaluating the safety and secondary outcome included measuring the seizure frequency during the treatment (day 1- 5) and at follow-up (day 12). Results: All patients enrolled completed the study. No significant changes in vital signs, or laboratory parameters, were observed at the end of treatment or follow-up when compared to baseline. The frequency analysis for all the components of electrocardiogram (ECG) was within the normal limits at all times. There were no serious adverse events (AEs) reported in this study. Overall, 26.66% of patients had mild to moderate AE intensity that resolved spontaneously without any other intervention. Most common AEs (frequency=5%) were abdominal pain upper, injection site pruritus, and nausea. The anti-seizure activity for lacosamide was maintained despite oral to i.v. switch of lacosamide. Conclusions: The study showed that i.v. lacosamide is a safe and effective strategy in patients with FOS and can be co-administered with other anti-epileptic drugs (AEDs), especially in acute settings with primary generalized tonic-clonic seizures. Registration: CTRI (CTRI/2011/07/001888; 14th July 2011).", "keywords": [ "Efficacy", "Epilepsy", "Focal onset seizures", "Intravenous Lacosamide", "Safety", "Switch" ], "content": "Introduction\n\nFocal onset seizures (FOS) are defined as seizures that originate within networks limited to one hemisphere of the brain that is often associated with increased morbidity and impaired quality of life (QoL), especially with associated or primary generalized tonic-clonic seizures.1 Epilepsy accounts for 0.5% of the global disease burden. In India, the overall prevalence of epilepsy ranges between 1.3 and 11.9 per 1,000 population, and the incidence varies from 0.2 to 0.6/1,000/year2 with a standardized mortality ratio of 2.56-7.6%.3–6 As per recent epidemiological studies conducted in India between 2018-2019, 17-47% of patients with epilepsy were suffering from FOS with or without focal to bilateral tonic-clonic seizures.7–9 Increased incidence of FOS was more predominant with advanced age.\n\nAcute seizures and/or status epilepticus cases often require an infusion strategy during the ictal phase to prevent neurologic damage that is likely to happen within the first 15 to 20 mins of uncontrolled seizures. In most of these cases, a switch to oral strategy is often recommended to prevent recurrence while improving patient compliance and adherence to therapy.\n\nLacosamide is an amino acid derivative from the novel class of anti-epileptic drugs (AEDs), termed functionalized amino acids. It is licensed for use as an adjunctive and monotherapy for FOS and associated focal to bilateral and primary generalized tonic-clonic seizures in the US as well as many European countries.10,11 The complementary action on slow voltage-gated sodium channels and collapsin response mediator protein 2 (CRMP2) results in stabilization of hyper-excitable neuronal membranes and inhibition of repetitive neuronal firing.10,12\n\nThe favorable pharmacokinetic/pharmacodynamic (PK/PD) profile includes high solubility (≈25 mg/ml), improved bioavailability and bioequivalence to intravenous infusion, minimal drug-drug interaction, low plasma protein binding (<15%), and it can be easily converted from oral to intravenous (i.v.) formulation without the need for dose modification.13,14 This is especially beneficial in conditions when oral administration of the drug is temporarily not feasible such as when patients undergo surgery, are hospitalized, have swallowing difficulties, or experience acute gastrointestinal disorders. Also, in case of a seizure emergency, such as refractory convulsive status epilepticus, where the quick onset of action is required, intravenous administration is essential for immediate seizure control.\n\nTo assess the clinical safety, tolerability, and efficacy of i.v. lacosamide when prescribed in real-world settings for patients with FOS, this clinical study was a single arm, ‘Switch’ study from oral to i.v. lacosamide therapy.\n\n\nMethods\n\nThis was an open label, single arm, multicentric clinical trial conducted in adult patients with FOS enrolled from nine sites across India. All investigational sites were approved by respective Independent Ethics Committees, or Institutional Review Boards (IRBs), before the enrolment of any patient in the trial.\n\nThis trial followed the principles outlined in the Declaration of Helsinki and the trial protocol was approved by the Institutional Ethics Committees and the Drug Controller General of India, the head of the department of the Central Drugs Standard Control Organization of the Government of India (BR/DCGI/LACO-INJ/10/648-I), which is a national body that provides authorization to conduct studies and approve products for marketing in India. The trial was registered on 14th July 2011 with the Clinical Trial Registry of India (Registry identifier: CTRI/2011/07/001888).\n\nThis trial was conducted from July 2011 to January 2012. The study was conducted according to the finalized protocol and no changes were done during the entire study period.\n\nAll male and female patients, ≥18 years of age with a confirmed diagnosis of FOS, on a stable dose of oral lacosamide (Torrent Pharmaceuticals Limited, India (batch no. IT11003)) and willing to sign informed consent were enrolled. Patients were excluded if they had abnormal platelet count, high liver enzymes, bilirubin and/or serum creatinine, seizures occurring in clusters, status epilepticus within three months of enrolment, history of non-epileptic seizures, allergy to the study drugs/excipients, cardiac conduction defects and on drugs that can prolong P-R interval, clinically significant ECG abnormalities, structural lesions in the central nervous system (CNS), progressive neurological disorders or psychiatric disorders, use of neuroleptics, monoamine oxidase (MOA) inhibitors, barbiturates, or narcotic analgesics within 28 days before screening. Pregnant or lactating women were also excluded from the study.\n\nWe conducted an open-label, single-arm, multi-centric safety and tolerability study across multiple sites in India between July 2011 and January 2012. Patients with stable FOS were enrolled after obtaining their written informed consent and a baseline evaluation was performed.\n\nDuring this phase, a thorough clinical history including the presence of concomitant disease, treatment with current AED regime, medical and surgical history, or allergies were noted. Laboratory investigations—ECG, hematology and biochemistry, urine examination, urine pregnancy test for women of childbearing potential—were done to exclude ineligible patients. Enrolled patients were given i.v. lacosamide and outcomes were assessed as per study protocol. A detailed flowchart of the study protocol is given in Figure 1.\n\nAED, antiepileptic therapy; ECG, electrocardiogram; AE, adverse event.\n\nEnrolled patients were switched from oral to i.v. lacosamide (10 mg/ml). The study medication was supplied by Torrent Pharmaceuticals Ltd., India in a single use vial i.e., lacosamide injection 200 mg/20 ml. The total daily intravenous dosage of lacosamide was optimized per patient and made equivalent to the total daily dosage and frequency of oral lacosamide. Lacosamide was infused intravenously for 30 to 60 minutes in 250 ml normal saline twice a day (morning dose, MD and evening dose, ED) for five consecutive days at 12-hr intervals.\n\nMedications other than the study drug that were considered necessary for the patients were allowed at the discretion of the investigator and an appropriate record was maintained in the “Concomitant medication Record Sheet” of CRF.\n\nThe individual patient’s observation period was 12 days, which included five days of treatment period with follow-up after seven days of the last injection.\n\nThe number of patients included in the study was on the basis of the first trial conducted with i.v. lacosamide.15 So, data of the 60 patients who completed the trial was deemed appropriate to meet the safety and tolerability objectives of the trial. Since, no sample size was determined in the study no statistical analysis was performed.\n\nSafety evaluation\n\nVital parameters\n\nVital signs (blood pressure, pulse rate, and temperature) were measured at baseline, day 1 to day 5 (end of treatment), and on day 12 (follow-up).\n\nLaboratory parameters\n\nHematology, biochemistry, and urine analysis were performed at the time of screening and day 5 (end-of-treatment).\n\nChanges in ECG\n\nECG was done at screening, day 3, day 5, and follow-up (day 12).\n\nAdverse events (AEs) assessment\n\nAEs were monitored for the entire study duration and the record was maintained.\n\nEfficacy evaluation\n\nA detailed history of seizures (frequency of seizure/week) was recorded during screening and after enrolment i.e., from day 1 to day 5 and also during follow-up to evaluate any change from baseline during treatment and also when patients were switched back to oral lacosamide.\n\nContinuous variables were summarized using mean, standard deviation (SD), and median. AEs and local AEs at the injection site were reported by frequency analysis with respect to incidence, severity, and seriousness. Seizures were calculated in terms of frequency. Maintenance of seizure frequency was calculated from baseline to follow-up. All statistical analysis were performed by using SAS® (RRID:SCR_008567) Version 9.1.3 (SAS Institute Inc., North Carolina, Cary, USA) (free alternative, R (programming language)).\n\n\nResults\n\nA total of 74 patients were screened for the study across all participating centers between July 2011 and January 2012. A total of 14 patients were ineligible for participation in the trial. Overall, 60 patients; 45 male and 15 female with a mean age of 29±9.5 years old were enrolled and completed the study (Table 1).36 No patient withdraw from the study during the entire study duration.\n\nAll enrolled patients were switched from oral lacosamide to i.v. lacosamide. The total daily intravenous dosage of lacosamide was equivalent to the total daily dosage and frequency of oral lacosamide. Intravenous treatment was administered for five consecutive days at 12-hr intervals. The dose range of i.v. lacosamide was 100-400 mg/day and majority of the patients were administered with a daily dose of 200 mg/day. Out of 60 patients, two patients were taking oral lacosamide three times a day and both received i.v. lacosamide twice a day for five days. However, the total daily dose of lacosamide injection in both patients was the same. Out of 60 patients, 49 patients received i.v. lacosamide as 30 min infusion and 11 received as 60 min infusion. Lacosamide dose and duration of the infusion for all patients are described in Table 2.\n\nConcurrent medications of all patients were recorded. The commonly used concomitant drugs were AEDs such as carbamazepine, clobazam, oxcarbazepine, phenytoin, sodium valproate, valproic acid, and levetiracetam.\n\nVital parameters\n\nThe comparison of vital signs—blood pressure (systolic and diastolic), pulse rate, and temperature—during baseline to follow-up visit is shown in Figure 2. No significant change in the blood pressure, pulse rate, and temperature were seen between days 1-5 or at follow–up.\n\nBP, blood pressure; MD, morning dose; ED, evening dose.\n\nLaboratory parameters\n\nMinimal changes were observed in the laboratory parameters from baseline to end of treatment i.e., day 5. Urine analysis & microscopy showed no abnormality in any patient. Urine pregnancy test was done in 14 female patients at screening and day 5; all tests were negative. There were no alterations in hematology, clinical biochemistry, and urine analysis in patients after study drug administration (Table 3).\n\nChanges in ECG findings\n\nECG was done at screening day 3, day 5, and follow-up (day: 12). The frequency analysis was within the normal limit for all the visits.\n\nAdverse events (AEs) assessment\n\nThere were no serious AEs reported in this study. Among all patients (n=60), 26.66% (n=16) patients reported a few AEs. A total of 24 AEs were reported; 16 systemic and 8 due to reaction at the local injection site. All AEs were mild to moderate and did not result in patient’s withdrawal from the study. The more commonly reported systemic AEs were upper abdominal pain upper (n=3), and nausea (n=3; 5% of the total population), whereas dizziness, insomnia, thrombophlebitis, upper respiratory tract infection (n=1; 1.7% of the total population) were less commonly observed (Table 4). All AEs were reported during lacosamide injection (days 1-5) except one that was reported during follow-up. A total of eight local AEs (pain, pruritus, erythema, thrombophlebitis and rash erythematous) were reported at the injection site out of 600 lacosamide infusions administered to 60 patients over a period of five days (Table 5). There was good tolerability with i.v. lacosamide as all the patients completed the study without a single withdrawal due to AEs.\n\nThe frequency of seizures in patients at baseline (screening), day 1 to day 5 (end of treatment), and from day 1 to day 12 (follow-up) is described in Figure 3.\n\nIt was observed that oral to i.v. switch of lacosamide either reduced or maintained the seizure frequency in patients with FOS. As compared to only 13 subjects who did not show seizures during baseline evaluation, 40 and 37 patients remained seizure free between days 1 to 5 and day 12, respectively.\n\n\nDiscussion\n\nThe purpose of this study was to evaluate the safety and tolerability of short-term i.v. lacosamide in patients with FOS.\n\nEthnic variability is an important factor that may influence both the pharmacokinetics and pharmacodynamics of a drug resulting in varied responses to drug therapy.16,17 Therefore, while similar studies were conducted in Western countries,15–20 this study was performed to establish the safety and efficacy of i.v. lacosamide in the Indian cohort. The findings reported herein showed that the patients on the established dose of oral lacosamide can be safely switched to i.v. lacosamide for up to five days across a broad range of doses (100-400 mg/day) without compromising the efficacy.\n\nThe primary assessment of safety and tolerability showed no alteration from baseline, in laboratory parameters (hematology, biochemistry, and urine analysis), vitals (blood pressure, pulse rate, and temperature), and ECG, after i.v. lacosamide administration and at follow-up. The AEs reported were mild to moderate in nature and mostly limited to upper abdominal pain, headache, nausea, vomiting, and pyrexia; none of the AEs resulted in patient withdrawal. The nature and frequency of these AEs were consistent with other similar studies conducted in the past.15,18,21 No events of bradycardia, hypotension or post-dose sedation were observed except for dizziness that was reported in one patient. Similarly, other studies reported no deleterious cardiovascular effect or QR/PT prolongation in patients treated with i.v. lacosamide,22,23 which suggests cardiovascular safety. This study also reported a few local reactions at the injection site (pain, pruritus, pain pruritus, erythema, thrombophlebitis, and rash erythematous) occurring in only 1.3% of the total infusions administered. These local AEs were mild and consistent with those found in other similar studies, suggesting no new safety signals with the use of parenteral lacosamide.15,18,21\n\nThe secondary outcome of the study evaluating the efficacy, in terms of seizure frequency, during i.v. lacosamide treatment did not show any negative change in the pattern of seizures over for 12 days. The majority of the patients remained seizure-free while others showed either a reduction in seizure frequency or were stable during the treatment period and even at follow-up after 12 days indicating no seizure deterioration with i.v. lacosamide. Similar efficacy was reported in other studies in patients with FOS.15,18 Besides FOS, the efficacy of i.v. lacosamide was also reported in status epilepticus and seizure clusters in some studies24–26 suggesting its use as a potential alternative to standard AEDs in seizure emergencies.\n\nCombination therapy is a common practice in the treatment of epilepsy. In the current study, many patients were on two or more AEDs such as carbamazepine, clobazam, oxcarbazepine, phenytoin, sodium valproate, valproic acid, and levetiracetam including lacosamide. However, no drug interaction was reported. This can be attributed to the low or no potential of lacosamide to inhibit or induce Cytochrome (CYP) 450 isoforms indicating that lacosamide can be safely co-administered with other AEDs. The finding was supported by two other studies, which showed no pharmacokinetic interaction with the use of lacosamide with other AEDs.27,28 Similarly, Baulac et al. (2017), also demonstrated the safety as well as the efficacy of i.v. lacosamide, in combination with levetiracetam in patients with FOS, is not adequately controlled by the combination of levetiracetam and sodium channel blockers AEDs (e.g., lamotrigine, carbamazepine, oxcarbazepine).29 Overall, the study suggests that i.v. lacosamide can be used as a suitable alternative in FOS and is devoid of severe AEs like respiratory depression, hypotonia, or cardiac arrhythmia, often associated with the use of conventional AEDs such as phenytoin, phenobarbital, benzodiazepines and valproate,30 or post-dose sedation (somnolence) commonly reported with newer AEDs such as levetiracetam and brivaracetam.31–35\n\nOur study had a few limitations. Seizure control and seizure freedom could not be assessed due to the short duration of the study. Long-term follow-up with observation for seizure frequency and seizure freedom at 4 to 12 weeks with better-matched safety assessment for QTc prolongation could be explored in further randomized controlled clinical trials in this line.\n\n\nConclusions\n\nIntravenous injection or infusion is essential to control seizures in an emergency or when oral administration is not feasible, to prevent neurological complications that may occur due to seizure cluster or status epilepticus. In such conditions, switching to an i.v. formulation of an already established oral AED regime is the safest and most convenient option but requires dose titration. With a favorable pharmacokinetic profile, minimal drug interaction, and dual mechanism of action, lacosamide is an ideal drug that can be safely switched to its i.v. form without dose modification to prevent seizure deterioration and to avoid unexpected side effects of using an alternative AED. The study showed that patients with FOS and on a stable oral dose of lacosamide can be safely switched to i.v. lacosamide for up to five days, alone or in combination with other AEDs without compromising the efficacy.", "appendix": "Data availability\n\nFigshare: Multi-centric Phase III, single-arm, open-label clinical study to assess clinical safety, tolerability, and efficacy of I.V. Lacosamide for Focal Onset Seizures (FOS). https://doi.org/10.6084/m9.figshare.21563541. 36\n\nThis project contains the following underlying data:\n\n- Demography.doc\n\n- Laboratory Parameters.doc\n\n- Vitals.doc\n\n- Completed CONSORT Checklist\n\nIt should be noted that Authors are allowed to share these documents and so the ‘confidential’ on the files can be ignored.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgments\n\nWe thank the participants, caregivers, and families for their participation in the trials; the site investigators and their staff for performance of the trial. The writing of this article was supported by a medical writer at Medwiz Healthcare Communications Private Ltd.\n\n\nReferences\n\nSarmast ST, Abdullahi AM, Jahan N: Current Classification of Seizures and Epilepsies: Scope, Limitations and Recommendations for Future Action. Cureus. 2020; 12: e10549. 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PubMed Abstract \| Publisher Full Text\n\nRudd GD, Haverkamp W, Mason JW, et al.: Lacosamide cardiac safety: clinical trials in patients with partial-onset seizures. Acta Neurol. Scand. 2015; 132: 355–363. PubMed Abstract \| Publisher Full Text\n\nLachuer C, Corny J, Bézie Y, et al.: Complete Atrioventricular Block in an Elderly Patient Treated with Low-Dose Lacosamide. Cardiovasc. Toxicol. 2018; 18: 579–582. PubMed Abstract \| Publisher Full Text\n\nKakkad A: Multi-centric Phase III, single-arm, open-label clinical study to assess clinical safety, tolerability, and efficacy of I.V. Lacosamide for Focal Onset Seizures (FOS). [Dataset]. figshare. 2022. Publisher Full Text" }	[ { "id": "208170", "date": "11 Feb 2024", "name": "Nobutsune Ishikawa", "expertise": [ "Reviewer Expertise pediatric neurology and epilpesy." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 that i.v. lacosamide is a safe and effective strategy in adult patients with FOS and can be co-administered with other anti-epileptic drugs based on 12 days-period phase III study in India. This is an overall well organized study, and the result is reasonable.\nThere are some points which can potentially improve this manuscript as follows:\nHow did the authors confirm seizure types of participants? The authors should clarify it because this is the basis of the study.\n\nThe authors should describe the length of observational period which is needed to determine seizure frequency, and authors should elucidate the length of period in which anti-seizure drugs were kept unchanged.\n\nIf authors did, blood concentration data including LCM and other anti-seizure medicine before and after change of LCM administration routes should be provided.\n\nLooking at Table 1, some patients suffering from infection and/or inflammatory condition were included in this study. Please give some comments in the manuscript in terms of this.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "11056", "date": "13 Apr 2024", "name": "Ashutosh Kakkad", "role": "Author Response", "response": "There are some points which can potentially improve this manuscript as follows: How did the authors confirm seizure types of participants? The authors should clarify it because this is the basis of the study. Response: In this study already known cases of focal seizures who were taking oral lacosamide were enrolled. The authors should describe the length of observational period which is needed to determine seizure frequency, and authors should elucidate the length of period in which anti-seizure drugs were kept unchanged. Response: The individual patient’s observation period was 12 days, which included five days of treatment period with follow-up after seven days of the last injection. If authors did, blood concentration data including LCM and other anti-seizure medicine before and after change of LCM administration routes should be provided. Response: Blood concentration was not done as the same was not required as per the objective of the trial. Looking at Table 1, some patients suffering from infection and/or inflammatory condition were included in this study. Please give some comments in the manuscript in terms of this. Response: Enrolled patient characteristics is elaborated at length in \"Patients\" & \"Study Design & Plan\" Section" } ] }, { "id": "203888", "date": "26 Apr 2024", "name": "Sudhir Shah", "expertise": [ "Reviewer Expertise Stroke", "Epilepsy", "Cognition", "Demyelination", "Ataxia" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “Multi-centric Phase III, single-arm, open-label clinical study to assess clinical safety, tolerability, and efficacy of intravenous lacosamide in focal onset seizures” by Dr Ashutosh Kakkad et al, authors have described the results of safety efficacy and tolerability of intravenous lacosamide in 60 adults more than 18 year of age. It is a good study and we do need such trials to assess intravenous antiepileptics in scenarios like status epilepticus or while switching from oral to IV in patients who need to be kept nil per mouth. The manuscript meets the stated goals and is written clearly. We have few comments: 1. It has been mentioned in study design and plan section that stable FOS patients were enrolled. However, exact definition of the same is missing in inclusion criteria as to what was seizure frequency, when was the last recorded seizure and how long the patients have been seizure free or what was the longest duration of seizure free interval. 2. In Table 1, some patients were on antibiotics, mannitol, artesunate. Please comment regarding the status of such patients if they were having any acute condition. 3. We could not find an explanation or basis of why two regimes of iv infusion i.e.; 30 min in 49 patients versus 60 min in 11 patients have been taken up and what was the basis of selection of a particular candidate for either. 4. Patients who were on concomitant other antiepileptics like carbamazepine, topiramate, zonisamide phenytoin and lamotrigine which have significant drug interaction with lacosamide. Although lacosamide does not induce or inhibit CYP 450 enzymes, other drugs may decrease or increase the lacosamide levels. Thus, therapeutic drug level monitoring may be required, specially in those patients who were having seizures while on antiepileptics. 5. In result section, in patients who developed adverse event, whether there was any relation with dose of lacosamide or duration with respect to 30 versus 60 min infusion time.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1037
https://f1000research.com/articles/12-1036/v1	24 Aug 23	{ "type": "Systematic Review", "title": "Wait-and-see and other factors associated with recurrence risk in central serous chorioretinopathy patients: A systematic review and meta-analysis of cohort studies", "authors": [ "Andi Arus Victor", "Ari Djatikusumo", "Anggun Rama Yudantha", "Kemal Akbar Suryoadji", "Ari Djatikusumo", "Anggun Rama Yudantha", "Kemal Akbar Suryoadji" ], "abstract": "Background: Central serous chorioretinopathy (CSC) is a common macular disease characterized by serous retinal detachment and leakage spots at the level of the retinal pigment epithelium. While acute CSC is often self-limiting, chronic and recurrent CSC can lead to permanent vision loss. The identification of risk factors associated with recurrent CSC is crucial for preventive measures and appropriate management. This systematic review aims to determine the various risk factors for the recurrence of central serous chorioretinopathy, with a particular focus on the \"wait and see\" approach and other contributing factors. Methods: The review protocol was registered in PROSPERO (ID: CRD42023413852). A systematic literature search was conducted across four databases (PubMed, Cochrane, Embase, and ScienceDirect) using the keywords \"((Central serous chorioretinopathy) AND (Recurrence) AND (Risk))\". Meta-analyses were performed using Review Manager 5.4. Results: A total of five studies met the inclusion criteria for all factors, while three studies were selected for meta-analyses regarding the \"wait and see\" approach. The analysis revealed four significant risk factors for CSC recurrence: patient's clinical condition, therapy administration, age and gender, and patient history. Additionally, a meta-analysis based on three studies involving 1025 eyes demonstrated a statistically significant difference between CSC patients who received no therapy (wait and see) and those who underwent specific therapeutic interventions, with a relative risk of 1.82 (95% CI 1.49–2.22, I2 95%, P <0.00001). Conclusions: Early identification of risk factors, including the ‘wait and see’ approach and other factors, is crucial in preventing the recurrence of central serous chorioretinopathy. Patients at high risk of recurrence should receive vigilant treatment and routine monitoring to avoid permanent vision loss. This systematic review provides valuable insights for clinical decision-making and emphasizes the importance of individualized management strategies for CSC patients.", "keywords": [ "central serous chorioretinopathy", "recurrence", "risk", "systematic review" ], "content": "Introduction\n\nA well-circumscribed serous retinal detachment in the macular region, along with one or more leakage points at the level of the retinal pigment epithelium (RPE), is a common clinical symptom of the common macular disease central serous chorioretinopathy (CSC), which is often detected using fluorescein angiography (FA).1 Chronic and recurrent CSC can cause RPE atrophy and neurosensory retinal changes, which can lead to permanent vision loss. Acute CSC is typically a self-limiting process with few visible sequelae.2 Mrejen et al. (2019) recently reported that 12.8% of their 133 chronic CSC study participants developed bilateral legal blindness.3 Blurring of vision, relative central scotoma, micropsia, dyschromatopsia, metamorphopsia, hyperopic refraction change, and reduced contrast sensitivity are all symptoms of CSC. The onset of CSC can be acute or chronic, but there is no general agreement on the definition of chronic disease in CSC. Some ophthalmologists define chronic CSC as unresolved subretinal fluid after three months, whereas others use a duration of four or six months.4–6 Several risk factors for CSC development have been identified in previous studies, including stress, depression, male sex, allergic disease, hypertension, smoking, shift work, alcohol consumption, and sleep disturbance.7–12 Currently, there are only a few studies that report the risk factors for the recurrence of CSC, which we will discuss in this systematic review. The objective of this study was to evaluate the factors associated with recurrence risk in patients with central serous chorioretinopathy (CSC) through a systematic review and meta-analysis of cohort studies.\n\n\nMethods\n\nWe registered a protocol prior to authoring this review and meta-analysis, and it was registered in the International prospective register of systematic reviews (PROSPERO) on April 13, 2023 (CRD42023413852).\n\nOur study utilizes a systematic review design method and conducts a meta-analysis of cohort studies. The clinical research in this study focuses on prognosis, specifically investigating various exposures to recurrence in CSC patients. Subsequently, a meta-analysis is performed on a homogenous type of study. We specifically opted for cohort studies as they provide the highest level of evidence for prognostic studies, alongside systematic reviews and meta-analyses of cohort studies.\n\nThe inclusion criteria used in this study were: (a) Cohort research study type, (b) Suitability to the topic of recurrence risk in central serous chorioretinopathy patients, (c) The study had at least one control group and one exposure group, and (d) clear extraction and statistical analysis method.\n\nThe exclusion criteria used in this study were: (a) Full text was not available, and (b) Use of languages other than English.\n\nThis study used the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines.13 Four electronic databases were used including PubMed, ScienceDirect, Embase and Cochrane. We used the term “((Central serous chorioretinopathy) AND (Recurrence) AND (Risk))”.\n\nFour researchers screened the title and abstract independently and excluded the irrelevant studies. After conducting title and abstract screening, four authors (AAV, AD, ARY, KAS) independently evaluated each entry based on the inclusion and exclusion criteria, responding with ‘Yes’, ‘No’, or ‘Maybe’. Any discrepancies were resolved through discussion and mutual agreement. The final retrieved studies were screened for duplicates and systematically evaluated according to the inclusion and exclusion criteria. The subject characteristics (central serous chorioretinopathy patients), outcome of the study (recurrence risk), and other relevant information were assessed by the four researchers, each researcher worked independently. The search was conducted using a systematic review method using the PubMed, Cochrane, and ScienceDirect databases. The keywords “((Central serous chorioretinopathy) AND (Recurrence) AND (Risk))” were used. After synthesizing the various factors that cause recurrent central serous chorioretinopathy, a meta-analysis was carried out using the ReviewManager, Revman 5.4 (RRID: SCR_003581), application related to the wait-and-see factor for the risk of recurrence in CSC patients. The use of the term ‘wait-and-see’ refers to CSC patients who do not receive therapeutic interventions in the form of photodynamics, anti-vascular endothelial growth factor (VEGF), and laser therapy, or a combination of them.14 In our study, the measured effect is the risk ratio of wait-and-see compared to therapy, along with a 95% confidence interval, regarding the recurrence events of CSC patients. This will be combined through a forest plot using data from various studies. Additionally, the effect of adjusting measurement factors on CSC recurrence will be discussed in the study.\n\nResearchers assessed each risk of bias from the retrieved study using the ReviewManager (RevMan 5.4; Cochrane, London, UK) based on the Cochrane Risk of Bias for cohort studies. Each study was classified as having a low, unclear, or high risk of bias. Any dissent was discussed until a final decision was reached. The GRADE (grading of recommendations assessment, development and evaluation) approach was employed to evaluate the certainty of evidence in this study. The assessment was conducted independently by all authors, and any disagreements among assessors were resolved through discussions among them.\n\n\nResults\n\nA total of 625 studies were retrieved based on the keywords from PubMed (n = 50), ScienceDirect (n = 467), Embase (n = 107) and Cochrane Library (n = 1). After screening the abstract and title, 620 studies were excluded. The studies were then compared for duplicates and no studies were excluded. The remaining five studies were screened for the inclusion and exclusion criteria. The final screening resulted in five studies that met the inclusion and exclusion criteria. The flowchart of the study selection can be seen in Figure 1.\n\nIn this systematic review, a total of five studies met the inclusion criteria. A description of the search results can be seen in Table 1.14–18\n\nA meta-analysis was also performed based on three studies involving 1025 eyes. There was a statistically significant difference between CRC patients who did not receive therapy or were told to wait and see and those who received specific therapeutic interventions, with an RR of 1.82 (95% CI 1.49–2.22 I2 95%, P < 0.00001). The forest plot can be seen in Figure 2.\n\nFigure 3 shows details of the risk of bias of the included studies. Figure 3a is a plot of risk of bias according to the Cochrane risk of bias tool for cohort studies. Figure 3b is a summary table of the risk of bias of each study. The study by Yu et al.,15 has a high risk of bias in assessment of exposure due to its high-risk self-reporting bias method. The study by Mohabati et al.14 has a high risk of bias in adjusting the possible prognostic factors. Three studies by Mohabati et al.,14 Yu et al.,15 and Zhou et al.18 have a unclear method in outcome assessment due to unclear blinding methods. The study by Mohabati et al.14 also has an unclear co-intervention bias. The evaluation of evidence certainty for all outcomes in this meta-analysis yielded a moderate level of certainty.\n\n(a) Plot of risk of bias per category; (b) Summary table of risk of bias for each study.\n\n\nDiscussion\n\nIn this systematic review, five studies demonstrated the recurrence of CSC in patients in various populations. These five studies used a total of 1,325 patients with CSC including patients from the Netherlands, China, Hong Kong, and Japan.\n\nThe first study from Mohabati et al. (2020) was a prospective cohort study with a minimum follow-up of 12 months. This study showed that 29% of patients experienced a recurrence of symptoms of CSC. Of the population that experienced recurrences, 24% of them were patients who were not treated immediately or were advised to wait-and-see, whereas in patients who underwent initial therapy, only 4% experienced recurrences. Initial therapy for patients included photodynamic therapy, conventional lasers, and subthreshold micropulse diode lasers.14 Therefore, patients with CSC had a better outcome if they had initial therapy with these three options compared to no therapy or wait and see. The next study by Yu et al. (2019), which was a retrospective cohort study providing questions in the form of questionnaires to 138 patients relating to various risk factors for CSC. Of all the patients, 85.5% had recurrent or persistent conditions. Various factors that were significantly associated with recurrence or persistence compared to only acute conditions included male sex (OR = 5.63), older age (OR = 1.14), and a higher Insomnia Severity Index score with an odds ratio of 1.30.15 Therefore, this second study showed that male sex, age, and degree of insomnia are risk factors for the recurrence of CSC in patients.\n\nThe study by Lai et al. (2015) was a retrospective cohort study comparing two groups of patients, namely a group that received half-dose verteporfin photodynamic therapy (PDT) and the group that did not receive any therapy. It was found that the group of patients without therapy had a higher percentage of recurrences than those with the therapy (53.8% vs 20%).16 Therefore, this study showed an increased risk of recurrence of CSC patients without therapy. The next study by Zhang et al. (2022) was a prospective cohort study of 162 patients with CSC and 19.14% of patients had recurrences of CSC. Various factors were significantly associated (P < 0.001) with recurrence according to this study including pigment epithelium detachment (OR = 1.78), retinal pigment epithelium damage (OR = 1.13), baseline best-corrected visual acuity (OR = 0.96), and subfoveal choroidal thickness level (OR = 1.18).17\n\nThe last study by Zhou et al. (2022), a retrospective cohort study of 538 CSC patients, showed that recurrence had a significant relationship with corticosteroid use history (OR = 5.52), bilateral disease (OR = 3.94), chronic manifestations (OR = 7.12), non-intense fluorescein leakage (OR = 3.34) and initial central retinal thickness (OR = 0.997).18 This study showed four risk factors for recurrence and one factor that can reduce the risk of recurrence in CRC patients, namely initial central retinal thickness.\n\nThe meta-analysis study was carried out based on three studies comparing CSC patients with the wait and see treatment, which did not provide specific interventions to patients and controls in the form of patients receiving certain therapies such as PDT, conventional laser, micropulse laser, and anti-VEGF. The first study by Lai et al. (2015) showed a significant risk of wait and see with an RR of 2.69 (95% CI 1.66–4.36).16 The second study by Mohabati et al. (2020) also showed a significant risk with an RR of 5.33 (95% CI 2.93–9.68).14 The last study by Zhou et al. (2022) showed non-significant results with an RR of 1.04 (95% CI 0.82–1.31). However, after calculations from the forest plot, a statistically significant risk was found with an RR of 1.82 (95% CI 1.49–2.22 I2 95%, P < 0.00001).18 Therefore, patients with CRC should be given an alternative therapy to avoid recurrence.\n\nThe probability of CSC recurrence is strongly correlated with increased choroidal thickness, according to a recent study by Matet et al.19 Although there weren’t enough choroidal thickness measurements in our data set to evaluate this apparent association, we noticed a thicker choroid was commonly found in patients with recurrent CSC. When compared to the wait-and-see group, subretinal fluid (SRF) resolved more quickly in the treatment group, and the prevalence of SRF recurrence was lower in the treatment group.14 Moreover Ozkaya et al. observed a two-fold greater probability of recurrence in 41 wait-and-see treatments in acute CSC (aCSC) patients (51%) than in 36 aCSC patients who received low-fluence PDT treatment.20 Therefore, to prevent the disease re-occurring it is better for patients to be given initial therapy compared to no therapy especially observation only.\n\nOur study showed that there are four broad stroke risk factors for the recurrence of CSC in patients, including the patient’s clinical condition, therapy administration, age and gender factors, and patient history factors. The risk factors for the patient’s clinical condition include pigment epithelium detachment, retinal pigment epithelium damage, baseline best-corrected visual acuity, subfoveal choroidal thickness level, chronic manifestations, bilateral disease, initial central retinal thickness, and non-intense fluorescein leakage.17,18 The recurrence factor based on patient therapy, namely patients who receive initial therapy in the form of photodynamic therapy, conventional lasers, and subthreshold micropulse diode lasers, can reduce the risk of recurrence in CRC patients.14,16 Furthermore, the risk based on the patient’s age and gender, namely male sex and older age, can increase the incidence of recurrence in CRC patients.15 Finally, patient history factors of CRC recurrence include a higher degree of insomnia and history of corticosteroid use.15,18 Sleep disruptions have been linked to heightened functioning of the hypothalamic-pituitary-adrenal axis and the sympathetic-adrenal medullary system, leading to changes in the release of stress hormones such as cortisol and catecholamines.21 These hormonal imbalances have been identified as contributing factors to the underlying mechanisms of CSC.22\n\nOur study was a two-stage review consisting of a systematic review that synthesized five cohort studies regarding factors that influence the recurrence of CSC patients. The five studies were divided into three studies with a retrospective cohort design and two studies with a prospective cohort design. We subsequently performed a meta-analysis of three studies comparing the recurrence risk of patients on wait and see treatment and patients receiving initial therapy. The three studies consisted of two retrospective cohort studies and one prospective cohort study. In terms of level of evidence, cohort studies are a good reference for studies with prognostic question types. Our study also conducted a risk of bias analysis, which showed that there were studies that had low, high, and an unclear risk of bias in the five studies reviewed.\n\nThe study found results showing that the recurrence of CSC increases if patients only receive treatment in the form of wait and see compared to patients receiving various initial therapies including laser, anti-VEGF, and photodynamics. Therefore, as the application of this evidence, doctors who have patients with CSC should recommend patients for initial therapy rather than suggesting wait and see treatment to patients, which can risk CSC recurrence.\n\nOur study certainly has limitations. Several studies had a high risk of bias and some aspects were unclear. Our study also performed a meta-analysis but only comparing wait and see management with initial treatment of CSC patients. It is hoped that other studies will perform a meta-analysis of various other recurrence factors.\n\n\nConclusions\n\nTherefore, it is crucial to identify potential risk factors early to prevent CSC recurrence, and patients who are at high risk should receive cautious treatment and routine monitoring to prevent vision loss. Based on our meta-analysis, the wait and see treatment of CRC patients is a risk factor for future recurrence. As a result, alternate therapies should be offered to CRC patients to prevent recurrence.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: PRISMA checklist for ‘Wait and see and other factors associated with recurrence risk in central serous chorioretinopathy patients: A systematic review and meta-analysis of cohort studies’, https://www.doi.org/10.6084/m9.figshare.23551929. 13\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nLiu B, Deng T, Zhang J: Risk factors for central serous chorioretinopathy: a systematic review and meta-Analysis. Retina. 2016 Jan; 36(1): 9–19. Publisher Full Text\n\nNicholson B, Noble J, Forooghian F, et al.: Central serous chorioretinopathy: update on pathophysiology and treatment. Surv. Ophthalmol. 2013 Mar-Apr; 58(2): 103–126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMrejen S, Balaratnasingam C, Kaden TR, et al.: Long-term Visual Outcomes and Causes of Vision Loss in Chronic Central Serous Chorioretinopathy. Ophthalmology. 2019 Apr; 126(4): 576–588. PubMed Abstract \| Publisher Full Text\n\nChan WM, Lai TY, Lai RY, et al.: Safety enhanced photodynamic therapy for chronic central serous chorioretinopathy: one-year results of a prospective study. Retina. 2008; 28(1): 85–93. PubMed Abstract \| Publisher Full Text\n\nBousquet E, Beydoun T, Zhao M, et al.: Mineralocorticoid receptor antagonism in the treatment of chronic central serous chorioretinopathy: a pilot study. Retina. 2013; 33(10): 2096–2102. PubMed Abstract \| Publisher Full Text\n\nLim SH, Chang W, Sagong M: Efficacy of half-fluence photodynamic therapy depending on the degree of choroidal hyperpermeability in chronic central serous chorioretinopathy. Eye (Lond.). 2013; 27(3): 353–362. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaimovici R, Koh S, Gagnon DR, et al.: Central Serous Chorioretinopathy Case-Control Study Group. Risk factors for central serous chorioretinopathy: a case-control study. Ophthalmology. 2004 Feb; 111(2): 244–249. Publisher Full Text\n\nChatziralli I, Kabanarou SA, Parikakis E, et al.: Risk Factors for Central Serous Chorioretinopathy: Multivariate Approach in a Case-Control Study. Curr. Eye Res. 2017 Jul; 42(7): 1069–1073. PubMed Abstract \| Publisher Full Text\n\nJi Y, Li M, Zhang X, et al.: Poor Sleep Quality Is the Risk Factor for Central Serous Chorioretinopathy. J. Ophthalmol. 2018 Aug 1; 2018: 1–6. Publisher Full Text\n\nKim YK, Woo SJ, Park KH, et al.: Association of Central Serous Chorioretinopathy with Psychosocial Factors is Dependent on Its Phase and Subtype. Korean J. Ophthalmol. 2018 Aug; 32(4): 281–289. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSetrouk E, Hubault B, Vankemmel F, et al.: Circadian disturbance and idiopathic central serous chorioretinopathy. Graefes Arch. Clin. Exp. Ophthalmol. 2016 Nov; 254(11): 2175–2181. PubMed Abstract \| Publisher Full Text\n\nBousquet E, Dhundass M, Lehmann M, et al.: Shift Work: A Risk Factor for Central Serous Chorioretinopathy. Am J. Ophthalmol. 2016; 165: 23–28. PubMed Abstract \| Publisher Full Text\n\nVictor AA, Djatikusumo A, Yudantha AR, et al.: PRISMA Checklist for “Wait-and-see and other factors associated with recurrence risk in central serous chorioretinopathy patients: A systematic review and meta-analysis of cohort studies”. Dataset. figshare. 2023. Publisher Full Text\n\nMohabati D, Boon CJF, Yzer S: Risk of Recurrence and Transition to Chronic Disease in Acute Central Serous Chorioretinopathy. Clin. Ophthalmol. 2020 Apr 29; 14: 1165–1175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu J, Xu G, Chang Q, et al.: Risk Factors for Persistent or Recurrent Central Serous Chorioretinopathy. J. Ophthalmol. 2019; 2019: 5970659.\n\nLai TY, Wong RL, Chan WM: Long-Term Outcome of Half-Dose Verteporfin Photodynamic Therapy for the Treatment of Central Serous Chorioretinopathy (An American Ophthalmological Society Thesis). Trans. Am. Ophthalmol. Soc. 2015; 113: T8.\n\nZhang X, Zhuang X, Dong J, et al.: Factors for recurrence in acute central serous chorioretinopathy patients underwent one-third dose verteporfin photodynamic therapy. Photodiagn. Photodyn. Ther. 2022; 39: 102984. PubMed Abstract \| Publisher Full Text\n\nZhou X, Komuku Y, Araki T, et al.: A multicentre study of the risk factors associated with recurrence of central serous chorioretinopathy. Acta Ophthalmol. 2022; 100(8): e1729–e1736. PubMed Abstract \| Publisher Full Text\n\nMatet A, Daruich A, Zola M, et al.: Risk factors for recurrences of central serous chorioretinopathy. Retina. 2017; 38: 1403–1414. Publisher Full Text\n\nOzkaya A, Alkin Z, Ozveren M, et al.: The time of resolution and the rate of recurrence in acute central serous chorioretinopathy following spontaneous resolution and low-fluence photodynamic therapy: a case-control study. Eye. 2016; 30(7): 1005–1010. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeerlo P, Sgoifo A, Suchecki D: Restricted and disrupted sleep: effects on autonomic function, neuroendocrine stress systems and stress responsivity. Sleep Med. Rev. 2008 Jun; 12(3): 197–210. PubMed Abstract \| Publisher Full Text\n\nDaruich A, Matet A, Dirani A, et al.: Central serous chorioretinopathy: Recent findings and new physiopathology hypothesis. Prog. Retin. Eye Res. 2015 Sep; 48: 82–118. PubMed Abstract \| Publisher Full Text" }	[ { "id": "214600", "date": "26 Oct 2023", "name": "Niroj Kumar Sahoo", "expertise": [ "Reviewer Expertise Macula", "imaging", "CSC", "ARMD", "macular hole" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 an interesting meta-analysis of the comparison of wait-and-see and other factors associated with the recurrence of CSC. However, there are certain issues that need to be addressed.\nMethods, para 5, line 6: \"The search was conducted using a systematic review method ... AND (Risk))” were used.\" These 2 lines are duplicate statements. This has already been described in the previous paragraph and can be removed.\n\nMethods, para 5, line 10: \"The use of the term ‘wait-and-see’ ... and laser therapy, or a combination of them.\"\n\nWhat about patients started on systemic medications like eplerenone? Was it included in the search?\n\nKindly elaborate on the statistical tests/ model used in the methods section.\n\nDiscussion\n\"Of the population that experienced recurrences, 24% of them were patients ...\" The study mentions that '24 % of untreated eyes had recurrence', and NOT '24% of eyes that had recurrence, were untreated'. Kindly check and correct it.\nOther minor comments:\nMethods, para 5, line 14: \"This will be combined ... recurrence will be discussed in the study.\"\nKindly convert to past tense.\n\nResults: Para 3: \"CSC\" instead of \"CRC\"\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1036
https://f1000research.com/articles/11-1521/v1	15 Dec 22	{ "type": "Research Article", "title": "Factors affecting HBV DNA suppression in chronic hepatitis B patients treated with tenofovir disoproxil fumarate", "authors": [ "Darmadi Darmadi", "Dharma Lindarto", "Jelita Siregar", "Tri Widyawati", "Muhammad Rusda", "Mustafa Mahmud Amin", "Fauzi Yusuf", "Putri Chairani Eyanoer", "Masrul Lubis", "Imelda Rey", "Dharma Lindarto", "Jelita Siregar", "Tri Widyawati", "Muhammad Rusda", "Mustafa Mahmud Amin", "Fauzi Yusuf", "Putri Chairani Eyanoer", "Masrul Lubis", "Imelda Rey" ], "abstract": "Background: This study aims to determine the factors affecting HBV DNA suppression in chronic hepatitis B patients with tenofovir disoproxil fumarate (TDF). Methods: A case-control was carried out from October 2021 to August 2022 on 182 chronic hepatitis B patients who had TDF therapy regularly for 24 weeks at H. Adam Malik and USU Hospitals in Medan, Indonesia. The history of the samples was obtained, followed by physical examination, and blood collection. CTLA-4 polymorphism examination was carried out using real-time PCR, while the serum CTLA-4 levels were assessed with ELISA. Results: The results showed that CTLA-4, HBV DNA, ALT, and CTLA-4 -1661G>A polymorphisms have a relationship with HBV DNA suppression in chronic hepatitis B patients with TDF. Conclusions: The levels of CTLA-4, HBV DNA, ALT, and CTLA-4 -1661G>A polymorphism have a potential relationship with the suppression of HBV DNA in chronic hepatitis B patients with TDF.", "keywords": [ "CTLA-4", "HBV DNA", "Hepatitis B", "Polymorphism", "Tenofovir" ], "content": "Introduction\n\nHepatitis B (HBV) is a health problem affecting several countries in the world, including Indonesia. A national survey in 2013 showed the proportion of positive Hepatitis B surface antigen (HBsAg) was 7.1% (Indonesia Health Ministry, 2013). The ideal endpoint is HBsAg clearance, a satisfactory endpoint is HBeAg seroconversion, and the next most desirable endpoint is persistent inhibition of HBV DNA replication. In practical experience, the loss of HBsAg as an ideal therapeutic endpoint is often difficult to achieve, hence, treatment is only focused on the satisfactory and desirable endpoints. Tenofovir disoproxil fumarate (TDF) is one of the first-line drugs used for the treatment of chronic hepatitis B (INA ASL, 2017). Several studies have explored its antiviral mechanism, but the immunomodulatory mechanism is still being studied. TDF has been reported to have immunomodulatory activity, and it can reduce Treg cell levels in chronic hepatitis B patients (Yu et al., 2013). It can also reduce the release of proinflammatory cytokines, such as IL-8 and CCL3 (Melchjorsen et al., 2011). Treatment with NA enhances specific T-cell function and anergy by regulating CTLA-4 (Das et al., 2008; Thimme and Dandri, 2013). Previous studies showed that long-term NA therapy can increase peripheral T cell levels to enhance antiviral response (Zheng et al., 2012). Furthermore, their activation is strongly determined by genetic factors, such as CTLA-4 (Schurich et al., 2011; Zheng et al., 2012).\n\nCTLA-4 levels in the body are influenced by allele variations in the CTLA-4 gene, which is located on chromosome 2q33.3 and consists of 4 exons and 3 introns (Valk, Rudd and Schneider, 2008). Chen's study in China revealed that the CTLA-4 +49G>A gene polymorphism of the GG genotype increased the risk of developing liver cirrhosis and HCC, while CTLA-4 -318 T>C of CC genotype increased the risk of persistent hepatitis B virus infection (Chen et al., 2014). Alizadeh in Iran stated that the CTLA-4-318 T>C allele polymorphism causes increased susceptibility to chronic HBV (Alizadeh et al., 2006). Wang stated that chronic HBV patients with CTLA-4 +49A>G allele G and genotype GG were significantly more at risk of developing HCC in ethnic Chinese (Wang et al., 2018).\n\nThese studies have focused on the association of CTLA-4 polymorphism with the persistence and progression of chronic hepatitis B infection, but to our knowledge, no studies have explored its relationship with HBV DNA suppression. Therefore, this study aims to determine the association of the CTLA-4-1661G>A gene polymorphism and other factors, such as age, gender, ethnicity, obesity, baseline AST, baseline ALT, baseline HBV DNA, HBeAg, genotype, degree of fibrosis, and CTLA-4 levels with HBV DNA suppression in chronic hepatitis B patients treated with TDF. The DNA was suppressed to an undetectable level, namely <100 IU/mL (INA ASL, 2017).\n\n\nMethods\n\nA case-control study was carried out from October 2021 to August 2022 on 182 chronic hepatitis B patients who had already done 24 weeks therapy with TDF at H. Adam Malik and USU Hospitals in Medan, Indonesia. Medication adherence was observed by self-reporting diary. The case group was patients who did not achieve a virological response to TDF treatment. The control group was patients who achieved a virological response to TDF treatment. The research samples were taken using consecutive sampling technique.\n\nEthical approval was obtained from the Ethics Review Board of Universitas Sumatera Utara on October 18th, 2021 (Ethical clearance number: 1028/KEP/USU/2021). Written informed consent was obtained from participants before enrollment in this study (Darmadi, 2022c).\n\nTo determine the sample size, use the following formula:\n\nZα = 1.96, Zβ= 1.282, p1 (proportion of patients with the GG+GA genotypes of CTLA-4 -1661 G>A polymorphism who did not achieve a virological response)= 0.57, p2 (Proportion of patients with the GG+GA genotypes of CTLA-4-1661G>A polymorphism who achieve a virological response)= 0.23 (Wang et al. 2007). The minimum sample size for each group was 91 subjects. The minimum total sample size was 182 subjects.\n\nPatients who received Peg-IFN or other NA therapies, consumed immunomodulatory/immunosuppressant drugs for the past 1 month, have liver cirrhosis, hepatocellular carcinoma, systemic diseases, such as HIV, chronic kidney disease, autoimmune, malignancy, and pregnancy were excluded from the study.\n\nThe selected patients were interviewed using a form to obtain age, ethnicity, and comorbid diseases, after which a physical examination was carried out by a hepatologist on duty.\n\nAbdominal ultrasound was performed to evaluate signs of liver cirrhosis, such as reduced liver size, irregular surface, heterogeneous parenchyma, ascites, splenomegaly, as well as signs of hepatocellular carcinoma. Transient elastography with fibroscan is a non-invasive method to evaluate the degree of fibrosis. Furthermore, its degree in hepatitis B consists of 4 categories, namely F0-F1/normal – mild when < 6.0 kPa, F2/significant fibrosis > 6 kPa, F3/severe fibrosis > 9 kPa, F4/liver cirrhosis > 12 kPa (Bonder and Afdhal, 2014). ALT, HBV DNA, HBeAg, and anti-HBe data were collected before starting treatment. Patients were examined for ALT, HBV DNA, HBeAg, and anti-HBe after 24 weeks of TDF treatment.\n\nThe determination of serum ALT levels was carried out using standard biochemical tests with an Olympics analyzer AU400 (Hamburg, Germany). The HBeAg and anti-HBe antibody levels were examined using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Roche ELISA kit, Germany). The HBV DNA viral load was assessed with the COBAS TaqMan HBV Test, version 2.0 (Roche Diagnostics, Tokyo, Japan), with a measurement ratio of 2.1 – 9 log copies/mL. We used the kits according the manufacturer’s instructions.\n\nDNA extraction was carried out with Genomic DNA Mini Kit (Geneaid Biotech Ltd., New Taipei City, Taiwan), Cat GB100, Lot. FG02009) using whole blood approximately 5 ml. All the procedure for CTLA-5-1661 G>A were performed in Prodia Laboratorium, Medam, Indonesia. All the procedure under the working procedure according to the kit.\n\nThe Primer & Probe used in this study were TaqMan SNP Genotyping Assay CTLA-4 -1661 G>A (Applied Biosystems, CA, USA) cat:44033111, Lot. 00905960. Genotyping of the CTLA-4-1661G>A gene was performed with a forward primer: 5′-CTAAGAGCATCCGCTTGCACCT-3′ and a reverse primer: 5′ TTGGTGTGATGCACAGAAGCCTTTT-3′.\n\nAmplification was performed with a C1000 thermal cycler CFX96 real-time system (BioRad, CA, USA) using the following standard protocol. The reaction mixture for RT-PCR used the TaqMan GTXpress master mix (2x) reagent with a total volume of 25 ul TaqMan GTXpress Master Mix (2x), added 1.25 ul 20x working stock SNP genotyping assay, 6.25 ul ddH20, and 5 ul DNA template with a concentration of 1-10ng/well. Amplification was performed using a CFX 96 touchTM RT-PCR (Biorad) with the following standard protocol: 20 seconds of enzyme activation at 95°C, followed by 40 amplification cycles consisting of 15 seconds of denaturation at 95°C, and 1 minute of annealing/extension at 60 C.\n\nThe measurement of serum CTLA-4 levels was performed with the ELISA method (Quantikine(R) ELISA with catalog number HSCT40, Human CTLA-4 Immunoassay R&D System Inc., Minneapolis, USA). All the materials we need, namely human CTLA-4 capture antibody (lyophilized), human CTLA-4 detector antibody 10x, human CTLA-4 lyophilized recombinant protein, antibody diluent 4BI, cell extraction buffer PTR 5X, cell extraction buffer enhancer solution 50X, stop solution, TMB development solution, wash buffer PT 10X, and sample diluent NS. All the items store in 4 C.\n\nAfter preparing all reagents, standards, and samples (serum), open the seal plate, put it back in the foil pouch, and store it at 4°C. Then add 50 μL of sample or standard to each well. After that, add 50 μL Antibody Cocktail to each well.\n\nThe plate was closed with a seal, then incubated for 1 hour at room temperature on a shaker at 400 rpm. Afterward, wash each well with 3 x 350 μL 1X Wash Buffer. In the last washing, turn the plate over and dry, then add 100 μL TMB Development Solution to each well and incubate for 10 minutes in the dark on a shaker at 400 rpm. Adding the Stop Solution will change the color from blue to yellow and increase the signal intensity by about 3X. To avoid signal saturation, proceed to the next step before the high concentration of standard reaches a blue color OD 600 equals 1.0. Given the variability in laboratory environmental conditions, the optimal incubation time can vary between 5 and 20 minutes.\n\nIn the final stage, add 100 μL Stop Solution to each well. It alternates the plate so that it is homogeneous. Then read at a wavelength of 450 nm.\n\nData analysis was carried out using the chi-square test and binomial logistic regression. Hardy Weinberg equilibrium was tested by chi square analysis. The significance of the statistical test results was determined based on the p-value <0.05 using SPSS-22 Statistics for Windows (IBM Corp, Armonk, NY, USA).\n\n\nResults\n\nFigure 1 shows the participant flow. Table 1 shows the demographic and laboratory characteristics.\n\n* p<0.05, HBV:hepatitis B virus, AST: aspartate aminotransferase, ALT: alanine aminotransferase, CTLA-4: cytotoxic T-lymphocyte associated protein 4, OR: odds ratio\n\nThere was a significant relationship between low baseline ALT, high baseline HBV DNA, high CTLA-4 levels, and HBV DNA suppression (p<0.05) (Darmadi, 2022a, 2022b). Hepatitis B patients with low baseline ALT are 1.37 times more at risk of not achieving HBV DNA suppression (p=0.037), while others with high HBV DNA are 1.42 times more at risk (p=0.017). Furthermore, people with high CTLA-4 levels are 1.6 times more at risk of not achieving suppression compared to others with low levels (p=0.002). The results showed that there was no association between gender, age, ethnicity, obesity, baseline AST, HBeAg, genotype, liver fibrosis, and HBV DNA suppression (p>0.05), as shown in Table 1.\n\nCTLA-4 levels were significantly higher in chronic hepatitis B patients without HBV DNA suppression compared to others (p=0.016), as shown in Table 2. This study used a value of 52.2 pg/mL as the cut-off to distinguish high and low CTLA-4 levels based on the results of ROC analysis with an area under the curve of 0.603 (p=0.016), as shown in Figure 2.\n\n* p<0.05, HBV:hepatitis B virus, CTLA-4: cytotoxic T-lymphocyte associated protein 4\n\nThe accuracy of CTLA-4 in predicting HBV DNA suppression in chronic hepatitis B patients with TDF is presented in Table 3.\n\nCTLA-4 levels of 52.2 pg/mL were able to predict the suppression of HBV DNA by TDF with a sensitivity and specificity of 60.4% and 62.6%, respectively, as shown in Table 3.\n\nThe genotype frequency of the -1661G>>A of CTLA-4 polymorphism in the case group (p= 0.987) and control group (p= 0.879) in Hardy Weinberg equilibrium (p>0.05) as shown in Table 4.\n\nThere was a significant relationship between the CTLA-4-1661G>A polymorphism and HBV DNA suppression. The GG+AG genotype increased the risk of not achieving suppression by 1.42 times compared to the AA genotype (p=0.031). Furthermore, patients with the G allele were 1.27 times more at risk compared to others with the A allele (p=0.025), as shown in Table 5.\n\n* p<0,05\n\nThe multivariate analysis showed that the levels of CTLA-4, HBV DNA, ALT, and CTLA-4 -1661G>A polymorphisms are associated with the suppression of HBV DNA. Furthermore, high CTLA-4 levels significantly increased the risk of not achieving suppression by 2.28 times (p=0.009). High HBV DNA significantly increased the risk by 2.09 times (p=0.026), while patients with low baseline ALT are 1.95 times more at risk (p=0.036). People with polymorphism CTLA-4 -1661G>A genotype GG+AG are 1.52 times more at risk of not achieving HBV DNA suppression (p=0.041), as shown in Table 6. The value of Nagelkerke R Square was 0.584, which indicates that the ability of the variable levels of CTLA-4, HBV DNA, ALT, and CTLA-4-1661G>A polymorphism in explaining HBV DNA suppression is 0.584 or 58.4%. There are 100% – 58.4% = 57.4% other factors outside the model that explain the dependent variable.\n\n* p<0,05, HBV:hepatitis B virus, ALT: alanine aminotransferase, CTLA-4: cytotoxic T-lymphocyte associated protein 4\n\nBased on the B values obtained from the calculation above, the equation model formed was: Ln P/1-P = -7.815 + 2.055 high CTLA-4 + 1.722 high HBV DNA + 1.599 low ALT + 1.204 CTLA-4 polymorphism - 1661G>A. The derivative formula of the equation can also be used, namely Probability = exp (-7.815 + 2.055 high CTLA-4 + 1.722 high HBV DNA + 1.599 low ALT + 1.204 CTLA-4 polymorphism -1661G>A) /1 + exp (-7.815 + 2.055 high CTLA-4 + 1.722 high HBV DNA + 1.599 low ALT + 1.204 CTLA-4 polymorphism -1661G>A).\n\nThere were significant differences in the serum CTLA-4 levels between the genotypes of CTLA-4-1661G>A polymorphism. Furthermore, CTLA-4 levels were significantly higher in hepatitis B patients with genotypes GG and AG compared to others with AA (p<0.001). It was also significantly higher in people with the G allele compared to the A allele (p<0.001), as shown in Table 7.\n\n* p<0.05,\n\n# significant compared to the AG genotype,\n\n! significant compared to the AA genotype\n\n\nDiscussion\n\nChronic hepatitis B is a health problem affecting several countries in the world, including Indonesia and it has high morbidity and mortality due to its complications. Furthermore, the importance of management in patients with the disease is to achieve treatment endpoints and to prevent its complications. Tenofovir disoproxil fumarate (TDF) is one of the NA classes of drugs used as the first line of treatment (INA ASL, 2017). It also has a high antiviral potency as well as 0% resistance after 5 years of use (De Clercq et al., 2010). Apart from its antiviral activity, TDF can also increase the levels of T cells in the periphery. The activity of these cells is influenced by CTLA4, which can transmit inhibitory signals to them after binding to CD80 and CD86 in APC (Schurich et al., 2011; Zheng et al., 2012). CTLA-4 level can be influenced by the CTLA-4 gene polymorphism (Corvalan, Carrasco and Saavendra, 2012), but no study has explored its relationship with response to hepatitis B treatment. Therefore, this study was carried out to analyze the factors influencing HBV DNA suppression, including the CTLA-4-1661G>A polymorphism in chronic HBV patients with TDF.\n\nMen are more prone to chronic hepatitis B infection due to immune, hormonal, and risk factors (Klein, 2012). Kosinska revealed that male murine models were associated with a worse HBV-specific T-cell response (Kosinska et al., 2017). The effect of gender on treatment response is controversial, but several studies reported that there is a relationship between them (Belci et al., 2016; Villa et al., 2011). The incidence of NAFLD and its severity is higher in women, especially postmenopausal compared to men (Villa et al., 2011). This indicates that NAFLD has an effect on the progression of fibrosis from mild to moderate or severe, which can cause a decrease in the response to antiviral therapy (Codes et al., 2007; Fierbinţeanu-Braticevici et al., 2010; Villa et al., 2011). Meanwhile, other studies stated that women aged <50 years have a better virological response to therapy, but there was no difference in response between men and women >50 years (Belci et al., 2016). Our study showed that there was no relationship between gender and response to chronic hepatitis B treatment (p=0.542), but it does not stratify the association between the two variables based on age group. Previous studies revealed that gender has no effect on HBV DNA suppression in chronic hepatitis B patients with TDF (Chien et al., 2003; Kau et al., 2008; Lim et al., 2007).\n\nKennedy stated that the age group with the worse T-cell response to HBV was children (Kennedy et al., 2012). Another study on adults with chronic HBV showed that patients < 36 years had better immune and virological responses compared to others (Bonino et al., 2007). This study also found that older people did not achieve HBV DNA suppression, while younger people had more probability, but there was no statistically significant difference (p=0.299). Other studies showed that age and treatment response have no association with NA (Lim et al., 2007). Tan reported that epidemiological factors, such as age and sex do have an effect on HBV-specific T cell counts. The result also showed that response to treatment has no association with NA (Tan et al., 2008).\n\nThis study revealed that there was no association between ethnicity and HBV DNA suppression in chronic hepatitis B patients with TDF (p=0.647). This is in line with previous studies that ethnicity/race has no relationship with virological response to NA or Peg-IFN (Kau et al., 2008; Lim et al., 2007; Bonino et al., 2007). Asian people have been found to be infected with chronic hepatitis B through vertical transmission (Tan et al., 2008). Ethnicity and route of transmission do not affect the response to hepatitis B treatment for NA or Peg-IFN (Tan et al., 2008; Lin and Kao, 2013).\n\nAnimal studies showed that obese mice have lower levels of antibodies and T cells compared to non-obese mice (Liu et al., 2017). Studies from South Korea also reported that memory T-cell response function can be affected by obesity (Park et al., 2014). Furthermore, there was an increase in leptin, which can induce systemic inflammation and B cells in obese patients. Leptin is produced by fat cells and can modulate adaptive and innate immune responses. The effect of obesity on the immune system has been reported by several animal studies, while its effect on response to hepatitis B treatment has not been explored (Frasca et al., 2013; Papathanassoglou et al., 2006). This finding is consistent with this study, where obesity had no association with HBV DNA suppression in chronic hepatitis B patients with TDF (p=0.436).\n\nALT is an enzyme produced by hepatocytes and a highly specific biomarker for hepatocellular injury (Hall and Cash, 2012). Patients’ ALT level is an important factor in the initiation of chronic hepatitis B therapy. Increased levels of the enzyme indicate high immune-mediated inflammation to eliminate HBV-infected hepatocytes. It has also become an important aspect in predicting the virological response of chronic hepatitis B patients in terms of HBV DNA suppression and HBeAg seroconversion (Han and Kim, 2008; Lok, 2019). In the GLOBE study, ALT levels > 2x the upper limit of normal were predictors of virological response to NA treatment (Zeuzem et al., 2009). A 4-year case-management study of HBeAg positive patients showed that 78% of people with ALT >10 upper limit of normal had HBeAg seroconversion compared to 52% in the chronic inactive hepatitis group (Wong et al., 2008). Meanwhile, Perillo reported a loss of HBeAg, especially in patients with levels > 5 upper limits of normal (Perrillo et al., 2002). Higher pretreatment levels can facilitate viral suppression by NA (Zeuzem et al., 2009; Wong and Chan, 2009). The role of baseline ALT in virological response is more dominant in people with HBeAg positive status compared to HBeAg negative (Lim et al., 2007). Baseline ALT is also a determinant of HBeAg seroconversion in patients with NA (Chien, Liaw and Atkins, 1999). This study revealed that low levels of the enzyme increased the risk of not achieving HBV DNA suppression in chronic hepatitis B patients with TDF by 1.37 (p=0.037). The multivariate analysis showed that ALT levels were independently associated with the suppression (p=0.036), but there was no relationship between AST levels and suppressed HBV DNA (p=0.234). Although AST is an enzyme produced by hepatocytes, it is also found in the skeletal muscle, heart muscle, and kidney tissue. This indicates that its specificity as a hepatocellular marker is lower than ALT (Hall and Cash, 2012).\n\nThe viral clearance by the host was influenced by the low amount of HBV DNA in the serum. Furthermore, its baseline levels are associated with a higher possibility of HBeAg loss or seroconversion in HBeAg-positive patients (Zeuzem et al., 2009), while low/undetectable levels are related to a virological response (Lim et al., 2007; Lok, 2019). The amount of HBV DNA in the serum is a predictor of chronic hepatitis B prognosis, namely cirrhosis, HCC, and death (Chen et al., 2006; Iloeje et al., 2006). The baseline levels also have a correlation with the effectiveness of NA therapy, including histologic grade, suppression of hepatitis DNA to undetectable, normalization of ALT, and HBeAg seroconversion (Mommeja-Marin et al., 2003). This study revealed that high levels of HBV DNA increased the risk of not achieving its suppression in chronic hepatitis B patients with TDF by 1.42 times (p=0.017). The multivariate analysis showed that the levels were independently associated with the HBV DNA suppression (p=0.026).\n\nContinuous exposure to high concentrations of HBV DNA, including HBeAg, HBsAg, and HBx causes anergy in T cells due to increased expression of CTLA-4 (Ye et al., 2015). Peng revealed that HBeAg can increase the expression in T cells. This is closely associated with high HBV DNA levels, but the underlying mechanism is still unclear (Peng et al., 2011). Serum HBeAg quantification can be a marker to predict HBV DNA in response to antiviral therapy. Measurement of its levels correlated with the level of HBV DNA replication and liver inflammation. HBeAg seroconversion is also associated with decreased viral load, but this result is controversial. Ji et all reported that 87.5% and 75% of patients with a decrease in HBeAg > 2 logs have undetectable HBV DNA and HBeAg seroconversion, respectively (Ji et al., 2013). Previous studies revealed that its levels were more consistently associated with HBeAg seroconversion than that of HBV DNA (Fried et al., 2008; Yang et al., 2016). Furthermore, its quantification during therapy also has a good prediction of HBV DNA suppression with Peg-IFN therapy. A previous study showed that people with HBeAg > 100 PEIU/mL at week 24 had a 96% probability of not achieving a virological response. HBeAg correlates with virological response in terms of levels, and qualitative status. This study revealed that there was no relationship between HBeAg status and HBV DNA suppression in chronic hepatitis B patients with TDF (p=0.456). However, its levels were not evaluated because the test is not a standard for monitoring chronic HBV.\n\nHepatitis B can be divided into 10 genotypes based on >8% difference in the genome sequence, namely A-J. Their clinical and virological course also differs with geographies. They have been reported to influence treatment reaction to Peg-IFN. Previous studies revealed that genotypes A and B have a better virological response than C and D (Buster et al., 2008; Marcellin et al., 2009; Sonneveld et al., 2012); Sunbul, 2014). HBV is a strong predictor of virological response to Peg-IFN (Lok, 2019). It also affects the incidence of mutations in the precore and core promoter regions of the HBV genome in some chronic hepatitis B patients. The dominant precore mutation involves changing the G allele at the nucleotide 1896 (Al896) to A allele, which induces a mutant precore stop codon. This mutation prevents translation of the precore protein and completely inhibits HBeAg production. Furthermore, mutations in the promoter core region have been reported, and the most common type involves the substitution of two nucleotides, namely A-T and G-A at nucleotides 1762 and 1764, respectively. Changes in the promoter core region contribute to the loss of HBeAg. These precore and core promoter mutations have been reported to affect the therapeutic response to IFN treatment, but not to NA. These changes majorly occur in genotypes C and D of HBV (Alfaresi et al., 2010). Several studies showed that HBV genotypes had no effect on the virological response to NA (Zeuzem et al., 2009; Chan et al., 2003; Gane et al., 2008; Westland et al., 2003). This is in line with this study that they have no association with HBV DNA suppression in chronic hepatitis B patients with TDF (p=0.549).\n\nThe progression of fibrosis in the disease depends on the comorbidities and the degree of active inflammation. Assessment of fibrous liver is important to assess the prognosis and urgency of therapy (Parikh, Ryan and Tsochatzis, 2017). Sustained HBV suppression with NA is closely associated with decreased necroinflammatory activity and improvement/regression of fibrosis (Marcellin et al., 2013; Papachrysos et al., 2015). The Knodell, Isaac, and METAVIR histologic systems are often used to assess disease activity as well as to evaluate treatment response. The goal of the therapy is to stop the ongoing necroinflammation and prevent fibrosis progression (Fernández-Rodríguez and Gutiérrez-García, 2014). The stage of fibrosis determines the prognosis and initiation of treatment, but cannot predict treatment response (Chen et al., 2021; Grizzi and Desmet, 2014). Its degree is also associated with response to pegylated interferon therapy. Shindo showed that hepatitis B patients who were non-responders to pegylated interferon were closely associated with lighter grading and more severe staging. The grading used to describe the intensity of necroinflammatory activity includes the assessment of portal, periportal, and intraacinar inflammatory cell infiltration, as well as various forms of hepatocyte damage and necrosis. Staging is an evaluation of the degree of fibrosis, which is caused by the necroinflammatory process consisting of F0-F4 (Buster et al., 2008; Shindo et al., 2004). However, there is no relationship between the degree and NA therapy (Lim et al., 2007; Bonino et al., 2007; Shindo et al., 2004). These findings are consistent with previous studies that the fibrosis stage is not associated with HBV DNA suppression in chronic hepatitis B patients with TDF (p=0.347).\n\nBased on a study conducted by Cho on the effect of TDF administration on CTLA-4 level, it was discovered that an immune molecule that can inhibit T-cell function in chronic HBV patients. After 6 months of therapy, there was a significant decrease in its expression. The result showed that TDF has immunomodulatory properties by lowering CTLA-4 level, which causes T-cell anergy. It also has a functional T-cell recovery effect (Cho et al., 2020). Wongjitrat revealed that CTLA-4 in chronic HBV patients was significantly higher than the healthy controls. It was also reported to interfere with the immune response and cause chronic hepatitis B persistence (Wongjitrat et al., 2013). Furthermore, it has an association with HBV DNA suppression in patients with TDF. In the bivariate analysis, subjects with high content are 1.6 times more at risk of not achieving suppression (p=0.002), while the risk was increased by 2.28 times (p=0.009) in the multivariate analysis. Level > 52.2 pg/mL can predict virological response to TDF with sensitivity, specificity, PPV, NPV, and accuracy of 60.4%, 62.6%, 61.8%, 61.3%, and 61.5%, respectively.\n\nIn this study, the Hardy-Weinberg test was carried out. The allele and genotype frequencies in the CTLA-4 -1661G>A polymorphism of the subjects were in accordance with the Hardy-Weinberg balance (Li and Graubard, 2009; Sha and Zhang, 2011). The results showed that the polymorphism can affect the circulating level. There were significant differences in serum CTLA-4 level between genotypes of the CTLA-4 -1661G>A. The level obtained in chronic hepatitis B patients with genotype GG were significantly higher compared to AG and AA (p<0.001). Furthermore, its amount in the serum were significantly higher in people with the G allele compared to A (p<0.001). The G allele of CTLA-4 -1661G>A showed higher promoter activity than the A allele. The polymorphism was located in the promoter region, which is important for the activation of the transcriptional process. Nucleotide sequence analysis showed binding sites with potential transcription factors, namely nuclear factor of activated T-cells (NFAT), Activator Protein 1 (AP-1), signal transducer and activator of transcription (STAT) (Valk, Rudd and Schneider, 2008; Gibson et al., 2007). The allele variation of the CTLA-4-1661G>A gene can increase the transcription factors, including NFAT, AP-1, and STAT, which bind to the promoter site. This is the initial site for the attachment of the RNA polymerase enzyme, which perform transcription. Furthermore, transcription factors are proteins that control the rate of genetic information transcribed. These factors or their combination with other proteins in a complex can activate RNA polymerase recruitment and binding stabilization, as well as catalyze histone acetyltransferase (HAT) activity. This then causes an increase in the transcription rate and expression of CTLA-4 [9, 15,67]. Chen reported that the factor Foxp3 in the promoter region of CTLA-4 can recruit histone acetyltransferase enzymes, which stimulate histone acetylation processes. It can also regulate transcription through direct chromatin remodeling (Chen et al., 2006). The regulation of CTLA-4 cell surface expression can be explained by the functional variations in the amino acid sequence of the molecule located at the cytoplasmic tail Y201 motif. Furthermore, this motif is an important intracellular regulator of CTLA-4 protein level through its interaction with the clathrin adapter complexes AP-1 and AP-2, which mediate, degrade and endocytose the protein, respectively. The newly synthesized CTLA-4 does not remain in the Golgi or post-Golgi compartment. It is also not stored in cytoplasmic vesicles, but rather transported directly to the cell surface (Valk, Rudd and Schneider, 2008).\n\nThere is a correlation between CTLA-4 -1661G>A gene polymorphism with HBV DNA suppression in chronic hepatitis B patients with TDF. People with GG+AG genotype are 1.42 times more at risk of not achieving suppression (p=0.031), while patients with the G allele of CTLA-4 -1661G>A have a 1.27-fold increased risk compared to others with the A allele (p=0.025). Based on the multivariate analysis, people with genotype GG+AG are 1.52 times more at risk of not achieving HBV DNA suppression (p=0.041). The effect of the CTLA-4-1661G>A gene polymorphism on virological response to TDF is caused by an increase in CTLA-4 level. Furthermore, CTLA-4 molecules through their interactions with costimulatory molecules B7 can cause negative regulation of T cells. Their increased expression can also increase spontaneous T cell apoptosis (Radziewicz et al., 2008). CTLA-4 can influence infection progression and antiviral immune response (Stoop et al., 2005; Tavakolpour, Alavian and Sali, 2016; Xu et al., 2006; Yang et al., 2007). Treatment with NA enhances specific CD8+ T-cell function and improves anergy by up-regulating CTLA-4 (Das et al., 2008; Thimme and Dandri, 2013).\n\nThis study demonstrated a dose-dependent effect of the G allele in increasing serum CTLA-4 level as well as the risk of not achieving a virological response to TDF. Moreover, CTLA-4 production and risk of non-responders to TDF were high in patients with the GG genotype, intermediate in AG, and low in AA, while the A allele was protective.\n\nThis study had limitations, including not analyzing quantitative HBeAg, quantitative HBsAg, and polymorphisms of other genes that can affect the virological response to TDF. This study also only analyzed 1 location of the CTLA-4 gene, namely -1661G>A.\n\n\nConclusion\n\nThe results showed that the levels of CTLA-4, HBV DNA, ALT, and CTLA-4-1661G>A polymorphism have a relationship with the suppression of HBV DNA in chronic hepatitis B patients with TDF.", "appendix": "Data availability\n\nFigshare: Mater Data Hepatitis. https://doi.org/10.6084/m9.figshare.21471264 (Darmadi, 2022a).\n\nThis project contains the following underlying data:\n\n- Master Data.xlsx (Data consists of polymorphisms towards HBV DNA)\n\nFigshare: The result of CTLA4 1661GA and CTLA ELISA https://doi.org/10.6084/m9.figshare.21651587 (Darmadi, 2022b).\n\nThis project contains the following underlying data:\n\n- Hasil CTLA4 1661GA dan CTLA4 ELISA.csv (This is the result of CTLA4 1661GA and CTLA ELISA)\n\n- RT-PCR CTLA4 1661GA (standard curves from RT-PCR)\n\nFigshare: Informed consent for sample https://doi.org/10.6084/m9.figshare.21564474 (Darmadi, 2022c).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAlfaresi M, Elkoush A, Alshehhi H, et al.: Hepatitis B Virus genotypes and precore and core mutants in Uae Patients. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "168445", "date": "21 Apr 2023", "name": "I Ketut Mariadi", "expertise": [ "Reviewer Expertise Hepatology and gastroenterology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this paper is excellent and is an important addition to the literature. The new idea in determining the response to hepatitis B treatment is important. but I have some suggestions for this paper:\nThe results of the study in the abstract should be mentioned clearly with the data, not the same as the conclusions.\n\nSome of the reference literature is older than 10 years.\n\nThe aim of the study, the results, and the conclusion should be in line.\n\nPlease use the term relation, correlation, and association properly\n\nDescribe the study design clearly. This is a case-control study, how to find and determine the case and control to reduce selection bias. how to find 91 cases and 91 control out of 276 samples ( after doing exclusion ). And not clear when you did the test of CTLA-4, at the start of treatment or at the end of treatment?\n\nPlease state clearly, how to determine categorical variables from the numerical variable. ex: High vs Low in AST, ALT, DNA etc. or high vs normal?\n\nTable 5 should be presented clearly\n\nPlease make a clear statement in the text to avoid misinterpretations. ex. in the results: \"There was a significant relationship between low baseline ALT, high baseline HBV DNA, high CTLA-4 levels, and HBV DNA suppression (p<0.05) (Darmadi, 2022a, 2022b).\" \" low baseline ALT are 1.37 times more at risk of not achieving HBV DNA suppression (p=0.037),\"\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? Yes", "responses": [ { "c_id": "10076", "date": "17 Nov 2023", "name": "Darmadi Darmadi", "role": "Author Response", "response": "Thank you for your kindness to give us some input to improve our paper. We have revised it according to your comments. The results of the study in the abstract should be mentioned clearly with the data, not the same as the conclusions. The result in the abstract has been revised Some of the reference literature is older than 10 years. : Majority of old literature has been deleted, except for a few pieces of literature which are still widely used as references The aim of the study, the results, and the conclusion should be in line. There is a revision in the conclusion so that it’s in line with the aim of the study. Please use the term relation, correlation, and association properly. There are sentences that use correlation which has been revised to become association because it evaluates the relationship between categorical and categorical data. Describe the study design clearly. This is a case-control study, on how to find and determine the case and control to reduce selection bias. how to find 91 cases and 91 control out of 276 samples ( after doing exclusion ). And not clear when you did the test of CTLA-4, at the start of treatment or at the end of treatment? A description of the case and control groups and pretreatment/baseline CTLA-4 has been added to the methods Please state clearly, how to determine categorical variables from the numerical variable. ex: High vs Low in AST, ALT, DNA, etc., or high vs normal? A cut-off for classifying numeric data as categorical has been added in methods. Table 5 should be presented clearly. Sentences have been added to further clarify Table 5. Please make a clear statement in the text to avoid misinterpretations. ex. in the results: \"There was a significant relationship between low baseline ALT, high baseline HBV DNA, high CTLA-4 levels, and HBV DNA suppression (p<0.05) (Darmadi, 2022a, 2022b).\" \" low baseline ALT is 1.37 times more at risk of not achieving HBV DNA suppression (p=0.037),\" This sentence has been revised." } ] }, { "id": "174440", "date": "30 May 2023", "name": "Hendra Koncoro", "expertise": [ "Reviewer Expertise Hepatitis B", "Liver Cirrhosis", "Hepatocellular Carcinoma" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDarmadi et al have done a very good and useful study to find out factors affecting HBV DNA suppression in chronic hepatitis B treated with Tenofovir Disoproxil Fumarate (TDF). TDF is one of the first-line drugs used for treatment of chronic hepatitis B. Previous studies have shown longterm nucleotide analog therapy (eg: TDF) can increase peripheral T cell levels to enhance antiviral response. Their activation is strongly determined by genetic factors, such as CTLA-4. It's really interesting that Darmadi et al focused on the association of CTLA-4 polymorphism in the persistence and progression of chronic hepatitis B infection, with HBV DNA suppression. Although this study has so many good points, there might be some suggestions and comments to improve this research article.\nFirst of all, there is a similarity in the abstract at the results and conclusion section. Preferably, the results are presented with numerical data.\nThen, in the methods, it is stated that \"the case group was patients who did not achieve virological response to TDF treatment, the control group was patients who achieved a virological response to TDF treatment.\" The author need to give definition of virological response to NUCs (eg. Tenofovir). Inclusion criteria was also did not mention in the Methods section.\nIn the procedure section, CTLA-4 level was measured, but the author did not state when the measurement was done. The author should state whether the measurement was done before, after, or before and after the treatment started.\nIn the data analysis, it is important to explain the cut off and how to define each of the categorical variables, eg. the age is divided into old and young, the author need to state the criteria of old and young. The AST and ALT baseline is better categorized as normal and increased instead of low and high.\nIn the discussion section, the author has written down the basic theory and supporting studies that are advanced and complete. However, it is better to use the latest reference to optimize and support this study as there are several references dates up to 24 years ago. In the discussion it should be stated how this research may change the daily clinical practice of clinicians. Does CTLA-4 levels or CTLA-4 polymorphism need to be checked for every chronic hepatitis B patients naive-treatment to achieve ideal or satisfactory end point or just to measure HBV DNA suppression.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10077", "date": "17 Nov 2023", "name": "Darmadi Darmadi", "role": "Author Response", "response": "We are very grateful for your comments on our paper. We have added some revisions according to your comments. 1. Result in the abstract has been revised. 2. Has been revised The description of the case and control groups. Has been added regarding the inclusion criteria in the methods section. 3. CTLA-4 was checked before treatment (baseline) and has been added to the methods. 4. A cut-off for classifying numeric data as categorical has been added in methods. 5. Majority of old literature has been deleted, except for a few pieces of literature which are still widely used as references. There has been additional discussion on this matter." } ] } ]	1	https://f1000research.com/articles/11-1521
https://f1000research.com/articles/11-337/v1	21 Mar 22	{ "type": "Opinion Article", "title": "Surfing Corona waves – instead of breaking them: Rethinking the role of natural immunity in COVID-19 policy", "authors": [ "Andreas Kalk", "Joachim Sturmberg", "Wim Van Damme", "Garrett W. Brown", "Valéry Ridde", "Martin Zizi", "Elisabeth Paul", "Andreas Kalk", "Joachim Sturmberg", "Wim Van Damme", "Garrett W. Brown", "Valéry Ridde", "Martin Zizi" ], "abstract": "Current COVID-19 response policies have aimed to break Corona waves through non-pharmaceutical interventions and mass vaccination. However, for long-term strategies to be effective and efficient, and to avoid massive disruption and social harms, it is crucial to introduce the role of natural immunity in our thinking about COVID-19 control and prevention. We argue that any Corona control policy must appropriately balance five key elements simultaneously: balancing the various fundamental interests of the nation, as well as the various interventions within the health sector; tailoring the prevention measures and treatments to individual needs; limiting social interaction restrictions; and balancing the role of vaccinations against the role of naturally induced immunity. Given the high infectivity of SARS-CoV-2 and its differential impact on population segments, we examine this last element in more detail and argue that an important aspect of ‘living with the virus’ will be to better understand the role of naturally induced immunity in our overall COVID-19 policy response. In our eyes, a policy approach that factors natural immunity should be considered for persons without major comorbidities and those having ‘encountered’ the antigen in the past.", "keywords": [ "COVID-19", "SARS-CoV-2", "vaccines", "natural immunity", "health policy" ], "content": "Introduction\n\nThe ongoing COVID-19 pandemic caused by a novel coronavirus, SARS-CoV-2, emerged late in 2019 in Wuhan, China, and spread within a few months around the globe, causing epidemics of variable intensity in diverse contexts.1 Genetic mutations created thousands of new variants of the virus, some of them being more infectious than the original variant and have become principal drivers of the pandemic, recently the Delta variant, superseded by the Omicron variant, are so far the most infectious (but less virulent?).2 At the time of writing this paper (March 2022), close to 450 million COVID-19 cases and more than six million related deaths have been registered around the world.3\n\nThe initial policy response to the pandemic in most countries was characterised by two factors. On the one hand, very few governments explicitly defined an overall policy goal for coronavirus control. Implicitly, some governments (like China) seemed to strive for elimination of the epidemic (eradication of a zoonosis being next to impossible), others (like Brazil or the USA) depicted it ‘like a bad flu’ and tried – at least for a given period – to basically ignore it.4 On the other hand, activities to control the pandemic showed an increasing degree of conformity across countries, the main pillars being initially ‘non-pharmaceutical interventions’ (NPIs) including lockdown measures, travel restrictions and face mask mandates, but soon, incorporated mass vaccination of the population in order ‘to break the waves’ (peaks of the epidemic in terms of hospitalisations and deaths, the main acknowledged policy objectives in most countries).\n\nSimultaneously, and in the absence of an explicit overall policy goal, secondary objectives such as ‘flattening the curve’ and ‘protecting health services from being overwhelmed’ were intensively discussed and imposed. The logic was simple, but also a gamble – use NPIs to slow the virus while waiting for vaccine discovery, rollout and mass inoculation.5 Yet, the need for protecting services also reflected prior negligence, where austerity policies, disinvestment in primary healthcare, and poor preventative and preparedness measures left systems struggling to cope, while also widening pre-existing inequities. These further policy developments went hand in hand with the slow recognition that it was neither feasible to eliminate the disease nor to continuously ignore it. And slowly coincided with an emerging understanding that vaccines alone cannot offer the miracle solution many governments had hoped for. Although most countries continue to act without a clear definition of an overarching policy goal – instead re-imposing knee-jerk NPI policies to reduce risk – there is now seemingly increased recognition that humankind will have to ‘to live with the virus’ one way or another.6 This recognition is crucial, since SARS-CoV-2, like all coronaviruses, is a zoonosis, having numerous mammalian host species.7–10 Not all those hosts represent a principal reservoir of the virus, but species can de facto ping-pong mutations between them, enhancing genetic variations and thus jeopardising virus control.\n\nWhat remains unclear and highly elusive are articulated long-term strategies for ‘living with the virus’, which can be effective and efficient, without causing the massive disruption and social harms associated with current coronavirus policies. Toward that end, it is crucial to consider basic immune system behaviour, and secondly, to better factor the known specific immunological pathways of SARS-CoV-2. In this paper, we propose to introduce an additional policy component in response, namely, the role of natural immunity – stimulating polyclonal antibody (B-cell) and cellular (T-cell) responses – in our thinking about COVID-19 control and prevention. However, our starting point prior to the presentation of this argument is to suggest that any coronavirus control policy (including this our alternative offered here) must appropriately balance five key elements simultaneously.\n\nFirst, the interest to protect the population from Covid-19 has to be appropriately balanced against other fundamental interests of the nation\n\nThese interests include respect for personal liberty and human rights on one side, and the need to protect local and the global economies on the other. For example, the former aspect requires serious consideration of the right to education, since school closures are one of the least evidence-based NPIs11–14 and there is credible evidence that children and adolescents are far less susceptible to illness and serious disease.15 The latter (economic) aspect has to include often forgotten informal economies, since in many countries (particularly low- and middle-income countries (they represent a major source of economic activity and livelihood. Thus, policy making has to better consider the economic effects both of the pandemic as well as the control measures. This is particularly germane since there is growing evidence demonstrating the adverse long-term economic and health effects of many NPIs. Finding an appropriate balance between these elements is important since education and socio-economic development are crucial determinants of health in the medium term,16 while producing epigenetic changes for several generations to come.17 The respect of such a balance of coronavirus control measures against other fundamental interests can be understood as an essential element of the social contract between government and citizens, contributing significantly to trust in government authority and bonds of social solidarity.\n\nAs pointed out by Kass18 and later by Turcotte-Tremblay and Ridde,19 finding an appropriate balance between health, liberty and other social concerns should review the available scientific evidence in light of ethical frameworks that can better reflect concerns for equity and social justice, thus incorporating wider considerations than those provided merely through morbidity and mortality calculations. Such a process is not easy and requires consultative and deliberative processes that can reflect multifarious interests, population strata, and sectors, rather than relying on the view of a limited cohort of specialists20 – which often promotes ‘groupthink’, underrepresentation, and ‘enclave echo chambers’.21\n\nSecond, coronavirus control has to be balanced against other interventions within the health sector\n\nThis challenge again entails two aspects: The allocation of human, financial and physical resources must correspond to the relative burden of disease in a given community.22 As of now, in many countries (e. g. sub-Saharan Africa), other health threats such as neonatal disorders, lower respiratory infections, malaria, and tuberculosis cause a significantly higher burden of disease than COVID-19.23 Of equal importance is the consideration of the negative health-related effects of coronavirus control ranging from psychological damage, particularly from the promulgation of fear, the interference to supply chains resulting in malnutrition (again especially in LMICs), and the deterioration of other pre-existing diseases (and even death) caused by travel restrictions, health system lockdowns, and other measures.\n\nThird, preventive measures have to be balanced against the need to assure and improve – and tailor to individual needs – the treatment of patients affected by COVID-19\n\nWe insist that pharmaceutical interventions represent one of the four cornerstones of coronavirus control (the others being preventive: NPIs, vaccination and natural immunity). It is unprecedented how quickly researchers identified, over the past months, drugs to treat the disease. Some of them were known, sometimes even for similar indications (as dexamethasone,24 heparin – preventively25 and/or therapeutically26 – or Xylitol nasal spray as a preventative);27 some represent newer developments (antivirals as molnupiravir28 or monoclonal antibodies).29 Yet, these advances have so far been largely overshadowed by a policy focus obsessed with the use of NPIs and vaccines.\n\nFourth, within the toolbox of preventive measures, most NPIs rely on reduced contact between human beings which runs counter to our social natures\n\nLarge scale and blanket NPIs have been recommended based on models that did not take into account the fact that SARS-CoV-2, like all coronaviruses, can have numerous interspecific hosts.10 Moreover, the evidence for the efficacy of ‘NPIs in practice’ is lacking,30 while evidence for their negative consequences has been consistently growing.31 NPIs provoke social segregation and must thus be seen as being in tension with our social nature while amplifying emotional distress. Catastrophic consequences of psychological damage (particularly for frontline healthcare workers, the elderly, children and people working in the informal care sector) have been observed on all continents.32–35 Consequently, priority should be given to prevent COVID-19 for those at greatest physical and/or emotional risk by achieving immunity, be it induced ‘naturally’ or ‘artificially’ through vaccination.\n\nLastly, the role vaccinations should play now, and in the future of coronavirus control, has to be balanced against the role naturally induced immunity already plays and could play in the future\n\nGiven the high infectivity of SARS-CoV-2 and its differential impact on population segments, this particular question deserves greater attention. As a result, we examine this in more detail below, arguing that an important aspect of ‘living with the virus’ will be to better understand the role of naturally induced immunity in our overall COVID-19 policy response.\n\nInnate immunity, which is the first line of defence of the immune system, is key to combat a novel virus such as SARS-CoV-2.36 When analysing the factors inducing immunity against SARS-CoV-2, five different steps of increasing immunological specificity can be distinguished. These levels might be based on different immunological mechanisms and pathways, which are outlined below and summarized in Figure 1.\n\nSource: authors.\n\nFirst, most people have some kind of natural resistance against pathogens37 or ‘general immune competence’ and are thus distinguished from those with known immune deficiency diseases or immune system compromise. The macrophages are one of the responsible actors. ‘Generally immune competent’ individuals are less prone to become infected or to become sick once infected. Such competence (based partially on a rapid interferon response) might explain the fact that children are much less affected by COVID-19 than adults.38,39\n\nSecond, one’s immune system is likely to be ‘trained’ in the course of one’s life through a large variety of infections without a higher degree of genetic similarity among the infectious agents. Thus, previous helminthic infections as well as malaria are likely to protect gradually from other infections, most notably by SARS-CoV-2, and from more serious forms of COVID-19. It is possible that the slow progress of the coronavirus pandemic in most sub-Saharan African countries can partially be explained by this phenomenon.40\n\nThird, it is known since 1970 that coronaviruses provoke 10-20% of all ‘flu waves’ and ‘common colds’ experienced globally. Previous exposure to such ‘older’ coronaviruses most likely fosters some level of cross immunity against SARS-CoV-2 and its different variants.41,42\n\nFourth, a previous infection with SARS-CoV-2 creates a certain degree of immunity against reinfection. This immunity can result both from symptomatic as well as from asymptomatic infections, the latter possibly characterised by a higher degree of immunity, notably because the virus is polyclonal and not limited to the highly mutating spike protein, and because it fosters stronger mucosal and T-cell responses.43–52\n\nFifth, immunity against the virus can be induced by vaccines. Whilst the aforementioned forms of immunity are caused ‘naturally’, vaccine-induced immunity represents an artificially created defence mechanism, generally based only on parts of the virus, principally the spike protein (viral vector and m-RNA vaccines), and inactivated or attenuated vaccines. Available evidence is not yet conclusive and partially contradictory, but vaccine-induced immunity might be more susceptible to evasion by new virus variants.53–56\n\nMost countries have now experienced several waves of coronavirus infections, and with this evolving experience, the strategies should be adapted. If one agrees that the elimination of SARS-CoV-2 is untenable, and if one accepts that the continued use of NPIs is not sustainable over the long run, then, by deduction, the best epidemic exit strategy would need to be based on the two remaining preventive pillars, namely, ‘natural’ immunity and vaccination. In this context, it is highly surprising that most governmental policies still rely largely on vaccination and ignore the role other forms of immunity could play and do play already. This is particularly astonishing now that the evidence strongly suggests that the efficacy of vaccinations wanes quite rapidly.57–61 In addition, the arrival of the Omicron variant powerfully illustrates that such vaccine efficacy will be constantly challenged by mutations – like in the case of traditional influenza vaccines, but unlike the efficacy of vaccines against ‘stable’ viruses as mumps, measles or yellow fever.62,63 Omicron has also demonstrated that current policies remain disjointed according to national self-interest, that good science and reporting is not always rewarded (as in the case of South Africa reporting Omicron), and that outdated notions of national health security (particularly in the West) will only be as strong as their global weakest links.\n\nAs far as the role of natural immunity is concerned, it must be recognised that people having had a proven SARS-CoV-2 infection (as long as confirmed by a previous positive PCR/antigen test) are registered in many countries, and they enjoy privileges similar to those with a complete vaccination record (in terms of movement restrictions, access to public places etc.) – at least for a few months. Nonetheless, so far, no effort has been made to identify systematically those people having either contracted COVID-19 or an asymptomatic/undiagnosed infection. Following data from the Institute for Health Metrics and Evaluation (IHME), this proportion is more than 50% in most countries in Africa and South Americas, and surpasses 85% in countries such as Algeria, Bolivia, Iran, Kenya, Russia and South Africa.64\n\nWe defend the thesis that these figures have to be taken into account when promoting COVID-19 prevention through individual and collective immunity. Furthermore, the immunostimulant synergy between previous vaccinations and previous infections has to be better scrutinised and reflected upon within public health policies.\n\nThe importance of conducting such an analysis is underpinned by several empirical conditions: i) the aforementioned high degree of peoples’ natural exposure to the virus and presumed widespread natural immunity in many countries, ii) the recognition that vaccine-induced protection wanes quite rapidly (in the course of 3-8 months),57,58,60 and in consequence iii) the ever-increasing frequency of test-identified ‘breakthrough’ infections (infections of correctly vaccinated persons) presenting with limited or unspecific upper respiratory symptoms.\n\nHence, crucial questions arise: Do we really need anti-coronavirus ‘refresher jabs’ with the frequency promoted by many policy makers, media outlets and vaccine producers? Or should these ‘jabs’ be reserved for highly vulnerable people? Following such a booster strategy for the entire population is costly, intrusive, and hugely ignorant of the fact that current vaccine distribution has been inequitable, with many parts of the world yet unable to access vaccines to protect at least those most vulnerable people.65 It is hard to imagine how this will not continue under any booster reliant regime as it will operate within the already existing ‘vaccine apartheid (or inequity)’.66\n\n\nConclusion: a plea for a ‘mix’ of immune inductions by antigen exposure\n\nAntia and Halloran distinguish three interrelated forms of immunity, which can all be caused both naturally and by vaccination:67\n\n• Immunity against infection;\n\n• Immunity allowing infection, but reducing infectiousness;\n\n• Immunity against disease (reducing disease manifestation and preventing death).\n\nThey stipulate – and this presumption is at least plausible – that immunity against infection wanes more rapidly than immunity against disease, the latter one being a weaker, but more important form of immunity mediated by T-cells.51 This results in a time window labelled by Antia and Halloran as a ‘region of mild boosting’, namely, a period allowing re-infection or re-vaccination without developing (serious) disease. This window is depicted as most appropriate for refreshing the immunity to previous or even new levels. It might be open for a period ranging from approximately 3 to 24 months since the last antigen exposure, be it based on natural exposure or on vaccination. Moreover, Antia and Halloran insist that ‘… we also need to determine if multiple infections or vaccinations are needed to generate long-lasting protection against pathology, and whether this depends on the age of the individual’.\n\nWe propose that the role of pre-existing or induced natural immunity deserves immediate further attention. In relation to our argument to balance interests, this appeal includes the suggestion to recognise the extraordinary immune competence of children and adolescents as far as SARS-CoV-2 infections are concerned, and to seriously scrutinise the use of compulsory vaccination for the young. If the concept of a ‘region of mild boosting’ is backed by additional evidence, for people having had previous encounters with the virus antigen (be it an encounter via SARS-CoV-2 itself or from a vaccination), then striving for early and regular re-infections might represent a viable alternative to avoid continuous re-vaccination – at least if no personal risk factors such as age or co-morbidity are present. The crucial point here is the idea that a re-infection for a previously infected person might be as harmless as a re-vaccination, be possibly of higher efficacy than a vaccination49 as the immune system is interacting with the entire virus antigen, and finally, that such ‘mild boosting’ based on a re-infection could take place independently from the pharmaceutical industry and the high costs of universal vaccination (not to mention feasibility constraints). This last point is particularly important, since many low resource countries are already struggling to implement first round vaccines and have limited budgets in which to afford continuous boosters.\n\nIn our eyes, an approach that factors natural immunity as a policy consideration should definitely be considered for persons without major comorbidities and those having ‘encountered’ the antigen in the past. Although the pursuit of this alternative will require further research evidence, we believe it could constitute a necessary and fundamental paradigm shift in coronavirus control, allowing us to ‘surf’ the waves instead of desperately trying to break them. An additional benefit of such a ‘surfing approach’ via a greater reliance on natural immunity is that such continuity of ‘antigen encounters’ will most probably lead – as Antia and Halloran point out – to the shift of the epidemic towards a milder childhood disease with limited long-term impact on health. This approach effectively could allow us to ‘live with the virus’ without continuous harmful NPIs nor the herculean task of boosting a global population of nearly 10 billion people on a continuous six-month rolling basis. Most importantly, however, what this approach offers is a long-term alternative to current policies, which remain ad hoc, reactive, insufficient, inequitable, and devoid of any long-term exit strategy.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nVan Damme W, et al.: The COVID-19 pandemic: diverse contexts; different epidemics—how and why?. BMJ Glob. Health. 2020; 5: e003098. PubMed Abstract \| Publisher Full Text\n\nMaslo C, et al.: Characteristics and Outcomes of Hospitalized Patients in South Africa During the COVID-19 Omicron Wave Compared With Previous Waves. JAMA. 2021; 327: 583–584. 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Policy. 2022.\n\nMcIntyre PB, et al.: COVID-19 vaccine strategies must focus on severe disease and global equity. Lancet. 2021; 399: 406–410. PubMed Abstract \| Publisher Full Text\n\nAntia R, Halloran ME: Transition to endemicity: Understanding COVID-19. Immunity. 2021; 54: 2172–2176. PubMed Abstract \| Publisher Full Text" }	[ { "id": "136136", "date": "13 Jun 2022", "name": "Cassian Minguet", "expertise": [ "Reviewer Expertise Family practice" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article asks some good questions. The question of natural immunity could be further referenced. The point of living with the virus (which is true) does not logically imply in my view that it necessarily means promoting natural immunity. On the other hand, concentrating vaccination on groups at risk (internationally) is indisputable. Similarly, taking into account the secondary effects (in terms of the general health of the population) of policies \"breaking the waves\" in order to make efficient and scalable decisions based on the advancement of knowledge is a major argument deserving to be even better highlighted in this article.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "144183", "date": "15 Aug 2022", "name": "Prof. Michael Marx", "expertise": [ "Reviewer Expertise Health systems research", "global health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important paper that critically discusses the current COVID -19 policy on a global scale. A substantial part of the literature is being reflected to support the arguments.\nHowever, given the unprecedented speed and amount of research on SARS CoV 2 and the wealth of new literature that was produced since the article was submitted, some of the latest papers could be included, especially those dealing with immunological and virological features of new virus strains, e.g. Omikron BA.5,\n\nand their public health impact.\nMoreover, although mentioned, more differentiation between northern and southern countries is missing especially when it comes to the impact of the pandemic related to incidence rates, demography, and respective immune competences and health care provision.\n\nAt some points, it is not clear enough which arguments are grounded on scientific evidence or which are based on assumptions deducted from current empirical knowledge. For instance, I would be more cautious to compare SARS CoV 2 with features of influenza virus strains.\n\nIt would add to the value of the paper if some statements would be a bit softened by admitting limitations due to still prevailing grey knowledge zones, e.g. immunity, future waves, and specific features of SARS CoV 2 virus strains.\n\nLast but not least, speaking of non-pharmaceutical intervention the value of preventive measures like wearing FFP2 masks, and Hepa- air filters shouldn't be underestimated when high incidence and morbidity rates risk compromising health care and social systems.\n\nHowever, with some updates and amendments, this article has the substance to further induce the critical and systemic discussion of the impact and control of the current pandemic.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-337
https://f1000research.com/articles/12-1032/v1	23 Aug 23	{ "type": "Study Protocol", "title": "Comparative evaluation of postoperative dentinal hypersensitivity between Cention-N and resin modified glass ionomer cement (RMGIC) in class V cavity using Schiff scale (SS) – an in vivo study", "authors": [ "Rishika Dhimole", "Dr. Aditya Patel", "Dr. Amit Reche", "Rudra Bharate", "Dr. Aditya Patel", "Dr. Amit Reche", "Rudra Bharate" ], "abstract": "Aim – The study aims to assess dentinal hypersensitivity after restoring the tooth with Cention N and resin modified glass ionomer cement (RMGIC) in class V cavities using the Schiff scale. Objective – This study will be conducted to assess the extent of dentin hypersensitivity to the patient's stimulus-response. Methods – A total of 36 patients reporting to the sharad pawar dental college and hospital will be taken having class V cavities. All the patients were divided into two groups. Using a high-speed flat-end straight diamond bur, a cavity with the dimensions two millimeters mmm in depth, three millimeters in width, and two millimeters in height was produced in the occlusal-gingival direction. In group A (n = 18) patients Cention N will be given as class V restoration and in group B (n = 18) patients RMGIC will be given. Follow-up will be done after seven days for evaluation of post-operative sensitivity. Informed written consent will be taken from the patients and the aims and methodology of the study will be explained before enrolment. Demographic data of the patient will be recorded. A statistical analysis will be done based on the recorded data using paired t-tests using Schiff Scale (SS). Expected results – Teeth restored with recently advanced restorative material Cention-N are anticipated to be more susceptible to microleakage and reduce postoperative dentinal hypersensitivity. Class V patients' postoperative sensitivity may be affected by the restorative material used and their method sensitivity. Conclusion – The overall microleakage in Cention N is expected to be minimal or absent after evaluating. Cention N will be superior mechanical properties in comparison to resin-modified glass ionomer cement (RMGIC). This substance will have a substitute restoring agent.", "keywords": [ "Cention N", "Class V restorations", "Resin modified glass ionomer cement (RMGIC)", "Post-operative sensitivity", "Microleakage." ], "content": "Introduction\n\nFor an effective restorative material to reduce microleakage, excellent bonding of restoration with tooth surface is crucial. Poor adaptation can result in pulpal inflammation, secondary caries, bacterial penetration, postoperative sensitivity, marginal discoloration, and pulpal discoloration.1 A restoring material should not be bioincompatible and provide a durable tight seal against infection by bacteria to retain pulp vitality.2 For many years, amalgam has been the material of choice in clinical settings because of its excellent mechanical and financial characteristics. However, due to the toxicity of mercury and its unpleasant look, there have been concerns over the biocompatibility of amalgam.3,4 Due to their aesthetics, minimum tooth preparation requirements, and strong bonding capabilities to tooth structures, these drawbacks encourage further study and development of restorative materials, such as glass ionomers and composite resins.3 Wilson and Kent created glass ionomer cement (GIC) in 1972.5 The benefits of GIC include its capacity to attach to enamel and dentin, release fluoride continuously and stop cavities by creating a marginal seal.4 The mechanical properties of the new GIC, GC Fuji IX GP,6 have been improved, which may be advantageous for patients.6 GIC contains fluoride, binds to tooth structure without additional bonding agent, is strong enough, and may be polished and finished in a single appointment.6 Cention-N is one of the recently created materials that has grown in prominence.7 An “alkasite” restorative substance is cention-N. This new category makes use of an alkaline filler can release ions that can neutralize acids.7 In comparison to both amalgams and GIC, Cention-N, a novel filling material, offers these qualities in addition to additional benefits such as increases bond strength and decreasing micro leakage. It is a tooth-colored, dual-cured material that can be used to restore both class I, II, and V permanent restorations as well as deciduous teeth.7\n\nThis study will be conducted to assess the extent of dentin hypersensitivity in relation to the patient's stimulus-response.\n\nThis is a type of in vivo study.\n\n\nMethods\n\nThis is an in vivo comparative study which will be conducted on the patents reporting to the sharad pawar dental college and hospital.\n\nInclusion criteria\n\nOnly those individuals who will be interested in undergoing the suggested treatment technique were included in the study, and enough time was allotted for the procedure to be adequately completed.\n\nThe patients must also meet the following:\n\n• Patients must be more than 18 years.\n\n• All maxillary and mandibular teeth included in this study have class V cavities.\n\n• Teeth having cervical abrasion.\n\n• Patients having cavity depth of at least 3mm.\n\nExclusion criteria\n\nPatients will be excluded if they have: reversible or irreversible pulpitis, periodontitis and periapical conditions, who are on anti-inflammatory medications and painkillers, patients have allergies to the study's materials, especially resin, patients having para-functional activities (i.e. bruxism, tongue thrusting, clenching).\n\nThe preparation of class V cavity will be done under strict isolation due to gingival crevicular fluid (GCF) and saliva on the facial or buccal surface of the tooth having a depth not more than 3 millimeters, using a high-speed airotor (Allure airotor handpiece by prime dental) with water cooling. Teeth having cervical abrasions will cleaned with the help of water using a three-way syringe. Both the materials (Cention-N and RMGIC) comes in powder and liquid form, powder and liquid will be taken on the mixing pad and liquid is mixed with powder. After achieving a restorative consistency the material will be placed into the cavity using cement carrier (API Dental carrier). Materials will be condensed into the cavity. Excess material is removed with the help of carver (API dental carver). Patients will recalled after seven days for evaluation. The Schiff Scale (SS) will be used to evaluate post-operative sensitivity.\n\nSingle blinding will be used in this study. Using patient's stimulus-response, this scale assesses the level of dental hypersensitivity pain.8 The individual receives a score between 0 and 3 on the SS.8 The score is assessed by the operator. The scoring criteria and interpretation are as follows.\n\nScoring criteria8\n\n0: No response\n\n1: The patient reacts to the stimuli, they do not ask for it to stop.\n\n2: Responding to the stimuli, patient either asks for it to stop or moves away.\n\n3: The patient reacts to the stimuli, experiences it as painful, and asks for it to stop.\n\nExpected outcomes\n\nWe expect that teeth restored with recently advanced restorative material Cention-N are likely to be more susceptible to microleakage and reduce post-operative dentinal hypersensitivity. It is also likely that class V patients' postoperative sensitivity may be affected by the restorative material used and their method sensitivity.\n\nSample size\n\nThis formula9 helps to derive the sample size.\n\nWhere,\n\nZ1 − α/2 confidence interval of 95% (as per reference article)\n\nConfidence interval Z1 = 1.96 (as per reference article)\n\nΖβ is the critical value of normal distribution at β = 0.95 (as per reference article)\n\nd = difference to be detected, 1.0-0.1 = 0.9 (as per reference article)\n\nσ, population standard deviation, SD1 = 0.37, SD2 = 0.48 (as per reference article)\n\nTotal sample size: 36\n\nWith the above-mentioned calculation, the sample size determination of both the groups is 18 in RMGIC group and 18 in Cention-N group. The total minimum sample size with 95% of the confidence interval. The total sample size was calculated to be 36, i.e. 18 in RMGIC group and 18 in Cention-N group. The first 18 patients who are enrolled in this study will be included in the Cention-N group and the second 18 enrolled will be in the RMGIC group.\n\nStatistical method\n\nData will be collected using Schiff Scale (SS) and paired t test will be done and data will be collected using Microsoft Excel (RRID:SCR_016137). Statistical analysis will be carried out using SPSS 23 Software (RRID:SCR_002865).\n\nDissemination\n\nIn this study we will be comparing the resistance to post-operative sensitivity and microleakage resistance after restoration with the two groups of restorative materials one is Cention-N and the other one is Resin modified glass ionomer cement (RMGIC). The alternative restorative material Cention-N is an excellent choice since it has promising mechanical characteristics (good bond strength in comparison with RMGIC) and minimal microleakage. As Cention-N is aesthetically pleasing and takes less chairside time it can be used as alternative restorative materials in class V cavities.\n\nStudy status\n\nThe study is not started yet. This trial protocol is not registered.\n\n\nDiscussion\n\nIn this study the expected postoperative sensitivity after restoration with a newer material which is Cention-N will be lesser as compare the conventional restorative material resin modified glass ionomer cement (RMGIC), According to multiple study literature Cention-N has less microleakage properties as compared to RMGIC so it will show less postoperative dentinal sensitivity. The expected chai side time will also be less in case of Cention-N as compared to RMGIC.\n\nIn class V cavities filled with the Cention-N which is a recent alkasite material, with and without the use of a bonding agent and flowable composite resin, Priyatama Meshram et al, examined microleakage at the enamel restoration and dentin restoration interface and concluded that each group's microleakage at the enamel-dentin repair interface was lower than the others, but the difference was not statistically significant.10 The Cention-N with adhesive had the least micro leakage, which was followed by flowable composite. When Cention-N was used without adhesive, more microleakage was observed.10\n\nPratima R Shenoi et al evaluated and compared marginal microleakage in the teeth in which restoration is done with flowable composite and Cention-N, Class V cavities. They concluded that Cention-N displayed reduced microleakage when compared to flowable composite, which was statistically significant (P = 0.005). In comparison to flowable composite, Cention-N demonstrated noticeably less leakage and improved adaptability.11\n\nSujith et al have done a study to differentiate between the physical characteristics and microleakage of Cention-N in relation to glass ionomer cement (GIC) and hybrid composite restorative materials. They observed that Cention-N had slightly lower compressive and flexural strength than glass ionomer cement (GIC), but comparable strength to hybrid composites.12\n\n\n\n• The research proposal has been approved by the Institutional Ethics Committee of data meghe institute of higher education and Research, sawangi meghe, Wardha. Ref. No- DMIHER (DU)/IEC/2023/720.\n\n• Informed written consent for the procedure will be obtained from all the participants and they are informed about the whole procedure.", "appendix": "Data availability\n\nNot applicable as this is a study protocol.\n\n\nReferences\n\nDiwanji A, Dhar V, Arora R, et al.: Comparative evaluation of microleakage of three restorative glass ionomer cement: an in vitro study. J. Nat. Sci. Biol. Med. 2014; 5(2): 373–377. Publisher Full Text\n\nSix N, Lasfargues JJ, Goldberg M: In vivo study of pulp reaction to Fuji IX, a glass ionomer cement. J. Dent. 2000; 28(6): 413–422. PubMed Abstract \| Publisher Full Text\n\nMickenautsch S, Yengopal V, Banerjee A: Atraumatic restorative treatment versus amalgam restoration longevity: a systematic review. Clin. Oral Investig. 2010; 14(3): 233–240. Publisher Full Text\n\nDhar V, Hsu KL, Coll JA, et al.: Evidence-based update of pediatric dental restorative procedures: dental materials. J. Clin. Pediatr. Dent. 2015; 39(4): 303–310. PubMed Abstract \| Publisher Full Text\n\nBerzins DW, Abey S, Costache MC, et al.: Resin-modified glass-ionomer setting reaction competition. J. Dent. Res. 2010 Jan; 89(1): 82–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNahlawi TA, Altaki Z, Abbood D: Post-operative sensitivity of class I, II amalgam and composite resin restoration: clinical evaluation in an undergraduate program. Int. Dent. Med. J. Adv. Res. 2015; 1(1): 1–4. Publisher Full Text\n\nMishra A, Singh G, Singh SK, et al.: Comparative evaluation of mechanical properties of Cention N with conventionally used restorative materials—an in vitro study. Int. J. Prosthodont. Restor. Dent. 2018; 8(4): 120–124.\n\nSchiff T, Dotson M, Cohen S, et al.: Efficacy of a dentifrice containing potassium nitrate, soluble pyrophosphate, PVM/MA copolymer, and sodium fluoride on dentinal hypersensitivity: a twelve-week clinical study. J. Clin. Dent. 1994 Jan 1; 5: 87–92.\n\nJohnston KM, Lakzadeh P, Donato BMK, et al.: Methods of sample size calculation in descriptive retrospective burden of illness studies. BMC Med. Res. Methodol. 2019; 19: 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeshram P, Meshram V, Palve D, et al.: Comparative evaluation of microleakage around Class V cavities restored with alkasite restorative material with and without bonding agent and flowable composite resin: An in vitro study. Indian J. Dent. Res. 2019 May 1; 30(3): 403–407. PubMed Abstract \| Publisher Full Text\n\nShenoi PR, Kokane VB, Thawale HV, et al.: Comparing marginal microleakage in Class V cavities restored with flowable composite and Cention-N using confocal microscope-an in-vitro study. Indian J. Dent. Res. 2021 Jul 1; 32(3): 348–353. PubMed Abstract \| Publisher Full Text\n\nSujith R, Yadav TG, Pitalia D, et al.: Comparative evaluation of mechanical and microleakage properties of Cention-N, composite, and glass ionomer cement restorative materials. J. Contemp. Dent. Pract. 2020 Jun 1; 21(6): 691–695. PubMed Abstract \| Publisher Full Text" }	[ { "id": "228550", "date": "11 May 2024", "name": "Parla Meva Durmazpınar", "expertise": [ "Reviewer Expertise Biomaterials", "endodontic microbiology", "root canal disinfection" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 highlights about dentinal hypersensitivity after restoring the tooth with Cention N and resin modified glass ionomer cement (RMGIC) in class V cavities using the Schiff scale. 1-The Introduction provides a clear overview of the topic and establishes the importance of understanding the restorative materials. However, there is no any null hypothesis. Introduction should have a null hypothesis. 2-There should be some tables or figures indicating the results. 3-The Discussion section of the manuscript lacks in-depth analysis and interpretation of the results. Overall, the authors should provide a more comprehensive and critical analysis of the study findings, linking them to existing knowledge and identifying areas for future research. 4-The authors should critically evaluate the limitations of the study.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-1032
https://f1000research.com/articles/12-370/v1	05 Apr 23	{ "type": "Research Article", "title": "The impact of smoking status on clopidogrel responsiveness in patients with coronary artery disease who undergo percutaneous coronary intervention", "authors": [ "Ali A. R. Aldallal", "Bassim I. Mohammad", "Ahmed N. Rgeeb", "Dina A. Jamil", "Hayder A. Al-Aubaidy", "Ali A. R. Aldallal", "Bassim I. Mohammad", "Ahmed N. Rgeeb", "Dina A. Jamil" ], "abstract": "Background: Previous studies have pointed out the disproportionate action of clopidogrel in inhibiting platelet aggregation due to smoking more than ten cigarettes per a day. This study was designed to examine whether smoking enhances clopidogrel responsiveness in patients who are clinically diagnosed with coronary artery disease (CAD), following percutaneous coronary intervention (PCI).\nMethods: A total of 324 IHD participants were enrolled in a case-control study. Platelet function test was performed to all participants two hours before PCI procedure to measure clopidogrel response. Participants were then categorized into a non-responder group (case group n = 111) and responder group (control group n = 213). Each group was subdivided into a smoker group and a non-smoker group. All participants received clopidogrel loading dose equivalent to 600 mg and scheduled for elective PCI. Participants’ age, gender, family history of chronic illnesses was recorded in this study.\n\nResults: Smoking participants displayed a significant higher level of hemoglobin as compared to the non-smoking participants among the responder and the non-responder study groups (14.6±0.55 vs. 13.12±0.38, P < 0.029; 14.3±0.31 versus 12.96±0.39, P < 0.033) but lower AUC level (17±9 vs. 45±6, P < 0.005; 62±3 vs. 95±7, P < 0.008). Additionally, smoking intensity enhanced clopidogrel responsiveness by odd’s ratio 0.4213 at 95% C.I. (0.259 - 0.684), P < 0.0002.\nConclusions: Current smokers had a good response to clopidogrel therapy which exerted a beneficial effect when undergoing PCI as compared to non-smokers. The marked difference in AUC between smokers and non-smokers could be related to the variance in hemoglobin level. The smokers’ paradox needs further justification to confirm this concept.", "keywords": [ "Clopidogrel responsiveness", "coronary artery disease", "smokers’ paradox", "and percutaneous intervention" ], "content": "Introduction\n\nClopidogrel is a known platelet inhibitor, which has been widely used for the prevention of major complications of heart disease following percutaneous coronary intervention (PCI).1 Previous studies have shown that there was some inter-individual difference in clopidogrel response2 and resistance to clopidogrel is related with an augmented risk of major adverse cardiac events (MACE).2 Among the several risk factors that affect the response of platelet to clopidogrel therapy, smoking is linked with enhanced clopidogrel responsiveness.3 Although various processes have been proposed (including those regarding cytochrome P450 enzyme system), the exact mechanism is still unclear.3\n\nClopidogrel is taken as an inactive drug; it then passes through a two-step oxidation process with the help of cytochrome P450 (CYP) to be activated, which leads to irreversible inhibition of platelet P2Y12 adenosine diphosphate (ADP) receptor.4 Smoking is identified as an inducer of CYP1A2, a major iso-enzyme controlling the first oxidation step in clopidogrel activation.4\n\nThe fundamental association between coronary arterial disease (CAD) and smoking is well established.4 Cessation of smoking is the only most significant intervention for prevention of CAD.4 However, the “smoking paradox” is a phenomenon which has been observed in smokers on clopidogrel treatment. In these patients, the pharmacodynamics of clopidogrel was enhanced by the intensity of smoking, and smokers who received clopidogrel had low platelets reactivity.5\n\nIn this study, we investigated whether smoking enhances clopidogrel responsiveness in patients with ischemic heart disease (IHD) following PCI.\n\n\nMethods\n\nThis was a prospective case-control study, conducted at Al-Najaf Heart Surgery and Catheterization Center, Al-Najaf, Iraq, between August 2020–June 2022. This study was approved by the Human Research Ethics, College of Medicine, University of Kufa (KUM 364 on 10 March 2022). The study protocol was explained to all patients and only participants who provided written informed consent to enroll in the study were included. A total of 370 participants were initially included in the study, however, 46 participants were later excluded because they did not meet the study criteria. Therefore, only 324 participants who had a platelet function test two hours prior to PCI to monitor platelet responsiveness were included in the current analysis. Participants were, subsequently, divided into two study groups based on their response to clopidogrel therapy: non-responder study cohorts (case group n=111) and responder study group (control group n=213). In each of the above two study groups, participants were further subdivided according to their smoking status (smoker study group and non-smoker study group).\n\nParticipants have been classified as “smokers” if they continued smoking cigarettes within a month of the enrolment period and had smoked at least a hundred cigarettes (or one hour shisha smoking) in their lives. Non-smokers were participants who have never smoked more than 100 cigarettes (or one hour shisha smoking) in their lives.\n\nAll patients received 75 mg of clopidogrel (Plavix, Sanofi Aventis, France), daily for 7 days before PCI procedure. Patients who had never taken them before were given a loading dose of aspirin (300 mg orally) and clopidogrel (300 mg orally).\n\nFive milliliters of venous blood sample were taken from the participants. Samples were placed in test tubes for 1 hour, then underwent centrifugation at 3000 revolutions per minute for ten minutes at 25°C. The platelet reactivity test was assisted by means of Multiplate analyzer (Verum Diagnostic, GmbH, Munich, Germany). The assay was quantified by the area under the aggregation curve (AUC) in units (U). In this study, the cutoff point for the ADP test used to determine the clopidogrel effect is value of UAC was 50 U, so any value ≥50 U was considered as clopidogrel resistance or non-responder, and values <50 U as non-clopidogrel resistance or responder.6\n\nAll data were analysed using SPSS (version 25, IBM, USA). In this study, descriptive statistics were used as numbers and percentages. To analyze categorical data or variables, we used the Chi-Square test. To analyze numerical data or compare between cases and control and among groups, we used the independent Student’s t-test. Body mass index (BMI) was calculated using the equation (BMI=kg/m2). A P-value ≤0.05 was deemed significant.\n\n\nResults\n\nTable 1 shows the demographic data and clinical features of all the participants in this study. A total of 324 patients with CAD were included in the study. Among the responder study group, 51.1% of the participants in this group were smokers and 48.9% of the participants were non-smokers. In the non-responder study group, only 30.6% of the participants were smokers, versus 60.4% of the participants in this group were non-smokers, Table 1\n\n* Significance (P≤0.05) among the pterostilbene and the atherogenic study cohorts.\n\n# Significance (P≤0.05) among the sitagliptin and the atherogenic study cohorts.\n\nIn addition, the mean value for participants’ age in the smoker cohort was 58.58±0.75 versus 56.63±0.77 (P value=0.07) in the non-smoker group for responder study group, whereas the mean value of the participants’ age for patients in the smoker cohort was 57.91±1.49 versus 55.20±1.05 (P value=0.14) for non-smokers in the non-responder study cohort (Table 1).\n\nOverall, 77.1% of the smoker responder study group were males as compared to 72.1% non-smoker males, P=0.477. In contrast, 50% of the smoker non-responder participants were male versus 71.4% were non-smoker, P=0.029 (Table 1).\n\nWith regards to the development of chronic illnesses, a non-significant difference was noticed in the prevalence of hypertension among the responder study cohort (67% in the smoking study sub-group versus 68.3% in the non-smoking study sub-group, P=0.772). Similarly, the prevalence of hypertension among the non-responder study group was not significantly different based on smoking status (70.6% smoking versus 64.9% non-smoking, P=0.56), Table 1.\n\nThe prevalence of diabetes mellitus was non-statistically different among the responder group (53.2% in the smoking sub-group versus 58.7% in the non-smoking study sub-group, P=0.42), as well as among the non-responder study group (55.9% in the smoking sub-group versus 48.1% in the non-smoking study sub-group, P=0.45), Table 1.\n\nIn addition, the prevalence of BMI varied insignificantly among the responder study cohort (28.84±0.47 in the smoking study sub-group versus 27.97±0.41 in the non-smoking study sub-group, P=0.16). Similarly, BMI level was not significantly different among the non-responder study cohort based on smoking status (28.91±0.50 smoking versus 30.02±1.00 non-smoking, P=0.47), Table 1.\n\nThe mean level of HbA1c was statistically significantly different in the responder study cohort (14.6±0.55 in the smoking sub-group versus 13.12±0.38 in the non-smoking study sub-group, P=0.029), While it was not significant within the non-responder study cohort (14.3±0.31 in the smoking sub-group versus 12.96±0.39 in the non-smoking study sub-group, P=0.33), Table 1.\n\nLikewise, the mean level of AUC was statistically significantly different in the responder study group (17±8 in the smoking sub-group versus 45±6 in the non-smoking study sub-group, P=0.005). A statistically significant difference was noted within the non-responder study cohort (62±3 in the smoking sub-cohort as compared to 95±7 in the non-smoking study sub-cohort, P=0.008), Table 1.\n\nSmoking status increased the response to clopidogrel therapy as manifested by the response to platelet function test with an odd’s ratio of 0.4213 (95% C.I. 0.259-0.684), P=0.0002, Table 2.\n\n\nDiscussion\n\nThe current study compared the clopidogrel response to smoking status in patients with CAD who were prepared to undertake a PCI procedure. In this study, we took into consideration all the modifiable and the non-modifiable risk factors such as the presence of chronic condition (hyperglycemia, hypertension, and hemoglobin level). We could not find a significant difference between smoking participants versus non-smoking participants in the responder study cohort nor in the non-responder study cohort. These findings were comparable to the findings of other studies.7,8\n\nAdditionally, this study illustrated a significantly higher hemoglobin level among the smoking participants when compared to the non-smoking patients, although Yun Gi Kim and his team found evidence that the AUC measured by Multiplate Analyzer was not affected by hemoglobin level.9 While there was a significantly lower AUC among the smoker participants as compared to the non-smoker in both study groups, these results are consistent with5,10 and contrary to results by Kim et al., 2016.9\n\nHowever, it is worth mentioning that cessation of smoking represents the first action for the secondary prevention of the development of ischemic events in people with CVD diseases because it lowers the risk of thrombotic cardiovascular processes.11,12 Although quitting smoking is the best strategy to lower thrombotic risk and save money on healthcare, many people with established CVD disease refuse to quit smoking. Accordingly, determining the best antiplatelet treatment plan for these patients is crucial.11,12\n\nWe also revealed that smoking enhanced clopidogrel responsiveness by 0.42 in responder study group and non-responder study groups. Previous studies concluded that smoking incudes enzyme activation of cytochrome P450 (primarily 2B6 and 1A2 isoenzymes), which are crucial for the biotransformation of the medication clopidogrel.5,13 Smoking increases clopidogrel’s anti-aggregative action and platelet inhibition since it speeds up the drug's metabolism. Platelet P2Y12 receptors may become more numerous and expressed when exposed to nicotine, which may improve their sensitivity to clopidogrel.5,13 Clopidogrel should be the drug of choice when it comes to long term prevention of CVD disease in smoker patients because of the larger number of clopidogrel responders and aspirin non-responders.14 This is especially important for patients who, in accordance with recommended practices, depend on a single antiplatelet therapy, typically aspirin, for their protection against the development of atherosclerosis and do not have a clinical rationale for a combined antiplatelet treatment in combination with clopidogrel.\n\nThe absence of clinical follow-up for outcome and complications and a genetic investigation to assess patients who did not respond to clopidogrel are among the study’s first limitations.\n\nThe inability to measure cotinine plasma levels to accurately depict smokers' health is a second limitation of our study. However, most studies addressing the smoking paradox and the increased cardiovascular risk associated with smoking have this drawback.\n\n\nConclusions\n\nThere is a marked variance in AUC between smoker and non-smoker participants in the responder study group as well as among the non-responder study group. Our results propose that cigarette smoking enhances clopidogrel responsiveness. These results suggest potential benefits of clopidogrel therapy based on the smoking paradox, but this theory needs further investigations.\n\n\nAuthor contributions\n\nThe authors declare that all data were generated in-house and that no paper mill was used. The authors responsibilities were as follows: Author Contributions: “Conceptualization, A.A., A.R., B.M., and H.A.; methodology, A.A., D.J., B.M., and A.R.; investigation, H.A., A.R., A.A., and B.M.; writing—original draft preparation, A.A., B.M., and A.R.; review and editing, H.A., and D.J.; supervision, B. M, and A.R. Authors have read and agreed to the final version of the manuscript.”", "appendix": "Data availability\n\nFigshare: The impact of smoking status on clopidogrel responsiveness in patients with coronary artery disease who undergo percutaneous coronary intervention, https://doi.org/10.6084/m9.figshare.22130228.v2. 15\n\nThis project contains the following underlying data:\n\n- Data Share - The impact of smoking on Clopidogrel Responsiveness in CAD.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors thank members of the Pharmacology and Therapeutic Department of University of Kufa.\n\n\nReferences\n\nPatti G, et al.: The Role of Clopidogrel in 2020: A Reappraisal. Cardiovasc. Ther. 2020; 2020: 8703627.\n\nKheiri B, et al.: CYP2C19 pharmacogenetics versus standard of care dosing for selecting antiplatelet therapy in patients with coronary artery disease: A meta-analysis of randomized clinical trials. Catheter. Cardiovasc. Interv. 2019; 93(7): 1246–1252. PubMed Abstract \| Publisher Full Text\n\nWestphal ES, et al.: Generic Clopidogrel: Has Substitution for Brand Name Plavix® Been Effective? J. Pharm. Pract. 2022; 35(4): 536–540. PubMed Abstract \| Publisher Full Text\n\nTunströmer K, et al.: Thrombus remodelling by reversible and irreversible P2Y(12) inhibitors. Platelets. 2023; 34(1): 2157805. PubMed Abstract \| Publisher Full Text\n\nRamotowski B, et al.: Effect of Smoking Cessation on the Pharmacokinetics and Pharmacodynamics of Clopidogrel after PCI: The Smoking Cessation Paradox Study. Thromb. Haemost. 2020; 120(3): 449–456. PubMed Abstract \| Publisher Full Text\n\nKweon OJ, et al.: Effectiveness of Platelet Function Analyzer-100 for Laboratory Detection of Anti-Platelet Drug-Induced Platelet Dysfunction. Ann. Lab. Med. 2019; 39(1): 23–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Z, et al.: Two common mutations within CYP2C19 affected platelet aggregation in Chinese patients undergoing PCI: a one-year follow-up study. Pharmacogenomics J. 2019; 19(2): 157–163. PubMed Abstract \| Publisher Full Text\n\nSiasos G, et al.: Genotyping, Platelet Activation, and Cardiovascular Outcome in Patients after Percutaneous Coronary Intervention: Two Pieces of the Puzzle of Clopidogrel Resistance. Cardiology. 2017; 137(2): 104–113. PubMed Abstract \| Publisher Full Text\n\nKim YG, et al.: Cigarette Smoking Does Not Enhance Clopidogrel Responsiveness After Adjusting VerifyNow P2Y12 Reaction Unit for the Influence of Hemoglobin Level. JACC Cardiovasc. Interv. 2016; 9(16): 1680–1690. PubMed Abstract \| Publisher Full Text\n\nCrimi G, et al.: Cigarette smoking reduces platelet reactivity independently of clopidogrel treatment in patients with non-ST elevation acute coronary syndromes. Platelets. 2018; 29(3): 309–311. PubMed Abstract \| Publisher Full Text\n\nLin GM, et al.: Secondhand tobacco smoke exposure, urine cotinine, and risk of incident atrial fibrillation: The multi-ethnic study of atherosclerosis. Prog. Cardiovasc. Dis. 2022; 74: 38–44. PubMed Abstract \| Publisher Full Text\n\nMulayamkuzhiyil Saju J, Leslie SW, Infarction R, et al.: StatPearls Publishing Copyright © 2022. Treasure Island (FL): StatPearls Publishing LLC; 2022.\n\nCai Y, et al.: Effects of cigarette smoking on older chinese men treated with clopidogrel monotherapy or aspirin monotherapy: a prospective study. Platelets. 2020; 31(5): 667–673. Publisher Full Text\n\nWiśniewski A: Multifactorial Background for a Low Biological Response to Antiplatelet Agents Used in Stroke Prevention. Medicina (Kaunas). 2021; 57(1). Publisher Full Text\n\nAl-Aubaidy H: Clopidogrel Responsiveness. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "184298", "date": "24 Jul 2023", "name": "Bogumił Ramotowski", "expertise": [ "Reviewer Expertise Clopidogrel responsiveness in smokers" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 by Aldalal et al. addressed the interesting problem of the effect of smoking on clopidogrel responsiveness, and the study group was adequately chosen; however, the study requires further improvement. I address the following issues:\nIntroduction\nThe effect of smoking on clopidogrel metabolism is well known and is mainly related to cytochrome P450 with regard to CYP1A2 polymorphism.\nThe smoking paradox is significant in trials with clopidogrel therapy but is also present before clopidogrel therapy and might be related to younger age and lower comorbidities.\nMethods\nI understand that 370 participants were screened and not initially included for study participation, and 324 patients matched the study criteria to include the study.\nThere may be a difference between patients receiving 75 mg clopidogrel therapy and those who received a loading dose of clopidogrel.\nResults\nI also propose to present groups of smokers and non-smokers and compare platelet responsiveness to clopidogrel therapy in these groups. There is no need to present non-significant data in the text that was previously presented in the table.\nIs there a difference in the number of active smokers between the responder and non-responder groups?\nThe paper requires English language proofreading.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10069", "date": "23 Aug 2023", "name": "Hayder Al-Aubaidy", "role": "Author Response", "response": "14 August 2023 Research article (The impact of smoking status on clopidogrel responsiveness in patients with coronary artery disease who undergo percutaneous coronary intervention) Dear Reviewer, Thank you for reviewing the manuscript and providing us with your comments. e confirm addressing all your comments (please see below) in the updated version of the manuscript (V2). Please see below for a detailed response to your comments: Reviewer Comments: The study by Aldalal et al. addressed the interesting problem of the effect of smoking on clopidogrel responsiveness, and the study group was adequately chosen; however, the study requires further improvement. I address the following issues: Introduction The effect of smoking on clopidogrel metabolism is well known and is mainly related to cytochrome P450 with regard to CYP1A2 polymorphism. Response: Thank you for your comment. We confirm that we have responded to this comment in the introduction section (Pages 3-4). The smoking paradox is significant in trials with clopidogrel therapy but is also present before clopidogrel therapy and might be related to younger age and lower comorbidities. Response: Thank you for your comment. We have expanded the section about the smoking paradox to discuss the potential mechanisms before the initiation of the treatment in the Introduction section (Page 4). Methods I understand that 370 participants were screened and not initially included for study participation, and 324 patients matched the study criteria to include the study. Response: Yes, we confirm that this is these are the accurate numbers for the initial recruitment and the active enrolment in this study (Methods section, Page 4). There may be a difference between patients receiving 75 mg clopidogrel therapy and those who received a loading dose of clopidogrel. Response: Thank you. We confirm that we have covered this in the Discussion section (Page 8). Results I also propose to present groups of smokers and non-smokers and compare platelet responsiveness to clopidogrel therapy in these groups. There is no need to present non-significant data in the text that was previously presented in the table. Is there a difference in the number of active smokers between the responder and non-responder groups? Response: Appreciate your input into this. Based on your suggestion, we have looked into the correlation between the platelet responsiveness to clopidogrel therapy between the smokers vs non-smokers in the two study groups and we have included the results (Table 2) in the Results section (Page 7). Noting that we have renamed the following Table to make sure that they are still in the correct order. Further changes to V2 of the manuscript include formatting the tables to include the extra table (Table 2), as well as including the additional references to support the additional changes to the Introduction section to address the reviewer’s comments. Best Regards Hayder Al-Aubaidy On behalf of all authors for this manuscript" } ] } ]	1	https://f1000research.com/articles/12-370
https://f1000research.com/articles/12-1027/v1	23 Aug 23	{ "type": "Study Protocol", "title": "Effect of Maitland and Mulligan mobilization on pain, range of motion and disability in patients with rotator cuff syndrome: a randomized clinical trial protocol", "authors": [ "Samiksha Vinod Sonone", "Deepali Patil", "Deepali Patil" ], "abstract": "Background: Rotator cuff syndrome (RCS) comprises partial or whole-thickness rotator cuff tears, rotator cuff tendonitis, subacromial impingement syndrome, and bursitis. Expansion of rotator cuff arthropathy and glenohumeral degenerative disease are both made more likely by chronic rotator cuff syndrome. The maximum well-supported nonsurgical treatment for individual with rotator cuff (RC) injury appears to be musculoskeletal physiotherapy, including manual treatments and exercises. Scapular stabilisation exercises, proprioceptive neuromuscular facilitation (PNF), range of motion (ROM), rotator cuff strengthening exercises, and stretching exercises are some of the therapeutic exercises that have been shown to be useful in recovering shoulder function. The passive, expert movements known as joint mobilisation and manipulation are used by clinicians to increase joint mobility and to reduce pain. Methods: In this study we aim to find out the effect of Maitland and Mulligan mobilization on improving ROM, functional disability and pain in individuals with rotator cuff syndrome through a randomized clinical trial. 80 individuals in total will be allocated equally into two groups, and both groups will be receiving treatment for six weeks with five sessions per week. After six weeks of therapy the results will be assessed for follow-up. By mobilization there will be increased range of motion, reduction in pain and functional disability, this technique may be used to patient with rotator cuff syndrome if our study's premise is found to be significant. Conclusions: In this study, we expect that the Maitland and Mulligan mobilization on pain, range of motion and disability in patients with rotator cuff syndrome will have significant effects. CTRI Registration: CTRI/2023/05/052643.", "keywords": [ "Rotator cuff syndrome", "Maitland Mobilization", "Mulligan Mobilization", "Visual analogue scale", "Disability of arm", "shoulder and hand and Range of Motion" ], "content": "Introduction\n\nThe teres minor, infraspinatus, subscapularis and supraspinatus are the muscular units that make up the rotator cuff (RC). Subscapularis is the powerful rotator cuff muscle. The teres minor and infraspinatus, which are located in the larger tuberosity on the inferior and middle faces respectively, are the other rotator cuff muscles.1 The greater tuberosity’s superior and middle faces receive the supraspinatus tendon, which has a breadth of about 23 mm. In order to abduct the arm past 15 degrees, the deltoid and supraspinatus must work together. The supraspinatus is largely engaged in shoulder abduction from 0 to 15 degrees. Anterior-most fibres of the infraspinatus tendon interdigitate with the posterior supraspinatus tendon, which has a comparable breadth and its insertion is on the middle facet. The teres minor, which insertion is on the inferior facet, and the infraspinatus work together to externally rotate the shoulder. With a strong internal rotator and adductor, the subscapularis is made up of four to six tendon slips that attach directly to the inferior third of the lesser tuberosity. Some of the subscapularis fibres cross the bicipital groove to attach to the lesser tuberosity, forming the transverse humeral ligament.2\n\nAmong the various complications of the shoulder, a rotator cuff injury is one of the most common injuries.3 In patients older than 50, rotator cuff tears (RCT) are a frequent source of shoulder discomfort and dysfunction. The aetiology is still up for question, however the prevalence rises with age. According to a number of authors, aging-related degenerative changes are the primary cause of the majority of tendon tears. As a result, this topic has received a lot of attention in study, and during the past few decades, many intrinsic and extrinsic causes of tendon degeneration have been proposed.4 Rotator cuff pathology might include inflammation, fibrotic alterations, partial- and full-thickness tears, and more. Subacromial impingement/pain syndrome is characterised by biomechanical dysfunction of the glenohumeral complex and the RC, which plays a role in its pathogenesis and development. Repetitive overhead activity is the highest frequent risk factor for subacromial discomfort and rotator cuff tendinopathy. Rotator cuff disorders frequently present with a gradual start of pain, but they can also be brought on by an abrupt injury.5 Any injury or declining condition affecting the RC is referred to as RCS. This comprises partial or whole thickness RCT, RC tendonitis, subacromial impingement syndrome, and bursitis. The probability of rotator cuff arthropathy and glenohumeral degenerative disease developing is increased by chronic rotator cuff syndrome. This exercise describes the diagnosis and treatment of RCS and the function of the multidisciplinary team in the care of individuals who have this condition.6\n\nPatients with full thickness RCT are frequently given conservative treatment, which includes exercise therapy, as their first line of defence. Surprisingly, clinical outcome scores and subjective satisfaction levels have been shown to be comparable between patients whose repairs fail following surgical repair and those with intact repairs. The authors of this study hypothesise that these results may be explained by the fact that the majority of the patients in these studies underwent some type of structured post-operative physical therapy, raising the possibility that post-operative rehabilitation is more responsible for the improvements in outcome. Reduced pain, improved function, and improved daily living activities are the main goals of non-operative therapy of a torn rotator cuff.7 Between the capsule of shoulder and coracoid process of scapula is where the subcoracoid bursa is situated. Anterior shoulder pain that snaps in a painful manner could be caused by subcoracoid bursitis.8\n\nThe most well-supported nonsurgical treatment for people with rotator cuff injuries appears to be musculoskeletal physiotherapy, which includes manual therapies. To restore shoulder kinematics the exercises are widely recommended as a therapy opportunity for patients who present with subacromial impingement syndrome and, in the worst cases, surgical decompression and acromioplasty are used to restore subacromial space. There is accumulating evidence in favour of these recommendations. It has been shown that therapeutic exercises, which combine scapular stabilisation, range of motion (ROM), RC strengthening, stretching exercises and proprioceptive neuromuscular facilitation (PNF) are useful in recovering shoulder function.9\n\nThe manual therapy technique of joint mobilisations, which involves realigning collagen, enhancing fibre slip, reducing adhesion, and re-establishing correct glenohumeral joint kinematics, can relieve pain by stimulating peripheral mechanical receptors, suppressing nociceptors, boosting synovial fluid nutrition, and reducing adhesion. Additionally, exercise training will establish shoulder proprioception, mobility, and stability. Exercise and manual treatment for corticosteroid injections and subacromial decompression techniques have the same outcomes, according to a recent Cochrane analysis.10 The passive, expert movements known as joint mobilisation and manipulation are used by clinicians to reduce pain and increase joint mobility. Furthermore, the management of tendon discomfort has been promoted for these methods. The use of joint mobilisation and manipulation close to or locally on the afflicted joint may lessen tendon pain, according to limited evidence, suggesting that these techniques could be used as analgesic interventions for the treatment of tendinopathy.11 Joints in the shoulder complex (acromioclavicular joint, glenohumeral joint, scapulothoracic joint and sternoclavicular joint), as well as the cervical or thoracic spine, may be subjected to joint mobilisations. Grades I, II, III, IV, and V — the most popular grading system — are used to categorise passive joint mobilisations based on the joint resistance which is influenced by the movement’s amplitude and the view in its range where it occurs. Grade V mobilisation, also known as manipulation, is a thrust technique that uses high velocity and low amplitude forces.12\n\nMulligan mobilisation is one of the techniques utilised for joint mobilisation. The Mulligan idea is a joint mobilisation approach that involves continuous active movement and continuous gliding when the joint is in a weight-bearing posture. In movement with mobilisation (MWM), the force application direction is chosen in accordance with the forces produced in the joints. Then, the ROM will be improved using the most suitable and efficient force.13\n\n\n\n1. To evaluate the effect of Maitland mobilization for pain (visual analogue scale), for range of motion (goniometer) and for functional disability (disability of arm, shoulder and hand) in individuals with rotator cuff syndrome.\n\n2. To evaluate the effect of Mulligan mobilization for pain (visual analogue scale), for range of motion (goniometer), and for functional disability (disability of arm, shoulder and hand) in individuals with rotator cuff syndrome.\n\n3. To compare the effects of Maitland and Mulligan mobilization for pain (visual analogue scale), for range of motion (goniometer) and for functional disability (disability of arm, shoulder and hand) in patient with rotator cuff syndrome.\n\n\nProtocol\n\nThis will be a single centric, two arm parallel equivalence randomized clinical trial.\n\n\nMethods\n\nThis is an interventional study, where participants will be recruited from the Physiotherapy OPD at Acharya Vinoba Bhave Rural Hospital, Sawangi Meghe, Wardha, Maharashtra, following approval from the institutional ethics committee of Datta Meghe Institute of Higher Education and Research. A total of 80 participants will be required. Participants who meet the inclusion and exclusion criteria will be made aware of the study’s objectives and procedures, and then they will be separated into Group A and Group B by means of simple random selection in a 1:1 allocation ratio; each group will consist of 40 participants. Group A will be the Maitland mobilization and conventional physiotherapy group, and Group B will be the Mulligan mobilization and conventional physiotherapy group. Randomization will be done using a computer-generated system. For sample distribution, we will use the sequentially numbered opaque sealed envelopes technique.\n\nThe outcome measures used in the study will be quantified at baseline and at the end of the six-week treatment by a postgraduate (PG) resident in musculoskeletal-physiotherapy with similar expertise who is aware of the study but blind to the intervention. The study will be monitored by the PG guide, head of department, and the principal and chief advisor of the research cell. Through routine treatment sessions, we will make sure that the patients follow the suggested treatment plan faithfully. If needed, individuals will receive counselling or telephone reminders about their therapy appointments.\n\nPrior to and following the analysis, the outcome measures will be assessed. The Visual Analogue Scale (VAS), Range of Motion (ROM) by Goniometer, Disabilities of the Arm, Shoulder, and Hand (DASH) and Short Form Survey (SF-36) are among of the tools used to measure pain, ROM, functional ability and quality of life. Along with conventional treatment for pain, ROM, and functional ability and quality of life, Group A will also undergo a Maitland Mobilization programme. Along with conventional treatment for pain, ROM, and functional ability and quality of life, Group B will also undergo a Mulligan Mobilization program. Figure 1 depicts the study’s design in visual form.\n\nCriteria for inclusion of subjects\n\n1. Both male and female\n\n2. Subjects having limited ROM of shoulder joint\n\n3. Between 30 and 70 years old\n\n4. Having a partial tear\n\n5. No surgery on the shoulder\n\n6. Willing to participate in the study.\n\nCriteria for exclusion of subjects.\n\n1. History of any neurological impairment\n\n2. Any orthopaedic injury and heart issues that make it impossible to use assessment methodologies\n\n3. A recent myocardial infarction or significant shoulder injury\n\n4. Patients with osteoporosis\n\n5. Not willing to participate in the study\n\n6. Infections/tumours allied with shoulder joint and upper limb implants.\n\nGroup A (interventional group)\n\nThe participants in this group will receive Maitland mobilization along with conventional physiotherapy. The patient will be lying with the arm bent at a 30-degree angle, and the therapist will be standing and gripping the proximal end of the humerus and keeping a lateral humeral distraction in the middle of its range. Conventional therapy will include: wand exercises, Codman exercises, shoulder capsule stretching exercises, stretching exercises, strengthening exercises, finger stairs, and shoulder hand wheel. They will also undergo 1.5 MHz of ultrasound (US) for 6 minutes and 100 Hz of transcutaneous electrical nerve stimulator (TENS) for 20 minutes. These exercises will be performed for 10 repetitions and gradually the repetition will be progressed week wise.\n\nPatients in this group will be given 60 minutes of overall treatment which will include 20 minutes of Maitland mobilization and 40 minutes of conventional physiotherapy (6 minutes of US, 20 minutes of TENS and 15 minutes of exercises). Glenohumeral caudal glide mobilisation will be administered for five consecutive weeks at a rate of 2-3 glides per second for 30 seconds.\n\nGroup B (conventional group)\n\nThe participants in this group will receive Mulligan mobilization along with conventional physiotherapy. The patient will be lying down, humeral head will be actively mobilised in the abduction, flexion, external and internal rotation directions on the individuals using the MWM technique. This control group will receive physical treatment using conventional techniques. Participants in this technique will be lying on a stretcher, and the position of the therapist will be across the patient’s upper limb. In order to treat the shoulder restrictions, the therapist will stabilise the shoulder girdle of the participants and with another hand, a glide of the humeral head over the thenar eminence will be given. The therapist will apply a gliding force to the head of the humerus. Throughout the full range of motion, the physical therapist will make an effort to keep the glide at a right angle to the plane of motion while urging participants to make an active movement. Conventional therapy will include the same activities as described above. These exercises will be performed for 10 repetitions and gradually the repetition will be progressed week wise.\n\nPatients in group B will need 60 minutes of overall treatment which will include 20 minutes of Mulligan mobilization and 40 minutes of conventional physiotherapy (6 minutes US, 20 minutes TENS and 15 minutes exercises). With a 30-second break in between each series, the MWM method requires approximately twenty minutes to complete in three sequences of 10 repetitions.13\n\nCriteria for discontinuing interventions for a given trial participant\n\nThe treatment will be discontinued if any patients in either of the assigned groups experience adverse effects such as shoulder subluxation. After that, the patients will get the necessary treatment and outpatient therapy. In order to collect follow-up data and avoid missing data, study participants will still be included in the experiment (unless they decide to withdraw their consent).\n\nAdherence and concomitant care\n\nExercise adherence will be measured as a percentage of exercises completed during the first follow-up (after the six-week intervention) for both groups, and will be taken into account when performing the pre-protocol analysis. Exercise adherence will be noted during the first consultation, where it will be stressed how important it is to follow the study guidelines and stay in the designated group.\n\nAll enrolled participants are encouraged to get in touch with the primary investigator immediately if they have any issues with the group intervention they were assigned. At these calls, the primary investigator will then complete a standard form. When necessary, patients can use the dietary supplements and medications that their doctor has prescribed. Patients will be urged to follow the prescribed rehabilitation intervention and refrain from using any other forms of healthcare while the trial is ongoing in order to prevent study contamination. Patients will be instructed to get in touch with the primary investigator or their general physician as needed.\n\nTime frames at baseline and six weeks later.\n\n1. Visual Analogue Scale (VAS)\n\nThis scale assesses intensity of pain and is used to follow a patient’s pain evolution or relate pain severity. The distance (mm) on a 10-cm line is marked by the patient which represents their pain. A greater score specifies a bigger degree of suffering. In order to score and interpret the VAS, four different pain thresholds are established no pain 0–4 mm; mild pain 5–44 mm; moderate pain 45–74 mm; and severe pain 75–100 mm. VAS has been determined to be patient-acceptable and requires little training to administer and score.14\n\n2. Shoulder flexion range of motion on universal goniometer (ROM)\n\nA goniometer is a device that assesses a joint’s available range of motion. A joint’s or bodily part’s range of motion is the amount of movement that can be made around it.\n\n3. Functionality (DASH)\n\nThe 30-question disabilities of the arm, shoulder, and hand (DASH) questionnaire measures a person’s ability to do particular upper extremity tasks. This questionnaire allows patients to self-report their responses and includes a 5-point Likert scale for measuring the degree of difficulty and interference with daily living. DASH is a measurement of physical restrictions and function in upper extremity issues.15\n\n4. Quality of life (SF-36)\n\nFor quality of life, commonly utilised outcome measuring tools include the 36-Item Short Form Survey (SF-36), a self-reported, well-researched, measure of healthiness. It is adapted from a study known as the Medical Outcomes Study for the objective assessment of life quality.16\n\nFormula using mean difference\n\nThe desired sample size will be calculated using mean difference\n\nPrimary variable (DASH – disability of arm shoulder & hand).\n\nMean ± SD (pre) result on DASH for Mulligan (control group) = 50.92 ± 11.56.\n\nMean ± SD (post) result on DASH for Mulligan (control group) = 17.87 ± 11.53.\n\nPooled standard deviation = (50.92 ± 17.87)/2 = 11.545.\n\nDifference = 33.05 (as per ref article).\n\nClinically relevant superiority = 20 % = (33.05 * 20)/100 = 6.61.\n\nAs per reference articles.\n\nTotal samples required = 37 per group.\n\nConsidering 10% drop out = 3.\n\nTotal sample size required = 37 + 3 = 40 per group = 80 in total.\n\nNotations:\n\nReference article: The effect of Mulligan mobilization on pain and life quality of patients with Rotator cuff syndrome: A randomized controlled trial.17\n\nThe participants will be screened in accordance with the inclusion and exclusion criteria prior to group assignment. The indicated outcome measures’ baseline evaluation will come next. All participants will be randomly assigned to either Group A or Group B after the randomization process. Five sessions each week for six weeks, each 60 minutes, will be devoted to the intervention. After the final day of the intervention session, the post-intervention data for the outcomes will be recorded. The information will be gathered and examined in order to study the outcomes. Throughout the course of the study, the guide will supervise the principal investigator as they administer the study interventions. In order to ensure that the patients’ adhere to the study, messages will be sent to their mobile phones informing and reminding them of the sessions in advance. Additionally, the standby days from the week will be used if the patient misses their appointments for any reason, ensuring that they receive a total of 30 intervention sessions over the course of the six-week period.\n\nAs per in the sample size calculation we have accounted for a 10% drop-out rate so that it does not interfere with the results of our study.\n\nThe data collection forms can be found in extended data.\n\nThe evaluation information will come from a pre-made spreadsheet with a variety of baseline characteristics. A secure database will be used to store the research data. The study environment will safely store paper copies of evaluation forms, signed informed consent forms, and other non-electronic paperwork. A full backup of the data entries will be made once per month till the trial is ended. The mechanisms for acquiring data and reporting will be under the direction of the lead investigators. The study papers’ accuracy has to be thoroughly examined. The Excel spreadsheet will published and sent to the statistician at the end of the study for the required analysis. Data loss caused by inefficient staff procedures can be prevented by using a checklist. The extensive follow-up assessment of this experiment indicates that participant retention and follow-up assessment completion will be relatively high. After six weeks, the participants in this trial will be invited to follow-up examinations.\n\nThe overall results will be calculated using R studio software 4.3 version. Descriptive statistics will be calculated for the quantitative assessment over the parameters mean SD, medium, maximal, and minimum for the demographic variables (age). For the qualitative assessment, frequency and percentage will be calculated over the variables (gender and hand dominance). All the results for inferential statistics will be tabulated and tested for the significance at 5% level of significance p ≤ 0.05. The outcome variables (primary) Visual Analogue Scale for Pain, Goniometer for measuring range of motion, disability of shoulder, arm and hand for physical limitations and function in upper extremity problems and (secondary) SF-36 for the measurement of quality of life will be evaluated pre and post-test using a paired-t test. Data for the outcome measure will initially be tested for normality using the Kolmogorov-Smirnov Test.\n\nIf the data fails to achieve normality, it will be attempted to transform into normal distribution by using a mathematical algorithm like log function, inverse mention, exponential function or boxcox transformation. If data persist with non-normal distribution then an alternative non-parametric test will be used for the parametric test result. For paired-t test, the alternative will be the Wilcoxon sign test. Unpaired t-test will be used to find the significance difference over the mean for both primary and secondary variables between control and experimental groups. Alternatively, the Mann Whitney test will be used in place of the unpaired t-test. Association analysis for finding significance of cofounding parameters will be appraised by using Chi-squared test or Fisher’s exact test, or a multi-variant analysis will be used to analyze association in order to determine the significance of confounding parameters.\n\nData monitoring\n\nWe will have a data monitoring committee led by the PI for maintaining and integrating the data.\n\nHarms\n\nThe entire procedure will be managed by a departmental committee and clinical staff. Any injuries or adverse events will be immediately reported to the committee during the trial. The completed dataset will be published on the institutional research website and made available to the appropriate authorities.\n\nAuditing\n\nEvery month, auditing of the trial is going to be conducted. Any deviation from the protocol will be documented and will be addressed.\n\nEthical considerations\n\nEthical approval was obtained from Datta Meghe Institute of Higher Education and Research (IEC no. DMIHER (DU)/IEC/2023/806; IEC approval date 21/03/2023). This protocol has been registered on CTRI (trial registration number CTRI/2023/05/052643, registration date 15/05/2023).\n\nThe study has been approved by the Scrutiny Committee at the college level on 01/03/2023 and by the IEC at the university level on 21/03/2023, following which, the CTRI registration was completed. As per the suggestions through these committees the study has already been modified and approved. So further changes could not be carried out.\n\nPatients will be given a translation of the study protocol into their local language. All study participants will provide their written consent after being fully informed.\n\nThe principal investigator will gather personal data as part of the procedure after explaining the study plan to the participant and a member of their family. The principal investigator, the subject, and two witnesses will all sign a confidentiality statement on the permission form. Consent from the patient will always be sought with full assurances of confidentiality whenever it is required to release information for the study.\n\nThe Principal Investigator will have access to the final trial datasheet.\n\nThe whole procedure is going to be held under the supervision of clinicians and the departmental committee i.e., Guide, Head of the department, Principal and member of Research Guidance Cell.\n\nAfter the trial session, the participations are going to be under supervision for about four weeks so that if there will be any harm, the Principal Investigator will take care of the participants.\n\nWe will present the work in International Conference Proceedings and publish in an indexed journal.\n\nThe study has not yet started.\n\n\nDiscussion\n\nRotator cuff syndrome refers to degeneration or other injury to the rotator cuff. This includes rotator cuff tendonitis, bursitis, rotator cuff tears, full- or partial-thickness subacromial impingement syndrome, and bursitis. The goal of the study is to determine whether Maitland mobilisation and Mulligan mobilisation have any impact on range ROM, pain, and functional disability in people with RCS. The overall number of people with subacromial shoulder discomfort included in this study was 358 across 11 RCTs, they compared the efficiency of MWM combined with exercise for rotator cuff mobilisation, isometric, ROM, strengthening and sham techniques. There was a non-significant difference in SPADI but a significant difference in VAS when MWM was compared to exercise, according to a meta-analysis by Mohamed Abdellatif Hassan et al. (2021).17 In another study, joint mobilisation paired with supervised exercise were examined in people with subacromial impingement syndrome (SAIS), between June 2014 and June 2015, 40 participants with SAIS for longer than six weeks were enrolled in this randomized-controlled research. The participants were split into two groups at random: group one (n = 20) underwent joint mobilisation and electrical stimulation, whereas group two (n = 20) received a supervised exercise plan and electrical stimulation. Pain intensity, DASH questionnaire, the Short Form-36, ROM, the Global Rating of Change Questionnaire, and the American Shoulder and Elbow Surgeons Standardised Shoulder Assessment Form (patient self-report part) were all used as outcome measures. Using the VAS, pain was assessed. After therapy, the range of motion dramatically improved while the mean VAS values in both groups greatly decreased. Both groups’ patient satisfaction with the course of treatment was comparable (p = 0.28). According to the findings of the study, patients with SAIS respond to both supervised exercise and mobilisation in a similar way, Ferit Pekgöz et al.18", "appendix": "Data availability\n\nNo underlying data are associated with this article.\n\nfigshare: Extended data - Samiksha.docx, https://doi.org/10.6084/m9.figshare.23909667.v1. 19\n\nThis project contains the following extended data:\n\n- Model consent form\n\n- Data collection form\n\n- Figure of study procedure\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 ‘Effect of Maitland and Mulligan mobilization on pain, range of motion and disability in patients with rotator cuff syndrome: a randomized clinical trial protocol’, https://doi.org/10.6084/m9.figshare.23909727.v1. 20\n\n\nAcknowledgements\n\nI would like to acknowledge Mr. Manoj Patil and Mr. Laxmikant Umate for their assistance in designing the data analysis and calculating the sample size.\n\n\nReferences\n\nGupta S, Patel MAR, Haneef M, et al.: Bilateral nasolabial flap and Buccal fat pad flap for reconstruction of fibrotomy defect in surgical management of stage III/IV Oral Submucous Fibrosis (OSMF) – A comparative study. J. Oral Med. Oral Surg. Oral Pathol. Oral Radiol. 2021 Jun 15 [cited 2021 Dec 21]; 7(2): 112–118. Reference Source\n\nFitzpatrick LA, Atinga A, White L, et al.: Rotator Cuff Injury and Repair. Semin. Musculoskelet. Radiol. 2022 Oct [cited 2023 Mar 1]; 26(5): 585–596. Publisher Full Text\n\nJobanputtra Y: PHYSIOTHERAPY REHABILITATION OF ROTATOR CUFF INJURY. J. Med. Pharm. Allied Sci. 2021 Nov 15 [cited 2023 Jun 7]; 10(6): 4057–4059. Publisher Full Text Reference Source\n\nNyffeler RW, Schenk N, Bissig P: Can a simple fall cause a rotator cuff tear? Literature review and biomechanical considerations. Int. Orthop. 2021 Jun [cited 2023 Mar 1]; 45(6): 1573–1582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVanBaak K, Aerni G: Shoulder Conditions: Rotator Cuff Injuries and Bursitis. FP Essent. 2020 Apr; 491: 11–16.\n\nVaracallo M, El Bitar Y, Mair SD: Rotator Cuff Syndrome. StatPearls. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2023 Mar 1]. Reference Source\n\nEdwards P, Ebert J, Joss B, et al.: EXERCISE REHABILITATION IN THE NON-OPERATIVE MANAGEMENT OF ROTATOR CUFF TEARS: A REVIEW OF THE LITERATURE. Int. J. Sports Phys. Ther. 2016 Apr [cited 2023 Mar 1]; 11(2): 279–301. PubMed Abstract Reference Source\n\nDeshmukh MK, Fating TB, Phansopkar PA: Comprehensive Physiotherapy Rehabilitation on a Complex Case of Combination of Subcoracoid, Subacromial, Subdeltoid, and Supraspinatus Tendinitis. J. Evol. Med. Dent. Sci. 2020 Oct 12 [cited 2021 Feb 14]; 9(41): 3075–3078. Publisher Full Text Reference Source\n\nRavichandran H, Janakiraman B, Gelaw AY, et al.: Effect of scapular stabilization exercise program in patients with subacromial impingement syndrome: a systematic review. J. Exerc. Rehabil. 2020 Jun 30 [cited 2023 Mar 3]; 16(3): 216–226. Publisher Full Text Reference Source\n\nZhang C, Wang J, Wang L, et al.: The effect of physiotherapy in rotator cuff injury patients with platelet-rich plasma: study protocol of a non-randomized controlled trial. BMC Musculoskelet. Disord. 2021 Mar 20 [cited 2023 Mar 1]; 22(1): 292. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSavva C, Karagiannis C, Korakakis V, et al.: The analgesic effect of joint mobilization and manipulation in tendinopathy: a narrative review. J. Man. Manip. Ther. [cited 2023 Mar 1]; 29(5): 276–287. Publisher Full Text Reference Source\n\nWang S, Chapple CM, Quinn D, et al.: Dosage of joint mobilisation for the management of rotator cuff-related shoulder pain: protocol for a scoping review. BMJ Open. 2022 Jun 3 [cited 2023 Mar 1]; 12(6): e056771. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMenek B, Tarakci D, Algun ZC: The effect of Mulligan mobilization on pain and life quality of patients with Rotator cuff syndrome: A randomized controlled trial. J. Back Musculoskelet. Rehabil. 2019 Jan 1 [cited 2023 Mar 1]; 32(1): 171–178. PubMed Abstract \| Publisher Full Text Reference Source\n\nHawker GA, Mian S, Kendzerska T, et al.: Measures of adult pain: Visual Analog Scale for Pain (VAS Pain), Numeric Rating Scale for Pain (NRS Pain), McGill Pain Questionnaire (MPQ), Short-Form McGill Pain Questionnaire (SF-MPQ), Chronic Pain Grade Scale (CPGS), Short Form-36 Bodily Pain Scale (SF-36 BPS), and Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP). Arthritis Care Res. 2011 Nov; 63: S240–S252. Publisher Full Text\n\nPhysiopedia: DASH Outcome Measure.[cited 2023 Mar 2]. Reference Source\n\nPhysiopedia: 36-Item Short Form Survey (SF-36).[cited 2023 Mar 2]. Reference Source\n\nMenek B, Tarakci D, Algun ZC: The effect of Mulligan mobilization on pain and life quality of patients with Rotator cuff syndrome: A randomized controlled trial. J. Back Musculoskelet. Rehabil. 2019 Jan 1 [cited 2023 Jun 27]; 32(1): 171–178. Publisher Full Text Reference Source\n\nHassan MA, Elanni EF, Hassan KA, et al.: Effect of Mulligan Technique on Subacromial Impingement Syndrome: A Systematic Review. Egypt. J. Hosp. Med. 2021 Oct 1 [cited 2023 Mar 2]; 85(1): 3043–3053. Publisher Full Text Reference Source\n\nSonone S: Extended data - Samiksha.docx. figshare. Journal Contribution. 2023. Publisher Full Text\n\nSonone S: samiksha SPIRIT_checklist.docx. figshare. Journal Contribution. 2023. Publisher Full Text" }	[ { "id": "212901", "date": "13 Oct 2023", "name": "Annegret Mündermann", "expertise": [ "Reviewer Expertise Rotator cuff pathologies", "movement science", "shoulder biomechanics", "patient reported outcomes", "clinical trials" ], "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 goal of the clinical trial described in this protocol is to compare the effects of Maitland and Mulligan mobilization on pain, range of motion and functional disability in patients with rotator cuff syndrome. The authors intend to randomize patients with rotator cuff syndrome to two different therapeutic interventions. While the topic of this manuscript is relevant, there are several aspects of the manuscript that require clarification:\n\nThe entire article requires copy-editing including, also for consistent use of US or OK spelling.\n\nThe authors start their introduction with a detailed description of shoulder anatomy which is basic knowledge and should be deleted from the article.\n\nMany statements in the introduction section lack references. The authors should cite appropriate literature.\n\nWhile the Mulligan mobilization is described in the introduction session, appropriate information should also be provided for the Maitland mobilization.\n\nThere are some open questions regarding the inclusion and exclusion criteria. One inclusion criterion is having a partial tear, and one exclusion criterion is osteoporosis. How will the authors confirm check these criteria? Will patients with glenohumeral or acromioclavicular osteoarthritis be excluded as these can affect the proposed outcome measures.\n\nThe authors state that shoulder subluxation is a reason for discontinuation. How often does this typically occur in standard intervention?\n\nThe authors should describe earlier in the manuscript how adherence will be recorded. In the current version it appears that this will be reported by the study participants. However, later in the manuscript it becomes clear that the therapists will know which sessions the participants will complete.\n\nShoulder flexion range of motion will be assessed using a goniometer. The authors describe what a goniometer is and what it can be used for. Please describe how and by whom the shoulder flexion range of motion assessment will be conducted.\n\nThe authors conducted a sample size estimation but should report the reference article earlier in the section. The calculation for the pooled standard deviation is incorrect. The pooled standard deviation is 16.3. With this value, 75 patients are needed for the study. The sample size estimation describes superiority, yet the statistical analyses aim at determining if both interventions have significant effects on the outcome parameters.\n\nThe authors assumed a drop-out rate of 10%. Is this realistic?\n\nThe authors should specify how missing data are handled and which intent to treat analysis is planned.\n\nAll participants will be expected to complete five sessions per week. Will these only be scheduled on weekdays? The authors state that missed appointments will be available on standby days. Are these on weekends? Please provide more details.\n\nAll data will be managed in excel. Excel spreadsheets are not appropriate for clinical trials for several reasons including that data can be lost. The authors are strongly encouraged to use a data management tool appropriate for clinical trials with a data log such as Redcap.\n\nThe authors state that data monitoring will be led by the PI. The data monitoring should be performed by an independent committee.\n\nThe discussion should address aspects related to the study design, expected results and implications of these results as well as the strengths and limitations of aspects of the study protocol\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? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-1027
https://f1000research.com/articles/12-1023/v1	22 Aug 23	{ "type": "Research Article", "title": "Ageism and Its Impact on Quality of Life Among Older Persons Experiencing Difficulties with Activities of Daily Living", "authors": [ "Felipe Alfonso Sandoval Garrido", "Timothy Bolt", "Yuta Taniguchi", "Peter Lloyd-Sherlock", "Timothy Bolt", "Yuta Taniguchi", "Peter Lloyd-Sherlock" ], "abstract": "Background: As the number of older persons increases globally, so does ageism, or being discriminated against because of their age. The overlap between ageism and ableism, which is discrimination based on impairments or disability may put persons having difficulties with their basic (ADL) or instrumental activities of daily living (IADL) at a higher risk of discrimination. Discrimination leads to poorer quality of life. However, ageism remains understudied, particularly in the UK. Methods: Data from the English Longitudinal Study of Ageing (ELSA) collected in the 2010-2011 period (Wave 5) was used to assess ageism, ADL/IADL status, and quality of life (CASP-19) at baseline. Multivariable linear regression was used to estimate the changes in the quality of life while adjusting for covariates and their baseline values in two following periods: 2016-2017 (Wave 7) and 2018-2019 (Wave 9). Results: At baseline, quality-of-life scores were significantly lower among individuals dealing with difficulties (33.6 points) compared to those without them (41.5 points), with a difference of 7.9 points (p < .001). In the following waves, age discrimination was found to have a negative effect on the quality of life of all the participants regardless of ADL/IADL difficulties (B: -1.106, p=<0.001), and also among those without difficulties (B=-1.401, p=<0.001). However, among those with difficulties, no such association was found in any of the analyses. Conclusions: Discrimination has a detrimental effect on quality of life, but this effect may be mediated by characteristics of the older persons suffering from loss of independence, such as coping mechanisms, resilience, social support, personality, and other latent characteristics that may be associated with ADL/IADL difficulties not included in this study. These findings underscore the importance of understanding the driving elements of quality of life among older persons to effectively inform future intervention strategies, especially for those at risk of ageism.", "keywords": [ "Discrimination", "Ageing", "Ageism", "ELSA", "England", "Older Adults" ], "content": "Introduction\n\nAge discrimination, often called ageism, is a term coined by Robert N. Butler in 1969.1 Ageism is a harmful type of discrimination that unfairly targets individuals, particularly older persons. It can take forms from prejudices to outright acts of exclusion, and its negative consequences are felt in multiple areas of life, such as work, healthcare and social engagement.2–4 Discrimination based on age significantly diminishes the well-being and quality of life of those affected.5–7\n\nThe capacity of an individual to independently perform Activities of Daily Living (ADLs) and Instrumental Activities of Daily Living (IADLs) may be impacted as they age due to a number of physiological and cognitive changes. IADLs and ADLs are categories of core self-care activities required for independent community living. ADLs encompass essential self-care activities like bathing, dressing, and eating,8 while IADLs encompass more complex activities necessary for independent living, such as shopping, meal preparation, financial management, and medication management.9 Difficulty in performing these activities implies a level of dependency. In the UK, 26% of individuals 65 and older needed assistance with ADLs in 2021, while 25% needed assistance with IADLs.10\n\nElderly individuals who face challenges in carrying out activities and independent living tasks are also more likely to encounter ableism. Ableism refers to discrimination against people with disabilities,11 indicating a connection between ageing and difficulties in performing activities for independent living tasks. Societal expectations for individuals to remain competent and fit, in the form of fitnessism, healthism, or lookism,12 coupled with the belief that typical abilities are superior,13 contribute to this discrimination. The societal emphasis on physical independence and the devaluation of dependency promotes the marginalisation of those who need assistance, reinforcing ableist norms and diminishing societal diversity and inclusion. Consequently, dependent older adults may internalise these societal values, leading to diminished self-esteem and lower quality of life.14\n\nWhile research has provided evidence of ageism and dependency independently, studies exploring the intersection of age discrimination and difficulties with activities of daily living are limited, particularly in the UK, where ageism remains an overlooked issue.15 A study showed that those who hold ableist views tend to discriminate against older people more.16 The number of elderly people is growing worldwide as people live longer,17 and ageism is more common than it has ever been. Using data from the English Longitudinal Study of Ageing, a prior study conducted in England looked at the relationship between ageism and older people’s health, but it did not explicitly look at those who had problems doing ADLs or IADLs. Although quality of life was not included as an endpoint, it did contain linked biological markers.18 The same authors used a quality-of-life indicator as an outcome variable in a later study. This study, however, did not isolate beliefs directly attributable to ageism and instead concentrated on visual impairment and a general feeling of discrimination.19\n\nThe primary goal of this study is to understand the extent of age discrimination older persons with ADL/IADL challenges experience and how it affects their quality of life. We specifically want to know if and how ageism, as experienced by older persons who are dependent, is related to shifts in their quality of life across time. By doing this, we hope to draw attention to the connection between ageism and dependence and offer useful data for formulating targeted interventions to improve this at-risk group’s quality of life.\n\n\nMethods\n\nFor the collection of the primary data, Ethical Approval for ELSA Wave 1 was granted from the Multicentre Research and Ethics Committee (reference number: MREC/01/2/91) approved on 7th February 2002.20 The participants were freely able to choose to participate or not to participate in the research without any repercussions. The participants’ informed consent could be withdrawn at any time without questioning the reason for the withdrawal. Additionally, approval from the Ethics Committee of the University of Tsukuba, Japan, was obtained for the secondary analyses of the data (approval number: 1817; R4/10/14). Our study was conducted in compliance with the ethical guidelines of the Helsinki Declaration and it complied with the Ethical Guidelines for Medical and Biological Research Involving Human Subjects.\n\nWe used information from the English Longitudinal Study of Ageing (ELSA), a recurring panel survey that gathers information from a representative sample of English people aged 50 and over, collected biennially across a variety of fields. Interviewers collected data by conducting Computer-Assisted Personal Interviewing (CAPI) and utilising self-completion questionnaires. At the participants’ homes, face-to-face first interviews were done. Telephone or in-person visits might be used to perform follow-up interviews.21 We used the 2010–2011 period (wave 5) for the original data set because, in contrast to other iterations of this panel survey, it contained explicit questions about perceived age discrimination. To see the effect on quality-of-life scores in subsequent periods, waves 7 (period 2016-17) and wave 9 (period 2018-2019) were also obtained.\n\nThe number of participants initially consisted of 10,274 people. Inclusion criteria were older adults who answered the questions themselves and with valid responses to the questions on perceived age discrimination and difficulties with ADL/IADL. A total of 537 participants (5.2%) were excluded due to their responses being provided by a proxy respondent, leaving 9,737 participants. The decision to exclude proxy respondents from the analysis was guided by the objective to understand and explore the direct experiences and perceptions of older individuals with respect to age discrimination and quality of life. While proxy respondents can provide valuable information, particularly when the older person is unable to respond due to cognitive or physical limitations, they may not fully capture the personal perceptions and experiences of the older individuals themselves. Given the subjective nature of perceived age discrimination and quality of life assessments, it was crucial to utilise first-hand responses to ensure the authenticity and accuracy of the data. Furthermore, studies have shown that proxy responses can sometimes be systematically different from self-reports, potentially introducing bias into the analysis.22\n\nOut of these respondents, data for perceived age discrimination (either positive or negative responses) was unavailable for 4,870 individuals (50%). The remaining 4,867 respondents had valid responses. Within this group, we further excluded 28 respondents (0.6%) for not having a quality-of-life measure, for a final sample of 4,839 participants. Among these, 3,531 respondents (73.0%) reported no difficulties with ADL or IADL, while 1,308 respondents (27.0%) reported having difficulties with either ADL or IADL. Details can be found in Figure 1.\n\nDependency\n\nDependency was defined as having difficulties with at least one ADL or IADL. The activities included were:\n\nActivities of Daily Living (ADLs):\n\n1. Difficulty dressing, including putting on shoes and socks\n\n2. Difficulty walking across a room\n\n3. Difficulty bathing or showering\n\n4. Difficulty eating, such as cutting up food\n\n5. Difficulty getting in and out of bed\n\n6. Difficulty using the toilet, including getting up or down\n\nInstrumental Activities of Daily Living (IADLs):\n\n1. Difficulty using a map to figure out how to get around in a strange place\n\n2. Recognizing when in physical danger\n\n3. Difficulty preparing a hot meal\n\n4. Difficulty shopping for groceries\n\n5. Difficulty making telephone calls\n\n6. Communication (speech, hearing, or eyesight)\n\n7. Difficulty taking medications\n\n8. Difficulty doing work around the house and garden\n\n9. Difficulty managing money, e.g., paying bills, keeping track of expenses\n\nGiven that most participants reported experiencing no difficulties, responses were dichotomised into categories delineating those without difficulties and those with one or more difficulties. This variable offers significant insights into the level of participant independence. To delve further into the compound effects of these difficulties, bivariate analyses were conducted, segregating the sample into three distinct groups: the first with a single ADL/IADL difficulty, the second with two such difficulties, and the third presenting three or more difficulties.\n\nThe English Longitudinal Study of Ageing (ELSA) incorporated questions concerning experiences of discrimination for a singular instance in its fifth wave. These queries have also been deployed in other longitudinal examinations, notably within the Health and Retirement Survey (HRS)23 and the Midlife in the United States (MIDUS) survey.24\n\nParticipants were asked about the perceived frequency of five discriminatory circumstances as follows: ‘How often do any of the following events occur in your daily life?’\n\n1. How often is the respondent treated with less courtesy than other people?\n\n2. How often does the respondent receive poorer service than others in restaurants?\n\n3. How often do people act as if they think the respondent is not clever?\n\n4. How often is the respondent threatened or harassed?\n\n5. How often does the respondent receive poorer service than others from hospitals?\n\nThe answers could be from 1, meaning ‘almost daily’, to 6, meaning ‘never’. We split the answers into two groups to see if participants faced discrimination in the past year (more than once a year vs. less than once a year or never).\n\nSubsequently, participants were asked a follow-up question about their perceptions of the reasons for the discriminatory experiences. The possible reasons were age, gender, race, weight, physical disability, and other. Participants could give multiple attributing reasons. For our analytical purposes, we focused exclusively on responses that attributed the experienced discrimination to age.\n\nThe CASP-19 scale, a well-known tool designed particularly for assessing quality of life in older individuals, was used to evaluate overall quality of life. It covers four domains: control, autonomy, self-realization, and pleasure.\n\nIn our study, we considered covariates found in previous studies, which were categorized as follows: Age was divided into four groups, less than 60 years old, 60-69 years old, 70-79 years old, and 80 years old or more. The participants were identified as either male or female for the variable sex. Excluding pensions, household wealth was segmented into five groups, or quintiles, with the 1st quintile representing the least wealthy and the 5th quintile symbolizing the most affluent households. The education level of the participants was classified into low, middle, or high. Marital status was categorized as either in a partnership, which includes those who are married or cohabiting, or not in a partnership, which includes those who are divorced, separated, or widowed. For ethnicity, participants were identified as either white or non-white. Physical health was assessed through the participants’ self-perception of their health, which was categorized as either excellent/very good/good or fair/poor, providing insight into the participants’ own evaluation of their physical health status. Finally, the Center for Epidemiologic Studies Depression (CES-D) scale25 was employed to assess mental health. A score of 4 or higher on this scale signifies the existence of depressive symptoms.\n\nThe study adopted both cross-sectional and longitudinal approaches for analysis. The cross-sectional analysis examined the data collected in ELSA wave 5 (2010-2011) to examine the relationship between perceived age discrimination, dependency, and quality of life at a single time point. Subsequently, the longitudinal analysis followed the same cohort across the subsequent waves in 2016-2017 (wave 7) and 2018-2019 (wave 9). These analyses enabled the tracking of changes in quality of life over time.\n\nThe analyses of the sociodemographic characteristics were carried out by analysing the data using bivariate statistics to summarize and organize the characteristics of the study population and to assess the distribution within the groups with difficulties with ADL/IADL characteristics and those without difficulties. At this stage, categorical variables were presented as frequencies and percentages.\n\nTo investigate the relationship between perceived discrimination and overall quality of life, we ran linear regression analyses. We considered a number of variables in these studies that may have an impact on the findings, including age, gender, ethnicity, income level, education level, marital status, physical and mental health issues, and employment status. We used the Hosmer Lemes how test26 to assess how well our models fit the data.\n\nOn the relationship between perceived age discrimination and quality of life, cross-sectional (wave 5) and longitudinal (wave 7 and 9) studies were carried out. Three cross-sectional logistic regression analyses were conducted utilising baseline data as part of the cross-analysis. Regardless of their ADL/IADL status, all individuals were included in the initial cross-sectional study. The second and third cross-sectional analyses were performed on people without ADL or IADL difficulties and those with difficulties, respectively. Finally, the prospective analyses to assess changes in quality of life were carried out in two subsequent periods: wave 7 (2016–2017) and 9 (2018–2019). For the prospective analyses the sample was restricted to those who reported having trouble performing ADL or IADL.\n\nAll models were adjusted for age, sex, wealth, education level, partnership status, ethnicity, perceived health, depressive symptoms, and current working status. The prospective models for waves 7 and 9 were also adjusted for baseline CASP-19 scores (wave 5).\n\nBefore the analyses, we verified the assumptions of normality, linearity, and homoscedasticity. Additionally, we examined multicollinearity among the variables as it could potentially impact the outcomes of the logistic regression. All tests were performed using a two-sided approach and any p value, below 0.05 was considered significant. Confidence intervals were set at the 95% level of certainty. We utilized IBM SPSS Statistics version 29.0.1.0 for all our analyses.\n\n\nResults\n\nIn our sample, 61% (2,966 participants) said they faced age discrimination. Table 1 shows that participants with ADL/IADL difficulties were more likely to experience age discrimination. Specifically, 29% (867 participants) of those with difficulties reported age discrimination, compared to 23.5% (441 participants) of those who did not.\n\nTable 1 additionally indicates a correlation between age and ADL/IADL challenges, with an age-related rising trend. It is observed that women encounter these difficulties more frequently than do males. Socioeconomic characteristics are also connected, with households in lower quintiles of wealth reporting greater difficulty than their counterparts in higher quintiles. Similarly, those with lower education levels report higher prevalence. This table also shows a connection with marital status, as individuals not in a partnership, including those divorced, separated or widowed, face these difficulties more often than those in partnerships. Ethnicity does not have a significant value in this context. Self-perception of physical health is linked to ADL/IADL difficulties, with individuals assessing their health as fair or poor reporting more difficulties. Mental health, assessed by the CES-D scale, shows a higher prevalence of ADL/IADL difficulties among individuals exhibiting depressive symptoms. Employment status shows that those who are not currently working face these difficulties more frequently.\n\nThose reporting one difficulty with ADL or IADL are 1.475 times more likely to experience age discrimination compared to the reference group with no difficulties (OR=1.475, 95% CI: 1.213-1.795). The odds rise to 1.674 for individuals with two difficulties (OR=1.674, 95% CI: 1.269-2.207). However, the increase in the odds of experiencing age discrimination for those encountering three or more difficulties is not statistically significant (OR=1.105, 95% CI: 0.915-1.335).\n\nTable 2 also analyses the different scenarios where discrimination was perceived. For those experiencing three or more difficulties, a significant correlation was observed in situations where they were treated with less courtesy than others (OR=1.335, 95% CI: 1.109-1.608).\n\nThe likelihood of receiving poorer service in restaurants increased significantly across all difficulty categories, with the highest odds ratio for individuals with three or more difficulties (OR=1.403, 95% CI: 1.136-1.732).\n\nAlso, the odds of respondents feeling that they were regarded as less intelligent increased significantly across all categories of difficulties, peaking for those with three or more difficulties (OR=2.289, 95% CI: 1.892-2.769) when compared to those without difficulties.\n\nA similar trend was observed for reporting that they were being threatened or harassed, with the highest odds ratio belonging to those with three or more difficulties (OR=1.788, 95% CI: 1.408-2.272).\n\nFinally, the odds of receiving inferior service from hospitals were significantly higher, particularly for those with two difficulties (OR=1.763, 95% CI: 1.352-2.299) and those with three or more difficulties (OR=1.715, 95% CI: 1.412-2.084).\n\nTable 3 displays the variance in the CASP 19 score, which measures quality of life, on a scale of 0 to 57 points between adults who do not have any difficulties with Activities of Daily Living (ADL) or Instrumental Activities of Daily Living (IADL) and those who face such challenges. The mean CASP 19 score for individuals without ADL/IADL difficulties (n=3531) is 41.5 points whereas the average score for individuals with these difficulties (n=1308) is significantly lower at 33.6 points. The difference between the two groups means is 7.9 points with an error of 0.29077. The t value of 27.207 indicates significance at p<0.001. The standard deviation of CASP 19 scores is slightly higher in the group facing ADL/IADL difficulties (9.3) compared to those without difficulties (8.1). This suggests a range of variability among individuals dealing with these challenges. Additionally, there is an error of the mean (SEM) in the group facing difficulties compared to those without them (0.257 vs 0.137) indicating greater variability in their scores. In summary these findings demonstrate that individuals experiencing ADL/IADL difficulties tend to have poorer scores than those without such challenges.\n\nTable 4 displays the mean CASP-19 score difference between those that and those who did not experience age discrimination. Participants who did not perceive age discrimination had an average CASP-19 score of 40.4 (n=1873). The mean score was slightly lower at 38.7 for those who reported age discrimination (n=2966), though. This shows a mean difference of 1.7 points between the two groups that is statistically significant.\n\nTable 5 shows the results from a cross-sectional linear regression analysis in wave 5. This analysis looked at the relationship between quality of life (measured using the CASP-19 score points) and perceived age discrimination in three distinct groups: all participants who perceived age discrimination, regardless of difficulties; participants without any ADL/IADL difficulties who perceived age discrimination; and participants with ADL/IADL difficulties who perceived age discrimination.\n\nOur main outcome variable is Quality-of-Life Scale, measured by CASP-19. The results indicate that participants who perceived discrimination tended to have a lower quality of life score. This was significant in the overall participant group (Unstandardized Beta Coefficients (B): -1.106; CI:-1.577~-0.634) with a p-value of <0.001, and among participants without difficulties (B: -1.401; CI: -1.934~-0.868) with a p-value of <0.001. However, there was no significant association for participants with difficulties (B: -0.401; CI -1.353~0.552) with a p-value of 0.409. A more detailed breakdown of all variables by group, including covariates, can be found above.\n\nAll participants who Perceived Age Discrimination (n=4246)\n\nAge was not a significant determinant of perceived age discrimination. Being female was significantly associated with increasing CASP-19 scores (B: 0.738; p=0.001). As wealth increases by quintile there is a significant increase in quality-of-life scores compared to the lower quintiles (B: 1.003; p≤0.001). Those who were married, or cohabiting had higher CASP-19 scores (B: 0.690; p=0.007). Being white had no significant association. Regarding health status, participants who rated their physical health as “fair/poor” had lower CASP-19 scores (B: -5.801; p=0.033). Participants in the group over four symptoms of depression had lower CASP-19 scores (B: -8.314; p≤0.001). The current working status showed no statistical significance.\n\nParticipants without ADL/IADL Difficulties who Perceived Age Discrimination (n=3056)\n\nAge was not a significant determinant of perceived age discrimination. Being female was significantly associated with better CASP-19 scores (B: 0.647; p=0.014). As wealth increases by quintile, there is a significant increase in quality-of-life scores (B: 0.992; p≤0.001). Those who were married, or cohabiting had higher CASP-19 scores (B: 0.756; p=0.010). Being white had no significant association. Regarding health status, participants who rated their physical health as “fair/poor” had lower CASP-19 scores (B: - 4.714; p≤0.001). Participants in the group over four symptoms of depression had lower CASP-19 scores (B: -8.299; p≤0.001). The current working status showed no statistical significance.\n\nParticipants with ADL/IADL Difficulties who Perceived Age Discrimination (n=1308)\n\nThe likelihood of increasing quality-of-life scores were higher for women compared to men (B:1.162: 0.872; p=0.011). Also, the higher the quintile of wealth the better the CASP-19 scores (B=0.714; p≤0.001). Regarding health status, participants who rated their physical health as “fair/poor” had lower CASP-19 scores (B: -4.749; p≤0.001). Participants in the group with 4 or more symptoms of depression had lower CASP-19 scores (B: -7.628; p≤0.001). Unlike other groups, being in a partnership had no positive effect on quality-of-life scores. Also, as mentioned before, the association of perceived age discrimination and CASP-19 score was not significant in this group (B: -0.401; p=0.409), suggesting that the effect of quality of life on perceived age discrimination may be less pronounced among individuals with ADL/IADL difficulties.\n\nTable 6 shows that there is no significant association of perceived age discrimination and quality-of-life scores among those with ADL/IADL difficulties. The lack of significance prevails on all models; cross-sectional linear regression analysis for wave 5, and prospective linear regressions for waves 7 and 9. The numbers analysed were 1,190 participants in Wave 5, 757 in Wave 7, and 530 participants in Wave 9.\n\n\nDiscussion\n\nOur study’s findings provide evidence to suggest a high prevalence of perceived age discrimination exists among dependent older adults, with this discrimination correlating with a lower quality of life for older persons. This detrimental association is perceived among all participants in our multivariable model. When participants were divided into those without ADL/IADL difficulties and those with difficulties, only the group without difficulties had a statistically significant association between experiences of discrimination and detrimental changes in the quality-of-life scores. This effect was not observed among older persons having ADL/IADL difficulties. The lack of statistically significant results for those having ADL/IADL difficulties was consistent across prospective observation waves.\n\nThe negative impact of age discrimination on the well-being of individuals aligns with the discoveries made by Sutin et al.,.4 They revealed that perceiving age discrimination is linked to emotional and cognitive health consequences, including reduced life satisfaction in older adults who have poor health or are burdened by illness. Our findings further support this understanding by demonstrating that these detrimental effects also affect quality of life.\n\nHowever, the lack of significance among those with ADL/IADL difficulties may seem counterintuitive. The absence of statistical significance among those with difficulties could potentially be attributed to the “disability paradox”. This phenomenon suggests that individuals living with disabilities may report a surprisingly high quality of life.27 Despite facing difficulties with daily living tasks, this group may have developed effective coping mechanisms or adapted to their expectations and values, maintaining their perceived quality of life. For them, age discrimination may represent just one among many challenges, and it might not significantly alter their overall quality of life. In contrast, individuals without ADL/IADL difficulties may have a lower threshold for disturbances in their quality of life and might perceive age discrimination as a significant negative experience.\n\nA study by Sutin et al.,4 argues that people who have lived many years with defining characteristics, such as disabilities and ensuing difficulties with ADL/IADL, may develop some resilience against discrimination. In line with the argument by Sutin, resilience and coping mechanisms have been suggested as critical elements in mitigating the negative impacts of stressors, such as discrimination, on quality of life.28 As such, the persistence of age discrimination without a significant fluctuation in quality-of-life scores could reflect the resilience mechanisms that some older adults have developed over time. This might explain why, in our study, only participants without ADL/IADL difficulties showed an impact on their quality of life due to age discrimination.\n\nIn contrast, in our bivariate cross-sectional analyses, individuals who perceived age discrimination had a higher percentage of ADL/IADL difficulties. This could suggest that the stress or marginalisation associated with age discrimination may impact functional health, but the effect is overshadowed in a smaller sample of only persons with difficulties or when adjusted by covariates. Overall, the findings may suggest that while discrimination could contribute to negative health outcomes, its impact on the broader, subjective measure of quality of life may not be straightforward. However, the absence of a negative impact among those facing difficulties could be linked to the research design, the scale used to measure quality of life, or the resilience factors inherent in the sample population.\n\nSeveral limitations within our study should be acknowledged when interpreting our findings. Firstly, the measure for age discrimination is subjective, meaning it is based on individuals’ perceptions rather than objective, external assessments. While subjective experiences are critical in understanding how age discrimination affects older adults, it does open the door for potential bias. Individuals’ responses could be influenced by personality characteristics, mood states, cognitive biases, or cultural norms. Therefore, these responses might not reflect the full extent or absence of actual discriminatory behaviours or attitudes experienced by the respondents.\n\nSecondly, it is important to note that the assessment of quality of life in our study is subjective. Although this approach provides insights into experiences and perspectives it can also be influenced by variations in optimism, resilience and other character traits. Consequently, there might exist a disparity, between individuals self-reported quality of life and objective indicators of health and overall well-being.\n\nThirdly, the measure for difficulties with ADL/IADL in our study is binary, meaning it does not fully explore the range of needs among participants. We categorized participants into those with one or more difficulties and those without, thus potentially grouping together individuals with diverse levels of need. For most of the analysis, someone experiencing potentially minor difficulties with only one activity was treated the same as someone with severe difficulties across multiple activities. This could obscure more nuanced associations between the extent of difficulties with ADL/IADL, age discrimination, and quality of life.\n\nAn additional limitation pertains to the temporal nature of our age discrimination measure. The question on discrimination asked participants to recall past instances of perceived discriminatory situations, ranging from daily occurrences to instances less than once a year, as well as an option for never experiencing such situations. This reliance on retrospective self-reporting could introduce recall bias, particularly for those participants who were asked to remember events that happened infrequently or long ago. This bias might distort the true association between age discrimination and quality of life.\n\nAlso, our analysis skipped the most immediate wave of data collection and used data every other wave, which took place every two years. This approach could potentially distort the strength of the observed association between perceived age discrimination and quality of life. Given the biennial nature of the data collection, our study might not capture changes in perceived discrimination and quality of life that occurred in the intervening years. It is plausible that the experiences and perceptions of participants may change within shorter time intervals, and this fluctuation may not be accurately captured by our measurements.\n\nFurthermore, our measure for difficulties did not discriminate between ADL, which includes basic self-care tasks, and IADL, which encompasses more complex activities related to independent living. This distinction could be important because difficulties with more simple but critical activities could potentially have a larger impact on quality of life and may be more closely tied to experiences of discrimination.\n\nLastly, while ADL/IADL difficulties can be an indicator of disability, it does not equate to a disability or dependency diagnosis. In our study, it is merely an indicator of a certain level of difficulty with daily activities that is not clearly defined. As such, this measure might not accurately capture the full range of functional capabilities or impairments among our participants. As mentioned by Gill et al.,29 there is no consensus on ADL/IADL as a measure of dependence. Most epidemiological studies agree with us in considering difficulties with ADL/IADL as dependence, but other argue that it should be defined as degree of difficulty.29\n\nFuture research should take these limitations into account. The integration of more objective measures, a distinction between types of difficulties and degrees of impairment, and a precise definition of difficulty levels could yield a more holistic understanding of the effects of age discrimination on the quality of life among older adults.\n\n\nConclusion\n\nOur research helps to examine the complicated relationship between age discrimination, difficulties with daily and instrumental activities, and quality of life among the elderly. These findings have implications for policymakers and healthcare professionals, emphasizing the significance of implementing strategies that address age discrimination to enhance the well-being of adults who require assistance with their daily activities. The widespread occurrence of age discrimination highlights the need for targeted efforts in preventing and addressing such actions.\n\nFurthermore, these findings shed light on how age discrimination impacts quality of life depending on level of difficulties with tasks. This provides an understanding of the relationship between age discrimination and quality of life highlighting the potential importance of resilience mechanisms and coping strategies among older adults. It is worth noting that while our multivariable model does not indicate an effect for persons with difficulties, our bivariate analyses demonstrate a difference in quality-of-life scores when such difficulties exist. This furnishes robust evidence for the influence of these difficulties on an individual’s quality of life. Specifically, the notable mean difference in quality of life between those facing difficulties and those without underscores a significant disparity, suggesting a compelling correlation between perceived age discrimination and the reported presence of ADL/IADL difficulties among older individuals. Given the distinct variations in our findings concerning who experience age discrimination and under what circumstances, further investigation into these disparities in future studies would be advantageous. Discerning why certain groups are more susceptible to perceiving age discrimination in differing contexts could facilitate the customisation of interventions to meet their distinct needs.\n\nFurthermore, the intersectionality of age discrimination with other demographic and health factors underscores the necessity for a more sophisticated approach to studying and addressing age discrimination among older adults, given the heterogeneity within this population. This strategy should prioritise vulnerable demographics such as women, individuals of lower socioeconomic standing, those with less education, those without partners, and individuals facing mental health challenges. Subsequent research should probe more deeply into these contributory factors underlying the observed disparities, which may lead to the design of more effective interventions for enhancing biopsychosocial conditions. A comprehensive methodology could guide the formulation of interventions that not only independently tackle ageism and functional difficulties but also their intersectional occurrences. This would significantly aid in fostering inclusive and equitable societies for all.\n\nThese intersectional interventions designed to alleviate ADL/IADL difficulties could significantly improve individuals’ quality of life but are also needed in routine care, particularly for populations known to be at risk for these difficulties. This might include older adults with chronic illnesses30 or those recovering from surgery.31\n\nFinally, the inconclusiveness of the present results calls for further research, specifically longitudinal studies that combine a more detailed assessment of disabilities and dependency, in combination with both subjective and objective outcome measures, which could shed light on the underlying dynamics of this relationship over time.", "appendix": "Data availability\n\nEnglish Longitudinal Study of Ageing (ELSA) http://doi.org/10.5255/UKDA-Series-200011. 32 This study contains the underlying data from three distinct waves:\n\n• Wave 5\n\n• Wave 7\n\n• Wave 9\n\nExcluding the categories of data which are deemed confidential or sensitive in nature, such as linked administrative records, geographically identifiable variables, or genetic material information, the data and pertinent documentation from Wave 5, 7 and 9 have been secured and are accessible via the archive provided by the Economic and Social Data Service. The data is freely available and to acquire access to the data, users need to agree to the data sharing policy.\n\n\nReferences\n\nButler RN: Age-ism: another form of bigotry. Gerontologist. 1969; 9(4): 243–246. PubMed Abstract \| Publisher Full Text\n\nNeumark D, Burn I, Button P: Is it harder for older workers to find jobs? New and improved evidence from a field experiment. J. Polit. Econ. 2019; 127(2): 922–970. Publisher Full Text\n\nLloyd-Sherlock PG, et al.: Institutional ageism in global health policy.2016; 354.\n\nSutin AR, et al.: Perceived discrimination and physical, cognitive, and emotional health in older adulthood.2015; 23(2): 171–179.\n\nGarstka TA, et al.: How Young and Older Adults Differ in Their Responses to Perceived Age Discrimination. Psychol. Aging. 2004; 19(2): 326–335. PubMed Abstract \| Publisher Full Text\n\nKang H, Kim H: Ageism and Psychological Well-Being Among Older Adults: A Systematic Review. Gerontol. Geriatr. Med. 2022; 8: 23337214221087023.\n\nChang ES, et al.: Global reach of ageism on older persons’ health: A systematic review. PLoS One. 2020; 15(1): e0220857. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatz S, et al.: Progress in development of the index of ADL.1970; 10(1_Part_1): 20–30.\n\nLawton MP, Brody EM: Assessment of older people: self-maintaining and instrumental activities of daily living. J. Gerontol. 1969; 9(3_Part_1): 179–186. Publisher Full Text\n\nCentre, H.a.S.C.I: Health Survey for England 2021, N. Digital, Editor.2023.\n\nWolbring GJD: The politics of ableism.2008; 51(2): 252–258.\n\nAyalon L, Tesch-Römer C: Contemporary perspectives on ageism. Springer Nature; 2018.\n\nWolbring G: Expanding Ableism: Taking down the Ghettoization of Impact of Disability Studies Scholars.2012; 2(3): 75–83.\n\nMarquet M, et al.: Understanding the mechanisms underlying the effects of negative age stereotypes and perceived age discrimination on older adults’ well-being. Aging Ment. Health. 2019; 23(12): 1666–1673. PubMed Abstract \| Publisher Full Text\n\nRippon I, et al.: Perceived age discrimination in older adults. Age Ageing. 2014; 43(3): 379–386. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFriedman C, VanPuymbrouck LJP, Geriatrics OTI: Ageism and ableism: Unrecognized biases in occupational therapy students.2021; 39(4): 354–369.\n\nGaigbe-Togbe V, et al.: World Population Prospects 2022; United Nations. New York, NY, USA; 2022.\n\nJackson SE, Hackett RA, Steptoe A: Associations between age discrimination and health and wellbeing: cross-sectional and prospective analysis of the English Longitudinal Study of Ageing. Lancet Public Health. 2019; 4(4): e200–e208.\n\nJackson SE, et al.: Association of Perceived Discrimination With Emotional Well-being in Older Adults With Visual Impairment. JAMA Ophthalmol. 2019; 137(7): 825–832. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBanks J, et al.: English longitudinal study of ageing: waves 0-9, 1998-2019.2022.\n\nSteptoe A, et al.: Cohort Profile: The English Longitudinal Study of Ageing. Int. J. Epidemiol. 2012; 42(6): 1640–1648. PubMed Abstract \| Publisher Full Text\n\nSneeuw KC, Sprangers MA, Aaronson NK: The role of health care providers and significant others in evaluating the quality of life of patients with chronic disease. J. Clin. Epidemiol. 2002; 55(11): 1130–1143. PubMed Abstract \| Publisher Full Text\n\nFisher GG, Ryan LH: Overview of the Health and Retirement Study and Introduction to the Special Issue. Work Aging Retire. 2017; 4(1): 1–9. PubMed Abstract \| Publisher Full Text\n\nRadler BT: The Midlife in the United States (MIDUS) Series: A National Longitudinal Study of Health and Well-being. Open Health Data. 2014; 2(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRadloff LS: The CES-D Scale:A Self-Report Depression Scale for Research in the General Population.1977; 1(3): 385–401.\n\nHosmer DW, et al.: A comparison of goodness-of-fit tests for the logistic regression model. Stat. Med. 1997; 16(9): 965–980. Publisher Full Text\n\nAlbrecht GL, Devlieger PJ: The disability paradox: high quality of life against all odds. Soc. Sci. Med. 1999; 48(8): 977–988. PubMed Abstract \| Publisher Full Text\n\nWindle G: What is resilience? A review and concept analysis. Rev. Clin. Gerontol. 2011; 21(2): 152–169. Publisher Full Text\n\nGill TM, Robison JT, Tinetti ME: Difficulty and dependence: two components of the disability continuum among community-living older persons. Ann. Intern. Med. 1998; 128(2): 96–101. PubMed Abstract \| Publisher Full Text\n\nPhelan EA, et al.: Activities of Daily Living Function and Disability in Older Adults in a Randomized Trial of the Health Enhancement Program. J. Gerontol. 2004; 59(8): M838–M843. Publisher Full Text\n\nHopkins RO, et al.: Instrumental Activities of Daily Living after Critical Illness: A Systematic Review. Ann. Am. Thorac. Soc. 2017; 14(8): 1332–1343. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nResearch, N.S., U.C. London, and I.f.F. Studies: English Longitudinal Study of Ageing. UK Data Service; 2023." }	[ { "id": "200102", "date": "19 Sep 2023", "name": "Robert Applebaum", "expertise": [ "Reviewer Expertise Research methods", "social policy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall this is a well done article. The writing and analysis are solid. I do offer the following comments on the paper:\nThe ADL and IADL results that show a higher proportion of the sample with IADL limitations is very unusual. I have analyzed ADL and IADL results in many studies and have never seen this result. This is doubly surprising in that the study used a higher number of IADL items than is typically used. I would encourage the authors to examine this result carefully as I believe something is not correct, possibly in how items were coded. This could also be impacting the major study finding.\n\nThe results on discrimination are confusing. The overall study reports that 60% of respondents report age discrimination, but then the study says of those with ADL 29% report discrimination and with no disability it is 23.5%. If a different measure is being used for this comparison the authors should explain. Right now these results are very confusing.\n\nIn a related note mentioned in point 1, I would suggest that the ADL and IADL disability measures be examined separately when looking at discrimination.\n\nThe limitations section is good, but it does not address the problem that half of the sample had missing data. The authors should examine the characteristics of the analytic sample compared to the overall study sample.\n\nThe authors should make a bigger deal out of the fact that 60% of respondents have reported age discrimination. This is a really big finding, that is glossed over. The discussion section should talk about this really important finding much more extensively.\nOverall this paper can make a nice contribution to the literature.\n\nIs the work 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": "278317", "date": "06 Jun 2024", "name": "Ayelet Dunsky", "expertise": [ "Reviewer Expertise Quality of life among elderly" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 commendably well-written, making it accessible and understandable. However, I have several comments. One of the variables on which the study is based is the level of disability based on an unfamiliar scale of dependence based on a list of tasks that represent (according to the authors) ADL, and IADL. Based on previous literature, there is a difference between the inability to perform one of the IADL tasks and the inability to perform the ADL tasks, which is considered a more severe level of dependency. Thus, in my opinion, it is not correct to unite the ADL indices with the IADL indices, and the division into three groups, as the researchers chose to do, is not accurate for defining disability (the bivariate analyses were conducted, segregating the sample into three distinct groups: the first with a single ADL/IADL difficulty, the second with two such difficulties, and the third presenting three or more difficulties). Following my previous comment, the discussion about the lack of statistical significance regarding the association between discrimination experiences and detrimental changes in quality-of-life scores among older persons having ADL/IADL difficulties seems less relevant.\nIn addition, a dramatic finding in the data analysis is that 61% of the participants experienced age discrimination. I think this finding should be discussed in detail at the beginning of the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1023
https://f1000research.com/articles/12-88/v1	23 Jan 23	{ "type": "Policy Brief", "title": "The road to success: drawing parallels between 'road' and 'research data' infrastructures to foster understanding between service providers, funders and policymakers", "authors": [ "Rob W.W. Hooft", "Corinne S. Martin", "Corinne S. Martin" ], "abstract": "Background: The work of data research infrastructure operators is poorly understood, yet the services they provide are used by millions of scientists across the planet. Policy and implications: As the data services and the underlying infrastructure are typically funded through the public purse, it is essential that policymakers, research funders, experts reviewing funding proposals, and possibly even end-users are equipped with a good understanding of the daily tasks of service providers. Recommendations: We suggest drawing parallels between research data infrastructure and road infrastructure. To trigger the imagination and foster understanding, this policy brief contains a table of corresponding aspects of the two classes of infrastructure. Conclusions: Just as economists and specialist evaluators are typically brought in to inform policies and funding decisions for road infrastructure, we encourage this to also be done for research infrastructures", "keywords": [ "Research Data", "Research Infrastructure", "Infrastructure Funding", "Sustainability" ], "content": "Introduction\n\nData-intensive research depends on data and data services, such as databases, software and tools, and standards. These are often made available to end-users through research infrastructure. Such a research infrastructure for biological data and bioinformatics service in Europe is ELIXIR.1\n\nAs is common for many types of infrastructure (especially those that are free at the point of use), the existence of research infrastructure, like the services provided by ELIXIR, is often taken for granted by their users. Their importance is only noticed when they are (temporarily) unavailable, or worse, could disappear due to discontinued funding.2,3 In the Kano model, infrastructure services are must-be qualities: their proper functioning does not make the users happy, but service disruptions are strong dissatisfiers.\n\nAs research infrastructures and their services are typically funded through the public purse, it is essential that policy makers, research funders, experts reviewing funding proposals, and possibly even end-users are equipped with a good understanding of the daily tasks of service providers. We have noticed that this is not often the case, and this becomes an issue when this lack of understanding affects the funding of research infrastructures. To foster better understanding, this policy brief provides a comparison table of features of data infrastructure and their relatable counterparts in road infrastructure. We believe that using this approach can help, first because the change in mind frame makes it possible to see consequences of certain choices more clearly, especially those linked to funding of research infrastructures. Second, many people, even those working in research, are much more accustomed to road infrastructure setup and road infrastructure disruptions than to research infrastructure setup and research infrastructure disruptions; this increased familiarity increases the chances that consequences of policy decisions are foreseen.\n\nFor the actual feature comparison, see Table 1.\n\n\nPolicy outcomes and implications\n\nThe comparison between research data infrastructure and road infrastructure has many hooks to support productive discussions, and decisions, on research infrastructure funding and sustainability governance. Given that we, the co-authors, all work in research infrastructures, there is an inherent bias in the approach presented, just like when Sutherland et al.4 published their “20 things politicians need to know about science”. Yet, we also do not intend to be counterproductive: policymaking is complex and multifaceted, as astutely explained in “20 things scientists need to know about policy”. The comparison table is simply our contribution to fostering longer-term sustainability of infrastructures that already exist, that are widely used across the world, and that have typically received significant public financing over the years.\n\nFurthermore, the comparison table is likely to support efforts of both research infrastructure operators and policymakers in more accurately conveying to taxpayers the public value of research infrastructures, in addition to their role as enablers of scientific discovery and applications of societal benefit. The word ‘enablers’ is perhaps the most important message to convey: just like a road enables travel (and a research infrastructure enables research), it is questionable whether it is right to ask the road construction/maintenance company (and the research infrastructure operator) whether the road (and the research infrastructure) bring(s) value to society. Economists and evaluation specialists are very well placed and qualified (and likely unbiased) to answer complex questions around the public value of financing roads and research infrastructures.5,6\n\n\nActionable recommendations\n\nRecommendation 1: When formulating opinions and decisions on research data infrastructure funding and sustainability governance, compare them with that of road infrastructure. The change of frame may bring new insights.\n\nRecommendation 2: Consider informing policies and funding decisions relating to existing and future research infrastructure with support from economists and specialist evaluators.\n\n\nConclusion\n\nWe welcome any additional rows for the comparison as well as discussion on improving the existing table in the original Google Document. We have found it difficult to broaden the set of comparisons to also include a sustainable travel angle (e.g. examples covering public transport versus private car travel). Considering the climate emergency, this would be a useful and still relatable expansion of the approach.", "appendix": "Data availability\n\nNo data are associated with this article\n\n\nReferences\n\nHarrow J, Drysdale R, Smith A, et al.: ELIXIR: providing a sustainable infrastructure for life science data at European scale. Bioinformatics. 15 August 2021; 37(16): 2506–2511. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerali Z, Giles J: Databases in peril. Nature. 2005; 435: 1010–1011. Publisher Full Text\n\nE.g. Figure 6 in Drysdale R, Cook CE, Petryszak R, et al.: The ELIXIR Core Data Resources: fundamental infrastructure for the life sciences. Bioinformatics. 15 April 2020; 36(8): 2636–2642. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSutherland W, Spiegelhalter D, Burgman M: Policy: Twenty tips for interpreting scientific claims. Nature. 2013; 503: 335–337. PubMed Abstract \| Publisher Full Text\n\nMartin CS, Repo S, Arenas Márquez J, et al.: Demonstrating public value to funders and other stakeholders—the journey of ELIXIR, a virtual and distributed research infrastructure for life science data. Ann. Public Coop. Econ. 2021; 92(3): 497–510. Publisher Full Text\n\nVignetti S, Pezzati L, Striova J, et al.: E-RIHS PP D6.1 Cost Benefit Analysis and socioeconomic impact assessment. Zenodo. 2020. Publisher Full Text" }	[ { "id": "164512", "date": "28 Mar 2023", "name": "Frédéric Sgard", "expertise": [ "Reviewer Expertise Science 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 policy brief addresses a very important element of research policy, which is the understanding by decision-makers of the role, relevance, and impact of research infrastructures. In this particular case, of research data infrastructure, by using a comparison with a much well known type of infrastructures: roads. This comparison with a much more mundane example (roads) is aptly supported by a strong argument regarding the usefulness of the infrastructure mostly appears when its activity is disrupted, as normal operation is often taken for granted and thus invisible. The comparison between data research infrastructures and roads is clearly and convincingly argumented. This brief is also timely as funders are increasingly stretched to provide support to existing and new research infrastructures and have to make selective choices in their investments.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Yes\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Yes\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "9981", "date": "29 Nov 2023", "name": "Rob Hooft", "role": "Author Response", "response": "We are very grateful for your kind words on the usefulness of this policy brief, especially to funders of research infrastructures. We hope that version 2 has strengthened the brief." } ] }, { "id": "161289", "date": "13 Apr 2023", "name": "Silvia Vignetti", "expertise": [ "Reviewer Expertise Public policy analysis and evaluation", "infrastructure appraisal", "economic evaluation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 sheds light on the important and often poorly understood world of research infrastructures providing data and other research services to scientific communities around the world to perform excellent science. It does so by showing the features a research infrastructure has with a road infrastructure, with the aim of making clear why and how public funding must be understood and justified.\n\nThe article is well-written and to some extent illuminating. The parallel with road infrastructures is fitting quite well with the purpose of the authors. The parallel may go even further in depicting the parallels with other infrastructures delivering public services such as water supply, sewage system and so on. The article however does not fully exploit the potential of this parallel and remains a bit open and generic in the explanations.\nFirst, the table, which is pretty much the core of the article, presents some elements that should be better qualified, explained and perhaps even reconsidered in a few cases (e.g. \"data service\" corresponds perhaps more to 'transport service' than to \"road\"; it is not clear why \"research project\" corresponds to \"car\" and \"project\" to \"trip, journey\"; \"oversubscribed service\" may be 'crowded road' more than \"traffic jam\"), so it would be better to add a short explanation or a narrative section where the content of the table is described, justified and put in context.\nSecond, the policy options and implications do not come immediately from the description of the main common features, especially for those who are not familiar with both research and more traditional infrastructures. Perhaps the table could be split in two to distinguish the key features (first half) from the policy implications on their funding and public justification (second half more or less).\n\nOverall, the article is very interesting and well-conceived, but it would benefit from a small additional effort to better explain the main claim and its implications.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Yes\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Partly\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "9982", "date": "29 Nov 2023", "name": "Rob Hooft", "role": "Author Response", "response": "We thank you very much for this accurate and positive description of our work. You make a very good suggestion relating to expanding the analogies to other public service infrastructures. Along this line and during the writing process, we had actually been tempted to introduce a climate change element to some of the analogies, e.g. how mass transit (train) may not satisfy all the specific transportation needs of all users, just like an international database might not either, yet these are far more cost-effective and climate-friendly than the proliferation of 100s of very specific databases and 1,000s of jeeps on the roads. We have added your suggestions at the end of the article. We agree with your suggestions on the table. We have split the table as you suggested in version 2 of the article, and have added relevant text in the [now two] tables, which are more readily understandable as a result. We have also invited an additional co-author (Elaine Harrison), with science communications specialism, to reformulate and strengthen the text, in line with the your comments." } ] } ]	1	https://f1000research.com/articles/12-88
https://f1000research.com/articles/12-1021/v1	22 Aug 23	{ "type": "Research Article", "title": "Effects of white matter hyperintensities on isolated executive function assessed by the Trail Making Test", "authors": [ "Tomiko Nagayama", "Shuhei Yamaguchi", "Takuya Nakayama", "Sunghoon Yang", "Seiichi Inagaki", "Masao Nagayama", "Shuhei Yamaguchi", "Takuya Nakayama", "Sunghoon Yang", "Seiichi Inagaki", "Masao Nagayama" ], "abstract": "Background: White matter hyperintensities (WMHs) on MRI are associated with cognitive dysfunction, particularly slow processing speed and executive dysfunction. However, it is not clear whether WMHs burden affects isolated executive function independent of aging when WMHs are assessed separately in periventricular hyperintensity (PVH) and deep and subcortical white matter hyperintensity (DSWMH). Purpose: To assess the relationship between the degree of WMHs and the performance on the Trail Making Test (TMT), which can evaluate isolated ability of set-shifting and working memory. Methods: 74 participants who visited our memory clinic and underwent the TMT subtests (TMT-A and TMT-B) and the Mini-Mental State Examination (MMSE). All subjects performed the TMT within the time limits and their MMSE scores were 24 or higher, and they were diagnosed as having normal cognition or mild cognitive impairment. The extent of PVH and DSWMH was graded from 0 to 3 using the Fazekas scale. We obtained testing time to complete the TMT-A and TMT-B, and calculated TMT-B minus TMT-A. We performed correlation analyses between the degree of WMHs and the time measures of the TMT subtests with adjustment of age. Results: Average scores of the MMSE were not different among the groups either by PVH grade or by DSWMH grade. In contrast, average time required for the TMT-A, TMT-B, and TMT-B minus TMT-A increased along with exacerbation of PVH and DSWMH grade. After the adjustment of age we found significant association between only DSWMH grade and the time difference of TMT-B minus TMT-A. Conclusions: Exacerbation of PVH and DSWMH differentially affected isolated executive functions assessed by the TMT subtests independent of age and general cognitive function.", "keywords": [ "cerebral white matter hyperintensities", "brain MRI", "Trail Making Test", "cognitive function", "age adjustment" ], "content": "Introduction\n\nCerebral white matter hyperintensities (WMHs) appear as hyperintense areas on T2-weighted magnetic resonance image (MRI) and increase with aging.1,2 While WMHs are often seen in healthy elderlies, they are commonly found in patients with brain diseases such as stroke and dementia. The spread of WMHs is associated with progression of cognitive impairment,3,4 although the significance of WMHs on different cognitive domains still remains under discussion. Because the forms of WMHs are variable, WMHs are divided into two broad categories depending on the difference in their locations, one is periventricular hyperintensity (PVH) and the other is deep subcortical white matter hyperintensity (DSWMH). The functional significance of these distinct lesions on cognitive functions are also unclear.\n\nRecent studies have revealed that WMHs predominantly affect attention5 and executive function6 rather than general cognitive function or memory function. The Trail Making Test (TMT) was developed as a part of the army individual test battery,7 and has been used for the assessment of visual scanning, spatial attention, psychomotor speed, and executive function. The TMT consists of two parts (TMT-A and TMT-B). The time required to complete each part of the TMT and the time difference (TMT-B minus TMT-A) are used for quantitative measures, which increase with age. In clinical practice the TMT is used as an indicator of neuropsychological impairment associated with various diseases of central nervous system. However, there has been controversy regarding the relationship between distribution and severity of WMHs and measures of the TMT performance.\n\nIn this study we investigated the relationship between the degree of PVH and DSWMH and the performance on the TMT and the Mini-Mental State Examination (MMSE) in a clinical population with mild cognitive impairment (MCI) or without cognitive impairment.\n\n\nMethods\n\nA total of 74 subjects (55 women), who visited the department of neurology in the Atami hospital in Shizuoka with a chief complaint of forgetfulness or memory disturbance between July 2015 and August 2022, participated in this study. All participants agreed to perform brain MRI scans and cognitive function tests including the MMSE and TMT. The inclusion criterion for the study was that they performed the TMT within the time limits (i.e. 180 sec for the TMT-A and 300 sec for the TMT-B) and their MMSE score was 24 points or higher. Their clinical diagnoses were as follows: 35 subjects without objective cognitive impairment and 39 subjects with MCI. MCI was diagnosed according to the criteria proposed by Petersen.8 The age of the patients ranged from 51 to 89 years old, mean 75.5±6.5 years old.\n\nPatient data were de-identified, and therefore written informed consent was not required. Moreover, information on this study, including the purpose of the study, was published on our hospital’s homepage to guarantee subjects the opportunity to refuse to participate in the study. The study was approved, and this consent protocol was reviewed and the need for written and informed consent was waived by the International University of Health and Welfare Atami Hospital’s Ethics Committee, Shizuoka, Japan (Authorization No.21-A-191).\n\nWe used a 1.5T-MRI scanner (Excelart MRI, Canon, Japan) from July 2015 to July 2018, and a 3T-MRI scanner (Vantage Galan MRI, Canon, Japan) after August 2018. T1-weighted image, T2-weighted image, fluid-attenuated inversion recovery (FLAIR) image, and T2*-weighted image were obtained. For WMHs evaluation we used FLAIR images.\n\nWe divided WMHs into two categories: PVH that is symmetrical and adjacent to the lateral ventricles, and DSWMHs that is asymmetrical and apart from the ventricles. According to the Fazekas classification scale,9 PVH was graded from 0 to III; grade 0: absent, grade I: “cap” or pencil-thin lining, grade II: smooth “halo”, and grade III: irregular PVH extending into the deep white matter. DSWMH was also graded from 0 to III; grade 0: absent, grade I: punctate foci (maximum size, <3 mm), grade II: beginning confluence of foci (the size was 3 mm or more), and grade III: large confluent areas. If the left and right cerebral hemispheres showed different grades, the higher one was adopted. The grade was determined based on the consensus of three different neurologists.\n\nThe TMT and MMSE were administered to all participants by the rehabilitation staffs. We used the Japanese version of the TMT and measured the time to complete the TMT-A and TMT-B separately. The Japan Society for Higher Brain Dysfunction advocated the criteria for clinical use of the TMT. The criteria for clinical judgment include the testing time required for completion of the TMT and the number of false responses, which were stratified according to age10 (Table 1). The time limit for completion was 180 s for the TMT-A and 300 s for the TMT-B according to the TMT manual. We judged the results using these criteria for the TMT performance as normal, borderline, or abnormal. We also calculated the time difference of TMT-B minus TMT-A as an index of isolated executive function (i.e. set-shifting and working memory).\n\nWe conducted a one-way analysis of variance test without and with age adjustment (ANOVA and ANCOVA) to determine significant differences of continuous variables between the groups with Bonferroni post-hoc comparisons. The chi-square tests were used to determine significant differences of the categorical frequencies between the groups, and also of each ratio of clinical judgement for the TMT-A and the TMT-B. All statistical analyses were performed using SPSS version 23 (IBM Corporation). A value of p < 0.05 was considered statistically significant.\n\nGender differences were not taken into account in our study design because the number of male patients was smaller than that of female patients, and so the subjects number for each grade of cerebral white matter lesions in the male patients group ranged from 2 to 10, which made the statistical analysis difficult. Also, the TMT is generally reported to have no gender difference.11,12 Furthermore, the standard criteria for clinical judgment which we used describe the testing time required for completion of the TMT only by age, but not by gender (Table 1). As a reference, the gender differences results were disclosed in the extended data of the repository.\n\n\nResults\n\nThe demographic data and the performance data in the neuropsychological tests for each grade of PVH and DSWMH are shown in Table 2. There was no subject who showed grade 0 of PVH or grade 0 of DSWMH. The mean age tended to increase as the grade of PVH and DSWMH increased (The DSWMH grade I vs. III groups: p=0.045). The prevalence of hypertension and dyslipidemia increased in the groups of DSWMH grade II and III compared to the grade I group (the grade I vs. II groups: p=0.003, grade I vs. III groups: p=0.027 for the prevalence of hypertension). The education periods were comparable among all groups for PVH and DSWMH.\n\nThe relationship between neuropsychological test scores and WMHs are shown in Table 3. There was no significant difference in general cognitive function by the MMSE among three groups for either PVH grade or DSWMH grade before and after age adjustment.\n\nAs the grade of PVH increased, the rate of “abnormal” clinical judgement increased for the TMT-A and the rate of “normal” decreased and the rate of “abnormal” increased for the TMT-B. However, there were no significant differences in the rate of three clinical judgements among three PVH groups. The testing time required for the TMT-A tended to increase as the PVH grade increased. The testing time required for the TMT-B also showed an increasing trend as the PVH grade increased. Post-hoc analysis showed that there were significant differences in the testing time required for the TMT-B between the PVH grade I and grade III groups without age adjustment (p=0.026). After age adjustment, the difference was not significant, but the testing time required for the TMT-B was marginally longer only for the PVH grade III group compared to the grade I group (p=0.07). The time difference of TMT-B minus TMT-A did not show significant difference among three PVH groups after age adjustment.\n\nRegarding the influence of DSWMH on the TMT subtests, the rate of “normal” clinical judgement for the TMT-B decreased and the rate of “abnormal” increased, but there were no significant differences in the rate of three clinical judgements among three DSWMH groups. The testing time required for the TMT-A was not different among the three DSWMH groups. The testing time required to complete the TMT-B showed an increasing trend as the grade of DSWMH increased. Post-hoc analysis showed significantly longer testing time required for the TMT-B between the DSWMH grade I and grade III groups (p=0.017) without age adjustment. After age adjustment, the testing time required for the TMT-B was marginally longer only for the DSWMH grade III group compared to the grade I group (p=0.079). The time difference of TMT-B minus TMT-A showed an increasing trend as the grade of DSWMH increased. Post-hoc analysis revealed that the time difference was significantly longer for the DSWMH grade II and III groups compared to the grade I group (p=0.037 and p=0.003, respectively) without age adjustment and this difference was still significant after age adjustment between the DSWMH group I and group III (p=0.011).\n\n\nDiscussion\n\nThe present study demonstrated that the severities of both PVH and DSWMH were related to poor performance in the TMT subjects, especially in the TMT-B compared to the TMT-A. Furthermore, isolated executive function assessed by the time difference of TMT-B minus TMT-A was selectively impaired by progression of DSWMH rather than PVH independent of age and general cognitive function.\n\nThe finding that the burden of PVH and DSWMH has strong impact on cognitive function in particular attentional and executive function is generally consistent with previous studies. Bolandzadeh et al.13 reviewed 14 reports that studied the relationship between WMHs and cognitive function. They reported inconsistent results regarding the distinct influence of PVH and DSWMH on processing speed/executive function. Six14–19 out of the 14 studies directly compared involvement of PVH and DSWMH in impairment of processing speed/executive function. Two studies15,16 among them reported stronger impact of PVH compared to DSWMH on processing speed/executive function, but one study14 showed an opposite finding. In addition, three studies17–19 found no clear difference in the effect of PVH and DSWMH on executive function. Moreover, one different study showed that executive function was more related to DSWMH rather than PVH.20 The exact reason for these conflicting results is not clear. The variability in methods for assessing processing speed/executive function and in clinical characteristics of studied sample might account for the discrepant results. In this study we focused on the distinctive effects of PVH and DSWMH upon the performance in the TMT subtests, which enabled to evaluate the contribution of each type of WMHs to executive function independent of processing speed.\n\nSeveral studies have revealed the associations of WMHs with isolated components of executive function estimated in the performance of two TMT subtests. Porcu et al.21 analyzed the effects of distributed WMHs burden on cognition assessed by the TMT subtests. Significant correlations were obtained between total WMHs burden and scores of the TMT subtests including TMT-B minus TMT-A. They demonstrated that total WMHs burden was correlated with the impairment of all three measures of the TMT subtest scores (i.e. TMT-A, TMT-B, and TMT-B minus TMT-A). The association was the strongest for the TMT-B, followed by TMT-B minus TMT-A, and the TMT-A in this order. Unfortunately, their correlation analysis did not include age as a covariate although age showed the highest correlation coefficient with WMHs burden and all measures of the TMT subtests. Han et al.22 also reported a significant correlation between total WMHs volume and the poor performance on TMT-B minus TMT-A and the TMT-A after adjusting age effect. In both reports, however, the distinctive effects of PVH and DSWMH on performance in the TMT subtests were not examined. MacPherson et al.23 reported that WMH burden at anterior thalamic radiation and uncinate fasciculus slowed the performance of the TMT-B, but this association was attenuated when controlling the processing speed by other neuropsychological measures. Thus, the current study is the first one that examined the individual influence of PVH and DSWMH on processing speed and executive function separately assessed by the TMT-A, TMT-B, and TMT-B minus TMT-A after adjusting age effect in a clinical population with MCI or without cognitive impairment.\n\nWhy does different type of WMHs selectively affect executive function independent of general cognitive function? Respino et al.24 demonstrated that disrupted structural connectivity of brain regions caused by WMHs was associated with poor performance in the TMT subtests. The TMT-A score was correlated with connectivity of the supramarginal gyrus, paracentral lobule, thalamus, and pallidum. The TMT-B score was correlated with connectivity of the supramarginal gyrus, pre- and post-central gyri, thalamus, and pallidum. The ratio of TMT-B/A representing isolated set-shifting ability was correlated with the anterior and posterior cingulate gyri, middle frontal cortex, and putamen. Thus, it is plausible that WMHs-related region-specific disrupted structural connectivity may contribute distinctively to behavioral measures of attentional set-shifting and processing speed assessed by the TMT subtests.\n\nOur study has several limitations. In addition to relatively small sample size, the participants were recruited from a single hospital and limited to the subjects who had normal cognitive function or MCI. It will be valuable if the effects of WMHs burden are studied in patient groups including dementia with degenerative or vascular type, which might reveal the relationship between impairment of executive function in dementia and pathological mechanism associated with WMHs. Furthermore, the severity of WMHs were grouped using the Fazekas grading system, but quantitative analysis of their distribution and severity would clarify further the relationship between WMHs and executive function.\n\nIn conclusion, exacerbation of PVH and DSWMH differentially affects processing speed and executive function, and DSWMH is more associated with isolated executive functions such as set-shifting and working memory. The TMT subtests may be useful for assessing impairment of isolated executive function in clinical subjects with WMHs.\n\n\nAuthor contributions\n\nTomiko Nagayama and Shuhei Yamaguchi contributed to the concept and design of the study. Tomiko Nagayama, Sunghoon Yang, and Masao Nagayama were involved in the evaluation of patients. Tomiko Nagayama, Shuhei Yamaguchi and Takuya Nakayama analyzed and interpreted the results. Seiichi Inagaki guided the statistical analyses. Tomiko Nagayama and Shuhei Yamaguchi wrote the manuscript. All authors read and approved the final manuscript.\n\n\nORCID iD\n\nTomiko Nagayama iD https://orcid.org/0000-0003-3430-8663\n\nShuhei Yamaguchi iD https://orcid.org/0000-0001-8363-6407\n\nSunghoon Yang iD https://orcid.org/0000-0003/1655/8060\n\nMasao Nagayama iD https://orcid.org/0000-0002-0332-2027", "appendix": "Date availability\n\nFigshare: Effects of white matter hyperintensities on isolated executive function assessed by the Trail Making Test; Underlying data, DOI: https://doi.org/10.6084/m9.figshare.23788503.v3. 25\n\nThis dataset contains the following underlying data:\n\nData of MMSE and TMT for PVH\n\nData of MMSE and TMT for DSWMH\n\nDemographic data of the participants for PVH\n\nDemographic data of the participants for DSWMH\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).\n\nFigshare: Effects of white matter hyperintensities on isolated executive function assessed by the Trail Making Test; Extended data, DOI: https://doi.org/10.6084/m9.figshare.23788539.v1. 26\n\nThis dataset contains the following underlying extended data:\n\nPVH and TMT in each gender\n\nDSWMH and TMT in each gender\n\nTables of TMT and WMH in each gender\n\nData are available under the terms of the Creative Commons Zero “No rights reserved“ data waiver (CC BY 4.0 Public domain dedication).\n\n\nReferences\n\nBreteler MM, van Swieten JC , Bots ML, et al.: Cerebral white matter lesions, vascular risk factors, and cognitive function in a population-based study: the Rotterdam Study. 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PubMed Abstract \| Publisher Full Text\n\nSmith EE, Salat DH, Jeng J, et al.: Correlations between MRI white matter lesion location and executive function and episodic memory. Neurology. 2011; 76(76): 1492–1499. Publisher Full Text\n\nDelano-Wood L, Abeles N, Sacco JM, et al.: Regional white matter pathology in mild cognitive impairment: differential influence of lesion type on neuropsychological functioning. Stroke. 2008; 39: 794–799. Publisher Full Text\n\nPorcu M, Cocco L, Cocozza S, et al.: The association between white matter hyperintensities, cognition and regional neural activity in healthy subjects. Eur. J. Neurosci. 2021; 54: 5427–5443. PubMed Abstract \| Publisher Full Text\n\nHan SH, Chung MS, Kim S, et al.: Can the trail making test black and white predict white matter hyperintensity on MRI? J. Clin. Neurosci. 2019; 64: 155–159. PubMed Abstract \| Publisher Full Text\n\nMacPherson SE, Cox SR, Dickie DA, et al.: Processing speed and the relationship between Trail Making Test-B performance, cortical thinning and white matter microstructure in older adults. Cortex. 2017; 95: 92–103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRespino M, Jaywant A, Kuceyeski A, et al.: The impact of white matter hyperintensities on the structural connectome in late-life depression: Relationship to executive functions. Neuroimage Clin. 2019; 23: 101852. Publisher Full Text\n\nNagayama M: Effects of white matter hyperintensities on isolated executive function assessed by the Trail Making Test; Underlying data. Dataset. figshare. 2023. Publisher Full Text\n\nNagayama M: Effects of white matter hyperintensities on isolated executive function assessed by the Trail Making Test; Extended data. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "234256", "date": "12 Jan 2024", "name": "Tomohide Adachi", "expertise": [ "Reviewer Expertise Neurologist", "cerebrovascular disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is important in confirming the relationship between cognitive function and cerebral white matter lesions, particularly between frontal lobe functions such as processing speed and executive functions and white matter lesions, divided into PVH and DSWMH. The fact that there was a relationship between the degree of lesions and cognitive decline in DSWMH may help clarify the functional aspects of DSWMH. However, as the authors have stated, the number of cases is small, and it isn't easy to accept these results immediately. Furthermore, the composition of the cases targeted is likely to be diverse. In the future, it is considered necessary to conduct research that increases the number of cases and organizes the background of the target group.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10894", "date": "13 Apr 2024", "name": "Tomiko Nagayama", "role": "Author Response", "response": "Dear Dr. Adachi T. We greatly appreciate all of your valuable comments regarding our manuscript (F1000Res139557), entitled “Effects of white matter hyperintensities on isolated executive function assessed by the Trail Making Test”. We agree with your comments. As the Reviewer pointed out, the number of participants is small. We would like to increase the number of participants and conduct further research. Also, we would like to organize the background of the target group (for example, analysis will be conducted separately into a group with normal cognitive function and a group with mild cognitive impairment.) Again, thank you very much for your valuable suggestions. Tomiko Nagayama, MD, PhD Department of Neurology International University of Health and Welfare Atami Hospital" } ] }, { "id": "212610", "date": "08 Feb 2024", "name": "Aiko Osawa", "expertise": [ "Reviewer Expertise Neuropsychology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting and important study that shows an association between working memory and cerebral white matter lesions in patients seen in a memory clinic.\nIn a future paper, we would like to develop this study in the following directions:\nThe sample size for each age group may be small and should be increased for further study.\n\nPatients seen at the memory clinic may be affected not only by white matter lesions, but also by Alzheimer's pathology and other degenerative diseases. Therefore, it would be desirable to conduct a similar analysis on data from healthy older adults 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": [ { "c_id": "11050", "date": "13 Apr 2024", "name": "Tomiko Nagayama", "role": "Author Response", "response": "Dear Dr. Aiko Osawa We greatly appreciate all of your valuable comments regarding our manuscript (F1000Res 139557), entitled “Effects of white matter hyperintensities on isolated executive function assessed by the Trail Making Test”. We agree with all of your comments. As you pointed out, the number of participants is small, and we would like to increase the number of participants and conduct further research. Also, older people’s brains appear to have mixed pathology. So, we would like to conduct an additional analysis on data from healthy older adults and compare the results. It might reveal the relationship between impairment of executive function in dementia and pathological mechanism associated with white matter hyperintensities. Again, thank you very much for your valuable suggestions. Sincerely yours, Tomiko Nagayama MD, PhD Department of Neurology International University of Health and Welfare Atami Hospital" } ] } ]	1	https://f1000research.com/articles/12-1021
https://f1000research.com/articles/12-1014/v1	21 Aug 23	{ "type": "Case Report", "title": "Case Report: Post herpes zoster comedones: A new entity under Wolf’s isotopic response", "authors": [ "Kunal Gupta", "Arshiya Khan", "Yash Kashikar", "Sugat Jawade", "Bhushan Madke", "Arshiya Khan", "Yash Kashikar", "Sugat Jawade", "Bhushan Madke" ], "abstract": "The emergence of a completely new skin condition at the precise location of a different, unrelated and previously healed skin disease is referred to as a \"Wolf's isotopic response\". It is also referred as post-herpetic isotopic response because it typically manifests after contracting the varicella-zoster virus causing herpes zoster. Wolf’s isotopic response is a rare, unique and widely accepted phenomenon. In this study, we discuss the various theories proposed behind its etiology. Further studies can help us understand the pathomechanism behind the localization of skin diseases thereby, helping us manage the condition effectively. Here, we describe a case of a 36-year-old male who presented in the outpatient department with multiple open and closed comedones distributed along the site of T2 dermatome previously infected by herpes zoster infection. Dermoscopic examination revealed multiple, round, brown-colored follicular keratinous plugs with peri-lesional erythema. Histopathological analysis showed a large dilated follicular canal containing orthokeratotic stratum corneum consistent with closed comedone formation. The patient was diagnosed with post-herpetic zosteriform comedones and was prescribed oral isotretinoin 20 mg, topical tretinoin 0.05% cream with a moisturizer and asked to follow up regularly. The lesions gradually healed over a five-month period. In our case, timely medical intervention helped preventing the further progression of the disease. However, further studies involving large sample sizes can help us identify the underlying mechanism behind this phenomenon.", "keywords": [ "Wolf’s isotopic response", "herpes zoster", "post herpetic isotopic response", "Ruocco’s immunocompromised district", "comedone" ], "content": "Introduction\n\nThe term Wolf’s isotopic response is described as the occurrence of a new skin disorder at the exact site of another, unrelated and healed skin disease. It is usually noticed after herpes zoster infection caused by varicella-zoster virus; hence, it is also known as post-herpetic isotopic response.1 This is a rare and unique phenomenon as much still needs to be understood about the compromise of regional immunocompetence post herpes infected dermatomes making them susceptible to future dermatoses. Herpes zoster is a condition that causes painful, grouped vesicular eruptions, which are unilateral and only affect a dermatome innervated by a single sensory ganglion. This is caused by reactivation of varicella-zoster virus present in dormant form within the sensory ganglia.2 The most widely accepted hypothesis at play behind Wolf’s isotopic response is that of neuro-immune destabilization.3 Langerhans’ cells, a key mediator in neuro-immune balance are decreased in post-herpetic lesions. Sensory nerve fibers not only conduct sensorial stimuli but also modulate the dermal immune response by secreting neuromodulators such as substance P, vasoactive intestinal peptide and calcitonin gene-related peptide which interact with membrane receptors of immune cells.3 Thus, post herpetic viral damage to these sensory nerve fibers alters the neuro-immune homeostasis making the site involved susceptible to other dermatoses.\n\n\nCase report\n\nA 36-year-old male presented to the Dermatology Outpatient Department of the Datta Meghe Institute of Higher Education and Research affiliated tertiary care teaching hospital at Sawangi, Wardha, Maharashtra on 28th March 2023 with complaints of multiple pigmented papular eruptions extending from the midline of the chest to the right upper limb and right side of his back, for the last three months [Figures 1(A) and (B)]. The lesions were not associated with any burning, pain or itching sensation. He had no history of significant medical or surgical comorbidity. Past medical history revealed that he had an episode of herpes zoster involving the same region four months before for which he was treated with Valacyclovir 1 g three times a day for five days along with topical calamine lotion.\n\n(A) Multiple, grouped comedones on the right side of back. (B) Multiple, grouped comedones on the midline of chest.\n\nOn cutaneous examination, multiple, grouped open and closed comedones were noted on the midline of the chest, and the right side of the upper chest and back. The lesions were distributed in accordance with the thoracic T2 dermatome. Dermoscopic examination revealed multiple, round, brown-colored follicular keratinous plugs with peri-lesional erythema (Figure 2). A skin biopsy was performed, and histopathological analysis showed a large dilated follicular canal containing orthokeratotic stratum corneum consistent with closed comedone formation (Figure 3). Based on these findings, the patient was diagnosed with post-herpetic zosteriform comedones. The patient was prescribed oral isotretinoin 20 mg, topical tretinoin 0.05% cream with a moisturizer and asked to follow up regularly. The lesions gradually healed over a five-month period [Figure 4(A) and (B)].\n\nPolarized dermoscopic image showing multiple, round brown colored follicular keratinous plugs with peri-lesional erythema.\n\n(H&E, ×40)\n\nGradual resolution of lesions over five months. (A) Right side of back. (B) Midline of chest.\n\n\nDiscussion\n\nPost-herpetic isotopic response was first described in 1955 by a British neurologist, Wyburn Mason, who reported twenty-six patients having developed skin lesions at the locations of previously treated herpes simplex or herpes zoster infections.2 The majority of these secondary lesions were breast, squamous or basal cell carcinomas. Wolf et al. in 1985, described the first non-cancerous skin disorder as tinea corporis occurring at the site of previously healed herpes zoster infection in two patients and subsequently devised the term “isotopic response” in 1995 from the previously known “isoloci response”.2,4 At present, the most widely accepted term Wolf’s post-herpetic isotopic response is used when describing this phenomenon since herpes infection was reported to be the primary disease in the majority of the cases studied.3 The time elapsed between the primary and secondary diseases can be anywhere between a few days to several years.5 This phenomenon also falls under the umbrella term immunocompromised districts described by Ruocco et al. as a skin area more vulnerable than other sites as a result of either acquired (chronic lymphatic stasis, herpetic infections, UV radiations, burns, trauma, tattooing, intradermal vaccinations etc.) or genetic (primary lymphedema and skin mosaicism) etiology resulting in regional immune dysregulation. Failure in function of blood or lymphatic circulation, cytokines, immune-competent cells, neuropeptides or peripheral nerve fibers compromises the local immune homeostasis making the area susceptible to other infections, tumors or disorders of keratinization. Therefore, immunocompromised districts can develop even in an immunologically stable individual.6\n\nThere are multiple theories proposed behind the etiology of the Wolf’s post-herpetic isotopic response including viral, immunological, neural and vascular hypotheses.4 An interplay between these different factors leads to neuro-immune destabilization caused by viral damage to the sensory nerve fibers causing release of various neuropeptides and immune modulators that alter the local immune cells (macrophages, lymphocytes and Langerhans’ cells) making the involved site more susceptible to subsequent skin diseases.2,3 Substance P, a neuropeptide released from the damaged nerve endings post herpes infection can stimulate lipogenesis of the sebaceous glands causing increased activity of Propionibacterium acnes leading to the formation of comedones.7\n\nIn our case, timely medical treatment along with regular follow up and histopathological analysis enabled us to manage the disease effectively by preventing its further progression. Since this was a single case study, similar case series involving multiple patients will be required for further investigation to improve our understanding of this phenomenon. The primary takeaway lesson from our case is as follows: Since Wolf’s isotopic response is a rare clinical phenomenon, physicians need to be highly observant and take a comprehensive clinical history especially when encountering any dermatoses in a dermatomal distribution pattern in order to make the diagnosis and initiate treatment. Patients need to closely monitor the affected site on a regular basis to assess the development of any new lesions. Patients should be followed up regularly for effective and timely medical management.\n\n\nConclusions\n\nWolf’s isotopic response is a rare clinical phenomenon and much still needs to be learned about the underlying etiopathogenesis to help us understand the various factors responsible for the localization of skin diseases.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient in their vernacular language for voluntary participation. Confidentiality and privacy were ensured.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nWang B, Zheng J, Wang HW: Postherpetic Comedones in Two Chinese Han Patients. Chin. Med. J. 2017; 130(13): 1615–1616. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang T, Zhang M, Zhang Y, et al.: Wolf’s Isotopic Response after Herpes Zoster Infection: A Study of 24 New Cases and Literature Review. Acta Derm. Venereol. 2019; 99(11): 953–959. PubMed Abstract \| Publisher Full Text\n\nRuocco V, Ruocco E, Brunetti G, et al.: Wolf’s post-herpetic isotopic response: Infections, tumors, and immune disorders arising on the site of healed herpetic infection. Clin. Dermatol. 2014; 32(5): 561–568. PubMed Abstract \| Publisher Full Text\n\nWolf R, Brenner S, Ruocco V, et al.: Isotopic response. Int. J. Dermatol. 1995; 34(5): 341–348. Publisher Full Text\n\nYoon JH, Jang YJ, Park EJ, et al.: A Case of Herpes Zoster Granulomatous Dermatitis: Report of Wolf’s Isotopic Response. Ann. Dermatol. 2021; 33(2): 186–189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuocco V, Ruocco E, Piccolo V, et al.: The immunocompromised district in dermatology: A unifying pathogenic view of the regional immune dysregulation. Clin. Dermatol. 2014; 32(5): 569–576. PubMed Abstract \| Publisher Full Text\n\nSeo JK, Jeong KH, Shin MK: A Case of Post-Herpetic Nevoid Comedones. Ann. Dermatol. 2019; 31(Suppl): S36–S38. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "204056", "date": "09 Feb 2024", "name": "Ghazal Ahmed", "expertise": [ "Reviewer Expertise Dermatology", "venereology", "leprology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA well written article with all required details given.\nA point to ponder is that the discussion says that it is postulated that the neuro- immune dis-balance caused by viral damage to the sensory nerve endings might lead to secretion of substance-P which leads to lipogenesis causing comedone formation as a manifestation of isotopic phenomenon. If Substance P is secreted, it will also cause pain which is usually seen as post-herpetic neuralgia in post zoster cases and is quite common. Paradoxically, there was no pain associated in the reported case.\nThis brings us to a thought that there might be some other mechanism working in asymptomatic post-herpetic comedones. It would be great if the authors could share their take on this.\nAlso, it would be helpful if it is elaborated as to how common or uncommon, is this phenomenon of post-herpetic comedones and specifically to mention as to how many such cases has been reported till date.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1014
https://f1000research.com/articles/12-1013/v1	21 Aug 23	{ "type": "Research Article", "title": "An integrated RNA-proteomic landscape of drug induced senescence in a cancer cell line", "authors": [ "Maha Al-Roshdi", "Thomas Stevenson", "Franziska Görtler", "Sushma Nagaraja Grellscheid", "Maha Al-Roshdi", "Thomas Stevenson" ], "abstract": "Background: Senescent cells are characterized by an arrest in proliferation. In addition to replicative senescence resulting from telomere exhaustion, sub-lethal genotoxic stress resulting from DNA damage, oncogene activation or mitochondrial dysfunction also elicits a senescence phenotype. Methods: Senescence was induced in an osteocarcinoma cancer cell line in response to sub-lethal doses of a genotoxic chemotherapeutic agent, followed by quantitative SWATH proteomics and RNA-seq analyses. Results: We present here an integrative multi-omic analysis of proteomic and RNA-seq from proliferating and senescent osteosarcoma cells. Senescence is a controlled program affecting a wide variety of biological processes with some core hallmarks of senescence as well as cell type specific changes. Conclusions: This study presents an integrated analysis and makes available both RNA-seq and proteomic data from proliferating and senescent cells in appropriate FAIR data repositories to aid reuse by the community.", "keywords": [ "cell senescence", "RNA-seq", "quantitative proteomics", "integrative analysis", "U2OS" ], "content": "Introduction\n\nCellular senescence describes the irreversible loss of cells’ ability to proliferate. This phenomenon was first described by Hayflick and colleagues six decades ago when they showed that cultured human fibroblast cells have a limited capacity to proliferate.1 This durable cell cycle arrest is resistant to mitogenic stimuli and distinct from other hyporeplicative states such as quiescence or terminal differentiation. First considered an artefact of in vitro cell culture, senescence is now considered a fundamental cellular process associated with a broad range of developmental and pathological processes and an irrefutable hallmark of organismal ageing.2,3\n\nReplicative senescence is induced through telomere attrition; however, senescence may also be induced by several other intrinsic stressors such as oxidative stress, oncogene activation, or genomic instability.4 Senescence is also induced through extrinsic stimuli including viral infection, radiation, and chemotherapeutics.4,5 The onset of senescence is associated with a range of molecular and morphological traits including the expression of several senescence markers and a significantly enlarged and flattened appearance.6,7 The most commonly employed method to identify senescent cells is to stain for senescence-associated β-galactosidase activity (SA-β-gal). This assay exploits the unique pH of senescent cell lysosomes (pH 6.0), which can be detected using X-gal staining. Additional molecular characteristics include the formation of senescence-associated heterochromatin foci (SAHF) and the activation of p53 and p21, occurring at the onset of cell cycle arrest, which is subsequently maintained by the constitutive activation of p16.8,9\n\nCellular senescence is classically considered an anti-tumour mechanism, acting as a barrier to proliferation in the event of significant cellular damage.10 Although unable to divide, senescent cells remain metabolically active allowing them to participate in a range of physiological processes and secrete a range of potent inflammatory proteins known as the senescence-associated secretory phenotype (SASP).11 The SASP is thought to have evolved to aid in eliminating senescent/neoplastic populations through the recruitment of immune cells.3,12 However, as the number of senescent cells increases with age, the constant production of these inflammatory proteins promotes tissue dysfunction and contributes to a range of age-related diseases such as cardiovascular disease, diabetes, and cancer.11,13–15 Thus, senescence is highly pleiotropic and exists as part of a complex balance to maintain the function and health of cells, tissues, and organisms.\n\nSenescence and apoptosis are closely linked and together form the primary protective mechanisms to suppress tumorigenic events. Dysregulation in the apoptotic apparatus is well described during neoplastic transformation, and there is increasing evidence that pathways of senescence induction are also inhibited.16 Several chemotherapeutic drugs have been shown to induce senescence in cancer cells. An example is the topoisomerase inhibitor doxorubicin, used clinically to treat cancers of the blood, stomach, lungs, and ovaries (amongst others), which disrupts the re-ligation of DNA strands and leads to the activation of the DNA-damage response.17–19 Although inducing a non-proliferate state in tumour cells may appear to be a favourable outcome, it is likely to be a heterogeneous response with one population entering complete senescence whilst others continue to proliferate.16 Moreover, the induction of senescence and subsequent secretome produce a microenvironment conducive to tumourigenesis and/or disease relapse.16 It is also possible that this heterogeny gives rise to a more aggressive tumour population.\n\nUnderstanding the mechanisms through which cancer cells can escape proliferative arrest, which is otherwise expected in surrounding normal cells, is essential to understanding how malignant cells resist genotoxic drug therapy.16 Here we have integrated RNA-Seq and proteomic analyses to investigate the transcriptome of a model of chemotherapy-induced senescence in a common cancer cell line. We have induced senescence using doxorubicin, tracked the development of senescence and compared our data to existing data in commonly used models of cellular senescence.\n\n\nMethods\n\nWild type U2OS cells were a kind gift from Dr Nancy Kedersha, Harvard medical school, USA.20 Cells were maintained at 37°C, 5.0% (v/v) CO2, and 95% humidity and passaged when they reached ∼90% confluency. All cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% foetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin.20\n\nSenescence was induced via incubation in 200 nM doxorubicin for 48 hours. The media was then exchanged and the cells cultured for an additional 5–7 days. Senescent cells were identified via staining with senescence associated β-galactosidase (SA-β-Gal) staining solution (150 mM NaCl, 200 mM MgCl2, 40 mM citric acid, 12 mM sodium phosphate, 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide, adjusted to pH 6.4). Cells were fixed in 4% PFA prior to incubation with the staining solution overnight at 37°C. Cells were imaged using a bright field Evos XL Core Cell Imaging microscope.\n\nCells were washed in PBS and lysed in RIPA buffer (150 mM NaCl, 1 percent Nonidet P-40, 0.1 percent SDS, 0.1 percent sodium deoxycholate, 50 mM Tris (pH 7.4)) and centrifuged at 20,000×g for 20 min at 4°C. The protein concentration of the supernatant was measured using Pierce™ BCA assay kit (ThermoFisher Scientific, 23225). Preparation of peptide samples for proteomic analysis and mass spectrometry was performed by the proteomics facility at Durham University Biosciences as described before21 using a FASP Protein Digestion Kit (Expedeon #44250) and sequencing grade-modified trypsin (Promega, #V5111). Spin-filter eluates were de-salted using C18 ZipTips (Millipore) following freeze-drying and resuspension in 3 percent acetonitrile, 0.1 percent TFA. Each sample fraction analyzed contained 5μg peptides. Samples were loaded and washed on a TriArt C18 Capillary guard column 1/32”, 5μm, 5×0.5 mm trap column (YMC) and online chromatographic separation performed over 57 min on a Triart C18 Capillary column 1/32”, 12 nm, S-3 μm, 150 × 0.3 mm (YMC) at a flow rate of 5 μl/min with a linear gradient of 3â€“32 percent acetonitrile, 0.1 percent formic acid over 43 min, then to 80 percent acetonitrile, 0.1 percent formic acid over 2 min, held for 3 min before returning to 3 percent acetonitrile, 0.1 percent formic acid and re-equilibrated. Analysis was carried out on an Ekspert™ nanoLC 425 with low micro gradient flow module (Eksigent), coupled to a quadrupole Time-Of-Flight (QTOF) mass spectrometer (TripleTOF 6600, SCIEX, MA) with a DuoSpray source (SCIEX) and a 50-micron ESI electrode (Eksigent). SWATH acquisition was for 55 min with a 3.2 s cycle time. Each cycle consisted of MS-spectrum acquisition at 400 to 1,250 m/z for 250 msec followed by MS/MS (100 to 1500 m/z) using 100 variable SWATH windows (parameters downloaded from22), 25 msec accumulation for each in high sensitivity mode with rolling CE and 2+ ions selected. Samples were spiked with iRT peptides (Biognosys) at a ratio of 1μg protein to 0.1μl 10 × RT peptide mix. The data acquired from each scan cycle (400–1250 m/z) was processed using SCIEX version 1.7.1 software.23 Three biological replicates for each condition (young vs senescent) were prepared for analysis. For each biological replicate, three technical replicate LC_MS runs were undertaken. This resulted in nine total replicates for each condition.\n\nPeacView 2.224 was used to obtain raw counts of peptide distribution which were normalized on peak areas using MarkerView 1.2.25 To calculate the fold change between young vs. senescent cells, a t-test of the nine senescent cell samples against the nine young samples followed by a two-sample t-test for each experiment and per gene was carried out. This output contains fold changes as well as p-values. The p-adjusted values were then calculated using the Benjamini-Hochberg procedure in R (package stats (version 3.6.2), R version 4.1.226).\n\nStatistical testing for overrepresentation or enrichment of REACTOME terms was performed using the R package Reactome Pathway Analysis version 1.38.027 with the conditions pvalueCutoff = 0.05, pAdjustMethod = “BH”, qvalueCutoff = 0.2, minGSSize = 10 and maxGSSize = 500.\n\nCells were then fixed in 4% paraformaldehyde for 15 min at room temperature prior to permeabilisation in 0.5 pc Triton X-100 for 20 min. Cells were incubated in blocking buffer (3 pc BSA in PBS) for a minimum of 30 min. Primary (SAHF, 1:500 (Proteintech catalog number AB8898) and secondary antibodies (Cy3 conjugated anti-rabbit (Jackson Immunoresearch, catalog number 711-165-152) were diluted in blocking solution (3% BSA in PBS) and incubated with the cells for at least one hour. Cells were stained with DAPI staining solution for 15 min (40 ng/ml DAPI in PBS) and the coverslips were subsequently mounted with Vectashield mounting media (Vector Labs catalog number H-1900). Imaging was performed using a Zeiss 880 line scanning confocal microscope.\n\nTotal RNA was isolated from young and senescent U2OS cell cultured using Trizol reagent (Sigma, T9424). Cells were seeded at 3×105 per well in 6 well tissue culture plates and harvested in 300 μl Trizol after washing twice in PBS. Samples were incubated for 5 minutes at room temperature. 200 μl chloroform was the added to each sample, incubated for 5 minutes at room temperature and then centrifuged at 12,000×g for 15 minutes at 4 degrees Celsius. The upper aqueous phase was carefully removed. RNA was precipitated via the addition of 250 μl isopropanol and pelleted via centrifugation at 12,000×g for 10 minutes at 4 degrees Celsius. The pellet was washed twice in 70pc ethanol, air dried, and re-suspended in RNAse-free water. RNA yield and purity was determined using a nanodrop spectophotometer (ThermoScientific ND-1000). Library preparation and sequencing was performed at the DBS-Genomics sequencing facility, Durham University. Extracted RNA was further purified using DNAse TURBO and RNA purity and concentration was evaluated on TapeStation (Agilent Technologies). Library preparation was undertaken using 1 microgram purified RNA using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (E6310) followed by the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760). The indexes were also from NEB, NEBNext Multiplex Oligos for Illumina (Set 1, E7335). Libraries thus prepared were run on an Illumina NextSeq 4500 at 100 cycles 100bp paired end module. The quality of the raw data was controlled with FastQC 0.11.9,28 reads found to be sub-optimal were removed from the analysis pool, for example adapters and bases with an overall quality below 15 in a sliding window of size 4bp were trimmed with Trimmomatic 0.38.29 Annotation of the data to the human genome (GRCh38) was carried out using STAR 2.7.0f.30 Fold changes with corresponding p-adjusted values were calculated using DESeq2.31\n\n\nResults\n\nWT U2OS cells were harvested on Day 0 (when Doxorubicin was added), Day 2 (when doxorubicin was removed), Day 5, Day 7 and Day 9 prior to staining with SA-β-Gal solution. The blue colouration is indicative of a senescent phenotype with the intensity increasing with time after dox treatment. The first senescent cells were detected at Day 2 and the increased intensity of the stain indicated that most cells were senescent by Day 7 (see Figure 1a). At Day 7 the cells showed a significant increase in SA-β-Gal staining (fold change of four compared to day 5). The percentage of SA-β-Gal positive cells was analysed using ImageJ32 with version number 2.0.0-rc-69/1.52p. By Day 9 a decline of 1.25-fold in the SA-β-Gal positive cells was detected compared to Day 7.\n\n(a) SA-β-Gal positive cells were detected in WT U2OS cells. Cell senescence was induced using 200 mM doxorubicin. Cells were fixed and incubated with SA-β-Gal staining solution overnight at 37°C. Blue stains were detected in WT U2OS cells at Day 2 post-doxorubicin treatment. Gradual increase in the intensity of blue stain was observed. The blue stain indicates SA-β-Gal positive (senescent) cells. Scale bar = 100. (b) WT U2OS cells were treated with 200 mM doxorubicin for 48 hours. Cells were collected at Day 0, 2, 5, 7, 9, fixed and stained with SAHF antibody (red). WT U2OS cells showed no/few SAHF (less than 5 foci per cell) at Day 0, 2 and 5. On Day 7 and 9, they showed an increased number of SAHF. Scale bar = 20 um.\n\nWe employed a second method to assess the senescence induction protocol by measuring senescence-associated heterochromatin foci (SAHF). SAHF are formed when the chromatin in the nucleus of senescent cells undergoes remodeling by forming domains of heterochromatin.33 SAHF formation in U2OS cells was examined following the treatment routine with Doxorubicin. This was followed by immunofluorescence microscopy using a histone H3 (tri methyl K9) antibody that targets the nucleosome and provides a widely used read-out for SAHF. SAHF formation was monitored on Day 0, 2, 5, 7, and 9 post-doxorubicin treatment in Figure 1b. Cells that are forming five or more SAHF are counted and considered senescent. WT U2OS cells showed fewer than five structures on Day 0, 2 and 5. On days 7 and 9 the number of SAHF increased and were quantitatively analysed using ImageJ. At Day 7, cells showed a 3.75-fold change increase in the number of SAHF-forming cells of compared to Day 5 cell cultures and a further increase of 2.5-fold change at Day 9 compared to Day 7. Indeed, by Day 9, approximately 47% of U2OS cells were detected to form more than five SAHF foci per cell.\n\nA total of 5335 proteins were quantified in our measurements, these are listed in Table S1 in Ref. 34. Of these 2672 had a p-adjusted value below 0.05, and are visualized in Figure 2 as a volcano plot. Data points in grey represent genes that show less than two-fold differential expression, i.e. have a \|log2FC\|<1 and are therefore not significantly different between proliferating and senescent cells. Genes with a regulation between 1<\|log2FC\|<2 are significantly expressed and are depicted as small black dots. Highly differentially expressed genes (\|log2FC\|>2) are indicated by large black markers and are also labeled with their gene name. We found 211 up-regulated (log2FC>1) proteins of which 17 were highly up regulated (log2FC>2). Interestingly, only 91 proteins were downregulated (log2FC<−1), among these were six which were strongly downregulated (log2FC<−2). A list of the highly expressed proteins can be found in Supplementary Table S2 in Ref. 34.\n\n(a) Volcano-plot for all U2OS proteins with significant p-adjusted value (p-adjusted value >0.05). Proteins with a \|log2FC\|<1 are not significantly regulated and shown in grey; expressed proteins are significantly regulated (1 < \|log2FC\| < 2) and marked with small black points. Highly expressed proteins (\|log2FC\| > 2) are shown with big dots and are labeled with the gene name. (b and c) Identification of significant over-represented/underrepresented processes in senescence. Part a shows subsumption of Reactome terms of importance for the upregulated proteins (log2FC>1). Part c shows the significantly underrepresented processes in senescence (underlying proteins have a log2FC<−1).\n\nThe list of significantly enriched proteins (\|log2FC>1\|, p-adjusted value below 0.05) were investigated with Reactome35 to uncover functional pathways impacted during senescence. Figure 2b and c show the pathways, which significantly change in response to senescence induced with doxorubicin. Due to the large numbers of pathway hits, we organised the results such that the parent pathway is emphasised and child pathways that share an enriched parent pathway are ignored. Furthermore, two or more child pathways were merged together in a parent pathway when the parent pathway sufficiently describes the merged child pathways. A list of all pathways can be found in Ref. 34: Supplementary data S3 for the upregulated and S5 for the downregulated proteins. A shortened list where all child pathways are excluded when a parent pathway is enriched can be found in Ref. 34: Supplementary S5 (upregulated) and S6 (downregulated), and the list for the Figure 2b and c in Ref. 34: Supplement S7 and S8, respectively.\n\nAs seen in Figure 2b, several pathways are enriched in the group of genes upregulated in senescence, such as translation, immune system, extracellular matrix, and metabolism. The numbers of pathways in the downregulated group were more modest and surprisingly included cellular senescence itself. In addition, the downregulated group included apoptosis regulating genes. It has previously been reported that apoptotic pathways are suppressed in senescence.\n\nWe next sought to compare the list of senescence related genes from U2OS cells reported here with those previously reported from other cell types. Alvelar et al. developed a comprenhensive database of genes associated with cellular senescence called CellAge. This integrative database utilizes a systems biology approach to the analysis of senescence and has developed gene expression signatures for cellular senescence.36 We compared our most significantly changed proteins to the CellAge senescence gene expression list. These results are shown in Figure 3 and in Table S9 in Ref. 34.\n\nProteins that have the same direction of regulation (over/under-expressed) in the CellAge database as well as in our data are marked in blue. Proteins with the opposite regulation (i.e. overexpressed in CellAge database but under-expressed in this study) are marked in red. A list with all significant CellAge/HAGR genes in our data can be found in Ref. 34: Supplementary S9.\n\nGenes that have the same regulation pattern in our U2OS comparison as well as the CellAge list are marked in light blue, while genes that regulate differently (upregulated in one list and downregulated in the other one) are marked in red. Expressed genes (\|log2FC\| > 1) not included in the CellAge list are marked in black. For highly regulated genes (\|log2FC\|> 2) the gene name is added. As seen in Figure 3 the genes included in the Cell Age database, especially the highly regulated genes, act as expected. The genes that have a contrary regulation mostly have smaller fold changes. We also see several highly regulated genes in our data that are not included in the CellAge list, and are likely cell type specific senescence changes for U2OS cells.\n\nThe results from quantitative proteomics studies presented thus far were compared with those from RNA-Seq data generated under identical conditions using polyA selected mRNA. Following Illumina sequencing, quality control and further analyses including alignment, gene counts and differential expression, we retained only those genes with a mean count higher than 20 in the young and senescent conditions, fulfilled by 11835 genes. Figure 4 shows a comparison of the fold changes in RNA and protein measurements. In total, we had 4927 genes common between RNA and protein datasets. Grey dots depict genes that have a p-adjusted value above 0.05 and therefore do not have enough statistical power to be considered in the analysis. In this group there are 4122 genes. Genes with a p-value below 0.05 which change in both RNA and protein in the same direction (568 genes) are in black. Genes marked in red are statistically significant genes which are upregulated in senescence in RNA but downregulated in the protein measurements (74 genes). Genes marked in blue are regulated the opposite way, downregulated in senescence in RNA but upregulated in the protein measurements (163 genes). Genes with a \|log1FC\|>1 in RNA or in protein of the antiregulated genes are labeled in Figure 4 a with gene name. A list with antiregulated genes in either direction can be found in Ref. 34: Supplementary Table S10. Genes with a \|log1FC\|>1 in both conditions, RNA and protein, i.e. highly differentially regulated are few. In the red group, there is only one such gene, COMMD8, which is upregulated more than two-fold at the RNA level but downregulated more than two-fold in proteomics data. COMMD8 is a potential inhibitor of NF-KB signalling that controls senescence associated inflammatory signalling, hence this disparity in RNA versus proteomics data is interesting. In the blue group, there are nine genes that are highly upregulated at the protein level but more than two-fold downregulated in the RNASeq data. These are PHLPP1, ANLN, RACGAP1, KIF23, ITGB4, CCNB1, CAV1, WRAP53 and FAM83D with roles in insulin signalling, cell migration and cell growth.\n\n(a) All genes are included that have a corresponding protein measurement. Grey means that the p-adjusted value was higher than 0.05 in RNA or protein. Black genes behave consistently in RNA and protein measurements. Red: A gene is up-regulated in the RNA but down-regulated in the protein measurements. Blue: Genes which are up-regulated in protein but down-regulated in RNA. Red and blue genes that have a \|log2FC\|>1 in at least one condition are labeled. (b) Reactome pathways that are overexpressed in genes, which are up-regulated in proteins and down-regulated in RNASeq measurements. There are no enriched pathways for the genes that are down-regulated on protein and up-regulated on RNA level (red genes in a).\n\nA Reactome analysis for the two gene groups with anticorrelated behaviour in Figure 4 was also carried out. No enriched functional pathways were found for the red group in Figure 4a). A summary of the Reactome results for the opposite condition, i.e. genes where RNA levels are downregulated but protein levels are upregulated (blue group in Figure 4a) can be seen in Figure 4b. The pathways and underlying genes can be seen in Table S14 in Ref. 34.\n\n\nDiscussion\n\nCellular senescence is defined by irreversible growth arrest and profound changes in gene expression.2,37 Unsurprisingly, proteins involved in senescence and cell cycle progression are amongst the most highly up-regulated proteins in our data set. These include p21, a modulator of cell cycle progression and commonly employed marker of senescence, and RRM2B, a ribonucleotide reductase essential for DNA repair in non-proliferating cells38 (see Figure 2). In addition to the positive SA-β-Gal staining and identification of SAHF, the up-regulation of these proteins confirms the onset of cellular senescence in our model. Among the highly up-regulated pathways in the protein data (see Figure 2b), many are connected to SASP and cellular senescence. Furthermore, there are extracellular matrix alterations that are associated with cellular senescence.39 A further hallmark of cellular senescence is mitochondrial dysfunction which plays important roles not only in the senescence growth arrest but also in the development of SASP and resistance to cell death.40\n\nOur data also shows that proteins commonly associated with cancer, including prognostic indicators such as SERPINB5, are significantly up-regulated in DNA damage-induced senescence. Examples include WRAP53, which is known to be over-expressed in a variety of cancer cell lines of different origins and promotes cellular transformation.41–44 The proteins KRT17,45 KRT8,46 KRT20,47 LCP1,48 and VAMP849 are also reported to be associated with cancer development/metastasis and cellular proliferation. Functional pathway analysis revealed terms related to translation, inflammation, mitochondrial dysfunction and cell migration to be significantly up-regulated; all of which are typical hallmarks of cancer cells in addition to some being common with senescence. Although considered a bona fide supressor of neoplastic transformation, our data suggests that senescence builds a transcriptional/translational landscape that may promote malignancy. Interestingly, we observe a significant enrichment in proteins involved in rRNA processing which is in contrast to previous studies.50 Ribosome biogenesis and protein translation are finely coordinated and essential for cell growth, proliferation and differentiation. Multiple RP proteins have extra-ribosomal functions including activation of pathways in response to stress, resulting in cell cycle arrest and apoptosis. In cancers, these functions are often misregulated.51 Several studies have provided evidence for active keratin involvement in cancer cell invasion and metastasis. The keratinization pathway is here driven by the highly up-regulated KRT proteins.52 Tissue remodeling is promoted by MET tyrosine kinase receptor, which underlies developmental morphogenesis, wound repair, organ homeostasis and cancer metastasis.53 Several other upregulated pathways are related to cancer. Among these is the metabolism of amino acids and derivatives (GPT), which is here driven by the RPL and RPS gene group and the GTP group of our highly expressed genes.\n\nThe most significantly down-regulated proteins in our dataset include DHRS2, H1, and ENPP1. Decreased expression of DHRS2 contributes to p53 stabilization thus promoting the onset of senescence. In mice, Enpp1 has been shown to play a crucial role in regulating aging via Klotho expression and its down-regulation has been shown to be associated with aging.54 We were initially surprised to find the cellular senescence term to be significantly down-regulated in our data set (see Figure 2c). Among the genes in this pathway is MAP 2K6, which is involved in stress-induced cell cycle arrest, transcription activation and apoptosis.55 Another significant example is ERF, which is involved in development, apoptosis, and regulation of telomerase, a key regulator in age-related or replicative senescence. We believe this change may reflect a very late stage of senescence wherein the expression of pro-senescence proteins has reduced and which may represent an incomplete senescent phenotype.\n\nTo benchmark this study, we compared the proteomic results with known datasets on senescence through the Cell- Age database.36 Generally, our data is in agreement with that published by Alvelar et al. We believe that any discrepancies observed are likely to be due to differences in cell line and method of senescence-induction, suggesting that this is a useful resource that can support other studies on senescence in this model.\n\nA large number of pathway terms relating to translation and rRNA processing appeared amongst the proteins up-regulated in senescence in U2OS cells. This led us to compare proteomic data with RNA-Seq which highlights significant irregularities between the detected level of proteins and the expression of their corresponding genes suggesting altered mechanisms of translational regulation between proliferating and senescent cells. Interestingly, our data highlights that genes/proteins where RNA and protein levels are highly anti-regulated (Figure 4a) are typically associated with aging and senescence. Examples include PHLPP1, which protects against age-related intervertebral disc degeneration,56 and ANLN, the depletion of which induces cellular senescence.57 Expression of ITGB4 is reportedly down-regulated under oxidative stress or upon inflammatory stimulation leading to the induction of senescence in epithelial cells, mediated through p53 activity.58 CCNB1 silencing inhibits cell proliferation and promotes cell senescence via activation of the p53 signaling pathway in pancreatic cancer,59 CAV1, which has been shown to induce senescence in resting human diploid fibroblasts,60 and WRAP53 and FAM83D, which are commonly over-expressed in a variety of cancers and known to trigger apoptosis.41\n\nThe pathways driven by genes which are up-regulated at the protein level but down-regulated at the RNA level are varied and involve multiple cellular processes. An interesting pathway comprises genes associated with the assembly of the primary cilium, a sensory structure that interprets extracellular signals to stimulate a range of cellular pathways including growth, response to nutrient deprivation, and cellular development.61\n\nAdditional examples include proteins involved in a range of signal cascades and the stimulation of transmembrane receptors. Depending on the cellular context, this may impact cellular proliferation, differentiation and survival. Additional pathways following the same pattern including those related to autophagy, DNA repair and those related to cellular senescence. Cytokines (key components of SASP) and inflammatory mediators are an interesting example of proteins that play a significant role in the senescent phenotype but exhibit striking differences between mRNA and protein levels. These observations may suggest that mechanisms of translational regulation play a more significant role in the induction of the senescent phenotype than transcriptional regulation alone.\n\n\nConclusions\n\nWe have employed a cellular model of senescence in a cancer derived cell line to investigate broad transcriptomic and proteomic changes. By combining RNA-seq and proteomic datasets, our data demonstrate a dramatically altered translational landscape at both the protein and RNA level, whilst demonstrating that the model exhibits all the characteristics and markers of the senescent phenotype. The model can be easily implemented and utilised to study senescence in cancer cells in a wide range of contexts. Our data reveal a range of age and disease-relevant proteins and pathways that are altered in senescent cancer cells, such as the assembly of the primary cilium, and highlights the emerging role of lipids in the senescent phenotype. Our data are also suggestive of very significant regulatory changes in translation that warrant further investigation.\n\n\nAccession numbers\n\nENA.EMBL: RNA-Seq data for young and senescent U2OS cells. Accession number RJEB59999. https://identifiers.org/ena.embl: PRJEB59999\n\n\nAuthor contributions\n\nConceptualization by TS, MA-R, SNG. Data Curation was done by TS and FG. Funding Acquisition, Supervision and Project Administration was undertaken by SNG. Investigation was done by TS and MA-R. Methodology was developed by FG and SNG. Computational analysis was carried out by FG. Visualization was done by MA-R and FG. The original draft was written by by MA-R and FG and subsequent review & editing by TS and SNG.", "appendix": "Data availability\n\nZenodo: Underlying data for’An integrated RNA-proteomic landscape of drug induced senescence in a cancer cell line’, https://www.doi.org/10.5281/zenodo.7737499. 34\n\nThis project contains the following underlying data:\n\n\n\n• S0.csv contains information on the file content of the supplementary files.\n\n• S1.csv contains the protein data.\n\n• S2.csv contains the proteins with high absolute fold change listed.\n\n• S3.csv contains all upregulated Reactome pathways.\n\n• S4.csv contains all downregulated Reactome pathways.\n\n• S5.csv contains the upregulated Reactome pathways where the child pathways are deleted which have also an upregulated parent pathway.\n\n• S6.csv contains the downregulated Reactome pathways where the child pathways are deleted which have also a downregulated parent pathway.\n\n• S7.csv contains the upregulated Reactome pathways where subpathways are grouped together.\n\n• S8.csv contains the downregulated Reactome pathways where subpathways are grouped together.\n\n• S9.csv contains a list of the significant proteins and their HAGR regulation.\n\n• S10.csv is a list of genes which have an anticorrelated fold change in protein and RNA, belongs to figure 4 a.\n\n• S11.csv contains a list of all significant genes which are upregulated in RNA and downregulated in protein data or the other way round.\n\n• S12.csv contains a list of all all Reactome pathways of blue genes in Figure 3 a.\n\n• S13.csv contains a list of all all Reactome pathways of blue genes in Figure 3 a where the child pathways are deleted which have also an upregulated parent pathway.\n\n• S14.csv contains a list of all Reactome pathways of blue genes in Figure 3 a where subpathways are grouped together.\n\n• S15.csv contains a list of the fold changes in RNA data.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank Adrian Brown at Durham University, UK, proteomics service for assistance with proteomics. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Z, Jun F, Li N, et al.: Quantitative proteome analysis identifies MAP 2k6 as potential regulator of LIFR-induced radioresistance in nasopharyngeal carcinoma cells. Biochem. Biophys. Res. Commun. October 2018; 505(1): 274–281. Publisher Full Text\n\nZhang C, Joseph KM, Khan NM, et al.: PHLPP1 deficiency protects against age-related intervertebral disc degeneration. JOR SPINE. September 2022; 5: e1224. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMagnusson K, Gremel G, Rydén L, et al.: ANLN is a prognostic biomarker independent of ki-67 and essential for cell cycle progression in primary breast cancer. BMC Cancer. November 2016; 16(1): 904. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYuan L, Xizi D, Tang S, et al.: ITGB4 deficiency induces senescence of airway epithelial cells through p53 activation. FEBS J. February 2019; 286(6): 1191–1203. PubMed Abstract \| Publisher Full Text\n\nZhang H, Zhang X, Li X, et al.: Effect of CCNB1 silencing on cell cycle, senescence, and apoptosis through the p53 signaling pathway in pancreatic cancer. J. Cell. Physiol. August 2018; 234(1): 619–631. PubMed Abstract \| Publisher Full Text\n\nVolonte D, Galbiati F: Caveolin-1, a master regulator of cellular senescence. Cancer Metastasis Rev. April 2020; 39(2): 397–414. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSánchez I, Dynlacht BD: Cilium assembly and disassembly. Nat. Cell Biol. June 2016; 18(7): 711–717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStevenson T, Al-Roshdi M, Görtler F, et al.: An integrated RNA-proteomic landscape of drug induced senescence in a cancer cell line.March 2023. Publisher Full Text" }	[ { "id": "202373", "date": "23 Nov 2023", "name": "Ines Heiland", "expertise": [ "Reviewer Expertise Biochemistry", "mathematical modelling" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study presents a comparative analysis of proteomics and RNASeq data from induced senescence in U2OS cells. The authors find that some of the genes show anticorrellation of RNASeq and Proteomics and analyze pathway association of these proteins. Although they highlight some of these pathways and their role in senescence it remains a bit unclear what the meaning of their observations is. It has of course been previously observed that RNA and protein can appear to be counterregulated also for other processes. Maybe the authors could speculate a bit on why certain RNAs are increased while the corresponding proteins are decreased.\n\nIs the work 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": "280666", "date": "04 Jul 2024", "name": "Kotb Abdelmohsen", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study explores the senescence phenotype in osteosarcoma (U2OS) cells induced by the chemotherapeutic agent doxorubicin. By integrating RNA sequencing (RNA-seq) and quantitative proteomics (SWATH-MC), the authors provide a comprehensive overview of the molecular changes associated with drug-induced senescence.\nKey findings include:\nProteomic and Transcriptomic Changes: The study identified significant changes in the expression of numerous proteins and genes. Key pathways affected include those related to translation, immune response, extracellular matrix organization, and metabolism.\nFunctional Enrichment: The analysis highlighted pathways significantly enriched or depleted in senescent cells, providing insights into the molecular mechanisms underlying senescence.\nComparison with Other Datasets: The findings were compared with existing senescence-related datasets, revealing both commonalities and cell-type-specific differences.\nComments\nThe introduction provides a good overview but could benefit from a clearer statement of the research question and objectives. The methods section is detailed, but the protocol for RNA isolation and sequencing could include more specifics about quality control measures. The use of volcano plots and pathway enrichment analyses is effective. However, including more detailed legends and explanations for each figure would improve clarity. The statistical methods are appropriate, but the criteria for selecting significant changes (e.g., fold change thresholds) should be explicitly justified. While the comparison with the CellAge database is valuable, discussing discrepancies in more detail would strengthen the conclusions. The discussion could further explore the functional implications of the identified pathways and their relevance to cancer therapy. What specific criteria were used to define significant changes in gene and protein expression? How do the observed changes in proteomic profiles compare with those in other cancer cell lines treated with doxorubicin? The authors should provide more details on the quality control measures taken during RNA isolation and sequencing? How were the doxorubicin concentrations and treatment durations determined? Could the observed changes in the extracellular matrix-related proteins contribute to changes in cell adhesion and migration? How do the identified SASP factors in U2OS cells compare to those in primary senescent cells? What are the potential implications of the identified translation-related pathways for cancer therapy? Did the authors observe any changes in non-coding RNA expression, and if so, what might their roles be in senescence? How robust are the findings across biological replicates, and were any outliers observed? Can the authors discuss the significance of the observed downregulation in apoptosis-related pathways in senescent cells? How might the identified changes in mitochondrial function influence the overall metabolic state of senescent cells? Were there any unexpected findings that contrast with previous studies on senescence? How do the findings contribute to our understanding of senescence as both a tumor-suppressive and pro-tumorigenic mechanism? What are the limitations of using U2OS cells as a model for senescence, and how might this impact the study’s conclusions? The authors should validate the key findings from this study in other cell types or in vivo models? What are the potential therapeutic implications of targeting the pathways identified in this study? How do the findings align with current theories on the role of senescence in cancer progression and therapy resistance? Can the authors elaborate on the potential role of the identified keratinization pathway in cancer and senescence? What are the next steps for this research and how might it be expanded? How do the authors envision the integration of RNA-seq and proteomics data advancing our understanding of cellular senescence? It is essential to perform validations by RT-qPCR and Western blot analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1013
https://f1000research.com/articles/11-1439/v1	06 Dec 22	{ "type": "Case Report", "title": "Case Report: The first reported case of pulmonary alveolar proteinosis with myasthenia gravis in a 27-year-old patient", "authors": [ "Lenda Ben Hmida", "Islam Mejri", "Maroua Kacem", "Mariem Msalmani", "Hana Blibech", "Houda Snène", "Aida Ayadi", "Jamel Zaouali", "Zied Moatemri", "Islam Mejri", "Maroua Kacem", "Mariem Msalmani", "Hana Blibech", "Houda Snène", "Aida Ayadi", "Jamel Zaouali", "Zied Moatemri" ], "abstract": "Background: Pulmonary alveolar proteinosis is a very rare diffuse lung disease characterized by the accumulation of amorphous and periodic acid Schiff-positive lipoproteinaceous material in the alveolar spaces due to impaired surfactant clearance by alveolar macrophages. Three main types were identified: Autoimmune, secondary and congenital. Pulmonary alveolar proteinosis has been previously reported to be associated with several systemic auto-immune diseases. Accordingly, we present the first case report of pulmonary alveolar proteinosis associated with myasthenia gravis. Case: A 27-year-old female patient, ex-smoker, developed a dyspnea on exertion in 2020. The chest X-ray detected diffuse symmetric alveolar opacities. Pulmonary infection was ruled out, particularly COVID-19 infection. The chest scan revealed the “crazy paving” pattern. The bronchoalveolar lavage showed a rosy liquid with granular acellular eosinophilic material Periodic acid-Schiff positive. According to the lung biopsy results, she was diagnosed with pulmonary alveolar proteinosis. The granulocyte macrophage colony-stimulating factor autoantibodies were negative. Nine months later, she was diagnosed with bulbar seronegative myasthenia gravis, confirmed with the electroneuromyography with repetitive nerve stimulation showing significant amplitude decrement of the trapezius and spinal muscles. She was treated with pyridostigmine, oral corticosteroids and azathioprine. Given the worsening respiratory condition of the patient, a bilateral whole lung lavage was performed with a partial resolution of symptoms. Thus, this previously unreported association was treated successfully with rituximab, including improvement of dyspnea, diplopia and muscle fatigability at six months of follow-up. Conclusions: This case emphasizes on the possible association of auto-immune disease to PAP, which could worsen the disease course, as the specific treatment does not exist yet. Hence, further studies are needed to establish clear-cut guidelines for PAP management, particularly when associated to auto-immune diseases.", "keywords": [ "Pulmonary alveolar proteinosis", "Myasthenia gravis", "GM-CSF antibodies", "Bronchoalveolar lavage", "Whole lung lavage", "Rituximab" ], "content": "Introduction\n\nPulmonary alveolar proteinosis (PAP), also known as pulmonary alveolar phospholipoproteinosis, is a very rare chronic diffuse lung disease.1 It is characterized by the accumulation of amorphous and Periodic acid-Schiff (PAS)-positive lipoproteinaceous material in the alveolar spaces due to impaired surfactant clearance by alveolar macrophages.2 However, the underlying lung structure is preserved.2 The lipoproteinaceous material is principally composed of abnormal surfactant phospholipids and apoproteins.1 The accumulated substances filling the alveoli results in damaging gas exchange.1 The classic symptoms are dyspnea and hypoxemia, ultimately leading to respiratory failure and death.1 There are three main types of PAP: Autoimmune (previously named primary or idiopathic), secondary and congenital.1 PAP has been previously reported to be associated with several systemic auto-immune diseases.2 Accordingly, we present the first case report of PAP associated with myasthenia gravis (MG).\n\n\nCase report\n\nA 27-year-old, North African female patient, had a medical history of allergic rhinosinusitis and asthma. She is working as a web editor, mainly as a work from home employee. Her tobacco consumption amounted to one pack per day over two years. She had no particular exposure to toxics. On July 2020, she had an acute onset of respiratory symptoms, which consisted of productive cough, dyspnea on exertion and fever. She was, empirically, treated as a respiratory infection with antibiotics. One month later, she remained with a dyspnea occurring with effort. The physical examination and the laboratory findings were unremarkable. The chest X-ray detected diffuse symmetric alveolar opacities. Pulmonary infection was ruled out, particularly COVID-19 infection.\n\nThe chest scan revealed bilateral ground-glass peri-broncho-vascular opacities with interlobular and intralobular septal thickening, defining the “crazy paving” pattern (Figure 1). The bronchoalveolar lavage showed a rosy liquid with granular acellular eosinophilic material PAS positive. The lung biopsy confirmed the diagnosis of PAP showing alveoli filled with eosinophilic, acellular, granular and PAS-positive material containing foamy macrophages and some cholesterol crystals (Figure 2). At the time of diagnosis, the granulocyte macrophage colony-stimulating factor (GM-CSF) autoantibodies were not available. Lung function tests were normal, apart from a declined diffusing capacity of lung for carbon monoxide (<68% of the predicted value). Laboratory results, including quantitative immunoglobulins, proteins electrophoresis and autoantibody screening were normal, apart from an elevated c-antineutrophil cytoplasmic antibodies and anti-Mi-2 antibodies without clinical signification.\n\nThe yellow * indicates lipoproteinaceous material containing foamy macrophages. (a): low magnification (×4); (b): high magnification (×40). PAS, Periodic acid-Schiff.\n\nNine months after the onset, the patient presented with asthenia, muscle fatigability and right diplopia with worsening of symptoms later in the day. An electroneuromyography with repetitive nerve stimulation showed a significant amplitude decrement of the trapezius and spinal muscles. The patient, thus was diagnosed with bulbar MG. The acetylcholine receptor antibodies were negative. The patient was treated with pyridostigmine (60 mg pill, three times a day, for life), oral corticosteroids (prednisone 50 mg/day, once a day, for 5 weeks followed by progressive degression to 15 mg/day) and azathioprine (50 mg pill, twice a day, for 8 months). At that time, GM-CSF autoantibodies were negative.\n\nDuring the follow-up period, the patient’s respiratory condition worsened. She presented with oxygen desaturation at the level of 70% after a 6-min walk of 100 meters. Lung function tests degraded further, with a severe alteration of alveolocapillary diffusion (diffusing capacity of lung for carbon monoxide at 33% of the predicted value). After excluding the main differential diagnosis, the patient underwent a whole lung lavage (WLL). The left lung was washed with 10 liters of saline (Figure 3). Six weeks later, the right lung was washed with 20 liters (Figure 4). There was a partial resolution of symptoms. Following a multidisciplinary discussion, the patient was treated with rituximab (two intravenous injections of 1,000 mg, 14 days apart), after 6 weeks of azathioprine washout. An improvement of dyspnea, diplopia and muscle fatigability was noted at six months of follow-up.\n\n\nDiscussion\n\nPAP is an ultra-rare alveolar filling process with an estimated prevalence of 6.87 per million in the general population.1 PAP was first described by Rosen in 1958 and back to 2009, only 500 cases were reported in the literature.1,3 PAP occurs mainly in men with a sex ratio of two and a typical age of 40 to 50 years old in adults, which is contrasting with our case.2 About 50 to 80% of patients with PAP have a smoking history, as it was reported in our patient.2 The symptoms are non-specific, subacute and mild, resulting often in delaying the diagnosis by months, even by years.3 The positive diagnosis of PAP is suggested by the chest scan findings with the classically known “crazy paving” pattern.3 Bronchoalveolar lavage and the transbronchial biopsy with the characteristic features, establish the diagnosis of PAP.3 In the present case, PAP was confirmed by the pathological typical results of a lung surgical biopsy. Based on the pathogenic mechanism, PAP can be grouped into three types.1 Firstly, primary PAP as the most frequent form found in 95% of patients, is an autoimmune disease caused by elevated levels of the GM-CSF autoantibodies.1 In our patient, the GM-CSF antibodies were negative. However, their assessment was conducted under corticosteroids and azathioprine. The autoimmune hypothesis, though, was not definitely ruled out, especially when an associated auto-immune disease appeared during the course of PAP. Secondly, secondary PAP, occurring in 5% of patients, results from alveolar macrophage dysfunction caused by immune dysregulation, hematopoietic disorders, environmental exposure and pharmaceutical agents.1 In our case there was no other associated underlying illness or exposure, which eliminated a secondary PAP. Thirdly, congenital PAP occurs due to genetic variations, usually observed in children, which is not our case.1 In the current case, the onset of symptoms began after a respiratory infection that was very likely the initial trigger causing probably abnormal response in surfactant uptake.4 In the literature, there have been several cases of PAP associated with systemic auto-immune diseases; such as hemolytic anemia, polymyalgia rheumatica, ulcerative colitis, granulomatous polyangiitis, systemic lupus erythematosus and dermatomyositis.4,5 In this regard, our case is noteworthy as it demonstrates the first case in the literature of PAP and MG association.\n\nOur patient had a confirmed seronegative MG and was undergoing treatment with corticosteroids and an immunosuppressant. Whether the MG or its treatment has any bearing on the progression of PAP is questionable. Indeed, the worsening condition of the patient after MG treatment, suggested that the immunosuppressant therapy could be the exacerbating factor of PAP. This hypothesis is plausible, as it was reported in the literature. In fact, three cases of patients with collagen disease developing autoimmune PAP during the immunosuppressant therapy were reported.5 Nagasawa et al., outlined the development of autoimmune PAP, in a patient previously diagnosed with systemic lupus erythematosus, under glucocorticoid therapy and its worsening under immunosuppressive therapy.6 As for the treatment of PAP, WLL is the standard of care.1 However no randomized controlled trials have been reported on WLL due to the extreme rarity of PAP.1 New therapeutic strategies for PAP have emerged, including GM-CSF, rituximab and plasmapheresis.1 Rituximab is an anti-CD20, already used in several autoimmune diseases with a proven efficiency.7 A clinical trial with rituximab, conducted by Kavuru et al., included 10 patients with PAP, had shown promise in seven out of nine patients.7 In our case, a bilateral WLL was performed with a partial resolution of symptoms. Considering the exercise intolerance persistence after WLL and taking into account the negative level of GM-CSF antibodies; our patient was treated with rituximab, which resulted in promising outcomes. This result supports the auto-immune PAP hypothesis.\n\nIn conclusion, this case emphasizes the possible association between auto-immune diseases and PAP, which could worsen the disease course, as the specific treatment does not exist yet. Hence, further observational studies and randomized controlled trials are needed to establish clear-cut guidelines for PAP management, particularly when associated with auto-immune diseases.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nLiu S, Cui X, Xia K, et al.: Efficacy and safety of whole-lung lavage for pulmonary alveolar proteinosis: a protocol for a systematic review and meta-analysis. BMJ Open 20 avr 2022; 12(4): e057671. PubMed Abstract \| Publisher Full Text\n\nMutlu MY, İçaçan OC, Çelik S, et al.: ANCA Associated Vasculitis and Related Pulmonary Alveolar Hemorrhage in a Patient with Pulmonary Alveolar Proteinosis. Clin. Ter. 29 sept 2021; 172(5): 389–391. PubMed Abstract \| Publisher Full Text\n\nHuizar I, Kavuru MS: Alveolar proteinosis syndrome: pathogenesis, diagnosis, and management. Curr. Opin. Pulm. Med. sept 2009; 15(5): 491–498. PubMed Abstract \| Publisher Full Text\n\nSamuels MP, Warner JO: Pulmonary alveolar lipoproteinosis complicating juvenile dermatomyositis. Thorax. nov 1988; 43(11): 939–940. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamasue M, Nureki SI, Usagawa Y, et al.: Elevated Serum Anti-GM-CSF Antibodies before the Onset of Autoimmune Pulmonary Alveolar Proteinosis in a Patient with Sarcoidosis and Systemic Sclerosis. Tohoku J. Exp. Med. sept 2017; 243(1): 77–83. PubMed Abstract \| Publisher Full Text\n\nNagasawa J, Kurasawa K, Maezawa R, et al.: Systemic lupus erythematosus complicating autoimmune pulmonary alveolar proteinosis that was worsened by immunosuppressive therapy. Lupus. sept 2013; 22(10): 1060–1063. PubMed Abstract \| Publisher Full Text\n\nKavuru MS, Malur A, Marshall I, et al.: An open-label trial of rituximab therapy in pulmonary alveolar proteinosis. Eur. Respir. J. déc 2011; 38(6): 1361–1367. PubMed Abstract \| Publisher Full Text" }	[ { "id": "158819", "date": "05 Jan 2023", "name": "Helmi Ben Saad", "expertise": [ "Reviewer Expertise Physiology and pulmonary function" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read with a great interest the case report entitled “Case Report: The first reported case of pulmonary alveolar proteinosis with myasthenia gravis in a 27-year-old patient”.\nThe case report is very interesting. However, some minor changes should be applied before the acceptance of the paper:\nPOINT 1. I strongly recommend applying the CARE guideline1. For that reason, authors are asked to:\nConsult the guidelines (see PDF sent as an appendix) Submit (as an appendix) the CARE checklist (see the PDF) Write (in the beginning of the section Case report, page 3/7): This case reported was presented according to the CARE guideline (add the reference inside the paper and in the references list)\n\nPOINT 2. Abbreviations misuse: all abbreviations should be explained the first time they are used - unless it is a standard unit of measurement - and thereafter the use of abbreviations should be consistent throughout the paper. Example 1: Abstract Please abbreviate Pulmonary alveolar proteinosis as PAP at first use (1st line of the abstract) and therefore use always PAP. Example 2: Abstract and text Define COVID-19 (and do not use the abbreviations COVID).\nPOINT 3. Key words Please avoid citing as keywords some terms previously used in the title or the abstract. Please opt for MeSH terms and classify the keywords in alphabetical order.\nPOINT 4. Case report section Line 17 page 3: In the sentence “The physical examination and the laboratory findings were unremarkable”: please name the laboratory tests that were performed. Line 27 page 3: can you add the numerical values (and normal range) of c-antineutrophil cytoplasmic antibodies and anti-Mi-2 antibodies? Last line of page 3: change the sentence “of 70% after a 6-min walk of 100 meters” by “of 70% after walking 100 meters during the 6-min walk test”.\nPOINT 5. Discussion The first sentence should be a reminder of the main result (to take home message from this case report) Page 5 line 7: why 2009 why not 2023? In other terms, how many cases were published in PubMed until 2023? Add some limitations of this case-report: for example “assessment under corticosteroids and azathioprine”, other limitations.\nPOINT 6. Consent (page 6) Change “Written informed consent for publication of their clinical details and clinical images was obtained from the patient” By “Written informed consent for publication of her clinical details and clinical images was obtained from the patient.”\nPOINT 7. References (page 6) Standardize the way you present the journal short name (sometimes use of point, sometimes no use of point).\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": "10059", "date": "29 Nov 2023", "name": "LENDA BEN HMIDA", "role": "Author Response", "response": "I read, with a considerable attention, your review of the case report entitled “Case Report: The first reported case of pulmonary alveolar proteinosis with myasthenia gravis in a 27-year-old patient”. Thank you for revising our article and improving its quality. We addressed the comments mentioned in the review and we made some changes as stated. We ensured that the case report adheres to CARE guideline. We submitted accordingly our new version with great expectations. Here we respond to the changes that should be applied before the acceptance of the paper: POINT 1: We applied carefully CARE guideline: We re-consulted the CARE guidelines of 2013. We submitted the CARE checklist (as an appendix). We Wrote (in the beginning of the section Case report, page 3/7): “This case reported was presented according to the CARE guideline”. We added the reference inside the paper in the references list. POINT 2: We deeply regret our inattention concerning abbreviations misuse. Example 1: We defined our abbreviation PAP referring to Pulmonary alveolar proteinosis in the abstract. Example 2: We defined “COVID-19” by changing the term to “Coronavirus disease 2019” (without use the abbreviations). POINT 3: We opted for MeSH terms and changed the keyword (used in the title or abstract) from: “Pulmonary alveolar proteinosis” to “Pulmonary alveolar lipoproteinosis” “Myasthenia gravis” to “myasthenia” “Rituximab” to “anti-CD20” We classified the keywords in alphabetical order. POINT 4: Line 17 page 3: In the sentence “The physical examination and the laboratory findings were unremarkable”: We named the laboratory tests that were performed. Line 27 page 3: We added the numerical value (and normal range) of c-antineutrophil cytoplasmic antibodies and anti-Mi-2 antibodies. Last line of page 3: We changed the sentence “of 70% after a 6-min walk of 100 meters” by “of 70% after walking 100 meters during the 6-min walk test”. POINT 5: We added a first sentence in the discussion part as a reminder of the main result (to take home message from this case report). Page 5 line 7: In PubMed, there was no systematic review of pulmonary alveolar proteinosis until 2023. There was no article or review specifying the number of cases published in PubMed until 2023. We added another limitation to our case report: The follow-up period was short (only 6 months). There was not enough hindsight to assess the long-term effectiveness of rituximab. POINT 6: We changed “Written informed consent for publication of their clinical details and clinical images was obtained from the patient” by “Written informed consent for publication of her clinical details and clinical images was obtained from the patient.” POINT 7: We standardized the way we present the journal short name (references list)." } ] } ]	1	https://f1000research.com/articles/11-1439
https://f1000research.com/articles/12-1009/v1	21 Aug 23	{ "type": "Study Protocol", "title": "Comparative analysis for variation of skeletal and dental parameters in bilateral cleft palate in central India population – A Nemoceph study", "authors": [ "Shivani Rawat", "Vikrant Jadhav", "Priyanka Paul Madhu", "Amit Reche", "Aachal Lande", "Vikrant Jadhav", "Priyanka Paul Madhu", "Amit Reche", "Aachal Lande" ], "abstract": "Introduction: An area of dentistry called orthodontics focuses on repositioning crooked teeth and regulating facial development. Orthodontists and oral surgeons use cephalometric analysis as a planning tool for their patient care. Conventional film-based radiography equipment has been replaced with direct digital devices, which have advantages such as reduced patient radiation exposure, immediate radiographic image generation, the removal of the need for darkrooms, and simple image sharing. The second-most typical craniofacial abnormality that results in birth abnormalities is cleft lip and palate. Such patients frequently do not receive cephalometric testing because of their hypoplastic growth and limited development. Research is needed to help plan a standard treatment regimen, perform orthognathic operations, and use a low-radiation analysis technique. Objectives: To evaluate skeletal and dental parameters for cleft lip and palate using Down analysis, Steiner's analysis, and Tweed's analysis using Nemoceph. Methods: Around 50 cases from cleft lip and palate and non-cleft lip and palate will be selected from the orthodontics department of Sharad Pawar Dental College and Hospital. Steiner's, Down's, and Tweed's analyses will be evaluated using Nemoceph. Further analysis will be conducted using the observational data from the readings. There will be millimetre-based measurements made. Comparative analysis will be done with one-way ANOVA and post hoc statistics and regression analysis will be done for association of demographic variables. Expected outcomes: This study will use the nasion as a stable landmark to evaluate point A in cleft lip and palate patients, making it easier to perform normal cephalometric analysis. This will enable the evaluation of the probable position of point A for performing cephalometric analysis in orthodontics.", "keywords": [ "Cleft Lip & Palate", "Cephalometric analysis", "Down’s analysis", "Steiner’s analysis", "Tweed’s analysis", "Nemoceph", "Orthognathic surgery", "Point A" ], "content": "Introduction\n\nOrthodontics is a branch of dentistry that not only deals with correcting misaligned teeth but also modifying facial growth and is a guide to reconstructing the architecture of the face.1\n\nOrthodontists and oral surgeons can also use cephalometric analysis as a tool for arranging their patient’s care.\n\nOne of the primary procedures in every orthodontic facility is the acquisition, tracing, and interpretation of cephalometric head films or radiographs. Cephalometry is a tool that orthodontics utilizes to aid in diagnostics and comprehensive treatment planning. In comparison to conventional film-based equipment, these devices have several benefits, which involve less radiation exposure to the patient, immediate production of radiographic images, the removal of the need for darkrooms, associated costs and time for development, storage, and handling, and ease of image sharing with the appropriate professionals.2\n\nThe Steiner analysis and the Downs analysis are two of the more widely utilized methods of analysis in orthodontics.3\n\nThe second-most common type of craniofacial defect known to cause birth deformities is cleft lip and palate (CLP) which is known to be brought about by varying factors, including smoking, and alcohol consumption during pregnancy. Feeding issues, speech issues, hearing issues and recurrent ear infections can arise from such illnesses.4,5\n\nMaxillae abnormalities are among the abnormal characteristics present in people with craniofacial malformations such as CLP. This reduces the precision of the data because it is challenging to locate specific landmarks on cephalometric radiography. Finding the “Gonion,” “point A,” or points connected to the maxillary incisor pairs of landmarks can be particularly challenging.6–8\n\nPatients with CLP frequently do not undergo cephalometric analysis since it is challenging to quantify the landmarks due to their hypoplastic maxillae and constrained growth. It has been found that patients who inquire about the necessity of a cephalogram are unable to get such an analysis.9\n\nVery few studies have used cephalometric analysis due to the limited availability of sample data, prospective data, difficult treatment protocols, lack of comprehension, and lack of system development for cleft patients. Therefore, research is essential because it will aid in developing a standard treatment plan, carrying out orthognathic procedures, and employing a low-radiation analysis method.\n\nTo assess the reliability of Down’s analysis, Steiner’s analysis, and Tweed’s analysis in bilateral CLP in a central Indian population using lateral cephalogram.\n\n\n\n1. To evaluate skeletal and dental parameters for CLP using Down’s analysis, Steiner’s analysis, and Tweed’s analysis using Nemoceph.\n\n2. To compare skeletal and dental parameters for CLP using Down’s analysis, Steiner’s analysis, and Tweed’s analysis with non-CLCP patients using Nemoceph.\n\n\nMethods\n\nThe study design is a simple cross-sectional study and analytical study. The patients for the study will be selected from randomized control trails. A complete clinical examination will be done followed by case history, impression taking, model analysis, and radiographs such as lateral cephalograms. We are able to categorise the malocclusion into classes I, II, and III based on Angle’s malocclusion.\n\nThis study will employ nasion as one of the few consistent landmarks to establish the value for point A. This will support cephalometric study in patients with cleft lip and palate. On the contrary, the following mentioned modifiers can influence the outcomes of the study which are: patients with vertical and horizontal development patterns, non-syndromic instances such as isolated cleft lips, and patients with Angle’s class II and III subdivision. Patients undergoing presurgical nasoalveolar moulding (PNAM) (a non-surgical method of reshaping the alveolus, lips, and nostrils before primary cleft lip and palate surgery), and patients using chin cup facemask and headgear therapy during mixed dentition period are potential confounders of the study.\n\n\n\n1. The research proposal has been approved by the ‘Institutional Research Ethics Committee of Datta Meghe Institute of Higher Education and Research, Deemed University’ Ref No- DMIHER (DU)/IEC/2023/705 approved on 15/02/2023.\n\n2. Informed written consent will be obtained from all the participants.\n\nThe study will be carried out at Sharad Pawar DMIHER, Sawangi (M), Wardha, following approval from the ‘Institutional Research Ethics Committee of Datta Meghe Institute of Higher Education and Research, Deemed University’. Informed written consent will be obtained from all the participants.\n\nThe patients will be chosen using random sampling method from the Orthodontic Department’s Smile Train Outpatient Department (OPD). Around 50 patients will be invited to participate in the study based on the inclusion and exclusion criteria.\n\nThe cephalometric analysis and landmark recognition will be performed on a laptop using a mouse-controlled cursor. The landmarks will be notes and Down’s analysis, Steiner’s analysis, and Tweed’s analysis will be traced and measured by the same investigation to avoid interobserver errors. For examination, a proprietary programme called Nemoceph (version no. 8) will be employed. Open-access alternatives that can perform the equivalent function include Cephalopoint, Cephninja, and Oneceph. The photos will be saved in JPEG and PDF formats with a maximum resolution setting of 200 dpi.10\n\nFurther analysis will be conducted using the observational data from the readings. There will be millimeter-based measurements made. Once entered, these readings will appear on a Microsoft Excel page (version 2019 16.0.6742.2048).\n\nThe magnitude of competence and social support will be expressed in percentages along with a 95% confidence interval. Comparative analysis will be done with one-way ANOVA and post hoc statistics and regression analysis will be done for association of demographic variables.\n\n\n\n1. Data regarding the cases will be evaluated for the age group 10–18 years.\n\n2. Patients with maxillary deficiency in class I and III skeletal patients.\n\n\n\n1. Orthodontic treatment used in CLP instances.\n\n2. Syndromic cases.\n\nTo conduct the study, 50 digital lateral cephalograms of prospective orthodontic patients who reported to the outpatient department (OPD) of the Department of Orthodontics were taken. The cases that have visited the departmental OPD and smile train unit will be chosen based on the criteria for inclusion.\n\nDigital cephalograms were taken on Cephalostat machine (Planmeca Proline CC panoramic Xay, Planmeca OY Helsinki Finland). Kodak film size 8×10 inches (18×24 cms) and cassettes with in-built intensifying screen speed 400 were used.\n\nTo reduce measurement error and provide an unbiased assessment will be done by the same examiner every two weeks.\n\nUsing Fisher’s arctanh transformation\n\nANS-PNSC3VSNUNGL\n\nN = 25 samples needed in the study\n\nThe statistical methods employed in this study include the Student’s t-test and correlation analysis. These techniques will be used to analyze the data and uncover a pattern between cleft lip and palate patients and non-cleft lip and palate patients. The study’s approach is influenced by the research conducted by Philine H. Doberschutz.11 The formula for analysis is referenced from Lachin (1981), as documented in Controlled Clinical Trials.12 For the purpose of data analysis, the study will utilize SPSS version 27.0 (RRID: SCR_002865), a widely recognized software tool in the field of statistics. This software will aid in processing and interpreting the collected data effectively.\n\nDissemination\n\nDue to the abnormal anatomy of the patients with bilateral cleft lip and palate it is difficult to locate anatomic landmarks to study skeletal and dental parameters, and hence with this study, we will be able to evaluate point A with the help of stable landmarks which will be evaluated from the nasion. We will be able to determine the likely position of point A for performing cephalometric analysis in orthodontics. Evaluation of point A in CLP patients is challenging due to growth deformity in the maxilla, making it difficult to perform the normal cephalometric analysis.\n\n\nDiscussion\n\nCephalometry plays a vital role in deciding the diagnosis for the patient.13\n\nDue to restrictions in the utility of 2D cephalometry, the world is shifting to a digital mode of intervention. Digital cephalometry reduces intra and interobserver variations giving more accurate data for the study. CLP patients have an underdeveloped maxilla and due to this, we are not able to locate landmarks for determining the analysis.14 Ege Doğan et al. (2020), stated that the objective of his study’s was to assess the preciseness of cephalometric measurement data using lateral cephalometry in individuals with CLP present unilaterally.15 It is crucial to pay close attention to the marking of the anterior nasal spine, point A, nasion, sub nasal, and upper 1 points that are present in cleft regions. Reliability restrictions are essential in cephalometric studies of patients with CLP to guide doctors.16 Due to hypoplastic maxilla, we are unable to locate point A and the anterior nasal spine, and these points aid in assessing the growth pattern and malocclusion of the patients.17\n\nCatharina A.M. Bongaarts et al. stated that the landmarks point A, anterior nasal spine, and posterior nasal spine can be difficult to identify in unilateral cleft lip and palate patients with embryonic germs in the anterior maxilla, but no more accurate anatomical points were identified in this investigation. Caution should be used when interpreting cephalometric tests in unilateral CLP patients that use anterior nasal spine, posterior nasal spine, and point A.18\n\nManual Yudovich et al. (2015) described the importance of the cephalometric study as that it can help to optimize future treatment protocols, which helps to achieve the greatest potential function and aesthetics to raise the patient’s quality of life.19\n\nAs a result, a study was developed to evaluate the dental and skeletal characteristics of people with and without clefts to relocate to point A.\n\nNot started yet.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nProffit WR, Fields HW Jr, Sarver DM: Contemporary orthodontics. Elsevier Health Sciences. 2006 Dec 8.\n\nParedes V, Gandia JL, Cibrián R: Digital diagnosis records in orthodontics. An overview. Med. Oral Patol. Oral Cir. Bucal. 2006 Jan 1; 11(1): E88–E93. PubMed Abstract\n\nOria A, Schellino E, Massaglia M, et al.: A comparative evaluation of Steiner’s and McNamara’s methods for determining the position of the bone bases. Minerva Stomatol. 1991 Jun 1; 40(6): 381–385. PubMed Abstract\n\nQuintero JC, Trosien A, Hatcher D, et al.: Craniofacial imaging in orthodontics: Historical perspective, current status, and future developments. Angle Orthod. 1999; 69: 491–506. PubMed Abstract\n\nShrey S, Singam A: A Comparative Study of Atracurium and Cisatracurium in paediatric cleft Lip and Cleft palate surgeries. Res. J. Pharm. Technol. 2020; 13(2): 867–870. Publisher Full Text\n\nKragskov J, Bosch C, Gyldensted C, et al.: Comparison of the reliability of craniofacial anatomic landmarks based on cephalometric radiographs and three-dimensional CT scANS. Cleft Palate Craniofac. J. 1997 Mar; 34(2): 111–116. PubMed Abstract \| Publisher Full Text\n\nNeelapu BC, Kharbanda OP, Sardana V, et al.: Automatic localization of three-dimensional cephalometric landmarks on CBCT images by extracting symmetry features of the skull. Dentomaxillofac. Radiol. 2018 Jan; 47(2): 20170054. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMidtgard J, Bjork G, Linder-Aronson ST: Reproducibility of cephalometric landmarks and errors of measurements of cephalometric cranial distances. Angle Orthod. 1974 Jan; 44(1): 56–61. PubMed Abstract\n\nHagemann K, Vollmer D, Niegel T, et al.: Prospective study on the reproducibility of cephalometric landmarks on conventional and digital lateral headfilms. J. OrofacOrthop. 2000; 61: 91–99. Publisher Full Text\n\nKumar M, Kumari S, Chandna A, et al.: Comparative evaluation of CephNinja for Android and NemoCeph for computer for cephalometric analysis: a study to evaluate the diagnostic performance of CephNinja for cephalometric analysis. J. Int. Soc. Prev. Community Dent. 2020 May; 10(3): 286–291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDoberschütz PH, Schwahn C, Krey KF: Cephalometric analyses for cleft patients: a statistical approach to compare the variables of Delaire’s craniofacial analysis to Bergen analysis. Clin. Oral Investig. 2022 Jan; 26(1): 353–364. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLachin JM: Introduction to sample size determination and power analysis for clinical trials. Control. Clin. Trials. 1981 Jun 1; 2(2): 93–113. Publisher Full Text\n\nChen SK, Chen YJ, Yao CC, et al.: Enhanced speed and precision of measurement in a computer-assisted digital cephalometric analysis system. Angle Orthod. 2004 Aug 1; 74(4): 501–507. PubMed Abstract\n\nVerulkar A, Kamble R, Srivastav S, et al.: Evaluation of the awareness of different orthodontic treatment appliances in patients undergoing orthodontic treatment of Maharashtra–A survey. J. Datta Meghe Inst. Med. Sci. Univ. 2020 Jul 1; 15(3): 347. Publisher Full Text\n\nDoğan E, Çınarcık H, Doğan S, et al.: Is Cephalometric Analysis Reliable in Cases with Cleft Lip and Palate? Ege Üniversitesi Diş Hekimliği Fakültesi Dergisi. 2020; 41(1): 27–37.\n\nMidtgård J, Björk G, Linder-Aronson ST: Reproducibility of cephalometric landmarks and errors of measurements of cephalometric cranial distances. Angle Orthod. 1974 Jan; 44(1): 56–61. PubMed Abstract\n\nBjörk A: The face in profile. SvenskTandlaekare- Tidskrift, Vol. 40; No. 5B, suppl. Berlingska, Boktrykeriet, Lund.1947.\n\nBongaarts CA, van’t Hof MA , Prahl-Andersen B, et al.: Identification of cephalometric landmarks in unilateral cleft lip and palate patients: are there alternatives for point A, ANS, and PNS? Cleft Palate Craniofac. J. 2008 Jan; 45(1): 81–86. PubMed Abstract \| Publisher Full Text\n\nBurak MY, Ponglertnapakorn A, Calderón EG: Analysis of the cephalometric skeletal and dental characteristics of adult patients with cleft lip and palate who received orthopedic, orthodontic and/or surgical treatment during their childhood and adolescence. Revista Mexicana de Ortodoncia. 2015; 3(1): 22–32. Publisher Full Text" }	[ { "id": "248256", "date": "22 Feb 2024", "name": "Mohammad Abdelhamid", "expertise": [ "Reviewer Expertise Oral and maxillofacial surgery", "Cleft lip and palate", "Craniofacial abnormalities" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Thanks for submitting this study protocol to F1000Research. The study within hands is a cross-sectional analytical study that is proposed to assess the reliability of a software program; Nemoceph, in assessing the variation of skeletal and dental parameters in patients with bilateral cleft lip and palate, and correlating these results with measurements of patients without cleft lip and palate using digital cephalometric analysis. The protocol is neat and valid, but it may need some modifications so that the exerted efforts could be shown clearer.\nTitle: - I think the enrolled patients to be considered are cleft lip and palate patients and not just cleft palate patients. - The term \"patients\" should be added within the title. - In addition, I wonder about the expression \" A Nemoceph study\". Up to my knowledge NemoCeph is a software program and not a type of study. - Therefore, I recommend that the title would be \"Comparative analysis for variation of skeletal and dental parameters in bilateral cleft lip and palate patients in central India population: A cross-sectional analytical study using NemoCeph program\".\nIntroduction: - 5th text line: utilizes --> utilize. - 10th text line: more --> most. - 11th text line: the second most common : What is the first most common? If it's clubbed foot - as I think - then it's not a craniofacial anomaly. Thus, CLP is the first most common craniofacial anomaly and the second most common body anomaly among humans.\nObjectives: 2- To compare skeletal and dental parameters for CLP using Down’s analysis, Steiner’s analysis, and Tweed’s analysis with non-CLCP patients using Nemoceph: You mean to compare with data published in the literature, or with data acquired from enrolled patients without CLP. (This was not declared within the manuscript !) - CLCP: This abbreviation wasn't defined within the manuscript, I think you mean CLP instead.\nMethods: - The hypotheses, both null and alternative hypotheses, are missing. Please state. - Grouping identification is missing as well. How would you name each group within your study? - 2nd text line: trails --> trials. - 4th text line: A reference is needed at the end of the paragraph. - 11th text line: potential confounders of the study --> So, will they be excluded from the study? If yes, then please mention that in the exclusion criteria.\nSetting and study design: - 4th text line: using random sampling method --> Please define the method of randomization that will be used within your study. - 8th text line: notes --> noted. - 9th text line: investigation --> investigator. - 9th, 10th, and 11th text line: I think you need to state the corporations that have the mentioned program rights.\nExclusion criteria: - What if the enrolled patients have undergone alveolar cleft bone grafting (ABG)? Will they be excluded? As you know, ABG would affect the measurement parameters, especially when the included age that you stated in the inclusion criteria is referring to that when most CLP patients have undergone ABG (Secondary alveolar cleft bone grafting (SABG)). - Also, What about unilateral CLP patients? Will they be excluded as well? If yes, please mention.\nData sources/measurements: - 2nd text line: The cases that have visited ....etc. --> Referring to ‘patients’ as ‘cases’ throughout your manuscript is inappropriate. According to the AMA Manual of Style3, “the use of case is dehumanizing when referring to a specific person”. Please rephrase ‘cases’ into ‘patients’ whenever mentioned. Here we are not talking about AMA as a referencing style, but rather as a writing style.\nBias: - 1st text line: \" ... and provide an unbiased assessment will be ...\" --> \" .... and provide an unbiased assessment, measurements will be ...\". Please rephrase. - 1st text line: \" .... every 2 weeks.\" --> for how long? and, How many times? - If the same examiner will do multiple measurements for the same sample, then I think you'll need assessment for the intraobserver reliability.\nStudy size: - I think that the term \"Sample size\" is more appropriate than \"Study size\". - ANS-PNSC3VSNUNGL: Please clarify this expression. - N= 25 samples ... etc: --> Then, will you enroll 50 patients (as stated in the 'Setting and study design' section) or 25 patients (as stated here)? - I'm a little bit confused about the input values within the used formula. Please declare.\nStatistical methods: - 5th text line: SPSS version 27.0 --> I think you need to state the corporation that have the program rights (IBM Inc,).\nDiscussion: - 4th and 5th text lines: Ege Doğan et al. (2020), --> Doğan et al. (2020),. - 7th text line: upper 1 --> Please declare! - 8th text line: doctors --> Which doctors? Surgeons? Orthodontists? Or more broadly; cleft care providers? - 11th text line: Catharina A.M. Bongaarts et al. --> Bongaarts et al. (Please add a reference for this citation and the year of the mentioned publication). - 11th text line: \" ... nasal spine can be ...\" --> Please use the past tense. (i.e., \" ... nasal spine could be ... \" or \"... nasal spine were ...\"). - 15th text line: Manual Yudovich et al. --> By referring to the manuscript's bibliography, this citation should be cited as follows: Burak et al.\nAgain, endeavors of this manuscript authors are appreciated, and the above mentioned comments were highlighted from the reviewer's point of view for a better show of the already-shown efforts.\nBest regards.\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": "321965", "date": "25 Sep 2024", "name": "Marcela Scarpa", "expertise": [ "Reviewer Expertise Maxillofacial", "Cleft lip and palate", "craniofacial" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. In the received work, there are some conceptual errors:\n\n- Cleft lip and palate are the most common congenital facial defects (present in 1:650 live births) and the second most prevalent congenital defect (only less common than clubfoot).\n\n- Its primary causal condition is genetic, but it can also be influenced by environmental factors, epigenetics, and random causes.\n- Two-dimensional cephalometry is widely studied and described in the literature; however, it is incomplete due to its inherent limitations and parallax errors.\n\n- Cleft lip and palate are very common, and there is no shortage of data.\n\n- Treatment protocols are well-documented and extensively studied (up to the present day) and registered in large multicentric studies such as Eurocleft, Americleft, and Scandcleft.\n\n- Patients with cleft lip and palate may undergo cephalometry; however, three-dimensional evaluation is currently well-documented in the literature.\n2. Objective: - The objective of the study is unclear:\n\n- Is it to validate the Nemoceph software?\n\n- To evaluate cephalometric changes in patients with bilateral cleft lip and palate? If this is the objective, craniofacial changes have already been well-documented in the literature.\n3. Method: - A single evaluator is not adequate for the analysis. Even with an assessment at two different time points, evaluation by only one person is inherently biased. I suggest at least two expert evaluators at two distinct times to minimize errors. - If the objective is to validate the software, I suggest comparing the results with those of other recognized methods that can prove the effectiveness and accuracy of the Nemoceph analysis.\n\n- Regarding the age of the patients:\n\n- The range of 10-18 years is too broad, as mixed dentition can occur up to 12 years.\n\n- This period can interfere with facial growth and compromise the analysis.\n- If the objective is to validate the software, I would be happy to review the article again, provided the suggested changes are made.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] }, { "id": "321968", "date": "30 Sep 2024", "name": "Shreyasi Tiwari", "expertise": [ "Reviewer Expertise Cleft & Craniofacial 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\nSTUDY TITLE: Are the study subjects bilateral cleft palate or bilateral cleft lip & palate patients?\nABSTRACT:\nThe first sentence in the abstract introduction can be better framed. Please refrain from using layman's terms in a scientific paper. CLP is the MOST common congenital abnormality in the facial region. Precisely because of secondary maxillary hypoplasia, cleft individuals are usually advised to undergo serial lateral cephalometry, as opposed to what’s been mentioned by the authors. Please mention 'lateral cephalometric analysis' & 'Bilateral' CLP under objectives in the abstract. It’s unclear from the abstract methods whether there will be 50 subjects each in the CLP & non-CLP groups, or only 25 subjects per group. Will only millimetre-based measurements be made or angular measurements in degrees too? The science behind the reasoning under ‘expected outcomes’ of the abstract is quite obscure.\n\nINTRODUCTION:\nTweed’s analysis has been missed out. CLP is the most common craniofacial congenital deformity, not the second most common. The etiology of cleft remains unclear, although the combination of genetic and environmental factors is indisputable. The scientific basis for the claims made in the last two paragraphs of the introduction isn’t very clear. Individuals with CLP quite often are subjected serial radiography (especially in dedicated cleft centres) – cleft data is generally very well-documented in centres receiving funding for cleft orthodontic care.\n\nMETHODS:\nIt's not clear how 'randomized controlled trial' (RCT) is applicable in the research methodology of this protocol. The claim that Nasion landmark will be employed for better interpretation of point A, thereby making this current study protocol helpful in cleft cephalometry is repeatedly mentioned to somehow highlight the usefulness of this study. However, the scientific reasoning behind this is unclear. The second paragraph mentions ‘non-syndromic instances such as isolated cleft lips’ under study modifier, which makes the reader believe that the study subjects are only syndromic individuals. The last two sentences of the second paragraph regarding ‘modifiers’ & ‘confounders’ can be better phrased to convey the intended scientific meaning. The selection criteria of the present protocol is very obscure. The cleft and non-cleft groups must be matched with respect to parameters such as age, sex, etc. For selection within the BCLP group, there must be congruence in relation to the clinical presentation of cleft prior to the primary repair surgery, operating Surgeon & technique, ages at which primary lip & palate repair surgeries were performed, no. of revision surgeries, information about alveolar bone grafting etc. Also, the specified age group in the inclusion criteria is too vast for any fruitful data analysis. Reasoning behind using the said three cephalometric analyses has not been provided. It’s unclear if only millimetre-based measurements will be done or angular ones as well. Also, much stress has been laid on NemoCeph software more often than required (including in the study title). The study investigators must preferably be two or more well-experienced Cleft Orthodontists. The grammar in the methodology keeps changing from past to present to future tenses. Despite the elaborate sample size calculation equations provided, it’s unclear how the figure of 25 subjects was arrived at. The statistical analysis mentioned in the abstract and that under the study methodology are not in agreement. Intra-observer reliability has not been mentioned.\nDISSEMINATION & DISCUSSION require complete revision.\nIt isn’t clearly justified by the authors on how this study will add to the currently available scientific literature.\n\nThe grammar used in the study protocol can be improved, in general.\nThe use of more relevant references is encouraged.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1009
https://f1000research.com/articles/12-1005/v1	21 Aug 23	{ "type": "Research Article", "title": "Investigating how the electronic and optical properties of a novel cubic inorganic halide perovskite, Sr3NI3 are affected by strain", "authors": [ "Md. Abul Bashar Shanto", "Md. Ferdous Rahman", "Md. Rasidul Islam", "Avijit Ghosh", "Ahmed Azzouz-Rached", "Hind Albalawi", "Q. Mahmood", "Md. Abul Bashar Shanto", "Md. Rasidul Islam", "Avijit Ghosh", "Ahmed Azzouz-Rached", "Hind Albalawi", "Q. Mahmood" ], "abstract": "Background: Inorganic Perovskite materials have sparked the attention of the solar technology sector due to their remarkable structural, optical, and electrical capabilities. In the realm of efficient LEDs, inorganic perovskites have displayed considerable promise, showcasing various benefits such as exceptional color purity, the ability to adjust emission wavelengths, and cost-effective fabrication methods. Methods: The study extensively investigated the bandgap, density of states, electron charge density, structural properties, dielectric properties, loss function, and absorption coefficient of Sr3NI3 under strain using first-principles density functional theory (DFT). Results: At the Gamma (Γ) point, the unstrained flat structure of Sr3NI3 exhibits a direct band gap of 0.733 eV. Observing the spin-orbital coupling (SOC) effect reduces the bandgap to 0.711 eV in Sr3NI3 perovskite. Compressive strain minimizes the prevalence of the structure's bandgap, whereas tensile strain causes a slight elevation. The optical properties of this material, including the dielectric functions, absorption coefficient, reflectivity, and electron loss function, exhibit its excellent absorption capacity in the visible area because of its band characteristics. Conclusions: The research indicates that as the amount of compressive strain rises, the peak values of the dielectric constant of Sr3NI3 shift towards lower photon energy (redshift); meanwhile, when tensile strain is executed, it displays the behavior of altered photon energy with an increase towards higher energy levels (blueshift). Thus, the potential of utilizing Sr3NI3 perovskite in solar cells for energy production and light management is considered promising.", "keywords": [ "Perovskite material", "DFT calculation", "SOC effect", "Strain", "Optoelectronic properties" ], "content": "Introduction\n\nPhotovoltaic (PV) technology is a notable participant in the energy industry due to its significant ability to lower oil consumption, ameliorate greenhouse gas (GHG) emissions, and lessen environmental deterioration.1 The potential for developing conventional solar cells such as Si is limited due to their indirect, suboptimal bandgaps and costly fabrication processes.2 In 2009, halide perovskite was initially introduced.3 The solar technology field has been showing increased attraction in organic-inorganic perovskites because of their remarkable properties, including a significant bandgap, universal accessibility, exceptional optical absorption capabilities, low reflectivity, and cost-effectiveness.4–9 The power conversion efficiency (PCE) rapidly increased over the last thirteen years, from 3.8% to 25.8%.10 The PCE of CsPbI3 perovskite solar cells can achieve 17.9%11 without hole transport layer (HTL) in 2022 and 19.06%12 with HTL in 2023. The improvement of perovskite materials’ properties during the last 10 years has expedited this progression. Metal halide-based inorganic perovskites are attracting a lot of interest. The organic-inorganic perovskites’ difficulty with long-term longevity is a significant concern because of its susceptibility to moisture, wind, sunlight, and temperature when utilized in a natural environment.13,14 However, because of the low stability of lead (Pb) implemented in the perovskite materials, which causes rapid breakdown when exposed to specific circumstances like heat and moisture, the general use and improvement of perovskite solar cells are hampered. Another issue that restricts their employment is the device hysteresis effect, which results in instability and inconsistent outcomes under various measurement circumstances.15 Furthermore, lead toxicity is a significant concern during the production and disposal of perovskite solar cells, which impedes their sustainability and poses health and environmental risks.16 Zhu et al.17 revealed that inorganic halide perovskites had almost equal band edge carrier properties. Inorganic cubic perovskites have recently attracted interest as prospective options for LED, semiconductor, and solar technologies due to their straight bandgap nature and remarkable optical absorption characteristics.18–20 In addition, L. Zhang et al. discovered that inorganic perovskite solar cells may generate high open-circuit voltages in a consistent and long-lasting way.21 Perovskites with an A3BX3 type structure attract a lot of attention because of their extraordinary qualities, including the absence of lead, mechanical stability, superior electrical characteristics, and direct bandgap material. There have been no extensive studies on Sr3NI3 yet. The future applications of electronic and optoelectronic devices can greatly benefit from a thorough and comprehensive study of the inorganic perovskite Sr3NI3.\n\nThe bandgap of a solar cell significantly influences its PCE as it determines the generation of charge carriers and absorption of light. As per the Shockley-Queisser theory, a perovskite solar cell with a bandgap between 1.2 to 1.4 eV can achieve a PCE of up to 33%.22,23 Although inorganic halide perovskites are well-suited for optoelectronic and photovoltaic systems, they have a slight disadvantage of having a relatively higher bandgap.24,25 The ability to adjust the electronic bandgap using multiple methods is critical for achieving maximum PCE in inorganic halide perovskite solar cells. In recent times, there have been several types of research that have utilized strain engineering as a method to improve specific properties of a material.26–30 Strain engineering is an effective method for modifying perovskite materials’ atomic structure and physical characteristics, allowing them to be employed in solar applications. Recent investigations on the properties of materials resulting from strain have revealed an essential link between the material’s properties and its structural features.31–37 Applying a minor amount of pressure (less than 0.3 GPa) reduces the bandgap and improves the carrier lifetime by 70% to 100% in organic-inorganic tri-iodide perovskite materials.38 Rasidul Islam15 accomplished a thorough investigation on the characteristics of perovskites APbBr3 (where A represents Rb and Cs) and made notable conclusions regarding the impact of strain within the -6% to 6% range. Jing et al.39 conducted an extensive study on CsPbI3 and observed that by applying strain in the range of -5% to 5%, the bandgap of CsPbI3 may be adjusted to fall between 1.03 and 2.14 eV. Similarly, A. K. Hossain40 found that applying compressive strain on the inorganic perovskite CsSnCl3 can transform it from a semiconductor to a metallic substance, which possesses remarkable optical and electronic properties. By applying pressure to CH3NH3GeI3, it is possible to optimize the material’s potential for utilization in solar cells by attaining a more significant band gap energy, improved carrier mobility, and optimized absorption coefficients.41 Compressive and tensile strain can be used to successfully change some properties of CsGeI3, such as the bandgap and dielectric coefficient.42 Compressive and tensile strain considerably influence the structural, electrical, and optical properties of Sr3NI3 perovskite. Because of their larger atomic sizes, larger cations contain more nucleons than smaller ones, resulting in a modification in terms of the electronic band structure. The manipulation of electronic characteristics in various materials can typically be accomplished utilizing the spin-orbit coupling (SOC) phenomenon.43–45 When considering the SOC effect, the bandgap of halide perovskites may drop by approximately one electron volt (eV) and experience band splitting.45 Therefore, it is crucial to conduct a meticulous and systematic investigation of the SOC effect and biaxial strain variations in Sr3NI3 perovskite. Hence, performing a comprehensive analysis of the influence of strain and SOC effect on Sr3NI3 perovskite holds great significance.\n\nThis research aimed to execute FP-DFT computations to investigate how varying amounts of strain and SOC impact the structural, electrical, and optical characteristics of cubic Sr3NI3. Our investigation focused on a thorough analysis of the band structure and bandgap modification mechanism of Sr3NI3. Our primary focus was determining how the SOC phenomenon influences the electronic properties of the Sr3NI3 perovskite. We utilized two distinct methodologies to investigate the band structure of Sr3NI3 in our research. Our analysis specifically focused on the impact of strain and SOC changes on the bandgap, including both expansion and compression. According to our findings, Sr3NI3 has remarkable features that make it suitable for various semiconductor materials, including high-temperature superconducting materials, energy storage devices, and future solar cells.\n\n\nComputational method\n\nThe DFT computations were executed employing the Quantum Espresso simulation application package.46–50 The FP-DFT analysis of the Sr3NI3 perovskite structure was performed using a norm-conserving (NC) pseudopotential51–53 and the Perdew-Burke-Ernzerhof (PBE)54 exchange-correlation mechanism. The input data contained vital initial configurations, including the specification of the Brillouin zone grid, crystal structure arrangements, lattice constants, and the specified kinetic cut-off energy. To improve the performance and optimize the structure, adjustments were made to the kinetic energy cut-off and charge density cut-off parameters, setting them at 30 Rydberg (Ry) (approximately 410 eV) and 220 Rydberg (Ry) (approximately 2990 eV), respectively. The optimization of the lattice via vc-relax computation was carried out using a k-point (kx, ky, kz) dimension of (6×6×6). For the Self-consistent field (SCF) calculations, a convergence threshold of 10-6 atomic units and a maximum force tolerance of < 0.01 eV/was specified.15 A force convergence threshold of nearly 10-3 a.u was considered during the relaxation studies of ionic and structural adjustments. The use of modified PBE for metals was not implemented in our present study, despite the availability of approaches to control this inaccuracy.55,56 The biaxial compressive and tensile strain was reproduced by varying the astrained lattice parameter. To determine strain, we implemented a formula57:\n\nIn the formula, the unstrained lattice constant is denoted by the term “astrained.” The ε range is presented in 2% increments and spans -4% to +4%, whereas negative values indicate compressive strains, while positive values indicate tensile strains. Once the perovskite structure was dynamically stabilized, its optical properties were analyzed by calculating its complex dielectric functions, which are photon energy-dependent. We analyzed the optical properties of the material structures using the first-order time-dependent perturbation theory and verified their dynamical stability.58 The complex dielectric component was analyzed to determine the energy spectrum (measured in eV) at which it displays absorption peaks for photons. To calculate optical properties, a Monkhorst-Pack k-point grid with a 10 × 10 × 10 Gamma center was applied to sample the Brillouin zone. While determining the optical absorption coefficients, the complex dielectric function, represented by ε(ω) = ε1(ω) + jε2(ω), is considered the primary relationship.\n\n\nResult and discussion\n\nThe optimum structure of the inorganic perovskite of Sr3NI3 is shown in Figure 1(a) and the k-path of the first Brillouin zone in order to identify their electronic band structure is shown in Figure 1(b). Sr3NI3 is a metal halide perovskite compound that crystallizes in a cubic structure with a high degree of symmetry. Specifically, its crystal structure is associated with the space group Pm-3m, a highly symmetric space group corresponding to a simple cubic lattice.59,60 The strontium (Sr) and nitrogen (N) atoms in the SrN4 tetrahedra are bound together by covalent bonds with varying bond lengths ranging from around 2.23 to 2.51. Iodine (I) atoms generate weak van der Waals interactions with the surrounding atoms rather than being directly bound to strontium or nitrogen atoms. The average bond lengths in Sr3NI3 have been calculated to be 2.46Å for Sr-N bonds and 3.00Å for I-Sr bonds. The Wyckoff notation, which describes the symmetry-equivalent locations inside the unit cell, may be used to represent the fractional coordinates of nitrogen (N), iodine (I), and strontium (Sr) atoms in Sr3NI3. The Wyckoff locations for Sr3NI3 in the cubic space group Pm-3m are: (i) The Sr atom occupies a single place at the 1a site, with fractional coordinates of (0,0,0), (ii) The 3c site is defined by three places filled by an I atom, with fractional coordinates of (0.5,0.5,0), (iii) The 3d site corresponds to three N atom-occupied sites with fractional coordinates of (0,0.5,0). First, the structural characteristics of Sr3NI3 perovskite must be computed before analyzing its various features. PBE was used to obtain the structural properties, identical to the lattice constant a(Å) value shown in Table 1. The greatest sustainable lattice constant for Sr3NI3 has been identified by calculating the total amount of energy while considering the lattice parameter. The Sr3NI3 compound’s lattice constant has been reported to be a = 6.33 Å.\n\nMoreover, Cohesive and formation energy are valuable measurements for determining a structure’s stability. By calculating these energies for a given system, we can decide if the form is stable and use this information to validate its stability.61,62 The following formula may be used to compute these energies15:\n\nTable 1 shows the lattice constant and energy bandgap of Sr3NI3 were determined through a comprehensive analysis that included experimental data and earlier DFT computations. The findings of our investigation demonstrate that the formation energy of Sr3NI3 is approximately -12.6 meV per atom, which is a negative value. It indicates that Sr3NI3 has negative conjunctive and formation energies, suggesting that the perovskite structure is stable.\n\nStudying the electronic charge density of a component is a crucial aspect of analyzing its electronic characteristics. It involves creating a map of the charge density structure of the valence electrons in the unit cell, reflecting the overall charge concentration. By examining the total electronic charge density map, researchers can investigate the chemical bonding nature of the molecule. This charge density curve is composed of structural atoms that demonstrate how orbital electrons contribute to the electrical properties of atoms by accumulating charges.\n\nSubsequently, a color density map that highlights differences in charge is used to link electronic DOS spectra from individual elements. The mapping of the charge density in the Sr3NI3 compound is displayed in Figure 2(a) 2D view, Figure 2(b) bird’s eye view, and Figure 2(c) 3D view, respectively. The electron density analysis indicates that the N and Sr atoms interact weakly, which plays a role in stabilizing the crystal lattice. Or, to put it another way, the crossing of the outer electrons between these two components suggests the presence of a covalent link.64,65 Furthermore, the charge density distribution reveals that the I atoms possess low electron density due to their poor electronegativity and tendency to form weak covalent bonds.66,67 The charge distribution analysis provides strong evidence for the covalent bonding features of the Sr-N atoms. The researchers observed that the charge density surrounding the atoms has an almost spherical shape, a characteristic of ionic bonding similar to that seen in previously published perovskites.66,67 The bonding between the Sr and I atoms is consistent with an ionic bond, while the N-I bond exhibits negative population values, indicating an antibonding property.\n\nAfter structural optimization, we calculated the design of electronic bands and the highly symmetric point direction of Sr3NI3. A direct bandgap is usually necessary for a system to be an acceptable contender for optoelectronics device applications.68,69 The electronic properties of a substance are essentially dependent on its band structure, charge density, and density of states (DOS).70 The band configuration of the unstrained Sr3NI3 perovskite formation is shown in Figure 3(a). The Fermi levels have been zeroed out to evaluate the bandgap value conveniently. The crystal structure of the cubic Sr3NI3 is analyzed by considering the path Γ-X-M-R-Γ along the k-axis. Figure 3(a) illustrates the placements of the conduction band minimum (CBM) and valence band maximum (VBM) of Sr3NI3, both of which are situated near the Γ (Gamma) point. Based on the PBE calculations, it has been predicted that Sr3NI3 perovskite exhibits a direct bandgap structure with a bandgap value of approximately 0.733 eV. This result appears consistent with previously published values.71,72 When the bandgap of Sr3NI3 was calculated using the GGA method, it was significantly underestimated, which is a frequent disadvantage of the GGA method. Similarly, it was found that both the (LDA)+U and LDA techniques for local density approximation also underestimated the bandgap value of Sr3NI3.73 Several specialists have given several types of solutions to prevent this type of bandgap computing, including the GW methodology hybrid functional.74,75 Furthermore, a direct bandgap is necessary for crystalline substances to be suitable for advanced photothermal and sustainable energy applications. Because of their high direct bandgap value, these compounds are considered suitable for efficient solar cells and cells used in photovoltaic applications.\n\nThe partial density of states (PDOS) analysis explains how the distinct atoms and their various configurations in the structure impact the bandgap energy of Sr3NI3. Figure 3(b) illustrates the PDOS dispersion in Sr3NI3 for the -4 to 3 eV range. The PDOS analysis demonstrates that the forms of Sr and N, hybridized with I in the Sr3NI3 structure, extend throughout the entire energy range while maintaining the bandgap energy. It shows that the primary bond between Sr-I and N-I is covalent. Additionally, it was noticed in Sr3NI3 that there was electron charge transfer from Sr and N to I (illustrated in Figure 3b) due to the substantial difference in atomic states. According to the cubic phase analysis, the I-5p orbitals are essential in referring to the valence band of any perovskite structure. The investigation of the DOS in the vicinity of the valence band of Sr3NI3 in our work indicated that the I-2p orbital was the crucial factor. On the other hand, the N-2p orbital and a minor contribution from the Sr-3s orbitals significantly influenced the conduction band.\n\nThe SOC effect has been considered during the computation to precisely forecast the band structure because of the existence of nitrogen ions in the perovskite structure. The modification of CBM and VBM levels is significantly affected by the SOC effect, as illustrated in Figure 4(a) for both the conduction and valence band sections. The electronic features of Sr3NI3 perovskite were studied by factoring in the Hamiltonian equation that includes the SOC effect.76 The Hamiltonian equation is given by:\n\nWith regard to the SOC, HSOC represents the Hamiltonian operator that includes the effects of orbital angular momentum p→, potential energy or force F→, the mass of free electrons m0, and spin angular momentum s→, with ℏ representing the reduced Plank’s constant. The CBM shifted towards the Fermi level, whereas the VBM shifted upwards. Additionally, the only point in the crystal structure where the spin-degeneracy criterion is not satisfied is at the equally spaced point due to a band separation caused by inorganic perovskite crystals’ absence of inversion symmetry. The total angular momentum resulting from ls coupling can explain the band separation. The angular momentum of spin (s) and the angular momentum of orbital (l), where J is a value that falls between the absolute difference of l and s (\|l-s\|) and the sum of l and s (\|l+s\|). The values of l and j in s orbitals are 0 and 1/2 based on the molecular orbital theory, while the p orbitals have values of 1 and (1/2,3/2), and the d orbitals have values of 2 and (3/2,5/2). For each orbital, S equals +1/2 and -1/2. We saw the bandgap reducing when considering the SOC impact. The Sr3NI3 material has an energy band gap of 0.711 eV, which drives the valance and conduction bands closer to the Fermi level (Figure 4a). Table 2 displays the calculation of bandgap values for the Sr3NI3 perovskite both under and over SOC.\n\nTo better understand the band diagram of cubic Sr3NI3, we analyzed the PDOS in the presence of SOC. The PDOS revealed that the impact of the Sr atom is not limited by any predetermined rules when SOC is present, as demonstrated in Figure 4(b). The band edge remains unseparated at the high symmetry regions, even though the SOC effect divides I-2p and I-3p into p (j = 1/2) and p (j = 3/2). As depicted in Figure 4(b), the I-2p (j = 1/2) and I-3p (j = 3/2), atoms contribute the most significant portion of energy to the valence band (VB) within the range of -3.8 to -1.1 eV. The energy contribution to the CB side between 1 and 2 is mainly from N-2p (j = 1/2) and N-3p (j = 3/2).\n\nWe analyzed how the structures of Sr3NI3 are affected by applied strain (%), both under compressive and tensile stresses, while also considering the potential impact of the spin-orbit coupling (SOC) effect. We varied the amount of strain applied from compressive to tensile in steps of 1%, ranging from -4% to +4%. Throughout the compressive phase (-4% to 0%), we observed a displacement towards the Fermi level in the valence band maximum (VBM) and conduction band minimum (CBM) of perovskites, which is demonstrated in Figure 5(a). Similarly, as we applied tensile strain (+4% to 0%), we found that the VBM and CBM underwent a shift towards the Fermi level, while both VBM and CBM continued to remain at the Γ (Gamma)-point. Figure 6(a) shows the band configurations under compressive strain while considering the SOC impact of the Sr3NI3 compound. When considering compressive strain and including or excluding the SOC effect, the bond length of Sr, N, and I became shorter due to the clash between orbitals. Thus, the straight bandgap is established at the Γ (Gamma)-point whether or not the SOC effect is considered. Additionally, our observations revealed that higher levels of compressive strain lead to a decrease in the bandgap, both with and without accounting for the SOC effect.\n\nAs shown in Figure 5(c), we also investigated the effects of tensile strain application (0% to +4%) on the electrical band structure of Sr3NI3 perovskite. The band configuration of the Sr3NI3 structure under tensile strain, considering the SOC effect, is displayed in Figure 6(c). We found an increase in the bandgap as an impact of the applied tensile strain, which is indicated by the deviation of the CBM and VBM from the Fermi energy level. Figures 5(b), 5(d), 6(b), and 6(d) provide a closer view of the Γ (Gamma)-point when both the compressive and tensile strains are considered, both with and without taking into consideration the SOC effect. The increase in bond length due to tensile strain resulted in a decrease in the force between the atoms of Sr, N, and I and an increase in atomic distance. We observed that the straight bandgap is formed at the Γ (Gamma)-point, regardless of whether the SOC effect is comprised. Furthermore, we noticed that higher tensile strain levels lead to an increase in the bandgap, irrespective of whether the SOC effect is considered or not.\n\nThe energy bandgap of the Sr3NI3 structure with respect to the strain being applied, whether or not the SOC effect show in Figure 7. Compressive and tensile strain applied to the Sr3NI3 structure results in a direct bandgap in the electronic band structure. The bandgap variations of the Sr3NI3 structure during compressive and tensile strain are presented in Table 2 like previous study.15 We observed a shift in the bandgaps of Sr3NI3, from 0.582 eV to 0.879 eV (without SOC) and from 0.532 eV to 0.829 eV (with SOC) when the applied strain varied from -4% to +4%. Sr3NI3 maintains its direct bandgap characteristic across the whole range of applied strains is a remarkable observation. The Shockley-Queisser hypothesis suggests that this particular structure can potentially improve a solar cell’s efficiency.23\n\nThe PDOS of Sr3NI3 under compressive and tensile strains are illustrated in Figure (8a, 8b) and Figure (8c, 8d), respectively. As the applied strain changes from -4% to +4%, the 2p orbital of the I atom becomes active towards the VBM side, slightly lower than the Fermi energy level. The 2p orbital of N atoms controls the significant portion of the entire DOS in the conduction band region. The stable position and arrangement of the DOS represented by the 2p orbital of N are not significantly affected by the induced strain on the CBM section. However, the total DOS performance improves as the strain varies from -4% to +4%. The total DOS for the Sr3NI3 structure is estimated to be 18 electrons/eV under -4% strain and 23 electrons/eV under +4% strain in Figure 8. The hybridized N-I and Sr-I orbitals are visible across the entire energy spectrum, but they are not visible within the bandgap zone.\n\nThe PDOS of Sr3NI3 under both compressive and tensile strain is illustrated in Figure 9(a) and 9(b), and Figure 9(c) and 9(d), respectively, with the inclusion of the SOC effect. As the applied strain increases from -4% to +4% with the SOC effect, the 2p and 3p orbitals of the I atom become active towards the VBM side, appearing just below the Fermi energy level. Therefore, the 2p and 3p orbitals of the N atom control the majority of the TDOS in the region of CBM. The TDOS performance increases as the strain changes from -4% to +4%, with the SOC impact in consideration. Including the SOC effect leads to a change in the TDOS of the Sr3NI3 structure, as shown in Figure 9. Specifically, the TDOS is 17 electrons/eV at a strain of -4% and increases to 23 electrons/eV at a strain of +4%.\n\nFigure 10 illustrates the total density of states (TDOS) for Sr3NI3 under different compressive and tensile strains, both with and without the SOC effect. Specifically, Figure 10(a) shows the TDOS without SOC impact, while Figure 10(b) shows the TDOS with SOC impact. The electronic band structure of Sr3NI3 can be accurately understood by analyzing the Total Density of States (TDOS). When analyzing unstrained Sr3NI3, it becomes apparent that the orbitals of the iodine (I) atoms play a significant role in the TDOS of the valence band below the Fermi level (EF). In contrast, the Sr-5d and N-2p (j=1.5) orbitals have a negligible impact, whether the SOC effect is present or not. Compared to the valence band, the orbitals of the nitrogen (N) atoms have a significant contribution to the TDOS of the conduction band above the Fermi level (EF), while the Sr-5d and I-2p orbitals have a negligible impact regardless of the presence or absence of SOC effect. Under compressive strains (0% to -4%), the TDOS line shifts towards the Fermi level, with or without the presence of the SOC effect. This shift causes the conductive properties of Sr3NI3 materials to improve. Conversely, under tensile strains (0% to +4%), the TDOS line deviates from the Fermi level, both with and without considering the SOC effect. The conductivity of Sr3NI3 is reduced as a result of this divergence.\n\nAn investigation of the optical properties of a material involves analyzing complex dielectric functions, electron loss function, absorption coefficient, and reflectivity. Studying these properties makes it possible to determine whether the material is appropriate for photovoltaic and optoelectronic applications. The optical performance of a substance can be improved by using a thermodynamic method known as biaxial strain. This method involves adjusting the lattice parameter of the material, which can effectively modulate its optical properties and improve its performance. The stretch-ability of different optical properties of Sr3NI3 under compressive (0% to -4%) and tensile (0% to +4%) stresses were investigated in this work. The study demonstrated how the material’s optical characteristics could be improved under different strains to better its performance. The dielectric function of a material is represented by the symbol ε(ω) and is calculated as the sum of two components. The first component is the real component, denoted by ε1(ω), and the second component is the imaginary component, represented by ε2(ω).\n\nThe Kramers-Kronig transformation is applied to calculate the real component of the dielectric function, whereas the imaginary part is calculated by evaluating the momentum matrix components.77,78 The real part of the dielectric constant can be used to investigate polarization and dispersion effects in Sr3NI3.79,80 Figure 11a and 11b illustrate the real and strain-induced components of the dielectric permittivity of unstrained Sr3NI3. The graphs show the real dielectric function values of up to 7.5 eV for photon energy. The dielectric constant’s real component offers valuable insights into a material’s polarization and dispersion effects. The maximum frequency of zero, denoted as ε1(0), is an essential parameter in the real component of the dielectric function. This parameter represents the electronic contribution to the dielectric constant’s real part and is a fundamental factor in the ε1(ω) component. The calculated value of ε1(0) for cubic Sr3NI3 is 6.95, as shown in Figure 11(a). Under optical excitation, the value of ε1(ω) in the material starts increase from ε1(0) to its maximum value, after which it suddenly drops. This response indicates that the material has a high absorption capacity for light in this spectral region. The positive values of ε1(ω) for unstrained Sr3NI3 indicate that the material is highly refractive and exhibits semiconductor-like properties. The maxima of the real component of the dielectric constant in the Sr3NI3 perovskite have been shifted by applying biaxial strain. Typically, materials with higher bandgap have less intense peaks in their dielectric constant compared to materials with narrow bandgap. Therefore, due to the reduction in bandgap under increasing compressive strain, the Sr3NI3 perovskite structure exhibits a higher dielectric constant peak and experiences a shift towards lower photon energies (redshift). The Sr3NI3 perovskite structure displays a lower dielectric constant peak and a shift towards higher photon energy (blueshift) when tensile strain increases.\n\nThe behavior of the imaginary part of the dielectric function, ε2(ω), under unstrained and strained conditions, is illustrated in Figure 11(c) and 11(d), respectively. The dielectric function’s imaginary component is crucial for understanding optical absorption and the crystal structure’s capacity for storing energy caused by unbiased charge excitations. The imaginary dielectric function ε2(ω) provides information on the electronic bandgap and the energy of inter-band transitions near the Fermi level. This information can determine a material’s optical characteristics, such as its absorption and reflectivity. The absorption zone was mostly taken up of the values off ε2(ω) for Sr3NI3. Figure 11(c) shows that the most significant peaks of ε2(ω) for Sr3NI3 appear at an optical position of 6, indicating an energy of approximately 2.5 eV for absorption photons. As shown in Figure 11(d), when strain is applied, the imaginary portion of the dielectric function expands and moves toward longer wavelengths. The imaginary absorption peaks determine the movement of carriers from the valence band to the conduction band. The displacement of the peaks is attributed to fluctuations in the bandgap and lattice constant. Under compressive strain, the imaginary peaks show a red-shift, indicating a decrease in energy. Conversely, under tensile strain, the peaks exhibit a blue-shift, indicating an increase in energy, as depicted in Figure 11(d). The observed phenomenon implies that by applying either tensile or compressive strain to the Sr3NI3 compound, it may be possible to adjust the absorption spectral region, providing a means of tuning its properties.\n\nFurthermore, we found that the imaginary dielectric portion is zero when photon energy exceeds 10 eV. The absence of ε2(ω) at high photon energies (above 10 eV) implies that the material has low optical absorption and higher optical transparency.\n\nThe “electron loss function” refers to the amount of energy that electrons dissipate while traversing a dielectric substrate. The “electron loss function” refers to the amount of energy that electrons dissipate while traversing a dielectric substrate. In Sr3NI3, the peak observed in the plot of L(ω) in Figure 12(a) indicates the dissipation of energy when photons with energy higher than the material’s bandgap are emitted. The electron loss function, L(ω), can be calculated using the formula Lω=j(−1εω). Figure 12(a) illustrates that the L(ω) peaks for the cubic structure of Sr3NI3 improve within the energy range of 8.5 to 9.18 eV. The highest peak of the loss function was observed at 8.8 eV, while the lowest peak was found at 2.5 eV in the case of Sr3NI3. The negligible presence of L(ω) peaks below 2 eV in Sr3NI3 indicates that it could be an efficient optical absorption layer for optical photon spectra and IR. The strain-applied Sr3NI3 displays a loss function for up to 10 eV of photon energies. According to Figure 12(b), the energy losses of Sr3NI3 can be predicted under different biaxial compressive and tensile strain conditions. According to the findings, increased compressive strain causes a large redshift, suggesting decreased photon energy and optical loss for all structures. Conversely, increasing tensile strain leads to a blueshift, indicating an increase in photon energy and optical loss. The loss function of Sr3NI3 plays a crucial role in its overall performance and should be carefully considered during the design and fine-tuning of these materials for specific applications.\n\nThe absorption coefficient is an essential attribute of Sr3NI3 since it determines how much light the material absorbs at different wavelengths. The absorption coefficient of Sr3NI3 is impacted by various factors such as crystal structure, purity, and thickness of the material. The structure of the optical absorption coefficient for Sr3NI3 exhibits similar characteristics to that of the imaginary part of the dielectric constant for any given configuration. The visible region of the electromagnetic spectrum often contains the majority of the sun’s radiation, making it a region with a higher absorption coefficient. Figure 12(c) and 12(d) display the absorption coefficient of Sr3NI3 perovskite material as a function of photon energy, with and without biaxial strain, respectively. Under tensile strain, the absorption peak of Sr3NI3 exhibits a significant blueshift, while it displays a large redshift under compressive strain.\n\nThe compressed structure of Sr3NI3 has a more substantial absorption capacity in the visible spectrum, while the tensile structure has a lower absorption capacity than the unstrained material. Increasing compressive strain improves the absorption coefficient of Sr3NI3 in the visible range, which is crucial for solar cell applications. Conversely, increasing tensile strain causes the absorption coefficient of Sr3NI3 to decrease in the visible region. Optimizing the absorption coefficient of Sr3NI3 perovskite is essential in the design of photovoltaic devices based on this material. It can potentially improve the efficiency of these devices, making them more competitive with other types of solar cells.\n\nReflectivity refers to the amount of light reflected by Sr3NI3 perovskite when exposed to electromagnetic radiation, particularly visible light. The overall reflectivity of perovskite materials can vary significantly depending on factors such as composition, crystal structure, and surface shape. Additionally, the incident light’s wavelength and angle of incidence can also impact the reflectivity of Sr3NI3 perovskite. Figure 12(e) and 12(f) illustrate the reflectivity of Sr3NI3 perovskite as a function of photon energy in the absence and presence of biaxial strain, respectively. The reflectivity of Sr3NI3 perovskite shows the most variation between photon energies of 0 and 3.7 eV, with the peak reflectivity occurring at 0 eV. With increasing compressive strain, the reflectivity of the Sr3NI3 framework in the visible region increases, which is significant for the development of solar cells. Conversely, when tensile strain is applied, the reflectivity of the Sr3NI3 structure decreases in the visible region. These observations on the optical properties of Sr3NI3 are consistent with previous research. It is generally understood that materials with bandgaps lower than 3.1 eV tend to exhibit better performance in applications involving visible light.\n\n\nConclusion\n\nSr3NI3 has been researched for its possible use in optoelectronics and photovoltaics because of its good optoelectronic and electrical characteristics, such as a high absorption coefficient, high carrier mobility, and extended carrier lifetime. We thoroughly investigated the properties of Sr3NI3, an inorganic perovskite, using the first-principles DFT approach. We also studied the optical characteristics under various strains and found that the material displayed absorption peaks in the UV to visible range, which were either red-shifted or blue-shifted, depending on the type of strain applied. The analysis revealed that the direct bandgap of Sr3NI3’s structure was measured to be 0.733 eV. However, when considering the SOC effect, the electronic bandgap of Sr3NI3 decreased to 0.711 eV. The results clarify that with and without considering the SOC effect, the bandgap of Sr3NI3 rises as compressive strain increases, while it increases with induced tensile strain. Furthermore, it was observed that the peak value of the dielectric constant of Sr3NI3 changes towards higher photon energy (blueshift) when exposed to more significant tensile strain, while it shifts towards lower photon energy (redshift) when placed under tremendous compressive strain. These findings may open up possibilities for further exploration into the development of optoelectronic and photovoltaic devices based on Sr3NI3.", "appendix": "Data availability\n\nZenodo: Data and code for Sr3NI3. https://doi.org/10.5281/zenodo.7958201 81\n\nThis project contains the following underlying data:\n\n• With SOC (code that is used to generate the data files for the SOC effect)\n\n• Without SOC (Code that is that are used to generate the data files without SOC effect)\n\n• Data (In this folder, I included all the raw files that are used to generate all the figures)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgement\n\nThis research was funded by the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R29), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.\n\n\nReferences\n\nGong J, Darling SB, You F: Perovskite photovoltaics: life-cycle assessment of energy and environmental impacts. 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Publisher Full Text" }	[ { "id": "211713", "date": "01 Nov 2023", "name": "Congcong Wu", "expertise": [ "Reviewer Expertise Functional thin film materials" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a study on the influence of strain on Sr3NI3 perovskite using first-principles density functional theory (DFT). The authors investigate various properties of Sr3NI3, such as bandgap, density of states, electron charge density, structural properties, dielectric properties, loss function, and absorption coefficient. The results suggest that strain could be a promising approach in solar cell technology for enhancing energy production and managing light. The experimental evidence provided in the paper supports the authors' claims. Nonetheless, there are several language, expression, and graphic issues in the manuscript that somewhat hinder objective assessments of its accuracy.\nThe language in this paper could be more condensed. Besides, Please change all these expressions such as “a structure’s stability” and replace it with “the stability of a structure”.\n\nIn the first paragraph of page 5, “The strontium (Sr) and nitrogen (N) atoms in the SrN4 tetrahedra are bound together by covalent bonds with varying bond lengths ranging from around 2.23 to 2.51. Iodine (I) atoms generate weak van der Waals interactions with the surrounding” “may be used to represent the fractional coordinates of nitrogen (N), iodine (I) strontium (Sr) atoms in Sr3NI3 atoms rather than being directly bound to strontium or nitrogen atoms”. Similar abbreviations are repeated.\n\nIn the section “Electronic properties” and “Strain-induced electronic properties”, please pay attention to subscripts like “Sr3NI3”; In the section “Strain-induced electronic properties”, the abbreviations of valence band maximum (VBM) and conduction band minimum (CBM) came again. Please unify the expression “energy bandgap” or “bandgap energy” in this paper, such as in Table 1. In the section “Charge density”, “DOS” uses the abbreviation before the full name appears below (section “Electronic properties”). Similarly, TDOS in the section “Strain-induced electronic properties”. Please check all the details like these in this whole paper.\n\nThe textual information representing each line in Figure 9 is not clear.\n\nSome images are located before the first appearance of text in this paper (e.g., Figure 11).\n\nIn the last paragraph of page 13, the sentence \"The \"electron loss function\" refers to the amount of energy that electrons dissipate while traversing a dielectric substrate\" appears twice.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1005
https://f1000research.com/articles/12-1004/v1	18 Aug 23	{ "type": "Research Article", "title": "Identification of macronutrients by FT-IR analysis and physicochemical characterization of snacks elaborated from quinoa (Chenopodium quinoa Willd) and sacha inchi (Plukenetia volubilis)", "authors": [ "Sandra María Castillo-Guaca", "Karen Sofia Muñoz-Pabon", "Jesús Eduardo Bravo-Gómez", "Diego Fernando Roa-Acosta", "Juan Fernando Vergara Escobar", "Sandra María Castillo-Guaca", "Jesús Eduardo Bravo-Gómez", "Diego Fernando Roa-Acosta", "Juan Fernando Vergara Escobar" ], "abstract": "Background: Currently, the consumption of high-protein foods that replace animal sources is increasing, a trend that promotes the design of new food systems. Spectroscopy methods with physicochemical tests allow for rapid and reliable identification of macronutrients and bioactive compounds. Methods: Snacks were made using hyperproteic quinoa flour (HPQF) and sacha inchi flour (SIF) through an extrusion process and subsequent compression molding. Spectra infrared (IR) analysis was used to identify macronutrients such as starch, proteins, lipids, and fiber. Specific frequencies were selected that provided the greatest discrimination of the sample. Physicochemical measurements were performed using extractable (EPC) and hydrolyzable (HPC) phenolic compound analyses, carotenoid content, and antioxidant capacity through ABTS• + (2,2-azinobis- 3-ethylbenzothiazoline-6-sulphonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), and FRAP (ferric reducing antioxidant power) methods. Color and texture parameters of the snacks were also measured. Results: The identification of macronutrients using Fourier transform infrared spectroscopy – attenuated total reflectance (FTIR–ATR) was as follows: lipids showed two characteristic peaks at 2870 and 2960 cm−1; protein showed three peaks at 1540, 1630, and 1660 cm −1; starch showed two peaks at 1170 and 1155 cm −1. Regarding the content of free polyphenols, hydrolyzable polyphenols and carotenoids, the mixtures added with the highest inclusion of quinoa, i.e. 50%, showed the highest values of 3.05 mg GAE/g, 14.16 mg GAE/g and 14.06 µg-β carotene/g of dry base sample, respectively. The snacks showed significant differences (p<0.05) in the antioxidant properties determined by the ABTS and FRAP methods, with the highest values in the samples with 50% HPQF. The inclusion of HPQF was associated with a higher browning index, and snacks with a higher quinoa content exhibited greater hardness and crispness. Conclusions: Snacks were obtained with protein percentages between 26–33%, containing bioactive compounds, gluten-free, and without the addition of oil during their production.", "keywords": [ "Gluten-free foods", "Carotenoids", "Extrusion", "High-protein quinoa flour", "Plant-based snacks", "Polyphenols", "Antioxidant properties" ], "content": "Introduction\n\nAccording to the Good Food Institute, the majority of plant-based foods have a high demand in the market because consumers are seeking healthier and environmentally friendly options. In this category of plant-based foods, a food technology trend for 2023 will be to produce matrices with higher protein content. In this regard, products such as quinoa, amaranth, sacha inchi, and others are presented as alternatives for the production of vegan, gluten-free foods with high protein and bioactive compound contents.1–4 Quinoa has a protein content of 18%,5 and its digestibility after thermal and high-pressure treatment is above 90%.6 On the other hand, sacha inchi has a protein content of 25–30%7 and a digestibility of 41% after extrusion.8 Studies on these types of plant matrices show that processes like extrusion can improve the availability of nutrients such as protein, certain compounds affecting antioxidant activity, and the content of polyphenols.9–11\n\nWithin the food category, extruded snacks are an alternative with a market size of $48.3 billion in 2019, projecting a compound annual growth rate (CAGR) of 4.4%. Additionally, the launch of gluten-free products increased by 73% and vegan products by 196%, partly attributed to policies and claims of health properties associated with the food products offered to consumers.12 Therefore, it is necessary to promote research to evaluate the influence of new vegetable sources in the elaboration of snacks, the conditions for their processing and their physical, chemical and sensory characteristics. This technology is widely used to improve their nutritional quality, with an emphasis on protein content.13 In this type of food, textural properties such as hardness, shear resistance, tensile strength, freshness, and crunchiness are crucial for controlling processing operations and achieving the desired quality attributes of the finished product and consumer acceptability.14\n\nAccording to Ref. 15, these foods can contribute to increased energy intake and body weight gain in adults, considering that they are consumed at any time of the day during leisure activities (such as in front of the television or computer). Providing healthy snacks is an attractive alternative for the food industry. Studies in Europe have considered quinoa as part of a variety of protein-rich crops that have the potential to diversify global production systems focused on a limited number of species. This would help diversify diets and offer the possibility of generating new plant-based feeding alternatives that contribute to reducing greenhouse gas emissions and negative impacts on the environment.16 On the other hand, sacha inchi presents beneficial effects for various activities such as neuroprotective modulation, dermatological effects, antidyslipidemic effects, antioxidant properties, anti-inflammatory effects, antiproliferative effects, and antitumor effects. These benefits are associated with its bioactive compounds, particularly essential fatty acids, proteins, and phytochemicals.4\n\nFourier transform infrared spectroscopy – attenuated total reflectance (FTIR–ATR) analysis allows for the identification of the position, intensity, and shape of infrared peaks,17 which can reveal chemical bonds and quantify chemical structures of macromolecules such as carbohydrates, lipids, fiber, moisture, bioactive compounds, antinutrients, and other compounds of interest. This analysis helps assess the quality of the food matrix.18–21 Several chemical assays have been designed to measure the molecular or cellular level capacity for radical elimination, reducing power, and other specific attributes of antioxidants, as well as the overall inhibition of oxidation in foods and more complex biological systems.22 In this study, we measured antioxidant values using the, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) methods.\n\nThe objective of this study was to evaluate the inclusion of high-protein quinoa and sacha inchi cake in extruded and puffed snacks, resulting in a high-protein food with bioactive compound content, to offer vegan and gluten-free food alternatives.\n\n\nMethods\n\nThe cereals used in this study were provided by “SEGALCO” Company S.A.S. Popayán, Colombia. Quinoa was ground in a quinoa polishing machine (500-T, Mavimar, Colombia) with a processing capacity of 60 kg/h. This grain abrasion process allows obtaining a high-protein quinoa flour (HPQF). Sacha inchi almond was received in almond form and then subjected to a pressing process (CGLDENWALL, store model K28, Shanghai, China). The 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) diammonium salt, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) were acquired from Thermo Fisher Scientific (Massachusetts, USA). Ethanol (96%), acetone, hydrochloric acid, petroleum ether (98%), Butylhydroxytoluene (BHT), and Folin & Ciocalteu’s phenol reagent were obtained from Commercial Outsourcing (Manizales, Colombia).\n\nThe snack pellets were prepared according to the formulations shown in Table 1, treatments T1, T2 and T3 correspond to the different inclusions, which consisted of whole rice flour (WRF), HPQF, and sacha inchi flour (SIF) using a twin-screw industrial extruder (CY65-II TWN SCREW EXTRUDER, Qingdao, China). T1: WRF 55%, SIF 25% and HPQF 20%; T2: WRF 45%, SIF 25% and HPQF 30%; T3: WRF 25%, SIF 25% and HPQF 50%. The mixtures were moistened to a 32% moisture content by spraying water and continuous mixing. Then, the extrusion-cooking process was initiated at temperatures of 60°C, 80°C and 110°C, with a screw rotation speed of 200 rpm, and a nozzle diameter of 3 mm. The snack pellets were dried at 100°C for 30 minutes. Afterward, these pellets were moistened to a moisture content of 14%, cut to a length of 5 mm, and inflated using a machine (RICE CAKE MACHINE SYP4006, BUCHEON-SI, GYEONGGI-DO, Korea). The blowing conditions were a temperature of 230°C, a pressure of 100 bar, and a heating time of 10 seconds.\n\nThe proximate composition of the macronutrients protein, lipids, dietary fiber, ash and moisture of taro and sacha inchi flours was measured according to the methods proposed by the AOAC (Association of Official Analytical Chemists, 1990)15 and the carbohydrate content was estimated by difference. The details of the method are explained in the protocols uploaded to the repository.23\n\nProtein\n\nWe weighed 0.2 g of sample plus 1 g of Kjeldahl catalyst, added 10 mL of sulfuric acid and heated until the samples were completely clear and translucent, free of organic matter, in the laboratory Kjeldahl digester (Raypa MBC-6/N, Spain). After this process they were cooled to room temperature, then passed to the Raypa distillation unit, and each sample was titrated with 0.1 N HCl.\n\nLipids\n\nTo start the lipids determination, 1 g of sample was weighed into the extraction cartridges and 80 mL of petroleum ether was added, and this was transferred to the rack of the laboratory Soxhlet and Randall extractor (SX-6MP, RAYPA, Spain). After the extraction time the samples were placed in an oven at 60 °C for 1 hour to remove the remaining ether.\n\nFiber\n\nTo start the fiber measurement, 1–2 g of sample were transferred to the laboratory fiber extractor (F-6P Fibertest, Spain). Then, 150 mL of 0.255 N H2SO4 was added to an Erlenmeyer flask, the heating knob was adjusted to boiling point and left boiling for 30 min. After this time, it was filtered and washed with distilled water three times using 30 mL of water each time.\n\nThe FT-IR spectra of the flours and extrudates were evaluated using an FTIR spectrometer (IS50 Nicolet, Thermo Scientific, USA) with an ATR accessory. The intensity evaluation was performed on the spectrum after baseline subtraction, applying a 10-point smoothing, and normalizing the data from 0 to 1 in Origin v18 software. The height of the transmittance bands was determined from their baseline. The specific areas of interest studied included protein (amide I, II groups), protein secondary structure in the IR regions of approximately 1200–1800 cm-1. Additionally, the crystalline regions of starch were approached from 1200 to 800 cm-1 to evaluate changes in their relative intensities. The amide I region (1700–1600 cm-1) located within the protein’s spectral range was subjected to second derivative analysis to observe overlapped peaks in that region, corresponding to β-sheets and α-helix.\n\nThe extraction and analysis of EPC were based on the method described in Ref. 24 with slight modifications. Approximately 2 g ± 0.0500 g of sample sieved through a 200 μm sieve were weighed. Two extractions were performed, the first with ethanol/H2O (80/20, plus 1% formic acid) and the second with acetone/H2O (70/30), the supernatant from both extractions was combined and brought to a final volume of 20 ml with deionized water. The extract was kept at -80°C for colorimetric determination by the Folin-Ciocalteu reaction or evaluation of antioxidant capacity. The sediment was also kept at -80°C for the determination of hydrolysable phenolic compounds (HPC).\n\nThe extraction and analysis of HPC were based on the methods described by Ref. 24, with slight modifications. Approximately 0.8 g ± 0.0050 g of sediment from EPC was weighed, and then 10 mL of methanol/H2SO4 (90/10) was added. The tubes with the sample and solution were left for 22 hours at 85°C with magnetic stirring. The extract was kept at -80°C for colorimetric determination by the Folin-Ciocalteu reaction.\n\nThe content of EPC and HPC was expressed in mg of gallic acid equivalents per gram (GAE/g), of dry matter. All analyses were performed in triplicate.\n\nTo measure the antioxidant properties, EPP extracts were used.\n\nABTS\n\nIn a test tube, 4 mL of ABTS solution were placed and covered completely with aluminum foil. To initiate the process, 135 μL of standard solution was added and then mixed in a vortex for 5 s. The blank reagent consisted of 4 mL of acetate buffer and 135 μL of ethanol. The zero point was mixed with 4.5 mL of ABTS solution and 135 μL of ethanol. The test tube was closed and allowed to react for 30 min, and then the absorbance was measured at a wavelength of 729.7 nm using a UV-Vis spectrophotometer (GENESYS™ 10S Thermo Scientific, USA).\n\nDPPH\n\nIn a test tube, 3.9 mL of DPPH solution and 100 μL of standard solution were applied to initiate a reaction by vortex agitation for 5 s. The blank reagents (control) were carried out with ethanol. The zero point was adjusted by adding 3.9 mL of DPPH solution and 100 μL of ethanol. The sample was covered for 30 min to initiate the reaction, and then the absorbance was measured at 517 nm using a UV-Vis spectrophotometer (GENESYS™ 10S Thermo Scientific, USA).\n\nFRAP\n\nIn a test tube, 1.8 mL of solution containing (2.5 mL of 0.01 M TPTZ, 2.5 mL of 0.02 M FeCl3, and 25 mL of 0.3 M sodium acetate buffer pH 3.6), 180 mL of distilled water, and 60 μL of sample solution were applied, vortexed for 15 s to initiate a reaction, and then left at 37°C for 30 min. The blank reagents (control) were carried out with distilled water, and then the absorbance was measured at 595 nm using a UV-Vis spectrophotometer (GENESYS™ 10S Thermo Scientific, USA).\n\nThe extraction of carotenoid pigments was performed following the methodology described by Refs. 25–27 with modifications, in two stages: solid-liquid extraction and liquid-liquid extraction, of which the first extraction stage was adjusted.\n\nThe mechanical properties of the extrudates were determined following the method by Ref. 28 with slight modifications. They were analyzed using a texture analyzer (Shimadzu EZ TEST SM, model 500N-168, Japan). The area under the curve (S; N.mm) and the number of peaks (n) exceeding 1.5 N were calculated from the force-deformation curves and used to calculate the spatial frequency of ruptures (Nsr) (equation 1), average crushing force (Fcr) (equation 2), and crispness work (Wcr) (equation 3).\n\nThe color was determined using a Konica Minolta CM-5 Spectrophotometer colorimeter, controlled by SpectraMagic NX software, with illuminant D65 and an observer angle of 10°. The values of luminosity (L), green/red chromaticity (a), and blue/yellow chromaticity (b) were obtained, from which the chromaticity values (C) (equation 4), hue angle (h°) (equation 5), total color difference (∆E) (equation 6), whiteness index (WI) (equation 7), and browning index (BI) (equation 8) were calculated.\n\nA completely randomized design was employed to assess the incorporation of quinoa in high protein snacks formulation. Three inclusion levels were considered: 20%, 30% and 50%. The response variables included polyphenols, antioxidant activity, carotenoids, texture, and color.\n\nThe results were presented as the mean ± standard deviation of triplicate or more experiments, as appropriate. To compare the means, a one-way analysis of variance (ANOVA) was conducted. Significance of differences between means was determined at p < 0.05 by Tukey’s new multiple range test. Statistical data and curves were analyzed using Origin v 18, open-access alternatives that can perform an equivalent function are Oracle SQL, Matplotlib and R.\n\n\nResults and discussion\n\nThe following results were analyzed according to the data found in the repository.29\n\nTable 2 presents the proximate composition of the extruded and popped snacks containing HPQF, SIF, and rice.\n\nThe protein, lipid, ash, and fiber contents of snacks T1, T2, and T3 significantly increased (p < 0.05) with the inclusion of HPQF, while the carbohydrate content decreased. Quinoa flour contains a higher amount of protein, fiber, and lipids than other flours like rice and corn, which could be the reason for the increase in these macronutrients in the snacks. In previous studies,30 we reported that HPQF contained 30.12 g/100 g of protein, a higher amount than rice flour (9.4 g/100 g) and corn flour (7 g/100 g). On the other hand,31 reported a protein content of 11.45 g/100 g in defatted quinoa flour, higher than other cereals.\n\nAccording to studies, the removal of pericarp and aleurone layers of grains during milling affects the protein content.32 In this sense, quinoa, due to its size and structure, requires special conditions for milling and dehulling that allow extracting the endosperm without breaking it and obtaining a hyperprotein quinoa flour (HPQF) at a low cost and with reduced environmental impact. Based on this, in this study and our preliminary studies,33 we have proposed obtaining quinoa flour with a protein content close to 30%, allowing inclusions in food production with higher protein contents than those available in the market. Therefore, the addition of SIF is also an alternative that allows obtaining foods with a high protein content. Comparing the protein content of the snacks presented in Table 3 with others made from vegetables, we can observe a higher value in our snacks.34,35\n\nHowever, it is also important to highlight that the superior nutritional value of proteins from pseudocereals such as quinoa and sacha inchi seed over those from cereals also lies in their balanced composition; they are richer in some essential amino acids, particularly high in lysine, the limiting amino acid in most cereal grains, their essential amino acid balance is excellent due to a wider range of amino acids than in cereals and legumes.36 The sacha inchi cake on its part contains mainly amino acids such as glycine 201–215 mg/g protein, leucine 28–39 mg/g protein, threonine mg/g protein, 48–64 mg/g protein, and isoleucine 30–36 mg/g protein.37 It can be observed that, except for lysine and leucine, this would be meeting the amino acid requirements in the diet recommended by FAO for all age groups, except infants, so the union with quinoa is ideal to obtain the balance of amino acids in the snack.\n\nFourier transform infrared spectroscopy was used to study the composition of the snacks and to analyze the modifications induced by HPQF inclusions.\n\nInfrared spectroscopy is a physicochemical analytical technique used to identify conformational changes in functional groups and structural properties; however, the interpretation of a spectrum presented in a food sample can be complex due to the interaction and amount of compounds present in the sample, because the signals often overlap, which is why subtle changes are difficult to detect.18 For this reason, in this study, spectral analysis was performed on individual flours in order to detect the effect of the inclusion of the flours in the snack and the extrusion process.\n\nThe FT-IR spectra of quinoa, rice and sacha inchi flours and extrudates T1, T2 and T3 are shown in Figure 1. The FT-IR spectra show the three separate zones to analyze starch, proteins and lipids. The bands obtained by FT-IR spectroscopy were explained according to previous literature reported for similar flours.19\n\n(A) Complete spectra, (B) characteristic starch bands, (C) characteristic bands protein, (D) characteristic bands lips. (T1) treatment 1 with addition of 20% quinoa; (T2) treatment 2 with addition of 30% quinoa; (T3) treatment 3 with addition of 50% quinoa. WRF: Whole rice flour; SIF: Sacha inchi flour; HPQF: High protein quinoa flour.\n\nThe wavelength range from 800 to 1200 cm-1 corresponds mainly to the stretching vibrations that can be divided thus C-O, C-O-C; C-O, C-O-H; and C-O-C, C-O-H characteristic in starch (Figure 1B). The IR spectra of the snacks (T1, T2 and T3) and HPQF, WRF in this region are characterized by three main modes with maximum transmittance at 1078, 1014, 996 cm-1. The IR bands at 1078 and 1014 cm-1 are associated with the ordered and amorphous structures of starch, respectively and the band at 999 cm-1 is related to hydrated crystalline samples.18\n\nThe main band of raw quinoa flour at 996 cm-1 can be attributed to the contribution of starch and corresponds to the C-O bending of the glycosidic bond. As seen in Table 2, according to the statistical analysis the intensities in the 1078 band were significantly affected (p<0.05) by the addition of HPQF, presenting values of 0.218, 0.3535, 0.3747, 0.3822, 0.3974 and 0.3987 for SIF, HPQF, T3, WRF, T1 and T2, respectively. The intensity is significantly higher for SIF followed by HPQF i.e., for raw flours, this indicates a loss of the ordered structure in the starch fraction, which changes after the extrusion and compression process.\n\nAs for the protein analysis, the determination of the secondary structure of this macronutrient is mainly based on the analysis of the amide I band between 1700 and 1600 cm- 1. The peaks of the samples confirmed the typical characteristics of the protein spectrum, with amide I (1643 cm -1) and amide II (1533 cm- 1) (Figure 1C), of the protein samples, arising from specific stretching and bending vibrations of the protein backbone. Similarly, for sacha inchi flour, the maximum range for amide I was 1638 cm -1, whereas for amide II it was 1517 cm -1. The distinct peak in amide I was mainly caused by N-H stretching vibrations, whereas the single peak in amide III was caused by C-N stretching and N-H bending vibrations.38\n\nTo reveal a resolution of the molecular components of the proteins, a second derivative analysis on the ATR-FTIR spectra of the middle band I (Figure 2) and a Gaussian curve fitting method were employed to quantify each of the secondary components of the proteins. As shown in Figure 2, the extrusion process caused changes such as denaturation or hydrolysis, unfolding/unwinding of coiled/uncoiled structures, aggregation to form a new conformation and separation to form subunits in an unfolded/unwound manner. A similar situation was presented by Ref. 39, who performed a hydrolysis process through fermentation of oats. The strong vibrations of stretching, bending and the appearance of displaced or new peaks indicate that the quinoa, sacha and rice flours were modified by the extrusion and compression processes.\n\n(T1) treatment 1 with addition of 20% quinoa; (T2) treatment 2 with addition of 30% quinoa; (T3) treatment 3 with addition of 50% quinoa. WRF: Whole rice flour; SIF: Sacha inchi flour; HPQF: High protein quinoa flour. Values are ± standard deviation. a, b, c: different letters are significantly different (p < 0.05) between sample.\n\nAccording to Ref. 40 the region corresponding to amide I is divided into 1651–1660 cm-1 which is assigned to α-helix, between 1660–1700 cm-1 would belong to β-turns, between 1610–1640 cm-1 can be considered as β-sheets and between 1640–1650 cm-1 is assigned to random spiral conformation.\n\nThe percentages of the secondary structures of the pure and processed treatments were significantly affected by the addition of quinoa and by the technological process (extrusion and compression). The percentages of the secondary structures of the raw and processed treatments were significantly affected by the addition of quinoa and by the technological process (extrusion and compression). The percentages of the areas can be observed in Figure 2. With respect to the β-sheet conformations, T1 and WRF do not have significant differences and present the highest percentages of 36.39 and 27.96 respectively; T2, T3, and SIF do not present significant differences. Also, random coil conformation (RCC) is statistically different for WRF (6.31%) and HPQF (3.04%), and the other samples do not present significant differences. On the other hand, the α-helix is higher in HPQF (26.45%) statistically different from SIF (20.34%), T3 (19.65%) and WRF (14.66%). Finally, the β-turn presents the largest area in T3 (54.027%) and T2 (53.56%) without significant differences, WRF (47.27) and HPQF (47.06%) were classified in the same group, i.e., they do not present significant differences and T1 (40.26%) presented the lowest value.\n\nAccording to the above, the different conformations represent the changes that occur in the protein structures due to the flour mixture and the transformation process. The results of the FTIR analysis confirmed that the proteins of the flour and processed sample presented normal characteristics of a protein spectrum.\n\nThe lipid bands of the raw and extruded flours were at 1745, 2853 and 2923 cm-1 (Figure 1B), corresponding to the C=O, CH2 symmetrical stretching and CH2 asymmetrical stretching groups, respectively. As can be seen in Table 2, in the 1745 band, the highest intensity was recorded by the sacha cake flour followed by T3, T2 and HPQF; these last two do not present significant differences and recorded at 1745, 2853 and 2923 cm-1, which correspond to the C-H vibration.\n\nAccording to the results presented in Figure 3A, the EPC in the extrudates varied significantly (p < 0.05) from 1.29, 1.95, and 3.05 mg of GAE/g for T1, T2, and T3, respectively, with the highest being the one containing 50% quinoa. Similarly, the HPC in extrudates varied significantly (p < 0.05) between 11.08, 12.45, and 14.16 mg GAE/g for T1, T2, and T3, respectively. The highest values were presented by the mixtures with a higher content of quinoa. Polyphenols are active compounds produced during the secondary metabolism of plant matrices, containing one or more aromatic rings and hydroxyl groups in their structure. They exhibit properties such as antioxidant, anti-inflammatory, antimicrobial, anti-aging, cardiotonic, diuretic, laxative, hypoglycemic, and anticorrosive.41 According to Ref. 42, quinoa contains the following polyphenols: rutin, vanillic acid, ferulic acid, kaempferol, quercetin derivatives, p-coumaric acid, daidzein, caffeine, caffeic acid, pinocembrin, apigenin, and pinocembrin. In this type of plant-based matrices, polyphenols are divided into two types, EPC and HPC. The former is found in free form in the matrix and requires extraction with water or polar organic solvents such as methanol, ethanol, acetonitrile, acetone, or their water mixtures for their identification. The latter requires hydrolysis with acid or alkali to release phenolic compounds such as ferulic acid and lignans.43 In previous studies,6 we have shown how extrusion contributes to the release of phenolic compounds in snacks made from quinoa. If we compare those studies with the current one, the addition of sacha inchi may increase the content of both EPC and HPC, as the SI seed contains a level of polyphenols that can vary between 6.46–8.00 mg of GAE per gram of seed on a wet basis. Although these phenolic compounds can be altered by heat treatments and pressure, they are still present in the matrices.44\n\nA: extractable phenolic compounds (EPC) and hydroxidizable phenolic compounds (HPC). Values are ± standard deviation. a, b, c: different letters are significantly different (p < 0.05) between treatment.\n\nAntioxidant properties\n\nAs seen in Figure 3B, the inclusion of HPQF has an effect on the measured values through ABTS and FRAP (p < 0.05). There was no significant effect with the DPPH method; however, with all the methods, the inclusion of quinoa led to an increase in antioxidant activity.\n\nThe extrusion process, which combines high temperatures above 100°C and high pressures, can change the proportion of various phenolic compounds that enhance antioxidant activity. Vanillin and vanillic acid can be produced by thermal decomposition of ferulic acid, while p-hydroxybenzaldehyde can be formed from p-coumaric acid. The caffeic acid present in quinoa is heat-sensitive and may be reduced during thermal processes.45 Some phenolic compounds accumulate in the cellular vacuoles in cereal-based matrices, and it has been shown that the extrusion process can help release phenolic acids by breaking down cellular components and cell walls.10,46\n\nAs seen in Table 3, the BI of T3 is the highest (56.29). According to research, the formation of Maillard browning pigments (particularly melanoidins) during the thermal processing of foods has antioxidant activity and can contribute to an increase in polyphenols and the capacity for free radical scavenging. This could be due to the dissociation of the conjugated phenolic fraction during the thermal treatment, followed by polymerization and/or oxidation reactions, and the formation of phenolic compounds that are not present in any of the extruded products (rice/quinoa/sacha inchi).6,47,48 For example,49 developed cookies from quinoa flour, and at a cooking temperature above 180°C, they demonstrated increased stability of the molecules and an increase in antioxidant activity, attributed to the possible decomposition of phenols or their degradation products that could react with the DPPH reagent.50 made cookies from barley and wheat and also attributed the possible increase in antioxidant activity to the formation of proline-L-lysine during the thermal treatments.\n\nThe differences in antioxidant content measured through the three methods are due to the nature of each technique. The FRAP assay, in general, is a non-radical-based method based on electron transfer and has little relation to the process of radical scavenging that occurs in lipid systems. It also has little correlation with other measurements of antioxidant activity. On the other hand, the ABTS* assay has been used to measure the total antioxidant activity of pure substances, body fluids, and plant materials. The similarity between FRAP and ABTS may be due to the fact that the latter is also generally classified as an electron transfer-based method that applies the hydrogen atom transfer mechanism. However, the quantification through DPPH does not mimic the radical scavenging mechanism of antioxidants in real foods or biological systems due to the lack of oxygen radicals in the assay.22\n\nThe β-carotene content in the snacks significantly increased (p < 0.05) with the addition of quinoa and was found to be in the range of 7.31, 7.93, and 14.05 μg/g db (Figure 3C). The higher carotenoid content in T3 could be attributed to the incorporation of HPQF. Studies have revealed the presence mainly of α-tocopherol and β-tocopherol, 30.18 and 36.11 μg/g, respectively, in whole quinoa flour.51 According to these contents in raw quinoa, it is possible to observe that the extrusion process decreases the presence of carotenoids. Previous studies have shown that the carotenoid content of quinoa extrudates significantly decreases after extrusion and baking, possibly due to the thermal decomposition of these compounds, which are more prone to degradation due to their chemical structure with unsaturated covalent bonds.6 On the other hand, the addition of SI (ingredient) apparently does not contribute carotenoids.52 reported that the total carotenoids in different SI cultivars ranged from 0.07–0.09 mg/100 g of seed, which means that the addition of SI does not increase the β-carotene content in the snacks.\n\nTable 3 shows the textural properties of the extruded snacks, and it can be observed that the inclusion of quinoa has an effect on each parameter (p < 0.05). T3 presents higher values in hardness, indicating that more force is required to deform the food, resulting in lower Nsr values associated with the number of peaks and higher Wcr (crunchiness work). This could be attributed to the higher protein and fiber content and lower starch content (associated with carbohydrates) in T3. According to Ref. 53, the inclusion of protein above 20% has a positive correlation with hardness, which increases due to the reduced expansion caused by protein during extrusion, preventing the formation of air bubbles and resulting in thicker cell walls and therefore harder extrudates.54 developed snacks from soybean residues and also found that higher protein and fiber content, with lower starch content, result in harder and less crispy snacks.\n\nThe color parameters found in the snacks were L* values of 68.89, 64.95, and 61.73 for T1, T2, and T3, respectively; a* values of 6.87, 8.34, and 8.78; b* values of 22.66, 23.49, and 23.02; C* values of 5.43, 5.64, and 5.64; h° values of 1.28, 1.23, and 1.21; color difference ranging from 30.98 to 67.08; whiteness index of 60.89, 56.96, and 54.47; and browning index of 46.56, 53.52, and 56.29 (Table 3). Only the b* parameter was not significantly affected by the inclusion of quinoa (p > 0.05).\n\nThe increase of HPQF (20, 30, and 50 g/100 g) in the extrudates caused a decrease in L* and WI* as seen in Figure 4, as well as an increase in a* and BI. The highest values of L, h°, and BI were found in the product with 20% quinoa. The nature of the raw materials used was the main cause of the color changes in the obtained snacks, and this effect was mainly attributed to HPQF, as SIF remained constant in the formulation. This result was due to the effect of quinoa flour inclusion, which caused darkening of the color (browning index) of the mixture, probably due to the presence of Maillard reaction compounds. The color of the extruded products is the result of non-enzymatic browning reactions, and the degradation of pigments present in the raw materials. The caramel color produced during extrusion provides a brown color.\n\n(T1) treatment 1 with addition of 20% quinoa; (T2) treatment 2 with addition of 30% quinoa; (T3) treatment 3 with addition of 50% quinoa.\n\nThe lowest a parameter was found in the snacks with 20% quinoa flour (Table 3), exhibiting a weaker red tone than the other samples. The increase in a* with the increase of HPQF enhanced the red tone of the snacks. The b* parameter of all extrudates leaned towards yellow, and the most intense yellow tone was found in treatment 3. The increase of quinoa flour in the formulation decreased C* and h° parameters, thereby changing the color from yellow to red with the increase of HPQF flour. This browning effect is consistent with what has been reported by other researchers in extruded snacks using insects,55 and it is also in line with the findings reported by Ref. 56 in snacks made from wheat.\n\n\nConclusions\n\nThe inclusion of 50% quinoa flour significantly increased the protein content, EPC (extractable phenolic compounds), HPC (hydrolyzed phenolic content), antioxidant properties, and carotenoid content. However, texture properties such as Nsr (spatial frequency of ruptures) decreased, and Wcr (crispness work) increased with the inclusion of quinoa, resulting in harder and less crunchy snacks. The color was also significantly affected by the inclusion of HPQF. Snacks with 30% and 50% quinoa content exhibited a darker color, primarily due to the generation of pigments, mainly melanoidins, during the Maillard reaction. This reaction is common during the extrusion process, where carbohydrates and proteins are present.\n\nThe possible generation of products during the Maillard reaction could also contribute to the increased antioxidant content measured through ABTS and FRAP assays. However, the DPPH method did not show significant differences.\n\nThe inclusion of 25% sacha inchi cake contributed to an increase in protein content and the content of polyphenols and antioxidant compounds in the snacks.\n\nThe development of this new gluten-free, high-fiber, and high-protein snack can be considered as a bioactive compound-rich appetizer that meets the demand for nutritious snacks among consumers. At the same time, it would be a good way to increase the consumption of ancestral grains and healthy foods for individuals with gluten sensitivity.", "appendix": "Data availability\n\nZenodo: Artículo_sacha_v1. https://doi.org/10.5281/zenodo.8048635. 29\n\n• This project contains the following underlying data:\n\n• FTIR - HyperProteic Quinoa Flour (HPQF) R1: in this file you can find the FT-IR data of the “x” and “y” coordinates of the HyperProteic Quinoa Flour sample of replicate 1.\n\n• FTIR - HyperProteic Quinoa Flour (HPQF) R2: in this file you can find the FT-IR data of the “x” and “y” coordinates of the HyperProteic Quinoa Flour sample of replicate 2.\n\n• FTIR - HyperProteic Quinoa Flour (HPQF) R3: in this file you can find the FT-IR data of the “x” and “y” coordinates of the HyperProteic Quinoa Flour sample of replicate 3.\n\n• FTIR - HyperProteic Quinoa Flour (HPQF) R4: in this file you can find the FT-IR data of the “x” and “y” coordinates of the HyperProteic Quinoa Flour sample of replicate 4.\n\n• FTIR - Sacha Inchi Flour (SIF) R1: this file contains the FT-IR data of the “x” and “y” coordinates of the sacha inchi flour sample of replicate 1.\n\n• FTIR - Sacha Inchi Flour (SIF) R2: this file contains the FT-IR data of the “x” and “y” coordinates of the sacha inchi flour sample of replicate 2.\n\n• FTIR - Sacha Inchi Flour (SIF) R3: this file contains the FT-IR data of the “x” and “y” coordinates of the sacha inchi flour sample of replicate 3.\n\n• FTIR - Sacha Inchi Flour (SIF) R4: this file contains the FT-IR data of the “x” and “y” coordinates of the sacha inchi flour sample of replicate 4.\n\n• FTIR - T1R1: this file contains the FT-IR data of the “x” and “y” coordinates of the T1 sample of replicate 1.\n\n• FTIR - T1R2: this file contains the FT-IR data of the “x” and “y” coordinates of the T1 sample of replicate 2.\n\n• FTIR - T1R3: this file contains the FT-IR data of the “x” and “y” coordinates of the T1 sample of replicate 3.\n\n• FTIR - T1R4: this file contains the FT-IR data of the “x” and “y” coordinates of the T1 sample of replicate 4.\n\n• FTIR - T2R1: this file contains the FT-IR data of the “x” and “y” coordinates of the T2 sample of replicate 1.\n\n• FTIR - T2R2: this file contains the FT-IR data of the “x” and “y” coordinates of the T2 sample of replicate 2.\n\n• FTIR - T2R3: this file contains the FT-IR data of the “x” and “y” coordinates of the T2 sample of replicate 3.\n\n• FTIR - T2R4: this file contains the FT-IR data of the “x” and “y” coordinates of the T2 sample of replicate 4.\n\n• FTIR - T3R1: this file contains the FT-IR data of the “x” and “y” coordinates of the T3 sample of replicate 1.\n\n• FTIR - T3R2: this file contains the FT-IR data of the “x” and “y” coordinates of the T3 sample of replicate 2.\n\n• FTIR - T3R3: this file contains the FT-IR data of the “x” and “y” coordinates of the T3 sample of replicate 3.\n\n• FTIR - T3R4: this file contains the FT-IR data of the “x” and “y” coordinates of the T3 sample of replicate 4.\n\n• FTIR - Whole Rice Flour (WRF) R1: this file contains the FT-IR data of the “x” and “y” coordinates of the Whole Rice Flour sample of replicate 1.\n\n• FTIR - Whole Rice Flour (WRF) R2: this file contains the FT-IR data of the “x” and “y” coordinates of the Whole Rice Flour sample of replicate 2.\n\n• FTIR - Whole Rice Flour (WRF) R3: this file contains the FT-IR data of the “x” and “y” coordinates of the Whole Rice Flour sample of replicate 3.\n\n• FTIR - Whole Rice Flour (WRF) R4: this file contains the FT-IR data of the “x” and “y” coordinates of the Whole Rice Flour sample of replicate 4.\n\n• ITEM ABTS_1: in this file you can see information about the data of antioxidant properties measured by the ABTS method.\n\n• ITEM CAROTENOIDS_1: in this file you can see information about the carotenoid content data present in the snack samples.\n\n• ITEM DPPH_1: in this file you can see information about the data of antioxidant properties measured by the DDPH method.\n\n• ITEM FRAP_1: in this file you can see information about the data of antioxidant properties measured by the FRAP method.\n\n• ITEM Phenolic Compound (EPC y HPC)_1: this file contains information on the polyphenol content data present in the snack samples.\n\n• PARAMENTERS COLOR: in this file you will find the coordinates of the color parameters of the samples.\n\n• TEXTURE T1: this file contains the texture parameter data for the T1 treatment.\n\n• TEXTURE T2: this file contains the texture parameter data for the T2 treatment.\n\n• TEXTURE T3: this file contains the texture parameter data for the T3 treatment.\n\n• TEXTURE T1: this file contains the texture parameter data for the T4 treatment.\n\nZenodo: Article_sacha. https://doi.org/10.5281/zenodo.7625719. 23\n\nThis project contains the following extended data:\n\n• Bacillus cereus-the protocol for the determination of Bacillus cereus present in food according to the colombian regulations is presented.docx\n\n• Coliforms-the protocol for the determination of coliforms present in food according to the colombian regulations is presented.docx\n\n• Fungi and yeasts-the protocol for the determination of fungi and yest present in food according to the colombian regulations is presented.docx\n\n• Mesophiles the protocol for the determination of mesophiles present in food according to the colombian regulations is presented.docx\n\n• Protocol Fiber - The protocol for fiber determination is presented.docx\n\n• Protocol Lipid - The protocol for lipds determination is presented.docx\n\n• Protocol Protein - The protocol for protein determination is presented.docx\n\n• Salmonella Spp - The protocol for the determination of Samonella present in food according to the colombian regulations is presented.docx\n\n• Staphylococcus aureus - The protocol for the determination of Salmonella present in food according to the colombian regulations is presented.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nWang S, Zhu F: Formulation and Quality Attributes of Quinoa Food Products. Food Bioprocess Technol. 2016; 9(1): 49–68. Publisher Full Text\n\nRamos Diaz JM, et al.: Use of amaranth, quinoa and kañiwa in extruded corn-based snacks. J. Cereal Sci. 2013; 58(1): 59–67. Publisher Full Text\n\nVäkeväinen K, et al.: Potential of quinoa in the development of fermented spoonable vegan products. Lwt. 2020; 120(June 2019): 108912. Publisher Full Text\n\nMhd Rodzi NAR, Lee LK: Sacha Inchi (Plukenetia Volubilis L.): recent insight on phytochemistry, pharmacology, organoleptic, safety and toxicity perspectives. Heliyon. 2022; 8(9): e10572. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartinez D, et al.: LWT - Food Science and Technology Mixed sorghum and quinoa flour improves protein quality and increases antioxidant capacity in vivo. LWT- Food Sci. Technol. 2020; 129(May): 109597. Publisher Full Text\n\nMuñoz-Pabon KS, Roa-Acosta DF, Bravo-Gómez V, et al.: Quinoa Snack Production at an Industrial Level: Effect of Extrusion and Baking on Digestibility, Bioactive, Rheological, and Physical Properties.2022.\n\nTorres Sánchez EG, Hernández-Ledesma B, Gutiérrez LF: Sacha Inchi Oil Press-cake: Physicochemical Characteristics, Food-related Applications and Biological Activity. Food Rev. Int. 2021; 39: 148–159. Publisher Full Text\n\nJiapong S, Ruttarattanamongkol K: Development of Direct Expanded High Protein Snack Products Fortified With Sacha Inchi Seed Meal. J. Microbiol. Biotechnol. Food Sci. 2021; 10(4): 680–684. Publisher Full Text\n\nBrennan C, Brennan M, Derbyshire E, et al.: Effects of extrusion on the polyphenols, vitamins and antioxidant activity of foods. Trends Food Sci. Technol. 2011; 22(10): 570–575. Publisher Full Text\n\nOffiah V, Kontogiorgos V, Falade KO: Extrusion processing of raw food materials and by-products: A review. Crit. Rev. Food Sci. Nutr. 2019; 59(18): 2979–2998. PubMed Abstract \| Publisher Full Text\n\nAcosta DFR, Gómez JEB, Duque JFS, et al.: Antioxidant potential of extruded snacks enriched with hyper-protein quinoa flour and vegetable extracts. Food Sci. Technol. 2022; 42. Publisher Full Text\n\nTas AA, Shah AU: The replacement of cereals by legumes in extruded snack foods: Science, technology and challenges. Trends Food Sci. Technol. 2021; 116(April): 701–711. Publisher Full Text\n\nGomes KS, Berwian GF, Batistella VMC, et al.: Nutritional and Technological Aspects of the Production of Proteic Extruded Snacks Added of Novel Raw Materials. Food Bioprocess Technol. 2023; 16(2): 247–267. Publisher Full Text\n\nKhanna N, Singh M, Jain P: Chemical Science Review and Letters Effect of Extrusion Cooking on Textural Properties of Extrudates-A Review. Chem. Sci. Rev. Lett. 2019; 8(30): 276–279.\n\nSkoczek-Rubińska A, Bajerska J: The consumption of energy dense snacks and some contextual factors of snacking may contribute to higher energy intake and body weight in adults. Nutr. Res. 2021; 96: 20–36. PubMed Abstract \| Publisher Full Text\n\nAlandia G, Rodriguez JP, Jacobsen SE, et al.: Global expansion of quinoa and challenges for the Andean region. Glob. Food Sec. 2020; 26(March): 100429. Publisher Full Text\n\nLin H, Bean SR, Tilley M, et al.: Qualitative and Quantitative Analysis of Sorghum Grain Composition Including Protein and Tannins Using ATR-FTIR Spectroscopy. Food Anal. Methods. 2021; 14(2): 268–279. Publisher Full Text\n\nSiwatch M, Yadav RB, Yadav BS: X-ray diffraction, rheological and FT-IR spectra studies of processed amaranth (Amaranthus hypochondriacus). J. Food Meas. Charact. 2017; 11(4): 1717–1724. Publisher Full Text\n\nRoa DF, Santagapita PR, Buera MP, et al.: Amaranth Milling Strategies and Fraction Characterization by FT-IR. Food Bioprocess Technol. 2014; 7(3): 711–718. Publisher Full Text\n\nDave G, Modi H: FT-IR method for estimation of phytic acid content during bread-making process. J. Food Meas. Charact. 2018; 12(3): 2202–2208. Publisher Full Text\n\nMahesar SA, Kandhro AA, Cerretani L, et al.: Determination of total trans fat content in Pakistani cereal-based foods by SB-HATR FT-IR spectroscopy coupled with partial least square regression. Food Chem. 2010; 123(4): 1289–1293. Publisher Full Text\n\nShahidi F, Zhong Y: Measurement of antioxidant activity. J. Funct. Foods. 2015; 18: 757–781. Publisher Full Text\n\nMuñoz KS, Restrepo LF: Article_sacha.2023. Publisher Full Text\n\nPico J, Xu K, Guo M, et al.: Manufacturing the ultimate green banana flour: Impact of drying and extrusion on phenolic profile and starch bioaccessibility. Food Chem. 2019; 297(February): 124990. PubMed Abstract \| Publisher Full Text\n\nWaramboi JG, Gidley MJ, Sopade PA: Carotenoid contents of extruded and non-extruded sweetpotato flours from Papua New Guinea and Australia. Food Chem. 2013; 141(3): 1740–1746. PubMed Abstract \| Publisher Full Text\n\nRodriguez-Amaya DB, Kimura M, Godoy HT, et al.: Updated Brazilian database on food carotenoids: Factors affecting carotenoid composition. J. Food Compos. Anal. 2008; 21(6): 445–463. Publisher Full Text\n\nRodriguez-Amaya DB, Kimura M: HarvestPlus Handbook for Carotenoid Analysis.2004; no. 2068.\n\nKarkle EL, Alavi S, Dogan H: Cellular architecture and its relationship with mechanical properties in expanded extrudates containing apple pomace. Food Res. Int. 2012; 46(1): 10–21. Publisher Full Text\n\nMuñoz KS: Artículo_sacha_v1. [Dataset]. 2023. Publisher Full Text\n\nMuñoz KS, Parra AS, Roa DF, et al.: Physical and Paste Properties Comparison of Four Snacks Produced by High Protein Quinoa Flour Extrusion Cooking.2022; 6(March): 1–10. Publisher Full Text\n\nHuang R, et al.: Effect of defatting and extruding treatment on the physicochemical and storage properties of quinoa (Chenopodium quinoa Wild) flour. Lwt. 2021; 147(January): 111612. Publisher Full Text\n\nRoa DF, Baeza RI, Tolaba MP: Effect of ball milling energy on rheological and thermal properties of amaranth flour. J. Food Sci. Technol. Dec. 2015; 52(12): 8389–8394. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCadena Narváez AR, Zambrano Salas A, Gómez Bravo EJ, et al.: Characterization of sacha inchi (Plukenetia volubilis) and taro (Colocasia esculenta) flours with potential application in the preparation of both gluten-free and high protein foods. [version 1; peer review: awaiting peer review]. F1000 Res. 2023;1–17. Reference Source\n\nSciammaro L, Ferrero C, Puppo C: Physicochemical and nutritional characterization of sweet snacks formulated with Prosopis alba flour. Lwt. 2018; 93: 24–31. Publisher Full Text\n\nRamos-Diaz JM, Rinnan Å, Jouppila K: Application of NIR imaging to the study of expanded snacks containing amaranth, quinoa and kañiwa. Lwt. 2019; 102(May 2018): 8–14. Publisher Full Text\n\nAbugoch James LE: Quinoa (Chenopodium quinoa Willd.): Composition, chemistry, nutritional, and functional properties. Elsevier Inc.; 1st ed.2009; vol. 58(09).\n\nRuiz C, Díaz C, Anaya J, et al.: Aproximate analysis, antinutrients, fatty acids and amino acids profiles of seeds and cakes from 2 species of sacha inchi: Plukenetia volubilis and Plukenetia huayllabambana. Rev. la Soc. Química del Perú. 2013; 79(1): 29–36.\n\nMortensen JL, Anderson DM, White JL: Infrared spectrometry. Methods of Soil Analysis, Part 1: Physical and Mineralogical Properties, Including Statistics of Measurement and Sampling. 2015;743–770. Publisher Full Text\n\nTosun R, Yasar S: Comparing vibrational spectroscopic method with wet chemistry to determine nutritional and chemical changes in solid state fermented oats grain (Avena sativa L.). J. Food Meas. Charact. 2023; 17(1): 984–997. Publisher Full Text\n\nWang J, Yue Y, Liu T, et al.: Change in Glutenin Macropolymer Secondary Structure in Wheat Sourdough Fermentation by FTIR. Interdiscip. Sci. – Comput. Life Sci. 2017; 9(2): 247–253. PubMed Abstract \| Publisher Full Text\n\nNayak AK, Hasnain MS, Dhara AK, et al.: Plant Polysaccharides in Pharmaceutical Applications.2021; vol. 140.\n\nRen G, Teng C, Fan X, et al.: Nutrient composition, functional activity and industrial applications of quinoa (Chenopodium quinoa Willd.). Food Chem. 2023; 410(December 2022): 135290. PubMed Abstract \| Publisher Full Text\n\nTsao R: Chemistry and biochemistry of dietary polyphenols. Nutrients. 2010; 2(12): 1231–1246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang S, Zhu F, Kakuda Y: Sacha inchi (Plukenetia volubilis L.): Nutritional composition, biological activity, and uses. Food Chem. 2018; 265(April): 316–328. PubMed Abstract \| Publisher Full Text\n\nRagaee S, Seetharaman K, Ragaee S, et al.: The Impact of Milling and Thermal Processing on Phenolic Compounds in Cereal Grains The Impact of Milling and Thermal Processing on Phenolic Compounds. Crit. Rev. Food Sci. Nutr. 2014; 54: 837–849. PubMed Abstract \| Publisher Full Text\n\nNadeesha Dilrukshi HN, Torrico DD, Brennan MA, et al.: Effects of extrusion processing on the bioactive constituents, in vitro digestibility, amino acid composition, and antioxidant potential of novel gluten-free extruded snacks fortified with cowpea and whey protein concentrate. Food Chem. 2022; 389(April): 133107. PubMed Abstract \| Publisher Full Text\n\nSharma P, Gujral HS, Singh B: Antioxidant activity of barley as affected by extrusion cooking. Food Chem. 2012; 131(4): 1406–1413. Publisher Full Text\n\nJan KN, Panesar PS, Singh S: Textural, in vitro antioxidant activity and sensory characteristics of cookies made from blends of wheat-quinoa grown in India. J. Food Process. Preserv. 2018; 42(3). Publisher Full Text\n\nJan KN, Panesar PS, Singh S: Optimization of antioxidant activity, textural and sensory characteristics of gluten-free cookies made from whole indian quinoa flour. Lwt. 2018; 93(April): 573–582. Publisher Full Text\n\nSharma P, Gujral HS: Cookie making behavior of wheat-barley flour blends and effects on antioxidant properties. Lwt. 2014; 55(1): 301–307. Publisher Full Text\n\nEstivi L, Pellegrino L, Hogenboom JA, et al.: Einkorn Water Biscuits.2022.\n\nChirinos R, Zuloeta G, Pedreschi R, et al.: Sacha inchi (Plukenetia volubilis): A seed source of polyunsaturated fatty acids, tocopherols, phytosterols, phenolic compounds and antioxidant capacity. Food Chem. 2013; 141(3): 1732–1739. PubMed Abstract \| Publisher Full Text\n\nLuo S, Koksel F: Application of physical blowing agents in extrusion cooking of protein enriched snacks: Effects on product expansion, microstructure, and texture. Trends Food Sci. Technol. 2023; 133(January): 49–64. Publisher Full Text\n\nAussanasuwannakul A, Teangpook C, Treesuwan W, et al.: Effect of the Addition of Soybean Residue (Okara) on the Physicochemical, Tribological, Instrumental, and Sensory Texture Properties of Extruded Snacks. Foods. 2022; 11(19). Publisher Full Text\n\nRamírez-Rivera EJ, Hernández-Santos B, Juárez-Barrientos JM, et al.: Effects of formulation and process conditions on chemical composition, color parameters, and acceptability of extruded insect-rich snack. J. Food Process. Preserv. 2021; 45(5): 1–13. Publisher Full Text\n\nBhat NA, Wani IA, Hamdani AM, et al.: Effect of extrusion on the physicochemical and antioxidant properties of value added snacks from whole wheat (Triticum aestivum L.) flour. Food Chem. 2019; 276(April 2018): 22–32. Publisher Full Text" }	[ { "id": "298242", "date": "10 Sep 2024", "name": "Miguel Angle Garcia Parra", "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\nIt is noteworthy that the document presents a methodology that integrates techniques that allow the analysis of the physicochemical characteristics of two food matrices.\nThe paper presents the information in a precise and solid way. The document has references that support each of the premises established within its structure.\nIn accordance with the methodological design, it is presented in a very detailed manner. I consider it important to talk about seeds or grains, rather than cereals.\nOn the other hand, the research precisely details the methods used to study the two food matrices. Details of the equipment, reagents and supplies necessary for the respective analyzes are identified.\nThe data are evaluated through analysis of variance. However, it is important that the tables detail the significance of each of the variables.\nThe conclusions manage to specify the findings achieved with the research, especially in the physicochemical characteristics of the food matrices and their impact on the properties of the snacks.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1004
https://f1000research.com/articles/11-1565/v1	22 Dec 22	{ "type": "Study Protocol", "title": "Investigating the circulating sphingolipidome response to a single high-intensity interval training session within healthy females and males in their twenties (SphingoHIIT): Protocol for a randomised controlled trial", "authors": [ "Justin Carrard", "Thomas Angst", "Nadia Weber", "Joëlle Bienvenue", "Denis Infanger", "Lukas Streese", "Timo Hinrichs", "Ilaria Croci", "Christian Schmied", "Hector Gallart-Ayala", "Christoph Höchsmann", "Karsten Koehler", "Henner Hanssen", "Julijana Ivanisevic", "Arno Schmidt-Trucksäss", "Thomas Angst", "Nadia Weber", "Joëlle Bienvenue", "Denis Infanger", "Lukas Streese", "Timo Hinrichs", "Ilaria Croci", "Christian Schmied", "Hector Gallart-Ayala", "Christoph Höchsmann", "Karsten Koehler", "Henner Hanssen", "Julijana Ivanisevic", "Arno Schmidt-Trucksäss" ], "abstract": "Introduction: Growing scientific evidence indicates that sphingolipids predict cardiometabolic risk, independently of and beyond traditional biomarkers such as low-density lipoprotein cholesterol. To date, it remains largely unknown if and how exercise, a simple, low-cost, and patient-empowering modality to optimise cardiometabolic health, influences sphingolipid levels. The SphingoHIIT study aims to assess the response of circulating sphingolipid species to a single session of high-intensity interval training (HIIT). Methods: This single-centre randomised controlled trial (RCT) will last 11 days per participant and aim to include 32 young and healthy individuals aged 20-29 (50% females). Participants will be randomly allocated to the HIIT (n= 16) or control groups (physical rest, n= 16). Participants will self-sample fasted dried blood spots for three consecutive days before the intervention (HIIT versus rest) to determine baseline sphingolipid levels. Dried blood spots will also be collected at five time points (2, 15, 30, 60min, and 24h) following the intervention (HIIT versus rest). To minimise the dietary influence, participants will receive a standardised diet for four days, starting 24 hours before the first dried blood sampling. For females, interventions will be timed to fall within the early follicular phase to minimise the menstrual cycle's influence on sphingolipid levels. Finally, physical activity will be monitored for the whole study duration using a wrist accelerometer. Ethics and dissemination: The Ethics Committee of Northwest and Central Switzerland approved this protocol (ID 2022–00513). Findings will be disseminated in scientific journals and meetings. Trial Registration The trial was registered on www.clinicaltrials.gov (NCT05390866, https://clinicaltrials.gov/ct2/show/NCT05390866) on May 25, 2022.", "keywords": [ "Cardiometabolic health", "cardiovascular heatlh", "exercise", "physical activity", "sphingolipids", "ceramides", "exercise medicine" ], "content": "Introduction\n\nCardiometabolic diseases represent a growing socioeconomic burden and concern for healthcare systems worldwide.1,2 Improving cardiometabolic risk stratification should help clinicians better tailor prevention and treatment strategies to individual needs and thus combat this burden more effectively.3 Currently, lipid profiling is still mainly limited to total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, and triglycerides.4 However, technological advances in mass spectrometry now allow to quantify less abundant circulating lipids such as sphingolipids.5–7 Sphingolipids constitute a family of important bioactive lipids, which modulate numerous biological processes and are involved in the pathogenesis of coronary artery disease, type 2 diabetes mellitus (T2DM), and non-alcoholic fatty liver disease.8–10 This suggests that comprehensive sphingolipid panels should be considered surrogates of cardiometabolic health.8 Ceramides, the most studied sphingolipid class, have been shown to predict cardiovascular outcomes beyond LDL cholesterol.11–13 Ceramide-based scores were more accurate than the 2019 SCORE of the European Society of Cardiology in terms of cardiovascular risk prediction14,15 and are now used in clinical practice in the Mayo Clinic.16,17 In addition, specific ceramide species were proved predictive for T2DM 10 years before the disease was diagnosed.18 Mechanistically, excess caloric intake and inflammation stimulate sphingolipid synthesis.19 Indeed, tumour necrosis factor-alpha (TNF-α) and free fatty acids activate sphingolipid synthesis enzymes.20 Sphingolipids in excess are converted to ceramides, which accumulate on the surface of LDL, where they drive LDL transcytosis through the endothelium and uptake into macrophages.21–23 This results in foam cell formation, vascular inflammation, and atherosclerosis.24 Additionally, ceramides inhibit Akt/protein kinase B activity, which reduces the translocation of glucose transporters to the cell membrane, thereby contributing to peripheral insulin resistance and non-alcoholic fatty liver disease.25–27 Thus, sphingolipid metabolism might become a future therapeutic target in clinical medicine, as demonstrated by rodent studies demonstrating a reduction of arterial hypertension, T2DM, atherosclerosis, and heart failure following ceramide synthesis inhibition.28\n\nRegular physical activity is essential for maintaining general health29–31 and for preventing and treating cardiovascular diseases,32,33 insulin resistance, and T2DM.34,35 Exercise mitigates traditional risk factors and directly improves cardiometabolic health by optimising vascular endothelial function, stimulating myokines secretion, and other mechanisms which remain to be elucidated.33 Changes in sphingolipid metabolism might be one of the mechanisms through which exercise optimises cardiometabolic health (Figure 1). This hypothesis is supported by the fact that circulating sphingolipids have been reported to be negatively associated with cardiorespiratory fitness (CRF).36 Two preliminary studies investigated the effect of exercise on the circulating sphingolipidome. A single bout of moderate-intensity continuous training (MICT) increased circulating sphingolipids in endurance athletes, sedentary obese individuals, and patients with T2DM.37 Conversely, 12 weeks of MICT in patients suffering from obesity or T2DM decreased plasma sphingolipid levels.38 This duality could be mediated through inflammation as acute exercise tends to induce inflammation and regular exercise lowers it, while inflammation drives sphingolipid accumulation.19,39\n\nThe SphingoHIIT study aims to investigate the effect of a single session of high-intensity interval training on circulating sphingolipids, which are novel biomarkers of cardiometabolic health. Abbreviations: HIIT = high-intensity interval training. This figure has been adapted with permission from Carrard, J. et al. A. How Ceramides Orchestrate Cardiometabolic Health—An Ode to Physically Active Living. Metabolites 2021, 11, 675. https://doi.org/10.3390/metabo11100675\n\nThese preliminary studies investigated, however, a limited number of sphingolipid species (n=8 and 7, respectively), whereas targeted lipidomics has now matured into a high-throughput approach allowing for comprehensive analysis of lipid metabolism at the molecular species level.5,6 Moreover, these studies examined the sphingolipid’s response to acute or regular MICT, while many studies showed that regular high-intensity interval training (HIIT) is a safe40 and a more effective way to improve CRF41,42 and insulin sensitivity.43,44 Furthermore, lipid profiles are potentially subject to day-to-day variations, an element not considered in these two studies.45–47 Lastly, to investigate the physiological responses of sphingolipid species to exercise, it is necessary to examine healthy participants before investigating clinical populations, thereby avoiding confounding effects of chronic diseases on sphingolipid metabolism. To address these gaps, this randomised controlled trial will investigate the response of an extensive panel of circulating sphingolipids species to a single HIIT session within healthy individuals in their twenties while taking day-to-day variations into account. The results of this acute study will inform subsequent long-term exercise interventions. Moreover, investigating the effects of a single HIIT session is particularly relevant since signalling moieties, known as “exerkines”, are released in response to acute exercise.48,49 These molecules can positively impact various organs through endocrine, paracrine, or autocrine signalling pathways.50\n\n\nProtocol\n\nThe present research project aims to assess, at the molecular species level, the response of the circulating sphingolipidome to a single HIIT session in healthy individuals aged 20-29. We hypothesise that circulating sphingolipid levels will temporarily increase following a single HIIT session, apart from the sphingosine-1-phosphate level, which might decrease. Indeed, this metabolite could be cardiometabolically favourable as patients with multiple sclerosis taking the drug fingolimod, a sphingosine-1-phosphate receptor modulator, experienced cardiac side effects such as first-dose bradycardia.51\n\nThe primary endpoints will be changes from pre- to post-intervention levels of the four most studied sphingolipid species, which are also included in the ceramide-based scores (i.e. ceramide 16:0, ceramide 18:0, ceramide 24:0 and ceramide 24:1).14 The secondary endpoints will be changes from pre- to post-intervention levels of the resting sphingolipid species to be acquired (n=57).\n\nThis prospective two-arm, single-centre randomised controlled trial will be conducted at the Department of Sport, Exercise, and Health of the University of Basel. It will be carried out in accordance with the Declaration of Helsinki and the guidelines of Good Clinical Practice of the World Medical Association in 2013. The Ethics Committee of Northwest and Central Switzerland approved the study (project-ID 2022–00513). Substantial changes to the protocol will be submitted to the Ethics Committee for approval before implementation, as required by Swiss law. The study was registered on www.clinicaltrials.gov (NCT05390866) on May 25, 2022, and follows the SPIRIT reporting guidelines.52 All items from the World Health Organization Trial Registration Data Set are summarised in Table 1, according to the SPIRIT reporting guidelines. This is the first version of the protocol (22 November 2022). There will be no financial compensation for participation in the SphingoHIIT study.\n\nThis pilot study aims to include 32 healthy individuals aged 20-29 (50% females) from the Basel area (Switzerland) randomised to the HIIT or control group. Participants meeting the inclusion criteria (Table 2) will be eligible for the study.\n\n\n\n• Female or male sex.\n\n• Age = 20-29 years old.\n\n• BMI = 18.5-24.9 kg/m2.\n\n• Meeting the WHO guidelines on physical activity, i.e. at least 150–300 minutes of moderate-intensity aerobic physical activity per week as well as muscle-strengthening activities on 2 or more days per week.\n\n• Clearance for physical activity according to the 2022 PAR-Q+.53\n\n• Regular menstrual cycle.\n\n• Informed consent as documented by signature.\n\n\n\n• Females with known pregnancy or breastfeeding.\n\n• Females with known polycystic ovary syndrome.\n\n• Current exercise limiting conditions of the lower limbs (e.g., tendinopathy, fractures, or other musculoskeletal pathologies).\n\n• Known acute or chronic diseases: e.g. any active infectious diseases, past or current malignant tumours, lung diseases (e.g. bronchial asthma), cardiometabolic diseases (e.g. arterial hypertension, diabetes, dyslipidaemia), gastrointestinal diseases (e.g. coeliac disease, Crohn’s disease, ulcerative colitis), psychological disorders (e.g. depression, if medically diagnosed, anorexia, bulimia), endocrinological diseases (e.g. all types of diabetes mellitus, hyper- or hypothyroidism), nephrological diseases and neurological disorders.\n\n• Current or past smoking, current or past psychoactive drug use (alcohol excluded here, see below).54\n\n• Excessive alcohol consumption in the past two weeks, defined as either binge drinking (consuming five or more drinks during a single occasion) or heavy drinking (consuming 15 or more drinks per week).55\n\n• Current or regular medication use, including any kind of hormonal contraception.\n\n• Following diets such as: vegetarian, vegan, lactose-free and gluten-free, low-FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols).\n\n• Inability to follow the procedures of the study, e.g., due to linguistic or cognitive problems.\n\n• Concomitant or recent (last 4 weeks) involvement in another trial.\n\nParticipants will be recruited through advertisements on the website of the Department of Sport, Exercise, and Health of the University of Basel and on the social media channels of the Department mentioned above. Detailed information about study procedures, risks, and benefits will be given by telephone to all participants. Following the phone call, all potential participants will be provided via e-mail with a participant information sheet and a consent form. All participants will have to sign a consent form and be informed about their right to withdraw from the study without consequences or the need for providing reasons.\n\nParticipants will be randomly allocated either to the HIIT (n= 16, 50% females) or control (n= 16, 50% females) groups. Blocked randomisation will be used to reduce bias and achieve balance in allocating participants to both groups, as commonly done when the sample size is small.57 The principal investigator will be responsible for the randomisation and will inform participants and investigators about the group allocation only on the day of the intervention (i.e. opening of the sealed envelope). Therefore, participants and investigators supervising the intervention will be blinded for group allocation till the day of the intervention.\n\nOverview\n\nFigure 2 summarises the planned study procedure. Following eligibility criteria assessment, a maximal cardiopulmonary exercise testing (CPET) will be performed to determine peak oxygen uptake (V̇O2peak), heart rate, and power output. An eight-day washout period will be carried out until the intervention (HIIT vs physical rest). In the three days preceding the intervention, participants will self-sample fasted dried blood spots (DBS) to determine sphingolipid baseline levels. DBS will be collected at five additional fixed time points (2min, 15min, 30min, 60min, and 24h) following the intervention. To minimise the dietary influence, participants will be asked to uniquely consume the provided individualised, pre-packaged meals starting one day before the first dried blood sampling. The study will last 11 days for each participant.\n\nThe SphingoHIIT study will last 11 days per participant. Following the baseline examination (which includes cardiopulmonary exercise testing), participants will have to avoid any vigorous-intensity physical activity (≥ 7 Metabolic Equivalent of Task) to maximise contrast between the pre-and post-intervention dried blood spots. To avoid potential confounding effects of food intake on circulating sphingolipids, participants will be fed starting 24h before the first dried blood spot collection. Abbreviations: CPET = cardiopulmonary exercise testing, HIIT = high-intensity interval training\n\nEligibility screening\n\nPotential participants will be screened for initial eligibility during a first in-person visit at the Department of Sport, Exercise, and Health of the University of Basel, which will take place after potential participants return the signed consent form. Inclusion and exclusion criteria will be carefully reviewed during the first in-person visit. Height and weight will be measured, and body mass index (BMI) will be calculated.\n\nBaseline clinical assessment (day 1)\n\nParticipants included after the eligibility screening will be invited for a clinical baseline examination. Physical examination and vital sign measurements (blood pressure, heart rate, respiration rate, oxygen saturation, and body temperature) will be conducted. Body composition will be analysed by four-segment bioelectrical impedance analysis using the InBody 720 (Inbody Co. Ltd., Seoul, South Korea). InBody 720 provides reliable body composition measurements compared to dual-energy x-ray absorptiometry analysis.58,59 Before the measurement, participants must refrain from intense physical activity for 24 h, fast for a minimum of two hours, and void their bladder. Physical activity will be objectively monitored throughout the whole study duration (11 days) using a wrist-worn triaxial accelerometer (GeneActiv Activinsights Ltd., Kimbolton, UK). The device will be attached to the participant’s nondominant wrist and worn day and night continuously in their free-living conditions. Accelerometry data will be exported using the GENEActiv software version 3.2 (GeneActiv Activinsights Ltd. Kimbolton, UK). Participants will receive an oral and written introduction to the accelerometer’s use.\n\nA resting ECG will also be performed to rule out cardiac contraindications to maximal exertion. A maximal cardiopulmonary exercise testing (CPET) will be conducted on a cycle ergometer to determine V̇O2peak, peak heart rate, and peak power output (Ergoselect 200; Ergoline, Bitz, Germany). A 3-min warm-up will be performed either unloaded or with a load of 10, 20, 50, or 50 W for protocols 1 to 5, respectively. The warm-up will be followed by a ramp protocol 1-5 with a workload increase of 7, 10, 15, 20, or 30 Watts/min, respectively. The 3-min recovery phase will be performed on the same wattage as the warm-up. The protocol will be chosen based on the following formula by Hansen et al.60 The increment closest to the one evaluated with the formula will be used.\n\nParticipants will receive the HemaXis DB10 whole blood collection device (DBS System SA, Gland, Switzerland) for at-home capillary whole blood sampling. To ensure the proper performance of blood sampling, they will receive written and oral instructions on the HemaXis DB10 device.\n\nWashout period\n\nA washout period (days 2 to 9) will take place between the CPET and the intervention (HIIT versus rest), during which participants will be asked not to perform any vigorous-intensity physical activity (≥ 7 Metabolic Equivalent of Task).\n\nPre-intervention blood sampling\n\nFrom day 8 to day 10, participants will self-sample two DBS in a fasting state between 6 and 8 am to assess baseline values and day-to-day variations of the circulating sphingolipid species levels. For each time point of blood sampling, two DBS will be sampled. The same blood source will provide the two DBS so that only one skin puncture per blood sample will be necessary, as two droplets of blood suffice for two DBS. Participants will be instructed to let the blood spot dry for 10 minutes, put the DBS in a provided plastic bag containing a small silica gel bag to absorb humidity and store them in their fridge at around 4°C. On day 10, they will bring back the DBS from days 8-10. There is no need to refrigerate the samples for transport when bringing the samples from home to the Department of Sports, Exercise, and Health of the University of Basel.\n\nExercise intervention (day 10)\n\nA single HIIT session will be performed on day 10 between 6 and 8 am. It will consist of a 3-min warm-up, followed by four 4-min intervals performed at 85-95% of peak heart rate, interspersed with 3-min of active recovery periods at the rating of perceived exertion (RPE) 11-13 (i.e., fairly light to somewhat hard).61 A 2-min cool-down will follow the HIIT. This protocol was chosen because it has been extensively studied in healthy and clinical populations and its effects on CRF improvement are well-documented.41,62 This protocol also fulfilled the requirements of a high-volume HIIT.63 The guidelines for HIIT prescription and monitoring established by Taylor et al. will be strictly followed in this study.41 Heart rate will be recorded by the exercise professional supervising the session using a heart rate monitor at the end of each minute, and RPE will be assessed within the final 15 seconds of each minute. These data will then be used to assess adherence to the prescribed intensity.\n\nControl intervention (day 10)\n\nThe control group participants will undertake the same procedures except for the HIIT session. Participants of the control group will have to physically rest in the lab in a seated position for the duration of the HIIT (30 min) before post-intervention DBS can be collected to ensure similar daily times for sample collection between both groups. During the rest period, participants are allowed to read a book and use a smartphone or a computer.\n\nPost-intervention blood sampling\n\nTwo DBS will be collected at 2 min, 15 min, 30 min, 60 min (directly in the lab), and 24 h post-intervention (i.e., DBS of day 11, at home). DBS from day 11 will be returned on day 11.\n\nSphingolipidomics\n\nAll DBS will be vacuumed and subsequently stored at -80°C at the Department of Sport, Exercise, and Health of the University of Basel before being delivered to the Metabolomics Unit of the Faculty of Biology and Medicine at the University of Lausanne. To quantify an extensive panel of circulating sphingolipids (n=61), a high-coverage method using reversed-phase liquid chromatography coupled to tandem mass spectrometry (RPLC-MS/MS) will be applied, as previously described.7\n\nStandardised diet\n\nTo minimise the differential influence of food intake on sphingolipid levels between participants, each participant will be provided with individualised, pre-packaged meals for days 7 to 10. The meals will have to be consumed during predetermined time windows, i.e., between 7 and 9 am for breakfast (but after dried blood sampling in any case), between 11 am and 1 pm for lunch, between 4 and 5 pm for an afternoon snack and between 6.30 and 8.30 pm for dinner. All participants will be fed to energy balance to maintain weight stability throughout the study period. Energy requirements will be calculated with the formulas of Mifflin St. Joer64 and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Body Weight Planner.65,66 All diets will contain ~55% energy from carbohydrates, ~25% energy from fat, and ~20% energy from protein. To monitor diet adherence, participants will be instructed to return all non-consumed foods from the pre-packaged meals to the lab and take photos of additionally consumed foods for later analysis of food and energy intake. Participants will also be asked to refrain from alcohol consumption during the dietary control period and from caffeine intake before the HIIT on day 10.\n\nControlling for menstrual cycle\n\nFor females, the regularity of the menstrual cycle will be assessed during the screening procedure using items five and seven of the Reproductive Status Questionnaire for Menstrual Cycle Studies.67 The goal will be to time the study, so that day 8 coincides with the beginning of the early follicular phase. This phase is indicated by the onset of bleeding and corresponds to the lowest concentrations of oestrogen and progesterone.68 Combined with the exclusion of females taking any hormonal contraceptives, this will minimise the effect of oestrogen and progesterone on sphingolipid levels and reduce heterogeneity among female participants.68\n\nDue to the extensive number of targeted endpoints (n=61), a classical sample size calculation cannot be performed. Therefore, to estimate the required sample size, we based our analysis on the four most investigated sphingolipid species, which are also the species entering the ceramide scores used at the Mayo Clinic (i.e. ceramide 16:0, ceramide 18:0, ceramide 24:0 and ceramide 24:1).16,17 Further, we hypothesised for simplicity that log2-transformed pre-intervention sphingolipid levels are similar in both the intervention and the control groups (due to the randomisation). Next, we assumed a standard deviation of log2-transformed sphingolipid levels of 0.407, an effect size (expressed as a geometric mean ratio) of 1.19 and a correlation coefficient between pre-and post-intervention values of 0.8. To obtain these values, we used the raw data of the four sphingolipids mentioned above issued from a previous exercise intervention study.37 Finally, we analysed covariance (ANCOVA) to calculate the sample size. We obtained a result of 16 participants per group for a power of 80% (Figure 3). Lastly, it should be pointed out that the planned repeating measurements will enhance precision.69\n\nA sample size of 16 participants per group was obtained (expressed as a detectable geometric mean ratio), assuming a power of 80%, an effect size of 1.19 (expressed as a geometric mean ratio), a standard deviation of 0.407 and a correlation coefficient ρ between pre-and post-intervention values of 0.8.\n\nThe trajectory over time of each lipid species will be modelled using linear mixed models with a random effect for each subject. To identify variables we should include in the models, we drew a causal directed acyclic graph (DAG) using DAGitty.70 Sex, body fat mass, CRF, and habitual physical activity were identified as variables to be included in the linear mixed models to reduce the outcome variation and improve the precision of the average causal effect of the intervention on the sphingolipidome (Figure 4).69 As explained above, the design will control food intake as each participant will be provided with individualised, pre-packaged meals for days 7 to 10. It is also assumed that age difference will not play a significant role as all participants will be between 20 and 29 years old. Therefore, the models will include fixed effects for time points, sex, fat body mass, CRF, physical activity, and a group indicator, as well as an interaction term between the group indicator and time point. Planned contrasts between the groups at each time point will allow us to assess differences in lipid concentrations between the groups after the intervention. P-values and confidence intervals will be adjusted for multiple testing using the Benjamini-Hochberg method.71 We will use residual diagnostic plots to assess whether the model assumptions are met. If model assumptions are violated, lipid concentrations will be log-transformed. If the proportion of missing data is below 5%, a complete case analysis will be done. Otherwise, we will consider multiple imputations. Drop-outs will be replaced by recruiting new subjects to achieve the targeted sample size of 32 participants.\n\nThe intervention (HIIT vs physical rest) is defined as the exposure and sphingolipids as the outcomes. Due to the randomisation, the exposure has no ancestor. Thus, no variable influences simultaneously the exposure and outcomes, which implies that there is no confounding variable. Sex, body fat mass, CRF, and physical activity were identified as variables to be included in the statistical models to reduce the outcome variation and improve the precision of the average causal effect of the intervention on the sphingolipidome. Abbreviations: HIIT = high-intensity interval training, CRF = cardiorespiratory fitness, black triangle pointing to the right on green background = exposure, black bold vertical bar = outcomes, blue background = variables to be included in the statistical models to reduce the outcome variation and improve the precision of the average causal effect of the intervention. The figure was realised with http://www.dagitty.net.\n\nThe significance level is set at α = 0.05, and all tests will be two-sided. All analyses will be done according to the intention-to-treat principle. Statistical analyses will be conducted using R (version 4.0.2 or later). Study results will be reported in compliance with the CONSORT statement.72\n\nData will be anonymised, stored on the protected server of the University of Basel and accessible only for authorised personnel to fulfil the research objectives described in the present protocol. Biological material collected during the SphingoHIIT study will be stored at the Department of Sport, Exercise and Health for 10 years after study termination.\n\nAny adverse event will be classified and its severity assessed by the investigator according to the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use.73 The Ethics Committee will be informed in due time as required by Swiss law.\n\nFindings and data will be disseminated in scientific journals and meetings.\n\nRecruiting and data collecting.\n\n\nDiscussion\n\nOptimising patients’ metabolic risk stratification in clinical practice has the potential to improve personalised prevention and early treatment of cardiometabolic diseases. Sphingolipids in general, and ceramides in particular, are essential bioactive lipids and promising biomarkers to enhance patients’ phenotyping and pre-symptomatic management of cardiometabolic disorders. This preliminary RCT aims to reveal the physiological impact of an acute bout of high-intensity exercise on the circulating sphingolipidome in healthy individuals in their twenties, thereby avoiding the confounding effects of chronic diseases on sphingolipid metabolism. In addition, the brevity of this RCT enables us to strictly control nutrition, physical activity levels, and the menstruation cycle. Thus, this well-controlled setting will allow for robust effect estimates and subsequent power calculations for further studies aiming to investigate the effect of regular exercise on the sphingolipid profile in different clinical populations.\n\nInvestigating sphingolipid responses to a single HIIT session on the molecular species level could also highlight novel ways through which exercise orchestrates cardiometabolic health.19 While it is well established that regular exercise improves overall health, the mechanisms underlying exercise-mediated health benefits remain indeed ill-defined.49 As acute exercise releases signalling moieties (“exerkines”) that mediate short- and long-term effects of physical activity on the human body,50 this RCT may identify key sphingolipid species related to short-term exercise adaptation and ultimately pave the way for novel health-monitoring strategies. Additionally, this study may contribute to identifying novel therapeutic targets and open new horizons for creating safe sphingolipid-lowering pharmaceuticals.28 Altogether, improving the molecular understanding of exercise medicine will further contribute to establishing this novel medical discipline as a cornerstone in preventing and treating cardiometabolic disorders.\n\n\nAuthor contributions\n\nThis work has been developed with the contribution of each co-author. The manuscript underwent several revisions, with substantial contributions provided by each co-author. All authors provided critical feedback and have read and approved the final manuscript.\n\nConceptualisation: JC, TA, NW, IC, CS, HH, HGA, JI, and AST. Funding acquisition: JC and AST. Methodology: JC, TA, NW, JB, DI, LS, IC, TH, CH, KK, HGA, JI, and AST. Project administration: JC and TA. Resources: JC, CH, KK, JI, and AST. Software: JC and DI. Supervision: JC and AST. Visualisation: JC, JB, and DI. Writing - Original Draft Preparation: JC, TA, NW. Writing - Review & Editing: JC, TA. NW, JB, DI, LS, TH, IC, CS, HGA, CH, KK, HH, JI, and AST.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nOpen Science Framework: SPIRIT checklist for “Investigating the circulating sphingolipidome response to a single high-intensity interval training session (SphingoHIIT): Protocol for a randomised controlled trial”, https://doi.org/10.17605/OSF.IO/53QND. 74\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nLozano R, Naghavi M, Foreman K, et al.: Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012; 380(9859): 2095–2128. 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Lancet. 2011; 378(9793): 826–837. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHall KD, Chow CC: Estimating changes in free-living energy intake and its confidence interval. Am. J. Clin. Nutr. 2011; 94(1): 66–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmalenberger KM, Tauseef HA, Barone JC, et al.: How to study the menstrual cycle: Practical tools and recommendations. Psychoneuroendocrinology. 2021; 123: 104895. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElliott-Sale KJ, Minahan CL, de Jonge X , et al.: Methodological Considerations for Studies in Sport and Exercise Science with Women as Participants: A Working Guide for Standards of Practice for Research on Women. Sports Med. 2021; 51(5): 843–861. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCinelli C, Forney A, Pearl J: A crash course in good and bad controls. Available at SSRN 3689437.2020.\n\nTextor J, van der Zander B , Gilthorpe MS, et al.: Robust causal inference using directed acyclic graphs: the R package ‘dagitty’. Int. J. Epidemiol. 2017; 45(6): 1887–1894.\n\nBenjamini Y, Hochberg Y: Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological). 1995; 57(1): 289–300. Publisher Full Text\n\nSchulz KF, Altman DG, Moher D: CONSORT 2010 Statement: updated guidelines for reporting parallel group randomised trials. BMJ. 2010; 340: c332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuideline IHT, editor. Clinical safety data management: definitions and standards for expedited reporting E2A. International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use.1994.\n\nCarrard J, Angst T, Weber N, et al.: Investigating the circulating sphingolipidome response to a single high-intensity interval training session (SphingoHIIT): Protocol for a randomised controlled trial.2022, November 27. Publisher Full Text" }	[ { "id": "178102", "date": "22 Jun 2023", "name": "Stefan Gross", "expertise": [ "Reviewer Expertise More than 10 years of cardiovascular epidemiology incl. exercise testing", "biomarker research and sphingolipids/ceramides. More than 20 years of experience with statistical methods especially regression methods", "sample size calculation etc." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript of Carrard and colleagues describes the study protocol for an investigation on the relationship between S1p and exercise training and whether this biomarker is modified by exercise training. It is a pilot RCT. The rationale, study plan and also statistical plans are convincing in general. I highly appreciate that the authors also utilized DAGs for determining possible analysis adjustments. The whole manuscript is written in a comprehensive, straightforward way and easy to follow by the reader.\nHowever, I still have some minor remarks that should be addressed:\nSample size calculation: please provide additional details / information on the utilized software packages and if possible also the utilized code. It is not clear to me, whether the assumed effect size of 1.19 is only the post-treatment geometric group mean ratio? Or is it the assumed ratio between the pre-post changes of both groups? I tried to recapitulate the calculation made here and I always reached larger group sizes than 16.\n\nStatistical analysis: Since this is a randomized trial you should not include into the linear mixed effects model a main effect for the treatment group. The treatment group should be only included in the interaction term with timepoints itself. Randomization implies there is no group difference at baseline pre treatment, so both groups have the same intercept. Treatment can only causally act after the baseline timepoint.\n\nJust as a minor remark, sphingolipids and ceramides are presented in the introduction only as having detrimental effects especially in the cell/molecular mechanism part. The literature is not clear so far to my knowledge e.g. how chain length of ceramides are related to CRF and CV risk. It seems that short chain ceramides have more \"detrimental\" effects while very long chain ceramides seem to have also \"positive\" effects. Saturation seems to play also a role: C24:0 is more positively associated with CRF and inversely with CVD mortality while C24:1 seems to be different. C16/C24 ratio is inversely associated with mortality and risk of CV incident events. See e.g. Peterson et al., (2018)1. So maybe include 1-2 sentences into the introduction or discussion that the mechanistic picture is rather complex and it depends about which sphingolipid/ceramide in terms of chain-length and saturation we are speaking.\nAfter minor revision it should be accepted and I wish the authors good luck with conducting the study. I am highly looking forward to the upcoming and interesting results of this study.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "9801", "date": "29 Jun 2023", "name": "Justin Carrard", "role": "Author Response", "response": "Dear Dr Gross, We would like to thank you very much for your positive and insightful comments (in bold below). These have been addressed point-by-point (below) and in the revised manuscript via track changes. We hope that you will agree that these revisions have further strengthened our manuscript. Yours sincerely, The authors The manuscript of Carrard and colleagues describes the study protocol for an investigation on the relationship between S1p and exercise training and whether this biomarker is modified by exercise training. It is a pilot RCT. The rationale, study plan and also statistical plans are convincing in general. I highly appreciate that the authors also utilized DAGs for determining possible analysis adjustments. The whole manuscript is written in a comprehensive, straightforward way and easy to follow by the reader. Thank you for your positive and encouraging comments. However, I still have some minor remarks that should be addressed: Sample size calculation: please provide additional details / information on the utilized software packages and if possible also the utilized code. It is not clear to me, whether the assumed effect size of 1.19 is only the post-treatment geometric group mean ratio? Or is it the assumed ratio between the pre-post changes of both groups? I tried to recapitulate the calculation made here and I always reached larger group sizes than 16. We used the software R (version 4.0.2 or later) and the following packages: emmeans, lme4, nlme, ggplot2, tidyverse, visreg, and readxl. The R-code is now freely available on Open Science Framework (https://osf.io/53qnd?mode=&revisionId=&view_only=). To obtain 1.19, we first asked Bergman et al. if they would agree to share with us the circulating sphingolipid values pre- and post-exercise for the healthy participants of their studies (1). They kindly agreed to do so. Kindly note that we are, however, not authorised to publish these data along our study protocol as we do not own these data. We then calculated the geometric mean of the sphingolipid concentrations post- and pre-exercise for the four primary endpoints (i.e. ceramide 16:0, ceramide 18:0, ceramide 24:0 and ceramide 24:1) before we calculated the ratio of the geometric means post- to pre-exercise. Finally, we averaged the geometric mean ratios obtained for these four endpoints and obtained a value of 1.194. We then log2-transformed 1.194 and obtained 1.19. Therefore, 1.19 is the log2-transformed geometric mean ratios of the post- to pre-exercise sphingolipid concentrations. In other words, the geometric means of the sphingolipid concentrations differ by a factor of 1.19 post- to pre-exercise in healthy individuals in the study conducted by Bergman et al. (1). Similarly, we used the data provided by Bergman et al. to calculate a standard deviation of log2-transformed sphingolipid levels of 0.407 and a correlation coefficient between pre-and post-intervention values of 0.8. We rewrote the section on sample size calculation as follows to make it clearer: “Due to the extensive number of targeted endpoints (n=61), a classical sample size calculation cannot be performed. Therefore, to estimate the required sample size, we based our analysis on the four most investigated sphingolipid species, which are also the species entering the ceramide scores used at the Mayo Clinic (i.e. ceramide 16:0, ceramide 18:0, ceramide 24:0 and ceramide 24:1) (2, 3). Further, we hypothesised for simplicity that log2-transformed pre-intervention sphingolipid levels are similar in both the intervention and the control groups (due to the randomisation). Next, we assumed a standard deviation of log2-transformed sphingolipid levels of 0.407, an effect size (expressed as a geometric mean ratio) of 1.19 and a correlation coefficient between pre-and post-intervention values of 0.8. To obtain these values, we used the raw data of the four sphingolipids mentioned above issued from a previous exercise intervention study (1). Specifically, we calculated the geometric mean of the sphingolipid concentrations post- and pre-exercise for the four primary endpoints (i.e. ceramide 16:0, ceramide 18:0, ceramide 24:0 and ceramide 24:1). We then calculated the ratio of the geometric means post- to pre-exercise. Finally, we averaged the geometric mean ratios obtained for these four endpoints and obtained a value of 1.194. We then log2-transformed 1.194 and obtained 1.19. Therefore, 1.19 is the log2-transformed geometric mean ratios of the post- to pre-exercise sphingolipid concentrations. In other words, the geometric means of the sphingolipid concentrations differ by a factor of 1.19 post- to pre-exercise in healthy individuals in the study conducted by Bergman et al. (1).” Statistical analysis: Since this is a randomized trial you should not include into the linear mixed effects model a main effect for the treatment group. The treatment group should be only included in the interaction term with timepoints itself. Randomization implies there is no group difference at baseline pre treatment, so both groups have the same intercept. Treatment can only causally act after the baseline timepoint. Many thanks for pointing this out. We modified the model in consequence so that the two groups are assumed to be identical at the baseline measurements. The modified model allows for the groups to diverge solely at the time points following the intervention. Specifically, we omitted the fixed effects for treatment groups and included a three-way interaction term between the group, time point and an indicator variable for time points following the intervention. We rewrote the section on sample size calculation as follows to make it clearer: “The trajectory over time of each lipid species will be modelled using linear mixed models with a random effect for each subject. To identify variables, we should include in the models, we drew a causal-directed acyclic graph (DAG) using DAGitty.70 Sex, body fat mass, CRF, and habitual physical activity were identified as variables to be included in the linear mixed models to reduce the outcome variation and improve the precision of the average causal effect of the intervention on the sphingolipidome (Figure 4).69 As explained above, the design will control food intake as each participant will be provided with individualised, pre-packaged meals for days 7 to 10. It is also assumed that age difference will not play a significant role as all participants will be between 20 and 29 years old. Therefore, the models will include fixed effects for time points, sex, fat body mass, CRF, physical activity, and a three-way interaction term between the group, time point and an indicator variable for time points following the intervention. Planned contrasts between the groups at each time point following the intervention will allow assessing differences in lipid concentrations between the groups after the intervention.” Just as a minor remark, sphingolipids and ceramides are presented in the introduction only as having detrimental effects especially in the cell/molecular mechanism part. The literature is not clear so far to my knowledge e.g. how chain length of ceramides are related to CRF and CV risk. It seems that short chain ceramides have more \"detrimental\" effects while very long chain ceramides seem to have also \"positive\" effects. Saturation seems to play also a role: C24:0 is more positively associated with CRF and inversely with CVD mortality while C24:1 seems to be different. C16/C24 ratio is inversely associated with mortality and risk of CV incident events. See e.g. Peterson et al., (2018)1. So maybe include 1-2 sentences into the introduction or discussion that the mechanistic picture is rather complex and it depends about which sphingolipid/ceramide in terms of chain-length and saturation we are speaking. Thank you for this valuable comment. We added a sentence in the introduction, as kindly suggested, to better reflect the biological complexity of the sphingolipidome and its potential biological implications. These preliminary studies investigated, however, a limited number of sphingolipid species (n=8 and 7, respectively), whereas targeted lipidomics has now matured into a high-throughput approach allowing for comprehensive analysis of lipid metabolism at the molecular species level.5,6 This is particularly important as sphingolipids might have differential biological effects depending on acyl chain length and saturation, with shorter and unsaturated species being potentially more detrimental (4). After minor revision it should be accepted and I wish the authors good luck with conducting the study. I am highly looking forward to the upcoming and interesting results of this study. Thank you for your insightful review. References 1. Bergman BC, Brozinick JT, Strauss A, Bacon S, Kerege A, Bui HH, et al. Serum sphingolipids: relationships to insulin sensitivity and changes with exercise in humans. Am J Physiol Endocrinol Metab. 2015;309(4):E398-408. 2. Nicholls M. Plasma ceramides and cardiac risk. European Heart Journal. 2017;38(18):1359-60. 3. Vasile VC, Jaffe AS. An enhanced ceramide-based approach for primary prevention of atherosclerotic events. European Heart Journal Open. 2021;1(3). 4. Peterson LR, Xanthakis V, Duncan MS, Gross S, Friedrich N, Völzke H, et al. Ceramide Remodeling and Risk of Cardiovascular Events and Mortality. Journal of the American Heart Association. 2018;7(10):e007931." } ] } ]	1	https://f1000research.com/articles/11-1565
https://f1000research.com/articles/12-1001/v1	18 Aug 23	{ "type": "Clinical Practice Article", "title": "Case Report: HUGHES-STOVIN SYNDROME: A RARE ENTITY AND CHALLENGING THERAPEUTIC DECISION: CASE SERIES AND REVIEW OF THE LITERATURE", "authors": [ "Lobna Mahjoub", "Mohamed Yassine Kallala", "Ahmed Sami Hammami", "Jamel Saad", "Melek Kechida", "Hanen Dabbabi", "Syrine Dada", "Marwa Ben Brahim", "Habib Gamra", "Majed Hassine", "Sonia Ouali", "Lobna Mahjoub", "Mohamed Yassine Kallala", "Ahmed Sami Hammami", "Jamel Saad", "Melek Kechida", "Hanen Dabbabi", "Syrine Dada", "Marwa Ben Brahim", "Habib Gamra", "Sonia Ouali" ], "abstract": "Hughes-Stovin Syndrome is a systematic disorder generally revealed by deep venous thrombosis (DVT) and pulmonary artery (PA) aneurysms. Its pathogenesis is still unclear and there are no previously established diagnostic criteria or treatment guidelines. It is usually associated with Behçet disease. The aim of this study was to further enlighten this entity as it is often misdiagnosed. We report a case series of five patients with this rare pathology with a systematic literature review of all the previously reported cases.", "keywords": [ "Hughes-Stovin Syndrome", "Pulmonary artery aneurysms", "Deep venous thrombosis", "Behçet disease" ], "content": "Introduction\n\nHughes-Stovin syndrome (HSS) is a rare disorder with high morbidity and mortality rates that was first identified in 1959. It is characterized by the coexistence of deep in situ vascular thrombosis and pulmonary artery vasculitis. HSS is also associated with Behçet’s disease (BD). The main symptoms of HSS include recurrent episodes of deep venous thrombosis (DVT), typically affecting the lower extremities, and the development of PAA, which can lead to life-threatening complications such as pulmonary hemorrhage or aneurysm rupture.1–3\n\nThe occurrence rate of Hughes-Stovin syndrome is extremely low, with only a limited number of cases reported in the medical literature.2 It primarily affects young adults, predominantly males. The exact cause of HSS remains unknown, and various etiologies have been proposed. Interesting hypotheses suggested that Hughes-Stovin syndrome may be attributed to an autoimmune disorder, whereas others propose a genetic predisposition or a potential combination of both factors.4,5\n\nThe management of Hughes-Stovin syndrome is controversial due to its rarity and limited understanding. The primary objective of treatment is to prevent or control the complications associated with DVT and PAA. Anticoagulation therapy based on vitamin K antagonists is commonly used to prevent further DVT formation. Corticosteroids and immunosuppressive drugs like cyclophosphamide are also prescribed to reduce inflammation and prevent the development and progression of PAA as they are associated with a better outcome.2\n\nThese therapies are not based on robust scientific evidence due to the lack of large-scale clinical trials and the limited number of reported cases. The rarity of the syndrome makes it challenging to conduct robust research studies to evaluate different treatment strategies. Furthermore, the optimal duration of anticoagulation therapy and the appropriate dosages of immunosuppressive agents are still areas of debate.2\n\nWe report here five cases of HSS associated with BD (Table 1). All of our patients have been hospitalized in Fattouma Bourguiba University Hospital. An interesting variation of the nature and the severity of the symptoms is reported. This is linked to the primary lesions associated with each case. High doses of corticosteroids, immunosuppressants and anticoagulation are the three pillars of the treatment for each patient. We proposed embolization and embolectomy for the most extreme case.\n\nWe also included a systematic literature review of the reported cases in the literature and a brief discussion section presenting the different hypothesis for the pathophysiology of HSS.\n\n\nCase 1\n\nA 37-year-old North-African unemployed man with a family history of BD presented to the emergency department with a first episode of mild hemoptysis. Physical examination revealed bipolar aphthosis and pseudo folliculitis.\n\nSeveral artery aneurysms were detected by computed tomography (CT) angiography in both lungs, partially thrombosed, with the largest aneurysm measuring 24 mm (Figure 1a) involving the right lower lobe antero-basal segmental pulmonary artery with in situ mural thrombus. The main challenges were to rule-out the differential diagnosis and to start the IV treatment as soon as possible.\n\nGiven the association with BD and after exclusion of other differential diagnoses such as large-vessel arteritis, Wegene’s granulomatosis, systemic lupus erythematosus and connective tissue diseases, we highly suspected the diagnosis of HSS after clinical examination and pulmonary angioscan in the first day of presentation. We concluded to the diagnosis of HSS after receiving lab results ruling-out differential diagnosis. The patient was admitted to the Internal medicine department and treated by three pulses a day of methylprednisolone (1g) since the first day of presentation. These pulses were relayed by oral prednisone (1mg/kg/day). Colchicine (1mg/day per os) was also given as soon as the diagnosis was established.\n\nThe patient was readmitted six months later following another episode of hemoptysis accompanied by chest discomfort. His coagulation tests were within normal ranges, and his vital signs were normal. Both urine and sputum microbiological samples were negative for tuberculosis.\n\nChest CT angiography revealed a marked increase in the size of the right lower lobe antero-basal segmental artery aneurysm from 24 mmm to 116 mm in diameter, with intra-aneurysmal adherent in situ thrombosis and ipsilateral alveolar and pleural bleeding due to extra-luminal acute leak (Figure 1b). Chest CT showed also endoluminal defect in the posterior and latero-basal branches of the right pulmonary artery, related to acute pulmonary embolism. No additional testing or diagnostic challenges were noted as the patient was already being managed for this pathology. Treatment consisted of blood transfusion, antibiotics, and methylprednisolone 1g/day for three days, then cyclophosphamide (2000 mg/m2, IV). After stabilizing his vital signs, he had an embolization of the right lower antero-basal segmental pulmonary artery aneurysm, see Figure 2.\n\nA follow up chest CT angiography one year later showed multiple new pulmonary arteries aneurysms affecting the right upper lobe ventral segmental artery (Figure 3a) and the right lower lobe apical segmental artery, as well as the left upper lobe lingular superior segmental artery (Figure 3b).\n\nA cystic air-filled formation in the right lower lobe antero basal segment close to the embolization coils was also found. The patient had an embolization of the aneurysms of the right upper lobe ventral segmental and the right lower lobe apical segmental arteries partially thrombosed aneurysms (Figure 3c, d).\n\nDuring his last admission, he received three methylprednisolone (1g) pulses followed by oral prednisone (40 mg/day), IV periodic pulses of cyclophosphamide (1500 mg/m2 every four weeks during six months) in addition to oral anticoagulation (2 mg of acenocoumarol once daily per os).\n\nThe patient has since been lost to follow-up.\n\n\nCase 2\n\nThis was a 21-year-old North-African university male student who presented to our department seven months after being diagnosed with BD, with recurrent bipolar aphthosis associated with fever in the context of treatment noncompliance. Physical examination showed facial and neck edema, pseudo-folliculitis lesions and prominent superficial thoracic venous collaterals. Vital signs were unremarkable.\n\nFirst-line laboratory tests including complete blood count, basic metabolic panel, liver and thyroid functions tests, coagulation panel and urinalysis were within normal range except for a hemoglobin concentration of 9 g/dl without particular challenge reported.\n\nSeveral cardiac masses were detected by transthoracic and transesophageal echocardiography, including two in the right ventricle and one in the right atrium that protruded across the tricuspid valve.\n\nChest CT scan showed a brachiocephalic vein blockage and several thrombi in the right atrium and the right ventricle, and also in the superior vena cava.\n\nSeveral pulmonary infarcts and bilateral pulmonary emboli were present in the lower lobes of the lungs. In the right basal segmental artery, a 14 mm diameter aneurysm was observed. Considering the clinic-radiological data, the established association with BD and following the thorough exclusion of other potential differential diagnoses such as Takayasu arteritis, systemic lupus erythematosus, and various connective tissue diseases, the diagnosis of HSS was made.\n\nThe patient was admitted on the first day of presentation and given three daily doses of methylprednisolone (1mg/kg/day) based on an initial high suspicion of HSS. A total of 72 h after admission, laboratory tests ruled out the differential diagnosis and the oral corticosteroid was followed by oral prednisone (30 mg/day) over three weeks, in addition to anticoagulation (acenocoumarol 3 mg/day per os during nine months) and monthly cyclophosphamide pulses (2000 mg/m2 during six months), associated with colchicine (1mg/day during nine months).\n\nAt nine months follow-up, the patient’s symptoms had improved. CT scan showed no evidence of the previously mentioned thrombosis, as well as the regression of the pulmonary aneurysm, indicating a halt in the disease process with resolution of symptoms.\n\n\nCase 3\n\nA 55-year-old North-African daily laborer male patient with unremarkable medical history except for a chronic cough, presented to the emergency department with a pain and swelling of the left leg.\n\nPhysical examination revealed few folliculitis lesions on the back and edema of the left leg that was related to a deep vein thrombosis (DVT), confirmed by the doppler ultrasound.\n\nChest radiography demonstrated a widened superior mediastinum. Further investigation with chest CT scan showed an aneurysm of the ascending aorta of 48 mm.\n\nFirst-line laboratory tests including complete blood count, basic metabolic panel, liver and thyroid functions tests, coagulation panel, erythrocyte sedimentation rate and urinalysis, rheumatoid factor, antinuclear antibody and immunoglobulin levels were not in favor of thrombophilia or immunological abnormalities. HSS diagnosis was established on the basis of an aortic aneurysm and peripheral venous thrombosis with clinical findings consistent with BD.\n\nTherefore, the patient received three daily pulses of methylprednisolone (1mg/kg/day IV) followed by oral prednisone (40 mg/day) with gradual and monitored decrease in dosage over 6 months, and monthly pulses of cyclophosphamide (2000 mg/m2 IV during six months) relayed by oral azathioprine (2 mg/kg/day) associated with colchicine (1 mg/day per os). The patient also received three months of oral anticoagulation (acenocoumarol 2 mg/day per os).\n\nA follow-up thoracic CT scan performed in the same hospital at six months revealed a regression of the ascending aorta aneurysm.\n\n\nCase 4\n\nA 54-year-old North African medical professional male patient was referred to our department after an incidental discovery of an aneurysmal thrombosed postero-basal pulmonary artery during a thoraco-abdomino-pelvic CT scan. This scan was conducted for anatomical kidney assessment in the context of repetitive urinary tract infection. At that time, the patient did not have any relevant personal of family medical history.\n\nThe patient also had a history of neglected bipolar aphthae (buccal and genital). Therefore, the patient was admitted to the internal medicine department to assess those lesions. At admission, physical examination was unremarkable except for genital scars. Echocardiography showed a mild circumferential pericardial effusion (4 mm).\n\nA CT angiography of the aorta was subsequently performed two days after admission and showing a thrombosed saccular aneurysm of two right medial basal sub-segmental pulmonary arteries. BD was then retrospectively diagnosed and the treatment started during the third day of admissions based on three daily pulses of IV methylprednisolone (1 g) and a first pulse of a six-monthly cyclophosphamide 1 g treatment protocol were prescribed.\n\nThe patient was discharged a week later with oral prednisone (1 mg/kg/day) and colchicine (1 mg/day) and was monitored as an outpatient. Three months later, the patient presented to our tertiary care center with bilateral red swollen legs. At presentation, cutaneous examination revealed bilateral calves swelling, pitting edema and localized pain along vein distribution.\n\nCompression ultrasonography confirmed the diagnosis of bilateral deep venous thrombosis (DVT) of the anterior right tibial vein, and posterior left tibial vein, leading to the diagnosis of HSS. Anticoagulant therapy (acenocoumarol 2 mg/day orally) was then initiated in addition to a pulse of IV methyprednisone (500 mg) and cyclophosphamide (1g). The patient was then discharged and is still being monitored.\n\n\nCase 5\n\nA 21-year-old North-African male university student patient presented to our department with massive hemoptysis and dyspnea. He was diagnosed with BD at 16 years of age and treated with colchicine 1 mg/day.\n\nAt presentation, his vital signs were within normal limits. On physical examination the patient was pale and had genital scars with oral aphthae and pseudo-folliculitis over his thighs.\n\nAt blood examination, he had an elevated C-reactive protein (55 mg/dl) and erythrocyte sedimentation rate (ESR) (82 mm at 1 h). Management consisted of urgent intubation and blood transfusion. Chest CT scan with contrast revealed three aneurysms of the right lateral basal segmental branch and one aneurysm of the posterior segmental branch of the right upper lobe, as well as two aneurysms of the apical posterior segmental branch of the left upper lobe.\n\nA right lower lobe infiltrate consistent with blood was identified, and extravasation of contrast into the lung parenchyma was observed.\n\nEmbolization of the right lateral basal segmental branch was successfully performed. Afterwards, the hemoptysis ceased and the patient was extubated. Then, he benefited from three days of methylprednisolone IV pulses (1 g each) and 1 g of cyclophosphamide pulse part of a six-month treatment protocol.\n\nThe patient was then discharged home with oral prednisone 1 g/kg/ day and colchicine 1mg/day.\n\nSix months after discharge, the patient developed intermittent hemoptysis with approximately 10 cc per day for three weeks. He admits being non-compliant to his treatment. His records showed that he received only one immunosuppressant pulse.\n\nOn physical examination, the patient exhibited right heart failure signs (distension, positive hepato-jugular reflux). A repeat contrast chest CT showed eruption of two new aneurysms along with the previous findings and a left pulmonary artery thrombus.\n\nAn elective right lower lobectomy was performed, and anticoagulation treatment was initiated. The patient was also given immunosuppressant therapy (a second six-month cyclophosphamide treatment protocol), along with oral prednisone 1 g/kg/day and colchicine 1 mg/day.\n\nFinally, he was discharged home and did not report any other episode of hemoptysis. He is still being monitored.\n\n\nDiscussion and literature review\n\nHSS is a rare disorder with fewer than 90 cases reported to date in the literature. It was firstly described in 1959 by Dr. John Patterson Hughes and Peter George Ingle Stovin in four male patients.1 This disease is generally characterized by the combination of multiple pulmonary artery (PA) aneurysms and deep vein thromboses (DVT).3\n\nCurrent literature lacks formally established diagnostic criteria due to the scarcity of this syndrome. Although many hypotheses have been suggested to explain the exact etiology and pathogenesis of HSS, vasculitis remains the most relevant explanation6 similar to that involved in BD. In fact, HSS has been considered as “the cardiovascular manifestation of Behçet’s disease”,7 “incomplete Behçet’s”8 “rare case of Behçet’s disease”,9 and “variant of BD”,10 as both HSS and BD may develop PA aneurysms.11–13\n\nAn additional suggested way in which this disease operates is through autoimmune vasculitis inducing a hypercoagulability state.14 This increased tendency to clot may lead to preexisting blood becoming a focal point for bacteria when the patient contracts an infection. Consequently, those thrombi can transform into septic emboli, ultimately causing the characteristic PA aneurysms accentuated with the disease.2 Angiodysplasia has also been suggested as playing a key role in the pathophysiology of this syndrome.15\n\nThe course of this condition has been described as following three phases: initial thrombophlebitis, subsequent development of PA and/or bronchial aneurysms, and eventual rupture of the aneurysm, which can result in hemoptysis.\n\nUsing the terms “Hughes-Stovin syndrome,” we conducted searches on Google Scholar and PubMed. We looked at all of the relevant case reports and case series, excluding articles without patient information and studies on animal subjects, from the inception date to March 2023.\n\nWe identified 88 potentially relevant articles and we excluded 10 after going over the titles, abstracts and full texts. The following articles were excluded: exclusive review articles, articles with insufficient patients’ information, and articles about animal subjects. The results are summarized in Table 2.\n\nThe demographic characteristics of a total of 78 patients including our 5 patients were 64 males and 14 females. A male predominance was noted in the literature and all of our patients were male. The mean age was 32.1 (range 11–55 years), however the age of one patient wasn’t mentioned. In the present study, mean age was 40.4 (range 21–55 years).\n\nThis syndrome usually affects young adults.15 A total of 12 patients were identified as having BD associated to HSS and 14 others had some of the classical features of BD without fulfilling the diagnostic criteria. However, in the current case series, all the patients presented BD features.\n\nAt disease onset, patients may present with DVT, right heart failure, superficial thrombophlebitis, hemoptysis, headache, diplopia and seizures.2,16\n\nAll these clinical features are also encountered in many other diseases indicating the diagnostic difficulties. Consequently, special attention should be given to clinical or radiological manifestations allowing the differentiation.\n\nMoreover, the diagnosis might be fortuitous, as we described in the case number 4. Fortunately, for the first time since its initial report in 1959, a promising start has been made by the Hughes-Stovin Syndrome International Study Group (HSSISG), creating a comprehensive reference atlas of computed tomography pulmonary angiography (CTPA) images, a guide defining the wide spectrum of CTPA findings that have been observed in HSS.17,18\n\nTriggianese et al. presented an interesting approach linking HSS to HLAB51 in their case report, which is in contradiction with Manole et al.’s work suggesting that HSS is a mainly vascular disorder associated with certain minor genetic variations that can interfere with proper vascular function.4,5\n\nThese assumptions need further investigation but are hindered by the scarcity of patients. From the anatomical perspective, aneurysms may be single, multiple, unilateral or bilateral.19\n\nThey can arise anywhere in the central circulation as shown in our case series; however, the pulmonary region remains preferentially affected. In fact, our review indicates that nearly two thirds of cases presented with pulmonary aneurysms.\n\nThrombosis observed in HSS involves large vessels including the vena cava, jugular, iliac, femoral and the brachiocephalic veins (as we reported in the case number 2), as well as cardiac chambers and dural sinuses.6,8,20–23\n\nBeing an exceedingly rare disorder of unknown etiology, no randomized case control trials are available to help select the most appropriate and effective treatment for HSS. Recent literature reviews reported treatment success with corticosteroids and/or immunosuppressants. Cyclophosphamide is the most commonly used immunosuppressant followed by azathioprine.24 These treatments are considered by almost all authors as a first line medical treatment option of HSS.6,12 All of our patients were treated with corticosteroids, cyclophosphamide and oral anticoagulation.\n\nThe use of anticoagulants and thrombolytic agents remains controversial; they increase the risk of fatal hemorrhage while conferring a beneficial effect within a pre-embolic or embolic state. Consequently, clinicians should carefully consider case by case scenarios when recommending these therapies. Inferior vena cava (IVC) filter placement may be of interest in extreme cases.5\n\nAnti–tumor necrosis factor α (TNF-α) presents a new medical treatment option with promising perspectives. It provides a stable disease remission and cases of complete resolution of pulmonary aneurysms have been reported.25\n\nSurgical resection is still described as the gold standard treatment when the patient presents with severe or recalcitrant hemoptysis.26 However, surgery is not considered in cases of bilateral, extensive PA aneurysms. Thus, transcatheter arterial embolization is regarded as a promising alternative as it is less invasive and allows selective treatment of multiple and/or bilateral aneurysms,15 and prevents massive hemoptysis.\n\n\nConclusions\n\nHSS remains a diagnostic and therapeutic dilemma due to its uncommon occurrence and limited understanding of its pathogenesis.\n\nThrough our review of the literature and presentation of our case series, we have underscored the importance of considering HSS as a differential diagnosis in patients presenting with PAA and DVT. The association with BD and the exclusion of other potential differential diagnoses such as large-vessel arteritis, Wegener’s granulomatosis, systemic lupus erythematosus, and connective tissue diseases are crucial in making an accurate diagnosis.\n\nThe therapeutic management of HSS poses significant challenges. Our case series highlights efficient approaches undertaken, ranging from medical management alone based on corticosteroids, immunosuppressants and anticoagulation, to embolization and embolectomy in the most extreme cases, underscoring the individualized nature of therapeutic decisions.\n\nFurther research is warranted to enhance our understanding of the underlying pathogenesis, optimal diagnostic strategies, and treatment modalities for HSS. In order to improve patient outcomes and create evidence-based guidelines, collaboration between healthcare professionals is essential. Multidisciplinary teams should include internists, rheumatologists, cardiologists, radiologists, and surgeons.\n\nBy increasing awareness and understanding of this rare syndrome, we aim to improve early recognition, prompt management, and ultimately enhance the prognosis for patients affected by HSS.\n\n\n\n‐ Femoral vein thrombosis\n\n‐ Aneurysm of a left pulmonary artery (PA) segment.\n\n\n\n‐ Deep vein thrombosis (DVT) in the right leg\n\n‐ Right-sided lower lobe PA aneurysm\n\n\n\n‐ Thrombosis of the vena cava\n\n‐ PA aneurysms\n\n\n\n‐ Partially thrombosed aneurysm of the right lower lobe PA\n\n‐ Left lower lobe PA occlusion\n\n‐ DVT\n\n\n\n‐ PA aneurysm\n\n‐ Thrombus in the inferior vena cava (IVC)\n\n\n\n‐ DVT of the right leg\n\n‐ Pulmonary artery aneurysms\n\n‐ Lung thromboembolism\n\n\n\n‐ Multiple bilateral pulmonary segmental aneurysms with thrombosis\n\n\n\n‐ Multiple PA aneurysms\n\n‐ DVT\n\n\n\n‐ Celiac trunk aneurysm\n\n‐ Defuse thrombosis\n\n\n\n‐ PA aneurysm and thrombosis\n\n\n\n‐ Thrombus in the IVC\n\n‐ Multiple PA aneurysms\n\n\n\n‐ Thrombophlebitis in the saphenous vein\n\n‐ PA aneurysm\n\n\n\n‐ Thrombophlebitis in the basilic vein\n\n‐ PA aneurysm\n\n\n\n‐ DVT of the left femoral vein\n\n‐ Aneurysm and mural thrombosis of the left PA\n\n\n\n‐ Thrombophlebitis\n\n‐ bilateral PA aneurysms\n\n\n\n‐ Internal iliac artery aneurysm\n\n‐ Multiple bilateral PA\n\n‐ aneurysms partially thrombosed\n\n\n\n‐ DVT\n\n‐ Thoracic aorta aneurysm\n\n\n\n‐ Large aneurysms of the segmental branches of lower lobe arteries bilaterally and right upper lobe artery\n\n‐ Bilateral femoral vein thrombosis\n\n\n\n‐ DVT in the right iliac, right common femoral, right superficial femoral and right popliteal veins\n\n‐ Aneurysm of her right interlobar PA\n\n\n\n‐ DVT of the left external iliac and femoral vein\n\n‐ Aneurysm in the right lower lobe PA\n\n‐ Multiple chronic thromboses of the sclera mater venous sinuses\n\n\n\n‐ Multiple PA aneurysms\n\n‐ Thrombosis and intracardiac thrombi formation\n\n\n\n‐ Multifocal venous thromboses (IVC, right iliac and renal veins, hepatic veins, and left and right pulmonary veins)\n\n‐ Bilateral pulmonary aneurysms\n\n\n\n‐ Thrombosis of both pelvic veins and the IVC\n\n‐ Bilateral bronchial arteries aneurysms\n\n\n\n‐ Three partially thrombosed aneurysms\n\n\n\n‐ PA aneurysm\n\n‐ multiple venous thrombosis (vena cava, peripheral veins, or dural sinuses)\n\n\n\n‐ Bilateral pulmonary artery aneurysms\n\n‐ Intracardiac thrombi\n\n‐ Peripheral vein thrombosis (right distal subclavian, axillary, brachial, and proximal basilic vein thrombosis)\n\n\n\n‐ Lower limb deep vein thrombosis\n\n‐ PA aneurysms\n\n\n\n‐ Multiple venous occlusions in the superior vena cava and the left brachiocephalic vein\n\n‐ Bilateral PA aneurysms\n\n\n\n‐ Atrial and sigmoid sinus thrombosis\n\n‐ PA aneurysms\n\n‐ Aneurysms of femoral and humeral arteries\n\n\n\n‐ Femoral, popliteal, and small veins thrombosis\n\n‐ Bilateral multiple PA aneurysms with intraluminal thrombi\n\n\n\n‐ Multiple PA aneurysms\n\n‐ PA thrombi\n\n\n\n‐ Superficial thrombophlebitis\n\n‐ Peripheral PA aneurysms\n\n\n\n‐ Bilateral femoral DVT extending into the IVC\n\n‐ Multiple bilateral aneurysms\n\n\n\n‐ Left femoral vein thrombosis\n\n‐ Left PA aneurysm\n\n\n\n‐ Left sigmoid, transverse sinuses, both internal jugular veins and bilateral iliac vein thromboses and right femoral, right brachial\n\n‐ Pulmonary artery aneurysms\n\n\n\n‐ Two aneurysms: in a segmental branch of the inferior lobar right PA and in the inferior lobar branch of the left PA (perforated into the adjacent bronchus)\n\n‐ Thrombophlebitis in the lower limbs and left saphenous vein\n\n\n\n‐ Multiple PA aneurysms and thrombosis\n\n\n\n‐ Multiple PA aneurysms and thrombosis\n\n\n\n‐ Bilateral thrombosis of the deep venous trunks of the basal bronchi inferior extremities\n\n‐ Thrombosis of the iliac veins and of the IVC\n\n‐ Polylobular aneurysmal pulmonary pockets\n\n\n\n‐ Occlusion of the IVC partially thrombosed\n\n\n\n‐ DVT\n\n‐ Multiple PA aneurysms\n\n\n\n‐ Left femoral vein thrombosis\n\n‐ Large aneurysm in the left lower lobe PA\n\n\n\n‐ Multiple pulmonary aneurysms\n\n‐ Thrombotic obstruction of the inferior vena cava\n\n‐ Mural thrombosis in the right heart\n\n\n\n‐ Deep vein thrombosis (DVT) of the right popliteal and right common femoral veins\n\n‐ Bilateral PA aneurysms\n\n\n\n‐ Sagittal sinus thrombosis\n\n‐ Extensive DVT in the left iliofemoral veins\n\n‐ Bilateral pulmonary aneurysms\n\n\n\n‐ Deep venous thrombosis of both legs and the pelvic veins\n\n‐ Two large pulmonary aneurysms\n\n\n\n‐ Multiple pulmonary artery aneurysms -Inferior vena cava thromboses\n\n\n\n‐ Multiple pulmonary artery aneurysms\n\n‐ Thrombi in lower lobes\n\n\n\n‐ Multiple intrapulmonary artery aneurysms with systemic thrombophlebitis\n\n\n\n‐ Multiple aneurysms in the medium sized arteries\n\n‐ Dural venous sinus thrombosis\n\n\n\n‐ Deep veins of the right leg and the right femoral vein thrombosis\n\n‐ Multiple aneurysms of the right pulmonary arteries\n\n\n\n‐ Multiple pulmonary artery aneurysms -Left common iliac vein and right common femoral vein thrombosis\n\n\n\n‐ Bilateral iliac vein thrombosis extending to the vena cava\n\n‐ Aneurysm of the inferior\n\n‐ branch of right pulmonary artery\n\n\n\n‐ Multiple aneurysms in segmental and subsegmental pulmonary arteries\n\n‐ Small aneurysms in the upper and lower left bronchial arteries\n\n‐ Superior mesenteric artery aneurysm -An embolized splenic aneurysm\n\n\n\n‐ DVT\n\n‐ PA emboli\n\n‐ Pseudoaneurysm of the right iliac artery\n\n‐ Two PA aneurysms\n\n\n\n‐ Giant aneurysms of the PA\n\n‐ DVT\n\n\n\n‐ Multiple PA aneurysms partially thrombosed\n\n\n\n‐ Right superficial femoral artery aneurysm\n\n‐ Left posterior tibial artery pseudoaneurysm\n\n‐ Multiple PA aneurysms\n\n‐ -Deep venous thrombosis\n\n\n\n‐ PA occlusions and aneurysm\n\n\n\n‐ Pulmonary artery aneurysms\n\n‐ Recurrent thrombosis of systemic veins and dural sinuses\n\n\n\n‐ Right lower extremity deep vein thrombosis\n\n‐ Right intraventricular thrombus\n\n‐ Multiple pulmonary artery aneurysms\n\n\n\n‐ Multiple pulmonary artery aneurysms\n\n‐ Pulmonary embolism\n\n‐ Thrombus into the right atrium\n\n\n\n‐ -Pulmonary thrombosed aneurysms\n\n\n\n‐ Thrombosis of the left femoral vein and the greater saphenous vein\n\n‐ three separate aneurysms of the fight lower lobe branches of the pulmonary artery\n\n\n\n‐ Multiple pulmonary arterial aneurysms\n\n‐ Occlusion of the left upper lobe pulmonary artery\n\n\n\n‐ Left pulmonary artery aneurysm\n\n‐ Chronic in situ thrombosis in the descending branch of the right PA\n\n‐ Right atrial thrombus\n\n\n\n‐ Large PA aneurysm\n\n‐ DVT\n\n\n\n‐ Right atrial thrombus\n\n‐ bilateral PA aneurysms\n\n\n\n‐ PA aneurysm and arteriovenous thrombi involving IVC and right heart cavities\n\n‐ Non-bacterial thrombotic endocarditis involving the tricuspid valve\n\n‐ DVT\n\n\n\n‐ Five bilateral segmental and subsegmental\n\n‐ IVC thrombosis\n\n‐ Left common iliac vein ectasia\n\n\n\n‐ PA aneurysm\n\n‐ DVT\n\n\n\n‐ multiple thrombosed PA aneurysms\n\n‐ Right ventricular thrombus\n\n\n\n‐ Multiple PA aneurysms\n\n‐ Left renal and superior mesenteric arteries aneurysms\n\n‐ Recurrent DVT\n\n\nEthical statement\n\nThe Declaration of Helsinki-Ethical Principle for Human Subjects in Medical Research guided the conduct of this paper.\n\nWritten informed consent was obtained from all participants prior to their inclusion. The patients also provided written informed consent for the publication of their clinical details and images.\n\nThe illustrations and scans utilized in this case series paper are permitted for utilization, with all identifying individual features eliminated. This was done to protect the privacy and confidentiality of the individuals involved and to comply with ethical standards in scientific research. All images presented in this paper have been edited to remove any identifying information.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nHughes JP, Stovin PG: Segmental pulmonary artery aneurysms with peripheral venous thrombosis. Br. J. Dis. Chest. 1959 Jan; 53(1): 19–27. PubMed Abstract \| Publisher Full Text\n\nEmad Y, Ragab Y, Kechida M, et al.: A critical analysis of 57 cases of Hughes-Stovin syndrome (HSS). 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PubMed Abstract \| Publisher Full Text\n\nde Vries W , Koppelman GH, Roofthooft MT, et al.: Pulmonary medium vessel vasculitis in an 11 year old boy: Hughes Stovin syndrome as a variant of polyarteritis nodosa? Pediatr. Rheumatol. Online J. 2011 Aug 4; 9: 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYagi T, Yamagishi F, Mizutani F, et al.: A case of Behçet’s disease presenting with Hughes-Stovin syndrome (multiple pulmonary arterial aneurysms remitting with corticosteroid therapy). Nihon Kokyuki Gakkai Zasshi. 2001 Feb; 39(2): 140–144. PubMed Abstract\n\nAmezyane T, Bassou D, Abouzahir A, et al.: Unusual right ventricular thrombus in a woman with Hughes-Stovin syndrome. Intern. Med. Tokyo Jpn. 2010; 49(2): 207–208. PubMed Abstract \| Publisher Full Text\n\nJaramillo N, Gómez-Bueno M, García Suárez A, et al.: Combined pulmonary vasodilator therapy and endovascular intervention for Hughes-Stovin syndrome. Int. J. Cardiol. 2015 Jan 15; 178: e5–e7. PubMed Abstract \| Publisher Full Text\n\nBalci NC, Semelka RC, Noone TC, et al.: Multiple pulmonary aneurysms secondary to Hughes-Stovin syndrome: demonstration by MR angiography. J. Magn. Reson. Imaging JMRI. 1998; 8(6): 1323–1325. PubMed Abstract \| Publisher Full Text\n\nAlí-Munive A, Varón H, Maldonado D, et al.: Giant aneurysms of the pulmonary artery and peripheral venous thrombosis (Hughes-Stovin syndrome): regression with immunosuppressant therapy. Arch. Bronconeumol. 2001 Dec; 37(11): 508–510. PubMed Abstract \| Publisher Full Text\n\nKetchum ES, Zamanian RT, Fleischmann D: CT angiography of pulmonary artery aneurysms in Hughes-Stovin syndrome. AJR Am. J. Roentgenol. 2005 Aug; 185(2): 330–332. PubMed Abstract \| Publisher Full Text\n\nKopp WL, Green RA: Pulmonary artery aneurysms with recurrent thrombophlebitis. The “Hughes-Stovin syndrome.”. Ann. Intern. Med. 1962 Jan; 56: 105–114. PubMed Abstract \| Publisher Full Text\n\nKably IM, Reveron C: Multimodal endovascular management of a Jehovah’s Witness patient with Hugues-Stovin syndrome presenting with ruptured pulmonary artery aneurysm and cardiopulmonary thromboembolism. Eur. J. Cardio-Thorac. Surg. Off. J. Eur. Assoc. Cardio-Thorac. Surg. 2015 Apr; 47(4): e158–e161. PubMed Abstract \| Publisher Full Text\n\nLee WY, Hoon CS, Kim HR: Massive Hemoptysis Caused by Atypical Behcet’s Disease. Korean J. Thorac. Cardiovasc. Surg. 2014 Apr; 47(2): 178–180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWakamiya R, Fujiwara Y, Hamada M, et al.: Beneficial effect of diltiazem on pulmonary hypertension in a patient with Hughes-Stovin’s syndrome. Kokyu To Junkan. 1991 Dec; 39(12): 1247–1250.\n\nHamdy F, Tharwat S, Abdelwahab HW, et al.: Hughes-Stovin syndrome without venous involvement – Unusual presentation of a rare disease: Case report and review of the literature. Egypt Rheumatol. 2020 Apr 1; 42(2): 159–163. Publisher Full Text\n\nMoussa N, Znegui T, Snoussi M, et al.: Recurrent Haemoptysis Revealing Hughes-Stovin Syndrome. Eur. J. Case Rep. Intern. Med. 2021 Oct 5; 15. Publisher Full Text\n\nVyas V, Bryant SM, Shah A, et al.: Disseminated Mycobacterium chelonae infection with Hughes-Stovin syndrome. Bayl. Univ. Med. Cent. Proc. 2021 Sep 3; 34(5): 595–596. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "243628", "date": "27 Mar 2024", "name": "Nashwa K El Shaarawy", "expertise": [ "Reviewer Expertise Rheumatology", "Vasculitis", "RA", "SLE" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTHE AUTHORS ARE TO BE CONGRATULATED For tracking a challenging rare clinical problem and attempting to improve the research with such collective data. The inclusion of a case series along with a systematic literature review adds depth to the study. However, it might benefit from a brief mention of the key findings of the study. Regarding the introduction, some citations in this section would enhance the information provided. Literature review: Each case is described in a good way, including clinical findings, diagnostic imaging, treatment modalities, and outcomes. As well the use of images enhances the clarity of the presentation. However, ensuring consistency in formatting and style across all case presentations would improve the review.\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/12-1001
https://f1000research.com/articles/12-206/v1	23 Feb 23	{ "type": "Research Article", "title": "Ketamine infiltration decreases the need for opioids after thyroid surgery", "authors": [ "Moncef Sellami", "Imen Zouche", "Mariam Ben Ayed", "Maroua Bouhali", "Khadija Ben Ayed", "Salma Ktata", "Boutheina Hammami", "Mohamed Amine Chaabouni", "Ilhem Charfeddine", "Imen Zouche", "Mariam Ben Ayed", "Maroua Bouhali", "Khadija Ben Ayed", "Salma Ktata", "Boutheina Hammami", "Mohamed Amine Chaabouni", "Ilhem Charfeddine" ], "abstract": "Background: Postoperative pain increases the risk of postoperative complications and may predispose patients to chronic post-surgical pain. This study aims to evaluate the impact of ketamine wound infiltration versus placebo at the end of thyroid surgery on postoperative pain and analgesic requirements. Methods: In this randomized controlled trial, we prospectively studied patients who underwent thyroid surgery. Patients were randomized into two groups: group S, where local infiltration was performed using 10 ml of a physiological saline solution; and group K, where 10 ml of a solution containing 2 mg/kg ketamine was infiltrated. Standardized thyroidectomies were performed in the 2 groups. Pain perception was measured using a visual analog scale (VAS) every 10 minutes in the post-anesthetic care unit (PACU) for 2 hours and thereafter every 6 hours during the first 24 hours. The opioid requirement in the PACU was evaluated. A comparison between the 2 groups was carried out. Results: Postoperatively, the mean VAS was higher in group S compared to group K during all PACU stay periods and the first 24 hours. Pain scores during swallowing were significantly lower for group K in the PACU at 0, 10, and 20 minutes. The mean morphine consumption in the PACU was 0.71 mg and 0 mg respectively in group S and group K (p=0.03). The incidence of nausea and vomiting was similar in both groups. Conclusions: Ketamine wound infiltration is an efficient modality to reduce postoperative opioid consumption compared to a placebo after thyroid surgery.", "keywords": [ "Ketamine", "thyroid surgery", "wound infiltration", "analgesia" ], "content": "Introduction\n\nThyroid surgery is known to be responsible for mild to moderate postoperative pain during the first 24 hours after surgery.1 Postoperative pain can result in significant discomfort, delay in hospital discharge, and the development of chronic pain.2 Postoperative pain control is fundamental for better recovery and quick return to daily activities. Opioids are effective for postoperative analgesia, but they cause sedation, respiratory depression, nausea, and vomiting.3 The modern concept in pain management includes a multimodal approach that involves the use of systemic analgesics associated with locoregional anesthesia techniques to reduce postoperative pain and opioid requirement.4,5 The locoregional anesthesia technique has mainly included bilateral superficial cervical plexus block and local wound infiltration with a local anesthetic agent.1,6,7 Bilateral block of the superficial cervical plexus is an effective technique that ensures better postoperative analgesia and has been widely used in thyroid surgery.6,8 Wound infiltration with a local anesthetic agent is a simple and safe procedure for reducing post-operative pain.6,7 In the current literature, different anesthetic agents for thyroid surgery wound infiltration were studied, including bupivacaine, diclofenac, ropivacaine, and, more rarely, ketamine.9–12\n\nKetamine has a high affinity for N-methyl-D-aspartate receptors; it can also bind to opioid mu and sigma receptors that provide central and peripheral analgesic effects.13\n\nThe purpose of this study was to assess the impact of local wound infiltration using ketamine at the end of thyroid surgery on postoperative pain and opioid requirement.\n\n\nMethods\n\nEthical approval for this study was given by the Committee for the Protection of People in Southern Tunisia (approval number 0117/18). This randomized double-blind study was achieved through collaboration between the ENT department and the anesthesia department of the university hospital, Habib Bourguiba in Sfax, Tunisia.\n\nPatients scheduled for thyroid surgery were enrolled after having provided written informed consent, which they also gave for publication of their data, in de-identified form.\n\nPatients aged 18 to 65 were included in this study if they had an ASA score (American Society of Anesthesiology) of I or II. Patients with unstable diabetes, an allergy to study drugs, a history of previous cervical surgery, or a history of cardiac or respiratory disease, as well as patients on long-term analgesics or corticosteroids, were not included in this study. Patients who had major complications, such as allergic reactions to anesthetic drugs and major bleeding, or patients whose surgery duration exceeded 3 hours were excluded from this study. Patients who underwent a neck dissection associated with thyroid surgery were also excluded from the study.\n\nThe patients were informed about the anesthetic protocols and were educated about the use of the visual analog scale (VAS) to evaluate the severity of the pain. All patients in the study were blinded to the drug they received for postoperative pain.\n\nThese patients were randomized into two groups: group K represents wound infiltration using 10 ml of a solution containing 2 mg/kg of ketamine and group S (placebo group) represents wound infiltration using 10 ml of normal saline solution. The wound injection was performed at the end of the surgery after the skin suture.\n\nAfter 3 minutes of preoxygenation, anesthesia was induced with an injection of 3 μg/kg of Fentanyl followed by 3 mg/kg of Propofol; and with intubation using a silicone wire-reinforced tracheal tube with 0.2 mg/kg Cisatracurium. Anesthesia was maintained using isoflurane with a minimum alveolar concentration (MAC) of 1% in a 50% oxygen/air mixture. A 0.03 mg/kg Cisatracurium bolus was administered every 40 min, and a Fentanyl bolus of 0.1 μg/kg was injected whenever there was an increase of 20% of the base values in heart rate or systolic arterial blood pressure.\n\nBefore surgery, an anesthesiologist, not involved in the study, prepared an unlabeled sterile syringe using 10 ml of ketamine (2 m/kg) or 10 ml of a saline solution. After wound closure, the infiltration was performed by a blinded surgeon. The needle was introduced to a depth of 0.5 cm, an aspiration was then performed to avoid an intravascular injection followed by infiltrating the wound. The content of the syringe was used for homogenous infiltration of the subcutaneous wound sides by the surgeon.\n\nThirty minutes before the end of the surgery, all patients received 1 g of paracetamol ad then were extubated before being moved to the post-anesthetic care unit (PACU) for close monitoring for 2 hours. In the PACU, intravenous morphine titration (2 mg every 5 min) was performed until the VAS value was less than 30. All patients were admitted for at least 24 hours postoperatively in the ENT department. They all received 1 g of paracetamol systematically every 6 hours during the first 24 hours. An anesthesiologist blinded to study groups collected intraoperative and postoperative parameters.\n\nThe primary outcomes were to determine the intensity of the pain using VAS from 0-100 in the first 24 hours. The VAS score was assessed every 10 minutes in the PACU for 2 hours and every 6 hours during the first 24 hours after the operation in the ENT department. Nefopam was administered in the cases where VAS exceeded 30. Opioid requirements were recorded during the PACU admission period. The occurrence of side effects of opioids and ketamine was noted.\n\nIn each group, a total number of 27 patients was required to obtain a difference on the VAS scale of 20 mm (standard deviation of 25 mm), with a power of 0.9 and an α=0.05. To allow for some margin of error in the underlying assumptions, the baseline sample size was increased by 10 % to 32 patients per group. χ2 test or Fisher statistical tests were used for the analysis of qualitative variables and the Student's T test for the analysis of quantitative variables (sample size of more than 30 patients per group; normality was typically ensured by the central limit theorem). A p-value < 0.05 was considered statistically significant (one-tailed test). Data entry and statistical analysis of data were performed with the software version 20 of Statistical Package for Social Sciences (SPSS) for Windows (SPSS Inc., Chicago, IL, USA).\n\n\nResults\n\nThe study lasted for a total of 6 months from September 2018 to March 2019. Sixty-four patients were included. Two patients were withdrawn from the study for prolonged surgery (more than 3 hours). The flow of patients is represented in the CONSORT diagram of the study, which is shown in Figure 1. Demographic and intraoperative characteristics of patients are represented in Table 1.\n\n* Student t-test.\n\n† Fisher exact test.\n\n‡ χ2 test.\n\nPain scores during swallowing were significantly lower for group K in the PACU at 0, 10, and 20 minutes (Table 2).\n\nRegarding the first 24-hour postoperative hospitalization period in the ENT department, the mean VAS values were higher in group S compared to group K; either at rest or during swallowing, but the difference was not statistically significant (p>0.05) (Table 3).\n\nNone of the patients in group K received morphine while it was administered to four patients in group S. The mean morphine consumption in the PACU was 0.71 mg and 0 mg respectively in group S and group K. The difference in morphine consumption between both groups was statistically significant (p=0.04). Nefopam was not administered to any of our patients.\n\nNo patient presented a hematoma at the injection points of the product. A patient in group K showed hallucinations during the stay in the PACU. Dizziness was recorded in three patients of group K and one patient of group S. No patients presented postoperative shivers.\n\nRespiratory distress was not recorded in any of our patients. Nausea and vomiting were observed in a total of 11 patients but without significant differences between both groups.\n\n\nDiscussion\n\nAcute pain is one of the most common complaints after surgery. Thyroid surgery is known to be responsible for mild to moderate postoperative pain during the first 24 hours, with a mean VAS score of 6.9 (±1.7).1,14 Pain following thyroid surgery has several origins: surgery-induced inflammatory lesions, intraoperative neck hyperextension, postoperative drainage, incision site, and laryngotracheal mobilization at swallowing.15,16\n\nThe attempted locoregional anesthetic approaches to reduce post-thyroidectomy pain have included mainly bilateral superficial cervical plexus block and local wound infiltration with a local anesthetic agent.1,6,7\n\nWound infiltration with a local anesthetic agent is a simple and safe procedure to reduce postoperative pain with fewer side effects in comparison with cervical plexus block.6,7 Several molecules were used to infiltrate the thyroidectomy wound. Bupivacaine has been reported to effectively reduce post-thyroidectomy pain.10,16,17 Sellami et al.10 concluded that infiltration of bupivacaine wounds is effective in reducing pain perception and opioid requirements after thyroidectomy. In a recent meta-analysis, Jiang et al.17 recommended performing a local infiltration using bupivacaine before or after skin closure, 20 to 75 mg, as it significantly reduced postoperative pain and decreased rescue analgesic requirements.\n\nDiclofenac has also been reported as an effective molecule to infiltrate the wound before surgery compared to Bupivacaine in reducing postoperative pain.11 However, the use of ropivacaine was not associated with an analgesic effect.12\n\nKetamine also belongs to the molecules whose local wound analgesic effect has been studied. The local ketamine analgesic effect is related to different mechanisms: blocking sodium, calcium, and potassium channels, as well as binding to opioid, cholinergic, D2, and 5-HTE receptors and monoamine transporters.9,18,19 It also decreases microglial activation and migration and prevents local inflammation extension and exacerbation through its action on the prototype inflammatory mediators, the adenosine receptor system, and the activation of the NMDA receptors.20 However, only a limited number of studies demonstrated the analgesic effect of ketamine in thyroid surgery, either locally or systemically.\n\nIn our study, ketamine (2 mg/kg) was injected into the wound after skin closure. It was associated with a significant reduction in VAS scores during swallowing compared to the saline solution group in PACU at 0, 10, and 20 minutes. In a prospective, controlled, double-blind, randomized study, Abd EL-Rahman et al.9 compared post-thyroidectomy analgesia in three different groups using local ketamine wound instillation, intramuscular ketamine injection, or placebo. He recorded that VAS scores at rest or during movement, as well as morphine consumption, were reduced in the local ketamine group compared to intramuscular ketamine and placebo. He then concluded that local ketamine instillation of 1 mg/kg of ketamine diluted in 10 ml of a saline solution was associated with a superior analgesic effect compared to the other groups. He also recorded that total morphine consumption and first analgesia demand were significantly lower in the local ketamine group.\n\nWe also demonstrated that local injection of ketamine significantly reduced the average morphine requirement compared to the saline solution group. The use of opioids can be associated with severe side effects such as nausea, vomiting, and respiratory distress.15 Thus, modern analgesic approaches aim to reduce both postoperative pain and opioid requirements.4,5\n\nKim et al.21 also studied the effect of intravenous ketamine infusion during the bilateral axilla-breast approach for robotic or endoscopic thyroidectomy and concluded that it significantly reduced postoperative pain scores compared to the saline solution group with no increase in adverse effects. Lee et al.15 used ketamine infusion after robotic thyroidectomy and demonstrated that it was associated with a lower VAS score 24 hours after surgery and with a decrease in opioid needs.\n\nThe use of ketamine as a local wound infiltration agent was studied more frequently in oral, tonsillar, and abdominal surgeries. In their meta-analysis, Cho et al.22 concluded that the use of ketamine locally or systematically could provide pain relief in children undergoing tonsillectomy without side effects. The addition of ketamine to ropivacaine for local tonsillar application was associated with better postoperative analgesia compared to the application of ropivacaine alone.23 Dal et al.24 proved that locally injected ketamine effectively reduced pain scores in patients undergoing adenotonsillectomy. However, in a recent prospect guideline for post-tonsillectomy pain management, Aldalmulij et al.25 did not recommend the use of ketamine infiltration in children due to the risk of systemic side effects after absorption, although different studies consistently concluded that it was effective in reducing pain and analgesic requirements after tonsillectomy in children.\n\nInfiltrating the surgical site using ketamine in pediatric patients undergoing cleft palate surgery was superior to Bupivacaine with respect to analgesic requirements, quality of sleep after surgery, and speed of recovery.26 In third molar surgery, a meta-analysis based on prospective clinical trials and randomized controlled trials demonstrated the analgesic effect of local administration of ketamine and its anti-inflammatory potential during the first 24 hours.27\n\nRegarding abdominal surgeries, Honarmad et al.28 conducted a randomized, double-blind, prospective, placebo-controlled study, and concluded that intravenous or subcutaneous infiltration prior to incision of 0.5 mg/kg of ketamine improved analgesia during the first six hours after appendectomy with no significant side effects. The same authors reported that subcutaneous ketamine infiltration or intravenous ketamine administration for patients undergoing open cholecystectomy before the surgical incision provided auxiliary analgesia 24 hours after surgery.\n\nOur study has several limitations. First, thyroid surgeries were performed by different surgeons. Second, the VSA remains a subjective scale, especially since its assessment during the PACU stay was relatively uneasy with the residual anesthetic effect. We did not consider the size of the resected thyroid gland which can reflect the extent of surgery and influence postoperative pain.\n\n\nConclusions\n\nIn conclusion, local wound infiltration using 2 mg/kg of ketamine is an effective approach to reducing opioid requirements after thyroid surgery without increasing the side effects of ketamine.", "appendix": "Data availability\n\nFigshare: Underlying data for ‘Ketamine infiltration decreases opioid requirement after thyroid surgery’, https://doi.org/10.6084/m9.figshare.21365703.v1. 29\n\nThis project contains the following underlying data:\n\n‐ data ketamine thyroidectomy.sav (SPSS format; all datasets have been de-identified in accordance with the Safe Harbor method)\n\n‐ CONSORT checklist\n\nData are available under the terms of the Creative Commons Attribution 4.0 International Public Licence (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe would like to thank all nurses and the personnel of the operating unit, in the otolaryngology and anesthesiology departments for their assistance and cooperation.\n\n\nReferences\n\nShih ML, Duh QY, Hsieh CB, et al.: Bilateral superficial cervical plexus block combined with general anesthesia administered in thyroid operations. World J. Surg. 2010; 34(10): 2338–2343. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBanasiewicz T, Meissner W, Pyda P, et al.: Local anesthesia in thyroid surgery – Own experience and review of the literature. Pol. Przegl. Chir. 2011; 83(5): 264–270. PubMed Abstract \| Publisher Full Text\n\nGan TJ: Risk factors for postoperative nausea and vomiting. Anesth. Analg. 2006; 102(6): 1884–1898. Publisher Full Text\n\nMotamed C, Merle JC, Yakhou L, et al.: Postoperative pain scores and analgesic requirements after thyroid surgery: comparison of three intraoperative opioid regimens. Int. J. Med. Sci. 2006; 3(1): 11–13.\n\nMayhew D, Sahgal N, Khirwadkar R, et al.: Analgesic efficacy of bilateral superficial cervical plexus block for thyroid surgery: meta-analysis and systematic review. Br. J. Anaesth. 2018; 120(2): 241–251. PubMed Abstract \| Publisher Full Text\n\nHoh SY, Doon YK, Chong SS, et al.: Randomized controlled trial comparing bilateral superficial cervical plexus block and local wound infiltration for pain control in thyroid surgery. Asian J. Surg. 2019; 42(12): 1001–1008. PubMed Abstract \| Publisher Full Text\n\nStamenkovic DM, Bezmarevic M, Bojic S, et al.: Updates on Wound Infiltration Use for Postoperative Pain Management: A Narrative Review. J. Clin. Med. 2021; 10(20): 4659. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoldegerima YB, Hailekiros AG, Fitiwi GL: The analgesic efficacy of bilateral superficial cervical plexus block for thyroid surgery under general anesthesia: a prospective cohort study. BMC. Res. Notes. 2020; 13(1): 42. 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PubMed Abstract \| Publisher Full Text\n\nMiu M, Royer C, Gaillat C, et al.: Lack of Analgesic Effect Induced by Ropivacaine Wound Infiltration in Thyroid Surgery: A Randomized, Double-Blind, Placebo-Controlled Trial. Anesth. Analg. 2016; 122(2): 559–564. PubMed Abstract \| Publisher Full Text\n\nZanos P, Moaddel R, Morris PJ, et al.: Ketamine and Ketamine Metabolite Pharmacology: Insights into Therapeutic Mechanisms. Pharmacol. Rev. 2018; 70(3): 621–660. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGozal Y, Shapira SC, Gozal D, et al.: Bupivacaine wound infiltration in thyroid surgery reduces postoperative pain and opioid demand. Acta Anaesthesiol. Scand. 1994; 38(8): 813–815. Publisher Full Text\n\nLee J, Park HP, Jeong MH, et al.: Efficacy of ketamine for postoperative pain following robotic thyroidectomy: A prospective randomised study. J. Int. Med. Res. 2018; 46(3): 1109–1120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDumlu EG, Tokaç M, Öcal H, et al.: Local bupivacaine for postoperative pain management in thyroidectomized patients: A prospective and controlled clinical study. Ulus Cerrahi Derg. 2015; 32(3): 173–177. PubMed Abstract \| Publisher Full Text\n\nJiang Y, Zhang Z, Liang B, et al.: The effect of bupivacaine on postoperative pain following thyroidectomy: a systematic review and meta-analysis. Minerva Chir. 2020; 75(3): 193–202. PubMed Abstract \| Publisher Full Text\n\nSchnoebel R, Wolff M, Peters SC, et al.: Ketamine impairs excitability in superficial dorsal horn neurones by blocking sodium and voltage-gated potassium currents. Br. J. Pharmacol. 2005; 146(6): 826–833. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHayashi Y, Kawaji K, Sun L, et al.: Microglial Ca(2+)-activated K(+) channels are possible molecular targets for the analgesic effects of S-ketamine on neuropathic pain. J. Neurosci. 2011; 31(48): 17370–17382. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Kock M, Loix S, Lavand’homme P: Ketamine and peripheral inflammation. CNS Neurosci. Ther. 2013; 19(6): 403–410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim DH, Choi JY, Kim BG, et al.: Prospective, randomized, and controlled trial on ketamine infusion during bilateral axillo-breast approach (BABA) robotic or endoscopic thyroidectomy: Effects on postoperative pain and recovery profiles: A consort compliant article. Medicine (Baltimore). 2016; 95(49): e5485. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCho HK, Kim KW, Jeong YM, et al.: Efficacy of ketamine in improving pain after tonsillectomy in children: meta-analysis. PLoS One. 2014; 9(6): e101259. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHong B, Lim CS, Kim YH, et al.: Comparison of topical ropivacaine with and without ketamine on post-surgical pain in children undergoing tonsillectomy: a randomized controlled double-blind study. J. Anesth. 2017; 31(4): 559–564. PubMed Abstract \| Publisher Full Text\n\nDal D, Celebi N, Elvan EG, et al.: The efficacy of intravenous or peritonsillar infiltration of ketamine for postoperative pain relief in children following adenotonsillectomy. Paediatr. Anaesth. 2007; 17(3): 263–269. Publisher Full Text\n\nAldamluji N, Burgess A, Pogatzki-Zahn E, et al.: PROSPECT Working Group collaborators*: PROSPECT guideline for tonsillectomy: systematic review and procedure-specific postoperative pain management recommendations. Anaesthesia. 2021; 76(7): 947–961. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJha AK, Bhardwaj N, Yaddanapudi S, et al.: A randomized study of surgical site infiltration with bupivacaine or ketamine for pain relief in children following cleft palate repair. Paediatr. Anaesth. 2013; 23(5): 401–406. Publisher Full Text\n\nEsparza-Villalpando V, Ascencio-Padilla R, Pozos-Guillen A, et al.: Local Ketamine Improves Postoperative Analgesia After Third Molar Surgery. J. Oral Maxillofac. Surg. 2019; 77(12): 2386–2400. PubMed Abstract \| Publisher Full Text\n\nHonarmand A, Safavi M, Karaky H: Preincisional administration of intravenous or subcutaneous infiltration of low-dose ketamine suppresses postoperative pain after appendectomy. J. Pain Res. 2012; 5: 1–6. PubMed Abstract \| Publisher Full Text\n\nSellami M: Underlying data for ‘Ketamine infiltration decreases opioid requirement after thyroid surgery’.Publisher Full Text" }	[ { "id": "187834", "date": "31 Jul 2023", "name": "Chihebeddine Romdhani", "expertise": [ "Reviewer Expertise Regional anesthesia", "Anesthesiology", "critical care", "cardiac anesthesia", "artificial intelligence" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, researchers investigated the effects of local ketamine wound infiltration versus saline placebo on postoperative pain and opioid consumption in patients undergoing thyroid surgery. After randomizing 64 patients to receive either ketamine or saline infiltration, the researchers found that the ketamine group had lower VAS pain scores during the PACU stay for static pain at rest and dynamic pain on swallowing. The ketamine group consumed significantly less morphine in the PACU than the saline group. No adverse effects were reported. The authors concluded that local ketamine can reduce postoperative opioid use without increasing side effects. This study is well-designed and addresses an important clinical question.\nWe recommend you to :\nProvide more details regarding the randomization process: Was a computer random number generator used? Were block randomization or stratification techniques used?\n\nPresenting VAS scores graphically could better visualize trends.\n\nIs the work 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": "10074", "date": "18 Aug 2023", "name": "Moncef Sellami", "role": "Author Response", "response": "We thank reviewers for the quality and relevance of the feedback. In this study, researchers investigated the effects of local ketamine wound infiltration versus saline placebo on postoperative pain and opioid consumption in patients undergoing thyroid surgery. After randomizing 64 patients to receive either ketamine or saline infiltration, the researchers found that the ketamine group had lower VAS pain scores during the PACU stay for static pain at rest and dynamic pain on swallowing. The ketamine group consumed significantly less morphine in the PACU than the saline group. No adverse effects were reported. The authors concluded that local ketamine can reduce postoperative opioid use without increasing side effects. This study is well-designed and addresses an important clinical question. We recommend you to : Provide more details regarding the randomization process: Was a computer random number generator used? Were block randomization or stratification techniques used? Author response: The patients were randomly assigned to the two groups through an online random list generator (https://www.random.org), ensuring an unbiased allocation. The randomization method was specified. Presenting VAS scores graphically could better visualize trends. Author response: Although we appreciate the suggestion to present VAS scores graphically, we found that tabulated data provide a more precise and interpretable representation in this particular context." } ] }, { "id": "187838", "date": "10 Aug 2023", "name": "Mohamed Kahloul", "expertise": [ "Reviewer Expertise Anesthesia and intensive care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle:\n\nPostoperative opioid requirements were assessed only in the PACU. Postoperative pain is your primary outcome. Please rephrase your title.\nMethods:\nDifficult airways should be considered as an exclusion criteria because you had assessed VAS during swallowing.\n\nPlease precisely include which ketamine concentration was used.\nResults:\nBaseline characteristics should be comparable between the two groups. Please verify your data (age, performed surgery).\n\nYou have many VAS assessments exceeding 3/10. What was your rescue analgesia?\nDiscussion:\nThe choice of ketamine dosage should be discussed.\n\n\"However, the use of ropivacaine was not associated with an analgesic effect.12\" Others studies had found different results and they should be considered1.\n\nVAS was considered as a limitation while it is a validated tool.\nConclusion:\nPlease put “to reduce” instead of “to reducing”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10075", "date": "18 Aug 2023", "name": "Moncef Sellami", "role": "Author Response", "response": "We express our gratitude to the reviewer for his meticulous assessment, which has contributed significantly to the refinement of our study. Title: Postoperative opioid requirements were assessed only in the PACU. Postoperative pain is your primary outcome. Please rephrase your title. Author response: The title has been revised as 'Ketamine infiltration improves analgesia after thyroid surgery'. Methods: Difficult airways should be considered as an exclusion criteria because you had assessed VAS during swallowing. Please precisely include which ketamine concentration was used. Author response: In effect, difficult airways were considered an exclusion criterion. We specified the ketamine concentration used, which was 50mg/ml Results: Baseline characteristics should be comparable between the two groups. Please verify your data (age, performed surgery). You have many VAS assessments exceeding 3/10. What was your rescue analgesia? Author response: The patients were randomized and the results were statistically verified. Our data, including age and type of surgery, have been carefully validated. We specified that the rescue analgesic during hospitalization was Nefopam in the case of VAS values greater than 30 Discussion : The choice of ketamine dosage should be discussed. Author response: In our study, we infiltrated a solution containing 2 mg/kg ketamine based on established literature and clinical experience, in order to achieve effective analgesia while minimizing potential adverse effects. We discuss the chosen dose. \"However, the use of ropivacaine was not associated with an analgesic effect.12\" Others studies had found different results and they should be considered1. Author response: We discussed this issue and reported the study by Li X et al. VAS was considered as a limitation while it is a validated tool. Author response: We have considered the reviewer's comment and revised the limitations section of the study. Indeed, the VAS is a validated tool despite its subjective nature. Conclusion: Please put “to reduce” instead of “to reducing”. Author response: We have made the requested change, replacing \"to reducing\" with \"to reduce\" in the relevant section." } ] } ]	1	https://f1000research.com/articles/12-206
https://f1000research.com/articles/12-1000/v1	18 Aug 23	{ "type": "Study Protocol", "title": "Evaluation and comparison of anxiety levels after using Guided Imagery technique for relaxation and modified Guided Imagery-Hypnotherapy technique in 6 to 14 year-old children undergoing local anesthesia administration", "authors": [ "Himani Parakh", "Nilima Thosar", "Nilima Thosar" ], "abstract": "Anxiety has been defined as a “vague, unpleasant feeling accompanied by a premonition that something undesirable is about to happen”. Dental anxiety is driven by a strong negative or unpleasant emotion towards a dental office or dental procedures. Children with a high level of dental anxiety are more resistant to treatment, and due to this avoidance in dental care can lead to needing more dental treatments. The quality of a treatment being carried out, as well as what materials that are to be used, is directly influenced by a child’s level of cooperation and ability to follow instructions. Behaviour management techniques play a major role during all paediatric treatment procedures, and it is the clinician who is responsible to properly and safely guide the child’s behaviour. Guided Imagery is a visual and sensory-based complementary and alternative medicine (CAM) technique. In hypnotherapy which is a type of mind-body intervention; increased suggestibility is used in the treatment of a medical or psychological disease or condition. Therefore, there is a need of a newer modality based on guided imagery and hypnotherapy to be used in as a whole or in tandem with other conventional behaviour management techniques. The aim of this study is to evaluate and compare anxiety levels after using Guided Imagery for relaxation and modified Guided Imagery-Hypnotherapy techniques in 6-14 year-old children undergoing local anesthesia administration.", "keywords": [ "Guided Imagery", "Hypnotherapy", "Anxiety", "Local Anesthesia", "Children" ], "content": "Introduction\n\nAnxiety has been defined as a “vague, unpleasant feeling accompanied by a premonition that something undesirable is about to happen”.1 Although the terms “anxiety” and “fear” are frequently interchanged, anxiety is usually described as a general feeling, whereas fear is described as a reaction to a specific event or object.1 Dental anxiety is a phenomenon comprising of various physical, mental as well as social aspects. In the terms of dental fear and anxiety (DFA), dental anxiety is characterized as significant negative or unpleasant feeling about a dental office and dental procedures, whereas dental phobia is an irrational form of dental anxiety.2 Dental anxiety is a major contributor to poor oral health. People mainly delay dental treatment because they are afraid of the discomfort.3\n\nDental injection fear continues to be a critical concern that frequently requires cognitive behavioural psychology counselling and anesthesia in order to complete necessary dental treatment.3 Trypanophobia is an unreasonable fear of medical treatments involving needles (such as injections), whereas belonephobia is an irrational fear of needles and pins, which can cause unusual behaviour and anxiety. Receiving injections is one of the most frequently expressed fears. Since the delivery of local anaesthetic via injection is the main procedure in pain reduction techniques, needle anxiety is of big concern.4 Children who have a high level of dental anxiety are more resistant to treatment and require more time on dentist’s chair. DFA has the potential to have long-term detrimental consequences on individuals due to avoidance of dental care which results in the need for more dental treatments.5\n\nComplementary and Alternative Medicine (CAM) refers to a range of techniques that are commonly used in conjunction with mainstream medicine, rather than as a replacement for it. Integrative medicine refers to the use of conventional medicine in combination with complementary and alternative therapies.\n\nGuided Imagery is one of the complementary and alternative medicine (CAM) practice that involves a visual and sensory orientation. For a specific intention (such as relaxation, health, performance, or emotional release), in guided imagery one can be directed through a visual experience involving all five senses: imagined sight, smell, hearing, touch, and taste.6 These techniques are suitable for pediatric patients because they are more receptive to suggestions as their imaginations are more active than adult patients. Although children as young as age six to early adolescence can respond to hypnosis, these techniques are most effective when employed with children aged 8 to 12 years.7\n\nHypnosis a natural altered state of consciousness. The hypnotic state is a deeply relaxed condition similar to that which occurs just before falling asleep.8 In dentistry, hypnosis is referred to as ‘hypnodontics’. The human mind can create extremely real responses from imaginary events and if they are imagined vividly and with enough detail, our body experiences a physical reaction even at a simple thought. Suggestibility is defined as an attitude or collection of behaviours that allows a person to respond to a wide range of stimuli.9 Humans are suggestible by nature, and suggestion as per Heap and Aravind is “a communication delivered verbally by the hypnotist that leads the subject’s imagination in such a way as to elicit intended changes in sensations, perceptions, feelings, thoughts, and behaviour”.10 Hypnotherapy is a type of mind-body intervention in which hypnosis is used to create a state of focused attention and increased suggestibility in the treatment of a medical or psychological disorder or concern.11 Hence, hypnotherapy can be used alone or in combination with other behaviour modification strategies.\n\nThe stress response is influenced by cortisol levels, a steroid hormone produced by the adrenal glands. An increase in serum cortisol synthesis by the adrenal cortex lead to a consequent rise in salivary cortisol levels. Cortisol is released in reaction to stress, and it interacts with the hormonal and immunological systems in complex ways. It activates the hypothalamus–pituitary axis, causing the hormone ACTH to be secreted, which then acts on the adrenal cortex, resulting in an increase in salivary cortisol levels.12\n\nHence the focus of this research, according to the available literature, is to evaluate and compare relaxation guided imagery and modified guided imagery-hypnotherapy for assessment of anxiety levels during local anaesthetic administration in 6 to 14 year-old children.\n\n\nProtocol\n\nThe aim of this study is to evaluate and compare anxiety levels after using Guided Imagery for relaxation and modified Guided Imagery-Hypnotherapy techniques in 6–14-year-old children undergoing local anesthesia administration.\n\nObjectives are:\n\n1. To evaluate the levels of anxiety after using Guided Imagery technique for relaxation in 6–14 year-old children undergoing local anesthesia administration.\n\n2. To evaluate the levels of anxiety after using modified Guided Imagery-Hypnotherapy technique in 6–14-year-old children undergoing local anesthesia administration.\n\n3. To compare the effect of anxiety levels after using Guided Imagery for relaxation technique with modified Guided Imagery-Hypnotherapy technique on in 6–14-year-old children undergoing local anesthesia administration.\n\nThis randomized clinical trial will be conducted in an isolated setup over a period of two years (June 2022–June 2024). It will be performed within a single visit. Children from six to 14 years old who are healthy, co-operative but anxious and children undergoing dental treatment requiring administration of local anesthesia at the Department of Pediatric and Preventive Dentistry, Sharad Pawar Dental College, Datta Meghe Institute of Higher Education and Research will be included in the study.\n\nChildren with past exposure to local anesthesia, known allergy to local anaesthetic agent, psychological abnormalities, children with special health care needs, children suffering from systemic diseases and with parents who are unwilling to participate will be excluded from the study.\n\nThe Guided Imagery technique for relaxation is designed for children undergoing dental treatment. This approach is the Relaxation-guided imagery method based on Vagnoli, L., Bettini, A., Amore, E. et al.13 By taking deep breaths, the child can focus on their body and gradually release muscle tension from the feet to the head. A mental recreation of sights, sounds, smells, tastes, and feelings as though they were truly happening while instructing to imagine a favourite place, real or imagined, using the same terms for each participant; and after the child remains in the chosen location for a while, contact with the surrounding world is gradually reestablished until the child opens his or her eyes.\n\nModified Guided Imagery-Hypnotherapy is a combination of guided imagery, hypnotherapy along with colour psychology and it only requires relatively low levels of hypnosis to help the child calm. Modified guided imagery technique is based on general information on creating positive mental imagery (Stein, 2013),14 as well as the guidelines of the PETTLEP (Physical, Environment, Task, Timing, Learning, Emotion and Perspective) model of imagery (Holmes and Collins, 2001)14 and colour psychology.15 In this, a combination of above concepts will be used. The child will be shown a colour pinwheel while taking a few slow and deep breaths (about five to 10 times).\n\nThe child will then be told to close their eyes and imagine themselves becoming peaceful and relaxed, as well as smiling, feeling pleased, and having fun. The dental chair will be utilised as an anchor item, and the child will be encouraged to open their eyes and feel safe around the dental. This procedure will be repeated two to three times until the child is completely relaxed. The child will then be asked to gently open their eyes.\n\nIntervention of conventional behaviour management techniques like tell-show-do, ask-tell-ask, positive reinforcement, modelling, systemic desensitization and voice control will be done.7\n\nThe study is a randomized clinical trial, and this is the version one of the protocol. Sequences by random numbers will be computer-generated. The co-investigator of the department will generate the allocation sequence and will assign participants to interventions. Trial participants, and data analysts will be blinded. Trial participants will not be aware in which group they will be allotted hence they will be blinded. The data analysts will be provided with the data chart without revealing the intervention groups to maintain blinding. Data management by double data entry will be done. Personal information about potential and enrolled participants will be collected and maintained in order to protect confidentiality before, during, and after the trial. All study-related information will be stored securely at the study site.\n\nAll participant information will be stored in locked file cabinets in areas with limited access.\n\nAll laboratory specimens, reports, data collection, process, and administrative forms will be identified by a coded identification number only to maintain participant confidentiality.\n\nComparators guided imagery and conventional behaviour management method will be taken into consideration for comparing as all of them are interventions aiding for reducing the anxiety levels.\n\nThe sample size of a 45 in total was calculated with reference of study by Oberoi et al (2016)6 using the sample size formula for difference between two means.\n\nn = 15 patients needed in each group\n\nVFAS score using the Visual Facial Anxiety Scale for scoring anxiety16 and salivary cortisol levels as a biological indicator using salivary cortisol ELISA kit are the primary outcomes to be measured. VFAS score will be noted before and after the intervention. The salivary samples will be collected at the start and end of the intervention for measuring the salivary cortisol levels. The difference between the given two time points will be used to assess the clinical significance of the primary outcomes. Physical resistance, verbal resistance, tension in the muscles, co-operation during local anaesthesia administration are the secondary outcomes to be measured. All the outcomes will be measured before, during and after intervention.\n\nAnalytical tests like the Chi square test and student’s t-test will be performed for analysing data collected from the VFAS and salivary cortisol samples. All the statistical analysis will be performed using SPSS software, version 27.0. p<0.05 will be considered as the level of significance. This randomized clinical trial will be conducted at the Department of Pediatric and Preventive Dentistry, Sharad Pawar Dental College, Datta Meghe Institute of Higher Education and Research, Wardha, India. The allocation of participants will be done using computer generated numbers.\n\nOnce completed the study will be published in a PubMed and Scopus indexed journal.\n\nThis trial is yet to be started.\n\nEthical approval for the study was obtained from the Institutional ethics committee of Datta Meghe Institute of Medical Sciences (Deemed to be University) [ref no: DMIMSU (DU)/IEC/2022/756].\n\nThe study protocol will be explained to the participating children and their parents. Further, written informed consent for participation will be obtained from the parents.\n\nTrial Registration is done under The Clinical Trials Registry - India (CTRI reference no- REF/2022/06/055009)\n\nDate of Registration: 06/09/2022\n\nhttps://ctri.nic.in/Clinicaltrials/showallp.php?mid1=70398&EncHid=76235.25564&userName=Effectiveness%20of%20guided%20imagery%20and%20modified%20guided%20imagery%20in%20reduction%20of%20anxiety%20in%20children\n\n\nDiscussion\n\nThere are various studies done for using integrative medicine techniques. A study by Oberoi et al. (2016) included 200 children aged 6 to 16 that checked the anxiety states of the child by using the parameters – patient’s heart rate, oxygen saturation, physical and/or verbal resistance during local anesthesia administration. Patients were divided into two groups – hypnotic group and control group. The difference in behaviour between hypnotized and non-hypnotized children was related to the child’s calm frame of mind. Hypnosis could improve patient compliance, reduce resistance during unpleasant operations, and lower heart rates.6\n\nA study analysed the effects of H (hypnosis) and progressive muscle relaxation therapy (PMR) on anxiety, pain in a randomized control experiment. A total of 60 children were divided into three groups between the age group of eight to 12 years. There was a reduction in anxiety levels validated by reduction in blood pressure, heart rate and in reduction in pain validated by lowered need for analgesics, whereas the SPO2 remained constant throughout in the experimental group of hypnosis “H” and progressive muscle relaxation “PMR”. Hypnosis and PMR were effective for anxiety reduction and pain control in pediatric dentistry patients.17\n\nA link between stress and anxiety with salivary cortisol (SC) and salivary alpha-amylase (SAA) levels was evaluated in a study with 20 children in the age group of 5–12 years. Pre- and post-extraction saliva samples were collected and a positive co-relation was seen against a graph plotted for post extraction salivary cortisol and salivary amylase levels. It was indicative of activation of adrenal medulla by the signals from sympathetic system leading to increase in salivary secretions containing high levels of cortisol and alpha amylase in stressful and anxious situations. Saliva can be used as a new non-invasive approach of evaluating anxiety and stress by analysing the levels of stress indicators.12\n\nA randomized clinical study that included 48 subjects in total evaluated nature-based guided imagery (GI), urban-based GI and GI as a process within itself for reduction in state anxiety levels. The process used a set of 20 questions indicating trait anxiety to determine participant appropriateness for the study. The interventions in this research were two GI sessions, one of which was held in a natural setting and the other in an urban setting. Each subject was exposed to both conditions. Anxiety can be effectively treated with GI itself, and at the same time nature-based GI and urban-based GI was useful too. But the most effective GI, was said to be nature-based and hence the study concluded that nature-based GI interventions are effective anxiety management interventions which have the added benefit of being cost-effective and easily accessible.18", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: SPIRIT checklist for “Evaluation and comparison of anxiety levels after using Guided Imagery technique for relaxation and modified Guided Imagery-Hypnotherapy technique in 6 to 14 year-old children undergoing local anesthesia administration”, https://doi.org/10.5281/zenodo.8146049. 19\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nHare J, Bruj-Milasan G, Newton T: An Overview of Dental anxiety and the non-pharmacological management of dental anxiety. Prim Dent J. 2018; 7(4): 36–39. PubMed Abstract \| Publisher Full Text\n\nPorritt J, Buchanan H, Hall M, et al.: Assessing children’s dental anxiety: a systematic review of current measures. Comm Dent Oral Epidemiol. 2013; 41(2): 130–142. PubMed Abstract \| Publisher Full Text\n\nArmfeild JM, Milgrom P: A clinician guide to patients afraid of dental injections and numbness. SAAD Dig. 2011; 27: 33–39.\n\nOosterink FMD, de Jongh A , Aartman IHA: What are people afraid of during dental treatment? Anxiety-provoking capacity of 67 stimuli characteristic of the dental setting. Eur J Oral Sci. 2008; 116: 44–51. PubMed Abstract \| Publisher Full Text\n\nCarlsson SG, Wide Boman U, Lundgren J, et al.: Dental anxiety – a joint interest for dentists and psychologists. Eur J Oral Sci. 2013; 121: 221–224. PubMed Abstract \| Publisher Full Text\n\nOberoi J, Panda A, Garg I: Effect of Hypnosis during administration of Local Anesthesia in Six- to 16-year-old children. Pediatr Dent. 20l6; 38(2): 112–115.\n\nAmerican Academy of Pediatric Dentistry: Behavior guidance for the pediatric dental patient. The Reference Manual of Pediatric Dentistry. Chicago, Ill: American Academy of Pediatric Dentistry; 2022; pp. 321–339.\n\nAndrews EK: Complementary and alternative medicine techniques available for dentistry ADAA continuing education. Dent Assist. 2021.\n\nHeap M, Aravind KK: Hartland’s Medical and Dental Hypnosis. 4th ed.Edinburgh: Churchill Livingstone; 2002; pp. 3–14.\n\nWatson JB, Morgan JJB: Morgan Emotional Reactions and Psychological Experimentation. Am J Psychol. 1924; 28(6): 463–477.\n\nHogan K: The New Hypnotherapy Handbook: Hypnosis and Mind Body Healing.2001.\n\nChaturvedi Y, Chaturvedy S, Marwah N, et al.: Salivary Cortisol and Alpha-amylase-Biomarkers of Stress in Children undergoing Extraction: An in vivo Study. Int J Clin Pediatr Dent. 2018; 11(3): 214–218. PubMed Abstract \| Publisher Full Text\n\nVagnoli L, Bettini A, Amore E, et al.: Relaxation-guided imagery reduces perioperative anxiety and pain in children: a randomized study. Eur J Pediatr. 2019; 178: 913–921. PubMed Abstract \| Publisher Full Text\n\nStein T: 7 Tips for Creating Positive Mental Imagery and Holmes P. S., Collins D. J. (2001). The PETTLEP approach to motor imagery: a functional equivalence model for sport psychologists. J Appl Sport Psychol. 2013; 13: 60–83.\n\nBubna K, Hegde S, Rao D: Role of Colors in Pediatric Dental Practices. J Clin Pediatr Dent. 2017; 41(3): 193–198. PubMed Abstract \| Publisher Full Text\n\nCao X, Yumul R, Lazo E, et al.: A novel visual facial anxiety scale for assessing preoperative anxiety. PLoS One. 2017; 12: e0171233. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSabherwal P, Kalra N, Tyagi R, et al.: Hypnosis and progressive muscle relaxation for anxiolysis and pain control during extraction procedure in 8–12-year-old children: a randomized control trial. Eur Arch Paediatr Dent. 2021; 22: 823–832. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNguyen J, Brymer E: Nature-based guided imagery as an intervention for state anxiety. Front Psychol. 2018 Oct 2; 9: 1858. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParakh H, Thosar N: SPIRIT checklist for “Evaluation and comparison of anxiety levels after using Guided Imagery technique for relaxation and modified Guided Imagery-Hypnotherapy technique in 6 to 14 year-old children undergoing local anesthesia administration”. [Data]. 2023. Publisher Full Text" }	[ { "id": "281221", "date": "29 May 2024", "name": "Mohamed Jaber", "expertise": [ "Reviewer Expertise Oral Cancer", "Precancer", "Epithelial dysplasia", "Facial Injuries", "Dental Education", "Medically compromised patients." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe protocol outlines clear objectives, inclusion and exclusion criteria, and the methodology for conducting the study. The study design, a randomized clinical trial, is appropriate for evaluating the effectiveness of the interventions. The blinding of participants and data analysts enhances the rigor of the study. Ethical considerations, including obtaining informed consent and ethical approval, are appropriately addressed. The introduction provides a clear definition and differentiation between anxiety and fear, particularly in the context of dental anxiety. It outlines the significance of dental anxiety, its impact on oral health, and the necessity of addressing it. The introduction effectively introduces the concept of Complementary and Alternative Medicine (CAM) and its relevance in managing anxiety. The research focuses on evaluating and comparing relaxation-guided imagery and modified guided imagery-hypnotherapy is clearly stated.\n\nThe sample size justification based on a single reference study might not adequately consider the variability in the population or other relevant factors. A more comprehensive sample size calculation considering effect size, power, and potential dropout rates would strengthen the study. While the protocol mentions primary and secondary outcomes, it could benefit from a more detailed discussion on the rationale behind selecting these outcomes and their clinical relevance. The description of interventions lacks specificity in terms of duration, frequency, and standardization, which could affect reproducibility and comparability of results.\n\nThe protocol lacks a discussion on potential confounders or variables that might influence the outcomes, such as age, gender, or previous dental experiences. The introduction lacks a comprehensive literature review. While it briefly mentions previous studies, it could benefit from a more detailed discussion on existing research, especially regarding the effectiveness of CAM techniques in managing dental anxiety. The introduction lacks a clear statement of the research gap of the study. What specific aspect of dental anxiety management is this study addressing that hasn't been sufficiently explored before? The introduction could be more concise and focused, avoiding repetitive statements.\n\nSuggestions for Improvement:\nConduct a more comprehensive sample size calculation considering various factors and ensure an adequate sample size to detect meaningful differences between groups. Provide more detailed descriptions of interventions, including standardized protocols for implementation, to ensure consistency and reproducibility across different settings. Discuss potential confounders or variables that might impact outcomes and consider strategies to control for them during analysis or interpretation. Consider including a pilot study to refine the protocol and address any practical or methodological issues before conducting the main study. Expand the literature review to provide a more thorough overview of existing research on dental anxiety management, including various CAM techniques. Clearly articulate the research gap of this study to the existing body of knowledge. Streamline the introduction to maintain clarity and conciseness, avoiding unnecessary repetition.\n\nThe introduction lays a foundation for an important area of research, but it requires further refinement and expansion to meet the standards of a high-impact journal like F1000. Additional analysis of existing literature would enhance the paper. The protocol demonstrates a sound methodology for conducting a randomized clinical trial, but it would benefit from further refinement and clarification, particularly regarding sample size justification, intervention descriptions, and consideration of potential confounders.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "281227", "date": "06 Jun 2024", "name": "Vivek Padmanabhan", "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\nDear authors I would like to begin by congratulating the authors on their initiative in undertaking this study. I hope my feedback will be helpful for further development.\nInitially, the article appears to aim at comparing the Guided Imagery technique for relaxation with the modified Guided Imagery-Hypnotherapy technique. However, as the article progresses, it delves into evaluating salivary cortisol, which is not clearly included in the stated objectives. Clarifying the relevance and necessity of salivary cortisol evaluation in the objectives would enhance the coherence of the study.\nThe sampling methodology needs further attention to align with these objectives. Specific details about the sample size and the number of samples in each group are missing and should be provided.\nFinally, the clinical implications of this study should be clearly outlined once the aforementioned points are addressed.\nIn summary, the article requires a comprehensive revision to ensure clarity and completeness. I look forward to seeing the revised version.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] } ]	1	https://f1000research.com/articles/12-1000
https://f1000research.com/articles/10-944/v1	20 Sep 21	{ "type": "Research Article", "title": "Variability in soil properties influencing pigeonpea (Cajanus cajana L.) yield: a multivariate statistical analysis", "authors": [ "Rajesh N L", "Narayana Rao K", "Sathishkumar U", "Wali V B", "Basavaraj K", "Rudramurthy H V", "Desai B K", "Narayana Rao K", "Sathishkumar U", "Wali V B", "Basavaraj K", "Rudramurthy H V", "Desai B K" ], "abstract": "Aims: The aim of the study was to reveal the variability in soil properties influencing pigeonpea (Cajanus cajana L.) seed yield under semi-arid rainfed condition. Methods: Soils were initially classified into series level and further these series were divided into soil-phase units. For two site years viz., 2018-19 and 2019-20, surface soil samples from each soil-phase unit were collected before sowing of pigeonpea and subsequently crop growth parameters at critical stages were recorded. Results: The principal component analysis with varimax rotation resulted in seven components for both the site years, having eigenvalues greater than one, explained more than 80% of the variability. The step wise linear regression analysis showed that the pigeonpea seed yield was linearly correlated with PC3 (p<0.01), PC4 (p<0.01) and PC7 (p<0.05) of soil properties with R2 = 0.679, during 2018-19. Whereas, during 2019-20, the seed yield was linearly correlated with PC1 (p<0.01), PC3 (p<0.01) and PC6 (p<0.05) with R2 = 0.677. In site year 1, the available P2O5, Fe, Zn, S, Cu, number of pods, surface soil moisture determined the yield. In site year 2, the available K2O, P2O5, Fe, Zn, S, clay, CEC and available water content determined the yield. All these variables together explain variability in yield.", "keywords": [ "Soil-phase unit", "Soil-plant relationship", "Principal component regression analysis" ], "content": "Introduction\n\nDespite environmental perturbations, agricultural landscapes provide diverse ecosystem services and maintain crop yields (John, 2020). The resilience of agricultural landscapes is underpinned by complex interactions between environmental factors (precipitations, temperature, pollutants, and sunlight) and agricultural inputs provided during crop cultivation (FAO, 2013). Inherent soil properties, namely soil texture, type of clay, soil depth to bed rock and drainage class which result primarily from the soil-forming factors such as climate, topography, parent material, biota and time, influence the land suitability to cultivate a given crop to produce maximum yield (Moore, 2001). Therefore, the relationship between soil and crop yield mostly depends on the prevailing climate and the landscape. The process of deriving spatial or statistical models to establish a relationship between soil and crop yield involves a systematic inventory of land resources, specifically soil (physico-chemical properties) and plant variables (growth and yield parameters). Efforts have been made using multivariate statistical analysis to reclassify the soil variability into spatial management units, based on principles of similarity which are analogous to crop yield.\n\nIn this study, soil-phase was used as a functional unit designed by differentiating landscape properties (such as slope, erosion, gravelliness and stoniness that are not used as criteria in soil taxonomy) and soil series (which were categorized by soil depth). Although a soil-phase is a unit of soil outside the system of soil taxonomy, understanding the variability of landscape and soil properties and their effect on crop yield is a critical component of site-specific and sustainable management interventions (Juhos et al., 2015). Therefore, soils were initially classified into series level, and these series further divided into soil-phases, which are soil management units differentiated according to soil profile depth and land surface characteristics.\n\nMultivariate analysis would be most appropriate to understand the specific variables influencing crop yield. Principal component analysis (PCA) is an extremely powerful analytical technique and can often indicate which variables in a data set have a significant effect on the response variables, and which are not significant (Hair et al., 2003). PCA can simplify the structure of a set of variables by replacing those with a few uncorrelated linear combinations of original variables. As soil parameters are correlated with each other due to their multicollinearity, it makes sense to examine them jointly (Zsigrai, 1999); therefore, in the present study both soil physico-chemical properties and crop growth parameters were subjected to PCA. PCA is a mathematical linear transformation of the original variables, which accounts for the maximum share of the variability present in the original set of variables, with a minimum number of composite variables and makes no assumption of a model (Jolliffe & Cadima, 2016). Therefore, the resulting components were subjected to a stepwise linear regression analysis, to check the significance of soil and plant growth attributes on the resulting yield. Further, these derived significant variables could be considered as primary targets for interventions to address the site-specific soil variability and improve the crop yield.\n\nThe study was planned for two site years viz., during kharif (cropping season, between June to October) 2018–2019 and kharif 2019–2020, to assess the variability in surface soil properties for each soil phase and crop growth parameters influencing pigeon pea yield.\n\n\nMethods\n\nThe study area was located in the Northern dry zone of Karnataka State, India (Figure 1), between latitudes and longitudes of 17°40’17.956’’ N - 76°59’46.836” E and 17°38’12.689” N - 77°1’17.049” E, covering 645.20 ha, at an altitude of 511 - 637.86 m above mean sea level.\n\nKalmandari Tanda-1 micro-watershed (MWS) reported mean monthly minimum and mean monthly maximum temperatures of 19.08°C and 31.97°C, respectively, during January 2018 and May 2018, whereas the reported values for these variables were respectively 18.89°C and 33.7°C for January 2019 and May 2019. The average annual rainfall measured by Kalmandari Tanda-1 MWS was 442 mm and 831.50 mm in 2018 and 2019, respectively (Figure 2 and Figure 3).\n\nA comprehensive methodological workflow adopted in this study is presented in Figure 4. To divide the entire micro-watershed into different soil phase units, a detailed land resource inventory was initially carried out at a 1:8000 scale, using Indian Remote Sensing Satellite-P6 (IRS P6) Cartosat-1 merged LISS-IV satellite imagery (2.5 m spatial resolution) as base map to interpret the soil physiographic unit map (Figure 1). The soil (soil profile depth, number of horizons, soil color, soil structure, texture, consistency, presence of carbonates, and soil pH) and site (slope, erosion, drainage, runoff, gravelliness, stoniness, presence of rocks on surface, lithology/parent material and current land use) characteristics were recorded for all soil profile sites on a standard pro forma (Pedon description form which consists of a list of soil and site parameters that need to be recorded during a field study. A copy is provided in the Data availability section (see Extended data, Rajesh, 2021)) as per the guidelines given in Field Guide for Land Resources Inventory (Natarajan et al., 2016).\n\nIn this micro-watershed, both Vertisol and Alfisol soils were found, which are soils derived from basalt and basalt-laterite intrusion parent materials, respectively (Government of India, 2008). Pedons that showed similar soil profile horizons in soil color, soil texture, soil pH, and consistence, mineral and chemical compositions were grouped into soil series (FAO, 2006) and further, these soil series were divided into 23 soil phases (Figure 5), based on surface characteristics with respect to soil texture, slope, erosion and gravelliness.\n\nA total of 462 surface soil samples (0-30 cm) were collected in two site years. Each year, before sowing of pigeon pea, 231 soil samples were collected from the same locations (recorded using Trimble Juno 3D, a hand-held GPS) of each soil phase of the micro-watershed. Physico-chemical properties of all collected surface soil samples were analyzed, using standard procedures presented in Table 3. Erosion hazards were judged through the visible soil erosion method, by assessing the presence of rills and gullies within a field, as well as their associated deposits (Evans, 2012). Soil texture was evaluated using the feel method, and slope with the help of a dumpy level. Organic carbon (OC) was determined using the Walkley & Black (1965) wet oxidation method. Available N was determined by a modified alkaline potassium permanganate method as described by Subbiah and Asija. Available P2O5 was determined using Olsen’s method. Available K2O was estimated using a flame photometer after extraction with ammonium acetate. Soil reaction (pH) was determined in 1:2.5 soil water suspensions using a glass electrode (Piper, 1966). Electrical conductivity was measured in the soil water (1:2.5) suspension using a conductivity bridge (Jackson, 1973). The level of free calcium carbonate ions in soil samples was determined using a rapid titration method ith standard HCl (Piper, 1966).\n\nCationic micronutrients like iron, copper, manganese and zinc were extracted using diethylene triamine penta acetic acid (DTPA, 0.005 M and 0.01 M CaCl2 + 0.1N tri-ethanol-amine at pH 7.3), and the concentration was measured using an atomic absorption spectrophotometer (ContrAA 700 Make) as outlined by Lindsay & Norvell (1978). Available boron in soil was estimated with a colorimetric method using hot water as extractant and expressed in mg kg-1 (Page et al., 1982). Available sulphur in soil was extracted with CaCl2.2H2O (0.15%), and the extract was reacted with barium chloride crystals. The intensity of the resulting turbidity was measured using a spectrophotometer at a wavelength of 420 nm (Jackson, 1973)\n\nThe bulk density was estimated using Keen’s cup method for the disturbed soils (Piper, 1966). Particle size distribution of soil samples was determined using the \"International Pipette\" method (Piper, 1966); the soil’s cation exchange capacity (CEC) was determined by equilibrating the soil with 1N sodium acetate solution using a flame photometer, to calculate the CEC. The exchangeable sodium percentage (ESP) was calculated by dividing the exchangeable sodium by CEC and exchangeable bases (Ca, Mg, and K, Na), which were measured by titration and flame photometer respectively.\n\nPigeon pea growth and yield parameters were recorded using a crop cutting experiment, where observations were regularly made in the farmer’s field at specific locations of the study area and at different crop growth stages. The growth parameters viz., plant height (in centimeter), number of branches per plant, and leaf area index (AccuPAR Ceptometer model LP-80) were recorded for each soil phase. Five pigeon pea plants were labeled in a 1 m2 area of each soil phase unit and the growth parameters were recorded at 30, 60, and 90 days after sowing (DAS), as well as during the crop harvesting stage. Similarly, pigeon pea yield parameters, namely the number of pods per plant and grain yield (kg ha-1), were recorded at the harvest stage and after the harvest respectively. Details of pigeon pea varieties and fertilizer doses (urea and di-ammonium phosphate manufactured by Zuari Agro Chemical Ltd., India) used are given in the Extended data (Rajesh, 2021). Meanwhile, soil moisture and available water content (AWC) at each soil-phase were measured using a theta probe (Stevens Water Monitoring Systems Inc, S/N 238825) and pressure plate membrane apparatus (SOILMOISTURE Equipment Corp, Model #1500F2), respectively.\n\nDescriptive statistics and principal component regression analysis of soil and plant parameters were carried out in SPSS v.16. An assessment of the normality of the data is pre-requisite for PCA. A Z-scale test was applied for normality test using skewness and kurtosis (for n < 300). The distribution is approximately normal if skewness and kurtosis are between -1 and 1. The right and left skewed data (Table 1 and Table 2) were subjected to logarithmic and square root transformation to improve the normality. These transformed variables were subjected to a PCA with varimax rotation (Ayoubi et al., 2009; Cox et al., 2003; Shukla et al., 2004b). The descriptive statistics of physico-chemical properties of soil and plant growth parameters are presented in Table 1 and Table 2, respectively.\n\nInference from multivariate analysis: During 2018-19, yield contributing variables such as soil available P2O5, Fe, Zn, Cu and soil moisture (% AWC) and whereas during 2019-20 soil available K2O, S, Fe, Zn, Mn, Ca, Mg, CEC, clay, and available water content (AWC), a higher availability of these variables will be more beneficial. Exch: exchangeable; ESP: exchangeable sodium percentage ; CEC: cation exchange capacity; BD: bulk density; PWP: permanent wilting point; FC: field capacity.\n\nNote: Plht: Plant height, Br: Number of branches, LAI: Leaf area index, DAS: Days after sowing.\n\nInference from multivariate analysis: In both the site years “number of pods at harvest” have contributed to the yield and is the one which explains yield variability among plant growth & yield parameters\n\nEC: electric conductivity; DTPA: diethylene triamine penta acetic acid;\n\nPCA was used as a data reduction technique. The independent variables such as soil properties (soil pH, EC, CaCO3, OC, available N, P2O5, K2O, S, Zn, Fe, Cu, Mn, B, exchangeable Ca, Mg, Na, ESP, CEC [Cmol(p+)kg-1], Sand [%], Silt [%], Clay [%], bulk density [Mgm-3], permanent wilting point [PWP in %], field capacity [FC in %], AWC [%]) and plant growth parameters (plant height, number of branches per plant, LAI, number of pods at harvest stage) were subjected to a PCA; variables with factor loadings greater than the measured Kaiser-Meyer-Olkin (KMO) values, were chosen as highly correlated variables from the derived principal components (PCs) with eigenvalues greater than one (Suryanarayana & Mistry, 2016). The KMO test is a measure of sampling adequacy and factorability. The KMO tests whether the partial correlations within the data are close enough to zero to suggest that there is at least one latent underlying factor among variables (Vijayamohanan Pillai & Asalatha, 2020). As a KMO value of < 0.5 is unacceptable (Hair et al., 2003), the results of the PCA with a KMO > 0.5 loadings indicate that the chosen correlation matrix was appropriate for factor analysis.\n\nThe Kaiser Meyer Olkin (KMO) test is given by;\n\n\n\nWhere, r = [rij] - Correlation matrix\n\nU = [uij] - Partial covariance matrix\n\nThe eigenvalue is the variance explained by a component or factor and is denoted by lambda.\n\n\n\nWhere, A2ik is the factor loading for variable i on component k, and n is the number of variables. A low eigenvalue represents a lower contribution to the variance in the analyzed set of variables. The component matrix table shows the component loadings, which represent the correlations between the variables and the components. As a rule of thumb, the larger the size of the component loading for a variable, the more significant the variable is in the interpretation of the component (Nargundkar, 2005). The first component is generally more highly correlated with the variables than the second components, and so on. A varimax rotation enhances the interpretability of the uncorrelated components; therefore, only the outcomes of the rotated component matrix are presented in our results.\n\nThe derived PCs were subjected to a stepwise linear regression analysis, which is an alternative to multiple regressions analysis. In LPCR, instead of regressing the dependent variables on the explanatory variables directly, the principal components of the explanatory variables were used as regressors, each time removing the weakest correlated variable. The highly correlated and significant soil and plant independent/explanatory variables from each PC were fitted to the dependent variable (pigeon pea crop yield). Further linear equations were drawn separately.\n\n\nResults and discussion\n\nThe soil phases GUTmB2g1, GUTmC2g1 and GUTmC3g1 from the Gutti soil series (Figure 6) showed maximum crop yield (pooled) during kharif (June to October) 2018–19 (4073 kgha-1) and kharif 2019–20 (4443 kgha-1). The GUTmB2g1 soil phase prevailed on gentle slopes (1-3%) could give rise to a maximum yield of 1394 and 1496 kgha-1 during kharif 2018–19 and 2019–20 respectively. However, of the two site years, crop yield was greater during kharif 2019–2020, due to a greater available soil moisture than that of kharif 2018–2019. The KKThE3g1S1 and KKThF3g1S1 soil phases of Kurukota series prevailed on stronger slopes (10–15 %), showed severe erosion (3), and led to a low yield in both kharif 2018–19 (respectively 1325 and 1295 kgha-1) and kharif 2019–20 (respectively 1368 and 1343 kgha-1). Thus, these soil phases would need intensive care and management, including fertilizer input and soil and water conservation strategies, so as to boost crop productivity.\n\n\nPCA & LPCR to assess the influence of soil properties on pigeon pea yield\n\nSeven components were extracted with total cumulative variances of 82.30% and 80.75% during 2018 (Table 4) and 2019 (Table 5), respectively. The principal component 1 (PC1) in each site year was identified as soil available nutrient content with high loadings of K2O (0.843 and 0.764, respectively), Mn (0.870 and 0.882 respectively), Ca (0.845 and 0.900), and Mg (0.835 and 0.919 respectively). The presence of these adsorbed cations on negatively charged clay surfaces (supported by high loadings of clay content 0.897 and 0.937) contributed to significantly high CEC loadings (0.897 and 0.937) (Schoonheydt et al., 2018). On the other hand, soil moisture retention was low due to high loadings of sand (-0.858 and -0.901) and bulk density (-0.927 and -0.743) which are inversely correlated with AWC loadings (0.822 and 0.625). This indicates that the water-soluble nutrients did not have sufficient soil moisture to dissolve, due to scarcer rains during 2018–19 (Figure 2). In 2018, PC2 was identified as humification and vegetative growth with high loadings of OC and available N. The high loading of EC during 2018 may be due to the retention of dissolved salts associated with low precipitation. In 2019, because of higher precipitation levels (Figure 3) the results of PC2 converged towards enhanced calcification with high loadings of CaCO3 (0.793) due to dissolution of CaCO3 in the presence of CO2 (Neina, 2019) which is normally found at ten times greater levels in soil (0.3 % CO2) when compared to the CO2 concentration (0.03%) in the atmospheric air above the soil. Further, PC2 in site year 2 was also identified as qualitative vegetative growth with high loadings of Cu (0.764), which enhances plant photosynthesis; this was also supported by an increased number of branches (0.595) per plant at the time of harvest. PC3 in 2018 and 2019 was identified as improved enzyme activity and pod formation stage, which was supported by high loadings of Fe (0.919 and 0.889), Zn (0.846 and 0.735) and number of pods (0.736 and 0.605) at harvest.\n\nBold typeface - Strong correlation coefficient\n\nTProbe: Surface soil moisture, Br_har: Number of branches at harvest, Pod_har:Number of pods at harvest, EC: electric conductivity, ESP: exchangeable sodium percentage, CEC: cation exchange capacity, BD: bulk density, AWC: average water content.\n\nBold: Strong correlation coefficient\n\nTProbe: Surface soil moisture, Br_har: Number of branches at harvest, Pod_har: Number of pods at harvest, EC: electric conductivity, ESP: exchangeable sodium percentage, CEC: cation exchange capacity, BD: bulk density, AWC: average water content.\n\nIn 2018 (site year 1), PC4 was identified as root proliferation stage, with high loadings of P2O5 (0.801) and surface soil moisture (0.727). Root interception of inorganic phosphorous increases with root proliferation. The diffusion of P2O5 from the soil through the roots increases with increased moisture content of dry soil (Lambers et al., 2006). Low rainfall (Figure 2) events during critical growth stages of pigeon pea (September 2018 to December 2018), led to dry soil and reduced the dissolution of readily available (water-soluble) nutrients, which are required for plant growth. During the draught, when soil moisture slightly increased from an uneven and low precipitation pattern during November and December 2018, the plant enhanced its root proliferation in the presence of high available soil P2O5, in search of soil nutrients (Basu et al., 2016; Peret et al., 2014). In 2019 (site year 2), PC4 was identified as humification with high loadings of EC (0.838), OC (0.558) and N (0.557) suggesting activation of humification (Doni et al. 2014). In site year 1, PC5 was identified as chemical changes in soil, and soil aeration supported by high loadings of soil pH (0.752) and silt (0.674). During site year 2, in PC5, soil ESP (-0.657) and silt (-0.583) showed a high negative factor loading, however available surface moisture (0.754) exhibited a high positive factor loading; this implies that when in water, dissociated calcium cations from CaCO3 in the presence of CO2, have replaced sodium ions and adsorbed on the soil colloids (van de Graaff & Patterson, 2001). In site year 1, PC6 had high negative factor loadings of CaCO3 (-0.781) and positive factor loadings of available boron (0.788), which are attributed to plant cell wall synthesis and root nodule formation for N2 fixation in leguminous crops. Growing plant cells responsd well to boron for dividing and synthesizing cell walls when compared to growth-limited plant cells, in which the synthesis of primary cell walls is negligible (Fleischer et al., 1998). In leguminous plant roots, the development of symbiosis with soil Rhizobium bacteria depends on the concentration of available soil boron and calcium and that both nutrients are essential for nodule structure and fixing atmospheric nitrogen in the plant roots (Redondo-Nieto et al., 2003). As CaCO3 is a source of calcium required by the plants during critical growth stages, the dissolution of CaCO3 in soil will be very rapid in the presence of carbon monoxide (CO), (Gallagher & Breecker, 2020), therefore the negative factor loading of CaCO3 was observed in PC6 of site year 1. In PC6 of site year 2, high factor loadings of available P2O5 (-0.715) and sulphur (0.719) were found, whereas in PC7 of site year 1, available copper (0.682) and sulphur (-0.688) had high loadings associated with enhanced enzyme activity essential for nodule formation and nitrogen fixation in the leguminous plant roots (Weisany et al., 2013). PC7 of site year 2 showed a high negative factor loading of available boron (-0.726). Legume crops required more amount of boron compared to most field crops as boron plays a vital role in the development of reproductive organs, pollen germination, flower and fruit setting. Boron being a required trace element, there is a narrow gap between deficiency and toxicity in soil–plant systems (Chatterjee & Bandyopandhyay, 2017). Therefore, one spray at the initiation of the reproductive phase is sufficient for optimum flowering and pod yield (Subasinghe et al., 2003); without initial boron spray, plant uptake will deplete boron in the soil, hence the factor loading in PC7 being negative for boron.\n\nAll seven PCs of site year 1, when subjected to stepwise linear regression with crop yield, explained the variance with an R2 = 0.68 (p<0.01). The F test of ANOVA was significant at the 1% level. The coefficients of PC3 and PC4 were significant at the 1% level independently, whereas PC7 was significant at the 5% level. Therefore, the variables explaining PC3, PC4 and PC7 such as Fe, Zn, number of pods at harvest, available P2O5, soil moisture, and copper significantly influenced the yield (Equation 1, Table 6). Available Zn and P2O5 help in seed formation, whereas available soil moisture supports crop growth, which was observed in terms of number of pods per plant and seed yield at the time of harvest. (Subrahmaniyan et al., 2008).\n\nYseed = Crop yield\n\nEquation 1:\n\nYseed = 1300.913 + 0.636PC3 + 0.397PC4 + 0.341PC7, F=13.414, R2=0.679\n\nAll seven PCs of site year 2, when subjected to stepwise linear regression with crop yield, explained the variance significantly at the 1 % level ((R2 = 0.677 p<0.01). The PC1 coefficient was significant at the 1% level. PC3 and PC6 coefficients were significant (p<0.05). Therefore, the variables constituting PC1, PC3 and PC6 such as K2O, S, Fe, Zn, Mn, Ca, Mg, CEC, clay, available water content and number of pods at harvest were significantly influencing the yield (Equation 2, Table 6). This may be due to increased availability of nutrients such as K2O, Fe, Zn, Mn, and the presence of high clay content, which enhances the available water and water holding capacity of the soil; this facilitates nutrient uptake, leading to the increase in number of pods (Rana & Badiyala, 2014; Zarei et al., 2011).\n\nEquation 2:\n\nYseed = 1421 + 0.698PC1 + 0.339PC3 - 0.275PC6, F = 13.264*, R2 = 0.677\n\n\nConclusion\n\nIn the present study, a systematic methodological approach was adopted, using geospatial and multivariate statistical techniques for spatial classification of soils, and to gauge the major variables influencing crop yield, which helps in deriving land- or soil-specific management interventions.\n\nOur study revealed that the predominant yield-contributing variables in rainfed agriculture depended on a number of factors, including distribution of rainfall and prevailing land slope. During a drought year (site year 1), the availability of P2O5, Fe, Zn, Cu in the soil, and number of branches and pods determined the yield. In contrast, during a high-precipitation monsoon year (site year 2), availability of K2O, Mn, Ca, Mg, Fe, Zn, S, CEC, clay content, available water content and number of pods per plant were the main yield-contributing factors. Therefore, in a semi-arid region, an increase in soil moisture/available water content to an optimum level (25-30% AWC) will help dissolve most soil nutrients, and increases CEC and nutrient uptake, thus leading to a higher crop yield.\n\nThe principal component regression analysis performs a reasonable dimension reduction and operates well with highly correlated variables. Crop growth and yield depend on multiple factors but are limited by the least-available nutrient present in the environment relative to demands of this nutrient for growth. Therefore, all the loaded PCs with a minimum of 80 % cumulative variance should be considered for principal component regression analysis. The variables which were identified from the principal component regression analysis can efficiently explain the yield variability; further cross-verification of these variables with their availability in each soil management/soil-phase unit, may help to identify limiting factors, such as land surface parameters, soil depth, slope, and available soil moisture. Interventions involving these variables would need to be applied in a site-specific way to increase crop yield.\n\n\nData availability\n\nMendeley Data: Support for improved program integration under rainfed areas, https://data.mendeley.com/datasets/ng5v92xht3/4 (Rajesh, 2021).\n\nThis project contains the following underlying data:\n\n- Table-1 2018–19-Before Sowing.xlsx (Physico-chemical properties of initial surface soil samples of Kalmandari Tanda-1 MWS during June 2018)\n\n- Table-1-2019–20-After harvest.xlsx (Physico-chemical properties of surface soil samples (after harvest) of Kalmandari Tanda-1 MWS during 2018–19)\n\n- Table-2-2018–19-Crop growth yield.xlsx (Pigeonpea plant growth and yield parameters of Kalmandari Tanda-1 MWS during kharif 2018–19)\n\n- Table-2-2019–20-Crop growth yield.xlsx (Pigeonpea plant growth and yield parameters of Kalmandari Tanda-1 MWS during kharif 2019–20)\n\nMendeley Data: Support for improved program integration under rainfed areas, https://data.mendeley.com/datasets/ng5v92xht3/4 (Rajesh, 2021).\n\nThis project contains the following extended data:\n\n- Annexure-1.pdf (Pedon description form)\n\n- Extended data-1.xlsx (pigeon pea growth variety and fertilizers used)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nGround Water Information Booklet Gulbarga District, Karnataka. 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Plant Physiol. 1998; 117(4): 1401–1410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGallagher TM, Breecker DO: The Obscuring Effects of Calcite Dissolution and Formation on Quantifying Soil Respiration. Global Biogeochem Cycles. 2020; 34(12): e2020GB006584. Publisher Full Text\n\nHair JF, Anderson RE, Tatham RL, et al.: Multivariate data analysis. Pearson Education, Singapore and India. 2003. Reference Source\n\nJackson ML: Soil Chemical Analysis. Prentice hall of India, New Delhi. 1973; 498.\n\nJohn WF: Biodiversity Conservation as a Strategy for Poverty Alleviation Around Protected Areas. A case of Bwindi Impenetrable National Park. Journal of Hospitality & Tourism. 2020; 18(2).\n\nJolliffe IT, Cadima J: Principal component analysis: a review and recent developments. Philos Trans A Math Phys Eng Sci. 2016; 374(2065): 20150202. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJuhos K, Szabo S, Ladanyi M: Influence of soil properties on crop yield: a multivariate statistical approach. Int Agrophys. 2015; 29: 433–440. Publisher Full Text\n\nLambers H, Shane MW, Cramer MD, et al.: Root Structure and Functioning for Efficient Acquisition of Phosphorus: Matching Morphological and Physiological Traits. Ann Bot. 2006; 98(4): 693–713. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLindsay WL, Norvell WA: Development of a DTPA soil test for zinc, iron, manganese and copper. Soil Sci Soc Am J. 1978; 42(3): 421–428. Publisher Full Text\n\nMoore GA: Soilguide. A handbook for understanding and managing agricultural soils. Agriculture Western Australia Bulletin. 2001; 4343. Reference Source\n\nNargundkar R: Marketing Research-text and cases. (Publishing: Tata McGraw Hill). 2005.\n\nNatarajan A, Reddy RS, Niranjana KV, et al.: Field guide for land resources inventory, Sujala III project,Karnataka. NBSS, (Publishing: ICAR-NBSS & LUP Bangalore). 2016; 154. Reference Source\n\nNeina D: The Role of Soil pH in Plant Nutrition and Soil Remediation. Hindawi Appl Environ Soil Sci. 2019; 2019: 5794869. Publisher Full Text\n\nPage AL, Miller RH, Kenay DR: Methods of Soil Analysis. Part-II Chemical and Microbiological properties. American Society of Agronomy. (Publishing: Madison, Wisconsin, USA). 1982. Reference Source\n\nPeret B, Desnos T, Jost R, et al.: Root Architecture Responses: In Search of Phosphate. Plant Physiol. 2014; 166(4): 1713–1723. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiper CS: Soil and Plant Analysis. Academic Press, New York, 1966; 367.\n\nRajesh NL: Support for improved program integration under rainfed areas. Mendeley Data, V4, 2021. http://www.doi.org/10.17632/ng5v92xht3.4\n\nRana R, Badiyala D: Effect of integrated nutrient management on seed yield, quality and nutrient uptake of soybean (Glycine max) under mid hill conditions of Himachal Pradesh. Indian J Agron. 2014; 59(4): 641– 645. Reference Source\n\nRedondo‐Nieto M, Wilmot AR, El‐Hamdaoui A, et al.: Relationship between boron and calcium in the N2‐fixing legume-rhizobia symbiosis. Plant Cell Environ. 2003; 26(11): 1905–1915. Publisher Full Text\n\nSchoonheydt RA, Johnston CT, Bergaya F: Clay minerals and their surfaces. Dev Clay Sci. 2018; 9; 1–21. Publisher Full Text\n\nShukla MK, Lal R, Ebinger M: Principal component analysis for predicting corn biomass and grain yields. Soil Science. 2004b; 169(3): 215–224. Publisher Full Text\n\nSubasinghe S, Dayatilake GA, Senaratne R: Effect of B, Co and Mo on nodulation, growth and yield of cowpea (Vigna unguiculata). Trop Agric Res Ext. 2003; 6: 108–112. Reference Source\n\nSubbiah BY, Asija GL: A rapid procedure for the estimation of available nitrogen in soils. Curr Sci. 1956; 25(8): 259–260.\n\nSubrahmaniyan K, Kalaiselvan P, Balasubramanian TN, et al.: Soil properties and yield of groundnut associated with herbicides, plant geometry, and plastic mulch. Commun Soil Sci Plant Anal. 2008; 39(7–8): 1206–1234. Publisher Full Text\n\nSuryanarayana TMV, Mistry PB: Principal component regression for crop yield estimation. (Publishing: Singapore: Springer). 2016; 65. Reference Source\n\nvan de Graaff R, Patterson RA: Explaining the Mysteries of Salinity, Sodicity, SAR and ESP in On-site Practice. In Proceedings of On-site ‘ 01 Conference: Advancing On-site Wastewater Systems. by R.A. Patterson & M.J. Jones (Eds). Published by Lanfax Laboratories, Armidale. 2001; 361–368. Reference Source\n\nVijayamohanan Pillai N, Asalatha R: Reliability, Validity and Uni-Dimensionality: A Primer. 2020. Publisher Full Text\n\nWalkley A, Black IA: An examination of the Degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. Soil science. 1934; 37(1): 29–38. Reference Source\n\nWeisany W, Raei Y, Allahverdipoor KH: Role of some of mineral nutrients in biological nitrogen fixation. Bull Env Pharmacol Life Sci. 2013; 2(4): 77–84. Reference Source\n\nZarei I, Khah EM, Mohammadi G, et al.: Assessment of growth and yield components following the application of different biological fertilizers on soybean (Glycine max.L.) cultivation. Aust J Crop Sci. 2011; 5(13): 1776–1782. Reference Source\n\nZsigrai GY: The effect of fertilization on the soil is some chemical and maize yield in long-term experiments. PhD. Dissertation. Uni. Debrecen, Debrecen, Hungary, 1999." }	[ { "id": "94826", "date": "18 Oct 2021", "name": "Sathish Ayyappa", "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 title of the article is precise and straight and does match with the research findings. Abstract and keywords are well framed with-in the 300 words. Methods adopted are very comprehensive and comprises a detailed soil survey at 1:8000 scale and classifying soils to series level and further dividing each series to soil phases/management units. Use of PCA and LPCR analysis to find out influence of soil properties on pigeonpea yield at soil phase level is also an indication of site specific management for various agronomic/soil operations. The technology adopted namely remote sensing data interpretation (for soil survey and classification), various lab standard analytical techniques/methods, GIS mapping, PCA, and LPCR are Apt. Therefore, the article can be approved for indexing with the following minor corrections;\n\nPage no. 4: last line of the first paragraph in the second column of the page “with standard HCl (Piper, 1966).” w letter is missing.\n\nPage no. 12 & 13: Expansion of AWC, just below the table 4 and 5, should be changed to Available water content instead of Average water content.\n\nPage No. 8: Under the sub heading ‘PCA & LPCR to assess the influence of soil properties on pigeonpea yield’ 2nd column of the page no. 8, 7th line, high loadings of clay content (0.897 and 0.937) these values should be changed to (0.905 and 0.929) as per the table 4 & 5 respectively.\n\nPage No. 8: Under the sub heading ‘PCA & LPCR to assess the influence of soil properties on pigeonpea yield’ 2nd column of the page no. 8, 9th to 14thth line (on the other hand …..(Figure 2)). Please rewrite the sentence as given below;\n\"High clay content supported high available water content with loadings of 0.822 and 0.625 during 2018-19 and 2019-20 respectively. Therefore, sand (-0.858 and -901) and bulk density (-0.927 and -0.743) are negatively correlated in PC1 of both the site years. Though the initial mean available water content is high (27.93 %) in soils during 2018-19 (Table 1), the scarcer rains in subsequent months during 2018-19 (Figure 2) has inferred that there was no sufficient soil moisture to dissolve the soil nutrients to make it readily available to pigeonpea plant when compared to the rains of 2019-20 (Figure 3).\"\n\nPage No. 13: 2nd column, line 2, correct the spelling of ‘responds’.\n\nPage 14: 2nd column of the page, under equation 1, line 5, The PC1 and PC3 coefficients were significant at 1 % (p<0.01) level. PC6 was significant at 5 % (p<0.05). Note that PC3 significant is only at 1 % and not at 5 %.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "157225", "date": "17 Feb 2023", "name": "Gabriel Oladele Awe", "expertise": [ "Reviewer Expertise Soil and Water Conservation", "Soil Physical processes", "Irrigation", "Soil tillage", "Cropping systems" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper sought to evaluate the variability of soil properties using multivariate analysis with a view to detecting which properties influence the yield of pigeon pea.\nThe study could contribute to knowledge in the area of site-specific agriculture. However, the Introduction requires thorough revision: it contains some statements that should appear in Methodology.\nA table (Table 3) appears repeating information already presented in the text. Also the Table inserted under Figure 6 is a repetition of same Figure, remove!\nFor other comments, please refer to the text.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-944
https://f1000research.com/articles/12-762/v1	28 Jun 23	{ "type": "Research Article", "title": "The impact of shift-work light conditions on tissue-specific circadian rhythms of canonical clock genes: insights from a mouse model study", "authors": [ "Bala S. C. Koritala", "Panshak P. Dakup", "Kenneth I. Porter", "Shobhan Gaddameedhi", "Bala S. C. Koritala", "Panshak P. Dakup", "Kenneth I. Porter" ], "abstract": "Background: The natural day-night cycle synchronizes our circadian rhythms, but modern work practices like night shifts disrupt this pattern, leading to increased exposure to nighttime light. This exposure is linked to various health issues. While some studies have explored the effects of night shifts on human circadian rhythms, there is limited research on the consequences of long-term exposure to shift-work light conditions. Rodents can provide valuable insights into these effects. This study aimed to examine how short- or long-term exposure to rotating shifts and chronic jetlag affects the core circadian oscillators in the liver and skin of mammals.\nMethods: C57BL/6J male mice were subjected to simulated shift-work light conditions, including short-term or long-term rotating shifts and chronic jet-lag conditions. Liver and skin samples were collected every four hours over a 24-hour period on the second day of constant darkness. RNA was extracted and qRT-PCR analysis was conducted to measure the circadian gene expression in liver and skin tissues. Circadian rhythm analysis using CircaCompare compared the control group to mice exposed to shift-work light conditions.\nResults: The liver's circadian clock is significantly altered in mice under long-term rotating shift conditions, with a lesser but still noticeable impact in mice experiencing chronic jetlag. However, short-term rotating shift conditions do not significantly affect the liver's circadian clock. Conversely, all three simulated shift conditions affect the skin's circadian clock, indicating that the skin clock is more sensitive to shift-work light conditions than the liver clock. Compared to the liver, the skin's circadian clock is greatly affected by long-term rotating shift conditions.\nConclusions: The study findings indicate more pronounced disturbances in the canonical clock genes of the skin compared to the liver under simulated shift-work light conditions. These results suggest that the skin clock is more vulnerable to the effects of shift-work.", "keywords": [ "Canonical clock genes", "mouse", "rotating shift", "jet-lag", "liver", "skin." ], "content": "Introduction\n\nThe natural day-night cycle is the primary synchronizer of circadian rhythms in humans.1 The advent of electricity has led to 24/7 work practices including night shift and rotating shift schedules, increasing nighttime light exposure in modern society. Unfortunately, these modern work habits have been associated with an elevated risk of various health conditions, including cancer, cardiovascular diseases, depression, diabetes, gastrointestinal problems, and metabolic syndrome.2–5 The disruption of circadian rhythms, both at the physiological and behavioral level, contributes to these modern work habit-associated health conditions. The few studies investigating the impact of night shift on the molecular circadian rhythms in humans have revealed significant changes in the circadian transcriptome and metabolome within a one-week study duration.6–8 However, in real-world scenarios, many shift workers experience prolonged exposure to rotating shifts that extend beyond a single week. Unfortunately, there is a lack of systematic research investigating the consequences of long-term exposure to shift-work light conditions on molecular circadian rhythms in humans. This absence may be attributed to logistical, financial, and facility constraints in setting up human sleep laboratories. Nonetheless, rodents can serve as valuable preliminary models for understanding the effects of different shift schedules or exposure to shift-work light conditions on molecular circadian rhythms, providing insights into the underlying molecular mechanisms associated with shift-work related disorders.9\n\nIn laboratory settings, researchers can replicate shift-work-like light environments using rotating shift and jet-lag protocols in rodents. Several studies10–14 have employed these protocols in rodents to investigate the mechanisms underlying tumor development, metabolic disorders, and other health outcomes associated with shift work. These studies have indicated that exposure to shift-work or jet-lag light environments can promote tumor progression,10,12,15–18 glucose intolerance,19,20 obesity,19,21 genotoxic stress mediated cardiovascular toxicity,22 and radiodermatitis.23 Although some research has shown abnormal activity/sleep rhythms in response to shift conditions,11 our understanding of how short- or long-term exposure to shift-work and chronic jet-lag conditions affects the mammalian core circadian oscillator, the canonical clock genes, remains limited. Rodents serve as a convenient model for studying tissue-specific circadian gene expression of these clock genes. Therefore, in this study, we utilized a well characterized circadian mouse model, C57BL/6J,24 to investigate the impact of short- or long-term exposure to rotating shifts and jet-lag conditions on the mammalian core oscillators in various tissues.\n\nThe primary focus of our study was to examine the effects of shift-work-like light conditions on the liver and skin. We specifically chose the liver because it is widely recognized as the primary organ model for studying the circadian clock in peripheral tissues.25 The liver plays a crucial role in carrying out various metabolic functions regulated by the circadian clock through transcriptional and posttranslational mechanisms.26–28 On the other hand, although the skin is not as metabolically active as the liver, it serves as accessible window for understanding the body’s internal clock in humans.29,30 Moreover, a study has shown that restricted feeding altered the phase and amplitude of clock genes in mouse skin, subsequently affecting the skin’s response to ultraviolet radiation exposure.31 Given these factors, the liver and skin represent distinct metabolic tissue models with robust circadian clocks, offering valuable insights into circadian rhythm disturbances caused by shift work.\n\nOur study employed a comprehensive design that enabled us to investigate the tissue-specific disturbances in circadian rhythms resulting from simulated short- or long-term exposure to rotating shifts and chronic jet-lag conditions. After subjecting C57BL/6J mice to simulated shift-work light conditions, we measured the circadian gene expression of canonical clock genes (Arntl, Npas2, Per1, Per2, Per3, Cry1, Cry2, and Dbp) in liver and skin samples. The measurements were taken over a 24-hour period on the second day of constant darkness (Figure 1). Our findings indicate more pronounced disturbances in the canonical clock genes of the skin compared to the liver under simulated shift-work light conditions. Notably, long-term rotating shift work significantly impacted the expression of these canonical clock genes in both tissues. Based on our observations, it is evident that the skin clock is more vulnerable to the effects of shift-work light conditions when compared to the liver clock.\n\nPanels from left to right represent simulated light conditions of control (left), short- and long-term rotating shifts (middle), and chronic jet-lag (right). After exposure to simulated shift conditions of light (yellow) and dark (Gray), liver and skin samples were collected for 24 hours with four-hour intervals on the second day of constant dark condition. 0th time was defined as the onset of the light phase before the animals entered constant darkness for tissue collection.\n\n\nMethods\n\nThe mice utilized in this study were procured from The Jackson Laboratory and housed in the Program of Laboratory Animal Resources (PLAR) of Washington State University - Spokane. To guarantee proper animal welfare, the guidelines set by the Institutional Animal Care and Use Committee (IACUC) were followed. Our program compiles with all regulations outlined in the NIH guide for the Care and Use of Laboratory Animals, encompassing procurement, conditioning and quarantine, housing, management, veterinary care, and carcass disposal. All efforts were made to ameliorate the suffering of the mice involved. There were no adverse events during the study and no humane endpoints were established. The animal experiments conducted in this study were approved by the IACUC under the protocol number: 6202 on March 27th, 2019.\n\nThis investigation took place from May through September 2019. Healthy immunocompetent wild-type C57BL/6J male mice at 3 weeks of age were purchased from the Jackson Laboratory (strain #: 000664). Mice were purchased to limit variability and maintain consistency in the genetic quality of the animals. Upon arrival, these mice were acclimatized to the controlled environment of the PLAR facility at Washington State University – Spokane for at least one week. The only inclusion criterion was for animals to be at least 4 weeks of age by day 1. To investigate the impact of shift-work light conditions on circadian rhythms of canonical clock genes, a group of C57BL/6J male mice were randomly assigned and housed (3 mice per cage) in a controlled environment that simulated shift-work light conditions. The mice were subjected to different simulated conditions: either short-term (15 days) or long-term (45 days) rotating shifts, where the light conditions changed every seventh day, or a jet-lag condition lasting 21 days with an 8-hour phase advance every two days. As a comparison, a control group of mice followed the standard light-dark cycle of the facility (Figure 1). A total of 72 mice were included in this study, to cover 24 timepoints across 4 light conditions. An n = 3 was chosen per time point as the minimum number required to identify statistical differences per time-point, which translates to n = 18 per experimental condition for circadian analyses. Following exposure to simulated light conditions with an intensity of ~590 lux, the mice were housed in constant dark for up to 48 hours. On the second day, mice were sacrificed at the age of 11 to 12 weeks old, and samples of liver and dorsal skin tissues were collected from three mice at each time point over a 24-hour period, with four-hour intervals (Figure 1). To manipulate the light conditions, the mice were housed in light-tight circadian chambers provided by Phenome Technologies, Inc. The light schedules were regulated by an external timer (XT-4, Chrontrol Corporation), and the light status and intensity were monitored using HOBO data loggers (UA-002-08, Onset Computer Corporation). All mice, regardless of the light conditions or constant darkness, were given ad libitum feeding. Throughout the time-course sample collection, red light was utilized, and animals were killed per housed group, where one cage of 3 mice represented one time-point. This was done to limit variability in any time-point. Mice were anesthetized with 3-4% isoflurane and euthanized by cardiac puncture blood collection followed by cervical dislocation as a secondary method of euthanasia. The collected liver and skin tissues were snap-frozen and stored at -80 °C for subsequent RNA extraction.\n\nFrozen liver and skin tissues were homogenized in liquid nitrogen using mortar and pestle. Next, we purified the total RNA from the homogenized tissues using a Trizol-based RNA extraction protocol. Approximately 100 mg of the ground tissue was combined with 1 ml of ice-cold TRIzol reagent (Thermo Fisher Scientific, #15596018). After thorough mixing, 0.2 ml of chloroform was added to the TRIzol tissue cocktail, mixed well, and incubated at room temperature for 3 mins. We separated the different phases of the mixture through centrifugation at 12,000 g for 15 mins. The aqueous phase (top layer) was carefully collected and transferred to a new tube. To precipitate the RNA, an equal volume of isopropanol was added to the collected aqueous phase and mixed by inverting. After a 10-minute incubation at room temperature, the samples were subjected to another round of centrifugation at 12,000 g for 10 minutes. The supernatant was discarded, and the RNA pellet was washed three times with 70% ice-cold ethanol. Subsequently, the RNA pellet was air dried for 5-10 mins and dissolved in 20 μl of ddH2O. To assess the quantity and quality of the extracted RNA, we utilized NanoDrop® 2000 Spectrophotometer, measuring the light absorbance at 260, 280 and 230 nm. Following the RNA purification step, we treated the RNA samples with DNase-I (New England Biolabs, #M0303S), and synthesized complementary DNA (cDNA) using TaqMan™ Reverse Transcription Reagents (Thermo Fisher Scientific, #N8080234). Real-time polymerase chain reaction (RT-PCR) was then performed using PerfeCTa® SYBR® Green Supermix with ROX™ (Quantabio, #95055-500) in a StepOnePlus™ Real-Time PCR System (Applied Biosystems™). The RT-PCR reaction included denaturation (3 minutes at 95 °C) and 40 PCR cycles (15 sec at 95 °C and 1 minute at 60 °C). The expression level of each clock gene was normalized to the geometric mean expression of two housekeeping genes,32 Actb and Ppib using the ΔCt method. Subsequently, the data were scaled to the sample with the highest expression level (ΔΔCt) for the time course analysis. Missing values in the raw data files resulting from technical issues with the qPCR plate were excluded from analysis. We specifically measured the expression of canonical clock genes (Arntl, Npas2, Per1, Per2, Per3, Cry1, Cry2, and Dbp) in both liver and skin tissues using validated primers. To ensure the accuracy of the PCR amplification, the primers were validated using cDNA template serial dilution, and their efficiency was calculated based on the slope value33 (Table 1). The primer design for this study was performed using Primer3 (v.0.4.0), a widely used primer design tool.\n\nWe used CircaCompare34 to analyze the differences in circadian rhythms of canonical clock genes in animals exposed to control and simulated shift-work light conditions. Circadian time, CT0 was defined as the onset of the light phase before the animals entered constant darkness for tissue collection. This alignment allowed us to effectively compare and assess the variations in circadian rhythms between control and simulated shift-work light conditions. We applied a p-value cut-off of <0.05 to determine the presence or absence of rhythms and to evaluate significant differences in phase, amplitude, and the Midline Estimating Statistic of Rhythm (MESOR). Sample resolution is a critical factor in circadian analysis as it directly affects the precision of phase estimates.35 In our study, we used four-hour sample resolution, therein phase differences were only considered if they were at least four hours apart. This approach was implemented to ensure the accuracy of our results.\n\n\nResults\n\nTo investigate the impact of shift-work light conditions on the liver clock, we examined the circadian rhythms of canonical clock genes (Arntl, Npas2, Per1, Per2, Per3, Cry1, Cry2, and Dbp) in the liver samples of mice exposed to simulated shift-work light conditions. Rhythmic gene expression and its significance was determined at p < 0.05 of CircaCompare. Our findings revealed significant rhythmic patterns in all analyzed clock genes, both in the control group and in the mice exposed to the simulated shift-work light conditions. However, Cry1 did not show significant rhythmicity in the long-term rotating shift scenario and Cry2 did not exhibit significant rhythmicity in the chronic jet-lag scenario (Figure 2, Extended Data Table 151). This suggests that these genes may be more sensitive to prolonged disruption of light-dark cycles. Furthermore, we examined various circadian properties such as phase, amplitude, and MESOR and compared differences between conditions (Figures 2 and 4A, Extended Data Table 151). In the short-term rotating shift and chronic jet-lag scenarios, we did not observe any discernible phase differences compared to the control condition. However, in the long-term rotating shift scenario, significant phase advance of at least four hours were detected in five out of eight clock genes: Npas2, Per1, Per2, Per3, and Cry2. Regarding amplitude, we did not find significant differences in the rotating shift conditions. However, we did observe a significant decrease in amplitude of Per1 and Dbp among mice exposed to chronic jet-lag compared to the control group. This indicates that chronic disruption of light-dark cycles may affect the robustness of circadian rhythms in these genes. We did not find any significant changes in MESOR with short-term rotating shift. However, we observed a significant increase in MESOR with the genes Arntl, Per2, Per3, and Cry2 in the long-term rotating shift condition. Conversely, we found a significant decrease in MESOR with the genes Per1 and Dbp in the chronic jet-lag scenario. Overall, these results suggest that the circadian clock in the liver is highly impacted in mice exposed to the long-term rotating shift condition with a clear but less dramatic impact in mice exposed to chronic jet-lag. In contrast, we found no significant impact on the liver’s circadian clock in mice exposed to a short-term rotating shift condition.\n\nThe graphs display 24-hour expression (Mean ± SE) of Arntl, Npas2, Per1, Per2, Per3, Cry1, Cry2, and Dbp in the liver under constant dark after exposure to simulated shift-work light conditions. The figure allows for a comparison between the control group and three experimental conditions: short-term rotating shift (left), long-term rotating shift (middle), and control vs chronic jet-lag (right) were represented in the figure. To ensure accurate analysis, the mRNA expression throughout the time course was normalized against the geometric mean expression of housekeeping genes Actb and Ppib. The statistical significance of the circadian rhythms and the differences in circadian characteristics between the control group and the simulated shift-work conditions were assessed using CircaCompare, with a significance level set at p < 0.05. Genes that exhibit a significant rhythmic pattern are represented by continuous lines, while arrhythmic genes are represented by dotted lines.\n\nIn addition to studying the liver, we also examined the expression of canonical clock genes in the dorsal skin of mice (Figure 3, Extended Data Table 151) collected under constant darkness after being exposed to simulated shift-work light conditions. We analyzed the circadian rhythms and determined their significance using CircaCompare with a p < 0.05. Our analysis revealed that majority of the analyzed clock genes in the skin exhibited rhythmic patterns. However, we observed a loss of rhythmicity in Cry1 after exposure to short-term rotating shift and chronic jet-lag conditions. Additionally, Cry2 became arrhythmic when subjected to long-term rotating shift and chronic jet-lag conditions. Furthermore, we compared the circadian rhythms between the control group and the simulated shift-work groups, focusing on differences in phase (at least four hours), amplitude, and MESOR (Figure 4B, Extended Data Table 151). Out of the eight genes analyzed in the skin, significant phase differences were observed in two genes (Arntl and Cry2) following the short-term rotating shift condition. Four genes (Arntl, Per2, Per3, and Dbp) exhibited significant phase differences following the long-term rotating shift condition, and one gene (Arntl) showed a significant phase difference following chronic jet-lag. Notably, most of these genes displayed a phase advance following simulated shift-work conditions compared to the control group. Arntl consistently exhibited dysregulation across all simulated shift conditions, particularly showing a phase delay in the long-term rotating shift scenario. Moreover, we observed alterations in amplitude: Cry2 and Arntl exhibited reduced amplitude following short-term and long-term rotating shift conditions, respectively. Per2 displayed reduced amplitude in both rotating shift conditions, while no significant differences in amplitude were noted with chronic jet-lag. Regarding MESOR, two out of eight genes analyzed in each shift condition exhibited MESOR differences compared to the control group. Arntl displayed a significant increase in MESOR following exposure to short-term rotating shift and chronic jet-lag conditions, while Per1 showed a significant increase following long-term rotating shift and chronic jet-lag. Additionally, Cry2 and Per2 exhibited a significant decrease in MESOR following exposure to the short-term and long-term rotating shift conditions, respectively. Overall, these results indicate that the circadian clock in the skin is impacted by all three simulated shift conditions and suggest that the skin clock is more sensitive to shift-work light conditions compared to the liver clock. Similar to our observations in the liver, the circadian clock in the skin is highly impacted in mice exposed to long-term rotating shift conditions.\n\nThe graphs in the figure illustrate the 24-hour expression (Mean ± SE) of Arntl, Npas2, Per1, Per2, Per3, Cry1, Cry2, and Dbp in the skin under constant darkness following exposure to simulated shift-work light conditions. The figure enables a comparison between the control group and three experimental conditions: short-term rotating shift (left), long-term rotating shift (middle), and control vs chronic jet-lag (right) were represented in the figure. To ensure precise analysis, the mRNA expression throughout the entire time course was normalized against the geometric mean expression of housekeeping genes Actb and Ppib. The statistical significance of the circadian rhythms and the variations in circadian characteristics between the control group and the simulated shift-work conditions were evaluated using CircaCompare, with a significance level set at p < 0.05. In the figure, genes that exhibit a significant rhythmic pattern are depicted by continuous lines, while genes lack a rhythmic pattern are represented by dotted lines.\n\nThe figure represents differences in phase (left), amplitude (middle), and MESOR (right) of canonical clock genes in the liver (top) and skin (bottom) of mice subjected to short-term, long-term rotating shift, and chronic jet-lag conditions, in comparison to a control condition. Significance testing was performed using CircaCompare with a threshold of p < 0.05, and the corresponding p-values can be found in Extended Data Table 1.51\n\n\nDiscussion\n\nOur study aimed to fill a significant knowledge gap regarding the effects of different durations and types of shift conditions on the circadian system. Shift-work has been hypothesized to disrupt the synchronization between the central and peripheral circadian machinery.36 Previous studies in humans have only examined the impact of mistimed sleep on molecular rhythms for about a week,6–8 limiting our understanding of the long-term consequences. We conducted a study using a well-established C57BL/6J mouse model to investigate the consequences of various durations and types of shift-work light conditions on the circadian system. By simulating shift-work light conditions, we were able to observe changes in the circadian expression of canonical clock genes in both the liver and the skin.\n\nLight is widely recognized as the primary synchronizer for maintaining the mammalian circadian system. Two photoreceptors, melanopsin (OPN4) and neuropsin (OPN5), have been identified as mediators of light entrainment from the retinal ganglion cells to the suprachiasmatic nucleus (SCN),37,38 which serves as the central pacemaker of the circadian system. Interestingly, recent studies have shown that OPN5 in murine melanocytes is involved in circadian photoentrainment and the expression of clock genes, a phenomenon not observed in the liver.39 On the other hand, the liver, being a metabolically active organ, can directly entrain its clock through feeding-fasting cycles.40–42 Prior research has demonstrated this ability, indicating that the liver is more influenced by metabolic cues compared to the skin. Therefore, in the current study, we examined the impact of simulated shift-work light conditions on the circadian gene expression of canonical clock genes in two different tissues: the liver and the skin.\n\nUnlike the skin, which directly senses external light stimuli,39 the entrainment of liver clocks by light serves as a secondary event. In addition to synchronization from the central clock, feeding has been well demonstrated as a mechanism to reset liver clocks.40–42 In our study, we provided mice with unrestricted access to food, allowing us to primarily observe changes arising from shift-work light conditions. The data from our study indicated that short-term exposure to rotating shift-light conditions did not significantly affect the circadian rhythms of liver clock genes. However, long-term exposure to rotating shifts resulted in circadian dysregulation, as evidence by loss of rhythmicity or significant differences in phase, amplitude, or MESOR in 7 out of 8 tested clock genes, and the jet-lag condition affected 3 out of 8 of the genes tested. These results suggest that liver clocks exhibit adaptability under less aggressive shift-work light conditions, possibly due to input from multiple environmental cues. Moreover, recent research has demonstrated that restricted feeding not only entrains liver clocks but also influences skin clocks in a distinct manner, highlighting an interesting interplay wherein a single stimulus can impact multiple systems.31\n\nIn our investigation, we observed significant disturbances in the circadian rhythm of canonical clock genes in the skin under various shift-work light conditions. Short-term exposure to rotating shift schedules and chronic jet-lag led to the disruption of circadian rhythmicity in 4 out of 8 tested canonical clock genes, while long-term exposure to rotating shifts affected 6 out of 8 of the genes, with most of these changes attributed to phase differences. Notably, unlike the liver, the circadian clock in the skin exhibited significant dysregulation across all shift-work light conditions examined in our study. This suggests that the skin’s circadian clock is more susceptible to the impact of shift-work light conditions compared to the clock in the liver. One potential explanation for this susceptibility is the involvement of opsins-mediated signaling. Opsins are a class of light sensing G protein-coupled receptors that trigger signaling cascades upon photosensation. Previous studies have identified the expression of various opsins in different models, including murine melanocytes,39 human melanocytes, keratinocytes, and dermal fibroblasts, and hair follicle stem cells.43 The presence of opsins within the skin cells indicates the possibility of light-sensing capabilities in the skin tissue and the potential influence of environmental light cues on core clock regulation. However, experimental verification of this hypothesis is still required.\n\nAt the level of independent clock genes, we found that the circadian rhythms of cryptochrome genes (Cry1 and/or Cry2), which are the primary repressors of the molecular clock and also involved in maintaining genome integrity of cells,44,45 were significantly disrupted under simulated shift conditions in both tissues. However, in the case of short-term rotating shift specifically affecting the liver, the rhythmicity of these genes remained intact. These findings are consistent with existing literature in humans, which has associated disrupted rhythms of Cry1 and Cry2 with circadian dysregulation of DNA repair and increased DNA damage following exposure to simulated night shift conditions.46 Additionally, we observed consistent dysregulation of the circadian transcription factor Arntl, which is involved in protecting skin cells and regulating UVB associated DNA repair response and melanin pigmentation.23,47,48 This dysregulation occurred following exposure to all different types of simulated shift-work light conditions utilized in this study.\n\nOur study has few limitations that should be considered when interpreting the findings. First, the use of a mouse model may limit the generalizability of the results to humans. Second, the simulation of shift-work light conditions may not fully capture the complexity of real-world shift schedules. Third, the sample size was relatively small, potentially affecting the statistical power. Fourth, the duration of exposure in our study may not fully reflect the long-term consequences of shift-work in humans.\n\nDespite these limitations, our study emphasizes the importance of recognizing the varying impacts of different shift conditions on the circadian system. We highlight the heightened susceptibility of the skin to circadian misalignment under shift-work light conditions, in comparison to the liver. Furthermore, we emphasize the significance of tissue-specific responses and the role of light regulating circadian rhythms, specifically focusing on canonical clock genes during shift-work. These findings offer valuable insights that can guide future research, potentially leading to the discovery of novel mechanisms and more effective treatment strategies for shift-work associated diseases.\n\n\nAuthor contributions\n\nBala S. C. Koritala: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft preparation, Writing – review & editing. Panshak P. Dakup: Conceptualization, Investigation, Methodology, Resources, Validation, Writing – original draft preparation, Writing – review & editing. Kenneth I. Porter: Investigation, Methodology, Resources, Validation, Writing – review & editing. Shobhan Gaddameedhi: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – review & editing.", "appendix": "Data availability\n\nfigshare: Raw data files with replicates titled as “Cycle threshold values of qPCR data for genes in the liver and skin of animals that were exposed to both control and simulated shift conditions”. https://doi.org/10.6084/m9.figshare.23269757.v1. 50\n\nfigshare: Extended Data Table 1. CircaCompare analysis of canonical clock genes in the mouse liver and skin following exposure to simulated shift-work light conditions. https://doi.org/10.6084/m9.figshare.23512155.v1. 51\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nfigshare: ARRIVE checklist for ‘The impact of shift-work light conditions on tissue-specific circadian rhythms of canonical clock genes: insights from a mouse model study’. https://doi.org/10.6084/m9.figshare.23519379.v1. 52\n\n\nReferences\n\nWright KP Jr, McHill AW, Birks BR, et al.: Entrainment of the human circadian clock to the natural light-dark cycle. Curr. Biol. 2013 Aug 19; 23(16): 1554–1558. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoenneberg T, Merrow M: The Circadian Clock and Human Health. Curr. Biol. 2016 May 23; 26(10): R432–R443. PubMed Abstract \| Publisher Full Text\n\nCosta G: Shift work and health: current problems and preventive actions. Saf. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoritala BSC, Dakup PP, Porter KI, et al.: Cycle threshold values of qPCR data for genes in the liver and skin of animals that were exposed to both control and simulated shift conditions. [Dataset]. figshare. 2023. Publisher Full Text\n\nKoritala B, Dakup PP, Porter KI, et al.: CircaCompare analysis of canonical clock genes in the mouse liver and skin following exposure to simulated shift-work light conditions. [Dataset]. figshare. 2023. Publisher Full Text\n\nKoritala B, Dakup PP, Porter KI, et al.: Author Checklist - F1000 Research.pdf. figshare. 2023. Publisher Full Text" }	[ { "id": "184816", "date": "13 Jul 2023", "name": "Tianpeng Zhang", "expertise": [ "Reviewer Expertise circadian clock and metabolism" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Koritala et al. report that shift-work light affects the circadian rhythms of canonical clock genes in the liver and skin of mice. Although the authors have utilized multiple approaches to test their hypothesis, there remain areas where the methods section should also provide detailed information. Please refer to the following comments for more details.\nDescribe in detail the process for collecting skin, which part of the skin tissue was collected?\n\nIs the selection of light intensity reasonable in the experiment? Multiple studies have shown that bright light can affect the circadian rhythm of activity, metabolism and clock gene expression (Bano-Otalora et al., 20211; Alves-Simoes et al., 20162; Fan et al., 20223). Meanwhile, the light intensity also affects the impact of chronic jet-lag.\n\nMore information should be provided on Circadian rhythm analysis, especially the setting of parameters.\n\nWhy use two housekeeping genes in the same tissue for clock gene relative quantification?\n\nThe author should discuss why Cry genes are preferentially altered under three light conditions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9999", "date": "29 Aug 2023", "name": "SHOBHAN GADDAMEEDHI", "role": "Author Response", "response": "Reviewer Comments In this manuscript, Koritala et al. report that shift-work light affects the circadian rhythms of canonical clock genes in the liver and skin of mice. Although the authors have utilized multiple approaches to test their hypothesis, there remain areas where the methods section should also provide detailed information. Please refer to the following comments for more details. 1. Describe in detail the process for collecting skin, which part of the skin tissue was collected? Authors' response: As per the suggestion of the reviewer, we have elaborated on the procedure of skin tissue collection within the 'Study Design and Sample Collection' section in our methods. 2. Is the selection of light intensity reasonable in the experiment? Multiple studies have shown that bright light can affect the circadian rhythm of activity, metabolism and clock gene expression (Bano-Otalora et al., 20211; Alves-Simoes et al., 20162; Fan et al., 20223). Meanwhile, the light intensity also affects the impact of chronic jet-lag. Authors' response: Our study maintained similar light intensities across control and shift simulation groups to focus on shift impacts, thereby reducing potential variation from light intensity differences. However, recognizing the significance of light intensity on chronic jet-lag, we will consider incorporating this factor into future experimental designs, perhaps by comparing the effects of different light intensities on chronic jet-lag. 3. More information should be provided on Circadian rhythm analysis, especially the setting of parameters. Authors' response: In response to the reviewer’s suggestion, we have clarified our use of the standard parameters of the CircaCompare R package (as per Parsons et al., Bioinformatics, 2020, PMID: 31588519). This approach includes a fixed period of 24 hours. 4. Why use two housekeeping genes in the same tissue for clock gene relative quantification? Authors' response: Vandesompele et al. (Genome Biol. 2002, PMID: 12184808) showed that normalizing with the geometric mean of carefully chosen control genes improves qPCR accuracy. Using a single universal control gene is less effective due to variations across different cells or tissues. Moreover, fluctuations within standard housekeeping genes depending on the time of day have been recognized in studies (Kosir et al., BMC Mol Biol. 2010; PMID: 20712867). Thus, the utilization of more than one housekeeping gene minimizes this variability. 5.The author should discuss why Cry genes are preferentially altered under three light conditions. Authors' response: In the discussion section of the revised manuscript, we covered the response of Cry genes to simulated shift conditions." } ] }, { "id": "184831", "date": "11 Aug 2023", "name": "Brian J. Altman", "expertise": [ "Reviewer Expertise Molecular cancer biology", "lung biology", "immunology", "circadian rhythms", "metabolism" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn “The impact of shift-work light conditions on tissue-specific circadian rhythms of canonical clock genes: insights from a mouse model study”, Koritala, Dakup, and colleagues explore the outcome of short- and long-term exposure to rotating shift work compared to normal conditions and jet lag in circadian rhythms of the skin and liver in mice. This aims to fill a knowledge gap where only short-term shift work experiments have been conducted on humans in laboratory settings, whereas human shift workers tend to engage in rotating shift work for many years. The Authors find that, in general, short-term shift work has a subtle effect on rhythmicity of molecular clock gene expression, particularly in the liver. In longer-term shift work and jet lag, the Cry genes were the most sensitive to disruption; however, long-term shift work and jet lag both dramatically affected the oscillatory characteristics of many circadian genes, often in a disparate fashion. The Authors conclude that different rotating work and sleep schedules can have outsize impacts on molecular clock oscillation that may vary between tissues.\nOverall, this is a well-designed and executed study with clear outcomes and interpretable results. I have several minor suggestions and comments that I believe will enhance the quality of the final manuscript.\nThis study was only carried out in male mice, which goes against the NIH policy “Consideration of Sex as a Biological Variable in NIH-funded Research” (NOT-OD-15-102). The Authors should discuss this in their Limitations section, and how the outcomes may be different in females.\n\nIn Figure 4, it is difficult to assess which oscillation characteristics are significantly different from control just by looking at the figure. I am aware that full statistics are available in Extended Data Table 1, but it would be best if the Authors also figured out a way to represent, perhaps with a different shape (a star instead of a circle?) which datapoints were significantly different in Figure 4.\n\nIn the Discussion, the Authors mainly focus on the observed differences between short-term and long-term rotating shift work, which is indeed important. Equally important is the fact that many papers and groups use the jet lag protocol as a proxy for shift work, and the Authors demonstrate here that the outcomes of jet lag can be quite different from either short-term or long-term rotating shift work. This is a very important finding that should be addressed in the Discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "10061", "date": "29 Aug 2023", "name": "SHOBHAN GADDAMEEDHI", "role": "Author Response", "response": "Reviewer Comments In “The impact of shift-work light conditions on tissue-specific circadian rhythms of canonical clock genes: insights from a mouse model study”, Koritala, Dakup, and colleagues explore the outcome of short- and long-term exposure to rotating shift work compared to normal conditions and jet lag in circadian rhythms of the skin and liver in mice. This aims to fill a knowledge gap where only short-term shift work experiments have been conducted on humans in laboratory settings, whereas human shift workers tend to engage in rotating shift work for many years. The Authors find that, in general, short-term shift work has a subtle effect on rhythmicity of molecular clock gene expression, particularly in the liver. In longer-term shift work and jet lag, the Cry genes were the most sensitive to disruption; however, long-term shift work and jet lag both dramatically affected the oscillatory characteristics of many circadian genes, often in a disparate fashion. The Authors conclude that different rotating work and sleep schedules can have outsize impacts on molecular clock oscillation that may vary between tissues. Overall, this is a well-designed and executed study with clear outcomes and interpretable results. I have several minor suggestions and comments that I believe will enhance the quality of the final manuscript. Authors' response: We appreciate the reviewer’s constructive and positive feedback on our manuscript. 1. This study was only carried out in male mice, which goes against the NIH policy “Consideration of Sex as a Biological Variable in NIH-funded Research” (NOT-OD-15-102). The Authors should discuss this in their Limitations section, and how the outcomes may be different in females. Authors' response: We thank the reviewer for highlighting this. We concur and have addressed the exclusive use of male mice in the limitations section, including potential differences in female outcomes. 2. In Figure 4, it is difficult to assess which oscillation characteristics are significantly different from control just by looking at the figure. I am aware that full statistics are available in Extended Data Table 1, but it would be best if the Authors also figured out a way to represent, perhaps with a different shape (a star instead of a circle?) which datapoints were significantly different in Figure 4. Authors' response: Great suggestion. We revised Figure 4 to visually highlight the significantly different datapoints. 3. In the Discussion, the Authors mainly focus on the observed differences between short-term and long-term rotating shift work, which is indeed important. Equally important is the fact that many papers and groups use the jet lag protocol as a proxy for shift work, and the Authors demonstrate here that the outcomes of jet lag can be quite different from either short-term or long-term rotating shift work. This is a very important finding that should be addressed in the Discussion. Authors' response: Thank you for highlighting this. We've updated our Discussion section to emphasize the differences between jet lag outcomes and the effects of both short-term and long-term rotating shift work." } ] } ]	1	https://f1000research.com/articles/12-762
https://f1000research.com/articles/11-1556/v1	22 Dec 22	{ "type": "Research Article", "title": "Circadian clock control of tRNA synthetases in Neurospora crassa", "authors": [ "Kathrina D. Castillo", "Emily D. Chapa", "Deborah Bell-Pedersen", "Kathrina D. Castillo", "Emily D. Chapa" ], "abstract": "Background: In Neurospora crassa, the circadian clock controls rhythmic mRNA translation initiation through regulation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2). Active CPC-3 phosphorylates and inactivates eIF2α, leading to higher phosphorylated eIF2α (P-eIF2α) levels and reduced translation initiation during the subjective day. This daytime activation of CPC-3 is driven by its binding to uncharged tRNA, and uncharged tRNA levels peak during the day under control of the circadian clock. The daily rhythm in uncharged tRNA levels could arise from rhythmic amino acid levels or aminoacyl-tRNA synthetase (aaRSs) levels. Methods: To determine if and how the clock potentially controls rhythms in aspartyl-tRNA synthetase (AspRS) and glutaminyl-tRNA synthetase (GlnRS), both observed to be rhythmic in circadian genomic datasets, transcriptional and translational fusions to luciferase were generated. These luciferase reporter fusions were examined in wild type (WT), clock mutant Δfrq, and clock-controlled transcription factor deletion strains. Results: Translational and transcriptional fusions of AspRS and GlnRS to luciferase confirmed that their protein levels are clock-controlled with peak levels at night. Moreover, clock-controlled transcription factors NCU00275 and ADV-1 drive robust rhythmic protein expression of AspRS and GlnRS, respectively. Conclusions: These data support a model whereby coordinate clock control of select aaRSs drives rhythms in uncharged tRNAs, leading to rhythmic CPC-3 activation, and rhythms in translation of specific mRNAs.", "keywords": [ "Circadian clock", "Neurospora cras", "tRNA synthetases", "translation control" ], "content": "Introduction\n\nAminoacyl-tRNA synthetases (aaRSs) play a fundamental role in mRNA translation by catalyzing the attachment of specific amino acids onto their cognate tRNAs. For accuracy, aaRSs employ chemical selectivity and proofreading capabilities (Rubio Gomez and Ibba, 2020, Roux and Topisirovic, 2012). Mounting evidence supports that aaRSs have functions beyond their role in charging tRNAs, including roles in immune signaling, cell cycle, nutrient metabolism and growth, and thus are linked to various human diseases (Pang et al., 2014, Nie et al., 2019, Park et al., 2008). Aminoacylation mechanisms are conserved across all kingdoms of life. While the regulation of aaRS expression in prokaryotes is well-described, less is known about their regulation in eukaryotes (Frugier and Giege, 2003). Several aaRSs were reported to have daily rhythms in abundance at the mRNA and/or protein levels in the filamentous fungus Neurospora crassa (Sancar et al., 2015, Hurley et al., 2018, Castillo et al., 2022), and mammalian cells (Pembroke et al., 2015, Barclay et al., 2012, Hughes et al., 2009, Miller et al., 2007, Vollmers et al., 2012, Eckel-Mahan and Sassone-Corsi, 2009, Geyfman et al., 2012, Yoshitane et al., 2014, Janich et al., 2015). These data suggested that the circadian clock imparts regulation on aars gene expression, which would impact rhythmic protein synthesis and clock-controlled cellular processes.\n\nThe circadian clock is an endogenous timekeeping mechanism that regulates diverse biological processes in many organisms, allowing them to anticipate and prepare for daily environmental cycles, and to organize cellular processes to the right time of day for improved fitness (Dunlap and Loros, 2017). Disruption of the circadian clock has profound effects on human physiology and behavior, and can lead to a wide range of diseases (Bass, 2017, Foster, 2020, Hernandez-Garcia et al., 2020). Depending on the organism and tissue type, the circadian clock regulates daily rhythms in mRNA and protein accumulation for up to 50% of the eukaryotic genome (Hurley et al., 2014, 2018, Zhang et al., 2014, Mauvoisin et al., 2014). Remarkably, most of the proteins that cycle in abundance under the control of the circadian clock are produced from mRNAs that are not clock-controlled (Reddy et al., 2006, Robles et al., 2014, Zhang et al., 2014, Hurley et al., 2018, Castillo et al., 2022). These data suggested a prominent role for clock regulation of post-transcriptional processes, including rhythmic mRNA translation.\n\nThe N. crassa circadian clock is composed of negative elements FREQUENCY (FRQ), FRQ-INTERACTING RNA HELICASE (FRH), and CASEIN KINASE 1 (CK1), and positive elements WHITE COLLAR-1 (WC-1) and WHITE COLLAR-2 (WC-2) (Baker et al., 2012, Dunlap and Loros, 2017). WC-1 and WC-2 heterodimerize to form the White Collar Complex (WCC) which binds to the promoters of frq and downstream clock-controlled genes (ccgs), including 24 transcription factors, to drive their rhythmic transcription (Froehlich et al., 2003, Smith et al., 2010). In addition, the N. crassa clock generates rhythms in the activities of the conserved eukaryotic translation initiation factor 2 (eIF2) and eukaryotic translation elongation factor 2 (e-EF2) (Caster et al., 2016, Karki et al., 2020, Ding et al., 2021). A central mechanism for translational control is the phosphorylation of eIF2α, as even partial phosphorylation is sufficient to inhibit protein synthesis (Baird and Wek, 2012). Furthermore, rhythms in activity of the N. crassa eIF2α kinase CPC-3, a homolog of the well-studied yeast and mammalian eIF2α kinase GCN2, are dependent on rhythmic uncharged tRNAVal, levels. The rhythm in uncharged tRNAVal, levels is driven, at least in part, by rhythms in valyl-tRNA synthetase (ValRS) levels (Karki et al., 2020). However, in addition to ValRS, several other aaRSs were found to be clock-controlled from genomic datasets (Sancar et al., 2015, Hurley et al., 2018, Castillo et al., 2022), suggesting that aaRSs may be coordinately regulated by the clock to control rhythmic translation.\n\nIn this study, we sought to independently validate clock control of two aaRSs and to determine the mechanisms of clock control of aaRS genes. Using aspartyl-tRNA synthetase (AspRS) and glutaminyl-tRNA synthetase (GlnRS) luciferase translational reporters, we confirmed that AspRS and GlnRS protein levels are rhythmic in WT cells with a peak in the subjective night, similar to the peak time of ValRS (Karki et al., 2020), and arrhythmic in clock mutant Δfrq cells. Furthermore, we identified clock-controlled transcription factors that bind to promoters of AspRS and GlnRS to mediate their rhythmic transcription. We found that AspRS::LUC and GlnRS::LUC are arrhythmic in the transcription factor knockouts, Δncu00275 and Δadv-1, respectively, supporting that the clock controls rhythms in AspRS and GlnRS through these transcription factors. Overall, these findings provide a basis for further studies investigating coordinate clock control of aaRSs and their roles in rhythmic mRNA translation.\n\n\nMethods\n\nStrains, key reagents, and oligonucleotide primers used in this study are listed in Table 1. N. crassa wild type 74-OR23-IV (FGSC 4200) was grown in Vogel’s minimal media with 2% glucose (V2G) (Davis and De Serres, 1970). All strains containing the hygromycin phosphotransferase (hph) construct conferring resistance to hygromycin B were maintained on V2G and supplemented with 200 μg/mL of hygromycin B (VWR). Strains containing the bar cassette conferring resistance to Basta were maintained on V2G lacking NH4NO3 and supplemented with 0.5% proline (Sigma-Aldrich) and 200 μg/mL of Basta (LibertyTM, Bayer).\n\nTo assay rhythmic translation of aspartyl tRNA synthetase (AspRS) and glutaminyl tRNA synthetase (GlnRS), an aaRS::LUC translational fusion to luciferase was generated by 3-way fusion polymerase chain reaction (PCR). Primers RSF1 and RSR1 were used to make fragment 1, and primers RSF3 and RSR3 were used to make fragment 3, both using wild type (WT) genomic DNA as template. Primers RSF2 and RSR2 were used to make fragment 2 using pRMP57, a plasmid containing the N. crassa codon-optimized luciferase gene as template DNA (Gooch et al., 2008). The three PCR fragments with overlapping regions were stitched via fusion PCR using primers RSF1 and RSR3, and the resulting PCR product was co-transformed with the plasmid pBP15 containing hph (Beasley, 2006) into WT (FGSC2489). Primers were designed using Serial Cloner version 2.6.1 and the sequences are given in Table 1. Lyophilized PCR oligonucleotide primers were obtained from Integrated DNA Technologies (IDT) and resuspended in 1x Tris-EDTA, pH 8.0 buffer to make 100 μM primer stocks. The PCR reaction mix was as follows: total reaction mix = 50 μl, water = 37.5 μl, 5X High Fidelity (HF) buffer = 5 μl, dNTP mix = 1 μl (10 mM), primers = 2.5 μl each (10 μM), Phusion® DNA polymerase = 0.5 μl (1.0 units/50 μl PCR), and template DNA = 1 μl (50 ng). Phusion® High Fidelity DNA polymerase kit (Cat. No. M0530) and dNTP mix (Cat. No. 4030) were purchased from New England Biolabs (NEB) and Takara, respectively. The following annealing temperatures and extension times were applied: AspRS fragment 1 = 65°C, 1:30 min, fragment 2 = 72oC, 1:30 min, fragment 3 = 59°C, 1:30 min, and fragments 1+2+3 = 59°C, 3 min; GlnRS fragment 1 = 63°C, 1:30 min, fragment 2 = 72°C, 1:30 min, fragment 3 = 64oC, 30 s, and fragments 1+2+3 = 63°C, 3 min. PCR cycling was performed with a C1000 Touch Thermal Cycler (Bio-Rad) using the following program: 30 cycles of denaturation at 98°C for 10 s, annealing at varying temperatures for 30 s (as described above) and extension at 72°C for varying times (as described above).\n\nHygromycin-resistant transformants were screened for luciferase activity and homologous insertion into the aars gene (primers RSF4 and RSR4) using the same PCR conditions described above with annealing temperatures and extension times for AspRS = 62°C, 4 min, and GlnRS = 59°C, 4 min. To generate aaRS::LUC in different mutant strains, aaRS::LUC, WT were crossed with the knockouts on synthetic cross medium supplemented with 0.25% biotin (Westergaard, 1947, Davis, 1970). AspRS::LUC transformants were crossed with Δfrq::bar (DBP1228) to generate AspRS::LUC, WT (DBP3999), AspRS::LUC, Δfrq::bar (DBP4000). GlnRS::LUC transformants were crossed with Δfrq::bar (DBP1228) (Bennett et al., 2013) to generate GlnRS::LUC, WT (DBP3991 mat A and DBP3992 mat a), GlnRS::LUC, Δfrq::bar (DBP3989). DBP3999 was crossed with Δclr-1::hph (DBP981) and Δncu00275 (DBP927) to generate AspRS::LUC, Δclr-1::hph (DBP4137) and AspRS::LUC, Δncu00275::hph (DBP4142), respectively. DBP 3992 was crossed with Δadv-1::hph (DBP917) to generate GlnRS::LUC, Δadv-1::hph (DBP 4137). DBP 3991 was crossed with Δncu00275::hph (DBP927) to generate GlnRS::LUC, Δncu00275::hph (DBP 4211).\n\nTo generate transcriptional fusions to luc, promoter regions of asprs (primers asprsF5 and asprsR5 to generate the 1.8 kb fragment Pasprs), and glnrs (primers glnrsF5 and glnrsR5 to generate the 1.82 kb fragment Pglnrs) were amplified by PCR using the cycling conditions described above with annealing temperatures and extension times for fragments Pasprs and Pglnrs = 68oC, 1:30 min. PCR products were digested with NotI and SpeI (NEB), and cloned into plasmid pRMP57 containing the codon-optimized luciferase gene. The resulting plasmids were linearized by PciI (NEB) digest, co-transformed with hygR pBP15 into WT (FGSC 4200) cells, and hygromycin-resistant transformants were screened for luciferase activity.\n\nTo examine bioluminescence rhythms arising from strains containing luciferase fusions, 1×105 conidia were inoculated into 96 well microtiter plates containing 150 μl of 1X Vogel’s salts, 0.01% glucose, 0.03% arginine, 0.1 M quinic acid, 1.5% agar, and 25 μM firefly luciferin, pH 6. After inoculation of conidia (1×105 conidia), the microtiter plate was incubated at 30°C in LL for 24 h and transferred to DD 25°C to obtain bioluminescence recordings using EnVision Xcite Multilabel Reader, with recordings taken every 90 min over 4–5 days. Raw reads were normalized to the mean to graph the data.\n\nRhythmic data from luciferase assays were fit to a sine wave or a line as previously described (Lamb et al., 2011). Nonlinear regression to fit the rhythmic data to a sine wave (fitting period, phase, and amplitude) and a line (fitting slope and intercept), as well as Akaike’s information criteria tests to compare the fit of each data set to the 2 equations, were carried out using the Prism software package version 9.4.0. The p-values reflect the probability that, for instance, the sine wave fits the data better than a straight line. Error bars in all graphs represent the standard error of the mean (SEM) from independent experiments. Raw and normalized luciferase activity reads were analyzed for period, phase, and amplitude values using BioDare version 2 (Zielinski et al., 2014). Heat maps were generated using the ggplot2 R package for genes with rhythmic RPF counts in WT, and sorted according to increasing peak phase of the oscillation (Wickham, 2016). RPF levels are standardized within each gene (row) (Z-scores).\n\n\nResults\n\nN. crassa circadian ribosome profiling (ribo-seq) data revealed rhythms in ribosome occupancy for 17 of 36 aaRS using the Extended Circadian Harmonic Oscillator (ECHO) rhythmicity detection tool (De Los Santos et al., 2020, Castillo et al., 2022). Genes with an adjusted p-value of < 0.05, and with circadian harmonic, damped, or forced oscillation types were considered rhythmic. A heat map of the phase-sorted fitted ribosome protected footprint (RPF) values obtained using ECHO showed robust rhythmic ribosome occupancy for 17 aaRSs in WT cells, with peak ribosome occupancy primarily during the late subjective day (DD40-44). As expected for circadian clock control, the rhythms were abolished in the clock mutant Δfrq cells (Figure 1A). Circadian rhythms in ValRS protein levels were previously validated using a luciferase (LUC) translational reporter. ValRS::LUC levels peaked in the subjective night (Karki et al., 2020), lagging the observed peak in ribosome occupancy (Figure 1A).\n\nA) Heat maps of the peak phase of aaRS mRNAs with rhythmic ribosome footprint reads (RFP) counts in WT, and arrhythmic RFP counts in Δfrq cells (N = 17) from samples grown in DD and harvested at the indicated times (Hrs). Genes are sorted by the peak phase in WT. Cyt (cytoplasmic), mt (mitochondrial) B-C. Plots show the normalized fitted RFP reads in WT (black line, ECHO p-value ≤ 0.05) and Δfrq (gray line, ECHO p-value > 0.05 for aspartyl tRNA synthetase (AspRS) and p-value = 0.04 for glutaminyl tRNA synthetase (GlnRS), but with a short 16 h period) cells for B) AspRS and C) GlnRS. Luciferase (LUC) activity from D) AspRS::LUC and E) GlnRS::LUC translational fusions in WT (black line) and Δfrq (gray line) cells. The average bioluminescence signal is plotted (AspRS::LUC, mean ± SEM, n=12 and GlnRS::LUC, mean ± SEM, n=24). Raw reads were normalized to the mean to plot the data. AspRS::LUC and GlnRS::LUC in WT cells were rhythmic as indicated by a better fit to a sine wave (dotted black line, p-value < 0.001). AspRS::LUC and GlnRS::LUC in Δfrq were arrhythmic as indicated by a better fit of the data to a line (dotted gray line p-value > 0.05). The bar at the bottom of the heat maps and graphs represents subjective day (gray) and subjective night (black) in this and all subsequent figures. Data from Bell-Pedersen (2022).\n\nIn higher eukaryotes, 9 aaRSs form a multisynthetase complex (MSC) that is proposed to aid translation by providing a channel through which tRNAs can pass to reach bound aaRSs (Hyeon et al., 2019). Interestingly, 5 of the 9 aaRSs in the complex (AspRS, GlnRS, GluRS, LeuRS, and MetRS) are clock-controlled based on our ribosome profiling datasets (Figure 1A), and we focused on validating circadian clock control of AspRS and GlnRS (Figure 1B & C). AspRS and GlnRS luciferase translational reporter fusions were generated (AspRS::LUC and GlnRS::LUC) and assayed for rhythmic luciferase levels from cells grown in constant darkness (DD) over 4 days. Bioluminescence rhythms were observed for both AspRS::LUC and GlnRS::LUC, with peak levels during the early subjective night (e.g. DD 48) and a period of 22.6 ± 0.5 h and 23.5 ± 0.6 h, respectively. Similar to ValRS::LUC, the peak in AspRS::LUC and GlnRS::LUC levels occurred a few hours after the peak in ribosome occupancy (Figure 1B-E). The AspRS::LUC and GlnRS::LUC rhythms were abolished in Δfrq cells, supporting that AspRS and GlnRS protein levels are clock-controlled (Figures 1D & E, Table 2).\n\nIn addition to rhythms in protein levels, several N. crassa aaRS mRNAs were reported in genome-wide studies to be clock-controlled (Hurley et al., 2014, 2018, Sancar et al., 2015, Castillo et al., 2022). Of these aaRSs, asprs and glnrs exhibited rhythms in mRNA levels in WT cells, with mRNA levels peaking in the subjective early evening (DD40-44) (Figure 2A & B). In support of these genomic data, asprs (Pasprs::luc) and glnrs (Pglnrs::luc) promoter luc fusions were rhythmic in DD peaking during the subjective night (Figure 2C & D), with no significant period and phase differences between the mRNA and protein levels (Table 2).\n\nA-B. Plots show the mRNA fragments per kilobase of exon per million mapped fragments (FPKM) levels in WT (black line, ECHO p-value < 0.05) and Δfrq (gray line, ECHO p-value > 0.05) cells for A) asprs and B) glnrs. C-D. Plots show the luciferase activity from C) Pasprs::luc transcriptional (black line) and D) Pglnrs::luc transcriptional (black line) fusions in WT cells grown in DD and recorded every 90 min over 4 d (Hrs DD). The average bioluminescence signal is plotted (mean ± SEM, n=12). Luciferase activities are rhythmic as indicated by a better fit to a sine wave (dotted black line, p-value < 0.0001). Data from Bell-Pedersen (2022).\n\nAlthough the levels fluctuated over time, the rhythms were abolished in Δfrq cells as shown by the ECHO-generated fitted values for normalized mRNA levels by FPKM (fragments per kilobase of exon per million mapped reads) (Figure 2A & B) (Castillo et al., 2022). Together, these data support that rhythmic AspRS and GlnRS protein levels arise, at least in part, from cycling mRNA levels.\n\nTo determine if clock-controlled rhythms in AspRS and GlnRS protein levels require clock-controlled transcription factors and rhythmic transcription, we examined AspRS::LUC and GlnRS::LUC rhythms in cells deleted for transcription factors that are direct targets of the WCC, and bind to downstream ccgs to regulate their rhythmic expression (Smith et al., 2010, Dekhang et al., 2017, Munoz-Guzman et al., 2021). We found, for example, that AspRS::LUC was rhythmic in WT and Δclr-1 (NCU07705) cells with a similar period and phase (Figure 3A, Table 2), but arrhythmic in Δncu00275 cells (Figure 3B). Also, AspRS::LUC levels were lower in Δncu00275 compared to WT cells (Figure 3C), suggesting that NCU00275 directly, or indirectly, activates asprs transcription. GlnRS::LUC was rhythmic in WT and Δncu00275 cells with no significant differences in period or phase between WT and transcription factor knockout cells (Figure 4A and Table 2). Previous ChIP-seq of the clock-controlled transcription factor ADV-1 (NCU07392) showed that ADV-1 binds to the promoter of glnrs (Dekhang et al., 2017). GlnRS::LUC levels in Δadv-1 cells became arrhythmic after 2 days in DD (Figure 4B). Examination of the raw bioluminescence signals showed that GlnRS::LUC levels were higher in Δadv-1 than in WT cells (Figure 4C), suggesting that ADV-1 negatively regulates glnrs transcription. Furthermore, the progressive dampening of GlnRS::LUC levels in Δadv-1 significantly reduced the amplitude of oscillation, leading to arrhythmicity (Figure 4D). Taken together, these data support that specific clock-controlled transcription factors contribute to circadian rhythms in the expression of aaRSs.\n\nLuciferase activity from AspRS::LUC translational fusions in WT (black line) and transcription factor knockouts A) Δclr-1 (blue line) and B) Δncu00275 (blue line). Raw reads were normalized to the mean to plot the data. The average bioluminescence signal is plotted (mean ± SEM, n=12). Luciferase activities are rhythmic as indicated by a better fit to a sine wave (dotted black line, p-value < 0.0001) or arrhythmic as indicated by a better fit of the data to a line (dotted blue line, p-value > 0.0001). C) Raw bioluminescence signals from AspRS::LUC translational fusions in WT (black line) and transcription factor knockout Δncu00275 (blue line).\n\nLuciferase activities from GlnRS::LUC translational fusions in WT (black line) and transcription factor knockouts A) Δncu00275 (blue line) and B) Δadv-1 (blue line). The average bioluminescence signal is plotted (mean±SEM, n=12). Luciferase activities are rhythmic as indicated by a better fit to a sine wave (dotted black line, p-value<0.0001) or arrhythmic as indicated by a better fit of the data to a line (dotted blue line, p-value>0.0001). C) Raw bioluminescence signals from GlnRS::LUC translational fusions in WT (black line) and transcription factor knockout Δadv-1 (blue line). The average bioluminescence signal is plotted (mean±SEM, n=12). D) Mean amplitude (mean±SEM, n=12; **** p-value < 0.0001) of GlnRS::LUC bioluminescence traces in WT (black bar) and Δadv-1 (blue bar) cells. P-values were calculated by an unpaired t-test.\n\nAll underlying data can be found in the Underlying data section (Bell-Pedersen, 2022, Castillo & Bell-Pedersen, 2022).\n\n\nDiscussion\n\naaRSs play a central role in translation and translation fidelity, yet the regulation of aaRS gene expression in eukaryotes is understudied. Genome-wide datasets revealed that several eukaryotic aaRSs are clock-regulated at the level of mRNA and protein (Sancar et al., 2015, Hurley et al., 2018, Castillo et al., 2022, Barclay et al., 2012, Pembroke et al., 2015, Hughes et al., 2009, Miller et al., 2007, Vollmers et al., 2012, Eckel-Mahan and Sassone-Corsi, 2009, Geyfman et al., 2012, Yoshitane et al., 2014, Janich et al., 2015). We previously showed that the levels of ValRS cycle under control of the circadian clock with peak levels during the subjective night (Karki et al., 2020). Here, we validated that asprs and glnrs mRNA and protein levels are also clock-controlled with a similar night-time peak in protein levels. The bulk of rhythmic protein accumulation occurs at night in N. crassa (Hurley et al., 2018) supporting that the night-time peak in RS protein levels serve to coordinately increase protein synthesis at night.\n\nWe observed that AspRS and GlnRS protein rhythms are dependent on the circadian clock through the activities of clock-controlled transcription factors. AspRS is arrhythmic in Δncu00275. NCU00275 is annotated as a hypothetical protein, but its homologs in other fungi suggest that it belongs to a C3HC4-type RING finger proteins involved in transcription, signal transduction, ubiquitination, and recombination (Basenko et al., 2018, Krishna et al., 2003). GlnRS rhythms were less robust in Δadv-1. ADV-1 has a role in connecting the circadian clock and light signaling pathways to developmental processes (Dekhang et al., 2017). Interestingly, AspRS and GlnRS are two aaRSs that were shown in Saccharomyces cerevisiae and humans, respectively, to bind specifically to their own mRNAs, providing another potential layer of regulation to their gene expression (Frugier and Giege, 2003).\n\nCharging tRNAs with the correct amino acid is the first step in translation, and therefore the levels and function of aaRSs are critical to translation fidelity (Yu et al., 2021, Hausmann and Ibba, 2008). Mistakes in translation are generally considered detrimental; however, during stress, mistranslation may be beneficial by increasing the levels of altered proteins that can perform new functions to aid the response (Pan, 2013, Ribas de Pouplana et al., 2014). In eukaryotes, some aaRSs form the MSC (Bandyopadhyay and Deutscher, 1971, Lee et al., 2004, Kerjan et al., 1994), with varying composition dependent on the organism. In S. cerevisiae, the MSC is comprised of MetRS, GluRS and the scaffold protein Arc1 (Galani et al., 2001). The MSC in mammals has 9 aaRSs, including 5 of the 9 aaRSs (AspRS, GlnRS, GluRS, LeuRS, and MetRS) that are rhythmic in ribo-seq data sets, and 3 scaffold proteins, AIMP1-3. The MSC helps the function of its components; for example, the Km for binding of tRNAMet to MetRS in the yeast MSC is about 100-fold lower compared to the Km for binding of tRNAMet to MetRS alone (Simos et al., 1998). MSC components are also involved in cell signaling, stress responses, metabolite sensing, and controlling gene expression by binding to specific RNA and DNA sites, supporting the idea that a key role of the MSC is to support alternative functions of aaRSs (Cui et al., 2021, Pang et al., 2014, Rubio Gomez and Ibba, 2020). Furthermore, aaRSs, either alone or in the MSC complex, participate in a wide variety of processes outside of their classical role in tRNA charging, including transcription regulation, splicing, and metabolism (Rubio Gomez and Ibba, 2020), and abnormal expression, localization, and molecular interactions of aaRSs are associated with a variety of human diseases, including cancer (Zhou et al., 2020). This widespread impact of aaRSs on host biology raises the intriguing idea that daily rhythms in the levels of aaRSs represent a missing factor linking the clock to a wide range of rhythmic biological processes that are critical to health, underscoring the need to better understand the mechanisms underlying their circadian regulation.\n\n\nData availability\n\nGene Expression Omnibus: Ribosome profiling and RNA-seq data used in Figures 1 and 2. Accession number GSE181566; https://identifiers.org/geo:GSE181566 (Bell-Pedersen, 2022).\n\nFigshare: Circadian Clock Control of tRNA synthetases in Neurospora crassa.\n\nhttps://doi.org/10.6084/m9.figshare.c.6209830.v2 (Castillo & Bell-Pedersen, 2022).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank the Fungal Genetics Stock Center for providing strains.\n\n\nReferences\n\nBaird TD, Wek RC: Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism. Adv. Nutr. 2012; 3: 307–321. PubMed Abstract \| Publisher Full Text\n\nBaker CL, Loros JJ, Dunlap JC: The circadian clock of Neurospora crassa. FEMS Microbiol. Rev. 2012; 36: 95–110. PubMed Abstract \| Publisher Full Text\n\nBandyopadhyay AK, Deutscher MP: Complex of aminoacyl-transfer RNA synthetases. J. Mol. Biol. 1971; 60: 113–122. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "159010", "date": "17 Jan 2023", "name": "Ruifeng Cao", "expertise": [ "Reviewer Expertise circadian rhythm", "translational control", "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\nIn the manuscript, Castillo et al. report circadian regulation of aminoacyl-tRNA synthetases (aaRS) in Neurospora crassa. The study started with genome-wide translational profiling around two circadian cycles and found 17 of 26 aaRS exhibited circadian rhythmicity in ribosomal occupancy. Next, they focused on two of the 17 rhythmic aaRS, AspRS and GlnRS. They found that their protein and mRNA levels were regulated by the circadian clock, and by clock-controlled transcription factors, e.g., NCU00275 and ADV-1. They conclude that select aaRS drive rhythms in uncharged tRNAs, which in turn leads to rhythmic CPC-3 activation and mRNA translation. Overall, this is an elegant study that nicely follows up with their previous work, and most results are compelling. The new findings will advance our understanding of translational control mechanisms by aaRS in the circadian clock. I have some moderate to minor comments to help improve the manuscript.\n\nIn Fig.1A, it would be informative and comprehensive if the heatmaps of the 19 aaRS that did not exhibit circadian rhythms in ribosome occupancy are also shown in the figure or in a supplementary figure.\n\nIn Fig.1 legend, RFP should be RPF (ribosome-protected fragments or footprints).\n\nThe Ribo-Seq experiment and data analysis were not described in Methods. What are the numbers of biological replicates for WT and Δfrq groups, respectively? N=17 is for which group? Are those 17 samples pooled or processed separately for Ribo-Seq? What are the sample sizes in Figs.1B, 1C, 2A, and 2B?\n\nIn all figures, the lengths of the bars indicating circadian day and night are slightly shorter than 24h. Are these determined based on the period data in Tab.2?\n\nRelated to Figs. 2C and D, do Pasprs::luc and Pglnrs::luc exhibit rhythms in Δfrq cells? It would be interesting to see these data since the data in A and B show weak (although not statistically significant) rhythms in asprs and glnrs mRNA levels in Δfrq cells.\n\nFor audiences who are not familiar with the Neurospora clock, it would be helpful if more background information can be given regarding why the mutants Δclr-1 and Δncu00275 were selected in the current study. Please also discuss potential mechanisms for why AspRS::LUC was repressed and arrhythmic in Δncu00275 cells but the rhythms of GlnRS::LUC were barely affected. Are there any data indicating ncu00275 directly regulates asprs transcription?\n\nIs the work 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": "9955", "date": "29 Nov 2023", "name": "Deborah Bell-Pedersen", "role": "Author Response", "response": "We thank the reviewers for their comments and suggestions. We have made all of the recommended changes in the manuscript, including new experiments showing that the clock controls the levels of asprs and glnrs RNA levels, as described below under each point in bold. In Fig.1A, it would be informative and comprehensive if the heatmaps of the 19 aaRS that did not exhibit circadian rhythms in ribosome occupancy are also shown in the figure or in a supplementary figure. We now include this data in Figure 1A. In Fig.1 legend, RFP should be RPF (ribosome-protected fragments or footprints). Corrected, thank you for catching this. The Ribo-Seq experiment and data analysis were not described in Methods. What are the numbers of biological replicates for WT and Δfrq groups, respectively? N=17 is for which group? Are those 17 samples pooled or processed separately for Ribo-Seq? What are the sample sizes in Figs.1B, 1C, 2A, and 2B? The Ribo-seq data were previously published and we reference the paper, but now also provide a very brief description of the datasets in the Materials and Methods. In the legend to Figure 1A, N=17 was to indicate that 17 tRNA synthetases were rhythmic. This was confusing and now removed from the figure legend. We include that the Ribo-seq datasets were n=2 in the legend. In all figures, the lengths of the bars indicating circadian day and night are slightly shorter than 24h. Are these determined based on the period data in Tab.2? Yes, the day night bars reflect subjective day and night based on the free running period in Table 2. Related to Figs. 2C and D, do Pasprs::luc and Pglnrs::luc exhibit rhythms in Δfrq cells? It would be interesting to see these data since the data in A and B show weak (although not statistically significant) rhythms in asprs and glnrs mRNA levels in Δfrq cells. We now include this data in Figure 2D and E, showing that both Pasprs::luc and Pglnrs::luc are arrhythmic in Δfrq cells. For audiences who are not familiar with the Neurospora clock, it would be helpful if more background information can be given regarding why the mutants Δclr-1 and Δncu00275 were selected in the current study. Please also discuss potential mechanisms for why AspRS::LUC was repressed and arrhythmic in Δncu00275 cells but the rhythms of GlnRS::LUC were barely affected. Are there any data indicating ncu00275 directly regulates asprs transcription? We now include the background on why these particular TFs were chosen for study and discuss that although the RS’s peak in the same phase, this is regulated by different clock-controlled transcription factors." } ] }, { "id": "160525", "date": "14 Mar 2023", "name": "Luis F. Larrondo", "expertise": [ "Reviewer Expertise Circadian rhythms", "Neurospora genetics and gene expression" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work by Castillo et al., provides new information on how the circadian clocks impact cellular proteostasis, particularly how several tRNA synthases are rhythmically expressed. The observations contained in the MS provide a mechanistic perspective on the prior events leading to rhythmic translation via eIF2a.\nOverall the MS is nicely written and easy to follow. There are some minor points that could by further clarified.\n“While the regulation of aaRS expression in prokaryotes is well-described, less is known about their regulation in eukaryotes (Frugier and Giege, 2003)”.\nSince this is a 20-year old ref, please provide additional references that exemplify that up to this date there is a knowledge gap in eukaryotes.\n\nTable 2 legend fails to mention that it also compiles data extracted from Fig. 4.\n\nIn Fig 2. It appears that both tRNA synthase genes show some residual rhythm in the delta frq strain. Moreover, when one goes to the heatmap on Fig1, one may have a similar “impression”. Could the authors further discuss this and, particularly, comment if the rhythmicity indexes obtained with the software yielded borderline rhythmicity?\n\n“We found, for example, that AspRS::LUC was rhythmic in WT and Δclr-1 (NCU07705) cells”.\nIt is unclear why the authors decided to examine these two particular KOs (NCU07705 and NCU00275) in AspRS expression. Was the choice guided by bioinformatic analyses or literature curation? The same applies to the GlnRS.\n\n“Furthermore, the progressive dampening of GlnRS::LUC levels in Δadv-1 significantly reduced the amplitude of oscillation, leading to arrhythmicity (Figure 4D).”\nWhile I agree that the absence of adv-1 has an impact on GlnRS expression, I am not so convinced that the end result is arrhythmic gene expression.\nEye-examination of Fig 4c reveals a distinct oscillatory pattern. I would even predict that if the rhythmicity analysis is limited to the first 72 or 96 h, the software will confirm circadian rhythms. Moreover, if one were to cross such strain with a frq7 allele, my prediction is that the expression of GlnRS in the absence of adv-1 will look indistinctively circadian (particularly aided by the overall higher amplitude of rhythms in frq7) and with a longer period.\n\n“ GlnRS rhythms were less robust in Δadv-1”\nIn the discussion, the authors are less categoric about the effect of adv-1 and don’t talk about arrhythmicity, while in table 2(and Fig 4) they use the latter concept.\n\nIs the work 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": "9956", "date": "29 Nov 2023", "name": "Deborah Bell-Pedersen", "role": "Author Response", "response": "We thank the reviewers for their comments and suggestions. We have made all of the recommended changes in the manuscript, including new experiments showing that the clock controls the levels of asprs and glnrs RNA levels, as described below under each point in bold. “While the regulation of aaRS expression in prokaryotes is well-described, less is known about their regulation in eukaryotes (Frugier and Giege, 2003)”. Since this is a 20-year old ref, please provide additional references that exemplify that up to this date there is a knowledge gap in eukaryotes. We included a more recent study demonstrating coordinate regulation of cytoplasmic aaRS gene transcription by amino acid starvation (Shan et al, 2016). Table 2 legend fails to mention that it also compiles data extracted from Fig. 4. Thank you, this was corrected. In Fig 2. It appears that both tRNA synthase genes show some residual rhythm in the delta frqstrain. Moreover, when one goes to the heatmap on Fig1, one may have a similar “impression”. Could the authors further discuss this and, particularly, comment if the rhythmicity indexes obtained with the software yielded borderline rhythmicity? We now include data in Figure 2D and E, showing that both Pasprs::luc and Pglnrs::luc are arrhythmic in Δfrq cells. “We found, for example, that AspRS::LUC was rhythmic in WT and Δclr-1 (NCU07705) cells”. It is unclear why the authors decided to examine these two particular KOs (NCU07705 and NCU00275) in AspRS expression. Was the choice guided by bioinformatic analyses or literature curation? The same applies to the GlnRS. We now include the background on why these particular TFs were chosen for study “Furthermore, the progressive dampening of GlnRS::LUC levels in Δadv-1 significantly reduced the amplitude of oscillation, leading to arrhythmicity (Figure 4D).” While I agree that the absence of adv-1 has an impact on GlnRS expression, I am not so convinced that the end result is arrhythmic gene expression. Eye-examination of Fig 4c reveals a distinct oscillatory pattern. I would even predict that if the rhythmicity analysis is limited to the first 72 or 96 h, the software will confirm circadian rhythms. Moreover, if one were to cross such strain with a frq7 allele, my prediction is that the expression of GlnRS in the absence of adv-1 will look indistinctively circadian (particularly aided by the overall higher amplitude of rhythms in frq7) and with a longer period. “ GlnRS rhythms were less robust in Δadv-1” In the discussion, the authors are less categoric about the effect of adv-1 and don’t talk about arrhythmicity, while in table 2(and Fig 4) they use the latter concept. We reanalyzed the data in Figure 4C, as suggested by the reviewer, from DD12 to DD96, and found that the data fit to a sine wave with a period of 22.5 h, but with a low amplitude compared to WT. This information is now included in Table 2 and in the text." } ] } ]	1	https://f1000research.com/articles/11-1556
https://f1000research.com/articles/12-999/v1	17 Aug 23	{ "type": "Research Article", "title": "Composition and hepatoprotective effect of geopropolis of Melipona subnitida", "authors": [ "Kaliane Alessandra Rodrigues de Paiva", "Antonio Salatino", "Giuseppina Negri", "Carmen Eusebia Palacios Jara", "Hélio Noberto de Araújo Júnior", "Kizzy Millenn de Freitas Mendonça Costa", "Geysa Almeida Viana", "Natanael Silva Félix", "Mariana Aquino Carvalho", "Hilgarde Ferreira Pessoa", "Tiago da Silva Teófilo", "Jael Soares Batista", "Kaliane Alessandra Rodrigues de Paiva", "Antonio Salatino", "Giuseppina Negri", "Carmen Eusebia Palacios Jara", "Hélio Noberto de Araújo Júnior", "Kizzy Millenn de Freitas Mendonça Costa", "Geysa Almeida Viana", "Mariana Aquino Carvalho", "Hilgarde Ferreira Pessoa", "Tiago da Silva Teófilo", "Jael Soares Batista" ], "abstract": "Background: Geopropolis produced by bees of the subfamily Meliponinae (Hymenoptera, Apidae) is used in Brazilian popular medicine for the treatment of different diseases. The aim of the present work was to evaluate the hepatoprotective potential of the hydroethanolic extract from geopropolis (HPE) produced by Melipona subnitida Ducke. Additionally, the chemical composition of HPE and antioxidant activity were also evaluated. Methods: The hepatoprotective potential of HPE was evaluated by serum biochemical profile and macroscopic and histological examination of the liver of 24 Wistar rats (Rattus norvegicus), distributed in four experimental groups: G1 (negative control), G2 (HPE), G3 (HPE plus acetaminophen), G4 (acetaminophen - positive control). The HPLC-DADESI-MS/MS method was used to evaluate the chemical composition of HPE and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used to measure the antioxidant activity of geopropolis. Results: While HPE alone (G2) caused no liver injury, the livers of G4 exhibited color and histological alterations, extensive hemorrhagic suffusions, friable consistence, yellowish-white multifocal areas, degeneration and diffuse hepatocyte necrosis, in addition to altered levels of urea and enzymes TGO (glutamic-oxaloacetic transaminase) and TGP (glutamic pyruvic transaminase). HPE was shown to exert hepatoprotective effect in G3 animals, only mild hemorrhage and focal areas of vacuolar degeneration and hepatocyte necrosis having been observed. The geopropolis analyzed contains a high diversity of phenolic compounds, including chalcones, flavones and flavonols. high antioxidant activity was determined (IC50=48 μg/mL). Conclusions: The hydroethanolic extract of the geopropolis produced by M. subnitida has a high diversity of phenolic compounds and a high antioxidant activity. It has hepatoprotective action, as evidenced by a reduction in the levels of enzymaraes and the severity of the macroscopic and histological changes in livers of Wistar rats subjected to acetaminophen induced liver changes.", "keywords": [ "Antioxidant activity", "Flavonoids", "Geopropolis", "Hepatoprotection", "Stingless bees", "Ecology and conservation", "Biochemistry and chemical ecology", "pollination" ], "content": "Introduction\n\nTreatment of liver diseases is presently one of the major medical challenges, and the development of alternative therapies is of paramount importance, with emphasis on the investigation of the potential liver protection from natural products (Cogliati et al., 2016). Propolis has great relevance in Brazilian popular medicine for the treatment of various diseases. The interest of the scientific community is growing and a wide variety of scientific experiments are in course, providing evidence that propolis contains bioactive compounds, such as phenolic compounds, which are known to exert antioxidant activity and thus protection against oxidative stress, besides anti-inflammatory, antineoplastic, immunomodulatory, and wound-healing properties (Silva et al., 2014; Bonamigo et al., 2017).\n\nMelipona subnitida Ducke (“jandaíra”; Meliponinae, Apidae) is a stingless bee endemic from the northeast Brazil, where it is important for the pollination of species of the caatinga, an exclusive dry forest ecosystem of Brazil. Melipona subnitida geopropolis has bioactive substances with antioxidant and anti-inflammatory activity (Souza et al., 2018). It is hypothesized that it may also prevent or minimize damages to liver cells subjected to hepatotoxic substances. Paracetamol, also known as acetaminophen, is widely used as an analgesic and antipyretic in Brazil. Prolonged use or even the ingestion of high doses of this drug may cause hepatotoxicity in humans and animals, due to increased lipid peroxidation in the liver, causing membrane disintegration and hepatocellular necrosis (Brayner et al., 2018).\n\nAcetaminophen intoxication is a useful experimental model capable of causing severe liver damage after acute administration, resulting in severe histological lesions and changes in the serum biochemical profile (Junapudi et al., 2018). The present study aimed to evaluate the effectiveness of geopropolis produced by M. subnitida as a hepatoprotector and preventive of liver damage experimentally induced in Wistar rats by acetaminophen.\n\n\nMethods\n\nThe experimental protocol was submitted to the Animal Ethics Committee (CEUA) of the University of the State of Rio Grande do Norte (UERN), under protocol number 006/15. We declare that all phytosanitary and environmental efforts were taken, aiming at the welfare of the animals involved in the research. We also emphasize that no effort was spared so that the animals were admitted for euthanasia in the most humane way possible, according to the National Council for the Control of Animal Experimentation (2015) Euthanasia Practice Guideline.\n\nGeopropolis produced by stingless bees M. subnitida Ducke was collected from apiaries in the city of Mossoró, Rio Grande do Norte, located at 37°20′39″ West and 05°11′15″ South. The climate of the region is warm semi-arid tropical type, with annual average temperature 27°C, average air humidity 50% and annual average rainfall 500 mm.\n\nPortions of geopropolis were collected from several hives and combined to make up a mass of 100 g. The pooled geopropolis was powdered with a blender and transferred to a flat bottom balloon. The extraction was performed by adding 300 mL of 80% ethanol and leaving the flask standing at room temperature in the dark, for 10 days (GARCIA et al., 2004). The extract obtained was then concentrated at 40°C under reduced pressure. The concentrated extract was dried in a ventilated oven at 50°C.\n\nThe dried hydroethanol geopropolis extract (HPE) was diluted in methanol HPLC-degree to a concentration of 1 mg/mL, then filtered through a 0.45 μm filter and an aliquot of 10 μL of the solution injected into a HP 1260 (Agilent Technologies) HPLC chromatograph equipped with diode array detector (DAD) using a Zorbax Eclipse Plus C18 column (4.6 × 150 mm, 3.5 μm) at 40°C. The solvents used were 0.1% acetic acid (A) and MeOH (B). The following gradient was used, based on the concentration the solvent B: 0-10 min, 10-20%; 10-20 min, 20-40%; 20-30 min, 40-50%; 30-38 min, 50-60%; 38-48 min, 60-65%; 48-55 min, 65-75%. The flow was set at 1,0 mL/min and the column temperature at 40°C. The DAD detector was adjusted for detection at 210 and 400 nm.\n\nThe constituents of HPE were characterized by the HPLC-DAD-ESI-MS/MS technique, according to Ferreira et al. (2017a). A Shimadzu DADSPD-M10AVP chromatograph was used, equipped with a degasser, two LC-20AC pumps, CTO-20A furnace column, SIL 20AC auto-injector and SPD-20A diode array detector, adjusted to 210 and 400 nm. The chromatograph was coupled to an Esquire 3000 Plus Bruker Daltonics mass spectrometer, equipped with a quadruple ion capture mass analyzer and hardware components controlled by the CBM-20A software. The analyses were performed with a Phenomenex Gemini C-18 reverse phase column (4.6 × 150 mm, 3.5 μm), protected by a guard column. The same gradient program, solvents and flow rate were applied, as described above.\n\nThe full mass acquisition was obtained by electrospray ionization in the positive and negative modes in the m/z interval 100-1200. Helium was used as collision and nitrogen as nebulizing gas, with assistance of a coaxial nitrogen coating gas, at a pressure of 27 psi. The desolvation was assisted using a counter-current nitrogen flow fixed at 7.0 L min-1 flow and capillary temperature of 320°C. Data dependent MS/MS events were performed on the most intense ions detected at full MS scan. The maximum time of ion capture accumulation and the MS numbers to obtain the mean MS spectra were set at 30 and 3 MS, respectively.\n\nTo evaluate the HPE antioxidant activity, the in vitro photocolorimetric method of the free radical DPPH (2,2-diphenyl-1-picryl-hydrazil) was used. A set of tubes containing ethanol solutions of HPE at concentrations: 100, 50, 40, 20, 5, and 2 ppm, and free radical DPPH at 220 μg/mL. After 20 min in the dark, absorbances were read at 518 nm, using a Shimadzu UV 1601 UV-vis spectrophotometer. All readings were performed in triplicate, and the percentages of antioxidant activity were calculated, using the formula: %AA = 100 - [(Aa - Ab) × 100]/Aa, where: %AA = percentage of antioxidant activity; Aa = absorbance of the DPPH solution; Ab = absorbance of the solution containing HPE plus DPPH (MENSOR et al., 2001).\n\nThe research was carried out in partnership with the State University of Rio Grande do Norte (UERN), developed in the Central Animal Facility, using 24 rodents of the species Rattus norvegicus Berkenhout, 1769, Wistar strain, from the institution’s own bioterium, the animals chosen were healthy, with 60 days of age and weighing approximately 200 g. The animals were divided into 04 groups and conditioned in boxes (06 animals per box) made of polycarbonate, lined with wood shavings and kept in a room with a 12 hour light cycle, controlled aeration, exhaustion and acclimatization (23°C), receiving balanced feed (23% crude protein, 4% total lipids, 5% fiber and 12% minerals) and water ad libitum.\n\nThe choice of sample size was made using as a basis the work of Tzankova et al. (2019), who also worked on hepatoprotective activity in this same animal model.\n\nThe animals were randomly distributed in four experimental groups with six animals each. In order to avoid potential confounding factors in the results, animals of the same age and weight range were used. In addition, raffles were carried out to compose the experimental groups, randomizing the animals to one of the intervention groups or the control group. GI was treated neither with HPE nor acetaminophen (negative control); G2 received 50 mg/kg HPE; G3 received 50 mg/kg HPE and 600 mg/kg acetaminophen; G4 received 600 mg/kg acetaminophen (control).\n\nThe control and identification of the animals was done by separating the groups into boxes, identifying them by numbering, so that each one contained a different group. Aiming at daily handling, other boxes were used to separate the animals from each group, ensuring safety in administering the substances to the animals, avoiding repetition. This control was under the supervision of the researchers responsible for the study.\n\nThe administrations of water, HPE, and acetaminophen occurred by gavage once a day on the laboratory premises. The methodology for the application of the acetaminophen and extract was based on Said (2001). For eight consecutive days, drinking water and HPE were administered, and on the eighth day acetaminophen was administered one hour after drinking water and HPE administration. The animals were divided into 04 groups consisting of 06 animals: G1 (Negative Control): drinking water; G2: 50 mg/kg of geopropolis hydroethanolic extract; G3: 50 mg/kg of geopropolis hydroethanolic extract, together with the last administration (1 hour after), received a dose of 600 mg/kg of acetaminophen (200 mg/mL, Paracetamol, EMS S/A, Brazil); G4: (Positive control): drinking water, next to the last administration (1 hour after), they received a 600 mg/kg dose of acetaminophen (200 mg/mL, Paracetamol, EMS S/A, Brazil). For administration of the hydroethanolic extracts of M. subnitida D., a pool was made with the six extract samples at a concentration of 45 mg/mL.\n\nThe animals were weighed on the first and last day of administration and their physical characteristics were monitored daily.\n\nThe animals were anesthetized with a dose of Ketamine (10 mg/kg, i.p.) and Xylazine (10 mg/kg, i.p.) 48 hours after the last administration of HPE and acetaminophen. Once the anesthetic procedure was completed, celiotomy was performed on the alba line to access the hepatic vein, 3 mL of blood from each animal was collected and transferred to tubes without ethylenediaminetetraacetic acid (EDTA) addition.\n\nThe material was packed in thermal boxes and sent for biochemical analysis, which was used to determine blood markers of liver damage: alanine aminotransferase (TGP), aspartato aminotransferase (TGO), albumin (ALB), urea (URE), total protein (TP), glucose (GLC) and total triglycerides (TRIG). The biochemical analyzes were performed in the clinical pathology laboratory of the Federal Rural University of the Semi-Arid - UFERSA, using commercial reagents (VIDA Bioteconologia®) and spectrophotometric analyses.\n\nThe animals were induced into deep anesthesia using ketamine (10 mg/kg) and xylazine (10 mg/kg) intraperitoneally, and after that they were euthanized by exsanguination according to the National Council for the Control of Animal Experimentation (2015) Euthanasia Practice Guideline. Upon death, the animals were submitted to necropsy using the technique proposed by Vasconcelos (1996). The livers were submitted to macroscopic examination, and observation of possible changes in size, color, consistency, contours, presence of hemorrhages, and necrosis.\n\nThe livers were washed with 0.9% NaCl solution, fixed in 10% formalin buffered solution and routinely processed for histology after paraffin inclusion and attainment of cuts 5 μm thick and hematoxylin-eosin (HE) staining. The histological sections were examined for the presence of vacuolar degeneration of the cells that compose the hepatic parenchyma, infiltration of inflammatory cells and hepatocyte necrosis. The macroscopic and microscopic analyses were performed by two experienced and independent evaluators.\n\nThe data obtained from serum biochemical parameters (TGO, TGP, albumin, urea, glucose, triglycerides and TP) were expressed as mean values ± standard deviation, as well as minimum and maximum values using SAS software statistics version 8.0. The influence of geopropolis concentrations (dry matter) on antioxidant activity, total phenols and flavonoids was determined by Spearman correlation. After analysis of parametric assumptions, statistical differences between the different experimental groups were obtained by analysis of variance (One-Way ANOVA) followed by Tukey’s test. The percentage data had an arc-sine transformation √x. When the Gaussian distribution was stopped, the values were compared by the Kruskal-Wallis test. Values of p < 0.05 were considered significant.\n\n\nResults\n\nHPLC-DAD-ESI-MS/MS analysis revealed that HTP has a great diversity of constituents, most of which belong to the class of phenolic compounds. such as chalcones (Isoliquiritigenin, 4,5′,6′-Trihydroxy-2′,4′-dimethoxy chalcone, 4,5′,6′-Trihydroxy-2′,4′-dimethoxy chalcone, 2′,4′-Dihydroxy-4-methoxy chalcone, 4′-Methoxy-4,2′,6′-trihydroxy chalcone, Mandelic acid acetyl-rhamnoside), flavones (Apigenin, Chrysoeriol, 5,3′,4′-Trihydroxy-6,7-dimethoxy flavone, 5,4′-Dihydroxy-6,7-dimethoxy flavone, Naringenin, 3-O-Methyl galangin), flavonols (Quercetin, Kaempferide, Rhamnetin, Myricetin-3,7,3′-trimethyl ether, Quercetin-dimethyl ether, Kaempferol dimethyl ether, Kaempferol dimethyl ether, Quercetin-dimethyl ether, Quercetin-3,7,3-trimethyl ether, Kaempferol methyl ether coumaroyl-arabinoside. The following non-flavonoid phenol compounds have also been identified (4-Demethyldeoxypodo-phyllotoxin-4-O-glucoside, 4-Methoxy-3-prenyl benzoic acid acetyl-glucoside, 4-O-Vinyl-3,5-diprenyl coumaric acid, 4-O-Ethoxy-3,5-diprenyl coumaric acid, 4-Methoxy-3-prenyl benzoic acid acetyl-rhamnoside).\n\nBiochemical analysis showed that the values of the enzymes TGO and TGP and urea increased significantly in groups G3 and G4 compared to groups G1 and G2. However, the levels of these biochemical parameters were lower in G3 compared to G4 (Table 1).\n\nHPE: hidroethanol geopropolis extract from Melipona subnitida Ducke.\n\n1 TGO: Aspartate aminotransferase; TGP: Alanin aminotransferase; TP: Total proteins.\n\n2 Means followed by the same letter on lines are not significantly different (p < 0.05).\n\n3 Based on Melo et al. (2010).\n\nThe rats in groups G1 and G2 had morphologically normal livers with no anatomical changes (Figure 1A and B). In the livers of the rats in the G3 group, focal pale areas and small spots of hemorrhage in the capsule were observed (Figure 1C). The rats in the G4 group had diffusely pale livers with marked hemorrhage in the capsule and a friable consistency (Figure 1D).\n\nNormal morphologic aspect is noted on livers A (G1) and B (G2); C (G3) has pale brown color and discrete hemorrhage; D (G4) is markedly pale and hemorrhagic suffusions are seen on the capsule.\n\nHistopathological evaluation showed that the liver tissue of G1 and G2 showed no changes and had normal histopathological aspects (Figure 2A and B). In G3, the histopathological examination revealed foci of swelling, vacuolization (hydropic degeneration) and necrotic hepatocytes, as well as mild infiltration of neutrophilic and monocytic inflammatory cells. Moderate dissociation of hepatocyte strands and sinusoidal congestion was also found (Figure 2C). Histological changes were more intense in the livers of G4, with loss of the architectural pattern of the organ, evidenced by diffuse degeneration and necrosis of hepatocytes, several foci of hemorrhage, marked dissociation of hepatocyte cords, and intense infiltration of inflammatory cells such as neutrophils, monocytes, and macrophages (Figure 2D).\n\nNormal histological architecture is seen on A (G1) and B (G2); on C (G3) swollen hepatocytes are visible; degeneration and diffuse hepatocyte necrosis is noticed on D (G4).\n\n\nDiscussion\n\nThe composition of HPE is similar to the composition of the geopropolis of Scaptotrigona aff. depillis (Ferreira et al., 2017b), in that both contain methoxylated flavonols and chalcones as predominant constituents. The present study represents the first report of the presence in geopropolis produced by the bee Melipona subnitida Ducke of constituted with lignan 4-desoxypodophyllotoxin 4-O-glucopyranoside. The literature reports that this chemical constituent presents antineoplastic activity (Zilla et al., 2014).\n\nThe antioxidant capacity of HPE, with IC50 48.0 μg/mL, represents high protection against oxidative stress. According to Melo et al. (2010), IC50 values below 50 μg/mL are considered highly active. Geopropolis of other species of Melipona also have shown high antioxidant activity; for example samples of geopropolis of Melipona fasciculata from the State of Maranhão (northeast Brazil) exhibited IC50 values in the range 2.24 μg/mL - 44.24 μg/mL (Silva et al., 2016).\n\nThe evaluation of serum biochemical parameters, as well as the macroscopic and histological aspects of the livers of Wistar rats showed that the HPE analyzed exerted a hepatoprotective effect by inhibiting hepatotoxicity induced by acetaminophen, a drug known to cause hepatocellular necrosis and elevation of serum levels of TGO and TGP enzymes (Lopes and Matheus, 2012). In the livers of G3 animals, it was possible to observe a reduction in the intensity of degenerative and necrotic lesions occurring in hepatocytes, as well as decreases in the mean values of serum levels of TGO, TGP and urea enzymes. The liver enzymes TGP and TGO can be measured in the blood and reflect the status of liver function. In cases of hepatocyte lesions, increases in their serum levels occur (Jesus et al., 2014).\n\nThe results of this investigation corroborate the previously reported results. Said (2001) demonstrated the hepatoprotective effect of the hydroethanol extract of Apis mellifera propolis. In this study, the author used acetaminophen to induce liver lesions in Swiss rats. The author found that the group of rats that received the extract at doses of 50-400 mg/kg showed no lesions in the hepatocytes and the liver tissue showed histological pattern within normality.\n\nThe macroscopic and histological evaluation of the livers of G2 animals shows that the oral administration of HPE does not exert hepatotoxicity, indicating that the product is safe at 50 mg/kg. A study using isoniazid and rifampicin (100 mg/kg, i.p.) for induction of hepatic lesion in albino rats revealed no toxicity after five days of treatment with ethanol extract of propolis of A. mellifera (200-440 mg/kg, orally). The experimental group of rats that received the extract showed no serum biochemical changes, as well as no histological changes on the liver (Wali et al., 2015).\n\nAmong the constituents of propolis that have proven medicinal effects are flavonols, such as quercetin and kaempferol, which were detected as constituents of the propolis evaluated in the present study. These compounds, in joint action, demonstrate high anti-inflammatory potential by modulating the action of cellular components involved in the mechanism of inflammation (Vaher and Koel, 2003). Chalcones is another chemical constituent found in HPE that should also be mentioned for its biological activity. Chalcones have been shown to exert a variety of cytoprotective and modulatory functions as well as beneficial roles in inflammatory processes (Kontogiorgis et al., 2008).\n\nAccording to Lahsasni et al. (2014) chalcones are among the promising substances for use as pharmaceuticals. Kontogiorgis et al. (2008) demonstrated that chalcones exert a variety of cytoprotective and modulating functions as well as inflammatory activity. The results obtained in the present study suggest that the observed hepatoprotective and anti-inflammatory effects are associated with HPE flavonoids.\n\n\nConclusions\n\nAfter analysis it was observed that the hydroethanolic extract of geopropolis produced by M. subnitida showed hepatoprotective activity in the liver of Wistar rats, indicated by a decrease in the severity of acetaminophen-induced macroscopic and microscopic tissue alterations, in addition to a decrease in the level of TGO, TGP and urea enzymes. Additionally, HPE showed a great variety of phenolic compounds including chalcones, flavones and flavonols besides excellent antioxidant activity. In this sense, in order to improve the methods of use of geopropolis in the treatment of liver diseases caused by different agents, further studies on this subject should be conducted to evaluate the safety and viability of the methods used, so that later the geopropolis produced by M. subnitida can be used in the treatment of humans and other animal species, after undergoing clinical trials.", "appendix": "Data availability\n\nFigshare: Seric biochemical parameters, https://doi.org/10.6084/m9.figshare.23596476.v1 (Paiva et al., 2023a).\n\nThe project contains the following underlying data:\n\n• Seric biochemical parameters.docx [Seric biochemical parameters (means ± SD) obtained with Wistar rats (Rattus norvegicus Berkenhout, 1769).]\n\nFigshare: Photomicrographs of livers of Wistar rats, https://doi.org/10.6084/m9.figshare.23600130.v1 (Félix et al., 2023a)\n\nThe project contains the following underlying data:\n\n• Photomicrographs of livers.docx [Photomicrographs of livers of Wistar rats (Rattus nervegicus Berkehout) stained with hematoxylin and eosin.].\n\nFigshare: Macroscopic aspect of livers of Wistar rats, https://doi.org/10.6084/m9.figshare.23600103.v1 (Félix et al., 2023b).\n\nThe project contains the following underlying data:\n\n• Macroscopic aspect of livers.docx [Macroscopic aspect of livers of Wistar rats (Rattus norvegicus Berkenhout, 1769), obtained from groups G1-G4.]\n\nRepository: ARRIVE checklist for ‘Composition and hepatoprotective effect of geopropolis of Melipona subnitida’, https://doi.org/10.6084/m9.figshare.23620488.v1. (Paiva et al., 2023b).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nBonamigo T, Campos JF, Alfredo MT, et al.: Antioxidant, cytotoxic, and toxic activities of propolis from two native bees in Brazil: Scaptotrigona depilis and Melipona quadrifasciata anthidioides. Oxidative Med. Cell. Longev. 2017; 2017(1): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n: Baixa o Capítulo “Introdução Geral” do Guia Brasileiro de Produção, Manutenção ou Utilização de Animais para Atividades de Ensino ou Pesquisa Científica do Conselho Nacional de Controle e Experimentação Animal - CONCEA. Brasília, DF: Diário Oficial da União; 2015.\n\nBrayner NF, Silva AA, Almeida FR: O risco do uso irracional do paracetamol na população brasileira e seus efeitos na hemostasia. Revista Científica da FASETE. 2018; 12(16): 138–153.\n\nCogliati B, Yanguas SC, Silva TC, et al.: Connexin32 deficiency exacerbates carbon tetrachloride-induced hepatocellular injury and liver fibrosis in mice. Toxicol. Mech. Methods. 2016; 26(18): 362–370. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFélix NS, Paiva KAR, Salatino A, et al.: Photomicrographs of livers of Wistar rats. figshare. Figure. 2023a. Publisher Full Text\n\nFélix NS, Paiva KAR, Salatino A, et al.: Macroscopic aspect of livers of Wistar rats. figshare. Figure. 2023b. Publisher Full Text\n\nFerreira JM, Fernandes-Silva CC, Salatino A, et al.: New propolis type from north-east Brazil: chemical composition, antioxidant activity and botanical origin. J. Sci. Food Agric. 2017a; 97(11): 3552–3558. PubMed Abstract \| Publisher Full Text\n\nFerreira JM, Fernandes-Silva CC, Salatino A, et al.: Antioxidant activity of a geopropolis from northeast Brazil: Chemical characterization and likely botanical origin. Evid. Based Complement. Alternat. Med. 2017b; 2017(1): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcia RC, Sá MEP, Langoni H, et al.: Efeito do extrato alcoólico de própolis sobre a Pasteurella multocida “in vitro”. Acta Sci. Anim. Sci. 2004; 26(1): 69–77. Publisher Full Text\n\nJesus GC, Sousa HHBA, Barcelos RSS: Principais patologias e biomarcadores das alterações hepáticas. Estudos. 2014; 41(3): 525–537. Publisher Full Text\n\nJunapudi S, Janapati YK, Bramhachari PV: Hepatoprotective activity of Talinumportulacifolium forsk, extract against paracetamol induced hepatic damage in rats. Jordan J. Pharm. Sci. 2018; 11(3): 171–179.\n\nKontogiorgis M, Mantzanidou M, Hadjipavlou-Litina D: Chalcones and their potential role in inflammation. Mini Rev. Med. Chem. 2008; 8(1): 1224–1242. Publisher Full Text\n\nLahsasni SA, Korbi A, Hamad F, et al.: Synthesis, characterization and evaluation of antioxidant activities of some novel chalcones analogues. Chem. Cent. J. 2014; 8(1): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLopes J, Matheus ME: Risco de hepatotoxicidade do Paracetamol (acetaminofem). Rev. Bras. Farm. 2012; 93(4): 411–414.\n\nMelo JG, Araújo TAS, Castro VTNA, et al.: Antiproliferative activity, antioxidant capacity and tannin content in plants of semi-arid northeastern Brazil. Molecules. 2010; 15(12): 8534–8542. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMensor LL, Menezes FS, Leitão GG, et al.: Screnning of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother. Res. 2001; 15(2): 127–130. PubMed Abstract \| Publisher Full Text\n\nPaiva KAR, Salatino A, Negri G, et al.: Seric biochemical parameters. Dataset. figshare. 2023a. Publisher Full Text\n\nPaiva KAR, Salatino A, Negri G, et al.: The ARRIVE guidelines 2.0: author checklist. Dataset. figshare. 2023b. Publisher Full Text\n\nSaid RA: Atividade hepatoprotetora de diferentes concentrações dos extratos aquoso e etanólico da própolis, nas intoxicações agudas por acetaminofen, em camundongos (dissertação de mestrado). Viçosa, Brazil: Universidade Federal de Viçosa; 2001.\n\nSilva GH, Barros PP, Gonçalves GMS, et al.: Avaliação da atividade hepatoprotetora do asiaticosídeo em modelo experimental de lesão hepática por paracetamol em ratos. Revista de Ciências Farmacêuticas Básica e Aplicada. 2014; 35(3): 489–496.\n\nSilva JB, Costa KMFM, Coelho WAC, et al.: Quantificação de fenóis, flavonoides totais e atividades farmacológicas de geoprópolis de Plebeia aff. flavocincta do Rio Grande do Norte. Pesquisa Veterinária Brasileira. 2016; 36(9): 874–880. Publisher Full Text\n\nSouza SA, Silva TMG, Silva EMS, et al.: Characterization of phenolic compounds by UPLC-QTOF-MS/MS of geopropolis from the stingless bee Melipona subnitida (jandaíra). Phytochem. Anal. 2018; 29(6): 549–558. PubMed Abstract \| Publisher Full Text\n\nTzankova V, Aluani D, Yordanov Y, et al.: Nanoformulação de própolis micelar de alta atividade antioxidante e hepatoprotetora. Revista Brasileira de Farmacognosia. 2019; 29(3): 364–372. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVaher M, Koel M: Separation of polyphenolic compounds extracted from plant matrices using capillary electrophoresis. J. Chromatogr. A. 2003; 990(1): 225–230. Publisher Full Text\n\nVasconcelos AC: Necropsia e conservação de espécimes para laboratório. Belo Horizonte: Centro de Extensão da Escola de Veterinária da UFMG; 1996.\n\nWali AF, Avula B, Ali Z, et al.: Antioxidant, hepatoprotective potential and chemical profiling of propolis ethanolic extract from Kashmir Himalaya region using UHPLC-DAD-QToF-MS. Biomed. Res. Int. 2015; 2015(1): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZilla MK, Nayak D, Amin H, et al.: 4’-Demethyl-deoxypodophyllotoxin glucoside isolated from Podophyllum hexandrum exhibits potential anticancer activities by altering Chk-2 signaling pathway in MCF-7 breast cancer cells. Chem. Biol. Interact. 2014; 224(1): 100–107. PubMed Abstract \| Publisher Full Text" }	[ { "id": "197905", "date": "04 Sep 2023", "name": "Steven Tandean", "expertise": [ "Reviewer Expertise Neuroscience", "natural and alternative medicine", "neurosurgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 interesting research, that can be a new input for hepatoprotector choice. During the review, I found two things that need to be discussed and revised.\n\nIn the results section, the author indicates significance using letters but doesn't elaborate further, such as whether the results are well-distributed or what the P-values are for each group. I suggest a qualified statistician should provide further input.\n\nIn the references, the authors use several references in Portuguese language and it making difficult to verify whether the citations are relevant or not. So I suggest using English-language journals as references.\nIn my opinion, this article was Approved with Reservations. Please consider to revise the article before it can be considered to be fully scientifically valid.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "229257", "date": "24 Jan 2024", "name": "Oriel Hlokoane", "expertise": [ "Reviewer Expertise Medicinal chemistry", "Pharmacy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have studied the chemical composition and hepatoprotective effects of hydroethanolic extracts of geopropolis produced by stingless bees in the West and South of the city of Mossoró, Rio Grande do Norte. The research is interesting in that it can lead to the formulation of the new hepatoprotector choice. Based on my review, I have found one thing that need to be revised. In the results section; to study chemical composition, the authors used HPLC-DAD-ESI-MS/MS analysis and detected phenolic compounds. My recommendation is that, the authors should include the data regarding peak assignments such as retention times (RT) and MS spectrum data as supporting information (SI).\n\nIs the work 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-999
https://f1000research.com/articles/11-1029/v1	12 Sep 22	{ "type": "Research Article", "title": "From pedagogical toward technological pedagogical content knowledge frameworks and their effectiveness in teaching mathematics: A mapping review", "authors": [ "Hashituky Telesphore Habiyaremye", "Celestin Ntivuguruzwa", "Philothere Ntawiha", "Celestin Ntivuguruzwa", "Philothere Ntawiha" ], "abstract": "Background: A study to reveal existing pedagogical or technological pedagogical content knowledge frameworks is crucial to inform and their effectiveness in teaching mathematics. This review study intended to explore the trends of the pedagogical content knowledge (PCK) framework, how it has changed over time until the most recent version of technological and pedagogical content knowledge (TPACK) was developed, and its effectiveness in teaching mathematics. Methods: We initially downloaded 273 articles from the first 30 Google Scholar pages and analyzed 229 journal articles. We got 24 frameworks from 64 journal articles since Shulman’s first model in 1986. About 52 out of 229 were mathematics studies. Among these studies, we found that 18 studies have extensively investigated the use of identified frameworks. Results: The frameworks were presented and descriptively discussed in chronological order. The empirical studies that compared the role of pedagogical and technological pedagogical content knowledge models among classrooms with teachers who possess and do not possess such skills were demonstrated. Conclusions: The gap in empirical studies was identified, and further studies about the intervention of PCK and TPACK models were suggested to gain more insight into the mathematics classroom.", "keywords": [ "Google Scholar", "framework", "mathematics", "pedagogical content knowledge", "technological pedagogical and content knowledge" ], "content": "Introduction\n\nAround the 1980s, a new era in subject matter and teacher pedagogy rose. Shulman (1986) argues that the emphasis on teacher material knowledge and pedagogy is conserved as special. He believes that teacher training should conglomerate these two areas of specialization. He introduced the concept of pedagogical content knowledge (PCK), which comprises pedagogical knowledge (PK) and content knowledge (CK), to address this dichotomy, amongst other classifications. His original portrayal of the teacher's knowledge encompassed information about the curriculum and knowledge of the educational context.\n\nAfter two decades, a major revolution has been proposed by various researchers in the field of technology. Mishara and Koehler (2006) established the concept of technological pedagogical and content knowledge (TPACK) in reaction to the lack of theory guiding technology integration into teaching. TPACK characterizes an extension of Shulman’s (1986) representation of what their colleagues looked for to teach explicit content (that is, PCK) by depicting the knowledge required to teach such content with technology (Mishara & Koehler, 2006).\n\nThe old review article we found was written in 2008 and discussed how English teachers across institutional networks develop PCK (Smith & Anagnostopoulos, 2008), while the latest review was published in 2020 from Njiku et al. (2020) and analyzes research instruments used and authenticated by 28 diverse studies to measure teacher TPACK.\n\nAmong 20 review articles we found, 15 were journal articles, two were conference proceedings, two were book chapters, and one was a book. Among these 15 journal articles, none investigated accumulating PCK framework over the years, or gave a clear analysis into teaching intervention through those frameworks, and teachers’ difficulties in attaining content knowledge. For instance, the current methods and instruments of TPACK were reviewed by Abbitt (2011) in the training of pre-service teachers, Aydin (2012) researched the field of science teacher training in the framework of the PCK in Turkey, and the validity and reliability of using TPACK as a reference framework for determining the extent to which teachers can teach using technology was done by Young et al. (2012). In 2013, Chai et al. reviewed 74 journal articles exploring information and communication technology (ICT) integration from the TPACK system, an investigation of the theoretical foundation and the functional use of TPACK (Voogt et al., 2013) was done, Depaepe et al. (2013) systematically reviewed PCK in the way the concept permeated mathematics pedagogical research, and Wu (2013) reviewed empirical TPACK studies that were published in prominent and international journals from 2002 to 2011.\n\nOne article gave a critical review of PCK’s nature, principles, and trends (HU, 2014), another gave an analysis of TPACK studies in Turkey using a meta-synthesis method and showed the types of trends in this field (Yilmaz, 2015), and another by Rosenberg and Koehler (2015) provided an inclusive and truthful picture of the degree to which background is encompassed in the TPACK. Evens et al. (2016) reviewed PCK in connection with teaching foreign and second languages; Hubbard (2018) summarized how PCK computing is conceptualized and investigated in computer pedagogical research. Rossi and Trevisan (2018) investigated how TPCK is defined and affected in the early career teaching profession. Grieser and Hendricks (2018) prepared a literature review on PCK, which is specific to the topic and to the music education environment in general to better understand problems that teachers with limited knowledge of string specific content may experience when teaching string students. Therefore, our study aimed at reviewing:\n\n1. Chronological trends in PCK models (from beginning to date, how did researchers modify the original model that Shulman started with? Which models, what was modified, why were the modifications made, which teachers and/or students benefited from that change?)\n\n2. Effectiveness of PCK and TPACK frameworks (empirical studies that have used these frameworks to upgrade students learning. Any level, primary-secondary-university. Any year. Descriptive and inferential statistics each study got, etc.).\n\n\nMethodology\n\nWe designed and employed a “one-term in one-source” method. This method involves using one term, a key, or search word in only one academic search engine. Thus, we have used “pedagogical content knowledge” as the search term in Google Scholar as an academic source. This search returned 20 review papers and 273 empirical studies at a glance (see Figure 1) on 19 August 2021.\n\nUsing the term “pedagogical content knowledge” in Google Scholar, we explored the results in the first 30 pages. We downloaded every article that contains the words “pedagogical content knowledge” in its title. We only included articles written in English. The first paper, “Pedagogical content knowledge in social studies,” was published in 1987, while the latest “A virtual internship for developing technological pedagogical content knowledge” was published in 2020. The first seven pages exhausted all articles written in review form, such as a review of literature, systematic review, meta-analysis, etc. This means that we checked pages eight to 10 and had no results and stopped checking after page 10. There were many more empirical studies (articles that investigate with primary data), so we limited the search to the 30th page (entry); as you can see, the last page still had seven articles. We did not limit ourselves to year of coverage, subject, or grades level. However, we intended to explore mathematical PCK explicitly. Thus, we retrieved all articles regardless of subject but then later excluded the non-mathematics articles. Literature review articles were visited only to frame our research problem, while empirical articles were focused on the analysis to answer our research objectives. The first author did initial screening and the co-authors checked the inclusion criteria.\n\nAlthough we used only one database, we found articles from other repositories. For instance, 24 articles were from the educational resources information center (ERIC), 17 from Academia, 13 from Research Gate, two from HAL (Hyper Articles en Ligne), and two from Durham University. However, all these were accessed via Google Scholar. Thus, articles in Google Scholar will take you to the website they are hosted on or repository they are deposited on. About the nature of articles selected, 229 were identified as journal articles, 22 were conference proceedings, 8 were book chapters, 2 were books, and 5 were generics (articles with unclear identification). Therefore, our analysis only considered journal articles.\n\nPCK models were cited, presented in figures, and described. Teaching interventions were presented in sections, and their statistics were presented in tables and descriptively discussed. Teachers’ knowledge was presented descriptively or in tables by discussing the type of study (survey, cases studies, interventions, etc.) that produced the results, the type of data such as quantitative or qualitative, the type of mode such as whether teachers’ knowledge was measured from him/herself or from his/her students, etc. Theoretical strands were also described accordingly. We used both NVivo 1.0 software and MS Excel 2016 to analyze data. Figure 2 is a typical example of the word analysis from NVivo using articles that were downloaded.\n\n12 subjects were identified across 229 articles. Articles discussing or investigating learning mathematics dominated the list (23% or 52 out of 229 articles). Science subjects and articles related to teacher education were 42 (18%) and 30 (13%) out of 229 articles, respectively, while unidentified articles or articles investigating PCK in a general sense accounted for 40 (17%). Figure 3 shows the distribution of articles among different subjects.\n\nAmong 229 retrieved journal articles, 110 (48%) were from 20 journals from various publishers. Table 1 shows that Teaching and Teacher Education, published by Elsevier, and the International Journal of Science Education, published by Taylor and Francis, were the two journals ranked first and second respectively among the top 20 contributors in this study (6% and 4%, respectively).\n\nFigure 4 displays the hierarchy of analysis. The blue color chart shows the number of all empirical studies downloaded across the years of publication. The first study that investigated PCK, after its launch from Shulman in 1986, was published in 1987, while the latest came out in 2020. After filtering out proceedings, books, book chapters, theses, and generics, 229 journal articles are shown in red. After analyzing these 229 articles, we found 64 articles that clearly investigated or used PCK or TPACK frameworks or models (see green color). Thus, others investigated these models. They did not formulate new models but used existing ones. Finally, among 229 articles, 52 articles were found to extensively investigate mathematics lessons, as shown in purple.\n\n\nResults and discussion\n\n65 (including 22 related to mathematics) out of 229 showed PCK trend models. About 24 articles (including 11 related to mathematics) showed original frameworks, while 41 referred to these 24 articles. Thus, Table 2 displays a review of 24 frameworks presented in the related 24 articles. Among these 24 frameworks, 17 are versions of PCK, and seven are upgrades of TPACK.\n\nTable 2 outlines the models and constructs for each study. We are now going to discuss each of the 24 concepts in more detail.\n\nStudents’ understanding, media for instruction, and subject matter are inter-related. At the same time, these components are connected to instructional processes that contain student focus, presentation focus, and media focus (Marks, 1990, p.5).\n\nThe subject-matter content knowledge (SmCK) contributes to teachable content knowledge (TCK), while PCK comes as a supplement to remedy misconceptions (for instance, using alternative representations). See Geddis (1993, p.677).\n\nThe author emphasized assessing students’ knowledge of mathematics and mathematical activities and representations, the teacher’s capacity to hypothesize the students’ knowledge and learning of particular content, and teacher’s capability to theorize the mathematics learning and teaching. These capabilities contribute to the learning trajectory that embeds the teacher’s learning objectives, the learning activity plan, and the hypothesis of the learning process (Simon, 1995, p.56).\n\nThe author outlined and described seven constructs of PCK (Jones & Moreland, 2003, p.82): nature and characteristics of the subject; conceptual, procedural, and practical features of the topic; knowledge of curriculum; knowledge of students (such as prerequisite knowledge, strenths and weaknesses of the student, and learning progress); special teaching and assessment practice for the subject (holistic, authentic, etc.); sympathetic of the role and place of context; and classroom environment and topic management (such as resources, hardware, and technical abilities).\n\nThe network is centered on students’ learning where both content, curriculum, and knowing students’ thinking (KST) are direct contributors. The network starts from beliefs that interchangeably corroborate with PCK in the center of three components (content, teaching, and curriculum) that directly interchangeably feed into the KST. KST has four other four outcomes apart from students learning. These are (a) remediating students’ misunderstandings, (b) make mathematics learning attractive to students, (c) constructing students’ mathematics ideas, and (d) endorsing students’ discerning mathematics (An et al., 2004, p.147).\n\nIn this model, four steps are involved in its development (Niess, 2005): an initial idea of the meaning of what to teach an actual topic with technology; understanding of teaching strategies and presentations for teaching a specific topic with technology; awareness of students’ understanding, thinking, and education with technology; familiarity of curriculum and its resources that fit in technology in students learning.\n\nThe authors emphasized the context of change. Existing PCK contributes to institutional intervention and expression of knowledge. The institutional intervention contributes to a new PCK that produces the expression of knowledge. Thus, this institutional intervention brings the context of change (Major & Palmer, 2006, p.626).\n\nThe three circles (content, pedagogy, and technology) overlap to create four more interrelated forms of knowledge (Mishara & Koehler, 2006, p.1025). These are pedagogical content knowledge (PCK), technological content knowledge (TCK), technological pedagogical knowledge (TPK), and technological pedagogical content knowledge (TPCK).\n\nThe authors unpacked subject matter knowledge from PCK (Hill et al., 2008, p.377). The PCK remained in three parts: knowledge of content and teaching (KCT), knowledge of content and students (KCS), and knowledge of curriculum (KC). On the other side, SMK comprised three components: specialized content knowledge (SCK), common content knowledge (CCK), and knowledge at the mathematical horizon (KMH).\n\nApart from PCK, the context of technology and engineering knowledge (ETK) was added (Miranda, 2008, p.3). Thus, the pedagogical context in engineering and technology education arises between pedagogy knowledge (PK) and ETK, while engineering and technology content in the context of representation of knowledge of content (CK) and ETK. The final product has emerged as PCK in engineering and technology (PCKET).\n\nAccording to the authors, PCK includes both subject matter knowledge (SMK) and pedagogical knowledge (PK), where PK (Kaya, 2009, p.964) is composed of (a) knowledge of instructional strategies and activities (KISA), (b) knowledge of curriculum (KC), (c) knowledge of students’ learning difficulties (KSLD), and (d) knowledge of assessment (KA).\n\nThree components (content, pedagogical, and technological) of knowledge intersect (Koehler & Mishra, 2009, p.63). The content knowledge (CK) and pedagogical knowledge (PK) contribute to PCK. CK and technological knowledge (TK) contribute to technological content knowledge (TCK). PK and TK contribute to technological pedagogical knowledge (TPK). The whole context (intersection of PCK, TCK, and TPK) produces TPACK (technological pedagogical and content knowledge).\n\nThe author described what CK, PK, and PCK involve during learning physics. CK involves knowledge of physics, thoughts, associations, and approaches to evolving novel information. PK contains information about brain development, cognitive science, collaborative learning, the classroom, and knowledge about leadership and school laws. PCK includes teaching orientation, knowledge of the physics curriculum, student ideas, effective teaching strategies, and assessment methods (Etkina, 2010, p.2).\n\nPedagogical content knowledge (PCK) is fuelled by the reason to teach, content to teach, learning difficulties, and methods of teaching (Saeli et al., 2011, p.76).\n\nThe authors cultured TPACK from socialization to internalization through problem-based learning (Tee & Lee, 2011, p.91). The first process is socialization (e.g., open-ended, in-class discussion, and asynchronous online debates). The second process is designing outsourcing, such as single and collective writing exercises, model or prototyping, discourse, and reflection from the group. The third process is a combination involving designing reliable or replicated multifaceted circumstances that challenge learners to fuse several facts is done. The fourth process is internalization (e.g., common written and oral replications and ideal explanations).\n\nThe general pedagogical and psychological knowledge (PPK) is composed of two components (classroom processes and students’ heterogeneity). Classroom controlling, teaching procedures, and classroom assessment reside on the side of classroom processes, while student learning progressions and individual physiognomies are catered for by the students’ heterogeneity component (Voss et al., 2011, p.954).\n\nPCK was divided into three components: subject matter understanding, knowledge of students’ development, and teaching techniques (McCray & Chen, 2012, p.295).\n\nThe terms used are Pedagogical Content Knowledge (PCK), content representation (CoRe), online PCK teachers’ analyzer (OTPA), and PCK textbook analyzer (PTA). CoRe portrays PCK and constructs OTPA to assess teachers while PTA assesses textbooks.\n\nThe normal PCK framework was divided into two segments where the upper part was referred to as learner perspective. In contrast, the lower part was split into two and referred to as teacher designs content (CK side), and teacher designs learning environment (PK side). Teaching and learning were emphasized on the PK side (Zepke, 2013, p.98).\n\nPCK has four components: knowledge of the mathematics curriculum, knowledge of instructing mathematics, knowledge of assessing mathematics, and students’ comprehending within mathematics (Lannin et al., 2013, p.406).\n\nContent knowledge (CK) is composed of (a) CcK (conceptual knowledge), such as conceptual understanding of fractions, language used, their uses and problem-solving; and (b) RK (representational knowledge), such as teacher illustrations of mathematical items (pictorial or figurative) about fractions. PCK is composed of (a) KTC (knowledge of the teaching of content), such as knowledge of the adaptation of mathematical facts, and (b) KSK (knowledge of students’ knowledge) (Olfos et al., 2014, p.919).\n\nTeacher practice directly influences students’ achievement (Gess-Newsome et al., 2019, p.948). There are three components: ACK, general PK, and PCK, which are interventions for teacher practice and, therefore, student achievement.\n\nTeachers’ professional knowledge has five bases; curricula, content, pedagogical, assessment knowledge, and students’ knowledge. This knowledge is directly related to topic-specific professional knowledge, teacher beliefs and orientation, context, classroom practice, students’ beliefs, prior knowledge, behaviors, and finally to students outcomes (Gess-Newsome et al., 2019, p.962).\n\nThe R-STLDM has two stages. The first stage is determining lesson objectives. It gathers, analyses, and diagnoses information or knowledge between instructional goals (within CK and TK) and learners’ context (within PCK and TPCK). This stage’s outcome is the design decision that connects to the second stage. The second stage connects planning of instructional and learning activities (PK and TPACK activities), the relevant choice of technologies (TPK, TCK, and TPACK resources), and assessment development (formative and summative) together (Chai et al., 2019, p.372).\n\nKnowledge was divided into two components; overt and inherent. Knowledge in overt context can be activated independently from the situation, can be uttered in technical semantic, can be methodically used to plan to learn, and can be used to perceive and spot juveniles’ knowledge or abilities. On the level of implicit knowledge, it can only be triggered and expressed in situational contexts, stimulate planning of learning conditions, and lead to adequate supportive reactions to children’s performance (Gasteiger et al., 2020, p.199).\n\nContent knowledge (CK), pedagogical knowledge (PK), and technological knowledge (TK) are drawn in a way that they contribute to TPACK. Also, PCK, TCK, and TPK contribute to TPACK. Then CK contributes to PCK and TCK, PK contributes to PCK and TPK, while TK contributes to TCK and TPK (Andyani et al., 2020, p.128).\n\n52 out 229 were identified as articles related to mathematics. Among 52 mathematics articles, 39 were related to TPACK, while 13 were investigating PCK. 18 of the 52 articles fit our analysis framework and spanned from 1988 to 2020. They showed the implications of PCK and TPACK frameworks. Except for these 18 articles, 34 out of 52 articles reviewed showed survey results that only provided the outcome of PCK or TPACK. These 18 articles were only articles that investigated the outcome of comparative studies such as pre-and post-test designs, variables such as teachers’ backgrounds, teachers’ gender, teachers’ geographical locations, etc. Most of the studies left out were surveys without comparison, and others were qualitative. Table 3 displays a review of 18 articles related to mathematics that showed the comparative outcome of PCK and TPACK.\n\nTable 3 presents trends of pedagogical content knowledge framework. We are now going to discuss them in more detail and reveal their effectiveness in teaching mathematics.\n\nThis study (Carpenter et al., 1988) examined the pedagogical content of 40 grade-one teachers about children’s explanations to word problems related to addition and subtraction in 27 schools located in Madison, Wisconsin, USA. Most teachers were able to identify many critical differences between the problems and the basic plans that children used to answer dissimilar types of issues. However, this information is often not prearranged into a cohesive linkage that connects the differences between difficulties, youths’ resolutions, and the severity of the problems. Instructors’ knowledge of whether their learners were able to solve a variety of problems correlated meaningfully with apprentice performance.\n\nTeacher training should be based on the knowledge base provided by trainee teachers (Van Der Valk & Broekman, 1999). A group of math and science teachers developed a methods for studying parts of knowledge from the pedagogical content of the subject. Pre-service teachers were asked to formulate a lesson on a topic, such as an area of the body. The authors concluded that the method is useable to fully make teachers prompt their PCK.\n\nThe development of PCK for custodial teachers was examined in terms of the integration of technology (Niess, 2005). The four gears of the PCK have been adapted to refer to the technology boosted PCK. This research studied the TPCK of student-teachers in a multidimensional training program for science and math teachers that integrates technology teaching and learning through the software package. Five gears defined hitches and the success of technology teachers in training the TPCK. Students’ sight of incorporation, taking into account the nature of technology and science, was acknowledged as a central part of the advance of the TPCK.\n\nThe 16 questions for the PCK of mathematics (MPCK) were established to size its aspects in the teaching of mathematics in primary school (Lim-Teo et al., 2007). The instrument was awarded to 113 of the 261 student teachers who entered the graduate program in July 2005. The MPCK was re-introduced in February 2006, shortly before teacher-students began their teaching internships. Between July 2005 and February 2006, MPCK development occurred in 96-hour computer science courses and in general education courses that include two other modules. The results show that teachers at the establishment of their education are poor in their knowledge of the pedagogical content of mathematics. There was a weighty development in all areas of the MPCK after the accomplishment of the pedagogy course.\n\nLee and Hollebrands (2008) provided examples of teacher training materials developed using an approach in which teachers' understanding of content, technology and pedagogy are prepared to teach data analysis and probability topics using specific technological tools. The fall 2005 control group (N=15) changed from 33% pre-test to 39% post-test score, spring 2006 execution of first draft of provisions (N=18) from 44% pre-test to 33% post-test score, and fall 2006 enactment after amendment for instruction (N=15) from 50% pre-test to 67% post-test score.\n\nIn the COACTIV (Professional Competence of Teachers, Cognitively Activating Instruction, and the Development of Students’ Mathematical Literacy) project, PCK and CK tests for secondary school mathematics teachers were established and implemented with German teachers who participated in the 2003/04 Programme for International Student Assessment (PISA) longitudinal evaluation (Krauss, Baumert, et al., 2008). The second COACTIV dimension point (2004), on which the PCK and CK tests were taken, involved 218 secondary school mathematics teachers; 198 teachers completed both exams. The sample results, examined in the context of the three growing information hypotheses, appear to be a marked upsurge in acquaintance in both PCK and CK during higher education and then a smoother rise in the next phase of teacher education. Outward associations with teachers’ subjective beliefs about math and math learning show that expert teachers discard the view that math is just a toolkit and that math can superlatively be educated through cautious listening. In addition, the outcomes of structural equation modeling show that PCK supports the learning conveyed by the lesson aspects.\n\nKow and Yeo (2008) examined the influence of knowledge of the pedagogical content of a mathematics teacher (MPCK) on the area and perimeter of teaching for four grades. Aspiring teachers’ classrooms were examined to identify the activities and teaching strategies used to identify the area and perimeter ideas. The observed results of MPCK in action are also explored through videos with a novice teacher. The complex interactions between the concepts of area and perimeter sometimes worked well for the novice teacher. At the same time, in other cases, gaps in knowledge about the content of pedagogical mathematics could mislead students.\n\nKrauss, Brunner, et al. (2008) describe the design of tryouts to test this knowledge and the application of these tests to the models of 198 German mathematicians. They assess whether knowledge of knowledge concepts and concepts of knowledge can make a difference and whether the average knowledge and level of connection between the two knowledge groups depend on mathematical skills. Studies have shown that educators with thorough mathematics training outperformed educators from other schools in both categories of knowledge and demonstrated a large grade of cognitive connection amid the two sets of knowledge.\n\n639 high-school students initially gave explanations about “a0 = 1, 0! = 1” and “a ÷ 0” where a ≠ 0 (Cankoy, 2010). Secondly, 58 high school mathematics teachers in Northern Cyprus wrote how they teach the abovementioned topics to high school learners. The study revealed that proficient teachers suggest more abstract teaching strategies than beginner teachers. These strategies were against PCK as they were mainly procedural, fostering memorization.\n\nThe results of this study recommended that a PBL-based classroom designed with an environment that supports socialization, externalization, pairing, and internalization can help teachers cultivate TPACK (Tee & Lee, 2011). The study found that teachers were better prepared to use TPACK more productively after their cerebral models switched to Biggs’ Level 2 and Level 3 styles. The course has created chances for teachers to understand that technology alone is unlikely to advance teaching skills, and teachers can consider their teaching skills and the nature of the subject when choosing a technology.\n\nThis article analyzed the extent to which primary teacher training can be considered effective and the possible causes of inequality in 15 nations (Blömeke et al., 2011). The target group for this study was demarcated as prospective teachers in the closing year of teacher education allowed to teach mathematics in basic education. Random sampling was used in a two-step process in each participating country. The study proved substantial cultural alterations in the success of teacher formation. The language was found to be divided into direct and indirect impacts. This presented an amalgamation of different options for teacher training programs rendering to teachers’ training and different achievements in these programs.\n\nIn a comparison study, Kleickmann et al. (2013) examined the CK and PCK of pre- and in-service teachers at different stages of their teaching careers. The results showed that there was a significant difference in the CKs of the teacher groups examined here. Confirmatory factor analyses revealed that reasonably invariant population reflected that PCK and CK measurement, the major variances in CK and PCK were established between the start and the close of preliminary teacher training.\n\nParticipants were randomly and proportionally selected by stratum in around 38% of the 144 schools in the three chief cities in Valparaíso, Chile (Olfos et al., 2014). At the beginning and end of the test, the authors used groundwork, academic, and socioeconomic formation in schools where they are grouped into the classroom. The constructivist sub-component of the authors’ framework was powerfully related with student attainment, approving the robust significance of the association with teaching experience. The CK of Socio-Economic Teachers presented an important link to learner learning, though these are divisions characteristic of the Chilean education system.\n\nThis empirical study was led in agreement with the ethical procedures of the German Psychological Society. Harr et al. (2014) assessed the duration of each participant’s working memory separately. In an experimental study of 60 mathematics students, authors examined the effect of giving aspects of overall pedagogical and psychological knowledge (PPK) as well as PCK in a combined or separate way. Under both experimental situations, participants were first given brief information about working with the computer program used in the treatment session. After a computer-assisted working memory task, participants worked on a previous PCK and PPK pretest. As a result, the integrated condition led to a wider application of the pedagogical and psychological features and growth in the concurrent submission of both sorts of data parallel to the separate condition. Overall, the results show the positive effects of integrated design on teacher training.\n\nDepaepe et al. (2015) researched the knowledge of potential teachers about the content (CK) and PCK in rational numbers, the connection between CK and PCK, and their dissimilarities among future teachers trained as primary and secondary school teachers. They found shortcomings in the perspective of CK and PCK teachers, a positive relationship amongst CK and PCK, and enhanced CK.\n\nKleickmann et al. (2015)’s comparison study of two nations provided additional proof on a two-dimensional edifice of the teachers’ knowledge on the subject. In addition to CK, PCK represents a special but related aspect of subject matter knowledge. The variances in CK and PCK among Taiwanese and German primary teachers after training presented in TEDS-M are more apparent than in experienced teachers who participated in this study. The teaching familiarity was not associated with the CK and PCK scores, which indicated the solidity of CK and PCK in the work stage.\n\nThis study developed and validated the TPACK scale to investigate the knowledge levels of mathematics teachers in TPACK constituents and to examine whether the TPACK stages of mathematics teachers differed (Ozudogru & Ozudogru, 2019). Gender, teaching experience, and grade level were demographic variables. Data was collected from 202 math teachers in the spring semester of the 2016-2017 school year. As a result, there were significant transformations in gender in favor of male teachers in terms of mastery of TK. It was pointed out that teachers who teach in primary schools feel more like their TK than teachers who teach in secondary schools. While male teachers teaching in elementary school with 16 to 20 years of teaching experience rated their highest TK, female teachers in high school with 1 to 5 years of teaching experience rated their TK lowest.\n\nDecolonization is a process of curriculum criticism and renewal (Khoza & Biyela, 2020). This study suggested the submission of personal knowledge to create a practical program as an elucidation to the decolonization of education. Counting algebra is learned by students from an early age in society. In other module cases, knowledge of technology and content dominated learning, but the pedagogical knowledge that arose from introspection to understand their identity always led the module. Therefore, the study recommended that students utilize their knowledge of content, pedagogy, and technology as a taxonomy of apprenticeship to meet the needs of mathematics, individuals, and society through technology integration.\n\n\nConclusion\n\nOur selected method of one-term in one-source was effective to dig into literature and find the relevant information required to answer the presented research questions. In the early 1980s, Shulman (1986) initiated the idea of pedagogy as an important construct to deliver an effective lesson. In both 1986 and 1987, he demonstrated that a teacher does not only need content knowledge, but rather pedagogical knowledge too (see Figure 5), such as the knowledge of curriculum, students, and instruction (Shulman, 1987).\n\nSince then, other researchers have continued to develop this idea. They started to implement it in the classroom and in different contexts such as various other subjects and ICT inclusion. Different contexts have prompted researchers to modify the initial model or framework to fit their needs. This is the reason that since the early 2000s, researchers have noticed a deficiency of relevant theoretical framework in the age of technology. Therefore, various researchers (mainly Koehler & Mishra, 2005; Mishara & Koehler, 2006) designed an experiment to understand teachers’ professional development via integrating ICT in their teaching.\n\nAs a result, technological, pedagogical, content knowledge (TPCK) was offered. The term TPCK was initially used in the literature until 2008 (Mishra & Koehler, 2008) when the academic world suggested the supplementary and easily pronounced term of technological, pedagogical, and content knowledge (TPACK). From then on, technology has been identified as an important player in the teaching and learning process as educational institutions move into the 21st century.\n\nIn this study, we opted to fill the gap in the literature of how the framework was hierarchically modified to fit effective teaching and to learn in different contexts and document their contributions to learning achievement. Therefore, we further investigated their effectiveness in refining mathematics teaching and learning by reviewing the outcomes of studies that tested PCK and TPACK frameworks. These were empirical studies that have used these frameworks as an intervention to upgrade students learning. The quick outlook showed that PCK was investigated more than TPACK – 13 out of 18 reviewed papers were related to PCK. We have found that many studies were surveying teachers on how they perform a certain component of these frameworks such as teacher subject knowledge, pedagogy, etc. Few studies focused on fostering PCK and TPACK skills to foster the students’ performance, attitude, and conceptual understanding. Thus, further studies are needed to illuminate these skills in the classroom and their outcomes on students’ learning. For instance, no study has investigated the difference between teachers with PCK skills and non-PCK skills and what the reflection of this is on their students. Future studies should also investigate the teachers’ lack of difficulties in attainment of content knowledge (CK) or pedagogical knowledge (PK) by looking into empirical studies – surveys, cases studies, interventions, etc. – that have investigated teacher knowledge (CK or PK).\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\nAuthor contribution\n\nAll authors have equally participated in designing and communicating the results.", "appendix": "Acknowledgments\n\nAll authors that produced reviews and empirical studies that helped us write our study are acknowledged.\n\n\nReferences\n\nAbbitt JT: Content Knowledge in Preservice Teacher Education: A Review of current methods and instruments. J. Res. Technol. Educ. 2011; 43(4): 281–300. Publisher Full Text\n\nAn S, Kulm G, Wu Z: The Pedagogical Content Knowledge of Middle School, Mathematics Teachers in China and the U.S. J. Math. Teach. 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Publisher Full Text\n\nDe Rossi M , Trevisan O: Technological Pedagogical Content Knowledge in the literature: how TPCK is defined and implemented in initial teacher education Technological Pedagogical Context Knowledge (TPCK) in letteratura: come viene definito e implementato il modello TPCK nei conte. Ital. J. Educ. Technol. 2018; 26(1): 7–23. Publisher Full Text\n\nSaeli M, Perrenet J, Jochems WMG, et al.: Teaching programming in secondary school: A pedagogical content knowledge perspective. Inform. Educ. 2011; 10(1): 73–88. Publisher Full Text\n\nSaeli M, Perrenet J, Jochems WMG, et al.: Programming: Teachers and Pedagogical Content Knowledge in the Netherlands. Inform. Educ. 2012; 11(1): 81–114. Publisher Full Text\n\nShulman L: Knowledge and Teaching: Foundations of the New Reform. Harvard Educ. Rev. 1987; 57(1): 1–23. Publisher Full Text\n\nShulman LS: Those Who Understand Knowledge Growth Teaching. Educ. Res. 1986; 15(2): 4–14. 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Publisher Full Text" }	[ { "id": "153309", "date": "07 Nov 2022", "name": "Joseph Njiku", "expertise": [ "Reviewer Expertise Mathematics education", "teacher education", "and technology integration in 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\nSummary: the study is a literature review. It is designed to discuss the trend in PCK and TPACK studies and how they influence effective learning, especially in mathematics. The study reviews different literature in the area to respond to two research questions. Data sources are obtained from the Google Scholar database and screened for relevance. Data analysis is done to uncover the trends in PCK and TPACK and their effectiveness in explaining learning. As such findings are presented first by listing and explaining studies that explain the PCK or TPACK framework and those that explain the use of the framework in explaining learning especially in mathematics. The topic is current and may inform future studies on the evolution and debate in PCK and TPACK and how they have been used to influence learning. Specific comments about possible areas for improvement of the paper are provided as follows:\nKeywords: although the authors used the Google Scholar database as the source of their articles, using google scholar as a keyword does not seem to fit what is discussed in the paper.\nStatement of the problem: It identifies the lack of studies that cumulatively explain the evolution of PCK from its inception. However, the authors would consider presenting the arguments logically instead of mixing up PCK and TPACK in a zigzag way by focusing their attention first on PCK when done then TPACK.\nResearch questions: Although the title and various places it is claimed that the study focuses on the learning of mathematics, none of the research questions is focused on mathematics.\nData selection: Selection of articles is well articulated. However, the authors have not explained why they chose google scholar as the sole database for their search. They may also explain why they ended on pages 10 and 30 in search of review articles and empirical studies respectively.\nNature of selected articles: The author concluded that “therefore, our analyses only considered journal articles” without explaining why so. Were other types of sources not relevant/fit for the study? They may consider explaining why they selected only journal articles.\nWriting style: The authors have written several sentences that begin with numerals. They need to rephrase them and ensure no sentence starts with a numeral. They also use one-sentence paragraphs, a style uncommon in academic writing.\nData analysis: This section is supposed to explain how the results presented in the following sections were obtained. However, the authors seem to give more space to what the articles were about – an explanation that fits in their preceding section named “extended sources and nature of selected articles”. For example, all the tables and figures in this section are about the articles selected (when they were published, where they were published, and their subjects) and not about how the selected articles were analyzed. Only the first paragraph of the section talks about how findings were presented but even this is not preceded by how such presented finding were found through analysis. The authors may consider focusing their attention on explaining how they went about analyzing the documents they selected.\nResults and discussion: The section seems to be divided into two major subsections namely PCK framework and PCK interventions. Under each subsection, there are presented lists of studies that the authors discuss one after another. The studies are discussed as isolated ideas that do not inform or complement each other. There is no explanation why the second study or the third study etc. are important in/or add to the idea presented in the preceding studies. Also, there is no synthesis as to what we cannot learn from the listed studies. What is (not) common about them? What do we now learn about PCK and TPACK from them? How about mathematics? What have the studies shown in general about the learning of mathematics? Is PCK or TPACK sufficient in explaining effective learning of mathematics? What component of the frameworks should/has not been well addressed by teachers for effective learning of mathematics? Also, the studies are presented separately from each other, each having its own heading, where some are discussed using one sentence paragraph. The authors may consider explaining in a logical flow how PCK for example, is understood from some two to three papers and how this understanding is corroborated/advanced/contradicted/etc. by other studies. They may do so by connecting the ideas from the studies to one another and explicitly showing what we can now learn from the cumulative trend.\nConclusion: Although the conclusion hits the point, especially in the last paragraph, the preceding paragraphs seem to supplement what may have been missed in the discussion. As such much of its content does not sound as concluding.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10053", "date": "17 Aug 2023", "name": "Hashituky Telesphore Habiyaremye", "role": "Author Response", "response": "Reviewer comments: Summary: The study is a literature review. It is designed to discuss the trend in PCK and TPACK studies and how they influence effective learning, especially in mathematics. The study reviews different literature in the area to respond to two research questions. Data sources are obtained from the Google Scholar database and screened for relevance. Data analysis is done to uncover the trends in PCK and TPACK and their effectiveness in explaining learning. As such findings are presented first by listing and explaining studies that explain the PCK or TPACK framework and those that explain the use of the framework in explaining learning especially in mathematics. The topic is current and may inform future studies on the evolution and debate in PCK and TPACK and how they have been used to influence learning. Specific comments about possible areas for improvement of the paper are provided as follows: Author response: Thank you so much for appreciating our study contribution! We have framed our study based on your suggestions. Keywords: although the authors used the Google Scholar database as the source of their articles, using google scholar as a keyword does not seem to fit what is discussed in the paper. Author response: We deleted the google scholar keyword. Statement of the problem: It identifies the lack of studies that cumulatively explain the evolution of PCK from its inception. However, the authors would consider presenting the arguments logically instead of mixing up PCK and TPACK in a zigzag way by focusing their attention first on PCK when done then TPACK. Author response: The problem statement was rewritten by focusing attention first on PCK and then TPACK. Research questions: Although the title and various places it is claimed that the study focuses on the learning of mathematics, none of the research questions is focused on mathematics. Author response: The problem statement was rewritten by focusing attention first on PCK and then TPACK. Author response: “in teaching and learning mathematics” was added. Data selection: Selection of articles is well articulated. However, the authors have not explained why they chose google scholar as the sole database for their search. They may also explain why they ended on pages 10 and 30 in search of review articles and empirical studies respectively. Author response: The choice of Google Scholar was influenced by its extensive coverage of scholarly articles and user-friendly interface, facilitating efficient searches to access a wide array of academic publications. The search ended on page 10 in search of review articles because the first seven pages exhausted all articles written in review form, such as a review of literature, systematic review, meta-analysis, etc. This means that we checked pages eight to 10, had no results, and stopped checking after page 10. Eventually, the search for empirical studies ended on page 30 because no mathematics-related paper was available. Nature of selected articles: The author concluded that “therefore, our analyses only considered journal articles” without explaining why so. Were other types of sources not relevant/fit for the study? They may consider explaining why they selected only journal articles. Author response: We considered journal articles due to their rigorous peer-review process that ensures higher academic scrutiny and quality. Writing style: The authors have written several sentences that begin with numerals. They need to rephrase them and ensure no sentence starts with a numeral. They also use one-sentence paragraphs, a style uncommon in academic writing. Author response: The journal editor suggested beginning with numerals in the pre-assessment review round. Data analysis: This section is supposed to explain how the results presented in the following sections were obtained. However, the authors seem to give more space to what the articles were about – an explanation that fits in their preceding section named “extended sources and nature of selected articles”. For example, all the tables and figures in this section are about the articles selected (when they were published, where they were published, and their subjects) and not about how the selected articles were analyzed. Only the first paragraph of the section talks about how findings were presented but even this is not preceded by how such presented finding were found through analysis. The authors may consider focusing their attention on explaining how they went about analyzing the documents they selected. Author response: This section refocused on explaining how we analyzed the documents we selected. We then moved other texts under the previous subsection of extended sources and the nature of selected articles. The following was added: “To reveal trends of the PCK framework, we identified the year of publication, author of the study, model of PCK, and construct (whether PCK or TPACK) for each study. To reveal the effectiveness of PCK and TPACK interventions, we identified the construct, theoretical framework used to frame the study, research design, teaching intervention, topic, and analysis method for each study.” Figure 1 was changed to Figure 4 and maintained in this section, showing data analysis software usage. Results and discussion: The section seems to be divided into two major subsections namely PCK framework and PCK interventions. Under each subsection are presented lists of studies that the authors discuss one after another. The studies are discussed as isolated ideas that do not inform or complement each other. There is no explanation why the second study or the thirdstudy etc. are important in/or add to the idea presented in the preceding studies. Also, there is no synthesis as to what we cannot learn from the listed studies. What is (not) common about them? What do we now learn about PCK and TPACK from them? How about mathematics? What have the studies shown in general about the learning of mathematics? Is PCK or TPACK sufficient in explaining effective learning of mathematics? What component of the frameworks should/has not been well addressed by teachers for effective learning of mathematics? Also, the studies are presented separately from each other, each having its own heading, where some are discussed using one sentence paragraph. The authors may consider explaining in a logical flow how PCK for example, is understood from some two to three papers and how this understanding is corroborated/advanced/contradicted/etc. by other studies. They may do so by connecting the ideas from the studies to one another and explicitly showing what we can now learn from the cumulative trend. Author response: Each article's discussion in more detail was changed to a more thorough connection, and discussion together to provide compromise, same trends, opposing trends, and contradiction from one to the other. Conclusion: Although the conclusion hits the point, especially in the last paragraph, the preceding paragraphs seem to supplement what may have been missed in the discussion. As such much of its content does not sound as concluding. Author response: The whole conclusion was revised." } ] }, { "id": "186613", "date": "03 Aug 2023", "name": "Jones Apawu", "expertise": [ "Reviewer Expertise Technology integration into the teaching of mathematics", "Algebraic thinking of learners", "Differentiated instructions" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAfter reading the article, which is a mapping review, there seems to be no pedagogical framework but rather PCK and TPACK frameworks. I suggest “From pedagogical content knowledge toward technological pedagogical content knowledge frameworks and their effectiveness in teaching mathematics: A mapping review. The article tried to give out the trend in both PCK and TPACK frameworks usage. The paper would be good for researchers who want to employ PCK and TPACK as their theoretical framework. However, there are some concerns under the following headings that I suggest the authors should look at.\nAbstract\n“Pedagogical or technological pedagogical content knowledge frameworks” seems as if pedagogical and TPACK are the same. Also, is crucial to inform what? It must be stated explicitly. So, this sentence “A study to reveal existing pedagogical or technological pedagogical content knowledge frameworks is crucial to inform and their effectiveness in teaching mathematics” needs recasting. “… and its effectiveness in teaching mathematics” is not specific. Is it PCK’s or TPACK’s effectiveness…? The last sentence under Background should be relooked at since it is not in relation with mathematics. The first sentence under Results, “The empirical studies that compared the role of pedagogical and technological pedagogical content knowledge model… seems like we have pedagogical model as well.\nProblem statement\nRossi and Trevisan (2018) investigated how TPCK is defined… seems like the authors are claiming Rossi and Trevisan (2018) tried to define TPCK. In 2005, it was TPCK and changed to TPACK in 2007 and context was added in 2008. The authors may wish to revisit the literature. The last sentence of 2 (see page 3), “Descriptive and inferential statistics each study got, etc.). Is it each study used or got? Look at that again.\nMethodology\n\nThe method “one-term in one-source” (see page 3) is not well known to the research community. You may wish to look at it again. The sentence, “There were many more empirical studies (articles that investigate with primary data), so we limited the search to the 30th page (entry); as you can see, the last page still had seven articles.” (see around line 5 under Figure, page 4) Should read “There were many more empirical studies (articles that investigate with primary data), so we limited the search to the 30th page (entry); as you can see, the last page still had seven articles (see Figure 1).\nData analysis\nIn Figure 4, the green colour is not showing well. It looks like grey colour. The authors referred to it as green colour (see sentence 3 under Figure 4, page 6)\nResults and discussion\nOn page 7, the first sentence under Table 2 should rather read the summary of the articles and not … in more detail.\n\nThe authors can consider looking at articles that relate to mathematics teaching as the title of the paper depicts.\n\nOn page 8, the first sentence under “Seven components of the framework”, from Table 2, it should be authors and not author.\n\nOn page 8, in the first sentence under “Components of the PSTs’ PCK investigated”, from Table 2, it should be author and not authors.\n\nThe effectiveness of using the frameworks did not show explicitly under “PCK interventions” as the title of the paper depicts.\n\nThe discussions were not done in relation to literature by connecting articles that use the frameworks to enhance effective teaching of mathematics and those that did not.\nReferences\nThe following cited works are not on the references list:\nRossi and Trevisan (2018)\n\nMiranda (2008)\nThe following references were not cited:\nDe Miranda MA: Pedagogical Content Knowledge and Technology Teacher Education: Issues for thought. Journal of the Japanese Society of Technology Education. 2008; 50(1): 17–26.\n\nDe Rossi M, Trevisan O: Technological Pedagogical Content Knowledge in the literature: how TPCK is defined and implemented in initial teacher education Technological Pedagogical Context Knowledge (TPCK) in letteratura: come viene definito e implementato il modello TPCK nei conte. Ital. J. Educ. Technol. 2018; 26(1): 7–23.\n\nIs the work 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": "10054", "date": "17 Aug 2023", "name": "Hashituky Telesphore Habiyaremye", "role": "Author Response", "response": "Reviewer comments: After reading the article, which is a mapping review, there seems to be no pedagogical framework but rather PCK and TPACK frameworks. I suggest “From pedagogical content knowledge toward technological pedagogical content knowledge frameworks and their effectiveness in teaching mathematics: A mapping review. The article tried to give out the trend in both PCK and TPACK frameworks usage. The paper would be good for researchers who want to employ PCK and TPACK as their theoretical framework. However, there are some concerns under the following headings that I suggest the authors should look at. Author response: Thank you so much for your guidelines. We have adapted to the suggested title by including “…content knowledge…” Abstract “Pedagogical or technological pedagogical content knowledge frameworks” seems as if pedagogical and TPACK are the same. Also, is crucial to inform what? It must be stated explicitly. So, this sentence “A study to reveal existing pedagogical or technological pedagogical content knowledge frameworks is crucial to inform and their effectiveness in teaching mathematics” needs recasting. “… and its effectiveness in teaching mathematics” is not specific. Is it PCK’s or TPACK’s effectiveness…? The last sentence under Background should be relooked at since it is not in relation with mathematics. The first sentence under Results, “The empirical studies that compared the role of pedagogical and technological pedagogical content knowledge model… seems like we have pedagogical model as well. Author response: The first sentence of the background was changed as follows: “pedagogical” was replaced by “pedagogical content knowledge”, and “is crucial to inform and their effectiveness in teaching mathematics” was replaced by “and their effectiveness in teaching mathematics is crucial to inform reader, teacher and researcher.” The last sentence under background was changed as follows” “its” was replaced by “their.” We added “content knowledge” after “pedagogical” in the sentence under results. Problem statement Rossi and Trevisan (2018) investigated how TPCK is defined… seems like the authors are claiming Rossi and Trevisan (2018) tried to define TPCK. In 2005, it was TPCK and changed to TPACK in 2007 and context was added in 2008. The authors may wish to revisit the literature. The last sentence of 2 (see page 3), “Descriptive and inferential statistics each study got, etc.). Is it each study used or got? Look at that again. Author response: The whole section was revisited to ensure clarity. “got” was changed to “used.” Methodology The method “one-term in one-source” (see page 3) is not well known to the research community. You may wish to look at it again. The sentence, “There were many more empirical studies (articles that investigate with primary data), so we limited the search to the 30th page (entry); as you can see, the last page still had seven articles.” (see around line 5 under Figure, page 4) Should read “There were many more empirical studies (articles that investigate with primary data), so we limited the search to the 30th page (entry); as you can see, the last page still had seven articles (see Figure 1). Author response: We invented the “one-term in one source” method. We changed the sentence as you suggested: \"There were many more empirical studies (articles that investigate with primary data), so we limited the search to the 30th page (entry); as you can see, the last page still had seven articles (see Figure 1).\" Data analysis In Figure 4, the green colour is not showing well. It looks like grey colour. The authors referred to it as green colour (see sentence 3 under Figure 4, page 6) Author response: Thank you! The grey replaced green, and the yellow replaced purple. Results and discussion ○ On page 7, the first sentence under Table 2 should rather read the summary of the articles and not … in more detail. ○ The authors can consider looking at articles that relate to mathematics teaching as the title of the paper depicts. ○ On page 8, the first sentence under “Seven components of the framework”, from Table 2, it should be authors and not author. ○ On page 8, in the first sentence under “Components of the PSTs’ PCK investigated”, from Table 2, it should be author and not authors. ○ The effectiveness of using the frameworks did not show explicitly under “PCK interventions” as the title of the paper depicts. ○ The discussions were not done in relation to literature by connecting articles that use the frameworks to enhance effective teaching of mathematics and those that did not. Author response: ○“summary” replaced “in detail.” ○Papers discussing mathematics teaching were identified in Tables 2 and 3. ○We added “s” on “author.” ○“The effectiveness of using…” was added to the “PCK interventions.” ○We removed “s” on “authors.” ○The discussion connected articles instead of discussing each article separately. References The following cited works are not on the references list: ○ Rossi and Trevisan (2018) ○ Miranda (2008) The following references were not cited: De Miranda MA: Pedagogical Content Knowledge and Technology Teacher Education: Issues for thought. Journal of the Japanese Society of Technology Education. 2008; 50(1): 17–26. ○ De Rossi M, Trevisan O: Technological Pedagogical Content Knowledge in the literature: how TPCK is defined and implemented in initial teacher education Technological Pedagogical Context Knowledge (TPCK) in letteratura: come viene definito e implementato il modello TPCK nei conte. Ital. J. Educ. Technol. 2018; 26(1): 7–23. Author response: There was an error in writing these names, Miranda instead of De Miranda and Rossi instead of De Rossi" } ] } ]	1	https://f1000research.com/articles/11-1029
https://f1000research.com/articles/12-996/v1	17 Aug 23	{ "type": "Research Article", "title": "Measurement uncertainty interval in case of a known relationship between precision and mean", "authors": [ "Steffen Uhlig", "Bertrand Colson", "Petra Gowik", "Bertrand Colson", "Petra Gowik" ], "abstract": "Background: Measurement uncertainty is typically expressed in terms of a symmetric interval y±U, where y denotes the measurement result and U the expanded uncertainty. However, in the case of heteroscedasticity, symmetric uncertainty intervals can be misleading. In this paper, a different approach for the calculation of uncertainty intervals is introduced. Methods: This approach is applicable when a validation study has been conducted with samples with known concentrations. In a first step, test results are obtained at the different known concentration levels. Then, on the basis of precision estimates, a prediction range is calculated. The measurement uncertainty for a given test result can then be obtained by projecting the intersection of the test result with the limits of the prediction range back onto the axis of the known values, now interpreted as representing the measurand. Results: It will be shown how, under certain circumstances, asymmetric uncertainty intervals arise quite naturally and lead to more reliable uncertainty intervals. Conclusions: This article establishes a conceptual framework in which measurement uncertainty can be derived from precision whenever the relationship between the latter and concentration has been characterized. This approach is applicable for different types of distributions. Closed expressions for the limits of the uncertainty interval are provided for the simple case of normally distributed test results and constant relative standard deviation.", "keywords": [ "In-house validation study", "reproducibility precision", "measurement uncertainty", "prediction interval", "uncertainty interval" ], "content": "Introduction\n\nIn this paper, a “top-down” approach for the calculation of measurement uncertainty is presented, in the sense that the estimate of measurement uncertainty is supported by precision data from a validation study.\n\nMeasurement uncertainty is defined in JCGM 100 as a parameter that “characterizes the dispersion of the values that could reasonably be attributed to the measurand”.1 This parameter is often expressed as a standard deviation which is then used to obtain a symmetric uncertainty interval around the measurement result. In the following, the approach yielding symmetric uncertainty intervals will be referred to as the y±U approach.\n\nAs will be discussed below, in the case of heteroscedasticity, the y±U approach can yield misleading uncertainty intervals. For this reason, a different approach for determining measurement uncertainty is presented. This approach is suitable when precision data from a validation study conducted with test samples with known concentrations are available. While the focus in this paper is in-house validation, the approach presented here can also be applied for data from an interlaboratory validation study. It will be shown how, under certain circumstances, asymmetric uncertainty intervals arise quite naturally. For this reason, the approach presented here will be referred to in the following as the asymmetric measurement uncertainty approach (short form: asymmetric approach).\n\nThe asymmetric approach is perfectly consistent with the JCGM definition given above. Indeed, in the asymmetric approach, the focus is explicitly on the “dispersion of values which could reasonably be attributed to the measurand.” Furthermore, it draws the same distinction between measurand (Y) and measurement (Ym) as JCGM 106.2 Even though the approach presented here is not Bayesian, there are important connections with JCGM 106, such as the definition of the best estimate of the measurand as EYYm.\n\nFirst, an experimental design for an in-house validation study and a statistical model are presented, allowing the calculation of in-house reproducibility precision as a function of concentration. Then it is shown how y±U uncertainty intervals are calculated from such precision data. The inconsistencies of the y±U approach are then discussed, and the asymmetric approach is presented. Throughout, the various concepts are illustrated with examples.\n\n\nMethods\n\nIt is assumed that an in-house validation study has been conducted with samples with known concentrations. In the simplest case, the samples with known concentrations are obtained by diluting certified reference material. For each concentration level, several measurement results are obtained under in-house reproducibility conditions. This is best achieved via a factorial design, such as described in ISO/TS 23471.3\n\nThe following table (Table 1) provides an example of a factorial design with 7 factors and 8 factor level combinations.\n\nMore generally, the number of factor level combinations is denoted n. With m (known) concentration levels and p replicates per concentration level, a total of m∙p∙n measurements are performed.\n\nThe statistical model (mixed linear model) is as follows:\n\nYijk denotes the measurement result at concentration level i=1,…,m for factor level combination j=1,…,n and replicate k=1,…,p\n\nxij denotes the known concentration,\n\nα and β−1 denote the absolute and the relative components of method bias. The α+β∙x curve represents the expected measured concentration at known concentration x and will be referred to in the following as the mean curve.\n\nAj and Bj denote the absolute and the relative components of the effect of factor level combination j (each of these terms results from summing effects of the individual factors).\n\nAj and Bj denote the absolute and the relative components of block effect j\n\naijk and bijk denote the absolute and the relative components of the repeatability error for measurement result Yijk.\n\nIn the following, two in-house validation data sets will be considered: Thiamphenicol in milk (Example 1) and Clopidol in egg (Example 2).\n\nThe following table (Table 2) provide test results for Example 1 (m=4 known concentration levels, p=1 replicate).\n\nThe test results for Example 1 are displayed in Figure 1.\n\nThe following table (Table 3) provides test results for Example 2 (m=6 known concentration levels, p=1 replicate).\n\nThe test results for Example 2 are displayed in Figure 2.\n\nFor each example, reproducibility standard deviation values are calculated by means of Equation 1. With the exception of the two fixed effects α and β, the terms on the right side of Equation 1 are modelled as random variables. The corresponding variance components are denoted as follows:\n\nA1s,…,Aqs are random variables with zero mean and variances σA,12,…,σA,q2, respectively\n\nB1s,…,Bqs are random variables with zero mean and variances σB,12,…,σB,q2, respectively and where\n\ns1j…sqj denotes the vector of factor levels for the q factors and for factor level combination j\n\nMoreover,\n\nA1,…,An are random variables with zero mean and variance σA2\n\nB1,…,Bn are random variables with zero mean and variance σB2.\n\na111,…,amnp are random variables with zero mean and variance σa2\n\nb111,…,bmnp are random variables with zero mean and variance σb2\n\nThe estimation of variance components in mixed linear models is described, for example, in Searle et al.,4 McCulloch et al.5 and Clarke.6 Estimates for Example 1 are provided Table 4.\n\nThe in-house precision parameters for Example 1 for four different concentrations are provided in Table 5. At a given known concentration x, the in-house reproducibility standard deviation is calculated as follows:\n\nThe following two tables (Table 6 & Table 7) provide variance and precision estimates for Example 2.\n\nThe estimate of reproducibility at concentration x (see Equation 2) can be used to derive an estimate of the standard measurement uncertainty for the measurand Y as a function of x:\n\nThe expanded measurement uncertainty is then obtained as follows:\n\nwhere k denotes the coverage factor.\n\nIt should be noted that it may be necessary to include the uncertainty of bias correction and the uncertainty of the (certified reference) values for the known concentrations used in the validation study, as appropriate. (see step 3 of Asymmetric measurement uncertainty approach).\n\nBased on the precision values from the previous section, and using k=2 (for the sake of simplicity), one obtains the following expanded uncertainty values (Table 8 & Table 9):\n\nTypically, the expanded measurement uncertainty is used to construct a measurement uncertainty interval of the form y±U. Such symmetric measurement uncertainty intervals are not always appropriate. This will be demonstrated on the basis of a theoretical example.\n\nIn this example, it is assumed that the relative measurement uncertainty is already known and is constant at 35% across the applicable range of concentrations as specified in the scope of the analytical method. If the measurement result is y=10 (in this theoretical example, the unit plays no role and is therefore suppressed), then, applying the y±U approach with k=2 (for the sake of simplicity) and with the standard uncertainty obtained from the known 35% relative standard deviation (RSD) value, a measurement uncertainty interval of [3, 17] is obtained. According to the JCGM definition of measurement uncertainty discussed in the introduction, this means that the value y=3 could reasonably be attributed to the measurand. However, if the measurement result had been y=3 (instead of y=10), applying the same 35 % relative measurement uncertainty would result in an uncertainty interval of [0.9, 5.1]. Since the original value of y=10 does not lie within this second interval, it can be seen that the y±U approach is inconsistent in such a situation.\n\nThe two uncertainty intervals just discussed are summarized in the following table (Table 10).\n\n(In this theoretical example, the unit plays no role and is therefore suppressed.)\n\nThe inconsistency of the y±U approach discussed above is illustrated in the following figure (Figure 3). Due to the constant RSD of 35 %, the width of the uncertainty interval (in the diagram: the height of the uncertainty interval) depends on the measurement result. Considered on its own, each uncertainty interval characterizes the dispersion of values that could be reasonably be attributed to the measurand on the basis of the measurement result. However, considered together, the different uncertainty intervals display inconsistencies. In particular, the value 10 does not lie within the uncertainty interval for the measurement result 3, even though the latter lies within the uncertainty interval constructed around the former.\n\n(In this theoretical example, the unit plays no role and is therefore suppressed).\n\nThe inconsistency of the y±U approach will now be shown on the basis of the uncertainty intervals for Example 2 provided in Table 9. At a Clopidol concentration of 4 μg/kg Clopidol, the y±U approach yields the uncertainty interval y ± 1.19 μg/kg. In routine testing, if the measurement result is y=4 is obtained, it will be concluded that values above 5.19 μg/kg cannot reasonably be attributed to the measurand. However, from the same Table 9, we know that the lower limit of the uncertainty interval for a sample with 5.5 μg/kg Clopidol concentration is 3.88 μg/kg (or 3.98 μg/kg if bias is considered). In other words, we know that a measurand value of 5.5 μg/kg is perfectly compatible with a measurement result of 4 μg/kg. It follows that the value 5.5 μg/kg should lie within the measurement uncertainty interval for a measurement result of 4 μg/kg. This is not the case for the uncertainty interval obtained via the y±U approach. This is illustrated in the following figure (Figure 4).\n\nFor a measurement result of 4 μg/kg (say in routine testing), values above 5.19 do not lie within the uncertainty interval. However, according to the evaluation of the data from the in-house validation study, measurement results ≤ 4 μg/kg were consistent with a measurand value of 5.5 μg/kg.\n\nAs has just been seen, in the presence of heteroscedasticity, the y±U approach leads to inconsistencies. For this reason, a different approach for the determination of measurement uncertainty in the case to heteroscedastic data is required. Such an approach will now be presented.\n\n\nAsymmetric measurement uncertainty approach\n\nStep 1: prediction range\n\nFor each known concentration, the distribution of test results can be characterized in terms of a prediction interval. This interval reflects the degree to which the test results agree with one another, at the given concentration level and under the specified conditions (e.g. repeatability or in-house reproducibility). For a chosen prediction probability level (e.g. ppred=95%), a subsequent test result will lie inside the prediction interval with probability ppred.\n\nIf measurements are performed at different known concentrations, then it is possible to construct a prediction range, rather than individual prediction intervals. This step involves applying one statistical model to all the data, as described the previous section. The construction of a prediction range is described in the following figure (Figure 5).\n\nFor each known concentration, the diamonds represent the measurement results and the solid vertical line represents the prediction interval. The prediction range is represented by the dashed lines.\n\nFor further information regarding the computation of a prediction range, the reader is referred to the discussion of variance functions in ISO 5725-27 and to the in-house validation approach described in Gowik et al.8 and Jülicher et al.9 [1].\n\nStep 2: measurement uncertainty interval\n\nOnce a prediction range has been calculated, the measurement uncertainty interval for a given test results (obtained e.g. in routine testing) can be determined. Step 2 no longer involves the data from the validation study. Rather, for Step 2, it is assumed that a prediction range has previously been calculated and is thus available.\n\nAccordingly, the meaning of the axes is now different. The vertical axis now represents the measurement result obtained, say, in routine testing and denoted ym, while the horizontal axis now represents the measurand (which is to be characterized via the measurement) denoted y (This notation is chosen so as to be consistent with JCGM 106, though, strictly speaking, ym is one realization of a random variable Ym and y is one realization of a random variable Y. However, a distinction between random variables and their realizations is not required in this paper.).\n\nThe starting point for Step 2 is a test result, displayed on the ym-axis. The intersection of the ym-value with the upper prediction curve is projected onto the y-axis to obtain the lower measurement uncertainty limit. Indeed, for measurand values below this y-value, measurement results can be expected to be less than the ym-value. Secondly, the intersection of the ym-value with the lower prediction curve is projected onto the y-axis to obtain the upper measurement uncertainty limit. Indeed, for a measurand values above this y-value, measurement results can be expected to be greater than the ym-value. The resulting y-axis interval (grey horizontal band hugging the y-axis) thus corresponds to the values which could “reasonably be attributed to the measurand.”\n\nThis procedure is illustrated in Figure 6.\n\nStep 3: best estimate for the measurand\n\nThe “best estimate of the measurand” is the projection onto the y-axis of the intersection of the measurement result (ym-value) with the mean curve α+β∙y (see previous section with the measurand y replacing the known concentration x). If the bias is negligible (i.e. α is close to zero and β is close to 1), then this y-value will be close to the ym-value. However, if a bias is present, then the two values will differ, and taking the y-axis projection corresponds to a bias or recovery correction. For this reason, the y-axis projection is denoted ycorr. If such a bias or recovery correction is performed, it is recommended to include the “uncertainty of the bias correction” (see Ref. 1) as an additional variance component in the calculation of the prediction intervals at each known concentration x during the validation study. The uncertainty of bias correction, in turn, may consist of various sources of uncertainty such as the statistical uncertainty of the parameters α and β and the uncertainty of reference values used as known concentrations.\n\nFinally, it should be noted that the symmetry of the measurement uncertainty interval is determined in relation to ycorr.\n\nIn this section, it is shown how to calculate the lower and upper limits of asymmetric measurement uncertainty intervals.\n\nIt is assumed that there is no bias and that, for each concentration level in the recovery experiment, the measurement results follow a normal distribution.\n\nLet yL denote the lower limit of the expanded uncertainty interval and yU the corresponding upper limit. Furthermore, let fUy denote the upper limit of the prediction interval at measurand value y, and let fLy denote the lower limit of the prediction interval at y. For a given measurement result ym, the two limits yL and yU can then be computed iteratively, using the implicit relationships\n\nIn the case of a constant relative standard deviation σrel across all concentration levels, and in the absence of bias, explicit expressions for yL and yU can be provided. Indeed, in such a situation, a given measurement result ym obtained to characterize the (unknown) measurand y will lie with 95 % probability [2] in the interval y−2∙y∙σrely+2∙y∙σrel, i.e.\n\nThe above inequality can be rewritten as\n\nThus, the measurand y will lie in the interval ym1+2∙σrelym1−2∙σrel with 95% probability.\n\nFor example, for a constant relative standard deviation of 40% (i.e. σrel=0.4) and for the measurement result ym=100, the lower and upper uncertainty limits are computed as:\n\nAs long as the exact quantile is used and the assumptions are valid, this uncertainty interval is statistically exact in the sense that the coverage probability is exactly 95%.\n\nWhen is the proposed uncertainty interval symmetric?\n\nIf the prediction limits run parallel to the mean curve α+β∙x (see Section 0), the uncertainty interval is perfectly symmetric. This is the case when it is the absolute rather than the relative standard deviation which is constant across concentration levels. It should also be noted that, when the prediction limits run parallel to the mean curve α+β∙x and when there is no bias (100 % recovery), then the prediction and measurement uncertainty intervals are identical. This is illustrated in the following figure (Figure 7).\n\nIf there is no bias and if the prediction limits run parallel to the mean curve α+β∙x, then the intervals obtained from the two approaches are identical.\n\nThe assumption that an uncertainty interval is symmetric is justified if the following two conditions are met:\n\nCondition 1: Measurement results are distributed symmetrically around the corresponding mean value at a given known concentration in the validation study.\n\nCondition 2: Heteroscedasticity is negligible.\n\nWhen is the proposed uncertainty interval asymmetric?\n\nIn the field of analytical chemistry, in most cases, the distribution of results obtained from measurements performed on one and the same sample is more or less symmetric, so that symmetry condition 1 above is met. Condition 2, however, is almost never met. In the case of weakly heterogeneous variances, uncertainty intervals may still be approximately symmetric. Similarly, if the spread of measurement results at any given concentration level remains small (i.e. the relative standard deviations are small), uncertainty intervals may still be approximately symmetric. However, if the dispersions vary considerably across concentration levels and the corresponding relative standard deviations are large (say, greater than 10 %), then it is necessary to take the asymmetry of the uncertainty intervals into account. Restricting the concentration range under consideration in order to avoid variance heterogeneity is common analytical practice. In some cases, this expedient may allow the symmetry assumption to be applied.\n\nThe following figure (Figure 8) illustrates the relationship between magnitude of dispersion and symmetry of the uncertainty interval.\n\nThe degree of asymmetry depends on the magnitude of the variance.\n\n\nApplying the alternative approach to the data from the factorial in-house validation studies (Example 1 and Example 2)\n\nThe uncertainty intervals for Example 1 and Example 2 are provided in the following tables (Table 11 & Table 12). The degree of asymmetry of a given uncertainty interval can be gauged by comparing the values of the differences ycorr−yL and yU−ycorr (See earlier sections for the notation yL, yU and ycorr.). The two values ycorr−yL and yU−ycorr can also be compared with the expanded uncertainty from the y±U approach. Accordingly, the U values from Table 8 and Table 9 are reproduced here for convenient reference.\n\nAs can be seen, for Example 1, the values for the differences ycorr−yL and yU−ycorr lie relatively close to one another and to the U values. By contrast, for Example 2, the difference values differ considerably from one another and from the U values. For instance, for ym=2 μg/kg, we have\n\n\n\nThe calculation of the upper and lower limits, as well as the best estimate of the measurand ycorr is illustrated in the following figure (Figure 9).\n\n\nConclusions\n\nAs has been seen, asymmetric measurement uncertainty intervals arise naturally in the case of heteroscedasticity. Accordingly, in such cases, the symmetric uncertainty interval y±U should be seen as a mere approximation of the exact measurement uncertainty interval. This approximation is adequate if variability is low (say, less than 10 % RSD). In the case of both high variability and heteroscedasticity, symmetric measurement uncertainty intervals can be misleading. An awareness of these issues is thus particularly important in fields where these two conditions are expected, such as chemical trace analysis.\n\nIn the examples considered in this paper, it is assumed that data follow a normal distribution. If data follow another distribution, the uncertainty interval is different. Lognormal data are a familiar case in point. Indeed, consider the case that for any given concentration x, the log-transformed test results can be expected to lie between lnx−2σ and lnx+2σ. Then, in the original domain (i.e. prior to the log-transformation), the RSD will remain constant across concentration levels and the dispersion will thus incre ase monotonically with the concentration.\n\nIn many cases a log-transformation stabilizes the variance, meaning that, in the log domain, all the data are normally distributed with one and the same standard deviation σ, independently of the concentration. Back-transformation (“anti-log”) then again provides asymmetric uncertainty intervals, from y/expkσ to y∙expkσ. These intervals are asymmetric, but to a lesser extent that the interval derived under the assumption that the original data follow a normal distribution. Take the case σ=RSD=0.25 and k=2. Then the asymmetric uncertainty interval based on the assumption that the original data follow a normal distribution (see Equation 5) is y1.52y, and the asymmetric uncertainty interval based on the log-normal distribution is y1.651.65y.\n\nHowever, a log-transformation does not always have this variance stabilization effect, even if the data follow a lognormal distribution. For instance, if the standard deviations in the original domain are constant across concentrations, then, in the log domain, the σ value will depend on the concentration.\n\nMore generally, this article establishes a conceptual framework in which measurement uncertainty can be derived from precision whenever the relationship between the latter and concentration has been characterized. Closed expressions for the limits of the uncertainty interval were provided for the simple case of normally distributed test results and constant relative standard deviation. In more complex cases, it may not be possible to provide closed expressions. However, iterative calculation procedures can be applied, and further work may be required to illustrate appropriate context-specific approaches.", "appendix": "Data availability\n\nNo other data were used than those provided in Table 2 and Table 3.\n\n\nReferences\n\nJCGM 100: 2008 Evaluation of measurement data – Guide to the expression of uncertainty in measurement.\n\nJCGM 106: 2008 Evaluation of measurement data – The role of measurement uncertainty in conformity assessment.\n\nISO/TS 23471:2022 Experimental designs for evaluation of uncertainty – Use of factorial designs for determining uncertainty functions.\n\nSearle SR, Casella G, McCulloch CE: Variance Components. John Wiley & Sons; 1992. Publisher Full Text\n\nMcCulloch CE, Searle SR, Neuhaus JM: Generalized, Linear and Mixed Models. 2nd ed.John Wiley & Sons; 2008.\n\nClarke BR: Linear Models: The Theory and Application of Analysis of Variance. John Wiley & Sons; 2008. Publisher Full Text\n\nISO 5725-2 (2002) Accuracy (trueness and precision) of measurement methods and results – Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method.\n\nGowik P, Jülicher B, Uhlig S: Multi-residue method for non-steroidal anti-inflammatory drugs in plasma using high-performance liquid chromatography-photodiode-array detection: Method description and comprehensive in-house validation. J. Chromatogr. 1998; 716: 221–232. PubMed Abstract \| Publisher Full Text\n\nJülicher B, Gowik P, Uhlig S: A top-down in-house validation based approach for the investigation of the measurement uncertainty using fractional factorial experiments. Analyst. 1999; 124: 537–545. Publisher Full Text\n\nCommission Decision 2002/657/CE of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results.\n\nCommission Implementing Regulation (EU) 2021/808 of 22 March 2021 on the performance of analytical methods for residues of pharmacologically active substances used in food-producing animals and on the interpretation of results as well as on the methods to be used for sampling and repealing Decisions 2002/657/EC and 98/179/EC.\n\n\nFootnotes\n\n1 This in-house validation approach is referenced in CD 657 10 and CIR 808. 11\n\n2 For the sake of legibility, the exact 95 % quantile has been replaced by the value 2." }	[ { "id": "197911", "date": "13 Nov 2023", "name": "Bertil Magnusson", "expertise": [ "Reviewer Expertise My area of research is quality if measurement . e.g. QC", "validation und uncertainty and quality of decision on a result comparing with a specification" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article introduces an important issue that a high relative uncertainties the uncertainty interval is not symmetric but asymmetric. It is well written and clear. The examples are easy to follow.\n\nSome minor comments:\n1 The conclusion as stated above is missing in the abstract In the article.\n2 It would be helpful for the reader if constant RSD, which is very common for instrumental techniques at higher concentrations, is described as one form of heteroscedasticity.\n3 The very important issue if uncertainty is related to the measured value or to the true value is just mentioned briefly and could have been expanded - it is important when the RSD is constant.\n\"Furthermore, it draws the same distinction between measurand (YY) and measurement (YmYm) as JCGM 106.2 Even though the approach presented here is not Bayesian, there are important connections with JCGM 106, such as the definition of the best estimate of the measurand as E(Y∣∣Ym)EYYm.\"\n4 The value of 10 % RSD is given without any examples for the reader to understand when it is critical .\"However, if the dispersions vary considerably across concentration levels and the corresponding relative standard deviations are large (say, greater than 10 %), then it is necessary to take the asymmetry of the uncertainty intervals into account.\"\n5 Table captions should be easy to read without referring to much to the text - now just example 1 and 2. One is low and one is higher RSD\n\nIs the work 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": "232307", "date": "17 Jan 2024", "name": "Chong Wang", "expertise": [ "Reviewer Expertise uncertainty quantification and propagation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 proposes a analysis framework to predict the measurement uncertainty interval in case of a known relationship between precision and mean. The research topic is interesting, and some minor comments are given as follows. 1. Many abbreviations and mathematical symbols have been used in the manuscript. For better understanding to the readers, a nomenclature presented in tabular form is suggested. 2. If possible, some existing methods for interval uncertainty prediction are suggested for comparison to verify the proposed method. 3. Some of the figures, e.g. Fig.5, can be further improved to present the results more clearly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "232361", "date": "24 Jan 2024", "name": "Hariyanto Gunawan", "expertise": [ "Reviewer Expertise Intelligent manufacturing system platform development", "Artificial Intelligence", "Internet of Things", "Big data analysis", "Industry 4.0 applications", "real time monitoring." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 introduced a different approach for the calculation of uncertainty intervals. This is an interesting topic due to the existence of uncertainty in measurement. However, I have some comments as follows: 1. Give the full name of the abbreviation JCGM in the beginning. 2. In Figure 1, the value of the horizontal and vertical axis must show the same number as in Table 2. Similar for Figure 2 and Table 3. 3. The author should add a state of the art so the readers can understand the recent related research. 4. In Figure 5, add units for the horizontal and vertical axis.\n\n5. The author should directly write Table 1 instead of using the following Table (Table 1). Same for other tables and figures.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": [] }, { "id": "232320", "date": "20 Feb 2024", "name": "Elcio Cruz de Oliveira", "expertise": [ "Reviewer Expertise Chemical Metrology", "focusing in measurement uncertainty", "measurement uncertainty arising from sampling and use of uncertainty information in compliance assessment." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 aimed to present a different approach for the calculation of uncertainty intervals in the case of heteroscedasticity. However, it is not clear in the abstract.\nAfter reading the entire document, I have some major comments:\n1 – I suggest that the keywords be placed in alphabetical order.\n2 – Introduction: Please clarify the relationship between “heteroscedasticity” and “asymmetric uncertainty intervals”.\n3 – Table 1. Factorial design with 7 factors, each with two levels. Did the authors use a Plackett–Burman design? Please clarify it better, explaining the reason and inserting more references.\n4 – Table 2. Example 1 – design and test results from an in-house validation study for Thiamphenicol in milk. It seems that the authors used a fractional factorial design, 24-1, resolution IV. If I am correct, I did not understand the correlation between Table 2 and Table 1. Please, explain it better.\n5 – Eqs. (1) and (2): Do the data present homoscedastic or heteroscedastic behavior? Did the authors use a weighted linear regression or an unweighted linear regression? See references: Farias et al (2015) [Ref-1] and Caldeira et al (2017) [Ref-2] 6 – Page 5: “… the in-house reproducibility…”, this term is not introduced in JCGM 200:2012. I assume the authors mean to refer to “intermediate precision”.\n7 – I recommend that authors include the Monte Carlo Method (MCM) to validate their study.\n8 – Conclusions should be more succinct and objective and future work requires greater clarification.\n\nIs the work 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": [] } ]	1	https://f1000research.com/articles/12-996
https://f1000research.com/articles/12-992/v1	16 Aug 23	{ "type": "Research Article", "title": "Predicting systemic lupus erithematosus disease activity index 2000 (Sledai 2k) score after pulse dose metyl prednisolone in severe systemic lupus erithematosus", "authors": [ "Ayu Paramaiswari", "Nyoman Kertia", "Umi Intansari", "Dhite Bayu Nugroho", "Ayu Paramaiswari", "Nyoman Kertia", "Umi Intansari" ], "abstract": "Background: Systemic lupus erythematosus (SLE) is a chronic multisystem rheumatic disease characterized by developing autoantibodies against nucleus antigen. It has a broad range of clinical symptoms and the potential to affect nearly all organs and tissues. Pulse dosage methylprednisolone (MEP) is the immunosuppression modality for life-threatening or organ-threatening SLE. However, it is challenging to estimate the MEP response rate. We combine clinical symptoms, routine laboratory examinations, and more specific protein examinations such as soluble B-cell activating factor (sBAFF), B-cell activating factor receptor (BAFF-R), and Interferon gamma-induced protein 10 (IP-10) to develop a formula that can predict the SLE Disease Activity Index 2K (SLEDAI 2K) score following a pulse dosage of methylprednisolone. Methods: In a prospective cohort study, patients with severe SLE with a SLEDAI 2K score of 12 or Lupus nephritis class III or IV according to WHO criteria were given methylprednisolone 500 mg/day for three consecutive days. Enzyme-linked immunosorbent assay (ELISA) tested blood samples for soluble (s) BAFF, IP 10, and flow cytometry for BAFF-R, CD-19. The SLEDAI 2K score was reevaluated after a pulse dose of methylprednisolone was administered. All statistical analyses were conducted using the Rstudio program. Results: Overall, 80 patients were included. Multivariate multiple regression analysis revealed that urine protein creatinine ratio (UPCR) (x1), CD19 percentage (x2), serum BAFF (x3), vasculitis (x4), and rash (x5) taken before MEP pulse were predictors for SLEDAI 2k score after pulse dose methylprednisolone in severe SLE with the formula 13.41+ (0.0008542 * x1) + -0.1829338 * x2) + (0.0008776 * x3) + (7.1801728 * x4) + (7.5429676 * x6). Conclusions: Formula to predict SLEDAI 2k score after MEP pulse was 13.41+ (0.0008542 * x1) + -0.1829338 * x2) + (0.0008776 * x3) + (7.1801728 * x4) + (7.5429676 * x6). Further validation is needed to be used in clinical practice.", "keywords": [ "Systemic Lupus Erythematosus", "SLEDAI 2K", "BAFF", "methylprednisolone pulse dose" ], "content": "Introduction\n\nSystemic lupus erythematosus (SLE) is a chronic multisystem autoimmune rheumatic disease characterized by autoantibody production towards nuclear antigen.1 Almost all patients with SLE have antinuclear antibodies (98%), and some also have autoantibodies against double-stranded DNA (dsDNA) (76%), hypocomplementemia (71%), and antibodies to Ro (SSA) (35%). SLE has a wide range of clinical symptoms and the potential to affect nearly all organs and tissues.2\n\nIn the 1970s, a pulse dose of 500-1000 mg/day for 1 to 3 days of MEP was established to treat life-threatening complications associated with SLE.3 Pulse-dose MEP is the fastest immunosuppression therapy for life- or organ-threatening SLE.3 It is effective for renal involvement and other severe symptoms such as pulmonary hemorrhage and neuropsychiatry.3 Conventional treatment with intravenous MEP can reduce activity and complement activation in lupus nephritis.3 However, some patients continue to develop increasing renal impairment, and even those with favorable responses are at risk for relapse. Currently, objective measures to estimate the rate of response to pulse-dose MEP are unavailable, and the safety profile for the increased risk of infection is high.\n\nSixty to seventy percent of SLE patients have elevated blood BAFF levels, which positively correlate with SLEDAI 2k and anti-dsDNA levels.4 In addition, the BLISS-52 clinical trial demonstrated that belimumab, an inhibitor of BAFF, can reduce serology activity, prevent flares, and reduce corticosteroid use.5 A study by Milz et al. showed that blood BAFF levels were elevated compared to healthy controls in different autoimmune diseases, such as Idiopathic Thrombocytopenic Purpura (ITP), and that Glucocorticoid (GC) therapy dramatically reduces serum BAFF levels.6 This may be related to GC receptor binding in the promoter region.6\n\nInterferon-gamma-induced protein 10 is a member of T-helper 1 lymphocyte chemokines produced and released in response to stimulation by infiltrating monocytes, macrophages, and endothelial cells.7 These cells subsequently bind to the T-lymphocyte-expressed CXCR3 receptor, essential for T-lymphocyte trafficking into SLE patients’ afflicted organs.8 IP-10 levels are significantly higher in the acute phase of SLE.9 Additionally, SLE treatment modalities such as prednisolone, mycophenolate, and hydroxychloroquine significantly lower serum IP-10 levels.10\n\nThe goal of this study is to use clinical manifestations, routine laboratory examinations, and more specific protein examinations such as sBAFF, BAFF-R, and IP-10 to predict SLEDAI 2k score following pulse-dose MEP, given that the spectrum of clinical manifestations and response to treatment varies from patient to patient.\n\n\nMethods\n\nThis study is a prospective cohort observational analysis that examines data on patients with systemic lupus erythematosus (SLE) who met the criteria of the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) 2019 or had class III or IV lupus nephritis (LN) according to World Health Organization (WHO) criteria. These patients underwent a follow-up period after receiving a daily dose of 500 mg MEP pulse for three consecutive days, during which their progress and response to the treatment were closely monitored and assessed.\n\nThe study included patients between the ages of 15 and 50 years who were willing to participate and did not have comorbidities such as pneumonia, tuberculosis, viral, fungal, parasitic, or protozoal infections, sepsis, diabetes mellitus, Graves’ disease, cardiovascular disease, or cancer. Additionally, patients were excluded if they had overlap syndrome with systemic sclerosis, rheumatoid arthritis, or dermatomyositis inflammation. Subjects were withdrawn from the study if they died or developed sepsis during the observation period. Patients who met the inclusion and exclusion criteria were informed about the study’s purpose, process, and potential risks and benefits, and asked to sign a consent form to participate. The explanation also highlighted the advantages and disadvantages of participating, allowing patients to make informed decisions about their involvement.\n\nThe determination of our study’s sample size was guided by the “rule of thumb” for multivariate analysis, which stipulates that a minimum of 15 samples per predictor is needed to ensure robust statistical analysis. Since we aimed to investigate five predictor variables in this study, the minimum required sample size was calculated as 75. To account for potential dropouts and missing data, which are common occurrences in research studies, we increased this number by an additional 10%. This precautionary measure resulted in a total minimum sample size of 83. By adhering to this method, we ensured that our sample was statistically adequate for the multivariate analysis of the five predictor variables.\n\nBefore administering the pulse dose of MEP, all information on the SLE Disease Activity Index 2K (SLEDAI 2K) form was completed, and the SLEDAI 2K score was recorded.11 Blood samples, typically ranging from 5 to 10 milliliters, were carefully collected from each patient using a standard venipuncture procedure. The samples were then processed and analyzed for the levels of sBAFF, IP-10, and BAFF-R. Specifically, the concentrations of sBAFF and IP-10 were measured using enzyme-linked immunosorbent assay (ELISA) techniques, while BAFF-R expression was analyzed through flow cytometry. Following the 3-day pulse dosage of MEP, blood samples were collected again using the same procedure, and these parameters were re-analyzed to assess and compare the changes in response to the treatment.\n\nMeasurement of sBAFF and IP-10 were measured using ELISA\n\nS BAFF was examined in PRODIA LABORATORIES, CAP certified, using Microplate Reader Biorad model 680Bio-rad Laboratories inc, CA, USA. Reagent kit used in this study was Quantikine® HS ELISA Human BAFF/BLyS/TNFSF13B (R&D Systems, Inc., Minneapolis, USA) according to standard protocols. The results were measured in pg/ml.\n\nMeasurement of R BAFF and B cells (CD19) using flowcytometry\n\nR BAFF and B cells (CD 19+) was examined in Clinical Pathology Laboratories of Gadjah Mada University Faculty of Medicine, Nursing and Public Health, ISO 17025 certified, using BD FACS Canto II 8 color Flowcytometry. Reagent used was PE anti human CD19 antibody (CD 19+) and CD 45 PerCP cy5.5 according to standard protocols. The results were measured in Mean Fluoresence Index (MFI), percentage of B cells CD 19+ to total lymphocyte, percentage of R BAFF to B cells CD 19+.\n\nThe Urine Protein Creatinine Ratio (UPCR) was determined at baseline, prior to the administration of MEP, by dividing the level of spot urine protein by the level of spot urine creatinine. Urine protein and urine creatinine were estimated by ERBA CHEM 5 V PLUS. In this study, the cutoff for normal UPCR is less than 150 mg/g and more or equal to or less than 500 mg/gr, consistent with diagnostic criteria for LN according to EULAR/ACR classification criteria.\n\nAll statistical analyses were performed using R Studio v. 4.1.1.1, employing the tidyverse and gtsummary packages.12,13 This study employed various statistical methods, including descriptive statistical analysis, univariate and multivariate logistic regression, and visualization using boxplots. Moreover, Pearson correlation analysis was conducted to evaluate the association between continuous variables by computing the Pearson correlation coefficient and its corresponding p-value, which provided insight into the magnitude and significance of any linear relationship.\n\nIn this study, we minimized potential biases through several strategies. To mitigate selection bias, clear inclusion and exclusion criteria were established, ensuring a representative study group. Measurement bias was addressed by using standardized assessment tools and established laboratory test methods. Multivariate analyses were conducted to control for confounding variables, such as age and sex. Attrition bias was tackled by carefully tracking all dropouts and withdrawals, and their potential impact on the study’s findings was thoroughly assessed. Additionally, all data and code were made openly available to promote transparency and reproducibility.\n\nThe sampling for this study was conducted from June 2021 to September 2022, after obtaining ethical approval from the Gadjah Mada University Faculty of Medicine, Nursing and Public Health ethics committee for human research (approval number: KE/FK/0359/EC/2021). The study was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent prior to their participation in the study.\n\nIn adherence to ethical guidelines, the inclusion of minor participants, aged between 15 to 17 years, in this study necessitated informed consent. In these cases, the consent was obtained through a two-step process: firstly, the minor’s assent was sought, which involved explaining the study’s purpose, procedures, potential risks, and benefits in an age-appropriate manner. Secondly, informed consent was also obtained from a parent or legal guardian. It was ensured that both the minor and their guardian fully understood the study before consent was provided. It was clarified that participation was voluntary, and they could withdraw from the study at any time without any negative consequences.\n\n\nResults\n\nA total of 100 SLE patients who met the inclusion and exclusion criteria were enrolled in this study, conducted from May 2021 to September 2022. Out of the 100 samples, 20 were excluded from the analysis due to the discontinuation of MEP, owing to side effects such as infection and elevated blood sugar levels, patient death prior to the completion of the procedure, disease activity assessment using the MEX-SLEDAI index, and the attending physician postponing the procedure due to discrepancies in calculating the SLEDAI-2K score between the researcher and the attending physician (Figure 1).\n\nIn this study, 80 individuals were eligible to participate, with a majority of female participants (97.5%).21 The average age of participants was 28 years, with a standard deviation of 9.5 years. Before receiving MEP treatment, the median SLEDAI 2K score for all patients was 28 (22, 38), indicating a high level of disease activity. In addition, all of the other variables including the median elapsed time between the onset of initial symptoms and the study’s completion, C-reactive protein (CRP), Erythrocyte Sedimentation Rate (ESR), leukocyte, thrombocyte, Neutrophil-to-lymphocyte (NLR), targeted protein level for sBAFF, proportion of BAFF R to B cell CD19 (BAFF R (%)), median fluorescence intensity (MFI) for BAFF R, percentage of B cells CD19 to lymphocytes (CD19 %), and serum IP-10 were also measured pre-treatment with MEP. Specifically, CRP, ESR, leukocyte, thrombocyte, and NLR levels were found to be 7 (5, 28) mg/dl, 62 (32, 100) mm/hour, 6,100/L (4,300, 9,250), 200,737/L (109,179), and 5.1/L (2.9, 9.3) respectively, while the targeted protein level for sBAFF was 1,584 pg/ml (860, 2,892), the proportion of BAFF R to B cell CD19 (BAFF R (%)) was 87% (66, 94), the MFI for BAFF R was 3,404 (2,024, 5,556), the percentage of B cells CD19 to lymphocytes (CD19 %) was 19%, and the serum IP-10 was 157 pg/ml (69, 526) (Table 1).\n\n1 Median (IQR).\n\n2 Mean (SD).\n\n3 n (%).\n\nAmong the neuro-psychiatry descriptors listed on the SLEDAI 2K form, seizures, psychosis, organic brain syndrome, visual disturbance, cranial nerve disorder (CND), lupus headache, and new onset stroke (CVA) were observed in 12 (15%), 8 (10%), 20 (25%), 2 (2.5%), 20 (25%), 5 (6.2%) of participants respectively. Mucocutaneous symptoms such as vasculitis, rash, alopecia, and oral ulcers were found in 7 (7.5%), 37 (46%), 46 (57%), and 31 (39%) of participants, respectively. Arthritis and myositis were observed in 51 (64%) and 12 (15%) musculoskeletal patients, respectively. Pleurisy and pericarditis were observed in 21 (26%) and 3 (3.8%) participants, respectively. Overall, 17 (21%) of individuals had a fever.\n\nThe median concentration of UPCR was 3,648.86 mg/g. 64% of individuals had a UPCR of 500 mg/gr. Median hematuria, cast cylinder, and leucocyturia values were 17/hpf (5, 123), 2.2/uL (0.5, 8.0), and 16 cells/mL (6, 39), respectively. Immunology parameters revealed a mean anti-dsDNA concentration of 175 U/mL (45, 200). The median concentrations of C3 and C4 were 45 mg/dL (26, 66), and 9 mg/dL,4,14 respectively.\n\nA correlation analysis revealed significant positive correlations between SLEDAI 2K scores, IP10 levels, sBAFF levels, and UPCR before treatment with response to treatment measured by SLEDAI 2K scores post-treatment (p < 0.05). Additionally, there is a significant negative correlation between C3 levels before treatment and SLEDAI 2K scores post-treatment (Table 2 and Figure 2).\n\n1 Pearson correlation.\n\nCRP: C-reactive protein; ESR: Erythrocyte Sedimentation Rate; PCR: Protein Creatinine Ratio; C3: Complement component 3; C4: Complement component 4; NLR: Neutrophil-to-lymphocyte ratio; SLEDAI 2K: Systemic Lupus Erythematosus Disease Activity Index 2000; CD19: CD19-positive B cells; BAFF R: B cell activating factor receptor; Mfi BAFF-R: Median fluorescence intensity of BAFF receptor; SBAFF: Soluble B-cell activating factor; IP-10: Interferon gamma-induced protein 10.\n\nIn our study, we performed a receiver operating characteristic (ROC) analysis to examine the relationship between various explanatory variables and the post-treatment SLEDAI-2K score. Our analysis determined that the optimal cutoff values for anti-dsDNA were 193.4 u/ml, for an NLR greater than 16.74/L, and a BAFF-R percentage greater than 48.8%.\n\nWe also conducted an independent t-test analysis, which revealed significant mean differences in SLEDAI-2K scores post-treatment based on NLR values greater or less than 16.74/L (p < 0.05). However, our analysis found no significant association between SLEDAI-2K scores and dsDNA levels greater or less than 193.4 u/ml or R BAFF levels more significant or less than 48.8% (as shown in Figure 3).\n\nNLR: Neutrophil-to-lymphocyte ratio; BAFF R: B cell activating factor receptor (BAFF R (%)); MFI BAFF R: Median fluorescence intensity of BAFF R on B cells; Anti dsDNA: antibodies against double-stranded DNA (IU/mL).\n\nWe conducted a linear regression analysis to identify the predictive impact of each pre-treatment MEP parameter on the SLEDAI 2K therapeutic score. The results showed a significant correlation between SLEDAI 2K and sBAFF (pg/mL), oral or nasal mucosal ulcers, and alopecia. We then used Stepwise analysis to determine the top five predictive characteristics: UPCR, CD19 (%), sBAFF, vasculitis, and rash.\n\n\nDiscussion\n\nA substantial positive correlation study between pre-treatment factors and SLEDAI 2K scores post-MEP pulse revealed that the decline in each level of these variables, as indicated by the pre-post analysis, cannot achieve normal values. This may explain why the higher the level of pre-treatment variables, the greater the post-treatment SLEDAI 2K score. A daily dose of 500 mg MEP for three days can reduce the values of each variable but not below normal. The same rationale applies to the negative correlation between post-treatment C3 levels and SLEDAI scores, as a low C3 level suggests a more active disease.\n\nWe observed substantial mean differences in clinical symptoms such as arthritis, rash, alopecia, oral ulcer, positive hematuria, and proteinuria in this study. This may be attributable to the high prevalence of these factors across all study participants: 63%, 46%, 57%, 38.8%, 73.8 %, and 80%, respectively.\n\nOur study found a strong correlation between the SLEDAI 2K pre-dose MEP and various markers of disease activity, including CRP levels, urine casts, and renal activity parameters. An increase in CRP levels and the presence of urine casts were both indicative of high disease activity. Additionally, we observed a positive correlation between Protein Creatinine Ratio, casts in the urine, hematuria, leukocyturia, and renal involvement. Immunology tests also revealed correlations, such as a positive connection between C3 and C4 levels and a negative connection between anti-dsDNA and C3 and C4 levels. We also found a substantial positive correlation between hemoglobin and leucocyte levels, as well as between thrombocyte and leucocyte levels, regarding hematological involvement. However, we discovered a significant negative correlation between anti-dsDNA and C3, C4 levels, thrombocytes, and leukocytes. Additionally, we observed a significantly negative relationship between ESR and hemoglobin levels.\n\nOur study found a significant positive correlation between the NLR and leucocytes, neutrophils, and CRP levels. This suggests that as the NLR level increases, leucocytes, neutrophils, and CRP levels also increase. On the other hand, we observed a significant negative correlation between the NLR and lymphocyte levels, indicating that the lymphocyte level decreases as the NLR level increases. This is in line with the calculation of the NLR, which is determined by dividing the neutrophil count by the lymphocyte count.\n\nIn the presence of systemic inflammation, it is common for leukocyte numbers to decrease and for there to be alterations in circulating leukocytes, with lymphopenia and neutrophilia being typical features. Despite this, the NLR is a more stable indicator of inflammatory processes than leukocytes. This is because leukocyte numbers can be more readily impacted by factors such as dehydration or overhydration, excessively dilute specimens, or the influence of the blood collection procedure.15\n\nThe study by Zaki et al. aimed to investigate the association between BAFF R levels and SLE activity markers. The study revealed a negative association between BAFF R levels and SLEDAI, ESR, CRP, and anti-dsDNA levels.12 Conversely, the study found a positive correlation between BAFF R levels and complement protein C3 and C4 levels (p < 0.001).12 Additionally, the study observed a rapid reduction in BAFF R levels with the progression of the disease, with the lowest expression frequency observed in the group with the most active disease.16 Similar to this study, we also found a strong negative correlation between BAFF R levels and disease activity markers such as CRP, sBAFF, and ESR. In our study, the MFI of BAFF R levels showed a significant positive correlation with leucocyte count. At the same time, MFI BAFF R, but not BAFF R (%), had a significantly negative association with anti-dsDNA. This study found that sBAFF levels correlated positively with SLEDAI pre- and post-dose MEP, demonstrating that sBAFF levels increased during severe SLE flares and remained elevated following a pulse dose of MEP. Conversely, the study found a significant negative connection between sBAFF and proteinuria, contrasting with another study that found a positive correlation between sBAFF and proteinuria.17 The authors noted that the significant chronicity of lupus nephritis (LN) in the study population may have influenced proteinuria results. Moreover, the accuracy of actively proliferative LN in the study is questionable, as the classification of LN was based on World Health Organization criteria and not by biopsy.\n\nAnother study by Suso et al. observed high glomerular expression of BAFF in the inflammatory infiltrate of patients with class II mesangial and class III lupus nephritis in their histopathological analysis of LN.18 The authors noted that the high expression of BAFF was strongly associated with activity scores but not chronicity ratings, making this one of the most intriguing findings of their study.18\n\nThere was a significantly positive correlation between IP 10 and CRP, NLR, and s BAFF. Qin et al. found significant positive correlations between IP 10 and CRP, NLR, and sBAFF.15 Specifically, there was a positive correlation between NLR and CRP (r = 0.471, p < 0.01), ESR (r = 0.610, p < 0.01), and SLEDAI (r = 0.471, p < 0.01). These findings suggest that IP 10 levels may be associated with markers of disease activity in systemic lupus erythematosus (SLE).\n\nThis study found that IP-10 levels were significantly negatively correlated with leucocyte, C-reactive protein (PCR), and BAFF R (%). This finding was consistent with the study by Hrycek et al., which showed a negative correlation between IP-10 levels and leucocyte (r = -0.423), polymorphonuclear cells (PMN, r = -0.303), lymphocyte (r = -0.386), and monocyte (r = -0.365) levels.19 However, the inverse association between IP-10 levels and PCR levels was inconsistent, as active lupus nephritis is expected to have higher PCR levels than inactive lupus nephritis.\n\nA meta-analysis confirmed this finding by Puapatanakul et al., which showed that serum IP-10 levels in active non-renal lupus did not differ significantly from inactive lupus nephritis and non-renal systemic lupus erythematosus (mean difference 22.6 pg/mL, p = 0.83).20 Another study found that IP-10 was a poor biomarker for predicting renal involvement.14 However, other investigations have reported conflicting findings. This discrepancy may be related to the varying levels of CXCR receptor expression on kidney-infiltrating cells. The current study’s authors hypothesized that the infiltration of IP-10 into renal tissue could increase its correlation with proteinuria in urine relative to serum levels.\n\nThe results of the univariate correlation test as shown in Table 3 indicated a strong positive association between the SLEDAI 2k, IP 10, and BAFF S variables prior to therapy and the post-therapy SLEDAI 2k score. This suggests that the post-therapy SLEDAI 2k score is proportional to the value of these variables prior to therapy. Additionally, the study found a non-significant negative connection between C3 levels and the post-therapy SLEDAI 2K score. This indicates that a lower C3 level is associated with a higher post-therapy SLEDAI 2K score. The correlation between the pre-and post-therapy SLEDAI 2K levels of CD19, C4, Hb, and platelets was modest and not statistically significant.\n\n1 CI = Confidence Interval.\n\nThe study also analyzed the difference in mean post-SLEDAI 2K scores between clinical manifestation variables that were present and absent, including seizures, psychosis, SOO, visual disturbances, cranial nerve disorders, lupus headaches, CVA, vasculitis, arthritis, myositis, rash, alopecia, mouth ulcers, fever, hematuria, and proteinuria. The results revealed a significant difference in the mean post-therapy SLEDAI 2K score for arthritis.\n\nA prediction model was generated using multivariate analysis with multiple linear regression after eliminating certain variables that were found to be correlated with each other (Table 3). This was done to ensure that the assumption criteria of multivariate linear regression analysis were met. The purpose of developing a formula that can predict SLEDAI 2K scores after MEP pulse dose is because SLE patients sometimes present with complicated conditions, the most common being flare-ups and infections. By predicting the SLEDAI score post-treatment, we can consider whether it will be beneficial or even worsen the infections before administering MEP pulses.\n\nThe resulting model was used to predict the response to MEP 500 mg (pulse) therapy for three days based on pre-therapy variables. The prediction formula was derived as follows: 13.41 + (0.0008542 * x1) - (0.1829338 * x2) + (0.0008776 * x3) + (7.1801728 * x4) + (7.5429676 * x5), where x1 is the UPCR (Urine Protein to Creatinine Ratio) prior to therapy, x2 is the CD19 (%) value prior to therapy, x3 is the sBAFF (B-cell activating factor) value prior to therapy, x4 represents the status of vasculitis prior to therapy (presumably coded as a binary value), x5 indicates the presence of a rash prior to therapy (also presumably coded as a binary value). This model is designed to predict the post-therapy SLEDAI 2K (Systemic Lupus Erythematosus Disease Activity Index 2000) score. The SLEDAI 2K score is a commonly used tool for assessing disease activity in patients with systemic lupus erythematosus. It incorporates a wide range of clinical and laboratory findings, and the prediction from the model above will be a crucial aspect of personalized treatment strategies for patients undergoing MEP 500 mg (pulse) therapy.\n\n\nConclusion\n\nIn conclusion, the study found that several variables, including the SLEDAI 2k, IP 10, and BAFF S prior to therapy, strongly correlate with the post-therapy SLEDAI 2k score. The study’s results can be used to predict the response to MEP 500 mg therapy for three days based on pre-therapy variables.\n\nWe construct the following formula to predict the SLEDAI 2k score after the MEP pulse: 13.41+ (0.0008542 * x1) + -0.1829338 * x2) + (0.0008776 * x3) + (7.1801728 * x4) + (7.5429676 * x5), where x1 is the UPCR prior to therapy, x2 is the CD19 (%) value prior to therapy, x3 is the sBAFF value prior to therapy, x4 is the status of vasculitis prior to therapy, x5 is the rash prior to therapy. Further validation is required for clinical use. Additional validation is required for usage in clinical practice.", "appendix": "Data availability\n\nFigshare: main dataset (sheet1), ip10 (sheet 2), baff (sheet 3). https://doi.org/10.6084/m9.figshare.22179919.v4. 21\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe author expresses their deep gratitude to the Boards of Directors of Sardjito General Hospital, Panti Rapih Hospital, and Karyadi General Hospital in Semarang for their approval and support in using patients in this study. The author extends their heartfelt appreciation to all participants who contributed to the success of this research. The author would also like to extend their thanks to Prodia Laboratory and the Clinical Pathology Laboratory of the Faculty of Medicine, Nursing, and Public Health at Gadjah Mada University for their invaluable support in conducting the laboratory examinations. The author acknowledges Mrs. Dewi and Mr. Adi for their assistance and support throughout the research.\n\n\nReferences\n\nMoulton VR, Suarez-Fueyo A, Meidan E, et al.: Pathogenesis of Human Systemic Lupus Erythematosus: A Cellular Perspective. Trends Mol. Med. 2017; 23(7): 615–635. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIppolito A, Wallace DJ, Gladman D, et al.: Autoantibodies in systemic lupus erythematosus: comparison of historical and current assessment of seropositivity. Lupus. 2011; 20(3): 250–255. PubMed Abstract \| Publisher Full Text\n\nBadsha H, Edwards CJ: Intravenous pulses of methylprednisolone for systemic lupus erythematosus. Semin. Arthritis Rheum. 2003; 32(6): 370–377. Publisher Full Text\n\nCarter LM, Isenberg DA, Ehrenstein MR: Elevated serum BAFF levels are associated with rising anti-double-stranded DNA antibody levels and disease flare following B cell depletion therapy in systemic lupus erythematosus. Arthritis Rheum. 2013; 65(10): 2672–2679. PubMed Abstract \| Publisher Full Text\n\nHui-Yuen JS, Li XQ, Askanase AD: Belimumab in systemic lupus erythematosus: a perspective review. Ther. Adv. Musculoskelet. Dis. 2015; 7(4): 115–121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamhieh-Milz J, Ghosoun N, Sterzer V, et al.: Effect of glucocorticoid treatment on BAFF and APRIL expression in patients with immune thrombocytopenia (ITP). Clin. Immunol. 2018; 188: 74–80. Publisher Full Text\n\nStumpf C, Auer C, Yilmaz A, et al.: Serum levels of the Th1 chemoattractant interferon-gamma-inducible protein (IP) 10 are elevated in patients with essential hypertension. Hypertens. Res. 2011; 34(4): 484–488. PubMed Abstract \| Publisher Full Text\n\nGroom JR, Luster AD: CXCR3 in T cell function. Exp. Cell Res. 2011; 317(5): 620–631. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrady MP, Chava S, Tandon S, et al.: Serum and Urine Interferon Gamma-Induced Protein 10 (IP-10) Levels in Lupus Nephritis. J. Clin. Med. 2022; 11(11): 3199. Publisher Full Text\n\nDima A, Jurcut C, Chasset F, et al.: Hydroxychloroquine in systemic lupus erythematosus: overview of current knowledge. Ther. Adv. Musculoskelet. Dis. 2022; 14: 1759720X211073001. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYee CS, Farewell VT, Isenberg DA, et al.: The use of Systemic Lupus Erythematosus Disease Activity Index-2000 to define active disease and minimal clinically meaningful change based on data from a large cohort of systemic lupus erythematosus patients. Rheumatol. Oxf. Engl. 2011; 50(5): 982–988. Publisher Full Text\n\nWickham H: Tidy Data. J. Stat. Softw. 2014; 59(1): 1–23. Publisher Full Text\n\nSjoberg DD, Haneuse S: gtsummary: Presentation-Ready Data Summary and Analytic Result Tables. R Package Version 142. Published online 2021. Reference Source\n\nKim ES, Choe PG, Park WB, et al.: Clinical Progression and Cytokine Profiles of Middle East Respiratory Syndrome Coronavirus Infection. J. Korean Med. Sci. 2016; 31(11): 1717–1725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQin B, Ma N, Tang Q, et al.: Neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) were useful markers in assessment of inflammatory response and disease activity in SLE patients. Mod. Rheumatol. 2016; 26(3): 372–376. PubMed Abstract \| Publisher Full Text\n\nZaki DSD, Ayoub NM, Mohammed ZAZ, et al.: Study of B Cell Activating Factor (BAFF) and BAFF-R in Systemic Lupus Erythematosus Patients. Am. J. Med. Med. Sci. 2019; 9(6): 203–209.\n\nVincent FB, Kandane-Rathnayake R, Koelmeyer R, et al.: Analysis of serum B cell-activating factor from the tumor necrosis factor family (BAFF) and its soluble receptors in systemic lupus erythematosus. Clin. Transl. Immunol. 2019; 8(4): e1047. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuso JP, Posso-Osorio I, Jiménez CA, et al.: Profile of BAFF and its receptors’ expression in lupus nephritis is associated with pathological classes. Lupus. 2018; 27(5): 708–715. PubMed Abstract \| Publisher Full Text\n\nHrycek E, Franek A, Błaszczak E, et al.: Serum levels of selected chemokines in systemic lupus erythematosus patients. Rheumatol. Int. 2013; 33(9): 2423–2427. PubMed Abstract \| Publisher Full Text\n\nPuapatanakul P, Chansritrakul S, Susantitaphong P, et al.: Interferon-Inducible Protein 10 and Disease Activity in Systemic Lupus Erythematosus and Lupus Nephritis: A Systematic Review and Meta-Analysis. Int. J. Mol. Sci. 2019; 20(19): 4954. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNugroho D: main dataset (sheet1), ip10 (sheet 2), baff (sheet 3). [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "197639", "date": "12 Sep 2023", "name": "Cesarius Singgih Wahono", "expertise": [ "Reviewer Expertise Rheumatology", "Autoimmunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis prospective cohort study goals is to develop a formula that can predict the SLE Disease Activity Index 2K (SLEDAI 2K) score after a pulse dosage of methylprednisolone. The subjects were patients with severe SLE with a SLEDAI 2K score of 12 or Lupus nephritis class III or IV according to WHO criteria, who were given methylprednisolone 500 mg/day for three consecutive days. Soluble BAFF, IP 10, BAFF-R, and CD-19 and SLEDAI 2K score were examined to develop the formula.\nThe conclusion: Formula to predict SLEDAI 2k score after MEP pulse was 13.41+ (0.0008542 * x1) + -0.1829338 * x2) + (0.0008776 * x3) + (7.1801728 * x4) + (7.5429676 * x6).\nMost of variable results are presented in Median and IQR (such as: SLEDAI 2K score for all patients was 28 (22, 38), CRP, ESR, leukocyte, thrombocyte, and NLR levels were f 7 (5, 28) mg/dl, 62 (32, 100) mm/hour, 6,100/L (4,300, 9,250), 200,737/L (109,179), and 5.1/L (2.9, 9.3) respectively, sBAFF was 1,584 pg/ml (860, 2,892), the MFI for BAFF R was 3,404 (2,024, 5,556), and the serum IP-10 was 157 pg/ml (69, 526), however the statistics in this manuscript are using parametric statistics (such as t test, ROC and logistic regressions). Could the authors explain about this?\n\nIt is stated that the authors performed ROC, but there is no ROC in this manuscript?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "296488", "date": "16 Jul 2024", "name": "Pamela Munguia", "expertise": [ "Reviewer Expertise Autoimmune Diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study presents intriguing findings and offers valuable insights. Various aspects need further refinement for clarity and completeness. It is recommended to provide a more detailed explanation of the procedural aspects. Important details such as the study location, whether it was a single-center study, and the setting of patient enrolment (consultation or emergency department) should be explicitly stated.\nAdditionally, the data collection procedure should be comprehensive, including whether patients were already diagnosed with SLE or were presenting with the disease onset. The duration of SLE diagnosis in the enrolled patients should also be specified.\nIn exploring the correlation of serum interleukin, it is essential to account for potential confounding factors like chronic damage ( SLICC score) or comorbidities, which were not thoroughly addressed in the study. Providing additional insights into these factors would enhance the study's depth and relevance.\nIn the statistical analysis section, it is advisable to specify the names of the specific statistical tests utilized in both univariate and multivariate logistic regression analyses. This practice enhances transparency and reproducibility, contributing to the credibility of the study.\nWhen including children under 16 years old, it is crucial to consider potential biases associated with a juvenile presentation of the disease. Acknowledging and addressing this factor can significantly impact the study's outcomes and interpretations.\nFurthermore, regarding the evaluation of treatment response after the third pulse of MPD, it is worth considering extending the assessment beyond seven days post-pulse to capture potential delayed treatment effects. Additionally, incorporating alternative disease activity indices such as BILAG alongside SLEDAI 2K could provide a more comprehensive evaluation of disease activity and treatment response. In the results: In the sentence \"However, our analysis found no significant association between SLEDAI-2K scores and dsDNA levels greater or less than 193.4 u/ml or R BAFF levels more significant or less than 48.8%,\" the phrase \"more significant or less than 48.8%\" seems unclear. It should possibly be rephrased for clarity.\nYou mentioned using Stepwise analysis to determine the top five predictive characteristics: UPCR, CD19 (%), sBAFF, vasculitis, and rash. This might need a bit more context or clarification on the exact process and the results obtained from this analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-992
https://f1000research.com/articles/12-551/v1	25 May 23	{ "type": "Research Article", "title": "An examination of the Petroleum Industry Act 2021: prospects, challenges, and the way forward", "authors": [ "David Oladeji Ehijie Borha", "Olusola Joshua Olujobi" ], "abstract": "Background: The study examines the gaps in the provisions of the Petroleum Industry Act (PIA) that could hinder the effective application of the Act in attaining its objectives. The repealed Petroleum Act of 1969 became obsolete and largely incapable of meeting the emerging global best practices in the industry due to inadequate sanctions, failure to address the aspirations of the people of the oil-bearing states, among others. Hence, the need for the PIA of 2021 to overhaul the industry to meet global standards though some controversial provisions that pose challenges to its proper implementation. Methods: The study examines the PIA to identify its prospects, challenges, and the way forward. The methodology the study utilises is doctrinal research with reports from existing literature and tertiary data sources such as newspapers, the Internet, and websites. Pertinent data collected from these sources were theoretically analysed and argued with current literature on the subject. Results: The finding is that the PIA does not make adequate provisions for the energy transition in line with Nigeria’s Nationally Determined Contributions (NDC) under the Paris agreement; the PIA was provided for weak institutions, which translates to weak implementation and enforcement of the law which further widening the gap between the law and reality. Conclusions: The study concluded that, although the Act delivered the much-needed stability in the petroleum industry in Nigeria, there is a need for an overhaul of the Act to further protect the interest of host communities and allow for co-ownership of petroleum resources by the state government.", "keywords": [ "Petroleum Industry", "Oil and gas", "Petroleum Industry Act", "Prospects", "Challenges" ], "content": "Introduction\n\nNigeria is blessed with numerous extractive resources such as crude oil, natural gas, gold, limestone, granite, and coal, among others. Hydrocarbon resources are some of the most valuable due to their high demand globally. Nigeria is currently the second-largest crude oil producer and one of the world’s top gas producers, possessing one of the Africa’s biggest gas reserves.1 Nigeria is set to benefit from the global demands for gas as a result of the ongoing conflict between Russia and Ukraine,2 as it is expected to benefit from the gaps in the global demands for gas and other supply chain caused by the ongoing war, hence the need to further overhaul the law.\n\nThe study examines the Petroleum Industry Act (PIA) to identify its prospects, challenges, and the way forward. The repealed Petroleum Act of 1969 appears obsolete and largely incapable of meeting the emerging global best practices in the industry due to inadequate sanctions, failure to address the aspirations of the people of the oil-bearing states due to a lack of measures that would have directly addressed the socioeconomic and environmental challenges affecting them, due to the detrimental effects of oil and gas exploration activities among others.3\n\nThe economy of Nigeria is heavily dependent on its hydrocarbon resources; however, as a result of the global transition to low-carbon energy sources, there is a need for Nigeria to look inward.4\n\nOn August 16, 2021, President Muhammadu Buhari signed the Petroleum Industry Bill (now the act) into law as the principal legislation regulating Nigeria’s oil and gas industry. Without a doubt, the PIA of 2021 is a landmark improvement in Nigeria’s oil and gas industry. Nonetheless, the act still has some flaws such as the recurring conundrums of gas flaring that have not been adequately addressed by the act.5 Likewise, the need for an energy transition in the context of the global demand and campaign against fossil fuels in an era when decarbonization is a global necessity.\n\nThe need for reform of the legislative and regulatory challenges confronting the act and to take full advantages of its prospects necessitate this study to bring the Nigeria’s petroleum legal in conformity with best practices and international treaties and agreements. The study will answer the following research questions:\n\na. What are the remedies to the gaps identified in the provisions of the PIA that could hinder the effective application of the act in attaining its objectives?\n\nb. What potential does the new PIA 2021 have in tackling the challenges in Nigeria’s petroleum industry when compared with international best practices?\n\nc. What are the key policies in Qatar, Kuwait and Norway’s oil and gas framework, that may be replicated in the Nigerian oil and gas regime?\n\nThe study will be useful to the Federal Government of Nigeria, policymakers in the oil and gas sector, energy practitioners, researchers, and scholars in policy formulation and enactment of legal regime regulating the oil and gas industry by offering guidelines for legislators on the best approach to employ when drafting a sustainable oil and gas legal framework to promote transparency, energy diversification by incorporating renewable energy and other low-carbon energy sources into the nation’s energy mix, and active citizen participation in the development of low-carbon energy technologies being a global clarion call for low-carbon energy utilisation.\n\nThe study is organized into five sections, with the introduction and statement of the problem in the first section. Section two is the literature review, including resource ownership theory and utilitarian theory. Section three outlines the methodology. Section four presents the results by deliberating on the challenges of the PIA 2021, analysing of the oil and gas legal regime in Qatar, Kuwait and Norway, and presenting other findings of the study. Section five contains the discussion, conclusions and recommendations.\n\nPreceding the discovery and production of crude oil in Nigeria, no detailed law had been enacted for the fiscal, regulatory, and administrative regime in the petroleum sector. Subsequent enactment of laws aimed at governing the petroleum sector was seen to be inefficient. The legal framework before the PIA lacked transparency and accountability due to its archaic nature, thus it was found ineffective and not in conformity with current international best practices and did not reflect the current economic reality. To address some of the flaws in the repealed Petroleum Act 1969 and the challenges in the Nigerian petroleum industry, the PIA 2021 was enacted.6 The PIA, though viewed as a panacea to the challenges in the petroleum sector, continues to be riddled with corruption, poor governance and poor management reflected by the appalling state of refineries capacity in Nigeria.7 This in turn occasions the importation of petroleum products with subsidy payment which gives room for endemic corruption in the sector. Crude oil theft issues and lack of effective metering have also been seen to be some of the challenges affecting petroleum production and national revenues, this is also exacerbated due to lax regulatory enforcement by relevant agencies. Nigeria has incurred a loss of over $ 2.1 billion as a result of oil theft.8\n\n\nLiterature review\n\nAccording to Umenweke and Chukwuma,9 the mandate of the PIA is to assist the government in attaining 40 billion barrels of reserves and production levels of four million barrels per day. This mandate was merely stated; they failed to state precisely how the act could assist the government in attaining this goal. Furthermore, the authors merely stated that the PIA now curtails the powers of the Minister in approving or revoking an oil license, without precisely stating the nature of the curtailment and the applicable sections of the act. This is one of the gaps in the existing literature that this study intends to fill among others, this can be done by stringent enforcement or implementation of the act’s provisions by its regulatory authorities with the backing of the executive arm of government and the strong political will of the president.\n\nUmenweke and Chukwuma noted that there is nothing in the act to show adequate commitment to climate change and energy transition. This position may also not be accurate because there are provisions in the act on the utilisation of the natural gas potential of the country. For instance, Section 52(1) of the PIA establishes a Midstream and Downstream Gas Infrastructure Fund (‘the fund’), which shall be a body corporate with perpetual succession and a common seal and reside in the authority as prescribed by this act. It shall have a governing council which shall supervise and make investment decisions for it. It shall be financed primarily from the 1% levy on the wholesale price of petroleum and natural gas sold and produced. It is expected that the fund shall be used to make equity investments in infrastructure related to midstream and downstream gas operations to increase the domestic consumption of natural gas in Nigeria in projects which are financed in part by private investment; encourage private investment; reduce or eliminate gas flaring. However, the provisions of the PIA are not adequate to tackle the issue of climate change given that even the role of gas in the energy transition mix and its production still have adverse effects on global warming and climate change.\n\nThe domanial ownership theory states that the sovereign possesses ownership of petroleum and other natural resources. According to this view of sovereign ownership, the state would have complete control and permanent sovereignty over all-natural resources, including petroleum.10 This ownership theory is considered to be the most prevailing theory on the ownership and control of petroleum resources. With the notable exception of the United States of America, this theory is favoured in Nigeria and virtually every other country with natural resources. The sovereign, state, or government owns and controls all mineral wealth in the country. These ownership and control rights are primarily established in the constitutions of most countries. Section 1 of the PIA confers the whole ownership and control of any petroleum in, under, or upon any land in Nigeria in the State. This authority allows the state to either explore resources directly or provide rights to third parties on any terms it considers appropriate.11\n\nThe endowment of natural resources has been described as a desirable benefit to the state and the people living within its geographical boundaries. The expected benefits of such endowment, however, have not always materialized, primarily because of several factors, such as the failure to use the revenues generated from the exploitation of natural resources to promote development, the abrogation of good governance, corruption, and a lack of accountability. This results in a “paradox of plenty”, with glaring underdevelopment and poverty in the presence of abundant resources. As a result, a state with abundant natural resources suffers from crippling underdevelopment, social unrest, and a lack of public accountability.12 The resource curse is a phenomenon that is pervasive in Nigeria and continues to be the country’s biggest obstacle to socioeconomic development. This is also a subject that has gained a lot of attention in the literature.\n\nUtilitarianism is a moral and ethical philosophy in political theory.13 Although it has its roots in Greek philosophy, it gained popularity because of the work of Jeremy Bentham and J.S. Mill (1748-1832). One of the strongest and most convincing theories of normative ethics in the history of philosophy is utilitarianism. Though not fully articulated until the 19th century, proto-utilitarian positions can be discerned throughout the history of ethical theory. The principle of utility is described by Bentham as ‘the principle that approves or disapproves of every action, according to the tendency that it appears to have to increase or decrease the happiness of the party whose interest is in question. The challenges affecting the Niger Delta communities are brought to light by a review of how the Nigerian government’s policies concerning oil and gas management are influenced by utilitarian dictates, which affect the host communities, while the Niger Delta communities continue to experience slow development.14\n\nAnother criticism levelled against the PIA is the meagre 3% allocation to the host communities, rather than the 10% that was demanded by the communities. This refers to the agitation of the Pan Niger Delta Forum (PANDEF) regarding the meagre 3% provision for the Host Communities Development Trust (HCDT) Fund and the allocation of 30% of Nigerian National Petroleum Corporation Limited (NNPC Ltd.) profit for the Frontier Oil Exploration Fund. The current researcher agrees with the authors that this 3% allocation to the host community has been condemned by activists, civil society organisations and host communities for failing to adequately cater for the host communities.15\n\nSimilarly, Bielu16 among other things, criticised the provision of a 3% contribution for the host community but 30% for the frontier Basin as being unfair. He also contended that due to the existence of the Niger Delta Development levy coupled with the 3% levy for the host communities there is a need for aggregation of the levies or scraping of one. This study also agrees with the author’s view that the PIA can benefit from an amendment to bring it to conformity with other legislations.\n\nBlythe and Todd17 also examined the fiscal changes introduced by the PIA. They rightly stated that the petroleum profits tax has been replaced by the hydrocarbon tax (HCT) and the companies’ income tax (CIT) and that the HCT only applies to crude oil, condensates and natural gas liquids produced from associated gas. However, they failed to state that this only applied to companies in the upstream oil and gas operations in onshore, shallow water and deep offshore. The authors stated the tax rates for HCT but failed to state the tax rate for CIT. This is also a gap in the existing literature that this current study intends to fill.\n\nMekwunye,18 similarly, on the issue of transparency in the petroleum industry, posited that the lack of transparency and accountability in the Nigerian petroleum industry is so pervasive that Nigeria is unable to account for the amounts of crude oil produced, exported, or the receipts of sales proceeds.\n\n\nMethods\n\nThe doctrinal research methodology will be adopted in this research. In carrying out this library-based research, primary and secondary sources of law and relevant existing literature will be consulted and relied upon, using sources obtained from internet and physical library searches for ‘Petroleum Industry Act 2021’ or ‘PIA’. Reliance will be placed on an examination of primary legal sources, including case law, judicial precedents, and domestic and international legislation, including the Constitution of the Federal Republic of Nigeria 1999 (as amended 2011), the PIA 2021, the repealed Petroleum Act 1969, the repealed Petroleum Profits Tax Act as well as other relevant statutes. Lessons will also be drawn from international legislation, including Law No 10 1974 of the establishment of Qatar’s petroleum, Law No 6 1980 of the establishment of Kuwait’s petroleum and Norway’s Petroleum Act 1996, as well as other enactments. These countries laws are chosen as a model because they comply with the international best practices on the regulation of petroleum industry as a comparison to Nigeria’s petroleum laws.\n\n\nResults\n\nThe prospects of the PIA are, among others, the introduction of a new regulatory and governance structure, the creation of new types of licences, the commercialization of NNPC Ltd., and the introduction of a new fiscal framework. These will be comprehensively examined. Some of the challenges of the PIA such as the allocation of 30% to the frontier basins fund, allocation of just 3% to the host communities; use of ambiguous and complex words, and lack of whistle-blowing provisions will also be comprehensively analysed.19 The act repealed approximately 10 existing petroleum laws governing the petroleum industry before the enactment of the act. The repealed Petroleum Act lacked measures that would have directly addressed the socioeconomic and environmental challenges affecting the inhabitants of the host communities as a result of the detrimental effects of oil and gas exploration by foreign and Nigerian oil corporations. The act establishes NNPC Ltd. as a profit-oriented legal entity. NNPC Ltd.’s new status as a limited liability company under the Company and Allied Matters Act (CAMA) 2020, the entity may be able to benefit from the capital market by being listed after satisfying the listing requirements and it raises capital for its operation by allotting its shares to the public. The act unbundled the industry by designing a well-organised governance structure with dynamic and distinct roles for the industry. The act further overhauls the fiscal and tax regime of the petroleum industry. It decreases the tax and royalty rates that operators must pay to encourage investments from both domestic and foreign investors.\n\nThe act introduces and specifies the regulations, procedures, and institutions which will ensure good governance, transparency, and accountability in the petroleum industry. However, there can never be perfect legislation. Certain provisions of the PIA have been deemed contentious, while others have been deemed potentially problematic that require legislative amendment to promote international best practices in the industry.20\n\nOwnership of natural resources\n\nSection 1 of the PIA, which deals with the ownership of petroleum by the Federal Government of Nigeria, should have been drafted to include the state, Local Governments, and the host communities in addition to the Federal Government as owners of the petroleum in the country. The three tiers of government and the host communities need to have been included in the vesting right to reduce incessant agitation for resources control, pipeline vandalization, crude oil theft and other oil related crimes. This is to reduce the Federal Government’s exclusive rights and total sovereignty over petroleum resources as it is practised in the United States and Canada.\n\nNorth and south division\n\nIn an effort to address the industry’s long-standing issues, the PIA 2021 created unintentional divisions or factions between the north and the south legislatures in the National Assembly. The bill was opposed by lawmakers and prominent leaders from the oil-rich Niger Delta states, and many southern legislators believe it favours the northern interests at the expense of the southern interests. According to the southern legislators, the law has been modified to benefit the northern part of the country. The southern lawmakers now believe that they are being cheated by the northern legislators. The host communities are also dissatisfied since they asked for 10% but the lawmakers rejected it. Pan-Niger Delta Forum (PANDEF), on the other hand, had also urged the lawmakers to immediately reverse the 3 percent they allocated to the host communities.21\n\nIn reality, the PIA still incites hostility in the Niger Delta. The Host Community Development Trust Fund (HCDTF)’s percent contribution, according to the PIA’s critics, is insufficient, while NNPC Ltd.’s 30% profit for the Frontier Basin Development Fund is unfair. Public remarks by certain Northern politicians have lent credibility to the Southern suspicions that the Frontiers Basin Fund is a tool for redistributing resources to the North. For instance, a northerner who works as the Group Managing Director of NNPC Ltd. recently claimed that the law will be more advantageous to the north since new crude oil reserves are being discovered in the region and the revenue derivable from exploration would accelerate additional discoveries in the north. Such utterances undermine efforts to get a national consensus on the petroleum policies that are interpreted in a way that is impartial to regions and that is in the national interest.22\n\nDouble taxation\n\nOil companies are required to pay a HCT under section 260 of the PIA. The same companies must also pay the CIT. Under section 302 of the act. These taxes may be seen as double taxation by the companies and investors may also be discouraged from investing in Nigeria’s petroleum industry by such taxation requirement.\n\nAmbiguous wording and imprecise language\n\nThe difficulty interpretation and legal ambiguities associated with the law is one of the biggest challenges of the act. For instance, it is not apparent if the duties of the host community development trust are separate from or in addition to current community levies such as the Niger Delta development levy. Similar to this, the act is silent regarding the definitions of “frontier basin” and “host community,” referring to the NUPRC for a definition of the former and settlors or licence holders for the definition of the latter. Frontier Basin Exploration does not have a proper definition under the PIA. These definitions affect revenue; they are not revenue neutral. This ambiguity breeds controversy and even the possibility of litigation, especially where stakeholders define them differently.23\n\nTensions over oil revenue sharing\n\nThe PIA has significant effects on the federation’s revenue as well as those of the states and the local governments. First, the three tiers of governments may see a significant drop in revenue accrued to them as a result of the reduction in taxes and royalties. Any revenue generating entity owned federation must deposit its profits in a fund called the Federation Account so that the three arms of governments can share them.24 More than 80% of the funds received by the states and local governments come from the Federation Account. Therefore, NNPC Ltd.’s contribution to the Federation Account may be significantly reduced as a result of the requirement that 30% of its revenues be set aside for frontier exploration. The Federation Account’s shared revenues with the different tiers of governments will decline in the near future. Numerous states and local governments, particularly those with extremely limited internal revenue-generation capabilities, will not be able to fulfil their responsibilities to provide social services to their populace. On the other hand, such a move would spur innovations at the state and local government to boost internal revenue-generating capacity and fiscal efficiency, therefore, this policy may have a good long-term outcome on the (3) three ties of governments.25\n\nThe success of the operations in Nigeria’s petroleum industry depends significantly on the host communities.26 The partnership may be the key to achieving profitable returns from the most capital-intensive ventures. However, throughout time, there has not been a friendly relationship between host communities and petroleum corporations.27 Petroleum operations have often been interrupted by the hostility and conflict. The significant theft of crude oil, the destruction of pipelines, and the ongoing suspension of large oil fields may also have been caused in part by the conflicts.\n\nThe government’s inability to satisfy its Organization of the Petroleum Exporting Countries (OPEC) crude oil output quota of around 1.8 million barrels per day in production has recently averaged 1.3 million to 1.4 million barrels per day, which indicates the severity of the issue. The tense relationship may in part be ascribed to these regions’ sluggish rate of growth. The government has attempted to solve this issue on several occasions with efforts that have varied in their degrees of effectiveness. Among these programmes is the establishment of the Derivation Fund, which is financed with 13% of oil revenues from the Federation Account and distributed to oil-producing regions; the establishment of the Niger Delta Development Commission (NDDC) in 2000, with an NDDC Levy of 3% of an overall annual budget of oil producing firms (Niger-Delta Development Commission [Establishment etc.] Act 2000 Act No 6 LFN). The establishment of the Ministry of Niger Delta Affairs in 2008, with a total budget allocation of NGN 584.6 billion between 2008 and 2022. and a number of others. In another effort to remedy the strained relationship between the host communities and petroleum corporations, the PIA mandated that petroleum industry operators to create a HCDT, as previously mentioned. The HCDT is a fund that will be established for the benefit of communities in the areas of operations of the petroleum corporation’s activities. The fund will support host communities’ infrastructure development and economic development and empowerment. The PIA allows operators to use their discretion when determining the host communities to include additional communities that may have an indirect effect on the HCDT’s success, although these other communities are not ’appurtenant’ to the operator’s area of operations. The PIA lays forth the formation of petroleum corporations’ development of the host communities activities with regard to the host communities, including the establishment of trusts for the domicile of companies’ contributions to community development.28\n\nThe host communities, on the other hand, are still disgruntled with the PIA’s requirement that petroleum corporations allocate 3% of their annual operational expenditure in the immediately preceding calendar year to the HCDTF, when they had requested at least 10%. Moreover, because indigenous oil firms hold the majority of onshore oil wells, host communities are unsure if this contribution will be given at all. The prior owners of the oil wells were foreign corporations, which meant that there were two sets of laws to ensure legal compliance Nigerian and those of the home country. With internal ownership of the majority of onshore oil wells, the risk of non-compliance with the host community contributions is high, particularly in Nigeria, where the judiciary is seen as weak, and court judgments are rarely upheld and political control of the industry is pertinent, because indigenous corporations dominate the onshore oil industry, there are legitimate concerns that the HCDTF will likely not receive any funding, which would leave the host communities underdeveloped.29\n\nLacunas in the HCDT provisions\n\nA few provisions have been discovered to be omitted from the HCDT under the PIA, some of these include:\n\ni. The midstream operators’ required contribution to the HCDT is not addressed by the PIA.\n\nii. A procedure for resolving disputes between the host communities and the operators does not exist under the PIA.\n\niii. There is no apparent framework for cooperation between the operators who may cover the same host community in order to avoid duplication of projects and achieve synergy.\n\nIt’s widely understood that corruption has plagued Nigeria ever since the country gained its independence in 1960, corruption persists now in every sector of the country’s economy, and unless it is addressed and eliminated at all levels.30 Corruption is an ever-present problem in the petroleum industry, from the initial stages of exploration and drilling through the latter stages of refining and distribution. Where a country mainly dependent on one single product or resources, like petroleum, for its social, economic growth, and development and corruption in that industry has a cumulative effect on the rest of the country’s economy and other industries.\n\nThe PIA is expected to attract investment and to boost production, but these cannot adequately come into play if the stakeholders do not work impartially by prioritising the interest of the nation over political apathy. Provisions of the law are ultimately intended to improve transparency in the petroleum industry while ideally significantly reducing revenue losses due to corrupt or sharp practices and lack of accountability in the sector. The Government of Nigeria’s revenues from petroleum products has decreased due to corruption.\n\nIt is alleged that government officials are responsible for this corruption and the misuse of oil revenues.31 Officials in the government’s petroleum industry have been accused of taking bribes from upstream petroleum corporations in exchange for illegal contract awards.\n\nQatar in perspective\n\nThe cornerstone of the Qatari economy is petroleum.32 The largest non-associated natural gas field and substantial oil reserves in the world are situated in Qatar. The country is a prominent stakeholder in the global energy market, the country is at the forefront of the gas-to-liquids and liquefied natural gas sectors, and quickly emerging as a key hub for hydrocarbon research and development.33 Until recently, Qatar was a member of OPEC and has had a significant impact on global energy markets since it assumed the Organization of Arab Petroleum Exporting Countries’ rotating annual presidency in 2015. Even as efforts to diversify the economy are made, the industry continues to dominate the national economy. The petroleum resources of Qatar account for over 70 percent of the country’s total revenue; 60 percent of the country’s gross domestic product and more than 85 percent of its export revenue come from the petroleum industry.34\n\nMost of Qatar’s hydrocarbon needs are satisfied by domestic production, however, limited volumes of oil with specified requirements are imported for a few purposes. As of 2022, Qatar’s oil reserves were 25.2 billion barrels, while its oil production dropped to 1.9 million production, and transport agreements, Qatar Energy serves as the government’s commercial arm. Similar to Nigeria, NNPC Ltd. is an independent commercial legal entity that operate like other business entity with statutory obligations such as payment of taxes to the Federal Government and declaration and payment of dividends to its shareholders if any. The state owns all-natural resources, including oil and gas similar to the practice in Nigeria under Section 44(3) 1999 Constitution of the Federal Republic of Nigeria (as amended). production, and transport agreements, Qatar Energy serves as the government’s commercial arm.\n\nQatar has enacted numerous laws regulating its natural resources, these include the following among others:\n\ni. Law No. 10 of 1974 (as amended by Law No. 15 of 1988) established Qatar Petroleum (now Qatar Energy), the national oil and gas company.\n\nii. Law No. 4 of 1977 on the conduct of petroleum operations and the conservation of petroleum resources in Qatar.\n\niii. Law No. 3 of 2007, deals with the exploitation of natural resources, which includes mining, oil and gas operations, and any related activities. (Exploitation of Natural Resources Law).\n\niv. Law No. 15 of 2007 awarding Tasweeq the sole right to sell and export petroleum produced in Qatar.\n\nv. Law No. 11 of 2012 giving Muntajat the sole authority to promote and market chemicals and petrochemicals produced in Qatar.\n\nIn Qatar, Qatar Energy has the sole and absolute right to explore for, produce, invest in, and develop any hydrocarbons, including petroleum, natural gas, and all others. Any corporation may apply for a licence or authorization from Qatar Energy to conduct petroleum operations within the country. Prior to undertaking any petroleum operations, a contractor commitment to provide Qatar Energy with a comprehensive overview of the proposed operations. This description covers plans, location, production capacity, operating modes, engineering data, cost estimates, varied computations, and other relevant documents, information, and statistics. Exploration and Production Sharing Agreements (ESPAs) and Development and Production Sharing Agreements (DPSAs) often provide for the formation of a management committee comprised of equal participation from the government and the contractor. the management committee has the capacity to assess and approve budgets and work plans, it contributes to concession-level decision-making on proposals for the submission of development plans, ceding of non-producing areas under the requirements of the EPSA/DPSA, and delineation of development areas. Under an EPSA, the contractor must submit a development and production programme and budget on commercial discovery and annually afterwards, outlining the development and production operations which the contractor plans to carry out throughout the year and any projected expenditure. The management committee must approve and may revise the development and production programme and budget.35\n\nThe Exploitation of Natural Resources Law requires Qatar Petroleum’s authorization before granting concession rights or transferring or assigning concession holders’ rights to third parties. Leases, licences, and concessions for the exploitation of hydrocarbons are granted following a decision by Qatar Petroleum and the State of Qatar. Previously, these agreements were made between Qatar Petroleum and one or more multinational petroleum corporations. using an instance of the Al Shaheen Field, when former Qatar Petroleum formed a joint venture with Total, and the government granted the concession to the new joint venture corporation.36\n\nDecommissioning requirements will be outlined in the applicable licence/concession agreement; however, these clauses typically result in additional agreement with Qatar Petroleum. The decommissioning must be consented to by the parties and Qatar Energy if the agreement does not address it. The Environmental Protection Law’s provisions will still apply to the parties, and any breach of them will subject them to penalties under the law. For this, a contractor often presents a plan for the abandonment, decommissioning, and disposal of any substantial fixed and mobile assets employed in conjunction with the conduct of the petroleum activities and including the restoration of site locations. The plan also specifies the duration for implementing each component as well as the expected decommissioning expenses. Sometimes a decommissioning committee will be created to oversee the technical and fiscal aspects of abandoning, decommissioning, and disposing of fixed and moveable assets. It will also be responsible for approving any decommissioning plans submitted by the contractor to the management committee. In most cases, the contractor is also in charge of removing, abandoning, decommissioning, plugging, and salvaging facilities and wells. In Nigeria, sections 232(1)-(14), 233(1)-(12) of the PIA 2021 and the provisions of the National Oil Spill Detection and Response Agency (Establishment) Act 2006 for decommissioning and abandonment of oil and gas infrastructure after caseation of operations in Nigeria. However, there is a need for stringent sanctions for infringement of the law. Therefore, there is a need for a specific law on decommissioning and abandonment of oil and gas assets or infrastructure in Nigeria with stringent penalties for non-compliance with the obligation. There are no regulations that particularly cover flares and vents in the case of gas flaring, although the Environmental Protection Law and its executive regulations generally apply to these activities. The executive regulations additionally specify the gas flaring operations’ permissible limits.\n\nIn response to concerns about climate change, the Qatari government has begun a number of initiatives to diversify its economy and reduce its near total reliance on the petroleum sector.\n\nA polysilicon manufacturing plant in Qatar with a capacity of 8,000 metric tonnes per year, Qatar Solar Technologies, stated in March 2017 that it had begun producing polysilicon. When giving concessions, Qatar now seems to be abandoning the usual Exploration and Production Sharing Agreements and Development and Production Sharing Agreements. The tendency is for foreign petroleum corporations and Qatar Petroleum or its affiliates to participate in contractual joint ventures.37\n\nHowever, Nigeria has not taken significant or concrete steps to diversify from oil and gas to a viable production sector, particularly the auto-mobile fabrication plants, cement, fabric industry, the mining, agricultural activities, information and communication technology and other significance segments of the country’s economy to promote energy diversification by integrating renewable energy and other low carbon energy sources into the national energy mix to promote technology development and to encourage active engagement of the citizens particularly the youths in the development of low carbon energy technologies being the current global demand due to adverse contribution of fossil fuel utilisation to global climate change.\n\nA polysilicon manufacturing plant in Qatar with a capacity of 8,000 metric tonnes per year, Qatar Solar Technologies, stated in March 2017 that it had begun producing polysilicon. When giving concessions, Qatar now seems to be abandoning the usual Exploration and Production Sharing Agreements and Development and Production Sharing Agreements. The tendency is for foreign petroleum corporations and Qatar Petroleum or its affiliates to participate in contractual joint ventures.\n\nKuwait in perspective\n\nKuwait is a major petroleum supplier and an OPEC member. Nearly half of Kuwait’s GDP, around 95% of exports and roughly 90% of government export revenue, are derived from petroleum. Kuwait now has a production capability of over 3.15 million barrels per day and has roughly 7% of the global oil reserves. The Kuwait Petroleum Corporation (KPC), a state-owned corporation, oversees the country’s petroleum industry. KPC has several corporations functioning under it which are known collectively as the “K companies”, for example, Kuwait Oil Company (KOC) is largely responsible for upstream operations. KPC manages the downstream petroleum operations.38\n\nKOC is the largest corporation in terms of revenue and is in charge of the production of petroleum. By 2040, the KPC plans to boost its capacity for oil production to 4.75 million barrels per day. Additionally, KPC declared plans to boost natural gas output to 4 billion cubic feet per day by 2030. Kuwait is one of the largest producers of oil and has one of the largest proven reserves of crude oil in the world, as previously stated. In the past, it flared off a considerable amount of its associated gases. It is also important to mention that KOC has made significant progress in recent years in reducing gas flaring in its operations. Kuwait has made efforts over the past decades to limit gas flaring in order to both cushion the effects of climate change, thereby executing a significant reduction in its carbon emissions, and provide its power plants with gas supplies to satisfy the country’s fast-rising energy demand. The residual gas flaring levels decreased by roughly 40% between 2014 and 2018. KOC contends that the reduction of associated gas flaring in Kuwait has been successful due to the following factors: a strong commitment from all levels of the corporation to make flare reduction a priority; significant financial investments in cutting-edge facilities and operations; joint efforts between KOC departments with downstream companies and customers to adapt to any unforeseen situations and limit the span of flaring; and a strong, beneficial collaboration with other organisations.39 This is unlike Nigeria, a country that is blessed with a vast deposit of gas but still flares a large percentage of its associated gas during upstream petroleum operations despite the country’s challenge with attaining a stable power supply. Flared gas could be used to supply gas to turbines to generate electricity. However, flared gas has occasioned the loss of billions of dollars in revenue to the Federal Government of Nigeria. Gas flaring has been a concern due to its deleterious effects on the ecosystem, aquatic resources, and human health hence the need to combat the menace to promote a healthy and sustainable environment in Nigeria.40\n\nKuwait also allotted a sizable budget to spend on technology initiatives that concentrate on reducing costs and improving operational processes since oil is the country’s main natural resource and there is a need to minimize the risk associated with declining oil prices.41 The digitization of oil fields is a top priority for KOC. Utilizing the most cutting-edge technology to remotely monitor and operate oil fields at four separate sites in West Kuwait, North Kuwait, South and East Kuwait, Kuwait Oil Company completed pilot projects to digitalize oil fields. This positive initiative is the Kuwait Integrated Digital Field, and which comprises a thousand fields, and roughly half of KOC’s oil fields.\n\nNorway in perspective\n\nNorway is a leading example of a developed, efficiently run hydrocarbon economy.42 The United Nations’ (UN) index of human development ranks Norway second after Switzerland as of 2022. The Norwegian Petroleum Act is consistent with the UN General Assembly Declaration on permanent sovereignty, and petroleum resources belong to the Norwegian people. In accordance with this clause, the Norwegian people will exercise complete control over their natural resources and use them to improve their welfare.43 The people of Norway, as a unitary state, own the country yet the monarch is responsible for administering it under section of the PIA 2021 the Federal Government in Nigeria, on the other hand, owns all of the petroleum found within the territory, including the country’s continental shelf and exclusive economic zone. In Norway, Statoil is the commercial arm of the petroleum industry representing the state.44 Statoil engages in exploration and production, natural gas supply, pipelines, decommissioning and research and development. In terms of transparency, Norway’s upstream petroleum industry has become more transparent as a result of Statoil, which operates like other for-profit oil corporations with no special status as a statutory conglomerate and where the government mainly serves a supervisory role and levies corporate taxes. The Norwegian Act also addresses domestic energy supply security through state participation. Norway dominates the retail product market because they prioritise its inhabitants before selling to other countries. The Norwegian energy policy supports the security of supply of petroleum directorate and aids the ministry with industry administration and regulatory issues. Policy concerns are separated from commercial management and regulatory challenges in this context. In contrast, the minister of petroleum in Nigeria fulfils commercial regulatory and policy functions.\n\n\nDiscussion\n\nNotwithstanding the huge potential offered by the PIA 2021, the study revealed the following gaps under the PIA 2021 which are not in line with the international best practices and which may impede the PIA’s effectiveness, among others:\n\n▪ Gas flaring and Energy Transition: There is silence on the subject of energy transition in the PIA. Nigeria’s government plans to increase the production.\n\n▪ Federal Control of the Petroleum Industry: NNPC Ltd. is established as a corporation under CAMA in accordance with Section 53 of the act in order to conduct commercial petroleum operations. Based on the phrasing of 53(3), it appears that the federal government will be the only shareholder in this company. State governments have no say in the administration or distribution of dividends from the corporation because the act stipulates that all shares are to be owned by the federal government and held by the ministry of finance on its behalf.\n\n▪ The act also appears to undermine Section 162 of the constitution, which specifies that federal revenues are to be deposited into a Federation account and distributed to the different tiers of government per the constitution. In this situation, the NNPC Ltd. would benefit from being fully unbundled to increase its viability, but as it is, this has just bolstered NNPC Ltd.’s attitude of non-accountability.\n\n▪ Host Community Development Trust: The PIA does not address the midstream operators’ contribution to the HCDT. However, the authority may issue regulations defining the contribution. The HCDT is to be financed at the rate of 3% of the operator’s upstream operations’ total operating expenses from the previous year. However, the PIA does not specify how much revenue midstream operators must put into the HCDT. The amount to be contributed is expected to be outlined in regulations issued by the Nigerian Midstream and Downstream Petroleum Regulatory Authority.\n\n▪ The PIA’s impact on the existing fiscal framework is its most crucial component. The PIA reduces royalties and introduces a new HCT to replace the Petroleum Profits Tax. It is possible that the new royalty system, which will allow the government to profit from rising oil prices, may reduce the likelihood of future alterations to the fiscal system.\n\n▪ One expected benefit of the PIA is a turnaround in Nigeria’s petroleum investment decline, attributable to the PIA’s potential to make the country more competitive relative to other petroleum-producing countries. The discovery of oil and gas in other regions of the world, notably West Africa, helps to explain the drop in Investment in Nigeria’s petroleum industry. Despite being one of the continent’s top producers and holding one of the highest reserves, Nigeria only received 4% of the $70 billion invested in Africa’s oil and gas sector between 2015 and 2019, as reported by KPMG. Data from Nigeria’s National Bureau of Statistics reveal that the industry represented 1.11 percent of total capital imports into the country in 2020, reflecting the decline in investment in the industry.\n\n▪ The PIA also attempting to shift responsibility for securing facilities to the surrounding host communities, noted that Damage to oil and gas infrastructure due to vandalism or sabotage will be repaired using this host community entitlement. As an alternative to relying solely on upper government action, this system establishes measures to have communities police themselves.\n\n▪ The PIA levies a one per cent charge on the wholesale price of petroleum products and threatens sanctions against petroleum corporations that misrepresent their financial standing. The new law, which replaces ten prior laws, prohibits residents from serving as trustees or executives for host communities. It stipulates that oil-producing regions must disperse revenues for capital projects within a fund established within 12 months.\n\nInvestors, especially those in the petroleum industry, in particular, will find that the policies of Qatar are not demanding. State ownership extends to all oil and gas reserves. There is a consensus that all-natural resources belong to the state. Domestic production accounts for the vast majority of Qatar’s hydrocarbon needs. Qatar Energy has the exclusive right to explore for, produce, invest in, and develop any hydrocarbons, including but not limited to petroleum and natural gas. Obtaining a licence or permit from Qatar Energy is a necessary step for any company interested in operating in the petroleum industry in Qatar.45 Following a decision by Qatar Energy and the State of Qatar, leases, licences, and concessions for the exploitation of hydrocarbons are issued. The contractor agrees to provide Qatar Energy with a detailed overview of the planned petroleum activities before beginning such operations. Plans, location, production capacity, operating modes, engineering data, cost estimates, various computations, and other pertinent documents, information, and statistics are all included in this description. Rights in produced petroleum can be transferred to oil and gas companies through net production sharing methods under the Exploration and Production Sharing Agreement and the Development and Production Sharing Agreement.46\n\nOil production in Kuwait is managed by the state-owned KPC. For KOC, digitising their oil fields is crucial to attaining their objectives. Pilot projects to digitalize oil fields were completed by KOC, which used state-of-the-art technology to remotely monitor and run oil fields at four different sites in West Kuwait, North Kuwait, South Kuwait, and East Kuwait. Kuwait has also set aside a substantial budget for technological efforts aimed at lowering costs and improving operational procedures, as oil is the country’s primary natural resource and there is a need to reduce the risk associated with falling oil prices.47\n\nWhen it comes to advanced, well-managed hydrocarbon economies, Norway is a prime example. The petroleum resources are the property of the Norwegian people, as stated in the Norwegian Petroleum Act and by the UN General Assembly Declaration on permanent sovereignty. As a result of taking full charge of their natural resources, the Norwegian people will be able to better their livelihood. Statoil is the state-backed commercial arm of Norway’s petroleum industry. Statoil is involved in a wide variety of industries, including exploration, production, natural gas supply, pipelines, decommissioning, and Research and development. In Norway, the gas pipelines are managed by a company called Gassco. While the Norwegian gas pipeline is a de facto monopoly, the country’s gas transport infrastructure is managed to increase productivity and decrease biases. Norway’s regime permits and encourages corporations that produce gas to sell it through a system that lets each licence sell its gas. This is not the situation in Nigeria, where the federal government maintains a monopoly.48\n\nSections 4 to 7 of the Norwegian Petroleum Act provide for joint and multiple liabilities for polluters, ensuring that victims of environmental damage receive adequate compensation, and also sets rigorous obligations on licence holders. Statoil in Norway has grown more transparent by functioning like other for-profit petroleum corporations with no special status as a statutory conglomerate and where the government primarily functions in a supervisory role and charges corporate taxes.49\n\nThe Norwegian Petroleum Act requires licensees or lessees to submit a two- to five-year decommissioning plan (including a thorough environmental impact assessment) when a licence expires, is relinquished, or the use of a facility is permanently discontinued. The Norwegian Act addresses domestic supply security through state engagement as well. Norway dominates the retail goods industry by prioritising sales to its citizens over sales to other countries.\n\nIt is obvious that, whereas all these nations have peculiar attributes that are in line with the global best practices in the petroleum industry, many of these provisions were not taken advantage of in the PIA 2021. Firstly, there is no state ownership of crude oil. This therefore possesses a peculiar disadvantage to investors in that the petroleum industry in Nigeria is regulated by several agencies. This is a major variance from what is obtainable in Qatar. Unlike Norway, Nigeria has not introduced the appropriate management of hydrocarbon economies. Likewise, Nigerian-owned corporations are not involved in the exploration, and production of crude oil, likewise, most of the natural gas supply are flared in a bid to increasing the exploration capacity of the crude oil. The Norwegian Act also contains adequate provisions for the compensation of victims of environmental degradation and transparency of the state-owned oil company. The Statoil company in Norway functions as a profit corporation which pays corporate taxes. The federal government merely performs a supervisory role in the administration of the company. The Norwegian Act also caters for supply security by prioritising the sale of retail goods by the industry to its citizens over sale to other countries. Kuwait on the final note, has policies invested at technological efforts which is aimed at lowering costs and improving operational procedures in the petroleum industry.50\n\nAll these are the various advantages of the petroleum industry in Qatar, Kuwait and Norway, advantages which the PIA 2021 could have incorporated. However, these were not incorporated into the PIA, whether directly or otherwise. This further diminished the utility of the PIA, in the light of global best practices, using the three countries of Qatar, Norway and Kuwait examples for analysis.51\n\nOil and gas administration by the government in Nigeria is also described as being purely utilitarian. The Nigerian government’s decision-making is heavily impacted by utilitarianism, as shown by an analysis of the country’s petroleum administration through the lens of utilitarian principles. Examining the effects of the Nigerian government’s utilitarian-influenced oil and gas management practises on the Niger Delta’s host communities, sheds insight into the problems that the region faces. Although progress has been slow in the Niger Delta region, the Federal Government controls most of the revenue on behalf of the country. Host Community members in the Niger Delta are directly affected by the petroleum industry’s lack of attention to sustainability, even when the majority of Nigerians gain from oil production. The administration of Nigeria’s oil and gas resources under the utilitarian approach did not effectively protect the human rights of the inhabitants of the Niger Delta.52\n\nThis study, however, concludes that the sovereign or domanial ownership theory is the best applicable theoretical framework for producing positive applications of the PIA. This must however be reflected in such a way that the federal government does not assume absolute control and authority over natural resources (petroleum included) but must cut across to the state government, a reality of which is not currently practised in Nigeria.\n\n\nConclusions\n\nThe PIA serves as a cornerstone upon which further progress can be made. While no legislation is perfect, Nigeria could benefit from commencing steps to work and improve various aspects of the PIA. The Niger Delta, which is responsible for most of Nigeria’s oil output, should not have to shoulder such a disproportionate share of the country’s oil revenues. This is inconsistent with the fact that 30% of NNPC Ltd.’s revenues, is set aside for frontier exploration. Despite the PIA’s many flaws, it did provide a strong foundation for the industry and is a step in the right direction. By highlighting some of the gaps in the PIA and petroleum operations in Nigeria, this study adds to the body of existing knowledge. The study will serve as a guide for the Nigerian government to reflect these practices by adopting consistent measures, and policies or making required modifications, while also identifying inadequacies of the existing regime and referencing best practices in other jurisdictions. The study will also aid lawmakers to make amendments to legislation, regulations, and policies when Nigeria’s Nationally Determined Contributions are not reflected in domestic laws.53 Given the significance of including Nigeria’s international climate change obligations in domestic legislation like the PIA, regulators in Nigeria would be better prepared to enforce the current laws, rules, and policies and fulfil the country’s international obligations; therefore, in view of the findings above, and in relation to achieving the intendment of the PIA 2021, this study recommends as follows: The PIA should be amended to include provisions which cater for the underlisted:\n\na. petroleum corporations must be compelled to install suitable gas recapturing and/or re-injection facilities, similar to what is performed in developed countries. Gas flaring must also be outrightly prohibited with no exceptions. Since most infrastructure currently in place condone gas flaring, a timeline should be introduced by the amendment, within which all gas flaring infrastructure must be phased out.\n\nb. To attain the desired objectives of the PIA, the HCDT resources must be appropriately administered. The PIA should therefore contain clear provisions on the appropriation of petroleum receipt quota for host communities, as well as the regulation of the actives of the HCDT. To address the different neglect, and environmental degradation of oil-bearing areas, the operator’s contribution of 3% of its yearly operating budget for the prior year in upstream petroleum activities to the HCDTF is incredibly low. At least ten per cent is feasible considering the irreversible environmental impact of operations. All parts of the country, but notably the host communities, should have their voices heard through a proper and thorough amending of the act. This should also include provisions to cater for collaborations between various operators in a host community, in order to prevent the duplication of efforts and cost maximisation in the development of host communities.\n\nc. Also, the PIA mandates that 30% of the profits of petroleum receipts be invested in the exploration and development of frontier basins, or remote areas of land along the borders of some Northern states and Lake Chad where there is little confidence in the presence of crude oil. Even though it is not guaranteed, NNPC Ltd. is required to invest 30% of its profit in developing the frontier basins, which are new places where oil is anticipated to be found. It appears that this provision is unnecessary and ambiguous. Therefore, it has to be modified or expunged.\n\nd. This study also addresses an important problem of why the host community should be held responsible for any act of sabotage on the corporations’ facilities located in the community, especially since most such acts of sabotage could be committed by strangers in connivance with some operational staff of the corporations. There is therefore the need to amend the PIA to include such provisions.\n\ne. Likewise, the PIA needs to establish a system for resolving disputes between host communities and corporations. This will ensure that the interests of the operators as well as the host community is adequately catered for.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAkpan MJD: Petroleum Industry Act in Nigeria: An Analysis of the Impact of the Novel Host Communities Development Trusts Provision. 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Mediterranean Journal of Social Sciences. 2012; 3(15): 98.\n\nOlujobi OJ: Nigeria’s Upstream Petroleum Industry Anti-Corruption Legal Framework: The Necessity for Overhauling and Enrichment’. Journal of Money Laundering Control. 2021; 24(4): 806–833. Publisher Full Text\n\nTagliapietra I: The Impact of the Global Energy Transition on MENA Oil and Gas Producers. Energy Strategy Reviews. 2019; 26: 100397. accessed 24 December 2022. Publisher Full Text\n\nDirioz AO, Erbıl E: The Prospects of Natural Gas Organization in Light of Qatar’s OPEC Exit: Some Critical Reflections’. The Extractive Industries and Society. 2023; 8(2). accessed 8 January 2023. Reference Source\n\nAlameen YMM: The Norwegian Oil Experience of Economic Diversification: A Comparative Study with Gulf Oil’ European. Journal of Business Management. 2016; 8(15): 94.\n\nUnited Nations General Assembly: Permanent Sovereignty over Natural Resources, General Assembly Resolution 1803 (XVII) New York, 14 December 1962, Audio-visual Library of International Law.1962. accessed 30 December 2022. Reference Source\n\nTadeo PE: A Comparative Study of Oil Resource Management in Norway and Nigeria: Lessons for Kenya’ (MA Thesis, Institute of Diplomacy and International Studies University of Nairobi).2016.\n\nPetroleum Industry Act 2021. accssed March 16, 2023. Reference Source\n\nBentham J: The Utilitarians. New York: Dolphin Books; 1789; 18.\n\nDepartment of State Investment Climate Statement: Executive Summary.2014. accessed 16 March 2023. Reference Source\n\nAmabipi AK: Understanding Host Community Distrust and Violence Against Oil Companies in Nigeria (PhD Dissertation School of Public Policy and Administration Walden University).2016.\n\nEditorial: Between OPEC quota and oil thieves’ The Guardian (2 January 2023).2023. accessed 4 January 2023 Abuja. Reference Source\n\nBudget Office: accessed 10 January 2023. Reference Source\n\nIjediogor G, Godwin A, Osayande M, et al.: Niger Delta tackles Buhari, may head to court over PIA. The Guardian. 21 August 2021. accessed 29 December 2022. Reference Source\n\nAchinike CB: Nigeria: Anti-Corruption Overview in The Oil and Gas Industry. Brickmans Law. 2021. (13 December 2021). accessed 20 December 2022. Reference Source\n\nOlujobi OJ: Nigeria’s Upstream Petroleum Industry Anti-Corruption Legal Framework: The Necessity for Overhauling and Enrichment’. Journal of Money Laundering Control. 2021; 24(4): 806–833. Publisher Full Text\n\nKPC: Kuwait Integrated Digital Field (KwIDF) project. accessed 6 January 2023. Reference Source\n\nTagliapietra I: The Impact of the Global Energy Transition on MENA Oil and Gas Producers. Energy Strategy Reviews. 2019; 26. accessed 24 December 2022. Publisher Full Text\n\nDirioz AO, Erbıl E: The Prospects of Natural Gas Organization in Light of Qatar’s OPEC Exit: Some Critical Reflections’. The Extractive Industries and Society. 2023; 8(2). accessed 8 January 2023. Reference Source\n\nMahmood S, Earley M: Oil and Gas Regulation in Qatar: Overview, Sultan Al-Abdulla and Partners.2021. accessed 19 December 2022. Reference Source\n\nAlagos P: Qatar Lauded for Diversifying Economy Away from Oil, Gas’ Gulf Times (13 September 2017).2017. accessed 19 December 2022. Reference Source\n\nEmbassy of the State of Kuwait: Economy.2022. accessed 29 December 2022. Reference Source\n\nEditorial: Kuwait Oil and Gas.2023. 2 January 2023. Reference Source\n\nState of Kuwait Environment Public Authority: Kuwait’s Initial National Communications under the United Nations Framework Convention on Climate. Change: UNFCCC November; 2012. Reference Source\n\nOlujobi OJ, Yebisi ET, Patrick PP, et al.: the Legal Framework for Combating Gas Flaring in Nigeria’s Oil and Gas Industry: Can It Promote Sustainable Energy Security? Sustainability. 2022; 14(13): 7626. 3. accessed January 30,2023. Publisher Full Text\n\nUnited Nations: Reducing Gas Flaring in Arab Countries; A Sustainable Development Necessity. United Nations Economic and Social Commission for Western Asia (ESCWA); 2020.\n\nAlameen YMM: The Norwegian Oil Experience of Economic Diversification: A Comparative Study with Gulf Oil’ European. Journal of Business Management. 2016; 8(15): 94.\n\nUnited Nations General Assembly: Permanent Sovereignty over Natural Resources, General Assembly Resolution 1803 (XVII) New York, 14 December 1962.1962.\n\nBentham J: The Utilitarians. New York: Dolphin Books; 1789; 18.\n\nNational Law Review: Nigeria’s New Petroleum Industry Act Signed into Law. Journal of Petroleum Technology. 2021. accessed 20 January 2023. Reference Source\n\nOlujobi OJ, Yebisi TE: Combating the Crimes of Money Laundering and Terrorism Financing in Nigeria: A Legal Approach for Combating the Menace. Journal of Money Laundering Control, Control. 2023; 26(2): pp. 268–289. accessed 16 March 2023. Publisher Full Text\n\nOlujobi OJ: Deregulation of the Downstream Petroleum Industry: An Overview of the Legal Quandaries and Proposal for Improvement in Nigeria. Heliyon. 2021; 7(4): e06848,1–e06848,10.\n\nOlujobi OJ, et al.: Conversion of Organic Wastes to Electricity in Nigeria: Legal Perspective on The Challenges and Prospects. International Journal of Environmental Science and Technology. 2022; 19: 939–950. Publisher Full Text\n\nAina-Pelemo AD, Olujobi OJ, Yebisi ET: A Socio-Legal Imperative of Domestic Violence Prohibition in Africa Vis-A-Vis Nigerian Legal Structure for Sexually Abused Women. F1000Research. 2023; 12. (in Press). Publisher Full Text" }	[ { "id": "183246", "date": "14 Jul 2023", "name": "Azubuike Hope Amadi", "expertise": [ "Reviewer Expertise Petroleum Engineering", "Production", "Petroleum Industry Policies & Politics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research demonstrated a smooth flow, and in my opinion, the paper presents an intriguing and informative contribution within its scope, which will enrich the existing literature and enhance policy-making in Nigeria. However, across different sections, there were poor citations of facts in different paragraphs. If these are improved on, then the manuscript is good for indexing.\n\nMajor points: Citation of facts on paragraphs, Making a conclusion on % allocated for frontier basins without adequate comparison with the case studies.\nMinor points: Typos, paraphrasing complex sentences, referencing the Acts introduced,\n\nBelow is the breakdown of my reviews on the different sections\nIntroduction: Citations needed P4 (Paragraph 4) Introduce the law first, before referring to it as \"the law\" At first instance, PIA should be referred to as Nigeria's PIA 2021\nLit reviews: Citations needed P4, P6 P3: section 1 of the PIA confers... \"to\" the state. 1 citation is not enough to justify a theory being introduced in this case.\nResults: Citations needed P3, P10, P16, P20, P23 P13: rephrase 1st sentence. Qatar and Norwegian Petroleum Acts were not referenced. Kuwait in perspective: Citations needed P2 Norway in perspective: Rewrite 5th sentence Discussion: cite 6th bullet point\nConclusion: Criticism of 30% to frontier basins would have been better compared with % kept for exploration by case studies used. Bullet d: do you mean \"should not be held\"?\nOverall The problem was identified, the method was clear, the findings were apt, and the suggested solutions were fair. However, evidence should be properly cited to avoid storytelling.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9910", "date": "16 Aug 2023", "name": "OLUSOLA JOSHUA OLUJOBI", "role": "Author Response", "response": "The research demonstrated a smooth flow, and in my opinion, the paper presents an intriguing and informative contribution within its scope, which will enrich the existing literature and enhance policy-making in Nigeria. However, there were poor citations of facts in different paragraphs across different sections. If these are improved upon, then the manuscript is good for indexing. AUTHORS' RESPONSE Thank you very much for reviewing our paper, providing candid comments and suggestions to strengthen our work. We appreciate your efforts. The citations of facts in different paragraphs or across different sections have been improved upon, and we have implemented all the suggestions to make the paper better and suitable for indexing. We have thoroughly reviewed the manuscript, paying special attention to grammar and editing in general. Major points: The citation of facts in paragraphs and making a conclusion on the allocation percentage for frontier basins without adequate comparison with the case studies. AUTHORS' RESPONSE We have corrected the citation of facts in paragraphs and the conclusion on the 30% allocation for frontier basins without adequate comparison with the case studies. We have now included adequate comparisons with the case studies of other countries. Frontier basins can be simply defined as areas where oil has not been found but there is a possibility of oil discovery, such as the Chad Basin, Sokoto Basin, Bida Basin, the Anambra platform, Calabar, and the Niger Delta areas, which have not been explored. Frontier basins hold significant reserves of oil and natural gas, with a projected volumetric ratio of 75% natural gas and 25% crude oil (Obaje, Adamu et al., 2022). However, the transition to low-carbon sources of energy has become crucial for achieving sustainable energy security. Therefore, it is essential for Nigeria to embrace clean energy sources, such as gas, and move away from fossil fuels (Olujobi et al., 2023). According to Nigeria's Petroleum Industry Act (PIA), 30% of the revenue from production sharing contracts, profit sharing, and risk service contracts is allocated for frontier basins exploration. This allocation affects the revenue that should be remitted to the federation, and it will reduce the oil revenue and monthly allocation to various government entities, especially the state governments that rely on it for their day-to-day obligations. In comparison to the selected case study countries, which have embraced clean and mixed energy sources to combat greenhouse gas emissions and their adverse effects on climate change, the allocation of 30% of NNPC LTD's profit for exploration is relatively excessive. These case study countries have allocated 0% for frontier basin exploration. Minor points: Typos, paraphrasing complex sentences, referencing the introduced Acts. Below is the breakdown of my reviews on the different sections. Introduction: Citations needed in P4 (Paragraph 4). Introduce the law first before referring to it as \"the law.\" At first instance, the PIA should be referred to as Nigeria's PIA 2021. Literature reviews: Citations needed in P4, P6, and P3: section 1 of the PIA confers... \"to\" the state. One citation is not enough to justify the introduction of a theory in this case. AUTHORS' RESPONSE We have meticulously corrected the typos, paraphrased complex sentences, and referenced the introduced Acts. The introduction has been completely reworked, introducing the law first before referring to it as \"the law.\" The PIA is now referred to as Nigeria's PIA 2021. We have provided the necessary citations in the Literature Review section, including in P4, P6, and P3 where section 1 of the PIA confers \"to\" the state. We have also included additional citations to support the introduction of theories in this case. In the Results section, we have provided citations in P3, P10, P16, P20, and P23. We have rephrased the first sentence in P13 as suggested by the reviewer. We have also referenced the Qatar and Norwegian Petroleum Acts in the respective sections and provided citations under the Kuwait in perspective section (P2). Additionally, we have reworked the fifth sentence in the Norway in perspective section. Under the Discussion section, we have provided citations for the sixth bullet point. The conclusion has been reworked as follows: The Petroleum Industry Act (PIA) 2021 allocates 30% of the revenue from production sharing contracts, profit sharing, and risk service contracts for frontier basins exploration in Nigeria. This allocation will reduce oil revenue and the monthly allocation to various government entities, especially state governments that rely on it for their day-to-day obligations. In comparison to the selected case study countries, which have embraced clean and mixed energy sources to combat greenhouse gas emissions and their adverse effects on climate change, the allocation of 30% of NNPC LTD's profit for exploration is relatively excessive. These case study countries allocate 0% for frontier basin exploration. The correction has been made for Bullet D, which now reads \"should not be held.\" Thank you very much." }, { "c_id": "9926", "date": "16 Aug 2023", "name": "OLUSOLA JOSHUA OLUJOBI", "role": "Author Response", "response": "We extend our sincerest gratitude for your dedicated effort in reviewing our paper. Your valuable feedback and candid comments have greatly contributed to enhancing the robustness of our work. We truly appreciate the time and expertise you have invested, and we wish abundant blessings upon you and your family. Once again, we express our heartfelt appreciation for your diligent contributions. We are delighted to inform you that we have diligently addressed all your suggestions, ensuring that the paper is now more suitable for publication and indexing. We have made significant improvements in the citation of facts, carefully placing them within paragraphs and across different sections. Additionally, we have rectified the issue of drawing conclusions without adequate comparison to the case studies by conducting thorough comparisons with the relevant countries. To provide clarity, we offer a concise definition of frontier basins as areas where oil has not yet been discovered, but there exists a possibility of finding oil. Notably, the Chad basin, Sokoto basin, Bida basin, the Anambra platform, Calabar, and the Niger Delta areas fall under this category, remaining unexplored. These frontier basins hold immense potential for vast reserves of oil and natural gas, with a projected volumetric ratio of 75% natural gas and 25% crude oil. In light of the urgent shift towards low-carbon energy sources for sustainable energy security, it is imperative for Nigeria to embrace cleaner energy sources, such as gas, and transition away from fossil fuels. Regarding the allocation of funds, the Nigeria Petroleum Industry Act (PIA) designates 30% of production sharing contracts, profit sharing, and risk service contracts to be allocated for frontier basins exploration. While this allocation supports the development of frontier basins, it is important to note that this revenue, which would otherwise be remitted to the federation, will reduce the overall oil revenue and monthly allocation to various government entities. State governments, in particular, heavily rely on these funds to fulfill their day-to-day statutory obligations. When compared to selected case study countries that have embraced clean and mixed energy sources, the allocation of 30% of NNPC LTD's profit for exploration in Nigeria appears relatively excessive. Notably, these case study countries allocate 0% for frontier basin exploration, emphasizing their commitment to combatting greenhouse gas emissions and addressing the adverse effects of climate change. We have meticulously addressed all typos, paraphrased complex sentences, and ensured proper referencing of the introduced Acts. Furthermore, the introduction has undergone a holistic rework, introducing Nigeria's PIA 2021 before referring to it as \"the law.\" Additionally, we have provided the necessary citations in various sections of the paper, including the Literature Review (citations under P4, P6, P3), Results (citations under P3, P10, P16, P20, P23), and Discussion (citation for the 6th bullet point). The corrections you suggested, such as rephrasing the first sentence in P13, referencing the Qatar and Norwegian Petroleum Acts, and overhauling the fifth sentence under the Norway in perspective section, have all been meticulously implemented. Finally, we have reworked the conclusion, incorporating your suggestions. The 30% allocation from production sharing contracts, profit sharing, and risk service contracts, as stated in Nigeria's Petroleum Industry Act (PIA) 2021, will lead to a reduction in oil revenue and the monthly allocation to various government entities, particularly state governments. Moreover, in comparison to the selected case study countries that have embraced clean and mixed energy sources, the allocation of 30% of NNPC LTD's profit for exploration in Nigeria appears relatively excessive. This is due to the adverse effects of greenhouse gas emissions on climate change. In contrast, the case study countries allocate 0% for frontier basin exploration. We are pleased to inform you that all the corrections, including the necessary adjustments to the bullet point you highlighted, have been diligently implemented. It now reads, \"should not be held.\" Once again, we extend our utmost gratitude for your invaluable feedback and guidance throughout this process. With warm regards" } ] }, { "id": "183238", "date": "19 Jul 2023", "name": "Enobong Mbang AKPAMBANG", "expertise": [ "Reviewer Expertise Oil and Gas Law", "Constitutional Law", "Comparative Constitutional Law", "and Women and the Law" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article discusses a contemporary issue relating to the Nigerian petroleum industry. It examines the Petroleum Industry Act 2021 vis-a-vis the prospects, challenges, and the way forward in addressing the problems identified in the article.\n\nThe background, methods, and results are okay but there are some minor editorial corrections to be made on page 1 and other pages of the article. For instance under 'Background' line 8 should read: Hence, the need for the PIA of 2021 to overhaul the industry to meet global standards; there are also some controversial provisions that pose challenges to its proper implementation.\nUnder Results on page 1, lines 5-6 should read ....the PIA was provided for weak institutions, which translates to weak implementation and enforcement of the law and thus, further widened the existing gap between law and reality.\nThe authors repeatedly used the word, 'act' to refer to the PIA. It is suggested that they should use \"Act\" instead.\n\nLiterature Review, paragraphs 1-4 (lines 1-20) on page five (5) of the article should be taken to and discussed as a continuation under the main Literature Review as the authors are still reviewing some available literature in the area.\nUnder ambiguous wording and imprecise language: it is not correct for the authors to state that PIA failed to provide definitions of the terms: \"frontier basin\" and \"host community\". On the contrary, section 318 of the PIA, which is the interpretation section, defines the terms. The said section defines \"frontier basin\" to \"mean basins where hydrocarbon exploration activities has not been carried out or previous commercial discovery oil and gas have not been made or an area that is undeveloped and includes Anambra, Dahomey, Bida, Chad and Benue trough or as may be declared by the Commission through a regulation.\" On the other hand, the section goes further to define \" host communities\" as \"communities situated in or appurtenant to the area of operation of a settlor, and any other community as a settlor may determine under chapter three\" of the PIA. The authors are accordingly referred to the said section.\nUnder corruption on page seven lines 1-2: the sentence is incomplete. The authors should also tell us what would happen if corruption is not tackled in the petroleum industry.\n\nUnder Discussion on pages 10-11, bullet three, lines 1-4: the authors should tell us how the PIA undermines section 162 of the 1999 Constitution of the Federal Republic of Nigeria (as amended). It is not enough for the authors to merely assert that the PIA seems to undermine section 162 of the Constitution.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9936", "date": "16 Aug 2023", "name": "OLUSOLA JOSHUA OLUJOBI", "role": "Author Response", "response": "AUTHORS’ RESPONSES We extend our deepest appreciation for the meticulous review and valuable suggestions provided by the esteemed reviewer. Your expertise and keen eye have undeniably enhanced the quality of our manuscript, and we are sincerely grateful for your time and effort. The article delves into a pressing contemporary issue, pertaining to the Nigerian petroleum industry, and meticulously examines the Petroleum Industry Act 2021 in relation to its prospects, challenges, and the way forward in addressing the problems identified in the article. We are deeply thankful for your insightful comments and suggestions that have undoubtedly strengthened the robustness of our work. May you and your family be blessed abundantly for your contributions. Once again, thank you, sir, for your unwavering support. We have diligently reviewed the manuscript, placing special emphasis on grammar and overall editing. Your feedback has been immensely valuable, and we truly appreciate it. 1. Thank you for your discerning assessment of our manuscript. We are grateful for the positive feedback on the background, methods, and results. Moreover, we acknowledge the need for minor editorial corrections on page 1. As per your suggestion, we have revised the 'Background' section accordingly, and line 8 now reads as follows: \"Hence, the need for the PIA of 2021 to overhaul the industry to meet global standards; there are also some controversial provisions that pose challenges to its proper implementation.\" Your attention to detail has been a tremendous asset, and we sincerely thank you for it. 2. Your sincere commentaries and efforts to strengthen our manuscript are highly appreciated. We have duly corrected the mentioned section under the results on page 1, lines 5-6, as per your recommendation. It now reads: \"The PIA was provided for weak institutions, which translates to weak implementation and enforcement of the law and thus, further widened the existing gap between law and reality.\" Thank you, sir, for your valuable input. 3. We express our gratitude for your astute observation. We understand the significance of consistency in writing style. While we initially attempted to change the word \"act\" to \"Act\" as suggested, the Journal's in-house style ultimately prevailed. Nevertheless, we have duly rectified the word, 'act,' to 'Act,' referring to the legislation made by the National Assembly under the PIA. Your attention to detail and commitment to excellence are truly commendable, and we are thankful for your kind understanding. 4. Your attention to the arrangement and continuity of the literature review is duly noted. We have incorporated your suggestion by taking paragraphs 1-4 (lines 1-20) on page five (5) of the article and seamlessly integrating them as a continuation under the main Literature Review section. Your keen eye for organization and clarity has been incredibly valuable, and we extend our heartfelt appreciation. 5. We sincerely appreciate your diligent analysis of the ambiguous wording and imprecise language in our manuscript. Your observation regarding the definitions of \"frontier basin\" and \"host community\" in the PIA is entirely accurate. We apologize for the oversight and have duly corrected the statement. Section 318 of Nigeria’s PIA 2021, which serves as the interpretation section, indeed defines these terms explicitly. As you mentioned, \"frontier basin\" is defined as \"basins where hydrocarbon exploration activities have not been carried out or previous commercial discovery oil and gas have not been made or an area that is undeveloped and includes Anambra, Dahomey, Bida, Chad, and Benue trough or as may be declared by the Commission through a regulation.\" Furthermore, \"host communities\" are defined as \"communities situated in or appurtenant to the area of operation of a settlor, and any other community as a settlor may determine under chapter three\" of the PIA. Your commitment to precision and accuracy is truly commendable, and we thank you for bringing this to our attention. 6. Your thorough review has been invaluable in identifying an incomplete sentence. We have now completed the sentence in the section discussing corruption on page seven, lines 1-2. Moreover, we have elaborated on the consequences of corruption if left unaddressed in the petroleum industry. Corruption hampers transparency and deprives Nigerians of the benefits of their abundant resources, leading to a decline in their standard of living, social, energy security, and sustainability, thereby promoting mass poverty. Your commitment to clarity and comprehensive understanding is deeply appreciated. 7. We are deeply grateful for your insightful suggestion regarding the discussion on pages 10-11. You have aptly pointed out that merely asserting the undermining of section 162 of the 1999 Constitution of the Federal Republic of Nigeria (as amended) by the PIA is insufficient. We have taken your advice and thoroughly explained how the PIA 2021 may undermine this constitutional provision. Specifically, by allowing the NNPCLTD to retain 30% from the production sharing contracts, profit sharing, and risk service contracts meant for frontier basins exploration, the PIA contravenes section 162 of the 1999 Constitution. This retention of 30% will reduce the oil revenue accruing to the Federal Government, states, and local governments, making it challenging for various government bodies to meet their statutory obligations. Furthermore, we have highlighted how other case study countries, which have embraced clean sources of energy and energy mixed to combat greenhouse gas emissions, have retained 0% for frontier basin exploration. These countries serve as valuable examples that could be emulated through constant reform under the PIA. Your exceptional insights and attention to legal aspects have significantly enriched our manuscript, and we sincerely thank you for it. 8. In conclusion, we are immensely grateful for your dedication to excellence, your comprehensive review, and your valuable suggestions. Your contributions have undeniably strengthened our work, and we are indebted to you for your guidance and support. Thank you once again for your unwavering commitment to ensuring the quality and integrity of our manuscript." } ] } ]	1	https://f1000research.com/articles/12-551
https://f1000research.com/articles/12-984/v1	15 Aug 23	{ "type": "Research Article", "title": "Efficacy of an external cold and vibrating device in reducing discomfort during the administration of an inferior alveolar nerve block in children: A split-mouth randomised crossover study", "authors": [ "Ananthu H", "Ashwin Rao", "Srikant Natarajan", "Karuna Yarmunja Mahabala", "Anupama Nayak", "Ananthu H", "Srikant Natarajan", "Karuna Yarmunja Mahabala", "Anupama Nayak" ], "abstract": "Background: Local anaesthesia is the backbone of pain management. However, the administration of a local anaesthetic injection itself is considered a painful procedure and triggers fear and anxiety in the patient. Methods: A split-mouth randomised controlled crossover trial was designed to study the efficacy of an “external cold and vibrating” device in reducing discomfort during the administration of an inferior alveolar nerve block (IANB) in children. A total of 40 children who fulfilled the inclusion criteria were evaluated for pain response following administration of IANB with and without the “external cold and vibrating” device. Randomisation was performed to determine whether the subject received the control intervention first or the test intervention first. The intensity of the pain response and discomfort were recorded using the Wong-Baker FACES Pain Rating Scale and the Faces Legs Activity Cry and Consolability scale. Results: There was a statistically significant difference in the pain response between the test group and the control group (p<0.001). Females reported a higher pain response than males, with a statistically significant difference, when the FLACC scores were compared. Conclusions: The “external cold and vibrating” device reduced discomfort during the administration of an IANB in children selected for the study.", "keywords": [ "Pain", "Pain management", "Analgesia", "Local anaesthesia", "Dental anaesthesia", "Nerve block", "Distraction", "Paediatric dentistry" ], "content": "Introduction\n\nPain management in patients has been one of the most influential factors affecting the success of any dental treatment. In fact, the fear and anxiety surrounding dental procedures has forced patients to resort to a more ‘wait and watch’ approach, delaying or avoiding the dental treatment as much as possible.1 Dental anaesthesia has always played a pivotal role in the management of pain in patients. However, the very procedure inducing anaesthesia, with the local anaesthetic injection, is considered a painful procedure and triggers fear and anxiety in the patient.2 Trypanophobia, or the fear for needles or injections, is considered as one of the major sources of pain in dental patients.3 This clinical problem becomes even more challenging in paediatric patients.\n\nThe search for methods to administer a painless dental anaesthetic injection has led to the discovery of various innovative techniques. Several devices and methods have been proposed to tackle this clinical problem, including the use of expensive computer-controlled local anaesthetic delivery systems.4 However, an ideal solution has not been found so far.\n\nOne of the techniques that developed over time to help reduce the discomfort during a local anaesthetic injection for dental treatment, is desensitising the injection site, through precooling, employing vibration techniques or a combination of both.5 Distraction is a safe and reliable behaviour management strategy that helps minimise pain and anxiety by directing the attention away from painful stimuli, during the anaesthesia administration. The use of distraction in paediatric patients during local anaesthetic injections, through the application of cold temperature and vibration techniques, has garnered much attention in recent times.6–11\n\nExternal cold and vibrating devices propose to reduce pain by altering the individual’s perception to pain. It is hypothesized that the distractive environment created by the vibrating device or buzzing device, coupled with cold, causes the brain cells to relay the non-pain signals (vibration or cold) generated, masking the pain signals triggered by the injection.12,13 This masking effect of pain is brought about by creating a confusion in the perception of signals by the pain pathway.13,14 Studies performed have shown the effectiveness of this concept of pain management in immunisations and venepuncture.12,13\n\nThe effectiveness of the “external cold and vibrating” device while administering the maxillary infiltration anaesthesia in children has been reported in literature.15 However, its efficacy to reduce discomfort during the administration of an inferior alveolar nerve block (IANB) in children has not been studied. The aim of the study was to evaluate the efficacy of an “external cold and vibrating device” (Buzzy Mini Personal Striped with ice wings, MMJ Labs, Atlanta, GA, USA), in reducing discomfort during the administration of an IANB in children.\n\n\nMethods\n\nThis randomized controlled clinical trial followed a split mouth, crossover study design with a 1:1 balanced allocation ratio. This study protocol was approved by the Research Ethics Committee of Manipal College of Dental Sciences (MCODS), Mangalore (Reference number 19086) and the trial was registered at the clinical trial registry-India (Registration number CTRI/2020/07/026702). The trial was carried out at the Department of Pediatric and Preventive Dentistry over a period of 23 months. Before enrolment, all participants and their parents/guardians signed written informed consent forms. All procedures were conducted in compliance with the Helsinki Declaration 1975.\n\nThe study was conducted on 40 children who reported to the outpatient department of Pediatric and Preventive Dentistry, MCODS Mangalore, MAHE (Deemed to be University), Mangaluru, Karnataka.\n\nBased on the key article by Alanazi et al.,15 the mean and standard deviation of pain response in test and control groups in males was 2 ± 0.32 and 7 ± 0.27 respectively. With 5% alpha error, 90% power of the study and a clinically significant difference of 0.2 units, the required sample in each group was calculated to be 40. The following formula was used to calculate the sample size (N),\n\nThe inclusion criteria for the study were children:\n\n• Aged six to 10 years with no prior experience of an intraoral local anaesthetic injection\n\n• Rated as definitely positive or positive on the Frankl behaviour rating scale\n\n• Classified ASA I according to the American Society of Anaesthesiologists Physical Status Classification System (ASA-PS)\n\n• With any mandibular tooth/teeth indicated for bilateral dental procedures that warranted the use of an IANB.\n\nThe exclusion criteria were children with a:\n\n• History of recent hospitalisation or surgery\n\n• History of central nervous system depressants or analgesics consumed within eight hours prior to the treatment\n\n• An existing orofacial oedema, infection, abscess or the presence of systemic comorbidities with special dental treatment considerations.\n\nWritten informed assent from the parents and written informed consent from the children were obtained respectively. The control intervention was carried on one side of the patient whereas the test intervention was performed on the contralateral side. The order in which the subjects received the interventions was allocated by randomisation. The right side of the patient received the intervention in the first visit (either control or test) whereas the left side received the intervention in the second visit (either control or test). The wash-out period between the two visits was one week.16\n\nRandomisation was performed to determine whether the subject received the control intervention first or the test intervention first. This was done by the statistician who was otherwise not involved in the study. A block randomization was done as it helped ensure a 1:1 sample allocation ratio, which meant that equal number of samples were subjected to both the test intervention as well as the control intervention on their first visit. Block randomisation was done using a varied block size of 2 and 4. Allocation concealment was done using opaque envelopes. The block sequences (AB, BA, AABB and so on) was computer-generated, following which the statistician performed random allocation of the samples to the blocks using the random number table. The treatment intervention group codes so generated, ‘A’ or ‘B’, wherein ‘A’ stood for the control intervention (administration of IANB without Buzzy) and ‘B’ stood for the test intervention (administration of IANB with Buzzy), were entered into cards along with the sample number, which was placed in numbered and sequentially arranged envelopes. The cards were wrapped in aluminium foil to confirm that the envelopes appeared opaque. The envelopes were sealed. In this way, the predictability of the sample allocation which is the disadvantage of block randomization was overcome. The envelopes were opened just prior to the introduction of the interventions, to avoid bias.17\n\nThe treatment protocol described in the envelope, was carried out on the right side of the patient while the alternative treatment was done on the contralateral side in the subsequent visit.\n\nThe site of needle penetration was topically anesthetized with 20% benzocaine topical anaesthetic gel (‘Precaine B, Pascal International, Bellevue, WA, USA’) four minutes prior to the injection in both the groups. The children assigned to the control group were administered the IANB with a conventional technique.18 The contents of the entire cartridge containing 1.8 mL of 2% lignocaine with 1:80,000 adrenaline was injected19 (Septodont Healthcare India Pvt. Ltd, Maharashtra, India).\n\nThe children assigned to the test group were administered the IANB coupled with the “external cold and vibrating” device (Buzzy Mini Personal Striped with Ice Wings, MMJ labs, Atlanta, GA, USA). The Buzzy (Figure 1) is a bee-shaped device which offers vibrations, powered by two alkaline AAA batteries and cold temperature offered by the detachable, refrigerable ice wings.20 Prior to the delivery of anaesthesia by injection, the refrigerated wings were attached to the bee shaped body of the device, following which the child was asked to hold and keep the device on the cheek area, corresponding to the ramus of the mandible (Figure 2). After thirty seconds, the administration of IANB was initiated, with the Buzzy device switched on manually. The device was switched off, upon completion of the administration of the anaesthetic solution.\n\nEach child was instructed to quantify the pain experienced during the injection using the “Wong-Baker Faces Pain Rating Scale” (WBFPRS) following administration of the IANB. The WBFPRS is a horizontal scale of six hand-drawn faces, scored from 0 to 10, that range from a smiling ‘no hurt’ face on the left to a crying ‘hurts worst’ face on the right. It is simple to use and implement, and it is well accepted by children, parents, and doctors.21 However, it being a self- reported scale, the child’s report of pain may be affected by developmental, cognitive and situational issues.22 To overcome this drawback, another scale was used as an adjunct to this scale. This was the Faces Legs Activity Cry and Consolability (FLACC) scale, which assesses five different aspects of the child’s behaviour. This scale is shown to have good validity and reliability. The five categories are assessed in order to rate pain wherein each category is ranked on a three-point scale (0-2), resulting in a summary score of (0-10).23,24 An independent precalibrated evaluator, rated the children on the FLACC scale during the injection.\n\nThe Statistical Package for Social Science (SPSS) (version 20.0) software package was used to analyse the data. The comparison between responses of the participants to pain on the WBFPRS at the test visit and control visit and the observed behaviour of participants using the FLACC scale at the test visit and control visit was measured by Wilcoxon signed rank test. The level of significance was set at 5% (i.e. <0.05).\n\n\nResults\n\nIn total, 40 children aged six to 10 years, with any mandibular tooth/teeth indicated for bilateral dental procedures that warrants the use of an IANB, were recruited for the study. The 40 recruited children comprised 13 (32.5%) females and 27 (67.5%) males with a mean age of 7.90 years (S.D ± 1.533). The percentage of males recruited for the study were almost double that of females.\n\n\n\n• Among females, on comparison of the median score values of WBFPRS control and WBFPRS test, the median score value of WBFPRS control was higher than that of the WBFPRS test by two units with a p-value of 0.002 (statistically significant).\n\n• Among females, on comparison of the median score values of FLACC control and FLACC test, the median score value of FLACC control was higher than that of test by three units with a p-value of 0.001 (statistically significant).\n\n• Among males, on comparison of the median score values of WBFPRS control and WBFPRS test, the median score value of WBFPRS control was higher than that of the WBFPRS test by two units with a p-value <0.001 (statistically significant).\n\n• Among males, on comparison of the median score values of FLACC control and FLACC test, the median score value of FLACC control is higher than that of test by two units with a p-value <0.001 (statistically significant).\n\n• Analysing the 40 participants, the median score value of WBFPRS control was higher than that of test by two units with a standard score (Z value) of -5.767. The WBFPRS score comparison gave a p-value <0.001 which was statistically significant.\n\n• Analysing the 40 participants, the median score value of FLACC control was higher than that of the test by three units with a standard score (Z value) of -5.655. The FLACC score comparison gave a p-value <0.001 which is statistically significant.\n\nThe inference from the above statements was that pain scores were higher for both the WBFPRS and FLACC scales in the control group.\n\n\nDiscussion\n\nThe results of the present study showed that the experimental Buzzy device demonstrated a statistically significant effect in reducing self-reported and observer-reported reaction to pain during the administration of the IANB. These findings support the results of previous studies, which had reported success with the combined use of cold and vibration to alleviate discomfort in children undergoing maxillary infiltration dental anaesthesia.15\n\nThe Buzzy is an economical, versatile, quickly vibrating plastic device designed like a bee, which combines high-frequency vibration and cold temperature to control sharp pain. The pain control mechanism is on the basis of the “gate control” pain relief theory by confusing the body’s own nerves, thereby dulling or eliminating sharp pain. The brain closes the gate on pain signals when nerves receive non-painful signals such as vibration or cold. Another mechanism by which the Buzzy device claims its effect is through “diffuse noxious inhibitory control” or DNIC wherein certain noxious signals are dampened out by the brain. The intense cold also activates a supraspinal modulation raising the body’s overall pain threshold.20,25\n\nAs pain and discomfort are subjective and vary from child to child, it’s difficult to compare the effectiveness of one approach to another when employed separately in different children.14 To avoid such variances, the split-mouth cross-over design was employed, which has been used in other trials investigating the effectiveness of the Buzzy device.9,10 Crossover trials are most appropriate for studies related to pain as well as when the treatment effects are reversible and short-lived.26\n\nRandomisation was performed to determine whether the subject would receive the control intervention first or the test intervention first. A block randomization was done as it helped ensure a 1:1 sample allocation ratio, which meant that equal number of samples were subjected to both the test intervention as well as the control intervention on their first visit.\n\nThis study included children aged six to 10 years old, as this has been suggested as the age at which cognitive growth begins to reveal itself.27 This will ensure adequate communication between the operator and the child for the purposes of the study. Inal et al. in 201228 and Moadad et al. in 201629 recruited a similar age group in their studies.\n\nIn the present study, two separate pain scales were used to ensure the reproducibility of the pain assessment.\n\nAlthough subjective pain assessment is typically deemed the gold standard for assessing pain, children’s perceptions of pain might well be exaggerated, especially soon after the treatment. Pain is a highly complex and multidimensional attribute that depends on various factors such as location, quality of sensory perception and the cognitive ability of the child. Use of only a subjective scale may not reflect the true pain and discomfort experienced by the child patient. Hence observational pain scales should be used in conjunction with subjective scales. For pain evaluation in young children, the FLACC scale has been proven to showcase great validity and reliability. Hence, in this study both self-report WBFPRS and the observational FLACC scale was used.\n\nThis study selected only children rated definitely positive and positive on the Frankl rating scale. This was done to negate the effect of behaviour on the results of the study. Lack of blinding, which could not be carried out due to the nature of the intervention, was also a shortcoming of this trial\n\nFuture studies can include anxious children or children of a younger age group to study the effects of the external cold and vibrating device.\n\n\nConclusions\n\nThe results showed that there was a statistically significant difference in the pain response when the control group and the test group were compared (p<0.001). Based on the inferences from the current study it can be concluded that the “external cold and vibrating” device reduced the discomfort during the administration of an IANB in children selected for the study.\n\nResearch trials should be conducted in the future to explore the effectiveness of the “external cold and vibrating” device during the various different techniques of local anaesthesia administration and among children belonging to different age groups.\n\n\nAuthors’ contributions\n\nAll the authors have equal contribution to this research in manuscript preparation, data collection and interpretation.", "appendix": "Data availability\n\nFigshare: Master data for the control group and the test group.xlsx, https://doi.org/10.6084/m9.figshare.23498491.v1. 30\n\nThis project contains the underlying following data:\n\nData file 1: Master data for the control group and the test group\n\nData file 2: Results table\n\nData file 3: Sample size calculation\n\nFigshare: Extended data, https://doi.org/10.6084/m9.figshare.23498508.v1. 31\n\nThis project contains the underlying following data:\n\nData file 1: Child assent form\n\nData file 2: Clinical trial protocol\n\nData file 3: FLACC Scale\n\nData file 4: IEC Approval Thesis\n\nData file 5: Informed consent sheet\n\nData file 6: Pain Rating Scale\n\nData file 7: Patient information sheet\n\nFigshare: CONSORT check list and flow chart for “Efficacy of an External Cold and Vibrating Device in Reducing Discomfort during the Administration of an Inferior Alveolar Nerve Block in Children: A Split Mouth Randomised Crossover Study”, DOI: https://doi.org/10.6084/m9.figshare.23498673.v1. 32\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe authors would like to express their gratitude to the Manipal College of Dental Sciences, Mangalore for all the help and support.\n\n\nReferences\n\nWiederhold MD, Gao K, Wiederhold BK: Clinical use of virtual reality distraction system to reduce anxiety and pain in dental procedures. Cyberpsychol Behav Soc Netw. 2014; 17(6): 359–365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCzarnecki ML, Turner HN, Collins PM, et al.: Procedural pain management: a position statement with clinical practice recommendations. Pain Manag Nurs. 2011; 12(2): 95–111. PubMed Abstract \| Publisher Full Text\n\nFriedrichsdorf SJ, Postier A, Eull D, et al.: Pain Outcomes in a US Children’s Hospital: A Prospective Cross-Sectional Survey. Hosp Pediatr. 2015; 5(1): 18–26. Publisher Full Text\n\nJälevik B, Klingberg G: Pain sensation and injection techniques in maxillary dento-alveolar surgery procedures in children--a comparison between conventional and computerized injection techniques (The Wand). Swed Dent J. 2014; 38(2): 67–75. PubMed Abstract\n\nAminah M, Nagar P, Singh P, et al.: Comparison of topical anesthetic gel, pre-cooling, vibration and buffered local anesthesia on the pain perception of pediatric patients during the administration of local anesthesia in routine dental procedures. Int J Contemp Med Res. 2017; 4(2): 400–403.\n\nAminabadi NA, Farahani RM: The effect of pre-cooling the injection site on pediatric pain perception during the administration of local anesthesia. J Contemp Dent Pract. 2009; 10(3): 43–50. PubMed Abstract \| Publisher Full Text\n\nGhaderi F, Banakar S, Rostami S: Effect of pre-cooling injection site on pain perception in pediatric dentistry: “A randomized clinical trial”. Dent Res J (Isfahan). 2013; 10(6): 790–794. PubMed Abstract\n\nHameed NN, Sargod SS, Bhat SS, et al.: Effectiveness of precooling the injection site using tetrafluorethane on pain perception in children. J Indian Soc Pedod Prev Dent. 2018; 36: 296–300. PubMed Abstract \| Publisher Full Text\n\nChing D, Finkelman M, Loo CY: Effect of the DentalVibe injection system on pain during local anesthesia injections in adolescent patients. Pediatr Dent. 2014; 36(1): 51–55. PubMed Abstract\n\nShilpapriya M, Jayanthi M, Reddy VN, et al.: Effectiveness of new vibration delivery system on pain associated with injection of local anesthesia in children. J Indian Soc Pedod Prev Dent. 2015; 33: 173–176. Publisher Full Text\n\nMenni AC, Radhakrishna AN, Prasad MG: DentalVibe® versus lignocaine hydrochloride 2% gel in pain reduction during inferior alveolar nerve block in children. J Dent Anesth Pain Med. 2020; 20(6): 397–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCanbulat N, Ayhan F, Inal S: Effectiveness of external cold and vibration for procedural pain relief during peripheral intravenous cannulation in pediatric patients. Pain Manag Nurs. 2015; 16(1): 33–39. Publisher Full Text\n\nCanbulat Şahiner N, İnal S, Sevim AA: The effect of combined stimulation of external cold and vibration during immunization on pain and anxiety levels in children. J Perianesth Nurs. 2015; 30(3): 228–235. PubMed Abstract \| Publisher Full Text\n\nBaxter AL, Lawson ML: Methodological concerns comparing buzzy to transilluminator device. Indian J Clin Biochem. 2014; 29(1): 114–115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlanazi KJ, Pani S, AlGhanim N: Efficacy of external cold and a vibrating device in reducing discomfort of dental injections in children: A split mouth randomised crossover study. Eur Arch Paediatr Dent. 2019; 20(2): 79–84. PubMed Abstract \| Publisher Full Text\n\nYogesh Kumar TD, John JB, Asokan S, et al.: Behavioral response and pain perception to computer controlled local anesthetic delivery system and cartridge syringe. J Indian Soc Pedod Prev Dent. 2015; 33(3): 223–228. PubMed Abstract \| Publisher Full Text\n\nPandis N, Chung B, Scherer RW, et al.: CONSORT 2010 statement:extension checklist for reporting within person randomised trials. BMJ. 2017; 357: j2835. Publisher Full Text\n\nMalamed SF: Basic injection technique. Handbook of Local Anesthesia. St. Louis, Mo: Mosby; 5th ed.2004; 159–169.\n\nSoxman JA, Malamed SF: Local anesthesia for the pediatric patient. Handbook of Clinical Techniques in Pediatric Dentistry. 2015; 27(1): 5–12.\n\nBaxter AL, Cohen LL, McElvery HL, et al.: An integration of vibration and cold relieves venipuncture pain in a pediatric emergency department. Pediatr Emerg Care. 2011 Dec; 27(12): 1151–1156. PubMed Abstract \| Publisher Full Text\n\nGarra G, Singer AJ, Taira BR, et al.: Validation of the Wong-Baker FACES Pain Rating Scale in pediatric emergency department patients. Acad Emerg Med. 2010; 17(1): 50–54. PubMed Abstract \| Publisher Full Text\n\nStinson JN, Kavanagh T, Yamada J, et al.: Systematic review of the psychometric properties, interpretability and feasibility of self-report pain intensity measures for use in clinical trials in children and adolescents. Pain. 2006; 125(1-2): 143–157. PubMed Abstract \| Publisher Full Text\n\nMerkel S, Voepel-Lewis T, Malviya S: Pain assessment in infants and young children: the FLACC scale. Am J Nurs. 2002; 102(10): 55–58. PubMed Abstract \| Publisher Full Text\n\nNilsson S, Finnström B, Kokinsky E: The FLACC behavioral scale for procedural pain assessment in children aged 5–16 years. PaediatrAnaesth. 2008; 18(8): 767–774.\n\nBaxter AL, Leong T, Mathew B: External thermomechanical stimulation versus vapocoolant for adult venipuncture pain: pilot data on a novel device. Clin J Pain. 2009; 25(8): 705–710. PubMed Abstract \| Publisher Full Text\n\nEvans SR: Clinical trial structures. J Exp Stroke Transl Med. 2010; 3(1): 8–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlomqvist M, Ek U, Fernell E, et al.: Cognitive ability and dental fear and anxiety. Eur J Oral Sci. 2013; 121(2): 117–120. PubMed Abstract \| Publisher Full Text\n\nInal S, Kelleci M: Relief of pain during blood specimen collection in pediatric patients. MCN Am J Matern Child Nurs. 2012; 37(5): 339–345. PubMed Abstract \| Publisher Full Text\n\nMoadad N, Kozman K, Shahine R, et al.: Distraction Using the BUZZY for Children During an IV Insertion. J Pediatr Nurs. 2016; 31(1): 64–72. PubMed Abstract \| Publisher Full Text\n\nAnanthu H: Master data for the control group and the test group.xlsx. [Dataset]. figshare. 2023. Publisher Full Text\n\nAnanthu H: Extended data. [Dataset]. figshare. 2023. Publisher Full Text\n\nAnanthu H: Reporting guidelines. figshare. [Reporting guidelines].2023. Publisher Full Text" }	[ { "id": "197890", "date": "04 Sep 2023", "name": "Vivek Padmanabhan", "expertise": [ "Reviewer Expertise Pediatric Dentistry", "Dentistry", "Epidemiological studies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGood research and may help to resolve this ambiguous part of pediatric dentistry, however the practical applicability remains to be seen.\n\nPlease note that the entire article has been split into a lot of paragraphs. Kindly revise it.\n\nThe part where it is mentioned consent and assent was taken please note that assents are taken from children and consent is taken from adults, so correct it accordingly.\nIn the statistical analysis, it has been mentioned Wilcoxon has been used, kindly elaborate how this test helps understand the study better, also give complete details of the SPSS as in brand name and associated details.\n\nIn the discussion please compare the results of your study with other studies done worldwide and then either defend the findings or agree with others. The first sentence of conclusion is not necessary. in conclusion also mention about differences between males and females as I believe that was one of your objectives.\nThe future recommendations need to be provided based upon the limitations of the present study also please note that the sample number should also be increased in future studies, this can be added as both drawback and address it in future recommendations.\n\nPlease note that the references can be kept within a maximum of 10 years else it is not clinically significant for today and need not be cited in the present study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "229569", "date": "28 Dec 2023", "name": "Meenu Mittal", "expertise": [ "Reviewer Expertise Local anesthesia in pediatric patients" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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's a good research but some details/corrections are required:\nMethods: 1.Correction regarding Assent/consent required. 2. Intervention- It is not mentioned why the injection was given and which procedure was performed post injection for treatment of which tooth.\nResults: 1.Needs to be compiled, written haphazardly with text matter repeated.\nDiscussion: 1.The description about pain scales etc. to be shortened 2. There is no comparison with other studies discussed, which needs to be added. Limitations: To be elaborated. Language: Small corrections in grammar needed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "197889", "date": "27 Mar 2024", "name": "Kavitha Ramar", "expertise": [ "Reviewer Expertise Nano dentistry", "special children", "behaviour management" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis clinical trial addresses one of the most important challenges faced by dentists around the globe - managing pediatric dental patients and it has done justice in validating an excellent practical solution. The research article has been well presented, and is scripted with detailed descriptions under all the sections. The Title of the study “Efficacy of an external cold and vibrating device in reducing discomfort during the administration of an inferior alveolar nerve block in children: A split-mouth randomised crossover study” is simple, understandable and outlines the theme of the research in a nutshell. The Abstract is precise, well-structured and has been summarized adequately. The Introduction provides an excellent opening to the theme of the research wherein the challenges faced by pediatric dentists and the techniques they employ to manage pediatric dental patients while administering local anaesthesia have been clearly outlined. The external cold and vibrating device Buzzy is a novel armamentarium which has been utilised by doctors and other healthcare givers while managing pediatric patients, however the limited research in the field of dentistry has triggered the researchers to come up with this proposed study, which they have substantiated with proper evidence. The authors have succeeded in clearly chalking out the necessary armamentarium utilised for the research. A step wise methodology has been presented, which would help fellow researchers who wish to pursue their research further in the same arena. A randomised clinical trial is evaluated by how comprehensibly the randomisation process has been described and followed. The authors have succeeded in this aspect by outlining the randomisation process in a very systematic manner. The split mouth design of the study has also been justified. Thus, the selection of the study design is justified and the work showcases academic merit. The results and interpretations are sound and supported by relevant and current evidence. The authors have stated the findings, highlighting key aspects in tables and figures. Commenting on the statistical analysis, by employing the Wilcoxon test, the authors have helped understand the study results better. The Discussion part is excellently structured and has been elucidated in a very appropriate manner justifying all the results and findings of the study. The discussion of the findings is suitable, gives answers to the research objectives, incorporating substantial support from literature. Cross references with similar as well as contrasting research have been appropriately described by the authors, which give a scope of better understanding for fellow researchers.\n\nThe authors have even mentioned the drawbacks of the trial, which is appreciable. One of the drawbacks of the study is the selection of only cooperative children which has also been mentioned by the authors. By mentioning the limitations of the study, authors have hinted at the scope for the improvement of the study. The conclusions are sound, justified and logically explained, and are supported by the results. No revisions are required.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-984
https://f1000research.com/articles/12-725/v1	21 Jun 23	{ "type": "Research Article", "title": "Antimicrobial efficacy of Kerr pulp canal sealer (EWT) in combination with 10% amoxicillin on Enterococcus faecalis: A confocal laser scanning microscopic study", "authors": [ "Madhureema De Sarkar", "Kundabala Mala", "Suchitra Shenoy Mala", "Shama Prasada Kabekkodu", "Srikant Natarajan", "Neeta Shetty", "Priyanka Madhav Kamath", "Manuel Thomas", "Madhureema De Sarkar", "Suchitra Shenoy Mala", "Shama Prasada Kabekkodu", "Srikant Natarajan", "Neeta Shetty", "Priyanka Madhav Kamath", "Manuel Thomas" ], "abstract": "Background: Sealers with antimicrobial properties play an important role in endodontic therapy success especially against Enterococcus faecalis infection found in failed root canal therapy. Addition of antibiotic agents to endodontic sealers may show significant increase in their antibacterial properties both against anaerobic and aerobic microbes. The purpose of the present study was to evaluate antimicrobial efficacy of Kerr pulp canal sealer (EWT) in combination with 10% amoxicillin against E. faecalis and post-root canal treatment viability of Enterococcus faecalis on the first and seventh day. Methods: A total of 60 extracted human mandibular premolar teeth were decoronated after initial decontamination with 1% NaOCl. Root length standardized to 12 mm. Canal instrumentation was done using ProTaper Universal file system till size F2 using 5.25% NaOCl. It was then infected with a pure strain of E. faecalis for a period of four days. Obturation was done using plain sealer, (n=30) and sealer-antibiotic combination, (n=30). Half of the teeth were sectioned at 24 hours (S, SA) and other half were sectioned seven days after obturation (S7, SA7). All samples were stained with SYTO9 and propidium iodide for imaging under Confocal Laser Scanning microscope. Statistical analysis was performed with the statistical software SPSS v. 17.0 (SPSS for Windows; SPSS Inc, Chicago, IL). Data was analysed using One Way ANOVA and post hoc Tukey test to determine statistical significance with p value < 0.01 considered significant. Results: Statistically significant differences were observed in green to red ratio between group S (9.561976) and S7 (0.435418) (p < 0.01). There was no difference found between SA (mean of green to red ratio, (0.70431) and SA7 (mean of green to red ratio, 0.85184). Conclusions: Antibiotics added to the sealer effectively eradicated of E. faecalis 24 hours post-obturation. However, after seven days, plain sealer was as effective as sealer-antibiotic combination.", "keywords": [ "Enterococcus faecalis", "Pulp Canal Sealer (EWT)", "Amoxicillin", "Confocal Laser Scanning Microscope", "Biofilm", "antimicrobial action", "Obturaion", "Eugenol" ], "content": "Introduction\n\nMicroorganisms, biofilm and irritants have been the principal causative elements associated with the pathogenesis and progression of pulp and periapical diseases. Eradicating them is the ultimate goal of endodontic therapy. This can be achieved through the combination of asepsis, chemical preparation mechanical preparation, antimicrobial irrigating solutions, intracanal medicaments and fluid tight seal of canal system by obturation.1 In many studies, Enterococcus faecalis has been identified as the most common species associated with persistent or secondary intraradicular infections that do not respond to treatment.2–6 It has been found that E. faecalis is isolated in 23-70% of positive cultures of obturated root canals that show signs of chronic apical periodontitis.2,7–12 It is associated more with asymptomatic cases and exhibit widespread genetic polymorphisms and they often occur in monoculture.13,14\n\nComplete elimination of microbes from root canal system is prevented by difficulty in negotiating complex anatomy, role of dentinal fluid in reducing efficacy of irrigants and intracanal medicaments. Also, high microbial virulence, biofilm formation and relative antimicrobial resistance of infecting bacteria prevent canal disinfection.15–19 In a biofilm, most antimicrobial agents and irrigants only act against microorganisms in its superficial layer, leaving those in the deeper layers unaffected.20\n\nRoot canal treatment failures can be prevented or at least minimized by following proper irrigation and obturation protocols. The obturating materials and root canal sealers should exhibit anti-microbial properties, sustained over a period of time, to prevent bacterial growth.21 These solid core obturating materials at times are unable to reach the irregularities of the root canal space such as accessory canals, apical ramifications, isthmuses, the fins, ramifications, and cul-de-sacs. Thus, root canal sealers are used in conjunction with these solid core obturating materials in order to fill these anatomical irregularities completely. The choice of a good sealer greatly affects the outcome of endodontic treatment.22\n\nSeveral anti-microbials are being added to improve antibacterial properties of sealers, including antibiotics. When conventional root canal treatment alone is insufficient, antibiotics such as penicillin and amoxicillin can be prescribed for treating endodontic infection.23 Using five antibiotics, Hoelscher et al. found Kerr pulp canal sealer (PCS) EWT enhanced antibacterial activity against Enterococcus faecalis.24 All of the sealer-amoxicillin combinations in the investigation displayed the highest zone of inhibition under both anaerobic and aerobic conditions.25 Amoxycillin at 10% volume showed the best result as an additive to PCS (EWT) sealer with the least mean apical leakage which is clinically significant.26\n\nAntimicrobials within dentinal tubules have a greater antibacterial effect on the seventh day than it did at 24 hours according to study by Heling et al.27Antimicrobial regimens can be evaluated during and post-treatment by culture from the site of infection, blood profile and powerful microscopic examination of the histopathological section. Recently developed Confocal Laser Scanning microscopy has gained popularity in the field of life sciences since it can be used to view and identify single cellular structures. It facilitates immediate fixation of live and dead bacteria, which is not possible in any culture-based method.\n\nThe present study was conducted to evaluate the antimicrobial efficacy of the addition of 10% amoxicillin to PCS (EWT) against E. faecalis, at 24 h and seven days following obturation, under Confocal Laser Scanning Microscope.\n\n\nMethods\n\nPrior to conducting the study, ethical clearance was obtained from Institutional Ethics Committee, Manipal College of Dental Sciences, Mangalore, Karnataka, India. Protocol approval with Ref. No. 15123. A total of 60 freshly extracted single rooted human premolars with Type I canal anatomy and mature root apex were selected and were stored in 1% sodium hypochlorite solution for 48 h for initial sterilization, and subsequently washed in sterile distilled water. Samples were decoronated to a length of 12 mm using a carborandum disc (Mani Inc. Japan) at a speed of 250 rpm attached to a slow speed handpiece (NSK Co., Japan) under water cooling. Canal orifices were enlarged with Gates Glidden drill size #5 (Mani Inc. Japan). Working length was determined by visual examination under 2.5X dental magnifying loupes (Pierson Surgical limited, Keeler, UK) using a 10 number K file (Dentsply-Maillefer, Ballaigues, Switzerland). All canals were then enlarged till size 25 hand K files using RC prep (Premier, India) for canal lubrication. Final preparation was done using rotary ProTaper Universal files (Dentsply, York, PA, USA, till File F2, using X-SMARTTM Electric Endo Motor. Throughout the instrumentation, 5.25% sodium hypochlorite was used to irrigate the canals, followed by irrigation with normal saline and 17% EDTA. Red nail varnish (Maybelline) was applied in double layers, to cover the outer surface of the samples except 3 mm from the apical end of roots. Samples were dried for 24 hours before being autoclaved (MELAG Euroklav 23 VS-S, Russia).\n\nInfection of the dentin blocks: The Enterococcus faecalis strain ATCC 29212 (ATCC, USA) was cultured and reactivated in Brain Heart Infusion broth (BHI, Difco, Kansas City, MO, USA), which was maintained at 37°C for 24 hours. Broth was transferred to another BHI flask and incubated again for another 24 hours to achieve exponential growth and adjusted to McFarland standard No. 1 (3 × 108 CFU/mL).\n\nOn the first day, as described by Andrade FB, et al.28 all dentinal blocks were infected over a period of 4 days. On the second day, following incubation, samples were agitated in a vortex for 10 s, and then inocula from the microtubes were discarded. One mL of sterilized BHI broth was inserted, following which a centrifuge cycle of 3,600 g for 5 min at 25°C was done. The microtubes were incubated again at 37°C for 24 hours. On the third day, a new inoculum of E. faecalis was inserted into the sample tubes, at exponential growth phase after seven hours of subculture in BHI broth. The centrifugation protocol was repeated twice at each speed, at 25°C. Procedures were repeated on the fourth day as described for the second day. On the fifth day, the samples were removed from the microtubes. Half of the samples were sectioned and then were stained with SYTO9 stain and the other half maintained in the incubator for another 7 days.\n\nSealer-antibiotic combination was prepared manually by mixing the antibiotic to the sealer 10% by weight using an electronic weighing scale (Sartorious Lab balance, Germany).29 The sealer was mixed according to manufacturer’s specifications for obturation. Root canals were irrigated with 17% EDTA for one minute and dried with paper points (Diadent, S. Korea) Sealer-antibiotic paste was lightly coated on to the canal walls twice with gutta-percha and canals were obturated with gutta-percha points (Diadent, S. Korea) with lateral compaction technique. Access cavity was restored with composite resin (3M ESPE, USA). All specimens were incubated at 37°C in humid conditions.\n\nPrior to obturation, the samples were randomly distributed into two groups of 30 specimens each. After the root filling, the samples were subdivided into two more groups of 15 teeth each, based on the time of assessment into the following:\n\nGroup 1: S - sealer group (24 h)\n\nGroup 2: SA - sealer antibiotic group (24 h)\n\nHalf if the samples sectioned on the seventh day were designated number ‘7’\n\nGroup 1 a: S7 - Seventh day sealer group\n\nGroup 2 a: SA7 - Seventh day sealer antibiotic group\n\nLow-speed hand pieces with small round burs were used to fracture each cylindrical dentin block by making thin vertical grooves in the middle (Dentsply Maillefer, Ballaigues, Switzerland). Following this, specimens were fractured using a chisel and mallet into two semi cylindrical halves. The outer convex surface was ground using low speed handpiece (NSK Co., Japan) attached to a water cooler and a fine carbide bur (Dentsply-Maillefer, Ballaigues, Switzerland) at 300 rpm, to achieve a standard thickness of 1 mm. Thirty specimens from each experimental group were sectioned 24 hours post-obturation and the rest on the seventh day and stained for analysis under a confocal microscope.\n\nThe sectioned dentin pieces were stained with SYTO 9 Green Fluorescent Nucleic Acid Stain (ThermoFisher Scientific, USA) and propidium iodide (Himedia, India), according to manufacturer’s instructions for 20 minutes in the dark at room temperature, and then rinsed with phosphate buffered saline for one minute. Each sample was then air-dried briefly and transferred into a micro centrifuge until used. Images were captured at magnification of 40×. In the case of SYTO9, the excitation/emission wavelengths were 480/500 nm, whereas in the case of propidium iodide, they were 490/635 nm. Image acquisition and analysis using CLSM at a resolution of 1024×1024 was carried out using Leica Application Suite (Leica Microsystems, Germany). Borders of the root canal were focused and two images per sample were obtained randomly. Background noise was then reduced in the images (Leica Application Suite software) and further quantitative analysis was carried out using ImageJ software (V ImageJ: Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA).30,31\n\nOne-way ANOVA and the Post hoc Tukey Test were used to analyse the data and establish its statistical significance. Significance was set at p < 0.05. The statistical programme SPSS v. 17.0 (SPSS for Windows; SPSS Inc, Chicago, IL) was used to conduct the analysis.\n\n\nResults\n\nThe results were interpreted as:\n\nGreen ratio was calculated by dividing the area of the living cells by the size of the entire magnified field of view at 40×.\n\nRed ratio was calculated by dividing the area of the dead cells by the size of the entire magnified field of view at 40×.\n\nGreen to red ratio: the value obtained by dividing the green ratio with the red ratio.\n\nCLSM images (40×) for 24 hours groups showed: [a] Baseline for Sealer group [b] Sealer group showing viable cells (in green color) at 24 h. [c] Baseline for Sealer-antibiotic group [d] Sealer-antibiotic group showing viable cells at 24 h (in green color) (Figure 1) After 7 days: [a] Baseline for Sealer group [b] Sealer group showing dead cells (Red color) and viable cells (in green color) [c] Baseline for Sealer-antibiotic group [d] sealer-antibiotic group showing dead cells (Figure 2).\n\n[a] Baseline for Sealer group. [b] Sealer group showing viable cells (in green color) at 24 hrs. [c] Baseline for Sealer-antibiotic group. [d] Sealer-antibiotic group showing viable cells at 24 hrs (in green color).\n\n[a] Baseline for Sealer group. [b] Sealer group showing dead cells (Red color) and viable cells (in green color). [c] Baseline for Sealer-antibiotic group. [d] Sealer-antibiotic group showing dead cells.\n\nThe first bar graph shows individual rd and green ratio of groups S7, SA7, SA, S (Figure 3). The second bar graph showthe green to red ratios of all groups along the x axis, while they axis shows different groups (Figure 4).\n\nBar graph showing in X axis green to red ratios of all groups. In Y axis showing different groups.\n\nIn the present study both, highest green and red ratio were seen in the S group followed by SA7, SA and least in S7 (Figure 3). There was no statistical difference among the groups in red ratio, but green ratio of S group showed statistically significant difference with other groups (Table 1). Ratio of green to red also shows that the mean value of S group was highest with lowest antibacterial effect on E. Faecalis followed by SA, SA7 and least in S7 with no statistically significant difference (Figure 4). The ratios were comparable between SA and SA7 with a slightly better ratio in the SA group. The SA, SA7, S7 showed marked reduction in the green ratio and live bacteria when compared with the S group. Comparison of the red ratio using Post hoc Tukey test between different groups showed no statistically significant difference (Table 2). Comparison of the green ratio using Post hoc Tukey test showed no statistically significant difference between different groups except between S7 and S showing significant reduction in live bacteria in S7 compared to S (Table 2).\n\n\nDiscussion\n\nThe S group (sealer without antibiotics at 24 hours) proved to have little antibacterial effect against E. faecalis at 24 hours compared to baseline. Even though highest red ratio is recorded for this group it could not outnumber the green ratio (Figure 1 [a] Baseline, [b] after 24 h) The reason for the highest red ratio in S group could be due to increase in the free eugenol release from freshly mixed sealer or cytotoxicity expressed within the first few hours. This hypothesis is supported by in vitro study, which showed inhibition of E. faecalis only within the first five hours of incubation due to the release of free eugenol.32 Eldeniz et al., also found that the antibacterial activity was higher at the 13th hour. Cytotoxicity of ZOE based sealers have been shown to sharply reduce after 24 hours of setting.33 The green ratio was highest in this group when compared to other groups because of the decrease in the release of eugenol from set sealer at 24 hours, allowing E. faecalis to grow. The results are in agreement with Zhang et al who evaluated the in vitro antibacterial activity of similar sealers and found no significant antibacterial activity one day after setting.34 According to Baer et al., sealers without amoxicillin did not inhibit the growth of E. faecalis. Additionally, they found no statistical difference between fresh mixed samples and those that had been set (p > 0.05).35 This is in agreement with the present study. Pizzo et al. in 2006 evaluated in vitro antimicrobial action of root canal sealers and found ZOE sealer to be equally effective in inhibiting bacterial growth until 24 hours after mixing.36 It is likely that the set materials released eugenol that contributed to this.37\n\nThe SA group (sealer with antibiotics at 24 hours) combination showed a marked positive inhibitory effect on E. faecalis. (Figure 1 – [c] Baseline, [d] after 24 h) A possible hypothesis is the alkaline pH of the combination enhancing the antimicrobial activity against E. faecalis and its susceptibility towards amoxicillin. The results are in agreement with Baer et al. who evaluated in vitro, the antibacterial effect of amoxicillin when added to different sealers and found that sealers mixed with amoxicillin were significantly more effective than without.35 According to Binoy et al., the reported, pH of the combination was 8.55, this high alkalinity might have deleterious effects on microorganisms in obturated canals.37 Vidulasri et al. stated that the anti-microbial activity against E. faecalis was improved when antibiotics like amoxicillin and clindamycin were added to zinc oxide eugenol sealer. Amoxicillin is beta-lactam bactericidal broad-spectrum antibiotic that acts by inhibiting bacterial cell wall synthesis.29 According to a previous study, E. faecalis is more sensitive to the antibiotics amoxicillin, amoxicillin-clavulanic acid, benzyl penicillin, vancomycin, and doxycycline while being less sensitive to the antibiotics erythromycin and azithromycin.38 An antibiotic-enhanced sealer that alters the environment of the microorganism and retains bactericidal qualities after setting time may be crucial for the success of initial endodontic therapy and for avoiding re-infection.39,40\n\nThe S7 group (sealer without antibiotics after seven days) showed significant improvement in antimicrobial effect compared to S group with a reversal in the green to red ratio. [Figure 2 – [a] Baseline, [b] after seven days] (p < 0.01). A possible hypothesis is a bactericidal effect of eugenol due to its sustained release on microbes causing protein denaturation. The results are in agreement with Pizzo G et al., who found that after seven days from mixing, ZOE containing sealer, still exerted antibacterial activity.36 Heling demonstrated the antibacterial property of Pulp Canal Sealer (EWT) at seven days owing to the bactericidal effect of eugenol as a part of the liquid.27 Hasheminia et al. stated that eugenol is a potent antibacterial agent, acting on microbes by protein denaturation.41 The findings of the present study demonstrating antibacterial activity of EWT after seven days are justified by the above mentioned studies. The results are contradicted by Smadi et al. who tested nine sealers against E. faecalis and found that most sealers had antibacterial properties immediately after mixing, but these properties deteriorated over time.42 Zhang et al. also did not see any antibacterial effect of eugenol based sealer beginning on the third day.34 This could be because of the use of different formulations of sealers or use of different microbiological techniques of assessment. In our study results were contrary to these findings probably because of the use of confocal laser scanning microscope for analysis.\n\nThe SA showed good antibacterial efficacy in 24 hours which could be because of the presence of antibiotics and maintained its efficacy even after seven days in the SA7 group (sealer with antibiotic after seven days) (Figure 2 – [c] Baseline, [d] after seven days) with no significant statistical difference with the S7 and SA. The results are in agreement with Baer et al.35 who concluded that the antimicrobial properties and inhibition of the E. faecalis growth even after seven days can be demonstrated in sealers combined with amoxicillin. However, a slight increase in the viable cell count is seen when compared with the 24 hour group, which could probably be because of multiplication of cell adjacent to the zone of killing or slow release of amoxicillin from a viscous mix.43 Our results are in agreement with Binoy et al. who reported the combination to have greater viscosity when compared with other antibiotic combinations. This finding further explains the reason for no enhancement in the antimicrobial property of 10% amoxicillin sealer combination after seven days when compared with 24 hours.37 However, more longitudinal clinical studies are needed to confirm the outcome of addition of different antibiotics to various sealers.\n\n\nConclusions\n\nWithin the limitations, the outcome of the current study concluded that adding antibiotics 10% amoxicillin to zinc oxide eugenol sealer increases its antibacterial effect against E. faecalis within 24 hours, while plain ZOE sealer start demonstrating good antibacterial property against E. faecalis only after seven days.\n\n\n\n• Adding antibiotics to sealer may help in preventing post obturation infection of the periapical region.\n\n• Since amoxicillin is reported as the drug of choice for endodontic infections in most countries, 10% amoxicillin is added to sealer in the present research, to prevent reinfection. Clindamycin and erythromycin can be alternative drugs for patients allergic to penicillin.\n\n• Adding amoxicillin to ZOE sealer, the antibacterial property of sealer will be improved within 24 h.", "appendix": "Data availability\n\nFigshare: master chart thesis.xlsx, https://doi.org/10.6084/m9.figshare.19180589.v1.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nFuss Z, Charniaque O, Pilo R, et al.: Effect of Various Mixing Ratios on Antibacterial Properties and Hardness of Endodontic Sealers. J. Endod. 2000 Sep; 26(9): 519–522. PubMed Abstract \| Publisher Full Text\n\nGomes BP, Pedroso JA, Jacinto RC, et al.: In vitro evaluation of the antimicrobial activity of five root canal sealers. Braz. Dent. J. 2004; 15(1): 30–35. 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J. Clin. Diagn. Res. 2014; 8(11): 6–11. Publisher Full Text\n\nRazmi H, Yazdi KA, Jabalameli F, et al.: Antimicrobial Effects of AH26 Sealer/Antibiotic Combinations against Enterococcus Faecalis. Iran Endod. J. 2008 Fall; 3(4): 103–108. PubMed Abstract \| Free Full Text\n\nKim HJ, Baek SH, Lee WC, et al.: Cytotoxicity of resin-based root canal sealer, adseal. Korean Acad. Conserv. Dent. 2004 Nov 1; 29(6): 498–503. Publisher Full Text\n\nSaha S, Samadi F, Jaiswal JN, et al.: Antimicrobial activity of different endodontic sealers: an in vitro evaluation. J. Indian Soc. Pedod. Prev. Dent. 2010 Oct -Dec; 28(4): 251–257. Publisher Full Text\n\nHasheminia M, Razavian H, Mosleh H, et al.: In vitro evaluation of the antibacterial activity of five sealers used in root canal therapy. Dent. Res. J. 2017; 14: 62–67. Publisher Full Text\n\nSmadi L, Khraisat A, Al-Tarawneh SK, et al.: In vitro evaluation of the antimicrobial activity of nine root canal sealers: direct contact test. Odontostomatol. Trop. 2008; 31: 11–18. PubMed Abstract\n\nAkhavan BJ, Khanna NR, Vijhani P: Amoxicillin. Amoxicillin. viewed on 17-02-2021. Reference Source" }	[ { "id": "181659", "date": "18 Jul 2023", "name": "Saravana Karthikeyan Balasubramanian", "expertise": [ "Reviewer Expertise Endodontics", "Irrigants", "Disinfection", "Dentin Tissue Engineering", "Biomaterials" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 interesting clinically significant study compared the antibacterial efficacy of a zinc oxide eugenol based sealer with and without 10% amoxicillin combination against E.faecalis. However, this manuscript requires minor modifications before acceptance for indexing. Please find my comments attached herewith:\nComments: Page 1, Abstract: Background, Line 2: \"Enterococcus faecalis\" and its abbreviation \"E.faecalis\" are used alternatively. It is recommended that the full form of the bacteria can be mentioned initially, followed by subsequent mention of its abbreviation.\nMethods, Line 4: Please mention the use of 17% EDTA during canal instrumentation.\nResults, Line 4: It is highly appreciable to mention the mean and SD values as well along with the statistical results.\nConclusions, Line 1: The conclusions can be very specific, addressing the effect of 10% amoxycillin rather than the mention of “Antibiotics” against E.faecalis.\nBased on the derived conclusions, the authors can make recommendations for inclusion of antibiotics to root canal sealers for potential eradication of the bacteria.\nPage 2, Introduction: As the effect of the combination of 10% amoxycillin and Kerr Pulp Canal Sealer (EWT) against E.faecalis, has been previously studied by Hoelscher et al., (2006[ref-1), I would recommend the authors to highlight the strong rationale of the current investigation before addressing the aim. (Please refer to: Hoelscher et al., 20061).\n\nNull/alternative hypothesis also must be mentioned.\nPage 2, Methods: How was the sample size of 60 arrived to? Details mentioning the pilot study or power calculation based on previous literature studies can be highlighted.\nInclusion and exclusion criteria of the extracted teeth including, the reasons for extraction, patient age group, canal curvature etc., can be mentioned.\nPage 2, Methods: Infection of dentin blocks: In the present study, the dentinal blocks were infected with E.faecalis for 4 days. However, literature evidence supports 4-weeks infection time period to ensure adequate penetration of E.faecalis into dentinal tubules. Please refer to:\nHaapsalo and Ørstavik (19872). Shabahang and Torabinejad (20033).\n\nPage 8 & 9, Discussion: The authors can highlight the virulence factors of E.faecalis substantiating the reason why E,.faecalis and the particular strain was studied? Role of NaOCl in inhibiting E.faecalis should also be discussed by the authors, which is clinically relevant. In the current study, 5.25% NaOCl was employed. There are contraindicatory findings reported in literature when different concentrations of NaOCl were tested against E,faecalis. The authors are encouraged to shed light on these observations as well. (Please refer to: Shabahang and Torabinejad (20033)).\nAdvantages of CLSM method can be highlighted. The authors can discuss in view of the methodology employed by Hoelscher et al., (2006[ref-1) who earlier tested the efficacy of 10% amoxicillin-Kerr Pulp canal Sealer EWT combination using agar diffusion method.\nBased on the results obtained, the authors can either accept or reject the proposed null hypothesis.\nFurther, the limitations and scope of the study should be highlighted. Limitations of the study may include sample size and infection time period. Future research investigations pertaining to the evaluation of the sealing properties such as shrinkage, setting time, diffusability and substantivity of the sealer-antibiotic combination needs detailed analysis. In addition, the antibiotic resistance and allergic host response to the antibiotic formulation also needs careful exploration.\nGeneral Comments: The authors are requested to check the language throughout the length of the manuscript. For instance: Page 1, Abstract: Methods section - “Root canal standardized to 12 mm”; Page 2, Keywords: “Obturaion”; Page 3, Introduction, Line 3: “This can be achieved through…. Chemical preparation mechanical preparation…….”- Can be mentioned as “chemo-mechanical preparation”.\nPlease mention the full form of technical terms, when they are cited in text for the first time, subsequently followed by the mention of their abbreviations. (e.g., E.faecalis, CLSM, NaOCl, EDTA etc.,).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9974", "date": "21 Aug 2023", "name": "Kundabala Mala", "role": "Author Response", "response": "Please find my comments attached herewith: Authors: Thanks a lot respected reviewer for going through the article in detail and giving scientific and very encouraging comments. Comments: Page 1, Abstract: Query: Background, Line 2: \"Enterococcus faecalis\" and its abbreviation \"E.faecalis\" are used alternatively. It is recommended that the full form of the bacteria can be mentioned initially, followed by subsequent mention of its abbreviation. Answer: It is done in resubmitted manuscript. Query: Methods, Line 4: Please mention the use of 17% EDTA during canal instrumentation. Answer: 17% EDTA is used for removing smear layer generated during instrumentation. Results, Line 4: It is highly appreciable to mention the mean and SD values as well along with the statistical results. Answer: The mean and standard deviations between green ratio, red ratio, green to red ratio and p values for all the four groups are mentioned in Table 1. Query: Conclusions, Line 1: The conclusions can be very specific, addressing the effect of 10% amoxycillin rather than the mention of “Antibiotics” against E.faecalis. Answer: Adding 10% amoxicillin to Kerr pulp canal sealer (EWT) against E. Faecalis may help in preventing post obturation infection of the periapical region. Query: Based on the derived conclusions, the authors can make recommendations for inclusion of antibiotics to root canal sealers for potential eradication of the bacteria. Answer: Conclusion is modified to 10% amoxicillin added to the Kerr pulp canal sealer (EWT) effectively eradicated of E. faecalis at 24 hours post-obturation. However, after seven days, plain sealer was as effective as sealer-antibiotic combination. Based on the derived conclusions, it can be recommended that inclusion of antibiotics to root canal sealers help in eradication of the bacteria. Page 2, Introduction: As the effect of the combination of 10% amoxycillin and Kerr Pulp Canal Sealer (EWT) against E.faecalis, has been previously studied by Hoelscher et al., (2006[ref-1), I would recommend the authors to highlight the strong rationale of the current investigation before addressing the aim. (Please refer to: Hoelscher et al., 20061). It is done in the resubmitted manuscript. Query: Null/alternative hypothesis also must be mentioned. Answer: The null hypothesis of the study is there is no difference in the antimicrobial efficacy of the Kerr pulp canal sealer (EWT) with or without addition of 10% amoxicillin against E. faecalis, Page 2, Methods: Query: How was the sample size of 60 arrived to? Details mentioning the pilot study or power calculation based on previous literature studies can be highlighted. Answer: Using G*Power 3.1.9.4 software, at 95% confidence level, 90% power, 0.5 effect size, and assuming 20% loss of sample during the process, the sample size for the present study was calculated to be 60 in each group. Query: Inclusion and exclusion criteria of the extracted teeth including, the reasons for extraction, patient age group, canal curvature etc., can be mentioned. Answer: Inclusion criteria: freshly extracted single rooted human premolars with Type I canal anatomy and mature root apex extracted for orthodontic purpose from patients of age group 18-25 selected Exclusion criteria: Fractured teeth, immature open apex, curved roots, root canals other than Type-I canal anatomy Query: Page 2, Methods: Infection of dentin blocks: In the present study, the dentinal blocks were infected with E.faecalis for 4 days. However, literature evidence supports 4-weeks infection time period to ensure adequate penetration of E.faecalis into dentinal tubules. Please refer to: Haapsalo and Ørstavik (19872). Shabahang and Torabinejad (20033). Answer: The present study followed the protocol suggested by Flaviana Bombarda de ANDRADE et. al. (REF.28) for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests for confocal laser scanning microscopy. Page 8 & 9, Discussion: Query: The authors can highlight the virulence factors of E.faecalis substantiating the reason why E,.faecalis and the particular strain was studied? Answer: E. faecalis was chosen as study microbe since it is found especially in failed to endodontic infections. It is because of its virulence factors such as aggregation substance, surface adhesins, sex pheromones, lipoteichoic acid, extracellular superoxide production, the lytic enzymes gelatinase and hyaluronidase, and the toxin cytolysin.REF: 32. The present study used CLSM for evaluating antimicrobial efficacy od sealer since it is one of the most commonly used fluorescence microscopic techniques recently in endodontics, particularly in three-dimensional (3D) studies involving biological cells and tissues. It is a flexibile approach, which makes it suitable for use in fast imaging of dynamic processes in living cells, sensitive morphological analysis of tissues. REF: 33 The Reasons or choosing the E. Faecalis as study microbe s already mentioned in the introduction, all the incorporated in the resubmitted manuscript Query: Role of NaOCl in inhibiting E.faecalis should also be discussed by the authors, which is clinically relevant. In the current study, 5.25% NaOCl was employed. There are contraindicatory findings reported in literature when different concentrations of NaOCl were tested against E,faecalis. The authors are encouraged to shed light on these observations as well. (Please refer to: Shabahang and Torabinejad (20033)). Answer: Sodium hypochlorite possesses an effective antibacterial action against E. faecalis its level of effectiveness depends mainly upon a)its concentration and form, b) different models of the microorganism and c) experimental procedures including manipulation and duration of contact with the microorganism Ref: Luddin N, Ahmed HM. The antibacterial activity of sodium hypochlorite and chlorhexidine against Enterococcus faecalis: A review on agar diffusion and direct contact methods. J Conserv Dent. 2013 Jan;16(1):9-16.. Advantages of CLSM method can be highlighted. The authors can discuss in view of the methodology employed by Hoelscher et al., (2006[ref-1) who earlier tested the efficacy of 10% amoxicillin-Kerr Pulp canal Sealer EWT combination using agar diffusion method. Advantages of CLSM are included in the discussion. Corrections are incorporated in the resubmitted manuscript. Query: Based on the results obtained, the authors can either accept or reject the proposed null hypothesis. Answer:Based on the results of the present study,authors reject the null hypothesis and conclude that addition of amoxicillin to sealersimproves the antibacterial efficacy of the Zinc oxide sealer. The corrections are incorporated in the resubmitted manuscript. Query:Further, the limitations and scope of the study should be highlighted. Limitations of the study may include sample size and infection time period. Future research investigations pertaining to the evaluation of the sealing properties such as shrinkage, setting time, diffusability and substantivity of the sealer-antibiotic combination needs detailed analysis. In addition, the antibiotic resistance and allergic host response to the antibiotic formulation also needs careful exploration. Answer: Corrections are incorporated in the resubmitted manuscript. General Comments: The authors are requested to check the language throughout the length of the manuscript. For instance: Page 1, Abstract: Methods section - “Root canal standardized to 12 mm”; Page 2, Keywords: “Obturaion”; Page 3, Introduction, Line 3: “This can be achieved through…. Chemical preparation mechanical preparation…….”- Can be mentioned as “chemo-mechanical preparation”. Please mention the full form of technical terms, when they are cited in text for the first time, subsequently followed by the mention of their abbreviations. (e.g., E.faecalis, CLSM, NaOCl, EDTA etc.,). Corrections are incorporated in the resubmitted manuscript. 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? Partly Are the conclusions drawn adequately supported by the results? Yes References 1. Hoelscher AA, Bahcall JK, Maki JS: In vitro evaluation of the antimicrobial effects of a root canal sealer-antibiotic combination against Enterococcus faecalis.J Endod. 2006; 32 (2): 145-7 PubMed Abstract \| Publisher Full Text 2. Haapasalo M, Orstavik D: In vitro infection and disinfection of dentinal tubules.J Dent Res. 1987; 66 (8): 1375-9 PubMed Abstract \| Publisher Full Text 3. Shabahang S, Torabinejad M: Effect of MTAD on Enterococcus faecalis-contaminated root canals of extracted human teeth.J Endod. 2003; 29 (9): 576-9 PubMed Abstract \| Publisher Full Text" } ] }, { "id": "181661", "date": "27 Jul 2023", "name": "Moksha Nayak", "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\nDisinfection of root canal system is a most important step for the success of endodontictherapy. During instrumentation irrigants and intracanal medicaments are used. Adding antibiotics to sealers is beneficial to prevent re-infection and impart antimicrobial property for an extended period of time at the post-endodontic period.\nThe work done by the authors is contemporary and need of the hour. The manuscript shows the study will be clearly understood by the readers and accurately presented. Regarding the citation of the current literature, I appreciate this manuscript is focused on synergizing the effect of these sealers by adding antibiotics to the sealer and its effect by studying their antimicrobial efficacy on microbes by one of the latest technologies-Confocal Laser Microscopy and most of the previous studies are done using culture and other older techniques. Moreover, live and dead bacterial status also has been studied. There are no sealer-antibiotics studies recently done using CLSM or any other recent technology trying to explore actual bacterial status of this combination.\nProper background for the study is established. Adequate justification is given for selecting this research question in the introduction. In the abstract section some corrections can be done regarding grammatical errors.\nAbstract: Conclusion: “Antibiotics added to the sealer effectively eradicated of E. faecalis 24 hours post-obturation.”. In the Materials and method section sample size is adequate and methodology is properly formulated and it ensures full reproducibility. Results are well documented and discussed well. All the sources of data underlying the results are available .\nConclusions drawn are adequately supported by the results.\n\nThe following are my suggestions after going through the article:\nThere are a few more papers published in the literature comparing various antibiotic-sealer combinations on endodontic microbes. Recent ones have studied the antibiotic combinations such as Triple (TAP) or Double antibiotic paste (DAP).\n\nThe authors could have included TAP or DAP combination with epoxy resin-based sealers or bioceramic sealers. Could the authors justify why they have not considered these options?\n\nKangarlou et al. (20161).\n\nBrezhnev et al. (20192).\n\nIn key points 2, Clindamycin and erythromycin can be alternative drugs for patients allergic to penicillin. The study has not included these antibiotics. Hence the authors need not mention about these.\n\nA focused review on antibacterial additives in epoxy resin-based root canal sealers by Brezhnev et al. (20192) could have been added in the discussion to justify the use of CLSM in the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10056", "date": "21 Aug 2023", "name": "Kundabala Mala", "role": "Author Response", "response": "The authors could have included TAP or DAP combination with epoxy resin-based sealers or bioceramic sealers. Could the authors justify why they have not considered these options? Authors' reply: Amoxicillin has been proved already to be one of the best antibiotagainst endodontic microbes. Hence we tried as additives to the sealer. However, we plan to conduct our future studies with TAP or DAP J. J. Segura-Egea, K. Gould, B. Hakan Şen, P. Jonasson, E. Cotti, A. Mazzoni, H. Sunay, L. Tjäderhane, P. M. H. Dummer.Antibiotics in Endodontics: a review.International Endodontic Journal .Volume 50, Issue12 December 2017 Pages 1169-1184 In key points 2, Clindamycin and erythromycin can be alternative drugs for patients allergic to penicillin. The study has not included these antibiotics. Hence the authors need not mention about these. Authors' reply: We have put it as recommendation and suggestions to the readers if the patient is allergic to amoxicillin; not the conclusion of our study. A focused review on antibacterial additives in epoxy resin-based root canal sealers by Brezhnev et al. (20192) could have been added in the discussion to justify the use of CLSM in the study. Authors' reply: It is future scope of our study." } ] }, { "id": "181658", "date": "28 Jul 2023", "name": "Arindam Dutta", "expertise": [ "Reviewer Expertise Endodontics", "Restorative Dentistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 to the authors for submitting this paper to the journal. The paper has used some sophisticated techniques to assess dead/live bacteria in response to root canal treatment procedures. However, the premise for the paper: using an antibiotic in a sealer does not represent a contemporary approach. Root canal infections have multiple micro-organisms and an antibiotic like amoxicillin alone will not suffice. Secondly, the experimental model used dentine which had a mono-infection of E. faecalis, which also does not represent the clinical scenario - a multispecies biofilm has been proposed for such in vitro studies. Nevertheless, as an initial step, in trying to understand whether an antibiotic may have some influence on E.faecalis mono-infection, this is an acceptable investigation which has shown the lack of benefit. This helps establish that further studies in this direction are not warranted.\n\nIs the work 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9986", "date": "21 Aug 2023", "name": "Kundabala Mala", "role": "Author Response", "response": "Thanks a lot for the respected reviewer for the pragmatic inputs. The premise for the paper: using an antibiotic in a sealer does not represent a contemporary approach. Answer: A series of studies are following. Invitro multiple microbic study will be slightly difficult at this moment since invitro conducive growth environment have to be created for each organism. Query: Root canal infections have multiple micro-organisms and an antibiotic like amoxicillin alone will not suffice. Answer: Penicillin VK and amoxicillin, both beta-lactam antibiotics, are the first line of antibiotics chosen as adjunct therapeutic agents in endodontics, according to AAE Guidance on the Use of Systemic Antibiotics in Endodontics. Hence we used in our study. Secondly, the experimental model used dentine which had a mono-infection of E. faecalis, which also does not represent the clinical scenario - a multispecies biofilm has been proposed for such in vitro studies. Amoxicillin demonstrates greater efficacy and therapeutic value even in multispecies endodontic infections. This drug acts by binding and inhibiting the activity of several bacterial proteins nvolved in the synthesis of the peptidoglycan cell wall in susceptible both gram-positive and gram-negative bacteria (23). This drug has been found to be highly effective against isolates from infected root canal systems that are composed primarily of facultative and obligate anaerobes. It is representation for multispecies since it is one of the most drug resistant organism for antibacterial agents used in endodontics. Hence for convenience we used single organism in our study. Thanks again for these good suggestions." } ] } ]	1	https://f1000research.com/articles/12-725
https://f1000research.com/articles/12-973/v1	14 Aug 23	{ "type": "Research Article", "title": "Popular image of students enrolled in Christian schools and the founding purpose of Christian schools – A study based on a survey of youth living in Tokyo/Kanagawa Prefectures", "authors": [ "Yoshihiro Goto", "Fumi Katayama", "Suzuretsu Sen", "Junko Teruyama", "Fumito Osawa", "Miyu Tsuchiya", "Mikiko Yokoyama", "Fumi Katayama", "Suzuretsu Sen", "Junko Teruyama", "Fumito Osawa", "Miyu Tsuchiya", "Mikiko Yokoyama" ], "abstract": "Despite the popularity of Christian schools in Japan, Christians account for only 1.6% of the nation’s population. In the attempt to explore this inconsistency, we conducted an online survey in January 2020 to those between the ages of 15 and 29 living in Tokyo and Kanagawa. Our subjects include those who have enrolled in Christian schools, those who have some sort of affiliation, and those who do not have affiliation. We attempted to measure the difference in attitude by the degree of their affiliation. We focused on the notion of 'triple faith' which was proposed by Inoue and others. Many people in Japan believe in both Buddhism and Shintoism because neither requires a rite such as baptism. Christianity requires baptism, but that aside, in practice, many Japanese have triple faith including Christianity. Prosperity of Christian schools, preferences towards Christmas, and popularity of Christian-style weddings, all testify to this. This argument by Inoue and others conflicts with the common belief that Christianity never got a foothold in Japan. The results of cross tabulation of key questions and affiliation with Christian schools, combined with our interview results, show that those who have enrolled in Christian schools are more likely to have what may be referred to as triple faith. We have also provided a discussion on the possible reasons behind this finding.\nキリスト教主義学校が一定の人気を誇るにもかかわらず、日本のクリスチャンは日本の人口の1.6％しかいない。我々はこのことのギャップを探ろうとしている。そこで2020年1月に東京都・神奈川県在住の15～29歳を対象としたウェブモニター調査を行った。対象者にはキリスト教主義学校通学経験者、それ以外の関与者、非関与者を含み、キリスト教主義学校関与度による意識の相違を測ろうとした。井上章一ほかが示した、トリプルな信仰という着想に焦点を当てた。仏教や神道は洗礼のような明確なプロセスを要さないので、双方をダブルに信仰する者も日本には多いが、キリスト教は洗礼を要する。洗礼を抜きに考えると多くの日本人は実質キリスト教も含めたトリプルな信仰をしている。その証拠がキリスト教主義学校の隆盛であり、クリスマスの受容であり、キリスト教式結婚式の人気であるという。この井上らの考え方は、日本でキリスト教は根づかなかったという通説と対立する。主要な設問とキリスト教主義学校関与度とのクロス集計結果を、インタビュー調査の結果で補うことで、基本的にキリスト教主義学校に通った者の方が、このようなトリプルな信仰に相当する意識を持っていることが分かった。またこのような結果となった理由についても併せて考察した。", "keywords": [ "Christian schools", "founding purpose", "popular image of students", "triple faith", "mission" ], "content": "はじめに\n\nキリスト教主義学校は、当事者も含めた一般の人びとから、どのような存在として受け取られているのであろうか。本稿はそのことをキリスト教主義学校の生徒・学生のイメージとキリスト教主義学校の設置目的の推測に焦点を当てて究明しようとした。\n\n本稿の筆頭筆者が研究代表者を務める、科研費挑戦的萌芽研究「キリスト教主義学校から見る日本人の寛容と洋化－ステークホルダーらの期待と文化資本」では、キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多いにもかかわらず、日本のクリスチャンの数は日本の人口比で 1.6% 前後しかいないことのギャップを探ろうとしている。そこで東京都・神奈川県在住の 15~29 歳を対象とした、ウェブモニター調査を行った。\n\n本稿はまずはこの調査全体の背景や目的を述べるが、紙幅の関係もあり、今回の調査のうちキリスト教主義学校の設置目的を推測させる問いと、キリスト教主義学校生徒・学生のイメージについて聞く問いがあるが、それらを中心に分析し、考察し、併せて3名のインタビュー結果を分析・考察の補強に使う。\n\n\n1. 先行研究と問題の所在\n\n本科研費研究の先行研究としては、國學院大學日本文化研究所教授（当時）で日本宗教学会長及び「宗教と社会」学会長を歴任した井上順孝を研究代表者にした科学研究費の研究、「現代日本における宗教教育の実証的研究」(1998~1999) がある。この研究は冊子体の報告書（井上順孝（研究代表者）科研費報告書『「現代日本における宗教教育の実証的研究」(1998~1999) 報告書』(2000) 國學院大學）が刊行されている。この研究は宗教系及び非宗教系の学校全般を対象とし、この研究の報告書である井上ほか (2000) には 10 編の報告論攷が載っているが、多くの論攷は非宗教系学校のみを対象としたり、宗教系及び非宗教系の学校全般を比較したりしている。また宗教系の学校についてもキリスト教主義学校に絞った研究ではない。しかしそれらのうち「宗教系大学別の分析」（磯岡哲也）と「宗教系高等学校の入学者と卒業生の傾向についての一考察」（市川誠）は本研究の直接の先行研究といえる。これら二つの論攷は、宗教系の高校あるいは大学についてその学校の宗教ごとに比較しているので、キリスト教主義学校の特徴も分析されているからである。ただ基本的にこの報告書は、当該科研費助成期間以前の 1995 年から「宗教と社会」学会と國學院大學日本文化研究所で共同で毎年行っていた、質問紙調査の分析に充てていて、これらの調査は高等教育機関の学生の宗教意識を探る目的のものである。出身高校名や在籍高等教育機関の名前を自由記述式で聞く問いがあるので、それらを設置母体の宗派でカテゴライズしクロス集計することによって市川や磯岡の上記論攷は成り立っている。また國學院大學日本文化研究所編井上順孝責任編集『宗教と教育』(1997)も宗教系及び非宗教系の学校全般を対象としているが、カトリック及びプロテスタントそれぞれの明治期の宗教教育についての章があり、また質問紙調査については宗教系大学生の宗教意識、宗教系高校生の宗教意識の章はあるが、これもキリスト教主義学校に絞った調査及び分析ではない。\n\n直接キリスト教主義学校に絞った研究は我々の科学研究費申請時には佐藤八寿子『ミッション・スクール―あこがれの園』 (2006) しか存在していなかった。この本は新書ではあるが竹内洋の指導の下に佐藤が京都大学大学院教育学研究科に提出した修士論文を加筆・修正したもので、キリスト教主義学校を見る周囲の視線に注目し、それが「運命の女性」という男性目線の理想の女性像に通じる点を明らかにした。またピエール・ブルデューの階層再生産の議論を援用しながら、良妻賢母を生むミッションスクールが卒業生の女性に、結婚を通じて良い社会階層を得る機会を創出してきたと論じた先駆的な研究である。しかしこの研究は小説、雑誌、漫画等の過去のメディア上のコンテンツの分析を主たる方法とするため、研究のバイアスとして佐藤自身が記しているように（佐藤 2006 p.89）、キリスト教主義学校の男子学生についての議論は充分でないし、実際に人びとのいだく意識等の調査はしていない。\n\nそのあと我々の科研費助成期間初年度の 2018 年に、現在、日本文化研究センターの所長を務める井上章一が筆頭著者の『ミッションスクールになぜ美人が多いのか』（井上ほか　2018）が出た。これも新書であるが、4 章で構成される同書のうち、井上担当の「まえがき」と第 1 章「プロテスタント校はあなどれない」は一般受けを狙ったエッセー風評論であるが、井上の後輩同僚日本文化研究センター（当時）の郭南燕（現在、東京大学グローバルリーダー育成プログラム推進室特任教授）による第 2 章「ミッション系大学の成功物語」、上智大の川村信三の第 3 章「変遷するキリスト教イメージ」第 4 章「『お嬢様学校』を生み出したカトリック」を含めた全体は学術的であるし、井上のエッセー的な章へのデータの裏づけも含めて今回の調査票を設計した。\n\nしかし佐藤同様、この本もキリスト教主義学校の女子のみに焦点が当てられ、キリスト教主義学校全般を知るには不足があるし、少なくとも第 1、2 章は小中高校ではなく大学に議論の焦点は絞られる。他方第 4 章に関してはタイトルにある「お嬢様学校」について、「『お嬢様』とは幼稚園、小学校という低学年層から私立学校に通わせる裕福な家庭出身者ということになろうか」（井上ほか 2018 p.222）と記され、小学校への目配りもある。とはいえ第 3 章は学校に限らないキリスト教イメージ全般を対象としているので、本全体のトーンは第 1、2 章のキリスト教大学の女子が決めている。\n\nしかし校種（あるいは課程）相互の定員の差から察せられるように、初等中等教育課程からの持ち上がりで高等教育課程に入った者の方が、少数精鋭でキリスト教主義学校の教育理念を身体感覚として身につけていることは、本稿では紙幅の関係であまり言及しないが本研究のインタビュー調査でも示されていることである。キリスト教主義に限らず、附属・系列学校等、大学より若い年齢の課程のある私立大学の場合、附属学校等の出身者の方がその学園独自のスクールカラーに染まって社会に旅立つ。他方ラサールや栄光学園のように東京大学合格者上位に名を連ねるカトリックの中高一貫男子校の多くは、大学附属ではない1。したがって井上らの対象としているキリスト教主義学校の校種、生徒・学生のカテゴリーには偏りがある2。\n\nまたこの井上らの本は第 2 章で統計資料、第3章、第4章で歴史的な文献資料を扱っているが、現在の人びとの意識についての実証に乏しい。「はじめに」や第1章で統計資料、文献資料以外で現代の人びとの意識を知る術は、ほぼ井上の非常勤先の学生との会話や、現代風俗研究会での報告会、ICU でのシンポジウム等で交わされた応答のみである。\n\nしかし井上の現代の人びとに対する知見には説得力はあるので、それも調査票の設計では勘案した。\n\nまたやはり本研究採択年の 2018 年にクリーグ波奈の「キリスト教主義学校の役割とその教育的意義」という論文が東大の教育学研究科の紀要に載った。この研究はキリスト教主義学校での価値の社会化、すなわち生徒が学び身につける価値に着目し、キリスト教主義学校の A 校の社会化装置を文献資料から検証する（クリーグ 2017）。この研究の存在自体、しばらく我々は見落としていたし、ここでレビューされる研究も見落としているものが多かったが、この研究もこの研究のレビューした研究も、キリスト教主義学校の教員や生徒等の当事者の認識のみに焦点が当てられている。他方、本調査は次項 1.2 で述べるように、当事者と非当事者を含む、普通の人びとのキリスト教主義学校への意識を問う中で、場合によって両者の比較をめざしている点で、クリーグ及びそこでレファーされる研究とは違う。\n\n「はじめに」で本科学研究費の研究全体の目的を概ね記したが、改めてここで確認しておく。日本のクリスチャンの数は日本の人口比で 1.6% 前後しかいないにもかかわらず、キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多いのはなぜかを究明することが、本研究の目的である。この科研費研究はウェブモニター調査とインタビュー調査、各種資料の内容の分析（B.ベレルソンのいう狭義の内容分析法に限らず、定性分析も含む）の 3 本立てで、この研究目的を達成しようとした。ただ折からの新型コロナウイルスの影響もあり、インタビュー調査、資料の内容の分析は充分にできているとはいいがたい。\n\n本稿は、この本科学研究費の研究全体のうち、ウェブモニター調査の結果の一部（ただし我々が最重要と思う部分）の報告に留める。\n\n当然本項第一段落の「日本の人口比で 1.6% 前後しかいない」という事実は、やはり本項第一段落にある文から引けば「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況と矛盾しうる。なぜならキリスト教主義学校が布教、ないしは布教の地ならしをそこでの教育でしているとすればクリスチャンは日本の人口比の 1.6% を遥かに越えるはずであるからである。ではキリスト教主義学校は何を目的として設立されているのであろうか。この「キリスト教主義学校は何を目的として設立されているか」ということの解明が、《「日本の人口比で 1.6% 前後しかいない」という事実が、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況と矛盾する状況》の解明に役立つ作業仮説といえる（これを作業仮説 1 とする）。\n\nもちろんこのことをキリスト教主義学校の経営陣に直接聞いたり、彼らの発言を文献で追ったりすることの方が、より直接的にこの問題の答えが出てくる。しかしそれだけだと人びとがどういう理由でキリスト教主義学校を現在、受け入れているかは見えてこない。キリスト教主義学校の強い当事者、弱い当事者、非当事者という層で人びとを分けて捉え、層ごとの意識の相違を探ることで、人びとがどういう理由でキリスト教主義学校を受け入れているかが朧気ながら浮かび上がる。\n\nしたがって、1.3 で後述する先行研究での議論とも関係してくるが、キリスト教主義学校の設置目的をどのように想像するかという問い (Q34) を聞き、その回答を、キリスト教主義学校の強い当事者、弱い当事者、非当事者という層で分けて捉えることで、《「日本の人口比で 1.6% 前後しかいない」という事実が、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況と矛盾する状況》の解明をしようと我々は考えた。このキリスト教主義学校の強い当事者、弱い当事者、非当事者という層で分けて捉えることは、1.3.1 や 2. で後述されるように、Q12 という変数を組変数にした Q12G によって行った。特にウェブモニター調査という性格上、キリスト教主義学校の通学経験者、通学経験者ならずとも家族に通学経験者がいるその他何らかの意味での当事者、双方の比率がウェブモニターの人びとに多い訳ではない。しかしある程度の比率、こういう当事者たちもモニターにいることが想定される。したがってキリスト教主義学校への強い当事者（通学経験者）、緩やかな当事者（家族に通学経験者がいる、受験したことがある、等）、非当事者、という三者での意識の差を探ることを、このウェブモニター調査の分析の軸にしようと考えた。\n\n具体的には Q34 でキリスト教主義学校の設置目的をどのように想像するかという問いを設けた。そこの選択肢のうち、単純に考えれば、強い当事者、緩やかな当事者、非当事者の順に選ばれ、その選ばれ方の頻度の差に有意差のあるものは、設置目的に相当するものであると、当事者たちから捉えられている選択肢と考えられる。ただし実際には緩やかな当事者、強い当事者、非当事者の順に選ばれるもの等、色々なケースがありうる。\n\nまた《「日本の人口比で 1.6% 前後しかいない」という事実は、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況と矛盾する事態》であるが、その理由の解明にもう一つ役立つのは、キリスト教主義学校の生徒・学生イメージを、人びとがどのように捉えているかを見ることでもあり、それも作業仮説であると考えられる（これを作業仮説 2 とする）。\n\n具体的には Q29 でキリスト教主義学校の生徒・学生のイメージを尋ねる問いを設けた。これも Q34 について述べたのと同様、そこの選択肢のうち、単純に考えれば、強い当事者、緩やかな当事者、非当事者の順に選ばれ、その結果に有意差のあるものは、キリスト教主義学校のイメージと、当事者から捉えられている選択肢と考えられる。ただし後述する結果の説明をやや先取りするが、こちらの問いの場合には Q34 の場合について以上に、実際には緩やかな当事者、強い当事者、非当事者の順に選ばれるものがあったので、その点についても結果の分析の箇所で考察を加えた。\n\nなお、これら Q34、Q29 の選択肢として具体的に何を選ぶかについては、先行研究の議論を踏まえて我々は考えたので、次の 1.3 でその議論を見て、そのうえで作業仮説について、もう少し言及する。\n\n1.3.1 概況\n\n前項でとりあげた井上ほか (2018) の内容の概略を、本稿に関連する限りで、以下に5つ列挙する。①日本にキリスト教徒は少ないが、日本人はダブルに信仰する民族であり、キリスト教に洗礼という手続きが不要であればキリスト教を含めトリプルな信仰をする人は多かったはずである。なお、このダブル、トリプルの問題は、本稿の分析・考察の軸になるものであるので、その詳細は 1.3.2 で説明し、また調査結果としては 3.3 で述べる。②その証拠はキリスト教式結婚式及びそれと類似したキリスト教主義学校の隆盛である。③女子アナウンサーにキリスト教主義学校出身率が高いのは、愛と奉仕の精神を育むとそれらの学校のウェブページに記載されていることに照応する。④皇族女子のキリスト教主義学校入学が多いのも、愛と奉仕の精神を育むとの期待があるからである。⑤キリスト教主義大学女子はきれい、金持ち、キリストの 3K といわれる。\n\nさらに「現代日本における宗教教育の実証的研究」 (1998~1999) のなかで指摘された、本研究に密接にかかわるものとして以下の⑥を挙げる。⑥キリスト教主義に限定した話ではないが、宗教系の学校の設立目的には、その宗派の信徒の子弟の教育機会の確保と、その宗派のシンパを作ることにある3。\n\n1.3.2 以下で必要に応じてこれらの補足説明を加えつつ、本稿にほぼ関わる限りでの本調査の設計に言及する。\n\nなお、今回の調査では 1.2 ですでに若干ふれ、また後で 2. で詳述するように、Q12 で「あなたのキリスト教主義学校への関与についてお聞きします。当てはまるものを選んでください」という設問を設けた。2. で後述するようにこの Q12 の回答を 3 つのカテゴリーに再グループ化した Q12G という変数を、他の多くの変数を分析する際の軸として考えて分析を進める。\n\n1.3.2 首尾一貫性かトリプルな信仰か、およびキリスト教式結婚式、等\n\nまず、1.3.1 の①についてであるが、郭は「宗派に帰属しない日本人のトリプルな信仰」（井上ほか 2018 p.136）という見出しの下、以下のように論じる。神道、仏教あわせた信者数は 1 億 7244 万人で日本の総人口より 5000 万人多い。これはダブルな信者が多いためであるが、入信等の儀式はほとんど受けてはない。他方、キリスト教では入信に洗礼を要するという知識を多くの日本人は得ていて、この人数の膨れ上がりにキリスト教は加担しない。しかし、狭義に入信したとはいわれないが、日本人の意識としては仏教、神道のダブルな信仰に加えてキリスト教も加えた「トリプルな信仰」という意識をもつのではないか、と郭はいう。その証拠がキリスト教式結婚式の隆盛とキリスト教主義学校の人気、そして国民的行事と化したクリスマスであるという。\n\n当然この郭の議論への反論として、クリスマスは言うに及ばず、キリスト教式結婚式も一つの風俗にすぎないとの見方もありうる。しかしながら濱田陽によるこの領域の研究（濱田 2001 pp.23−46）を読むと、郭の議論をある意味で裏づける事実に遭遇する。濱田のいうには、キリスト教式結婚式は、戦後間もなくYMCA が会員男性から非クリスチャンの一般信徒へとキリスト教式結婚式の門戸を開いたことが端緒であるが、これはその当時としては例外で（濱田 2001 p.25）、1960 年代末に、高度経済成長を背景に青学会館、東京 YMCA ホテル、等が一般人向けに開放され、またキリスト教徒も所属教会での挙式が手狭である場合には、これらを選択したことが事実上の始まりという。もっとも今日隆盛しているホテル等でのキリスト教式結婚式は、1980 年キリスト教ブライダル宣教団の牧師たちが日本人の若年層がキリスト教にふれるきっかけとして始めた（濱田 2001 p.33）。またカトリックについて濱田は日本のカトリック中央協議会が1973年にローマ教皇庁に提出した文書を紹介する。この文書では、キリスト教式結婚式を望む非キリスト教徒について、神社仏閣でなくあえてカトリックを選ぶことは彼らがカトリックを評価している証であると記し、またそういう申し出をする者の多くがカトリック系の学校の卒業生であると述べている点も濱田は指摘する（濱田 2001 p.29）。要するにキリスト教式結婚式は風俗の面も強いが、プロテスタント、カトリック共に聖職者の一部がそれを信仰の一歩手前のものとして肯定的に評価する動きもあり、またキリスト教主義学校との関連性も少なくともカトリックの聖職者たちからは意識されていることが、この濱田の研究で示される。もっともその一方でカトリックに関しては、日本側は宣教手段として結婚式を位置づけるべくお伺いを立てたが、バチカン当局は宣教ではなく霊的指導に留めるようにと指導し、他方、指導の後も日本のカトリックの側は霊的指導と共に宣教的方針を文書で明記しているという食い違いがあるとのことである（濱田 2001 p.30）。\n\nこのキリスト教式結婚式については本調査では Q22 で「Q22. キリスト教徒ではない日本人のカップルが、教会で結婚式を挙げることについて、あなたはどのように思いますか」という設問で 3 選択肢 1 択の SA で聞いている。\n\nまたトリプルな信仰という郭の指摘に対しては、「あなたが思う、キリスト教主義学校に対する設置母体のメリットに当てはまるものを上位 3 つまでの間で選び、中でも最もメリットだと思うものを 1 つ選んでください」とキリスト教主義学校の設立目的を聞くQ34 における、選択肢 9「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」や、入信していない場合の救済やご利益の有無について尋ねる Q49 や Q50 を聞くことで、直接に妥当性の有無が分かる。これらの質問と 1.3.1 の最後のパラグラフで記した Q12 とのクロス集計の結果に有意差があれば、郭のいうトリプルな信仰の側面から、キリスト教式結婚式を捉える可能性が拓けるといえるし、有意差がなければその可能性は低く、それは習俗の面に過ぎないといえよう。しかしいずれにせよキリスト教主義学校やキリスト教式結婚式についてはそれがトリプルな信仰の一環、つまり洗礼なき信仰心に通じる面がある可能性はありうるが、国民行事と化したクリスマスについては日本人の非信徒の場合、イエス・キリストとはほぼ縁もゆかりもないものと化しているというのが、多くの日本人あるいは日本に永く住んでいる外国人の偽らざる実感であろう4。とはいえ、この Q34 選択肢 9「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」とキリスト教主義学校関与度を示す Q12 に関連があればトリプルな信仰そのものの可能性は拓ける。\n\nまた同書は「まえがき」でつぎのように記す。「われわれは素行の悪いクリスチャンを、よくなじる。・・・しかし浄土真宗や日蓮宗の人びとが浮気をしても、宗教にからめて批判しない。・・・つまり、日本ではキリスト教の方が良い宗教であるかのように、思われている。・・・江戸期には、淫祠邪教だとみなされた。そんなキリスト教が百数十年の時を経て、倫理面では信頼される宗教になりおおせている。そのサクセスストーリーが、これまでのキリスト教受容史では、見すごされている。講壇学説の一般通念には、その意味でこまったかたよりがある」（井上ほか 2018 pp.10-11〔・・・は中略。特記しない限り、以下同様〕）。\n\nここで批判される「講壇学説」の内容を井上は次のように纏める。「近代の日本社会は、けっきょくキリスト教をうけいれなかったと、よく言われる。信者の人口は、日本人ぜんたいの二パーセントにも満たない」（井上ほか 2018 p.9）。「また、日本社会は、キリスト教を歪曲してうけとめたとも、評されやすい」（井上ほか 2018 p.9）。\n\nこの「はじめに」の文言を、先にあげた同じ著書の郭の先述の主張に照らしていい直してみると、次の《　》に入れた文章のようになろう。《キリスト教は日本社会に定着せず敗者のようにいわれるが、キリスト教式結婚式、キリスト教主義学校、クリスマスの隆盛等に示される、仏教徒でありつつ神道の信者でもありキリスト教徒でも（ほぼ）あるという、トリプルな信仰の表れの状況を踏まえると、日本においてキリスト教を敗者とみなす見方は誤りである》。\n\n井上章一自身は第 2 章末尾で、先行研究の佐藤 (2006) の日本でのキリスト教主義学校のもたらしたものは文化でありふるまいであったという発言を引きつつ、次のようにいう。「私は「ふるまい」もさることながら、キリスト教の倫理観に共鳴している日本人が数多くいると思っている。宗教や宗派にこそ帰属しないものの、仏陀の悟り、神道の霊魂とともに、キリスト教の神をも尊敬している」（井上ほか 2018 p.160）。このように佐藤の「ふるまい」としてのキリスト教の考え方を表向き肯定しつつも、それについて「さることながら」と述べて、「ふるまい」、習俗としての受容以上に倫理観としての受容があると論じ、その倫理観を「仏陀の悟り」「神道の霊魂」「キリスト教の神」という信仰の中核の言葉に置き換える。一見すると、井上に要約された「限りでの」佐藤の著書の見解を肯定するように書きつつも、信仰から切り離された習俗・「ふるまい」としてのキリスト教受容という見方を否定しようとしている。井上は参照頁を明示せず内容に言及するに留まるので、佐藤自身のテキストのどこが井上による言及の該当箇所なのか想像するしかないが、佐藤が佐藤卓己5と共訳したモッセに依拠しつつ「TPO に応じたリスペクタブルなふるまい」（佐藤 2006 p.25）と書かれた箇所のことだと推察される。仮にそこだとすると、ウェーバーの ethos、ブルデューの habitus 由来の、形が本質を決めることにも通じる考え方が強調されるが、ここで信仰と切り離した捉え方を佐藤が標榜している訳ではない。日本でのキリスト教受容は習俗であるとは述べるが、習俗が信仰を決定づける可能性は排除されない。「キリスト教に根ざした教育とは、すなわちリスペクタビリティの教育でもあった。洋風生活におけるマナー、作法といった、些末な行為の数々は、単に些末な行為の数々であるだけではない。神への敬虔は、現実の中では「つつしみ」がある生活態度として表出されねばならなかったのだ」（佐藤 2006 p.25）。佐藤自身はこのように型と精神の相互作用を想定する。\n\nその点で井上の佐藤への婉曲な批判は厳密にはミス・リーディングといえる。ただし結果的に両者とも、日本人のキリスト教受容に対して、それらは一見ふるまい、形としてのみの受容であるが、その根底に倫理や精神性、さらに信仰に通じるものを孕むと考える点では、むしろ共通する。\n\nここで井上章一らは丸山真男の名前を出していないし、彼を多少なりとも意識しているか否かも定かではない。しかしそもそもトリプルな信仰は、丸山の描くキリスト教の日本における受容の姿と真逆に相当する。というのも、首尾一貫性を重んじるものであるがゆえ、キリスト教は日本人あるいは日本文化の無限抱擁性と相容れず排除され、定着しなかったというのが、丸山の主張の骨子であるからである（丸山 (1961) の言い方は「マルクス主義やキリスト教」という言い方であって、キリスト教はややマルクス主義の添え物ではあるが）。\n\n丸山『日本の思想』は日本の固有信仰に由来する日本人の心性として無限抱擁性（丸山 1961 p.14）と中心性の欠如を指摘する。これは丸山の言葉ではないが、神道には、祝詞はあるし、経典として古事記、日本書紀、風土記等が列挙されるが、神道は明確な正典を欠く。伊勢神宮の簾の向こうは、初代文部大臣で明六社会長の森有礼が参拝のおり杖で示してみせ、彼の暗殺の一因になったとの説もあるように、不在で、中心がない。神道に典型的に示される日本の固有信仰の影響を受けた日本人は、神道に中心がないだけに、何でも受け入れる傾向にある。本地垂迹説や反本地垂迹説があるように、多様な宗教を受け入れる。別の著書である加藤周一・木下順二・丸山真男・武田清子『日本文化の隠れた形』（岩波書店）のなかで次のように丸山はいう。「神道というのは、はじめは仏教と習合して両部神道のような教義が生まれ、後には儒教と習合して、吉田神道とか吉川神道とかが出て来る。神道は、そういう他の世界観の助けを借りないと「教義」としての体系を持てないのです」（加藤ほか 2004 p.141）。以下の例も丸山にある訳でないが、洋食和食中華のいずれも好んで食べ6、年末年始の一週間はクリスマスを祝い除夜の鐘を衝き初詣に参拝し初日の出を拝むことを同じ人がやっても違和感なく受け入れられる等、文化の雑食性、「無限抱擁」性が日本人にはある。よくいえば思想的宗教的に寛容な文化である7。しかしそのような無限抱擁的な日本人の主に庶民が不寛容になる対象がある。丸山のいうにはそれが一神教のキリスト教であり、マルクス主義であるという（丸山 1961 p.15）。この言葉も丸山の言としては確認していないが、双方とも『聖書』『資本論』という正典とでも称すべき絶対的典拠がある。丸山の言葉を使えば、座標軸をもって物事を見ていく（丸山 1961 p.5）。他方、日本の固有信仰、神道は中心が欠如する（丸山 1961 p.21）がゆえに「寛容」であるのであるのだから、逆にそういった日本の宗教をはじめとする思想・文化は、正典という中心から首尾一貫性をもって世界を眺め解釈し正邪を判断するような一神教的な宗教や思想とは、まったく相容れないことになる。そういう首尾一貫性、いいかえるとある種の不寛容性をもつキリスト教、マルクス主義に対して、無限抱擁的な雑食文化のなかにいる日本人、とくに日本の庶民は、不寛容性を示す。さらに庶民がそうであるのと同時に、一人の人間の中に二つの傾向が併存しうるので、その点では知識人にも同様の側面がある（丸山 1961 p.52）と丸山はいう。\n\nこのような丸山の趣旨は《不寛容なものに対する不寛容》性を強く有する無限抱擁性（寛容性）と要約できようが、その議論からすると、ダブルな信仰は日本人の無限抱擁性に通じる。他方、郭はダブルとトリプルを連続して捉えるが、キリスト教をその一つに含めたトリプルな信仰という、郭を含めた井上ほか (2018) の着想は、丸山の文脈では、首尾一貫性を重視するキリスト教という位置づけから考えてあり得ないことになる。丸山からするとダブルと（キリスト教をその一つに含めた限りでの）トリプルとの間には断層があるが、井上ほか (2018) では、これらは連続する。ただしキリスト教の宗派や学校の側、クリスチャン自身の側がどう考えているかは今回の調査対象者の基本属性からひとまず措いて、日本の一般の人びとの対キリスト教意識は、丸山の時代とは大きく変わっている可能性もある。現代の人びとは首尾一貫した体系を有する宗教としてキリスト教を眺めない可能性がある。\n\n丸山を法学部助手に抜擢した南原繁は宗教的には内村鑑三の弟子で新渡戸稲造の影響も強く受けた。一方、唯物論研究会の発起人会議長も務めた長谷川如是閑が父親の親友で彼に幼少期より可愛がられ、第一高等学校在学中に如是閑の出る唯研の講演会を聞きに行ったがため警察に拘留された経験もある丸山に、当然クリスチャン歴はない。しかし丸山は「『イエスを信じる者』の契約」を誓った（内村 1958 p.28）札幌農学校二期生（新渡戸、内村も二期生）の孫弟子にあたる。長い禁教の時代を経た、明治期というキリスト教の事実上の復活期から数えると、まだキリスト教が事実上「新宗教」であった時代の息吹を、南原から伝え聞く機会は何度もあったはずである8。したがって丸山から幾世代も隔たった、令和のいわゆる既成キリスト教は日本においても既成宗教と化しており、その令和のさらに若年層に聞いた今回の調査結果は、丸山の意識と異なる可能性も否定できない。実際、クリスマス、キリスト教式結婚式、キリスト教主義学校は、習俗としてであれ倫理の表れとしてであれ信仰の入り口としてであれ、排他的ではないキリスト教というイメージを日本人の間に醸成するのに貢献した可能性のあることは否めない。\n\nその意味も籠めて今回の調査票を設計した。トリプルな信仰についての設問は先述の通りだが、それ以外にも首尾一貫したものへの寛容性についての設問もいくつか設けたが、そちらについては紙幅の関係もあり、別稿で論じる。\n\n1.3.3 愛と奉仕の精神、および一部女性皇族のキリスト教主義学校への入学について\n\n郭は女子アナウンサー出身校上位を占めるキリスト教主義大学の建学の精神を探るため、これらの大学のウェブサイトを探ると、いずれもそこで愛と奉仕の精神を謳っていると指摘する（井上ほか 2018 p.98）。しかし建学の精神がそのまま在校生や卒業生に浸透していると考えるのは、教育効果を楽観視し過ぎている。もっとも一般人ではなく、愛と奉仕の精神をご公務で日々示される皇族方にあらせられてはその限りではなく、皇族女子がキリスト教主義学校にご入学する、あるいはキリスト教主義学校出身者を男子皇族の配偶者にお迎えすることは、実際に行われてもきた。\n\nキリスト教主義学校の生徒・学生・卒業生の愛と奉仕の精神については、調査票の Q29 でキリスト教主義学校の生徒・学生のイメージについて聞いた選択肢のなかに含めている。Q29 の選択肢 4「他人を思いやる心が豊かである」、選択肢 7「社会的活動への関心が高い」でこれらについて測定しようとしている。\n\nまた女性皇族については、Q11 で「女性皇族にキリスト主義学校出身者が複数人いることについて、あなたはどのように評価しますか」と 5 者 1 択の SA で聞いている。ただし調査実施時期の 2020 年 1 月の時点で ICU 出身の秋篠宮眞子内親王（当時；現、小室真子氏）の婚約並びに結婚延期の報道がなされていたため、回答にそのことによるバイアスがあった可能性はある。\n\n1.3.4 きれい、金持ち、キリストの「3K」\n\n井上は上記の著書の「はじめに」でキリスト教主義大学の女子を「きれい、金持ち、キリストの 3K」であるという噂についてその信憑性を検証しようとしている。非常勤先の仏教系女子大生が、自己卑下するなかでキリスト教主義女子大生をこう評して井上に語ったことが井上の着想の契機である。「検証」といっても先にもふれたが、非常勤先や会合等での私的な会話のなかでその噂が確からしいことを確認しているにすぎない。\n\nとはいえキリスト教主義大学の女子のメディアへの登場頻度を独自に数え上げる等、マス・コミュニケーション学の内容分析的な作業をした資料は井上も作成し載せている。例えば 2005 年から 2014 年の「女性ファッション誌への学生モデル登場率の推移」という表（井上ほか 2018 p.74）では、読者モデル起用率で上位にキリスト教主義大学の女子学生が位置づけられていることを、数字をあげて説明している。\n\nしかし、ファッション誌編集者の眼にキリスト教主義大学の女子学生が 3K9 であると映っていることは、この表から証明されるが、実際に編集者以外の、ふつうの市井の人びとが 3K の意識でキリスト教主義大学女子を捉えているか否かはこれだけでは分からない。\n\n本調査ではこれもキリスト教主義学校の生徒・学生・卒業生のイメージを聞く、Q29 の選択肢に、この「きれい」「金持ち」のイメージの有無を測定するものとして、「裕福である」「清貧である」と「容姿端麗である（みめうるわしい）」「容姿端麗ではない（みめうるわしくない）」を加えた。ただし佐藤 (2006) や井上ほか (2018) と異なり、現代は女性に対するジェンダーバイアス（以下、女性バイアスと略す）を当然視できない時代ゆえ、生徒・学生等の性別を問わない表現にした。\n\n1.3.5 キリスト教主義学校の設立目的について\n\n「現代日本における宗教教育の実証的研究」(1998~1999) では、宗教系の学校全般の特徴として、信徒の子弟の教育機会の確保とその宗派へのシンパの獲得が、設立目的の例として示されていた10。このことはキリスト教主義学校に限定してもそのまま妥当するのかどうかを検証する必要がある。もっともこの研究ではキリスト教主義学校については、同志社の 1930 年頃までの卒業生の例を示して、現在とは比べ物にならない入信率を誇っていた時期のあることも示してもいる。この点については、後述するように佐藤 (2006 p.17) でもミッション系高等女学校についてのみだが、表に示している。また「現代日本における宗教教育の実証的研究」では教育基本法並びに学校教育法が国公立学校に対してであるが、戦前の国家神道への反省から特定宗教の宗教教育に否定的であることも、宗教系学校が特定の宗教への布教に結びつくような宗教教育に及び腰な理由として挙げられていた11。\n\nもっとも教育基本法の制定を推進したリーダーは南原繁で、南原は官立第一高等学校在籍時に校長新渡戸稲造に感化され、同高元教員内村鑑三に弟子入りし、その新渡戸と内村は官立札幌農学校二期生で、内村のいうには自身及び新渡戸ら二期生は、そのほとんどが入信した一期生の先輩たちに「『イエスを信じる者』の契約」をせよと、クリスチャンになることを迫られたとのことである。「カレッジの世論はあまりにつよく余に反対であった、それに対抗することは余の力におよばなかった。彼らは左に掲げる契約に署名するよう余に強制した、・・・余はついに屈した、そしてそれに署名した」（内村 1958 p.22）。少なくとも内村の入信は「上級生の圧力によっておこなわれた入信であった。自発的な入信ではなく、かなり抵抗したうえでのキリスト教入信であった」（生越達美「内村鑑三―苦悩と回心の軌跡」）（堀ほか 1996 pp.21-22）とされている。\n\nしたがって本研究では、信者の獲得という先行研究では一応否定されている選択肢も含めて、キリスト教主義学校の設立目的を幅広い選択肢で想定した Q34 を設けた。ここでは「あなたが思う、キリスト教主義学校に対する設置母体のメリットに当てはまるものを上位 3 つまでの間で選び、中でも最もメリットだと思うものを1つ選んでください」という MA、SA 複合の設問をし、選択肢は「その他」「答えたくない」「よく分からない」とそれら以外に 12 個用意した。選択肢の 1 は「信者の子弟の教育要求への対応」、選択肢の 7 は「キリスト教への共感者（シンパサイザー）を増やすこと」である。他に選択肢の 8 では「キリスト教の信者を増やすこと」を入れ、シンパを作ることより直接的な目的があると考えるか否かも問うた。他方、例えば選択肢 9 は「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」とした。これはある意味で「キリスト教への共感者（シンパサイザー）を増やすこと」にも近い内容であるが、それと同時に 1.2 の①や 1.2.2 で言及した、首尾一貫した信仰かトリプルな信仰かということに密接にかかわる選択肢として用意した。\n\nこの設問のように MA と SA 双方での答えを求める設問を本調査では多く用意したが、これらは MA ベースで回答者の比較的多い選択肢については、その選択肢を選んだ人を 1、選ばなかった人を 0 とした二択の変数を作り、他の多くの質問群とクロス集計させうる。このようにしてこの調査の軸となる変数を設定することも意図した。\n\n本稿のもとになったウェブモニター調査全体の概念図（図1）は以下の通りである。この図に記されている番号は我々の調査の問の番号で、調査票は https://doi.org/10.6084/m9.figshare.21367632.v1 (Goto, 2022c) にて閲覧でき、そこに調査のローデータも置いてある。\n\nこのうち今回の報告（本稿）では、分析の一番の軸としてキリスト教主義学校への関与を示す Q12 を想定し、分析の二番目の軸に女性皇族のキリスト教主義学校への関与の是非を問う Q11 を想定する。\n\nこれらを主な独立変数としながらキリスト教主義学校の設立目的を推測させる Q34 並びにキリスト教主義学校の児童・生徒・学生イメージについての Q29 の各選択肢を主な従属変数として分析していく。\n\n「1.2 本研究の目的」で本研究の目的とそれと係わらせてウェブモニター調査での作業仮説を示し、1.3 で本稿の調査票設計を先行研究との拘りで示してきたので、ここではそのことを受けて、改めて本稿での作業仮説を示す。\n\n1.2 でみたように、《「日本の人口比で 1.6% 前後しかいない」という事実は、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況と矛盾する状況》であるが、その理由の解明に役立つのは、「Q34 でキリスト教主義学校の設置目的をどのように想像するかという問い」と考えられる。よってその問いを設け、その結果をみることを考えた。特に「その Q34 の各選択肢のうち、強い当事者、緩やかな当事者、非当事者の順に選ばれ、その選ばれ方の頻度の差に有意差のあるものは、設置目的に相当するものであると、当事者たちから捉えられている選択肢と考えられる」と1.2で述べた。この 1.2 での記述では、Q12 あるいは Q12 をグループ化した Q12G を独立変数として、Q34 の各選択肢を選んだ者、選ばなかった者を 1、0 とする従属変数とをクロス集計することが想定されている。\n\n一方、1.3 でみた限りで、キリスト教主義学校と皇室女子との関係が、一般の人びとの、キリスト教主義学校のイメージ向上に関与しうる可能性が示唆された。したがって上述のように Q11 も独立変数として、Q34 の各選択肢を選んだ者、選ばなかった者を 1、0 とする従属変数とをクロス集計することをここで想定した。つまりキリスト教主義学校と皇室女子との関係を肯定的に捉える者は、キリスト教主義学校への通学経験等のキリスト教主義学校関与度とはある程度別の次元で（もちろん双方が完全に独立しているという訳ではない）、キリスト教主義学校に肯定的な可能性の高い人々と想定できる。したがってそういう人たちとそうでない人を Q11 でみた結果を独立変数として、Q34 の各選択肢を選ぶ選ばないを従属変数としてクロス集計でみていくことで、一般の人々のなかでのいわゆるファン層が、キリスト教主義学校設立目的をどう捉えているかが分かる（作業仮説1の具体的な形）。このように Q11 × Q34 の各選択肢を見ることで、Q12G × Q34 の各選択肢のクロス集計で得られた知見を補足することが可能となる。\n\nまた、これも 1.2 でみたように、《「日本の人口比で 1.6% 前後しかいない」という事実は、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況と矛盾する状況》であるが、その理由の解明にもう一つ役立つのは、キリスト教主義学校の生徒・学生イメージを、人びとがどのように捉えているかを見ることでもあり、それも作業仮説であると 1.2 で考えた。そこで具体的には Q29 でキリスト教主義学校の生徒・学生のイメージを尋ねる問いを設けた。これも Q34 について述べたのと同様、Q12G とクロス集計し、Q34 の選択肢のうち、単純に考えれば、強い当事者、緩やかな当事者、非当事者の順に選ばれ、その結果に有意差のあるものは、キリスト教主義学校のイメージと、当事者から捉えられている選択肢と考えられる。\n\nそしてこの Q29 についても、1.3 での検討結果を経て、上述の Q34 の場合と同様、Q11 の皇室女子のキリスト教主義学校への関与の是非の問いとのクロス集計も有効であると考えられる。要するにこれも上述のQ34 の場合とほぼ同様の記述をくり返すが、キリスト教主義学校と皇室女子との関係を肯定的に捉える者は、キリスト教主義学校への通学経験等のキリスト教主義学校関与度とはある程度別の次元でキリスト教主義学校に肯定的な可能性の高い人々である。したがって Q11 の回答で肯定的な人たちとそうでない人とを Q11 で独立変数として、Q29 の各選択肢を選ぶ選ばないをクロス集計でみていくことで、一般の人々のなかでのいわゆるファン層が、キリスト教主義学校の学生・生徒イメージをどう捉えているかが分かる。つまり Q11 × Q29 の各選択肢を見ることで、Q12G × Q29の各選択肢のクロス集計で得られた知見を補足することが可能となる（作業仮説2の具体的な形）。\n\n1.3 で示された、先行研究の最重要な知見は、トリプルな信仰があるか否かである。本科研費研究全般の目的、「日本のクリスチャンの数は日本の人口比で 1.6% 前後しかいないにもかかわらず、キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多いのはなぜかを究明すること」と、この「トリプルな信仰」の有無を照らしあわせてみる。\n\nいままで Q34 や Q29 での議論は、「「日本のクリスチャンの数は日本の人口比で 1.6% 前後しかいないにもかかわらず、キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多いのはなぜかを究明すること」という命題の後半部分、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多いのはなぜかを究明すること」にのみ焦点を当てていたといえる。他方、もしもトリプルな信仰が成立するとすれば、「日本のクリスチャンの数は日本の人口比で 1.6% 前後しかいないにもかかわらず」という部分を吟味することになる。\n\nつまり、井上ほか (2018) における郭の議論からすると、既成仏教や神社神道においては、入信というプロセスを経ないでも、それらの宗教の信仰者といえる。それと同様、キリスト教も、一般の日本人の意識からすると、もしも洗礼というプロセスが不要であれば、仏教や神道と同様、三つ目の信仰の対象としうるということになるからである。要するに、その場合には「1.6%」というのは見かけ上の数値に過ぎないということになるからである。\n\nそこでトリプルな信仰の有無について問う質問としてどのようなものを用意したかについていくつか例を述べる。前後の議論とやや重複するかもしれないが、例えばキリスト教主義学校設置目的の推測を問うQ34においては選択肢9として「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を置いた。この MA で選択肢を選んだ人を 1、選ばなかった人を 0 として、Q12G とクロス集計して、キリスト教主義学校への通学経験者が 1 の人が多ければ、トリプルな信仰的な意識の醸成に、キリスト教主義学校は寄与しているという可能性が拓ける。\n\nまた本稿では、幼少期から原家族でキリスト教以外の宗教行事にどの程度参加してきたかを聞いている(Q3)。もしも Q12G とクロス集計することで、もしもキリスト教主義学校通学経験者の方が仏教や神道の宗教行事に参加してきたという結果が出れば「トリプルな信仰」の存在をより強く裏づけることになろう。また Q49 では仏教、神道、キリスト教、イスラム教の四つの宗教について「宗教において、入信していなくても教えを守ることによって、救済やご利益（ごりやく）が得られると思いますか。当てはまるものをそれぞれ選んでください」という設問をしている。このキリスト教に関して「そう思う」という人がキリスト教主義学校通学経験者の方に多いとすれば、やはりトリプルな信仰的な意識の醸成に、キリスト教主義学校は寄与しているという可能性が拓けるといえる。\n\nこのようにトリプルな信仰に関わるこれらの設問を従属変数として、Q12G を独立変数としてクロス集計することを作業仮説3とする。\n\nなお、今回、インタビュー調査からの引用、並びに定性的な内容の分析を、これらウェブモニター調査の報告に交えている。しかしコロナ禍及び研究代表者の非力による研究の進捗の停滞もあって、まだインタビュー調査並びに各種コンテンツ及び文献の内容の分析は一定の仮説を証明する段階に至っていない。今回、これらの一部を引用しているが、それらはあくまでもウェブモニター調査報告の内容を具体的に肉付けするためのものであり、これら二種の調査の結果報告ではないことを申し添えておく。\n\n本稿の報告は科研費研究のウェブモニター調査が中心であり、そこでの調査方法は 2. 「ウェブモニター調査の概要」の章に委ねる。なおウェブモニター調査については分析方法に限っては、本節で記す。\n\nウェブモニター調査の結果報告に用いたインタビュー調査は、機縁法でインタビュイー（インフォーマント）をみつけている。今回 3 名についてのインタビュー結果を載せているが、B さんについては、ウェブモニター調査のプレ調査として倫理審査を受け筑波大学図書館情報メディア系倫理審査委員会の承認を得たものである。その倫理申請の際に提出した同意書において、プレ調査ではあるが調査結果を論文に使う場合もあるが、その場合には掲載の前に、内容を示し、事前にインタビュイーの許可を貰う旨、記してある。インタビュー調査前に B さんに同意書に署名してもらい、今回の論文投稿前に、内容を確認いただき、掲載の同意書も得ている。A さん、C さんについては、ウェブモニター調査のプレ調査兼インタビュー調査という形で倫理申請をし、筑波大学図書館情報メディア系倫理審査委員会の承認を得ている。A さん、C さんについても、インタビュー調査前に同意書に署名してもらい、さらに今回の投稿に際し、B さん同様、内容を確認いただき、掲載の同意書ももらっている。なお、A さん、B さん、C さん共に、調査時、20 歳を越えている。\n\nこのようにウェブモニター調査のプレ調査を兼ねたインタビュー調査なので、スノーボールサンプリングではなく機縁法でインタビュイーを得ているし、分析もグラウンデッド・セオリーやKJ法やベレルソン流の狭義の内容分析法によるのではなく、定性分析、つまり文学研究や思想哲学研究でテキストを読み込むのと同様の方法で分析し、あくまでもウェブモニター調査の結果分析の肉付け・補足として用いている。ただしウェブモニター調査の結果に対してそれに添う内容以外の発言も含め、事象を立体的・複合的に把握できるように努めた。COREQ のチェックリストの Data analysis に準じる記載をしておくと、24 番のコーダーの数であるが、そもそもコーディングをして積み上げる形をとっていないので該当せずというか0である。25 番のコーディングツリーについても該当せずである。26 番の、テーマが予めあったかこれらに由来するかについては、科研費申請時のテーマは当然これらのインタビュー前のものであるので、その点では「予めあった」といえるが、今回はウェブモニター調査のプレ調査を兼ねたインタビューであるので、ウェブモニター調査の作業仮説はこのインタビューから派生している部分もある。27番のソフトウェアは何を使ったかは該当しない。28 番のインタビュイーが知見をチェックさせたかについては、findingsという形で分離して示してはいないが、本論文草稿全体を渡し、インタビュイーの発言箇所を青字に記し、どことどこでそのインタビュイーの発言に言及したかの一覧表も渡し、さらに掲載についての承諾書も得ている。\n\n上述の、「文学研究や思想哲学研究でテキストを読み込むのと同様の方法で分析し、あくまでもウェブモニター調査の結果分析の肉付け・補足として用いている」という点はウェブコンテンツ、各種文献資料についての広義の内容分析の場合もほぼ同様である。当初はキリスト教主義学校のウェブページやキリスト教主義学校の〇〇年史を系統的に収集し、それらの傾向を量的な内容分析で測定しようと考えていたが、それらの進捗は芳しくはない。今回これらはあくまでもウェブモニター調査の補足として用いているに過ぎない。\n\nこのように「文学研究や思想哲学研究でテキストを読み込むのと同様の方法で分析し、あくまでもウェブモニター調査の結果分析の肉付け・補足として用いている」のは、本稿の筆頭著者が思想・哲学寄りの社会学者であることが大きい。そして 1.3 の先行研究の箇所で多く名前を出した、井上ほか (2000) の井上順孝は宗教学、井上ほか (2018) の井上章一は風俗論、佐藤 (2006) の佐藤八寿子は教育社会学、濱田 (2001)の濱田陽は宗教学である。風俗研究は色々な分野からのアプローチは可能であるが、社会学を日本では初期に世態学と訳したように（東京帝大の最初の Sociology の授業は美学者フェノロサによる「世態学」であった）社会学との繋がりは弱くない。そして社会学と宗教学は元々哲学から分かれた分野であり、本稿の先行研究として挙げた研究においても思想的な議論が多くなされている。また 1.4 でウェブモニター調査の分析の軸にすると述べた、井上ほか (2018) のトリプルな信仰という言葉は、丸山真男のキリスト教についての見解と大いに反することも、1.3 で述べたとおりである。また本稿 5. での「考察」の議論をやや先取りする感があるが、考察においても ICU の 50 年史の『未来をきり拓く大学―国際基督教大学五十年の理念と軌跡―』(2000) が、著名な思想史研究者の武田清子の単著のものであったので、思想の科学研究会のオリジナル・メンバーとして武田の古くからの友人である丸山の議論との接点も「考察」において記すこととした。\n\nこのように思想史的な文脈での研究報告であり、また 1.3 でウェブモニター調査の調査票の選択肢を決める際も、先行研究の議論を踏まえているので、今回は多変量解析等はせずに、ウェブモニター調査の集計・分析もクロス集計に留めることとした。なお今後本ウェブ調査のより多くの変数を取り上げながら多変量解析することも視野に入れている。\n\n具体的には 1.4 で記したように、キリスト教主義学校への関与度を示す Q12 あるいは皇族女子のキリスト教主義学校への関与への是非を問う Q11 あるいはそれをグループ化した変数を、分析の軸にする独立変数と考える。まず主な従属変数としては、キリスト教主義学校設立の目的への推測を問う Q34 とキリスト教主義学校の生徒・学生のイメージを問う Q29 を想定している。これら Q34 と Q29 は選択肢のうち最もそう思うもの 1 つを選ぶ SA と、そう思うものすべてを選ぶ MA、双方を聞いている。いずれも選択肢の数が多いので、SA はクロス集計に使うには度数が低い。したがって、MA でそれぞれの選択肢を選んだ人を 1、選ばなかった人を 0 とする離散変数を各選択肢ごとに想定し、それらを従属変数とし、Q12Gや Q11 を独立変数としてクロス集計をした。1.3 でみた先行研究に照らして各選択肢が選ばれているので、選択肢ごとの分析に努めた。Q34 については 3.1 で、Q29 については 3.2 で記述する。あと 1.4 で示したトリプルな信仰についての従属変数も Q12G とのクロス集計をした。これについては 3.3 で記される。\n\n\n2. ウェブモニター調査の概要\n\n本研究では株式会社サーベイリサーチセンターに委託したウェブモニター調査を実施した。なお、それに先立ち、筑波大学図書館情報メディア系倫理審査委員会の承認を得た（「科研費挑戦的萌芽研究「キリスト教主義学校から見る寛容と洋化―ステークホルダーらの期待と文化資本」(2018~2018 年度（代表:後藤嘉宏））のウェブモニター調査」（通知番号第 19-99 号））。調査対象者の属性は東京都・神奈川県に在住の 15 歳 ~29 歳の男女とした。\n\n同意はウェブモニター管理会社楽天インサイトの同意画面によって当人の意思確認をする形によって求めた。ウェブモニター管理会社楽天インサイトのデータ収集と同意を含むすべての手続きについて、筑波大学図書館情報メディア系倫理審査委員会の承認を得ている。\n\n調査は 2020 年 2 月 22 日に開始し、予定のサンプル数 700 に達した 3 月 3 日に終了した。なお、サンプル数は多ければ多いほど統計誤差が減るので望ましいが、この 700 という数値は、今回必要とされる、用意した質問数において今回使える予算の上限額目一杯のサンプル数であるので、この数値にした。\n\n東京都と神奈川県に居住地域を絞ったのは、これらの地域にキリスト教主義学校が多いからである。特に神奈川県はフェリス女学院等明治時代初期からキリスト教主義学校があっただけでなく、戦後 GHQ の政策で、旧海軍用地や農地改革等で接収した土地を横須賀の進駐軍との絡みで、キリスト教主義学校の校地のために優先的に払い下げたとの説もある（島本 1994）。以下の数字にカトリックは含まれないし、プロテスタントでも漏れはあるものの、一般社団法人キリスト教学校教育同盟の一覧では東京都 23 学校法人、神奈川県 11、愛知県 4、大阪府 7、兵庫県 8、福岡県 4 で、他の都道府県は 3 学校法人以下である（最終確認日 2022 年 10 月 17 日）。\n\nキリスト教主義学校が身近にない調査対象者だとイメージを浮かべにくい点が、これらの地域の住民に限定した理由である。さらにそのことと関連するが、それ以上に、本調査全体の枠組みにもこの限定は関わっている。再三申し上げているように本調査ではキリスト教主義学校に通ったことがある・あるいはいま通っている当事者をいわばキリスト教主義学校当事者として、それらの者の保証人等家族や志望したことがある者、兄弟姉妹に通学者のいる者、オープンキャンパスに行ったことのある者などを、当事者の周辺の者・緩やかな当事者とし、これらに該当しない者を部外者の者として、これら三者の間での意識の差の有無をみようとしている。これらの差を本調査の他の質問とのクロス集計の分析の軸として想定した。よって当事者や当事者周辺の者（緩やかな当事者）を一定数以上確保するためには、キリスト教主義学校が通学圏にほとんどない地域の調査対象者を減らすことが、有効と考えた。\n\n具体的には調査票 Q12 で「あなたのキリスト教主義学校への関与についてお聞きします。当てはまるものを選んでください」という設問をして、このキリスト教主義学校関与度を測定しようとしている。この設問の単純集計の結果は表2-1のようになっている。\n\nQ12. あなたのキリスト教主義学校への関与についてお聞きします。当てはまるものを選んでください。\n\nこれらのうち、選択肢 1 の「通ったことがある」を選んだ者、選択肢 2 から5までを選んだ者、選択肢 6 の「上記のいずれにも該当しない」を選んだ者、これら 3 つのカテゴリーに再グループ化した Q12G という組変数とした。この Q12G を独立変数として、他の諸変数を従属変数にした項目間クロス集計を行い、この Q12G を分析の軸にすることを想定した。要は上述のように、キリスト教主義学校に通ったことがある者、通ったことはないが何らかの当事者性のある者、関与のまったくない者の3カテゴリーに分けた Q12G を分析の軸とすることで、キリスト教主義学校関与者とそれ以外との意識の差が測れると考えた12。\n\n次に 15~29 歳の若年層に調査対象者を絞った理由は、まだ高校受験・大学受験・大学院受験の可能性のある者や、可能性は少なくなったが、進学を控えていた頃の記憶がまだ鮮明に甦る世代の意識を聞いてみる必要性を感じたからである。ただし今後、ほぼ類似の質問票で世代を替えて調査し、それと比較する可能性も想定してはいる。\n\n\n\nなお、今回の調査対象者の基本属性は表2-2 のようになる。\n\nまた今回の回答者の信仰する宗教についての設問の回答は表2-3 のようになっている。これは、Q1-9「あなたの信仰する宗教に当てはまるものを選んでください」という設問の回答（SA）である13。\n\n「よく分からない」の 11.7%、「答えたくない」の 2.8% を含めた中で、また「特にない」も 53.2% もある中で、「既成（伝統）キリスト教14」3.0%、「キリスト教系の新宗教」0.9% であわせると 3.9% に及び、全国平均のクリスチャンの比率 (1.0~1.6%) よりだいぶ大きい数値となっている。ただ Q12 に替えてクロス集計の軸にできるほどには大きい数値ではない。なおウェブモニター調査の性格上、モニター登録者のうち、このテーマに興味をもった者が答えるということから多めの数値になった可能性もある。\n\n「よく分からない」「答えたくない」「特にない」を合わせると 68.0% もいて、残りの 3 割のみが信仰をもつということは少ない数字とも感じられるが、井上順孝の『「現代日本における宗教教育の実証的研究」(1998~1999) 報告書』と較べるとむしろ大きい数字であるともいえる。同研究報告書では「信仰あり」が22.8% である（井上ほか 2000 p.40)。ただし同研究の調査票は本研究に比して、この数値が低く出る設計となっている。「あなたは宗教にどの程度関心がありますか。次のうちから選び、さらにそれぞれの質問に答えてください」という質問文で、枝問はとりあえず措いて選択肢は以下の四つである。「1. 現在、信仰をもっている　2. 信仰をもっていないが、宗教に関心がある　3. 信仰をもっていないが、宗教にもあまり関心がない　 4. 信仰はもっていないし、宗教にもまったく関心がない」。\n\nこの井上ほか (2000) の設問は、質問文の方が「関心」のみを聞いているのに、上記の選択肢は「信仰」の有無と「関心」の有無が混ざっている。本来「信仰」と「関心」は別次元であるので、別々に問うかファセット構造の質問にすべきで、おそらくファセット構造を目指しつつやや中途半端に終わっていると思われる。なぜならファセットなら質問文で「信仰」と「関心」の双方を聞く形に書くべきなのに「関心」のみを問うているし、選択肢の方もファセットで排他的かつ網羅的に回答者が選べるようにするには、「信仰をもっているが、宗教に関心がない」という選択肢が必要だが、それがない。また「1. 現在、信仰をもっている」はやや諄くなるがファセットであれば「1. 現在、信仰をもっているし、宗教に関心がある」の方がよい。したがってファセット構造が不充分であるので、幼児洗礼を受けたからキリスト教徒であるとか親が仏教徒だから何となく仏教を信じてはいるという者が、「信仰あり」から排除される可能性が生じる。\n\n先ほどこの井上ほか (2000) の枝問についてはとりあえず措くとしたが、選択肢 1.「現在、信仰をもっている」を選んだ場合、7 つの枝問を答えるように指示され、それらの枝問の 2 問目は、「その宗教には何歳のときに入信しましたか」とある。したがって何となく神道の信者であるとか、葬式仏教といわれるような先祖代々の宗教だから一応仏教の宗派を信じている人は、枝問まで眼に入ると、この本問の方の選択肢 1. 「現在、信仰をもっている」を選びにくくなっているのが、『「現代日本における宗教教育の実証的研究」(1998~1999) 報告書』の調査票であるといえる。なおこのように自覚的な信仰のある者に信仰のある人を絞り込むことはこの研究であえてそうしている可能性があり、そのことで、この研究では宗教系高校等の入信に対する効果を測定できている側面もあることを付言しておく。\n\nしたがって我々の調査の調査対象者で信じている宗教を答える者が『「現代日本における宗教教育の実証的研究」(1998~1999) 報告書』よりやや多いのは、ある意味当然ともいえる。\n\nただ井上ほか (2018) の「トリプルな信仰」の根拠は神道と仏教合わせて信徒数が日本の総人口を超えるということにあったが、井上ほか (2000) も我々も、それに比して信仰を持つ人が少ない。今後は自覚的な信仰と、親の信仰を何となく嗣ぐ場合のような緩やかな信仰を分けつつ、双方をきき取る調査票を設計したい。\n\nなお、ウェブモニター調査だと無作為抽出法の調査と違い、サンプルが母集団のほぼ正確な縮図になることはないという限界があることは我々も承知している。しかし、宗教に関する設問で郵送法・留置法だと回収率が期待できず、かといって新型コロナウイルスが流行りはじめた時期に、訪問面接法で、家の軒先で 40 分前後のやり取りを行うことは多くの調査対象者から敬遠されたと考えられる。また紙の調査とウェブモニター調査とを比較すると、それぞれに利点と欠点とがあり、紙の調査の回収率の低下している現代ではウェブモニター調査が学術目的の調査として有効ではないとは必ずしもいえず、特にセンシティブな内容の調査にはウェブモニター調査が適しているとの、日本学術会議社会学委員会の報告もある（最終閲覧日 2022 年 10 月 17 日）。\n\nなお今回、調査委託先のサーベイリサーチセンターを通じて、ウェブモニターの管理会社楽天インサイトの意向として、答えにくい設問については「よくわからない」や「答えたくない」の選択肢をつけるように指示されたため、無回答故の DKNA 以外に、「よくわからない」や「答えたくない」の選択肢の含まれる設問もある。なお、調査票は拡張データに含まれている (Goto, 2022b)。\n\nこの章の最後に STROBE のチェックリスト 12 番の Statistical methods についての記載をしておく。a)「交絡の制御に用いる方法を含む、すべての統計的手法を説明する」であるが、諸変数の交錯した影響を統制しつつ見るには多変量解析が最適であることは承知しているが、本稿は、まずは先行研究で指摘された事項それぞれを選択肢にし、それが妥当するかどうかを丁寧にみていくことが重要かつ先決な作業であると考え、クロス集計による分析に留めている。したがって、ここはクロス集計とカイ二乗検定という古典的手法のみによったという答えになる。b)「サブグループと相互作用を調べるために使用された方法を説明する」についてであるが、Q12 というキリスト教主義学校関与度によって全回答者をキリスト教主義学校の関与度でサブグループに分けてクロス集計した。c)「欠損データにどのように対処したかを説明する」についてであるが、欠損データは「無回答」として処理した。また基本的に上述のように「よくわからない」や「答えたくない」をあえて選択肢に設けた設問も多いので、「無回答」も生じにくい。d) については「コホート研究」「症例対照研究」「横断的研究」それぞれに該当する場合の質問がチェックリストにあるが、このいずれにも該当しない。e) は「感度分析があれば説明する」であるが該当しない。\n\n\n3. 調査結果とその分析\n\n以下で調査結果とその分析を記すが、説明の都合上 2.1 の①から⑥の順ではなく、順序は前後する。\n\n表3-1-1は「Q34. あなたが思う、キリスト教主義学校に対する設置母体のメリットに当てはまるものを上位3つまでの間で選び、中でも最もメリットだと思うものを 1 つ選んでください」と MA と SA 双方で聞いた質問の回答である。\n\n先述のように本調査では設問により「答えたくない」や「よく分からない」を入れることを委託業者から求められたが、この設問では選択肢の 15 においた「よく分からない」を選んだ者が 42.7% と最も多かった。\n\nこの選択肢を選ばなかった者を 0、選んだものを 1 として従属変数にして、キリスト教主義学校当事者指数を 3 段階にグループ分けした Q12G を独立変数として、両者をクロス集計する（表3-1-2）。\n\nキリスト教主義学校に通ったことがある者は「よく分からない」を選ぶ者が相対的に少なく21.3%、通ったことはないが緩やかな当事者性のあるグループが中間で 31.5%、関与のまったくない者は「よく分からない」を選ぶものが 55.4% に及んでいる(カイ二乗<0.01)。当事者性の強い者ほど、学校側の考えるものと合致しているか否かは措いて、キリスト教主義学校の設置母体の目的を何らかの意味で意識していることが、この結果から分かる。それと同時に、キリスト教主義学校の在校生・通学経験者やその周辺部分以外、つまり当事者性のない者の過半の者から、キリスト教主義学校の理念あるいは場合によってその存在自体がまったく意識されていない可能性も、この結果から示唆される。もっとも通ったことがある者でも「よく分からない」を選ぶ者が 21.3% いたが、進学・就職実績のみを理由に進路に選んだ者も相当数いるはずで、それがこの数値に示されていよう。\n\nこの「よく分からない」を除いた選択肢の度数の多い方から並べた図は図3-1-1のようになる。\n\nこの Q34の選択肢2.「教団組織の資金集め」が MA で 17.7% とつぎに多い、いいかえると「よく分からない」を除くと実質、一番多い。私立学校で理事や教職員が一般の俸給生活者より遥かに多額の報酬を得ることはあるにせよ、学校法人の法的性格から法人が上げた利益を外部に流すことは通常ないし15、またキリスト教主義学校の実態からも現実にはありえない。このようにこちらとしてはありえないはずの選択肢をあえて入れただけのつもりのこの選択肢が、なぜかこの設問の単純集計で実質最も票を集めた。これとQ12G をクロス集計すると（表3-1-3）、関与の全くない者は予想に反して、「教団組織の資金集め」を選ぶ者が 14.4% で、最も少ない。僅差で続くのが、キリスト教主義学校に通ったことがある者で、これらのグループで「教団組織の資金集め」を選ぶ者は 14.8% で、このグループが少ないことは予想の通りであった。一方、通ったことはないが何らかの当事者性のあるグループが、意外にもこれら3カテゴリー中で最も多くこの「教団組織の資金集め」を選んでいて、28.0% いた（カイ二乗<0.01）。\n\n特に Q12 をグルーピングする前の形で分布をみると、「通ったことはないが、受験したことはある」者が40.0%と特に多くなっている。なお、各セルの実数は少ないがこの表もカイ二乗<0.01 となっている（表3-1-4）。\n\n受からずに受験料のみ徴収された者や、合格して入学金を納めたものの志望順位のより高い学校に受かりキリスト教主義学校に通わなかった者が、「通ったことはないが、受験したことはある」となることを考慮すると、実際に通った者と、それ以外の緩やかな当事者性のある者との間にある、この温度差は、納得もゆく。\n\n次に選択肢 3「教会以外の、キリスト教に触れる敷居の低い舞台としての役割を果たすこと」を Q34 の MA で選んだ者は全体で 15.0% である。これについては、「よく分からない」を除いて Q34 の MA の単純集計で 2 番目になる。Q12G とクロス集計するとキリスト教主義学校に通った者はこの選択肢を 28.7% 選んでおり、ゆるやかな当事者は 22.6%、他方、まったく関与しない者は 8.5% である（カイ二乗<0.01）（表3-1-5）。\n\nこれはキリスト教主義学校の当事者性の大小ときれいに相関する。この選択肢3は「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」の選択肢9にも近いが、選択肢 7 の「キリスト教への共感者（シンパサイザー）を増やすこと」とも関係しうる。また先述のようにキリスト教式結婚式に関する濱田 (2001) による、信仰への入口と聖職者側が考えているという議論からも、この選択肢をキリスト教主義学校への通学者が多く選んだことは納得がゆく。1.2.2 で先述のように「キリスト教に触れる敷居の低い舞台」として聖職者の側がキリスト教式結婚式を提供していることが濱田 (2001) によって記されていたし、やはり先述のように濱田の研究において日本のカトリック中央協議会が1973 年バチカンに示した文書で、カトリック学校の卒業生にキリスト教式結婚式の利用希望者が多いと記されていた（濱田 2001 p.29）。このようなキリスト教式結婚式と同様の機能をキリスト教主義学校が果たしているとも、考えられる。\n\nQ34の選択肢1は「信者の子弟の教育要求への対応」であるが、これを選んだ者は MA での集計の全体で13.4% で、「よく分からない」を除いて MA の単純集計で 3 番目である。キリスト教主義学校に通ったことがある者は「信者の子弟の教育要求への対応」を選ぶ者が相対的に多く22.2%、通ったことはないが何らかの当事者性のあるグループが中間で 19.0%、関与のまったくない者は「信者の子弟の教育要求への対応」を選ぶものが 9.0% に留まっている（カイ二乗<0.01）（表3-1-6）。\n\nこれもキリスト教主義学校関与度指数と相関しているといえる。先行研究の『「現代日本における宗教教育の実証的研究」(1998~1999)報告書』における市川の分析によると、全体でみると、出身高校の宗派は非宗教系高校 87.9%、神道系 0.6%、新宗教系 0.6%、仏教系 3.3%、カトリック系4.2%、プロテスタント系 3.4% で、非宗教系高校が圧倒的に多い（井上ほか 2000 p.100）。他方、カトリックの信仰をもつ者についても非宗教系高校が多いことは多いが、58.3% で全体の 87.9% よりは遥かに少ない。そしてカトリック高校は 29.2% と平均の 4.2% より非常に多い。あと全体に自分の信仰と違う宗教系高校は避けると市川は分析するが、例外はカトリックとプロテスタントで、カトリック信者がプロテスタント高校、プロテスタント信者がカトリック高校というケースは少なくないという。「同じキリスト教ということで、相異点よりも共通点の方が強く意識されている」可能性を市川は推測する（井上ほか 2000 p.101）。要するに信徒の教育要求に応える機能を、宗教系学校はもっているとこの研究は示唆する。井上順孝らの先行研究から 20 年隔たっているが、基本的に今回の結果も、キリスト教主義学校について似た形になっている。\n\nまた Q34の選択肢8は「キリスト教の信者を増やすこと」であるが、この選択肢を選んだ者は全体で13.0% であった。これは「よく分からない」を除き MA の単純集計で 5 番目である。この選択肢を選ばなかった者を 1、選んだものを 0 として従属変数として、Q12G を独立変数として、両者をクロス集計」すると（表3-1-7）、非常に残念ながら通常の検定で有意であるとする基準である危険度 0.05 以下では有意差はなくp値=0.1346 の水準であった。だが、キリスト教主義学校に通ったことがある（あるいは通っている）者は「キリスト教の信者を増やすこと」を選ぶ者が少なく7.4%、通ったことはないが緩やかな当事者性のあるグループが中間で 12.5%、関与のまったくない者は「キリスト教の信者を増やすこと」を選ぶものが 14.6% と 3 カテゴリーで最も多くなっている。また選択肢8の「キリスト教の信者を増やすこと」と趣旨として全く逆になるであろう、選択肢 9 の「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」は、4パラグラフ後に申し上げるように、この選択肢 8 と全く逆の相関関係になっていて、そちらはカイ二乗 <0.01 である。したがって、この選択肢 8 で有意差がなかったのは、検出力不足（データ数の不足）のためであると我々は考察している。\n\n有意差はないとはいえ、キリスト教主義学校の当事者指数の高い者ほど、キリスト教主義学校が信者獲得を目的としていないという認識をもっている可能性が示唆されたことは、興味深い結果である。われわれは、同じ系列のキリスト教主義学校に幼稚園から大学院修士課程まで在籍した男子大学院Aさんにインタビューしたことがあったが、彼は高校時代、洗礼を考えたことがあり、宗教担当の教員に相談までしたが、急ぐことはないのでゆっくり考えるようにいわれたとの発言をしているが（調査日 2019 年 5 月 31 日）、そういう発言と、この調査結果は照合しうる。なお A さんの調査に先立ち、筑波大学図書館情報メディア系倫理審査委員会より「科学研究費（萌芽）「キリスト教主義学校から見る寛容と洋化―ステークホルダーらの期待と文化資本」（2018~2018 年度（代表:後藤嘉宏））の質的調査並びにウェブモニター調査のためのプレ調査」（通知番号第 19-10 号）の承認を得ている（以下、A さんの発言の引用の際は、この倫理審査についての記述を重複を避けるため控える）。\n\nキリスト教主義学校を通常、ミッションスクールと称するが、ミッションスクールではないと謳うキリスト教主義学校も少なくない。同志社などもその例ではある。例えば佐藤八寿子は「同志社は、キリスト教主義の学校ではありますが、キリスト教の伝達を目的とするミッションスクールではないのです」という同志社総長の入学式式辞に言及しながら、多くのミッションスクールが設立当初のキリスト教伝道団体から独立した日本のキリスト教主義の学校として運営されていると指摘する（佐藤 2006 pp.8-9）。\n\n他方、1951 年にはプロテスタント系の基督教学校教育同盟理事会が、キリスト教主義学校と呼称する申し合わせをしたが、キリスト教伝道団体から独立しているものの教育方針はそれ以前と同様であるため、ミッションスクールの呼称を容認する学校もあるという（佐藤 2006 p.11）。またミッションには「布教」と同時に「使命」の意味もあり、神に与えられたそれぞれの使命を生徒が果たせるような教育を行うという意味で、ミッションスクールを名乗っているカトリックの学校、長野清泉女学院の例も佐藤は紹介している（佐藤 2006 pp.12-13）16。しかしミッションスクールのミッションは通常日本では「布教」を意味する。キリスト教徒を増やすというこの狭い意味での「ミッション」の意図を表明している学校の少ないことは、前段落の佐藤の説明からも分かるが、それとともに、このような当事者と非当事者との比較からも一応示唆されるであろう。\n\n他方 Q34の選択肢9は「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」である。順番は前後するが、これは全体で13.3%で「よく分からない」を除いて MA の単純集計で4番目である。この選択肢は、選択肢 8 の「キリスト教の信者を増やすこと」が狭義の宣教・布教だとすると、より緩やかな、広義の布教となろう。神から与えられた「使命」を果たすという前々段落で佐藤により示された長野清泉女学院の例もこの類に近いといえるであろう。先にみた Q34.の選択肢 8 の「キリスト教の信者を増やすこと」より若干のみ多い数値であったが、こちらは Q12G とのクロス集計において選択肢 8 とは逆の分布になっている（表3-1-8）。\n\nこの選択肢を選ばなかった者を 1、選んだものを 0 として従属変数にして、キリスト教主義学校当事者指数の Q12G を独立変数として、両者をクロス集計すると、有意差<0.01 で、キリスト教主義学校に通ったことがある者はこの選択肢 9「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が相対的に多く、27.8% いた。他方、通ったことはないが何らかの当事者性のあるグループは中間で 11.3%、関与の全くない者は選択肢 9 を選ぶ者が 10.4% であり、この設問の回答はキリスト教主義学校当事者性の大小ときれいに相関する。\n\n1.2.2 で先述のように、井上ほか (2018) 第 2 章では「宗派に帰属しない日本人のトリプル信仰」（井上ほか 2018 p.136）に着目するが、この結果は、キリスト教主義学校の通学者はこのようなトリプルな信仰を想定している者が相対的に多いことを裏づける結果といえる。同書では「日本社会の「トリプル信者」を生み出す要素はいろいろあるだろうが、重要なのは中学校・高等学校の教育ではないかと思う」（井上ほか 2018 p.139）として、キリスト教主義の高校が全国の 4.2% と、キリスト教徒の比率よりは多く、しかも名門校が多いことも関連するとする。\n\n当然このようなトリプルな信仰は融通無碍で無限抱擁的な宗教の性格に親和性がある。他方、先述の丸山真男『日本の思想』で記されるように、キリスト教が日本において首尾一貫性を重んじる姿勢を貫いているとすれば、このようなトリプルな信仰に通じることはあり得ない、ということになる。\n\n基本的に「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校出身者に多いので、洗礼のような明確な入信のプロセスよりも、トリプルな信仰の方に、少なくとも比較的若い世代のキリスト教主義学校出身者は親和的であることを示唆している。われわれの行った質的調査において、現在の同志社は「徳育としてのキリスト教」という言葉を強調していると、中学から大学まで同志社に学んだCさんは語っていたが（調査日 2020 年 1 月 28 日）、この同志社の「徳育としてのキリスト教」という教育理念もこの選択肢の趣旨に近い発想であるといえよう。なおCさんの調査に先立ち、A さん同様、筑波大学図書館情報メディア系倫理審査委員会より「科学研究費（萌芽）「キリスト教主義学校から見る寛容と洋化―ステークホルダーらの期待と文化資本」（2018~2018 年度（代表:後藤嘉宏））の質的調査並びにウェブモニター調査のためのプレ調査」（通知番号第19-10 号）の承認を得ている（以下、C さんの発言の引用の際、この倫理審査についての記述は重複を避けるため控える））。なおこのトリプルな信仰については他の設問にて聞いているので 3.3 で詳述する。\n\nここまでみてきたこれら Q34 の選択肢 1，3，8，9 のそれぞれの単純集計及びそれらと Q12G とをクロス集計した結果を総合して考えてみると、キリスト教主義学校は、新たな信者を増やす場としてよりは、キリスト教的な徳育をする場として当事者たちから受け取られていると同時に、すでに信者である者の子弟に好個の教育施設としても位置づけられているといえる。そして狭義の布教（ミッション）をするよりはトリプルな信仰に通じる広義の布教(ミッション)をする場、さらには布教（ミッション）よりも神から与えられた使命（ミッション）を自覚させる場として意識されているといえる。要するにミッションという言葉の拡がり、偏差、バリエーションのなかで、狭義の意味以外のこの言葉の意味を選択している・あるいは模索していることが示唆されるといえよう。\n\n選択肢 9 についで MA で多いのは選択肢 5 の「教育を通じた地域貢献活動を行い、イメージの向上を図ること」で、全体で 14.9% である。これを Q12G とクロス集計すると表3-1-9のようになり、カイ二乗<0.01で有意差がある。キリスト教主義学校非該当者の方が、「教育を通じた地域貢献活動を行い、イメージの向上を図ること」を選ぶ者が少ない。他方実際に通ったことのある人と緩やかな当事者で比較すると、緩やかな当事者の方が「教育を通じた地域貢献活動を行い、イメージの向上を図ること」を選ぶ者がやや多い。\n\n緩やかな当事者の分布の実情を見るため、組変数にせずに Q12 そのものとクロス集計してみると、表3-1-10のようになる（カイ二乗<0.01）。「受験してみたい」「受験したことがある」者にこれを選ぶ者が多く、「通ったことはないが、家族に通ったものがいる」者は非当事者とほぼ結果が等しいほどに、これを選ぶ者が少ない。要するに、この選択肢についてはイメージ先行で、当人あるいは家族が通学していない者から選ばれやすいということになる。\n\nこの選択肢 5 についで MA で多いのは選択肢7の「キリスト教への共感者（シンパサイザー）を増やすこと」で全体で 10.9% である。また井上ほか (2018) ではキリスト教主義学校がどの社会階層をターゲットにするかという議論が過去にあったことを記しているが、そのこととも関係するといえる。カトリックは当初、救貧、慈善活動を強く意識し、孤児院の延長で白百合学園などの学校の前身施設を設立していったが（井上ほか 2018 pp.191-192）、正規の学校の条件として宗教教育に制限をかけた 1899 年の文部省訓令 12 号に圧力を受けたことや、プロテスタント系のキリスト教主義学校等によって、社会的に影響力のある社会階層の者が多くプロテスタントになったのを見ることで（井上ほか 2018 p.207）17、カトリックの方も社会的上層部出身にターゲットをシフトさせ、お嬢様学校に白百合や雙葉などが変貌したと、記している。その際考えられたのは、共感者を増やすことが、布教を巧みにする手段となるということであったと想定されている。しかしこの選択肢 7 と Q12G のクロス集計は有意差が出なかった。キリスト教主義学校通学経験者等の当事者たちから、特に「キリスト教への共感者（シンパサイザー）を増やすこと」は支持されているわけではない。\n\n次にキリスト教主義学校の生徒・学生のイメージを聞いた Q29 をみてみよう。この問いも MA で「イメージしたもの」3 つと SA で「最もイメージしたもの」1 つを挙げてもらった。「該当なし/答えたくない」を含め 29 個の選択肢を用意したが、その結果は表3-2-1 のようになる。\n\nQ29 で「該当しない/答えたくない」と Q12G をクロス集計すると、表3-2-2 のようになる（カイ二乗<0.01）。当事者の方が「該当しない/答えたくない」を選ぶ人が多いことは事前に予想された通りだが、実際の通学経験者よりも、緩やかな当事者の方にこの選択肢を選ばない傾向にあることは少し意外であった。その理由を想像するに、一部の通学経験者には、世間的なイメージ、色眼鏡で自分らを眺めることへの反撥があるのかもしれないが、そのあたりは今後インタビュー調査を重ねることで明らかにしたい表3-2-2。\n\nこの Q29 で「該当しない/答えたくない」を除いた選択肢の度数の多い方から並べると図3-2-2（下部）のようになる。\n\n最も多く回答を集めたのはこの「該当なし/答えたくない」の 31.9% である（MA、SAとも）が、それを除くと MA ベースで実質もっとも多く回答されたのは選択肢 1 の「外国語が得意である」の 22.0% である。キリスト教主義学校はその草創期から外国語教育に力点を置き、宗教教育との間で学内での緊張関係の可能性も胚胎してきた。なお佐藤 (2006) は 1980 年代に社会全体で立身出世物語が光を失うのに応じてその欲望の対象であったファム・ファタルもその輝きを失ったと指摘し、代わりにキリスト教主義学校がその存在意義を主張する役割を担い始めたのが、外国語教育であると指摘する（佐藤 2006 pp.187-189）が、外国語教育への期待は、古くからあったし、今回の調査結果でもまたその期待が裏づけられうる。\n\n次にいま Q29 の選択肢 1 の説明途上ではあるが、クロス集計の都合上、女性皇族のキリスト教主義学校への関与の是非をみた Q11 の単純集計結果を先にみておく。「女性皇族にキリスト教主義学校出身者が複数人いることについて、あなたはどのように評価しますか」という設問に 5 つの選択肢を用意した。選択肢 1「望ましいことだ」6.3%、選択肢 2「どちらかといえば望ましいことだ」15.1%、選択肢 3「どちらともいえない」70.7%、選択肢 4「どちらかといえば望ましくないことだ」4.4%、選択肢 5「望ましくないことだ」3.4%となっている（表3-2-3）。\n\n判断保留を意味する選択肢 3 が非常に多く7 割を超え、次に多いのは選択肢 2 の「どちらかといえば望ましいことだ」である。「どちらともいえない」の 70.7% の人びとがこれを選んだ理由は、自由記入欄を設けていないので不詳だが、望ましい部分と望ましくない部分の双方あるという意味での判断保留の場合、皇族とはいえあくまでも個人の選択の問題ゆえ評価・判断の対象でないという趣旨の場合、さらに「よく分からない」に相当する場合、いずれのケースもあると想定される。ただし望ましい部分と望ましくない部分の双方あるという意味で判断保留する者は一定数いると考えられる。神道と結びついているはずの皇室の一員がキリスト教主義学校に通うことに反撥する層がいるであろうし、他方、キリスト教主義学校で西洋文化の根底にあるキリスト教を学び、国際的に通用する教養と外国語力も身につけることで展開されるであろう皇室外交に期待する意識も強いであろうし、その双方の拮抗する側面が「どちらともいえない」の 70.7% という結果になっているとも考えられる。\n\nなお、この Q11 が本研究において重要である理由は、1.2.1 並びに 1.2.3 で井上ほか (2018) の文脈に添って説明はした。ただしそこでは愛と奉仕の精神を教育目標にしているがゆえに、皇室女子も、ということが記されているにすぎない。\n\nそこでここでは佐藤 (2006) の議論を少しみておく。佐藤は「美智子皇后、雅子皇太子妃が、カトリック系ミッション・スクールの出身であることは周知のとおりだ。では、ミッション・スクールには、本当に「身分の高い」方々が在籍していたのだろうか」（佐藤 2006 p.67）と問いかけ、第三高等学校と同志社の生徒の出身を華族、士族、平民に分けた明治 33 年のデータと、竹内洋の明治期の高等中学校のデータを比較する。そうすると竹内の示した全国平均の平民率よりも三高の平民率が高く、さらにその三高の平民率より同志社の平民率が高いという。そして当然のことながら華族は学習院に集中しているという。したがって事実としては、やんごとなき方々とキリスト教主義学校生徒とは数的には相関しない。しかしイメージとしては相関する。その二つを結びつけるのはリスペクタビリティ、ハビトゥスであると佐藤はいう。「近代に入って誕生した日本の華族は、率先してリスペクタブルな者となった。彼らの多くが「洋行し」欧米的教養を身につけたことは当然といえる。逆に華族でなくともその「ハビトゥス」を獲得することで、「リスペクタブル」な者となることは可能だった。ミッション・スクールは、その「プラティーク」を提供する場だったのである」（佐藤 2006 p.73)。\n\nまた佐藤は美智子上皇妃と雅子皇后を比較して、美智子妃の結婚時は大ブームになったのに雅子妃のとき比較的盛り上がりに欠けたのは、先述のように立身出世物語が衰退したのにあわせてファム・ファタルの輝きが失われたのと同時に、雅子妃が田園調布雙葉（小学校は編入学、小学校中学校卒業され高校生のときボストン移住）のあと、ハーバード、東大（学士入学）、外務省、オックスフォードと、立身出世の男性倫理を体現しているからであろうと述べている（佐藤 2006 pp.188-189）。そのことも、この「どちらともいえない」の多さに表われているのかも知れない。\n\nこの「どちらともいえない」の問題はそれを問う選択肢を用意する枝問を設けるであろう、今後の調査に詳細は委ねるとして、上述のように佐藤 (2006 p.67, pp.182-185) でも井上ほか (2018) でも女性皇族のキリスト教主義学校への通学が、キリスト教主義学校のイメージ形成に強く介在している点を指摘しているので、この Q11 を Q12 につぐ本研究の分析の軸になる変数と考え、次にこれを基にした組変数について記しておく。\n\nこの Q11 の選択肢 1，2 で 1 グループとし、選択肢 3 はそのままで、それに選択肢 4 と 5 で 1 グループとする、3 つのカテゴリーに纏めた Q11G を作ることにする。\n\nさて、ここで Q11 による分析の軸になる変数 Q11G の説明を終えたので、ようやく、また本項のテーマである Q29 の方に戻る。この Q29 の MA での選択肢のうち多く選ばれたものから順に、設問にその選択肢を配した趣旨を述べ、必要に応じてキリスト教主義学校当事者指数を示す Q12G 及び、女性皇族のキリスト教主義学校の関与の是非を示す Q11G、それぞれとのクロス集計結果をみていく。\n\nQ29 の単純集計はキリスト教主義学校のイメージをキリスト教主義学校当事者・非当事者あわせた本調査の回答者全体がどのように考えているかを示すものであるのに対して、Q12 のキリスト教主義学校関与度指数とクロス集計することで、特に当事者がそれをどのように捉えているかが分かる。当事者以外によって抱かれたイメージはイマジネール（想像上の>イメージ）という言葉に通じる、「想像されたもの」という意味でのイメージであるのに対して、キリスト教主義学校の通学者・通学経験者のいだくイメージは直接経験の「記憶」「記憶に残った像」という意味でのイメージであるといえる。他方、通学者・通学経験者以外の緩やかな当事者は、「期待」、「印象」レベルのイメージであるので、「イメージ」という言葉の広がりが、クロス集計の結果に反映されるといえる。\n\nそこで Q29 の各選択肢の検討の方、まずは先ほどみた選択肢 1 の方に戻るが、いまみた女性皇族のキリスト教学校への関与の是非についてグループ化した Q11G を独立変数として、この選択肢 1「外国語が得意である」を選んだ人と選ばなかった人を 1，0 にして従属変数としてクロス集計した結果は、以下のようになる（表3-2-4）。\n\n女性皇族のキリスト教主義学校への関与を Q11G で「望ましい」と考える者は、Q29 の選択肢 1 の「外国語が得意である」を選ぶ者が 32.0%、「どちらともいえない」と考える人で「外国語が得意である」を選ぶ者は 19.4%、「望ましくない」を選ぶ人では「外国語が得意である」を選ぶ者は 18.2% であった（カイ二乗<0.01）。つまり女性皇族のキリスト教主義学校への関与を「望ましい」と考える者ほど、キリスト教主義学校の生徒・学生のイメージについて「外国語が得意である」を選ぶ傾向にある。美智子上皇妃や雅子皇后がキリスト教主義学校出身で、また雅子皇后はハーバード大と外務省出身でもあり、国民からの皇室外交への期待も高いことと、この結果は関連して理解できよう。\n\n次にこの選択肢 1 と Q12G をクロス集計すると、以下の表3-2-5 のようになる。カイ二乗<0.01 であるが、実際のキリスト教主義学校の通学者よりも、緩やかな当事者の方がこの選択肢を多く選ぶ傾向にある。これは外国語が得意であるということが、イメージ先行になっている部分があることを示しているといえよう。Q12 そのものとクロス集計した結果は表3-2-6（カイ二乗<0.05）のようになっている。特にこの緩やかな当事者のなかのどのカテゴリーがこれを選ぶということはなく、どれも通学経験者よりは多く選んでいる。これはイメージ先行ということもあるが、実際語学に堪能な人は多くても自分はそうではないという場合もあり、そういう場合、実際に通っていても、この選択肢に〇をつけにくいという事情もあると考えられる。\n\nQ29 の MA で次に多かったのは選択肢 3 の「教養が豊かである」であり、MA ベースで 20.1% である。キリスト教主義学校はヨーロッパ文化の根底にあるキリスト教を教育の基盤理念とした学校であるだけに、教養への期待が高いことは納得がゆく。ヨーロッパの名門大学の多くが、修道院の大聖堂の付属施設を源流として発展し、神学部、法学部等の専門課程進学の前提としてリベラルアーツ、自由七学芸を学ぶことを義務づけてきた歴史を踏まえると、これは順当である。\n\nこの選択肢 3 を選んだ人と選ばなかった人を 1，0 にして従属変数とし、Q12G のキリスト教主義学校関与度を独立変数としてクロス集計すると表3-2-7 のようになる。キリスト教主義学校に通学経験のある者は「教養が豊かである」を選ぶ者が 31.5%、通学経験はないが、キリスト教主義学校に当人ないしは家族がかかわりある者で「教養が豊かである」を選ぶ者は 23.2%、かかわりのない人では「教養が豊かである」を選ぶ者は 16.0% であった（カイ二乗<0.01）。キリスト教主義学校にかかわりの高い者ほど、キリスト教主義学校出身者を「教養が豊かである」と自認している、といえよう。ここで「自認」と書いたが、周囲の友人に対する認識も含めた広い意味での自己認識、「われわれ」認識といえよう。佐藤 (2006 pp.147—149) はキリスト教主義学校のスクールカラーを端的に示すものとして「リベラル・アーツ」があるとし、リベラル・アーツを標榜し良妻賢母教育を批判した新渡戸稲造の講演を紹介するが18、そういうリベラル・アーツの伝統が現在も引き継がれているといえるであろう。\n\nまた女性皇族のキリスト教学校への関与の是非について、Q11G を独立変数として、Q29 の選択肢3を選んだ人と選ばなかった人を 1，0 にして従属変数としてクロス集計した結果は、表3-2-8のようになる。\n\n女性皇族のキリスト教主義学校への関与を「望ましい」と考える者は、「教養が豊かである」を選ぶ者が31.3%、「どちらともいえない」と考える人で「教養が豊かである」を選ぶ者は 17.0%、「望ましくない」を選ぶ人では「教養が豊かである」を選ぶ者は 18.2% であった（カイ二乗<0.01）。選択肢 1「外国語が得意である」同様、選択肢 3 についても、キリスト教主義学校出身の女性皇族への期待する人が、外国語や教養などの皇室外交と結びつく能力がキリスト教主義学校の生徒・学生にはあると捉えているといえよう。\n\nQ29 の MA ベースでつぎに多い（実質 3 番）のは選択肢 5 の「品がよい」で 16.3% である。先述の井上ほか (2018) においても記されていたように、キリスト教主義学校が情操教育をセールスポイントとしているので、この問いの答えが比較的多いのはその点で納得がゆくが、他方、後述する選択肢 4 の「他人を思いやる心が豊かである」の方こそが、井上ほか (2018) でいわれた先述の奉仕の精神に通じ、キリスト教主義学校の情操教育の成果として第一に妥当すべきであるが、そちらは 11.6% で 29 選択肢中 7 番目（実質 6 番目）と、高い順位ではない。この選択肢 4 の「他人を思いやる心が豊かである」という選択肢とここの「品がよい」はともに情操教育の成果ではあり、相互に密接に関連はするが、日本語の語感として「品が良い」はより外側に滲み出るもの、「他人を思いやる心が豊かである」はより内面的なものといえる。本調査では、キリスト教主義学校在校生卒業生の階層再生産戦略19について知ろうとしていて（その面のみに関していうと本研究の調査結果はあまり成功していない可能性があり、そこは別稿で論じるが）、階層再生産戦略に結びつくのは「品の良さ」の方であるともいえる。この選択肢 5 と Q11G をクロス集計したが有意差はなかった（表3-2-9）。\n\n一方 Q12G とクロス集計するとカイ二乗<0.01 で有意差があった（表3-2-10）。キリスト教主義学校関与度の高い者ほど、「品が良い」を選択する傾向にある。\n\nQ29 の MA ベースでつぎに多い（実質 4 番）のは選択肢 11「裕福である」で 14.7% である。1.2.3 で先述のように、井上ほか (2018) の先行研究ではキリスト教主義大学の女子がキリスト、きれい、金持ちの 3K であるといわれている点を強調するが、この本の第1章で論証もなくいわれるこの話のうち、きれいはひとまず措いて金持ちについては世間一般のイメージにおいても妥当することが、ここからうかがわれる。これについては井上ほか (2018) 同様、佐藤 (2006) も「京童が口にする三 K「かわいい、金持ち、キリスト教」あるいは「きれい、かしこい、金持ち」の、いずれのヴァージョンにも登場する「金持ち」という要素だ」（佐藤 2006 p.74）と記す。これについても佐藤は授業料でキリスト教主義学校が他より高いことはなく、最初期に進出した私立学校の多くがキリスト教主義学校であったことに金持ちイメージの理由があり、さらには「「羨望」の対象であった欧米文化あるいは都市文化というミッション・スクールの「徴」が、直接経済スケールに反映するものとして語られてきたという側面も無視できない」（佐藤 2006 p.80）と分析している。\n\nなおこれと Q11 の女性皇族のキリスト教主義学校の是非の問い及び Q12 のキリスト教主義学校関与度とクロス集計したが、いずれも有意差はなかった。\n\n我々のインタビュー調査で小中高とプロテスタントの女子校（小学校の際は若干男子生徒もいた）に通い公立大学に通っている（当時）B さんは、次のように語っている（調査日 2019 年 1 月 22 日）。なお、B さんの調査に先立ち筑波大学図書館情報メディア系倫理審査委員会より「科研費挑戦的萌芽研究「キリスト教主義学校から見る寛容と洋化―ステークホルダーらの期待と文化資本」（2018~2019年度（代表後藤嘉宏））初年次ウェブモニター調査のためのプレ調査」（通知番号第 30-84 号）の承認を得ている（以下、B さんの発言の引用の際は、この倫理審査についての記述を重複を避けるため控える）。\n\n後藤:キリスト教主義学校に行っていることで外の人の目が、例えばお母さまが職場の同僚に「うちの娘○○（学校名）で」って言ったときに「なかなかハイカラね」とか、そういう目で見られたという感じはどうですか?\n\nB さん:例えば、ここの大学入ったときに、自分の学校を説明する上で、女子高で私立でキリスト教というと、かなりお嬢様みたいなイメージを持たれたりするんですけど。でも私自身全くそんな意識はないし、何ならキリスト教、みんなが学んでいないことを学んでるという特別感を私的には感じたりしていたので。\n\n後藤:お嬢様という感じは公立大入ったときの周囲の目で……\n\nB さん:結構あったと思います。\n\n要は現実よりもイメージ先行ということになる。\n\nQ29 の MA ベースでつぎに多い（実質 5 番）選択肢は 8 の「国際的な問題への関心が高い」で 13.7% である。これは「該当なし・答えたくない」を除き実質 1 位の、先にみた「外国語が得意である」とも関連する選択肢である。キリスト教主義学校はキリスト教という西洋文化の根底を理解させることで、外国語と国際感覚を学ぶ場を提供しているという趣旨でこの設問を設けた。ただしこれを Q12G とクロス集計したが、有意差はなかった。キリスト教主義学校当事者において「国際的な問題への関心が高い」という自己認識は非当事者よりも多くも少なくもない。さらに Q11G ともクロス集計したが、これも有意差はなかった。女性皇族のキリスト教主義学校通学歴を是とする人は皇室外交への期待も大きいと予想し、それに関連する選択肢 1 の「外国語が得意である」については有意差がありそのことは裏づけられたが、それとともにその選択肢1と強く関連する「国際的な問題への関心が高い」の方については有意差がなかった。こちらは予想外の結果である。ただし皇室外交は憲法学的制約から政治的発言が制約されていて、そのことに鑑みると、皇室外交に求められるのは問題解決能力よりはコミュニケーション力である。その意味では、「国際的な問題への関心が高い」については有意差がなく「外国語が得意である」については有意差があることは制度上の面からすると一応納得がゆく。\n\n1.2.2 で先述のように井上ほか (2018) で、女子アナにキリスト教主義学校出身率が高いのは、愛と奉仕の精神を育むとそれらの学校のウェブページに記載されていることに照応する、と書かれている。しかしウェブで謳われていることと実際は必ずしも一致しないし、教育目標通りの効果もあるとは限らないが、井上ほか (2018) は調べていない。したがって Q29 の選択肢 4 として「他人を思いやる心が豊かである」を入れた。\n\n4 段落前に先述したようにこの選択肢 4 は MA ベースでは全体で 11.6% で 29 選択肢中実質 6 番目で、高い順位ともいえない。しかしキリスト教主義学校関与度指数を示す Q12G とクロス集計するとカイ二乗<0.01で有意差が出た（表3-2-11）。\n\nしかしこれには我々の事前の予想とは違う部分があった。キリスト教主義学校に全く関与しない者がこの答えを選ばない傾向にあることは予想の通りで、そのカテゴリーでこれを選んだ者は 8.5% であった。他方、キリスト教主義学校に実際に通った・通っている者でこの選択肢を選んだのは 14.8% であり、その他の関与者の17.3%よりもやや低い数値であった。\n\nそこで Q12 をグループ化せずに集計したところ表3-2-12 のようになり、各セルの実数は少ないので有意差は出にくいものの、カイ二乗<0.05で有意差を確認できた。これを見ると、キリスト教主義学校に「通ったことはないが、受験したい学校がある」者のみが、「他人を思いやる心が豊かである」を選択していることが分かる。これは「他人を思いやる心」を涵養させるだろうという受験前の期待が、キリスト教主義学校の生徒イメージとして数字に現れている面があると考察・想定される。他方、実際に通学してみると、キリスト教主義学校に入学する課程の、前の課程の学校のクラスメイトとさほど変わらなかったとか、あるいは自分自身をふり返ってみて他人を思いやっていないと考えると自省するとか等の諸事情が想定され、それは家族に通学者がいる者についても同様と推察される。\n\n内村鑑三はキリスト教主義国家のアメリカに行ってみて同行した友人が詐欺に遭い、キリスト教徒にも心のきれいでない人がいることに驚嘆したという話を『余は如何にして基督信徒となりし乎』で述べていたし（内村 1958 p.112）、内村の非戦論も自分にキリスト教を教えてくれたアメリカが参戦したことの衝撃が大きいとされる（若松 2018 pp.167-168）が、それと似た状況があるとも考えられるが、このあたりについてはキリスト教主義学校の学生や卒業生に今後質的調査をすることで確認をしていきたい。\n\nなお、この選択肢 4 と Q11G とクロス集計するとカイ二乗<0.01 で有意差が出た（表3-2-13）。\n\n皇室女子とキリスト教主義学校とを結びつける者ほど、キリスト教主義学校での思いやり教育に期待していることが分かる。これは本稿 1.2.1 で述べた、先行研究の④「皇族女子のキリスト教主義学校入学が多いのも、愛と奉仕の精神を育むとの期待があるからである」を、一般の人びとの意識の側から裏づける結果であるといえる。\n\nなお、ここまでのクロス集計結果は通学経験の有無よりは思いやりがイメージ先行であるというもので、それとはある意味、矛盾もしうるが、幼稚園から大学院修士課程まで一貫して同じ系列のキリスト教主義学校に通っていた A さんを我々はインタビュー調査したが、そこでは情操教育とキャリア形成について直接結びつけるような、次のような発言が得られている（調査日 2019 年 5 月 31 日）。\n\n「情操教育というところで、まず、○○学院の校訓の「人になれ、奉仕せよ」がひとつ、大きなものであって。小学校のときにも教訓というか、礼拝のときに毎朝暗唱するものがありまして、「自分に厳しく人に優しく」というのと「強く鍛える体と心」とか。あと「艱難に耐え、常に祈りなさい」っていう、そういうものを礼拝の前にみんなで言うところがありまして。ちょっとその「人になれ、奉仕せよ」も先生方が口々におっしゃるんですけど、私一回、結局どういう意味なんですかね、って小学校の時に訊いたら、先生たちも自分なりの考えはあるけど、正解はないんだということで、人生の中で見つけていってごらん、ということで、それを頭の中に置きながら普段生活しているところがあるんですけども。\n\nそれで、キャリアとかを考えた時に、私の大学の学部選びでも大きく関与しているところが、実はありまして、高校生の時に火事現場に遭遇したことがありまして、家におばあさんがいたんですけど、そこで入って助けたことがありまして、消防署で表彰をもらったときに、私の中での行動原理が、ついつい手を出してしまうみたいなところがありまして、それもあって、何かしらの人の助けとか役に立ちたいなというところから福祉の道を選びたいということで、大学選びで福祉を専攻したというところが私の中で大きなターニングポイントで、自分の中のキャリア形成じゃないですけれども、大きく土台としてあるものなんじゃないかと。道徳とか情操教育みたいなところを考えると、の中では「人になれ、奉仕せよ」とかキリスト教の校訓みたいなところはみんなの中に、何年間もいる中で根底に根付いてくるものなんじゃないかと」（は録音音声不明瞭な箇所）。\n\nこの発言の「キリスト教の校訓みたいなところはみんなの中に、何年間もいる中で根底に根付いてくるものなんじゃないかと」いうことからも、附属・系列校を通じて長くその学院・学園に在籍することで、思いやりの心、奉仕の精神は培われるものと考えられる。他方、ほとんどすべての法人で定員的には大学のみが突出して多いので、附属・系列校からいる人に比して相対的に大学の四年間は短期間ゆえ、大学生ではこれらが定着しない人の方が比率的には多いことも、この発言は暗に示しうる。また小学校から高校まで同じ法人のプロテスタントの学校に通い、大学で国公立大学の看護学科に進学した B さんに「聖書教育を受けてボランティアへの意識が高まったなどの、なんらかの影響はありましたか?」と聞いたところ、「小学校のときから、たとえばなしでキリストがゆるすお話を教えられてきたので、人に優しくあろうという感じの観念は小学校からみんなに対して伝えられてたのかなあと思います」と答えている（調査日 2019 年 1 月 22 日）。\n\nQ29 の選択肢で MA ベースでつぎの 7 番目だったのは、選択肢 7 の「社会的活動への関心」で 9.6% である。これも選択肢 8 の「国際的な問題への関心が高い」同様、Q12G 並びに Q11G とクロス集計したが、いずれも有意差はなかった。宗派問わず宗教人の多くがボランティア活動に熱心で、特にキリスト教はボランティア活動に積極的である印象が強いので、この選択肢自体がそこそこ選ばれたことは納得がゆくが、その反面、キリスト教主義学校当事者たちから特にこの選択肢が多く選ばれている訳ではないことからすると、このことはイメージ先行である可能性があるし、また「社会的活動」というワーディングが政治経済活動全般に及ぶ可能性があり、社会奉仕と必ずしも回答者の認識で直結しなかった可能性も、否めない。\n\nQ29 の選択肢で MA ベースでつぎの8番目だったのは、選択肢 2 の「話し上手である」で 8.6% である。コミュニケーション力の高さという点で、選択肢 3 の「教養が豊かである」とも結びつく内容といえる。\n\nこの選択肢と Q11G をクロス集計すると表3-2-14 のようになる。\n\nカイ二乗<0.01 ではあるが、女性皇族のキリスト教主義学校出身者がいることを「望ましい」と思う人たちと「望ましくない」と思う人たちにこの「話し上手である」という選択肢を選ぶ傾向にあり、「どちらともいえない」という多数派の人たちはこの選択肢を選ばない傾向にある。「望ましい」と思う人たちがこの選択肢を多く選ぶことは、コミュニケーション力という点からの皇室外交への期待という面からも容易に想像できることであり、ある意味予想どおりであったが、「望ましくない」と思う人にもほぼ同程度これを選ぶ者がいることは意外であったが、この理由については今後つめていきたい。\n\n次にこれと Q12G をクロス集計した結果は表3-2-15 の通りだが、これもカイ二乗<0.01 である。\n\n当事者（通ったことがある者）よりは緩やかな当事者の方がこの「話し上手である」を選ぶ傾向があり、当事者は非当事者よりは多いが、さほどそれとの差は大きくはない。\n\nよって度数が少ないので検定はしていないが、Q12 をグルーピングしない形でのクロス集計も示しておく（表3-2-16）。\n\n「通ったことがある」者以上に「上記いずれにも該当しない」者に近いのは、「通ったことはないが、家族に通ったものがいる」者である。要は当人や家族が通った者よりは、そうではなく受験しただけや受験したいだけ行ってみたいだけの者の方が、話し上手であるというイメージをもっている。現実よりもイメージ先行ということであろう。井上ほか (2018) の、女子アナにキリスト教主義学校出身が多いという記述があることは、1.2.2 で既出であるが、そういうイメージが共有されていれば「話し上手である」になるが、アナウンサーになれるのはどこの大学であろうとほんの一握りの学生であって、実際に通った当事者にとっては現実とは多少違うと感じられても不思議ではない。つまりアナウンサーになった人の集合で考えると、井上ほか (2018) でいうように、女子アナについてはキリスト教主義学校出身が多いとして、そのイメージは一般の人びとから共有されていることが、この結果からも分かる。他方、キリスト教主義学校の生徒・学生からすると、そのなかでアナウンサーになれるのはほんの一部で、周囲ではそのような人は見たことないという可能性も高い。\n\nQ29 の選択肢で MA ベースでつぎの 9 番目だったのは、選択肢 24 の「教えを忠実に信じる」で 8.1% である。ただしこの Q29 の選択肢 24 については「教えを忠実に信じる」ということで、信仰心が篤いとも受け取れられるし、批判力の欠如や盲信、ある意味洗脳されているというようなニュアンスでも受け取られ、ややワーディングが曖昧であった。そのことが 18 選択肢中、9 番目という多いとも少ないともいえない順位に表われているのかもしれない。これは Q12G とクロス集計してみたが、有意差はなかった。\n\nつぎに Q29 の選択肢で MA ベースでの単純集計でも回答者の少なかったものについて論じていく。\n\n1.2.3 で先述のように、佐藤 (2006 p.74) や井上ほか (2018) はキリスト教、きれい、金持ちという 3K のレッテルがキリスト教主義学校女子に張られているという。そのうちキリスト教は、キリスト教主義学校という言葉の反復になるので措くとする。金持ちについてはすでに選択肢 11 の「裕福である」のところで述べた通りである。他方、きれいについては選択肢 13 で「容姿端麗である（みめうるわしい）」を用意した。しかしこれは MA で 2.0%、SA で 0.3% しか選ぶ者がいなかった。MA ベースで 18 番目という低い順位である。またキリスト教主義学校当事者指数をグループ化した Q12G とも、女性皇族のキリスト教主義学校通学の是非を示す Q11G ともクロス集計してみたが、いずれも有意差はなかった。「容姿端麗である」という日本語がやや難しいので「みめうるわしい」という補足説明を括弧書きでつけたが、いずれも難しいということもあったかもしれない。また先述のように Q29 はキリスト教主義学校の男子も女子も含めたイメージを聞いているということの影響も大きいと思われる。しかし井上ほか (2018) にしても佐藤 (2006) にしてもキリスト教主義学校に男子学生が相当数いることは重々承知の上で、女性バイアスをあえて自分らの研究にかけてキリスト教主義学校を眺めているのであるから、そもそも当人の意識にバイアスのある回答者にとっては、男女合わせた聞き方を今回のようにあえてしてみても著しく結果が変わる訳ではないことも想定される。この数値は以上のワーディングの問題や女子に限定しない聞き方という問題を措いても極めて低い数値であるといえる。したがって佐藤 (2006)、井上ほか (2018) にある女性バイアス、キリスト教主義学校女子が「きれい」だという固定観念が、少なくとも本研究の調査対象とする若い世代では全くないことが分かった20といえよう。もっとも今回調査対象者を東京・神奈川に絞ったが、井上ほか (2018) も佐藤 (2006) も京都の事例を基にこの 3K について取り上げているので、関西の府県に調査対象者を求めたら、同じ世代であっても結果は大いに変わった可能性のあることは否めない。\n\n先ほど言及した、佐藤 (2006) のいう、立身出世のモチベーションがなくなったため、ファム・ファタルが欲望の対象でなくなったとの発言も、女性バイアスがほぼ過去のものになったことを示している。佐藤の著書は 2006 年ではあるが、佐藤の著書の述べた対象そのものは概ね 1980 年代の事象であり、そこから30 年以上経ていて、ファム・ファタルに通じる女性の容姿についての意識はそのとき以上に弱まっており、なおかつ今回の調査票のこの設問が男女問わないイメージを聞いたものであるので、なおのこと容姿についての選択肢が選ばれなかったと考えられる。\n\n他に Q29 の選択肢で MA ベースでも回答者の少なかったという点で特記すべきものとしては、選択肢 23 の「論理の整合性を尊ぶ」で、16 番目で MA ベースで 2.4% しか選ばれていない。これは丸山真男の『日本の思想』でキリスト教とマルクス主義が首尾一貫性を重んじる思想であるがゆえに、無限抱擁的に内外の思想宗教を受け入れてきた日本の固有の文化となじまずに、日本ではこれらキリスト教徒やマルクス主義者が知識人層以外に増えなかったという指摘を意識して入れた選択肢である。\n\nなお、この選択肢を選んだ者と Q12G をクロス集計したが有意差はなかった。実際この回答が少なかったのは、キリスト教の一神教としての首尾一貫性という丸山その他から従来いわれてきた通説を人びとが意識しない結果なのか、あるいはクリスマスやキリスト教式結婚式と類似した習俗のみの次元でキリスト教主義学校の生徒・学生を捉えた結果なのか、この問いだけからは分からない。しかしそもそもこの選択肢を含め多くの選択肢は、この調査における Q29 と類似した別の設問（次段落以降に記すように、Q26 は日本のキリスト教徒について同じ選択肢で聞き、Q27 は日本のキリスト教会の聖職者について同じ選択肢で聞いている）の選択肢と合わせて用意されている。キリスト教主義学校の生徒・学生とキリスト教徒とキリスト教聖職者、これら三者それぞれは関連するが別のものとして考える必要があるからである。\n\nそこでいまふれた Q26 であるが、これは「あなたは日本のキリスト教徒についてどのようなイメージを持っていますか。当てはまるものを上位 3 つまでの間で選び、中でも最もイメージしたものを 1 つ選んでください」という設問で、Q29 と同じ選択肢を配している。\n\nしかしここでも選択肢 23 の「論理の整合性を尊ぶ」は MA ベースで 1.4% しか選ばれていない。生徒・学生に対するイメージの Q29 以上に低い数値である。\n\nまた Q27 は「あなたは日本のキリスト教会の聖職者についてどのようなイメージを持っていますか。当てはまるものを上位3つまでの間で選び、中でも最もイメージしたものを 1 つ選んでください」という設問であるが、ここでも Q29 と同一の選択肢を配している。この設問においても選択肢 23 の「論理の整合性を尊ぶ」は MA ベースで 1.9% しか選ばれていない。\n\nキリスト教主義学校の生徒についてのイメージのみであれば、クリスマスやキリスト教式結婚式と同一の習俗、表層の次元で、キリスト教主義学校が捉えられうるから低い数値であるという説明も可能であろうが（リスペクタビリティの説明やトリプルな信仰についての箇所で先に申し上げたように、表層的であることが、精神性や宗教性に必ずしも通じない訳でもないが）、クリスチャンや聖職者のイメージについてもほぼ同様の数値しか得られていないことからすると、キリスト教というもの自体が、現代の若年層からは、そういう丸山のいうような、論理の首尾一貫性とは切り離されて捉えられている可能性が示唆される。\n\n3.3 キリスト教式結婚式の是非及びクリスマスの過ごし方と、キリスト教主義学校関与度指数\n\n井上ほか (2018) はクリスマスやキリスト教式結婚式の隆盛と、キリスト教主義学校の人気とを相同性のあるものとして関連づけるし、濱田の歴史研究でもキリスト教式結婚式とキリスト教主義学校の関連性は示されている。そこでまず本研究でキリスト教式結婚式について尋ねた設問についてみてみる。\n\nQ22. で「キリスト教徒ではない日本人のカップルが、教会で結婚式を挙げることについて、あなたはどのように思いますか」と SA で聞いて、表3-3-1 のような結果を得ている。\n\n「どちらともいえない」が 58.7% で最も多いが、「好ましい」が 36.0% で「好ましくない」の 5.3% よりかなり多い。全体として判断に迷う人が多いが、それにもかかわらず、肯定的な判断の方に傾いているといえる。なお、『「現代日本における宗教教育の実証的研究」(1998~1999) 報告書』では、1997 年度の調査から 1999 年度まで「次の事柄について、あなたの同意できる意見にすべて〇をしてください」という問いの 3. として「クリスチャンでない人が、キリスト教会で結婚式をあげるのはおかしい」という項目を設けているが、この結果について同報告書では日韓比較の箇所で一部ふれられるのみである。なおこの「現代日本における宗教教育の実証的研究」のこの設問では 1. そう思う　2. どちらかといえばそう思う　3. どちらかといえばそう思わない　4. そう思わない　の4択であるが、ここの報告での記述は、「そう思う」「そう思わない」の数値のみ示して以下の文章のように日韓比較をしている。「「そう思う」(+ +) は、日本は 8.2%、韓国は 16.6% で、やはりクリスチャン人口の多い韓国の方が習俗としてのキリスト教式結婚式に批判的なようだが、「そうは思わない」(- -)を見てみると、日本は 48.6% 、韓国は 45.2% で、それほど大きな差はなく、韓国においても、結婚式がイベント化している様子がうかがえよう」（井上ほか 2000 p.125）。\n\n本研究の Q22 を Q12G とクロス集計すると表3-3-2 のようになる。\n\nキリスト教主義学校の当事者ほど、キリスト教式結婚式に肯定的であることが、この表から分かる（カイ二乗<0.01）。\n\nこのことからもキリスト教主義学校とキリスト教式結婚式との相同性をいう井上ほか (2018) の指摘の妥当性が裏づけられる。\n\nまたこの結果は先述の濱田 (2001)とも照合する。なお『「現代日本における宗教教育の実証的研究」(1998~1999) 報告書』ではこの設問について高校や大学の宗派別の分析を載せていないので、単純には比較できないが、先に引いたように「「そう思う」(++) は、日本は 8.2%、韓国は 16.6% で、やはりクリスチャン人口の多い韓国の方が習俗としてのキリスト教式結婚式に批判的なようだ」とキリスト教徒がキリスト教式結婚式に批判的であるとの見立てを示しているが、今回の調査結果はむしろキリスト教主義学校とキリスト教式結婚式との親和性が示唆されている。\n\n一方、我々のインタビューにおいて中高大と同志社に通った C さんは、そもそも結婚式に興味があまりないし、本物を見ているだけにバイトによるキリスト教式結婚式には気が引けることを次のように語っている（調査日 2020 年 1 月 28 日）。\n\n後藤:教会で結婚式挙げるのが好ましいというのは、本人としてはどう?一般論でこれ聞いて。\n\nC さん:嫌ですよねー。でも、というのは、結婚式に出たことがあって、身内のやつとか。チャペルでやると、あれじゃないですか、バイトの人が読むじゃないですか。ちょっとさすがにあれは、礼拝とか受けてると抵抗ありますよね。\n\n後藤:本物がやるのもあるわけで、本物がやる場合はどう?\n\nC さん:それはいいんじゃないですかね。一応同志社の卒業生で、大学のチャペルで挙げられてましたよ。\n\n後藤:同志社のチャペルで結婚式挙げるのは?\n\nC さん:それならいいんじゃないですかね。\n\n後藤:したいかしたくないかでいうと?\n\nC さん:結婚式自体をやる気がないです。\n\n千:今の若者だ。\n\n後藤:そっか、そうだね。確かにね。\n\n（中略）\n\nC さん:まあ莫大なお金があればやってもいいかもしれないですけど。\n\n（中略）\n\n片山:白無垢よりは洋装?\n\nC さん:はい、神式にはもっと興味ないです。\n\n後藤:ああ、神式にはもっと興味ない。仏式は?\n\nC さん:仏式ってあるんですか?　聞いたことないですね。\n\nこの C さんの発言はキリスト教主義学校の出身者であっても、ストレートにキリスト教主義の結婚式を自分のこととしてはあまり積極的には捉えないということを示唆する。しかもCさんは同志社のキリスト教主義教育は 3.1 にて先述のように「徳育としてのキリスト教」であると語り、キリスト教への信仰心は全くないという人である。その意味では濱田 (2001) や井上ほか (2018) のキリスト教主義学校とキリスト教式結婚式をストレートに結びつける見方と逆の側面ともいえるし、本研究の Q22 と Q12G をクロス集計した分析結果への留保ともいえる。ただし本研究の Q22 は「キリスト教徒ではない日本人のカップルが、教会で結婚式を挙げることについて、あなたはどのように思いますか」という形で、一般論で聞いているので、身内の結婚式に言及したこのインタビューとは次元が少し違う点にも留意が必要であろう。ただこのインタビューでの発言は、井上ほか (2000) での日韓比較でのクリスチャン比率の高い韓国の人たちに対する分析のような方向性というか感覚も、日本のキリスト教主義学校当事者の側がもちうるということを示しているといえるであろう。\n\nつぎにクリスマスの過ごし方についてであるが、Q4 で「あなたが生まれ育ったご家族（原家族）あるいはそれに相当する環境では、クリスマスに何をしていましたか。以下の中から、1度でも行ったものを全て選んでください」という設問をした。単純集計の結果は表3-3-3 の通りである。なお今回の調査のクリスマスについての設問は比較的一般の質問文であったので、必ずしもクロス集計に向かないと考えられるし、キリスト教主義学校当事者指数と相関しそうな選択肢は軒並み少なくしか選ばれなかったが、度数が少ないながらもある程度有意差があるものもあったので、本稿では一応それらのクロス集計結果を示す。\n\n選択肢 5「クリスマスに聖書をみんなで読んでいましたか」については「読んだ人」「読まなかった人」で 1、0 で集計すると表3-3-4のようになる。緩やかな当事者に「聖書をみんなで読む」人が多いが、そもそも実数が少ないが、カイ二乗<0.05 で有意差はあった。\n\n選択肢 7「クリスマスにお祈りをしましたか」について同様の作業をした（表3-3-5）。これも実数が少ないもののキリスト教主義学校当事者指数が高いほど、クリスマスにお祈りをしている。これもカイ二乗<0.05 で有意差はある。\n\n選択肢 9「クリスマスに聖歌や賛美歌を歌いますか」についても表3-3-6 のように同様である。これはカイ二乗<0.01 で有意差がある。\n\n我々の質的調査において幼稚園から大学院修士課程まで一貫して同じ系列のキリスト教主義学校に在籍していたAさんは（インタビュー時、修士 2 年）、仏教徒の家庭に生まれ、キリスト教に入信はしていないが、賛美歌のメロディや歌詞からキリスト教への憧憬を強めたと語っている。「キリスト教になじみがあったのかなというところでは。それで他大学行って、中高で聖書・讃美歌やって「もういいや」ってなって他大学、全然宗教関係ない大学行った人たちも、やっぱり会うと、ちょっと礼拝が恋しくなるみたいなところは聞いていて。そういうアイデンティティじゃないですけど、帰属意識みたいなのはあるかな、と。未だに大学別のところ行っちゃった人、高校のとか、会うと聖句でこれ覚えてるとか言ってみんなで暗唱したりして。・・・親友の人たちは特に、好きな讃美歌は何番かとかで盛り上がってる感じで」（は音声不鮮明で録音の聴き取り不能箇所。多分「同窓会」。・・・は中略箇所）（調査日 2019 年 5 月 31 日）。一部の在籍生はクリスマスに限定されない日常生活の中に「聖歌や賛美歌を歌う」行動は定着していることが、この結果に示されているのかも知れない。\n\n選択肢 12 の「クリスマスに教会に行きますか」については今まで見てきた選択肢の中でも特にキリスト教主義学校通学者に著しいという形になっている（表3-3-7）。この p 値は 0.00031 なので、ほぼ確実にいえることであるといえる。ただ Q4 の質問文は「あなたが生まれ育ったご家族（原家族）あるいはそれに相当する環境では・・・」となってはいるものの、「以下の中から、1 度でも行ったものを全て選んでください」となっていることに引きつられて、キリスト教主義学校に通学していた時代に、学校行事として行ったものにも〇をつけた可能性のあることは否定できない。またクリスマスに限らず教会に行くことを在学生に強く推奨する場合もあることは、我々もインタビューを通じて、聴いてもいる。\n\n例えば小中高とプロテスタントの学校に通っていた大学生 B さんは、小学校の進学理由はキリスト教というより、家から近い私立という理由で受験したという。しかし毎週、小学校5年までは教会に通うようになったという（は音声不鮮明で録音の聴き取り不能箇所）（調査日 2019 年 1 月 22 日）。\n\nB さん:私一応小学校の頃は、学校に勧められて、小学校の先生になんとなく言われて日曜日に教会に行ってたんで、それも全然、一緒に送迎してくれたり。\n\n後藤:それは小学校の時は66人中何人ぐらい行ってたの?\n\nB さん:たぶん 30 人ぐらいは通ってたと思います。私も結構長い間、たぶん小5くらいまでは毎週日曜日行ってたと思うんですけど。\n\n後藤:小5まで?小 6 からはなんで通わなくなったの?\n\nB さん:一応、若干、内部とはいえ受験だよねという雰囲気になって。\n\n（中略）\n\n片山:どういう理由で先生には言われていたんですか?\n\nB さん:日曜……は教会にいくものとして教えられていて。なんでだろう。\n\n千:かといって洗脳ちっくな感じでは?\n\nB さん:ないです。全然ないです。なんだかんだで幼稚園から持ち上がりの子とかいたので。その子たちは元から日曜に教会に行く習慣があったりしたんで、その流れで、みたいな感じです。\n\n有意差はありつつ、選んだ人の実数が少ないということ、また質問文をやや勘違いして答えた者もいる可能性のあることを割り引いて考える必要があるが、クリスマスについても、キリスト教主義学校当事者指数の高い者の方が、信仰に結びつく行動をしているということは確認できる。\n\n他方、B さんは教会に行くことについては小学校時代習慣化していたと上述のように語る一方で、クリスマスについては家では極めて地味に過ごしていたという。また学校では 12 月 24 日にクリスマス礼拝があり、その日は授業がなかったという。\n\n千:逆にキリスト教の、例えばクリスマスを盛大にやるとかは?\n\nBさん:全然そういうのないです。普通でした。\n\n千:チキンとケーキ買ってくらいの。別にわけでもない。\n\nBさん:全然普通の、まったくないです。\n\n後藤:じゃあクリスマスは宗教行事でなく年中行事?\n\nBさん:はい、もうほんとに。\n\n後藤:普通日本人は宗教行事として捉える人は少ないけど。でも高校までは学校でクリスマスかイブの礼拝はあったんじゃないですか?\n\nBさん:もちろんありました。アドベントカレンダーも開けて、みたいな。\n\n後藤:それはみんな?\n\nBさん:みんな、はい。クリスマスあたりに「クリスマス礼拝」っていう日があって、礼拝のためだけに行ったりしましたね。\n\n後藤:それは 24 か 25 でない日に設定して?\n\nBさん:たぶん、土日かなんかだったりするとわからないですけど、大体 24 日に。\n\n後藤:じゃあ24日にクリスマス礼拝があると。1日?\n\nBさん:ちょうど冬休みに入る前くらいなので。\n\n後藤:授業なしにして?\n\nBさん:はい。\n\n以上から、井上ほか (2018) のいう、トリプルな信仰の現われとしてキリスト教式結婚式の人気とクリスマスの国民行事化それぞれが、キリスト教主義学校の隆盛に相関するという言説が、本稿の調査からもいえることが分かった。\n\nさらに本研究では「Q3. あなたは宗教に由来する年中行事に、何歳頃まで原家族単位で行っていましたか。当てはまるものを 1 つずつ選んでください。ここでは数年に 1 回程度の頻度でも、行っていたと考えてください」という設問を設け、お盆の先祖のお迎え、除夜の鐘付き、初詣について聞いている。\n\nこのそれぞれについて Q12G とクロス集計する（表3-3-8、表3-3-9、表3-3-10）。\n\nこれは（表3-3-8）カイ二乗<0.01 で有意差がある。キリスト教主義学校に通ったことのある者ほどお盆の迎え火を「今でも原家族と行っている」人が多く、キリスト教主義学校と関わりがない者は「原家族と行った記憶はない」者が多い。これも（表3-3-9）カイ二乗<0.01 で有意差がある。実数は多くはないが、キリスト教主義学校の当事者性が高い者ほど、原家族と除夜の鐘の鐘付きに今でも行っている。\n\nこの初詣についても（表3-3-10）カイ二乗<0.01 で有意差がある。「キリスト教主義学校に通ったことのある」者と「キリスト教主義学校に通ったことはないが、関わったことがある・関りのある人がいる」者の間には差があまりないが、それら何らかの意味でのキリスト教主義学校当事者と、キリスト教主義学校への関わりのない者との間では、差がある。\n\nなお、『「現代日本における宗教教育の実証的研究」(1998~1999) 報告書』では、「あなたは今年の初詣はどうしましたか」という問いに「家族と行った」「家族とは別に行った」「行った家族もいるが自分は行かなかった」「家族の誰も行かなかった」「その他」の選択肢を用意してある。宗教系大学生のみについてであるが、大学の宗教別のクロス集計をしている（井上ほか 2000 p.44）。宗教系大学の合計だと「家族と行った」24.5%、「家族と別に行った」22.4%、「行った家族もいるが自分は行かなかった」17.6%、「家族の誰も行かなかった」23.1% である。カトリック系大学生は「家族と行った」38.3%、「家族とは別に行った」21.7%、「行った家族もいるが自分は行かなかった」13.5%、「家族の誰も行かなかった」21.7% で、宗教系大学の合計の選択肢の 1，2 の合計が 46.9% であるのに対して、カトリック系大学では 60.0% である。プロテスタント系大学ではそれほど宗教系大学の合計と差はなかった。因みに神道系大学では「家族と行った」23.0%、「家族とは別に行った」25.4%、「行った家族もいるが自分は行かなかった」23.3%、「家族の誰も行かなかった」22.3%である。選択肢 1，2 の合計が 48.4% で宗教系大学の合計より少し多いだけで、カトリック系は神道よりも初詣に積極的という結果が出ている。同書の分析では「カトリック系が約 6 割、天理大と仏教系他学科（注記；この調査報告書では仏教系大学について一般学科と仏教学科を分けた集計を行っている）と神道系とプロテスタント系が約 5 割であるのに対して、仏教学科 2 割 5 分、創価大 1 割強とあとの 2 者が比較的割合が少ないことが理解できる」（井上ほか 2000 p.44）と記される。当然、これはキリスト教主義学校のみに焦点を当てた研究ではないので、この分析は妥当ではあるが、カトリック系が神道系を超えているというある意味非常に意外な結果については特にこだわっている印象はない。しかし我々の視点からこの 20 年前の研究を見返すと、「トリプルな信仰」に通じる寛容性の涵養をカトリック系の大学はこの時期既に行っていた可能性があるとも評せて、我々の調査結果に通じる結果がすでに出ていたともいえる。\n\n以上の 3 つの我々の調査による表から、キリスト教主義学校当事者の方が（通学経験者と緩やかな当事者との差はケースバイケースではあるが）、幼少期に原家族とお盆や除夜の鐘や初詣など、キリスト教「以外の」宗教行事に積極的に触れてきていることが分かる。つまり、この結果は排他的なキリスト教像とは逆とも評せるし、またキリスト教主義学校への期待も、キリスト教という特定宗教への期待というよりは宗教教育による情操の涵養への期待があることが想定できる。\n\n例えば幼稚園から大学院修士課程まで一貫して同じプロテスタントの学校法人の学校に通っていた大学院生（インタビュー当時）A さんは次のように語っている（調査日 2019 年 5 月 31 日）。\n\n片山:ご家庭でクリスマスには歌を歌うとか、キリスト教徒としてじゃなくて構わないんですけど、なにか行事としてありましたか?\n\nAさん:クリスマスは大体ケーキを買ってきて、料理も豪華にしたりしていて、やっぱり家族そろってるときはみんなで食べたりしてました。\n\n後藤:家族そろってクリスマスがあったのはいつ頃までですか?低学年のころ?段々ばらばらになってしまうというか、忙しくなってとか、友達と過ごすとかあったと思うんですけど。\n\nAさん:高校頭くらいまではみんなで祝っていたかな、という感じで。\n\n後藤:それはお兄さんも大学頭くらいまで?\n\nAさん:そうですね、大学入った頃くらいで。兄もそのときには結構忙しくなってたみたいで、あんまりもう一緒に祝うってことはなかったんですけど、大体クリスマスじゃないときの日曜日に、振替で何かうまいものでも食べようか、みたいな感じで格好つけてクリスマスっぽいことをしたことはありました。\n\n（中略）\n\nAさん:うちの親父の、○○県の実家がかなり浄土真宗の信仰が強くて、家族全員がお経を唱えられるので、私もそっちの方に行くとみんなと正座しながらお経を唱えて、教本もあって、数珠をつけてお経を唱えるというのが慣例なので。\n\n片山:初詣も?\n\nAさん:初詣も行きます。\n\n後藤:お経を読めるほどに熱心な信者ということ?\n\nAさん:そうですね。祖父がそういう浄土真宗のお寺の方で、住職ではないんですけど、一時期見習いみたいなことをやっていたころから、かなり熱心にやっていたみたいで。うちの父親も高校はそういう宗教の学校ということで、仏教というか、そっちの方で学んでたということで。\n\n後藤:仏教?真宗でない仏教系の学校?\n\nAさん:はい、学校で、高校は宗教を学んでたそうです。\n\n後藤:それは何宗の学校?\n\nAさん:それが浄土真宗……ちょっとすみません、詳しい学校までは聞いてなかったんですけど、だからお経とかは学校とかで普段やっていて、私たちが讃美歌歌っているのと同じくらいに、父親は経を唱えてたんだなって。\n\nこれまでも本稿で折々そのインタビュー記録を参照してきたように、A さんは入信してはいないが非常にキリスト教にシンパシーを抱いている。母親もお兄さんもAさんと同じ学校法人の学校への在籍経験もある。それゆえ、なんとなくというよりは積極的にこの学校法人を選んでいるといえる。そういうキリスト教主義学校のシンパサイザーである A さんの育った家庭は、キリスト教も仏教も神道もいずれにおいても宗教行事には積極的であり、その点で、井上ほか (2018) のトリプルな信仰を裏づける結果となっている。\n\nまた A さんは、このような父親がAさんのキリスト教への親和的な態度を肯定的に見守っていたともいう。\n\n後藤:お父さまは仏教徒としてのキリスト教への抵抗よりは、自分は浄土真宗の信仰心が篤かったから、同じようにキリスト教でも信仰心が篤い方がいいと考えていたんですか?\n\nAさん:どうなんですかね……完全にキリスト教徒になれとか、そういう感じではもちろんないんですけど。\n\n後藤:信仰心を養うことは悪いことではないと?\n\nAさん:そうですね、そうとは思ってると思います。なので教会とかも行けるなら行った方がいいんじゃないの、とかも言っていましたし、そういうところでせっかく関わるなら関わりを持ち続けたほうがいいと、そういうのは言ってくれるので、宗教を心の支えにじゃないですけど、自分の生きる道のひとつの支えにしていいんじゃないかということは言われたことがありますね。\n\n後藤:お父さまと、そういう人生とか将来について、お忙しそうだけど話す機会が時々あるんですか?\n\nAさん:そうですね、普段は忙しいんですけど、休みの日にはそういう話をしました。\n\n他方、小中高と同じ系列のプロテスタントの学校に通った B さんは、この A さんとはむしろ逆の側面も目立つ（調査日 2019 年 1 月 22 日）。\n\n後藤:お父様は仏教ということで、父方は仏式の葬儀とかをする訳ですか?\n\nBさん:そうでしたね、はい。それも別に父親にこだわりがあったわけではなかったと思います。\n\n後藤:こだわりはあまりない。よくある日本の仏教だと葬式は仏教で、葬式だけ仏教徒であることを自覚する、というそういう普通の日本人がそうであるような感じでお父様の家のお葬式は仏教?\n\nBさん:はい、たぶんそんな感じです。父方の祖母がこないだ亡くなったけど、その時は普通に仏式で、焼香あって、みたいな葬式でした。\n\n後藤:Bさんご自身は、お父様の家の方のお葬式が仏教で、浄土真宗か曹洞宗か真言宗かなにかわからないけど、それを特に信じてる、その教徒だという意識はあまりない?\n\nBさん:全然。たぶん、近くにあったお寺さんにあげてもらって、お墓買って、みたいな感じだったんで、そのお寺の宗教に則ってみたいな感じだったんだと思います。\n\n後藤: B さんご自身は基本的に無宗教に近い?\n\nBさん:そうですね。\n\n千:初詣などは家族で行く習慣があるとか?\n\nB さん:意識的には行かないですね。一応近くに観音様があるので、すいていれば行きますけど、大体混んでるので。\n\n千:お正月(?)とかに意気込んでいくみたいな感じでは\n\nB さん:全然ないですね。\n\n後藤:観音様が近くにあるというのは一人で?それとも家族で?\n\nB さん:普通に、親戚の挨拶回りとかで家族で出かけたりするので、そのときに寄ってったりみたいな感じですね。\n\n後藤:なるほど。親戚の挨拶回りの帰りに家族で行くことがあるが、（初詣に）行かないことも多いと。\n\nB さん:だいたい通り道に観音様があるので、チラッと覗いてみる。\n\n後藤:混んでるとやめると。\n\nB さん:ですね。\n\nまた B さんの家庭でのクリスマスの過ごし方も Q4 の選択肢 12 の箇所で見たように、比較的地味なものである。\n\n多分に葬式仏教的な緩やかな仏教徒の父親の下で育ち、宗教心はそれほどないということが大きいのかもしれない。A さんの場合は逆に父親は熱心な浄土真宗の信徒であり、そういう環境で育ったことが、クリスマスも初詣も積極的であるということになるとも考えられる、\n\nまた中高大と同志社だった C さんも家庭での宗教行事への取り組みは弱かったと語る（は音声不鮮明で録音の聴き取り不能箇所）（調査日 2020 年 1 月 28 日）。\n\n後藤:除夜の鐘付きはやったことがない?\n\nC さん:それはやったことがないです、はい。\n\n後藤:だよね。初詣は?\n\nC さん:初詣……お寺に初詣は、恵比寿さんが近くにあるので、父は商工会の付き合いで行きますけど、私も母もあんまりいかないです。妹は地元に友だちがいるので、友達と会うついでに行ったりします。\n\n後藤:お盆はやっていると。\n\nC さん:お盆は、お坊さんが来て、勝手に……\n\n後藤:お経読むの?\n\nC さん:勝手にって感じです。まあ誰かいると上がってお経聞くこともありますけど、まあ一応、御膳だけしてあって、田舎なので勝手に入って勝手に読んで帰るみたいなスタイルです。\n\n後藤:そうなんだ、勝手にお坊さんがそういうことするの。\n\nC さん:いや、ほんとは（家族にその場に）いてくださいね、っていうことで。\n\n後藤:棚経だよね、棚経を読む。\n\nC さん:はい、ほんとはいてほしいんでしょうけど、まあ誰も。\n\n片山:今何の話ですか?\n\n後藤:お盆。盆だな（精霊棚）にお経を読むのを棚経っていうんだよね。\n\nC さん:そうなんですね。全然知らないんですけど、お坊さんが来て。\n\n片山:勝手にやって帰ると。\n\nC さん:一応（実家の家業の）お客さんなんですけど。良い車乗ってるお坊さん、スカイライン乗ってるお坊さんが来ますね。〔……は発言者の口ごもり。は音声不明瞭〕\n\nこの C さんの同志社の中学入学理由は母親が仏教系の中高大出身で、仏教系高校の地味さや校則の厳しさから仏教系よりキリスト教主義の方が良いという親の判断があったということであるが、それと同時に、以下のようにCさん当人が、部活必須で校則も厳しい公立校の仕組みが非常に嫌で自由な校風の同志社を選んだという点が挙げられる。\n\n後藤:なぜ同志社を受けたかってことなんですけど、結局同志社以外で N 学園と C カレッジを受けたということで、C カレッジは仏教系なわけで。併願したところは行く気がなかったということなんですけど、それは両方ともですか?\n\nC さん:はい、両方共です。\n\n後藤:それぞれ受かってはいたんだよね?\n\nC さん:受かりましたね。\n\n後藤:結局、この間は時間の余裕がなくて急いできいちゃったけど、なぜそもそも進学しようと思ったんですか?\n\nC さん:制服が嫌だったのと……\n\n後藤:同志社は完全に自由なんだっけ?\n\nC さん:はい。\n\n後藤:でも自由といっても、都内だと立教女学院みたいに制服系のファッションでみんな着ていて、事実上制服化しているということもあると思うんだけど、同志社は本当に自由?要するに、スカートでなくカジュアルウェアで来ている人がいっぱいいた?\n\nC さん:はい、そうですね。\n\n後藤:その理由が制服のスカートが寒くて?\n\nC さん:そうですね、寒くて嫌でしたねー。寒いんですよ、すごい嫌だった。\n\n（中略）\n\n後藤: N 学園や C カレッジは制服?\n\nC さん:制服でした、はい。\n\n後藤:じゃあ結局同志社行きたかった理由は制服がなかったから、というのは分かるけど、進学しようとした理由はそこで、併願校を考えるとなんで\n\nC さん:そうですね……校則がゆるいところが良かった。\n\n後藤:同志社の理由だけじゃなくて、中学受験した理由はどうですか?\n\nC さん:中学受験した理由は……中学受験をした理由というか、えーと、地元の中学校に進学したくなかった理由ですよね?\n\n後藤:うん、まあ、結局は同じことになるよね。地元というか、公立の。\n\nC さん:公立校に行きたくなかった理由の方が大きくて、公立校に行きたくなければ私立に行くことになるんですよね。\n\n後藤:じゃあなんで公立の中学に行きたくなかったの?\n\nC さん:よくわかんない、公立校にはよくわかんない校則がいっぱいあるので、それがものすごく嫌だったのと……\n\n（中略）\n\nC さん:部活そもそもやる気が……それも結構共学、公立高に行きたくなかった理由で。\n\n後藤:部活強制だって前、言ってたね。\n\nC さん:部活強制なのは嫌だったので。\n\n後藤:部活強制はいやで、部活そのものもあまり入りたいと思ってなかった?\n\nC さん:思ってなかったです。\n\n後藤:だけど C カレッジなんて、行く気は最初からなかったにしろ、甲子園でよく活躍してるし。\n\nC さん:私そういうのすっごく嫌で、興味がなくて、未だに野球のルールもよく分からないんですよね。\n\n後藤:でもCだったら、C カレッジは C の本体とは違うかもしれないけど、いずれにしても、C は高校野球の名門だから、部活強制に近い学校でしょ?\n\nC さん:だから、応援とか行かされるって噂を聞いて、すっごい嫌だなぁと思ってました。\n\n片山:それは体育会系の部活が嫌なの?文化系も総じて嫌なの?\n\nC さん:うーん、まあ、体育会系の部活が嫌です。でも、吹奏楽部とかって実際は体育会系ですし。\n\n後藤:体育会系でなくても、吹奏楽もそうだし、合唱だって演劇だって、要するにランクをつけるというか、大会が、美術は大会あっても個人大会じゃん?全体の集合としての大会があるものは大体、体育会に準じるような言い方されるね。逆に言うと、スクールカーストはそういうところは文化部では一番高いらしくて、運動部に準じるんで。\n\n片山:そういう意味で?それとも上下関係みたいなのが嫌?\n\nC さん:それもあります。体育会系もいやだし、上下関係とか、たかだか 1、2 年生まれた年が違うだけなのに、先輩とか敬わなきゃいけないのが馬鹿みたいだって、真剣に思ってたので。\n\n後藤:なんでそう思うきっかけがあったの?\n\nC さん : うーん、一つは、私3歳くらいからずっと公文式に行っていて、結構その中ではよくできてた方だったんですね。なので、英検とか漢検とかも小学校のときから受けてて、英検の5級かなにか受けに行ったとき、私は小学生か幼稚園生だったんですけど、まわりはみんな大人の人とか中高生とかの環境だったので、年齢でできるできないを決めるのは馬鹿らしいなって、思ってましたね。あと、部活が嫌だったのは、私は習い事もやってて、書道とかピアノとか、たくさん習い事やってたんですよ。スポーツとか楽器とか、真剣にやるなら部活じゃなくて先生に習いに行けばいいのに、なんでこんな部活でやってるんだろう、ということが、真剣に意味がわからなくて。それは今も疑問に思ってるんですけど。\n\n後藤:そっか、そうだよね、確かに顧問はプロじゃないもんね。\n\nC さん:ほんとに上手くなりたいんだったら、そんなことしてるの時間の無駄じゃないですか。なのになんでわざわざ、って思ってました。\n\nこのように C さんは非常に積極的に同志社を選んではいるが、キリスト教主義に惹かれてではなく、公立学校が非常に嫌でたまらず公立に較べて自由な校風に憧れて私立を受け、私立のなかでも制服なしだったことが、受験して受かった複数の中学から同志社を選択した理由として挙げている。C さんは同志社の宗教教育も高く評価している。しかしそれは信仰とは一切関係なく、自分の大学や大学院での専門分野を究めるのに、キリスト教についての知識が役立つという点が強調される。その点で通学した学校法人の「人になれ、奉仕せよ」という校訓が今の福祉の仕事に通じているという、Aさんとは差がある。\n\nつぎに入信とご利益の関係を問うた Q49 と Q50 を見てみる。この二つは別のことを聞いたつもりであったが、調査終了より後の時点で気づいたが、実質ほぼ同じ内容となる。\n\nQ49 は「以下の宗教において、入信していなくても教えを守ることによって、救済やご利益（ごりやく）が得られると思いますか。当てはまるものをそれぞれ選んでください」という設問で神道と世界三大普遍宗教合わせて 4 宗教について同じ尺度で聞いている（表3-3-11）。「そう思う」の率が、神道、仏教、キリスト教、イスラム教の順に減ってはいるが、「そう思う」と「ややそう思う」を足しあげた数値でいうと、神道 41.1%、仏教 43.6%、キリスト教 32.6%、イスラム教 19.0%で、イスラム教に較べればキリスト教が目立って、仏教、神道より少ない訳ではない。\n\nこのキリスト教について Q12G を独立変数としてクロス集計すると、表3-3-12のようになる。キリスト教主義学校への通学経験者は「そう思う」が相対的に多く、緩やかな当事者は「ややそう思う」が多く、非当事者は「よく分からない」が多い。このようにキリスト教主義学校当事者指数が高いほど、キリスト教において、「入信していなくても教えを守ることによって、救済やご利益（ごりやく）が得られると思う」という人が多いということが分かる（カイ二乗<0.01）。またそれと同時にキリスト教主義学校への通学経験者は「よく分からない」が最も少ない。また大きな差ではないが「そう思わない」も最も多い。\n\nQ50 は Q49 と実質近い内容で、「以下の宗教において、入信していなければ教えを守っていたとしても、救済やご利益（ごりやく）を得ることはできないと思いますか。当てはまるものをそれぞれ選んでください」が質問文で、挙げた宗教および選択肢のカテゴリーは Q49 と同一である（表3-3-13）。\n\nこれも Q12G とキリスト教をクロス集計すると以下（表3-3-14）の通りである。これもカイ二乗<0.01 である。\n\n「そう思わない」「あまりそう思わない」はいずれもそれぞれ、当事者（通ったことがある者）、緩やかな当事者、当事者以外の順に多い。ただし「そう思う」も当事者が多く、緩やかな当事者、当事者はほぼ同じであった。「ややそう思う」は緩やかな当事者が多く、当事者、当事者以外は少なかった。他方「よく分からない」は非当事者が多く、当事者、緩やかな当事者は同程度に少ない。\n\n要するに、当事者や緩やかな当事者はこの見解に賛同しない者が非当事者よりも多いが、賛同する者も非当事者より多い。要は判断保留せずに、キリスト教主義学校に関与している者は、賛同しない側に偏っているものの両極に割れている。非当事者はキリスト教が首尾一貫性を重視し、洗礼というプロセスが重要であるという一般的知見、常識すらなく、そのため「よく分からない」が多いと推察される。他方何らかの意味での当事者はそういう一般的知見があるので「よく分からない」が減る分、そういう一般的知見に引きつられた「そう思う」が多いし、その一般的知見をあえて否定した、使命としてのミッションないしは徳育としてのキリスト教的な見解はさらに多いという結果になっていると考えられる。\n\n次に Q50 から Q49 にまた戻るが、キリスト教式結婚式の是非を問うた Q22 と Q49-3 のクロス集計は表3-3-15のようになり、カイ二乗<0.01 で有意差がある。キリスト教式結婚式を「好ましいと思う」人の方が、入信せずともご利益があるという見解に「そう思う」を選ぶ人が多く、キリスト教式結婚式が「好ましくない」と思う人の方が「そう思わない」を選ぶ人が多い。これは信者でない者がキリスト教式結婚式を行う場合に、事前講義があったり、本稿でも濱田 (2001) に即して説明してきた先述のようにバチカンの側でさえ日本のカトリックのキリスト教式結婚式に「霊的指導」という位置づけはしていることにも照応しよう。もっともキリスト教式結婚式について「どちらともいえない」という人は「好ましくないと思う人」よりも、Q49-3 で「そう思う」を選ぶ人が少ない（37.8%に対する26.0%）ことは、意外であったし、この理由はよく分からない。一方 Q22 と Q50-3 もクロス集計してみたが、こちらは有意差が出なかった。\n\nつぎに Q49 をグルーピングしたものと、「あなたは仮に人生をやり直せて、入学に必要な条件（学力、経済力、交通の便等）がそろっていれば、キリスト教主義の中学校あるいは高校に入りたいですか」という設問の Q35 の回答をグルーピングしたものをクロス集計すると表3-3-16のようになり、カイ二乗<0.01 であった。\n\nキリスト教について「入信していなくても教えを守ることによって、救済やご利益（ごりやく）が得られると思いますか」という問いに「そう思う・ややそう思う」と答えた者の方が、キリスト教主義学校に「入りたい・どちらかといえば入りたい」という者が多いことが分かる。\n\n以上の面から井上ほか (2018) らのいう「トリプルな信仰」は、我々の調査結果からも裏付けられるといえる。\n\n\n4. 調査結果のまとめとその限りでの結論\n\n今回は紙幅の関係もあり我々のウェブモニター調査の結果のうちの主にキリスト教主義学校設置目的を推測させた Q34 と、キリスト教主義学校の生徒イメージを聞いた Q29 の各選択肢に焦点を当てて、分析、論述した。分析の軸は主にキリスト教主義学校当事者指数を示す Q12G 及びキリスト教主義学校への女性皇族の通学への是非を聞いた Q11G を用いた。\n\nキリスト教主義学校設置目的の Q34 の分析結果としては、キリスト教主義学校の設置目的として信者の獲得を挙げる者が、キリスト教主義学校に通った者等、キリスト教主義学校への関与度の高い者の方が、有意差はないが、少ない傾向にある（p値=0.13）ことが示唆された。他方、「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校に通ったことのある者の方に多い（カイ二乗<0.01）ことも分かった。また「信者の子弟の教育要求への対応」、「教会以外の、キリスト教に触れる敷居の低い舞台としての役割を果たすこと」を選ぶ者も、キリスト教主義学校に通った者等、キリスト教主義学校への関与度の高い者の方に多いことが分かった。\n\nこのような結果からキリスト教主義学校の教育目的としては布教という狭い意味のミッションよりは、神に与えられた使命としてのミッションを果たすという広い意味で当事者たちから理解されていることが分かった。またこの「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校に通ったことのある者の方に多いということは、洗礼と非洗礼で一線を画し、首尾一貫性を重んじるという、従来いだかれてきた日本のキリスト教像とは異なった見方を、キリスト教主義学校当事者たちがとっているということにもなる。これは先行研究の井上ほか (2018) のいう「トリプルな信仰」としてのキリスト教という考え方に照応する結果であるともいえる。もちろん教育基本法の縛りの影響も勘案する必要はあるが。\n\n他方、キリスト教主義学校の生徒イメージを聞いた Q29 の分析結果としては、井上ほか (2018) のキリスト教主義学校の女子は「きれい、金持ち、キリスト」の 3K であるという点については、金持ちである点については「裕福である」という選択肢を設けたところ、それは、「該当なし /答えたくない」を含めた 19 個の選択肢のうち「該当なし /答えたくない」を除き 4 番目に選ばれ、ある程度妥当した。しかしきれいという点については「容姿端麗である（みめうるわしい）」という選択肢を用意したが、これは 19 個の選択肢のうち「該当なし /答えたくない」を除き 18 番目という低い順位で、なおかつこれは Q12G のキリスト教主義学校関与度指数とも相関しなかった。要するにこのきれいであるという点はまったく妥当しないことが分かった。このような結果となったのは今回 Q29 で女子に限らず、キリスト教主義学校の生徒・学生全般のイメージを聞いていることもさることながら、先行研究の佐藤 (2006) や井上ほか (2018) の言及対象とした時代よりも現在、キリスト教主義学校の女性バイアスが弱まっている可能性も大きいと考えられる。\n\nまた井上ほか (2018) ではキリスト教主義学校では愛と奉仕の精神を教育目標に掲げるところが多いとのことであったので、「他人を思いやる心が豊かである」を Q29 の選択肢に入れた。これは 19 個の選択肢のうち「該当なし /答えたくない」を除き 6 番目であまり多く選ばれているともいえないが、Q12G とクロス集計すると、キリスト教主義学校当事者の方が有意にこれを選ぶ者が多かった。しかし実際に通ったことのある者よりも当事者性の低い、緩やかな当事者の方がこれを選ぶ者が多かった。その点で、これは「他人を思いやる心」を涵養させうるだろうという家族等周囲の期待がその数字に表れていて、実際の通学経験者はそれほどでもないと思っていると考えられる。\n\n加えてこの Q29 で「該当なし/答えたくない」を除き最も選ばれたのは「外国語が得意である」である。女性皇族のキリスト教主義学校関与の是非を問う Q11G とクロス集計すると、キリスト教主義学校への関与を是とする者ほど、「外国語が得意である」を選んでいることが分かった。またこの Q29 でその次に選ばれたのは、「教養が豊かである」で、これも女性皇族のキリスト教主義学校関与の是非を問う Q11G とクロス集計すると女性皇族のキリスト教主義学校への関与を「望ましい」と考える者は、「教養が豊かである」を選ぶ人が「どちらともいえない」と考える人や「望ましくない」を選ぶ人より、有意に多かった（カイ二乗<0.01）。またこれを Q12G とクロス集計すると、キリスト教主義学校にかかわりの高い者ほど、キリスト教主義学校出身者を「教養が豊かである」と自認している（カイ二乗<0.01）ことが分かった。\n\nこれらの結果から、女性皇族のキリスト教主義学校関与を是と捉える層からの皇室外交への期待がうかがわれる。また「外国語が得意である」と「教養が豊かである」が、Q29 で「該当なし /答えたくない」を除き一番目、二番目に選ばれ、「他人を思いやる心が豊かである」が六番目であることを考察すると、キリスト教主義学校の意義を、宗教教育をはじめとする情操の涵養のレベルで捉えるよりも、外国語や教養などのより実利レベルで捉える人が多いということになる。もちろん、情操教育も広い意味で、教養に結びつくし、女性においては結婚戦略とあわせ、社会階層・地位上昇の契機になる可能性も、調査票の設計時には我々は考え、その限りでは実利に結びつくが、その前提となる女性バイアスそのものが弱くなっていることは、佐藤 (2006) に即して本稿でも先述した通りである。\n\n\n5. 考察\n\n1. で井上ほか (2018) の、日本人のトリプルな信仰志向にキリスト教も組み込まれるという指摘に対して、キリスト教は洗礼というプロセスを要するし、丸山らのいう首尾一貫性も求めるので、ダブルな信仰とトリプルな信仰とには断絶、距離があるという反論もある。いいかえると現実にはトリプルな信仰になり得てはいない可能性を吟味する必要があるのではということを述べた。また井上らはそのようなトリプルな信仰の根拠としてクリスマスとキリスト教結婚式とキリスト教主義学校の隆盛を挙げていた。\n\nこの三つが挙げられていることについてその妥当性は、3.3 で本稿の調査からもいえるということを示したが、ここでは文献的にそれを吟味する。\n\nクリスマスに関しては日本の場合、いっさい宗教色はなく、生活上の習俗と化しているといわれる。また、キリスト教式結婚式にしても先述の濱田 (2001) でも指摘されているように、キリスト教教会の側としては布教の一環として、人びとがキリスト教に触れる機会を提供しキリスト教のシンパを増やすという目的意識が、多かれ少なかれあるにせよ、利用者の方は習俗としてのクリスマスに毛が生えたようなものというレベルの認識である可能性は否めない。この習俗なのか宗教なのかは、本研究のテーマでもあり井上ほか (2018) のテーマでもあるが、習俗の延長上にある実利という部分も重要で、宗教教育と実益に直結する英語教育とは緊張を孕んできたし、今ではなおのこと、就職率の良さや進学先の良さ等の進路で選ばれている面が強くなっていることは間違いない。\n\nキリスト教主義学校もクリスマスとキリスト教式結婚式と併記して捉える限り、これらは総じてキリスト教という部分は習俗として及びその延長上にある実利のみのレベルで受け取られ、習俗としてのキリスト教のイメージ的な格好良さや進路といった実利面で選好されていると一応纏められるであろう。\n\nしかしキリスト教と鋭い緊張関係をもつもののキリスト教の源流であるユダヤ教が、習俗、生活習慣上の規律を重んじる宗教でもあったとされる点も考慮に入れてこの問題を再吟味すべきだろう。ユダヤ教では割礼その他独自の生活習慣を大切にし、それにより国土を失った散り散りの民族の統合を図っていた。そのような習俗の遵守を宗教上の最大の課題と考える律法主義者（ファリサイ人）たちに対して内面の信仰の重要性を唱えたのがイエス・キリストであり、イエスのそのような側面を強調したのがパウロで、そこから普遍宗教の側面を前面にだしていったという見方もある（波多野 1979）21。その点でキリスト教はユダヤ教の律法主義者的な側面のアンチテーゼとして出てきた部分はあり、それはキリストが磔刑に処せられる理由の一つともされている。しかし、ユダヤ教の聖典旧約聖書もキリスト教の聖典でもあり、習俗のみということは批判されるにせよ、1.2.2でリスペクタビリティについての佐藤 (2006) の言及への井上ほか (2018) のミス・リーディングに関連づけて先述したように、習俗などの形から入って内面の信仰に至るという筋道は一つの道、信仰の形として想定されてしかるべきということにはなる。\n\nキリスト教側からすると、首尾一貫性のあるキリスト教を理解し、一定の講義を受講し洗礼というプロセスを経ないとキリスト教徒になったとはいえず、そのような正真正銘のキリスト教徒は日本の総人口の1.0% ないしは 1.6% しかいない。そのことからキリスト教は日本において根づいていないと結論づけられることになる。しかしキリスト教にふれる、クリスチャン以外の一般国民の側からすると、洗礼のようなイニシエーションの儀式がなくても仏教や神道ならば信徒であると名乗れて、キリスト教には洗礼というプロセスがあるので正規のキリスト教徒でないにせよ、井上ほか (2018) が示唆するように、神道の信徒や仏教徒と近い水準でなら自分はキリスト教徒であるという意識をもつという可能性があるようにも思われる。それが先にみた、Q34 で「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校に通ったことのある者の方に多いことに表われている。\n\nまた Q29 のキリスト教主義学校の生徒・学生のイメージ、及び Q26 のキリスト教徒、Q27 のキリスト教の聖職者、いずれのイメージにおいても、「論理の整合性を尊ぶ」を選ぶ者が極端に少なかったことも、このことを補強する材料といえるであろう。というのも、論理の整合性を尊ぶことは首尾一貫性を求めることであり、唯一の聖典という基準があるので、洗礼後と洗礼前、自分たちと他者たちをその基準への意識によって明確に峻別することにもなる。そうすると、仮にその選択肢が多く選ばれていた場合、洗礼の有無という重要な基準をスルーする、トリプルな信仰という発想は、ロジカルにも否定されることになるであろう。一方、今回の調査結果はその逆になったため、そのことがトリプルな信仰という井上ほか (2018) の着想への補強材料になる。\n\nなお洗礼が、トリプルな信仰の考え方を妨げる障壁のように考えられる一方で、佐藤 (2006) は 1926 年~1930 年のキリスト教主義学校卒業生の洗礼率が平均 50% 前後で推移している（佐藤 2006 p.17）ことについて、学生時代に受洗したことがあるというもののいまでは仏教の法事を普通に執り行う佐藤自身の親類の老婦人の例を出しながら（佐藤 2006 p.15）、とりあえずは受洗してみたけれど、という人も少なからずそのなかに混ざっていたのではないのかと推測する22。したがってその意味で、このような人びとは、そもそも昔であっても、洗礼をしつつトリプルな信仰に近いスタンスをもっていた可能性もあるといえよう。そうはいっても日本において洗礼というプロセスの敷居は高い。家族と違う宗教を場合によって選び取る、また洗礼前に講義を受ける等々、諸々の手続きも要する。先祖の墓の維持・管理という問題まで生じうる。他方、欧米であればキリスト教徒になったからといって家族と違う宗教というケースは日本よりは少ないし23、それ以上に幼児洗礼のある宗派の場合、キリスト教徒であることは自明でそのことをさほど自覚せずに済むし、事前講義等の手続きもおそらくはなしで済む。その点でトリプルな信仰のひとつとしてのキリスト教というものは、洗礼という重大プロセスを無視しているとはいえるが、欧米のクリスチャンの場合、そもそも洗礼というプロセスを当人は意識せずに済むケースも少なくないという点にも留意が必要であろう24。\n\n先に 2.1 でみたように、キリスト教主義学校の設置目的の推測の設問である Q34. において選択肢 8 の「キリスト教の信者を増やすこと」を選んだ者は全体で 13.0% であったのに対して、選択肢 9「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」は全体で13.3% となっており、ごく僅かな差で有意な差とはいえないが、「キリスト教の信者を増やすこと」より多かった。しかも Q12G とそれぞれの選択肢を選んだ人、選ばなかった人とでクロス集計すると、これも先にみたように、選択肢 8 の「キリスト教の信者を増やすこと」を選ぶ人はキリスト教主義学校の通学者・通学経験者の方に有意差はない（なお 2.1 で先述のように検出力不足故、有意差が出なかったと我々は考察した）が少なく、逆に、選択肢 9 の「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」はキリスト教主義学校の通学者・通学経験の方が有意に多かった（カイ二乗<0.01）。キリスト教主義学校教師であるかどうかも本来聞くべきであるが、残念ながら今回それらに聞いた結果ではないものの、少なくとも通学経験者の回答の比率から推すと、キリスト教主義学校の教育において洗礼を受ける人という意味での狭義の信者を増やす意図よりは、キリスト教の精神を理解し、キリスト教的な徳目を守る人を増やすという意図が、強くなっていると考えられる。同志社は「徳育としてのキリスト教」を標榜しているが、他学も含めてそれに近い傾向があるといえよう。\n\nなお津城寛文はユニテリアンその他の「自由キリスト教」が 1880 年代後半に日本に入ってきて、それと東大宗教学講座の創設者姉崎正治の比較宗教学や万国宗教大会 (1893) の影響もあって、一部のキリスト教において、キリスト教の相対化、キリスト教の日本化の動きが生じたという（井上ほか 1997 p.124）。その流れの中に玉川学園の創始者小原国芳、桜美林の創始者清水安三も位置づけられるという。「小原国芳や清水安三らも、神道・仏教・儒教および習俗・習慣と断絶しない、日本化したキリスト教をとなえ、正統的なキリスト教教義にもとづく宗教教育ではない、超宗派的な、あるいは宗教色のうすれた人間教育（小原のいう「全人教育」など）をこころみた。ここには「一部のキリスト教」の自由キリスト教的な、あるいは日本化したキリスト教という姿勢が共有されているようにおもわれる」（井上ほか 1997 p.124~125）。ここで「一部のキリスト教」と記されているが、次に引く文でも津城は「一部の」という言葉を二度使う。「右にみたように、キリスト教は明治初年の直輸入なものから、明治なかば以降、その一部が「自由キリスト教」化あるいは日本化したが、それに応じて、キリスト教系の宗教教育の一部も、比較宗教的な教育へ変化している」（井上ほか 1997 p.125）。\n\n戦後間もなくの頃、カトリックの立場からは遠藤周作などが、またプロテスタントの立場からは ICU 教授の武田清子などが日本におけるキリスト教の受容と土着化の問題を考え抜いた。土着化、日本への定着の問題と日本のキリスト教主義学校の隆盛とを関連づけた研究は佐藤 (2006)、井上ほか (2018)、クリーグ(2017) 以外管見の限りないが、このような日本化したキリスト教という問題を、キリスト教主義学校はみずからの課題として考え、キリスト教主義学校の「ミッション」を洗礼する人を増やすという狭い意味での布教よりは、キリスト教的な徳目を守る人を増やすという意味、あるいは自らに与えられた「使命」を自覚する人を増やすという意味に変えていることが想定される。津城は慎重に前パラグラフでの引用で「一部のキリスト教」「キリスト教系の宗教教育の一部」と、控えめに主張するが、一部というよりは全般的な傾向として超宗派的な宗教教育がなされている可能性が窺われる25。\n\nそのことは今回のウェブモニター調査においても、Q34 において直接布教を目的とするという答えよりも、入信せずともキリスト教の教えを守ることで救われる人を増やすことという回答が多かったし、後者はキリスト教主義学校当事者ほどその傾向が強かったことからも、一応裏づけられるといえる。\n\nしかし布教・ミッションの目的は一つではなく複合している。したがってこういう複合している目的の問題を扱う際は、ロバート・キング・マートンの、順機能・逆機能、顕在機能・潜在機能の議論が有用である（マートン 1949=1961 pp.45-77）と思われる。\n\nミッションの初発の動機あるいは顕在機能としては救われる人を増やすという愛他精神が強くあると考えられる。\n\n他方、ミッションの副産物は当然あり得て、それらは初発の段階では潜在的機能といえる。およそほとんどの宗教において善行をすることは、みずからの救いへの一里程である。キリスト教に即していうと、仮に入信せずんば救済されずとみなされるとすれば、他者を入信させることはキリスト教の立場からすると他者の救済に貢献する善行の最たるものであろう。よってみずからの来世での救済をより確かなものにする可能性があると考えられる。\n\nさらにミッションの機能としては教団の経営基盤の確保の面もあり、これも当初は潜在機能であったと考えられる。というのも基本的にキリスト教も仏教もその初期において指導者層は托鉢のような形で布教しており、施しによって生活の糧を得ていたと考えられる。しかし聖職者が贅沢をしようが質素に生きようが、祈りと布教と学問と写本・写経のみに勤しんで、最低限の農作業はするもののほとんど生産活動をしない聖職者層が拡大すればするほど、またたとえ中世であっても完全な自給自足経済ではなく貨幣での取引が少しずつ浸透したであろうから、そうなるにつれ、聖職者層を養う財政基盤の確保は必須となる。\n\nまた日本でもヨーロッパでも教会や寺院が学問の中心であった歴史があり、庶民の子どもが知識を身につける数少ない術の一つが、宗教施設に入門することであった。また日本でもヨーロッパでも支配階級の嫡子以外の男子の子どもはお家騒動をさけるため修道院や寺院に入れさせられるケースも少なくなかった。したがって被支配階級の子弟の階層上昇と支配階級の嫡子以外の男子の階層再生産の舞台として宗教施設が機能した可能性はある。その場合のミッションを考えると潜在機能か顕在機能かは措いて、本音の部分では熱心な信者の数を増やし、教会や寺院の経営基盤を確かなものにするということが充分に想定されうる。なぜならそれに貢献した者は教団内での地位が向上する可能性が増えるからである。\n\nしかし伯爵家出身のトマス・アクィナスのように、親が階層再生産のために入信させようとしても当人は信仰心から親の望まない、伯爵の子息に似つかわしくない托鉢修道会のドミニコ会への入会を求める等、聖職者になる当人の初発の意識としては純粋に神のことを思う意思の貫徹が大きなミッション（使命）であったケースもあると考えられる。\n\nキリスト教主義学校設立当初の目的は、その意味で、救われる人を増やすというということが顕在的機能として高かったと考えられ、その点で、狭義の布教とミッションとが結びついていたと考えられる。しかし無限抱擁的な雑食文化の日本において、教義として首尾一貫性のある、あるいは首尾一貫した節操を信徒に要求するキリスト教は、根付きにくいと実感させられる体験を重ねるにつれ、あるいは丸山真男その他のそういった論述を耳にするにつれ、意識的にか無意識的にかは措いて、キリスト教主義学校関係者の側も自己利益ではなく他者の救済を考える場合には、入信までは求めず、キリスト教的徳目を守らせることで他者の救済を考えるようになったと考えられる。また前節で見た津城のいうような「自由キリスト教」や「比較宗教学」の影響も、そのように変わる要因として大きかった可能性もある。「学校経営として成功するためのキリスト教教育、宗教教育がいかなるものであるかは、あきらかではないにせよ、少なくとも小原国芳、清水安三と反対のことをそろえれば、経営として成功しないおそれがつよい、とはいえるかもしれない」（井上ほか 1997 p.140）。さらに、そのことと並行して、信者以外の人びとから認識される救いの形も、日本的なものになった（土着化した）のではないかと推察される。それらが先に確認したようにキリスト教主義学校の設置目的の推測として「キリスト教の信者を増やすこと」よりも、若干ではあるが、「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」が多いことにも表れていよう。しかも再三先述したように前者の回答はキリスト教主義学校の通学者・通学経験者に有意差はないが相対的に少なく（これについては 2.1 で先述のように、我々は有意差はないがその理由は検出力不足によるものと判断した）、他方、後者の回答はキリスト教主義学校の通学者・通学経験の方が有意差のある形で多い（カイ二乗<0.01）という結果にもなっている。\n\nこのことはキリスト教主義学校の存在が、正規の洗礼に比して緩い、事実上の入信に近いものを設け、ミッションのハードルを下げている可能性を示す。とはいえここで「緩い、事実上の入信に近いもの」というのが、キリスト教主義学校への在籍・卒業とか、聖書学とかキリスト教概論等の単位取得とか量に明確に指標化できる基準を想定してのものというわけではない。量的な基準によるものというよりは内面の感化、自覚といった質的な変化であろう。（そして少し後に後述するように、これはミッションの自覚ということであろうかと想定している）。もちろん今回の調査で分かるのは教会、教団や学校自身の意図ではなく、あくまでも一般の人びとからみる教団・学校の意図の推測に過ぎないが、意図はともあれ実際の行動において、仮に人びとによるこの推測と逆のことを実際にキリスト教主義学校がこれまでしてきたとすれば、キリスト教主義学校通学者・通学経験者とそれ以外との回答の分布は、今回の調査結果と逆になっているはずである。少なくともミッションという意味では直接の布教よりは、布教の地均しレベルでよしと考えていると推察され、先に佐藤 (2006) に依拠して申し上げた、ミッションを「使命」の意味に解するものもそこに含まれるといえるであろう。しかも佐藤は生徒自身が神から彼らに課せられた「使命」を自覚するという意味で「ミッション」に言及し、しかもそういう場合もあるというレベルでの言及であったが、キリスト教主義学校の側からすれば、そのような生徒自身の「使命」を自覚させることこそが学校の「使命」、あるいは教員のミッションと考えている可能性もあるといえるであろう。\n\n実際、佐藤 (2006 p.12) では、長野清泉女学院のウェブサイトでは「神様は、私たち一人ひとりに、何らかの使命をお与えになっています。その使命に気づき、その使命をはたすためによりよく生きる人を育てる学校が“ミッション・スクール”なのです」と書かれていると記される。なお、現在この学校のウェブを確認したが、すでにこの発言はないし、「使命」の言葉も見当たらない。一方、同校ウェブの「よくある質問」コーナーで「Q 宗教教育はどの程度行うのですか?」という質問に対して、「A 週 1 時間「宗教」という授業があり、キリスト教精神に基づいた“生き方”を学ぶことを主眼としています。決して信仰を強要するのではなく、思いやりの心を大切にする校内のあたたかな雰囲気は、宗教教育によって育まれてきます」と答えていて（最終閲覧日 2022 年 10 月 9 日）、情操教育の側面を強調している。\n\n他方、ミッション・スクールの「ミッション」とは結びつけた説明はしないが、「使命」については日本における代表的なカトリック女子中学校・高等学校のウェブにおいて強調されている。例えば白百合学園中学校・高等学校では「宗教教育」というタブの「宗教の授業」という項目において「中学校では、学園の歴史や聖書を学びながら、神に愛されている自分に気づき、思いやりの心、感謝と奉仕の心などキリストの教えに基づく価値観を身につけます。高校では、聖書を深く味わい、社会における自分の役割や使命について考えます」（最終閲覧日 2022 年 10 月 9 日）と記され、高校の課程で「使命」を学ぶとされる。あるいは学校法人雙葉学園のウェブでは「教育理念」のページで、「すべての人を愛されたイエス・キリストのように、自分を含めた一人ひとりを大切にし、その人にしか果たせない使命のあることに気づき、その人ならではの人生を歩めるようにする」（最終閲覧日 2022 年 10 月 9 日）と書かれ、やはりここでも「使命」に言及される。\n\nこのような「使命」としての「ミッション」を自覚した人は「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人」という Q29 の選択肢9にも通じうる。\n\nなお、プロテスタントの女子中高ではミッションに繋がる「使命」への言及は見た限りではない。例えばフェリス女学院中学校・高等学校では「キリスト教活動」という項目があるが「奉仕活動」や「宗教講演会」「パイプオルガンの練習」等、具体的な活動が示されているが、「使命」の文言はない（最終閲覧日 2022 年 10 月 9 日）。立教女学院中学校・高等学校でも「立教女学院のキリスト教教育」の項目では「礼拝」「ボランティア活動」「土曜集会プログラム」「キャンプ集会」などここの具体的な活動の小項目に分かれるが、「使命」への言及はない（最終閲覧日 2022 年 10 月 9 日）。ただ「よくある質問」の項目で「今までキリスト教と無縁だったのですが大丈夫ですか?」という問いに対して、「ご心配ありません。もともと私たちの学校はキリスト教とは無縁だった人のために始められました。学校生活の中で、少しずつキリスト教のことが身につくようになります」（最終閲覧日 2022 年 10 月 9 日）と記され、基本的に「布教」の地ならしに近いニュアンスの回答となっている。また先ほどの「立教女学院のキリスト教教育」の項目の「礼拝」において、「日曜礼拝のご案内」という小項目もあり、「聖マーガレット礼拝堂では、在校生・卒業生・学校関係者の方を対象に、月に一回、日曜礼拝を開催しています」と記されている。したがってこの学院では「使命」という意味でのミッションの側面は少ないが、「布教」の意味でのミッションの準備的な行動はなされているともいえる。\n\nなお中高ではなく系列の小学校の方であるが六十年史編集委員会編『六〇年の歩み』(1997、立教女学院小学校）によると、1947 年「日曜日には小学校の礼拝堂で矢崎（哲学・神学を大学で専攻した教諭）による子供のための礼拝が捧げられた。自由参加であったが、学齢に達しない在校生の妹や近隣の児童も参加して、毎主日百人近い出席があり、多い日には二百人を超えた」（六十年史編集委員会編 1997 p.184〔最初の( )は後藤ほか本稿執筆者による補記〕）とある。さらに 1949 年には任意参加ではなくなった。「一九四九（昭二四）年四月から学院は主日礼拝に出席することをキリスト教教育の基本方針として位置づけ、日曜日を自分の所属教会に出席するものの他は全員が学校での礼拝に出席する開放日とし・・・この制度は一九八二（昭和五七）年三月まで続いた」（六十年史編集委員会編 1997 pp.184-185）。\n\nなお同書では 1989 年の『立教女学院報』41 号を転載している。そこでチャプレンの杉山氏が「ご承知のように一九八二年度より小中高各校における、それまでの登校日としての日曜礼拝が廃止され、児童・生徒・勤務員はそれぞれの地域の教会の日曜礼拝に参加するようにすすめられ、日曜日は学校の休日となりました」（六十年史編集委員会編 1997 p.498）と語る。そのうえで廃止により参加者が減ったことを次のように表する。「現在、日曜日がキリスト教にとって生命線といえるほど大切な日であることを認識している生徒、また御父母がどれだけいるでしょうか。いや教職員の中にもどれだけいるでしょうか。私たちの学校がキリスト教の学校であるということ、ミッション（使命）をもった学校であるということを考えたとき、私たちはなんとしても日曜日には教会生活を送るよう啓発していく責任があるように思うのです」（六十年史編集委員会編 1997 pp.499-500 〔太字は後藤ほか本稿執筆者〕）。このように『六〇年の歩み』（1997 年、立教女学院小学校）では使命としてのミッションという言葉が転載再録の形ではあるが、記されている。\n\n以上を踏まえると、井上ほか (2018) の「トリプルな信仰」という見方も、日本式キリスト教というキリスト教の土着化の過程で、全否定はできないと考えられる。先述の話の繰り返しになるが、濱田 (2001) のキリスト教式結婚式の研究によると、日本における非クリスチャンを対象にした教会外でのキリスト教式結婚式の本格化はプロテスタントの一部の牧師たちによって始められたものもあるという。キリスト教を具体的に知ってもらう機会を得ることで、キリスト教入信への地均しをしようとする意図のあるものであるという。意図としてはこれはあくまでも地均しではあるが、これも広い意味でのミッション、伝道であり、日本における土着化の一つとしても考えられる。\n\nまた「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」という着想は、日本で内村鑑三の無教会主義がよく知られていて、内村が 1951 年に日本郵便の切手になるほどに著名であることとも関連している可能性がある。武田清子著『未来をきり拓く大学―国際基督教大学五十年の理念と軌跡―』ではクリスチャン条項をすべての教員に適用する ICU において、ICU 教会に無教会派の教員を加える際に問題になったことがある点が、記されていた。「洗礼を受けず、信仰告白もせず、無教会に「会員」として入会していない無教会信者と称する人たちを「キリスト教」と認めるということは、「キリスト者」の定義をあいまいにするのではないかという意見が教授会で述べられたことがあった」（武田 2000 p.111）。もっともこれはメンバーシップをアフィリエートといいかえることにより解決が図られたと記されている。実際「無教会を標榜する内村は、キリスト者になるためには「水のバプテスマ」すなわち洗礼を受ける必要はないと考えた」（若松 2018 p.180）。そうであれば信者であるなしは当人の自覚にかかわるようになり、公的に「信者になる」というプロセスは不要となる。武田は別の著書『背教者の系譜』で「内村鑑三を創始者とする無教会主義は、・・・日本型セクト運動ともいえると思うのであるが、日本のキリスト教会において長く異端視されてきた」（武田 1973 p.112）という。そのうえで「しかし、無教会グループは、今日、日本のキリスト教界の「正統」と一応考えられるキリスト教諸教派、諸グループの中に位置を占めつつある」とも述べる。武田の 1973 年の著書から半世紀近く経て一層のこと無教会派は一般的なキリスト教イメージとして定着している可能性がある。また内村は再臨運動をする中で、キリスト再臨の日には福音を信じない者にも「贖いの恩寵が光のごとく、万人にあまねくそそがれる」（若松 2018 p.180）と考えていた。あるいは堀孝彦は次のように内村の再臨運動を記す。「再臨を、ただ「教会問題」とみなしキリスト信者にのみかかわるものと見るのは大きな誤りだと言って、それが「宇宙問題、世界問題」であるから、万人に訴えて注意を喚起すべきだとしている。その根底には・・・万人救済論がある」（堀ほか 1996 pp.140-141 〔・・・は中略〕）。そのような内村の考え方がキリスト教の一般的姿として、日本のクリスチャンの内外から受け入れられ、それにあわせて武田 (1973) のいうように、プロテスタントの聖職者・研究者界隈でも当初は異端でありつつ次第に受け入れられてきたとするならば、「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」という回答は選ばれやすいといえる。\n\nまた東西宗教交流学会長の田中裕は西田幾多郎のクザーヌスへの着目に関連させて次のように語る。「カトリック教会の司祭（枢機卿）でもあったクザーヌスは、キリスト教以外の異教を排除せず、様々に異なる礼拝のなかに一なる宗教があるという立場を取った。・・・西田もまた、キリスト教を日本の国体に反するものとして排除しようとした幕末から明治にかけての多くの宗教者とは違って、キリスト教を異教として排除することをしなかった。福音書やパウロ書簡の宗教的経験の深さを洞察し、自らの禅の立場において、それを摂取し発展させた。西田とキリスト教を結ぶ媒介となったものは、ドグマに固執する神学ではなく、ドグマを越えるキリスト教の霊性的伝統であった」（田中 2020 pp.277-278）。実際、西田は東大で、同志社中退でジョン・ホプキンズ大学にて博士号をとりキリスト教に造詣の深い、倫理の教授元良勇次郎からの影響でウィリアム・ジェームスに強い関心を抱いた（苧阪 1998 p.349）という説もある26。\n\nこのように「ドグマに固執する神学ではなく、ドグマを越えるキリスト教の霊性的伝統」を、日本を代表する哲学者、西田がみいだしていたという点も、日本を代表するキリスト教徒の内村の無教会派と同様、多くの人びとに「入信」という壁を経ない、緩やかに「教えを守ることによって、救われる可能性のある人を増やす」という意識をもたせる背景として、想定できるといえるであろう。\n\nまた本研究では基本的に『日本の思想』での丸山真男の論旨に依拠しつつ、首尾一貫性を重んじるキリスト教という丸山の考え方とトリプルな信仰との矛盾の可能性の有無を述べてきたが、首尾一貫性を重んじるキリスト教という考え方は「執拗低音」を唱える後年の丸山の趣旨とは多少のずれも生じ得る。『日本文化のかくれた形』では神道等の、日本の古層を中心の不在とは表現しない。消去法によって外来文化の浸透部分を消していくと古層が現れるという。「そうすると、何もなくなるかというとなくならない。サムシングが残るのです。そのサムシングというものがつまり、原型―その断片をあらわしております。原型はそれ自身としては決して教義になりません。教義として体系化しようとすると外来世界観の助けをかりねばならない」（加藤ほか 2004 pp.142-143）。このように古層、原型があるから表層は変わりやすいが根本は何も変わらないのが日本文化の特質であると述べ、その特質はキリスト教の受容にも見られるとする。「絶えず新しいメッセージを求めるということと、新しい刺激を求めながら、あるいはそれ故にか根本的に驚くほど変わらないということ・・・この両面がやはり思想史的な問題としても重要なものになってくるのではないか。たとえばこのことをキリシタンの渡来とその「絶滅」の運命について早くからジョージ・サンソンという有名なイギリスの日本史家が指摘しています。「いかなる国民も新しい教えをこれほど喜んで受容する用意のある国民はなかった」。新しい教えというのは一五世紀に来たキリシタンのことです。「けれども他方、これほど伝統を頑強に固持した国民はなかった」」（加藤ほか 2004 pp.121-122)。\n\nこのサムソンの発言から丸山は中国韓国のキリスト教と比較して、日本のキリスト教が絶滅するときは綺麗に消えてしまう点を指摘する。「驚くほど変わらないということ」及び「伝統を頑強に固持した」という点からすると、キリスト教も日本思想の「古層」と結合する可能性もあったということを、丸山自身が示唆しているともいえ、そう考えると田中が描く上述の西田のキリスト教観、「ドグマに固執する神学ではなく、ドグマを越えるキリスト教の霊性的伝統」のようなもの、あるいは日本で土着化したキリスト教というものへの接点も丸山は想定していたとも考えられなくもない。\n\n実際この講演記録の著書の「あとがきにかえて」のつもりで当初著したものの丸山の薦めで岩波現代文庫版で一つの章にしたという IV 章において、ICU でのこの連続講演会の企画者で、また丸山同様、思想の科学研究会の7名のオリジナル・メンバーの一人として丸山との交流歴の非常に長い武田清子は、「外来宗教（思想）である“キリスト教の土着化”などということは、ありえないことだという断定にぶつかる時、私は戸惑いを覚える。日本人の内発的思考様式、価値意識には、超越的なもの、普遍的なるものを志向する要素が希薄だということは首肯できることであろう。しかし、それが、全く内在していないと果して断定できるだろうか?」（加藤ほか 2004 pp.121-122）とも述べている。\n\nあるいは同書の「まえがき」で武田は「ここに収録した丸山真男氏の講演は、第二次大戦後の氏の日本思想史研究における「方法論」についての歩みを、「原型」から「古層」へ、そこから更に「執拗低音」（バッソ・オスティナート）へと移行・発展する学問的自伝の一側面をたどるものであ」（加藤ほか 2004 p.9）るとも記されている。「「方法論」についての歩み」と武田は評するが、方法論のみならず不寛容なものへの不寛容として排除される存在としてのキリスト教から、日本思想の「古層」と結合する可能性もあるキリスト教への「移行・発展」も場合によってありうるといえよう。\n\nもう一つ考えるべきは、キリスト教主義学校という制度の自己目的化、いいかえるとキリスト教主義学校という制度が物象化し、当初の設立目的から自立して動いていくことである。結局これもマートンの機能の類型論で理解できる。\n\n先ほどはマートンの着想を援用して、信者を増やすという狭義の布教・ミッションについて顕在機能と潜在機能とを分けて論じたが、そもそも2.1でみたキリスト教主義学校の設立目的においても、機能の類型論を併せみる必要性があろう。布教のみならず「使命」の自覚も含めた広い意味でのミッションあるいは徳育としてのキリスト教を広めることが、顕在機能としてある。しかしひとたび学校が設立されれば、その学校の維持・発展が第一の目的に転じ、それが理事会、教職員、同窓生等、関係者たちから意識される。それらが潜在機能となり、場合によって新たな顕在機能となり、制度の自己目的化が起こる。学校の維持・発展には入学希望者という潜在的な生徒・学生の評価が重要で、これらは競争率や入試の偏差値に典型的に示されるが、それら潜在的な生徒・学生の評価を高める最も大きな要因は、卒業生の進路の良さであろう。高等学校まででいえば進学先の良し悪しであろうし、大学・大学院であれば就職先の良し悪しである。情操教育など数字に直接現れない教育の良し悪しも生徒・学生の評価を高める大きな要因ではあるが、進路の良さに較べれば副次的なものに留まる可能性も否定はできない。特に男女共同参画社会の定着によって、主に女性においてだが情操教育によって良い結婚相手をみつけ、階層の上昇や再生産を図るという戦略の有効性が低下した現代において、ますます進学・就職状況が、受験対象校として選ばれる際の重要な決め手となっていく。例えば廣瀬つぎ子編『雙葉小学校創立三十年記念誌　小百合』(1941) では、卒業生の動向を記すコーナー（「一回生消息」等）が設けられていたが、そこでは結婚相手の職業も記述されることがほぼ常であったし、子どもの在学学校も記されていた。例えば「一回生の消息」の二番目に記された人は以下のようになっている。「○○様（△△）女子学習院御卒業後・・・御主人さまには南洋長官、拓務次官と官界に御活躍其の御内助ぶりも伺われ誠に御立派な奥様でいらつしやいます。御令息様は慶應に、御嬢様は母校女学校二年に御在学でございます」（廣瀬 1941 p.32〔○○は人名、△△は県名。元々は匿名ではない〕）。今は男女平等のみならず個人情報保護の観点からも、このような記述はあり得ない。そうするとキリスト教主義学校の「キリスト教」の部分の比重は女性については従来男性より強く意識されたのに、男性並みにその意識は弱まらざるを得ないし、男性についても今まで以上に弱まるであろう。\n\n井上順孝は、「戦前に宗教立の学校が設立された場合、その教育に、宗教的理念を生かそうとする傾向はかなり強かった」（井上ほか 1997 p.19）と記す一方で、「しかし、戦後は、そうした創立時の宗教的情熱は一般に薄れる傾向に」あるという。また「戦後は、一九五五年頃までは、宗教教育に熱心な場合も少なくなかったが、やがてそうでもなくなった」（井上ほか 1997 p.19）とも記す。確かにキリスト教主義学校の連盟のウェブサイトにおいて、クリスチャンの教員の比率が経年的に漸次減少している点を議論する情報が掲載されている。進学実績を向上させ、偏差値や競争率をアップさせるには、教員のクリスチャン条項を外すことはいうまでもなく、場合によってはキリスト教主義に共鳴する人材を教員に迎えることさえも断念し、受験指導や進路指導に長けた人材、そうでなくても授業の巧みな人材を雇用した方が有利になる。しかも競争率が上がれば、定員も増やしやすくなるし、大学院博士課程・大学から中高の一貫（系列）校の場合には、アカデミック・ポストの恒常的不足のなか、大学院生の進路の面からも機会があれば定員は増やそうとする圧力は常時あると考えられる。定員が増えていけばそれに合わせて教員増となり、アカデミック・ポスト不足を多少なりとも緩和する。漸次定員が増えていくなかで、設立当初のキリスト教主義に強く共鳴した教員の比率が下がるのは当然である。\n\n先に言及した佐藤 (2006 p.17) の挙げた 1926~1930 年の例は、卒業生の 50% 程度が入信しているが、往時は定員も今よりは遥かに少なく、教員のクリスチャン率も現在より高かったことが推察される。この辺りは今後史料や文献を通じて明らかにしていきたい。例えば 2018 年 10 月にカトリック社会問題研究所が主催した、少子化・公立志向・教育改革、非信徒教員の増加の状況を踏まえて行われたセミナーで、日本カトリック学校連盟事務局長（当時）の品田典子は、信徒教員の減少については、特に信徒教員が非信徒教員から IS（イスラム国）ならぬ CS (Christian State) 呼ばわりされることが多い一方で、カトリックに理解を示す非信徒教員も多くいる現状から、非信徒教員との連携体制の構築が肝要という（「少子化、信徒教員の減少、迫る教育改革　カトリック学校の現状と将来 (2018 年 11 月 29 日 23 時 05 分）」最終閲覧日 2022 年 10 月 9 日）。\n\nまたこれはプロテスタントの方であるが、我々のインタビューに答えてくれた、小中高とキリスト教主義の学校に通った学生Bさんは、次のように答えている（調査日 2019 年 1 月 22 日）。\n\n「後藤:それから、先生の中でクリスチャンは、小学校と中高で違うかもしれないんですけど、自分がクリスチャンですと言うかも色々だと思うんですけど、雰囲気としては小学校の時はどのくらいクリスチャンの先生がいましたか?\n\nB さん:小学校のときは、結構いたと思います。礼拝のときにお話しする先生が小学校の先生とかもいらっしゃって、その人たちは大体クリスチャンだったと思うので。小学校はたくさんいました。でも、中高だと人数も増えるので、クリスチャンじゃない先生も普通にいらっしゃいました。\n\n後藤:小学校はクリスチャンが多いということなんだけど、礼拝のときに話をするのは、宗教の先生とかじゃなくて、普通の先生がお話をするということ?\n\nB さん:はい。\n\n後藤:大体の先生がそこでお話をしてた。そうすると、小学時代のほとんどの先生がクリスチャンかどうかさておき、キリスト教について教えられるほどに、キリスト教の素養がほとんどの先生にあったということ?\n\nB さん:はい、小学校の時はそうでした。\n\n後藤:中高はそうでない人もそこそこ、結構いただろうなって感じで。じゃあ例えば親しい先生、歴史部の顧問の先生は違いましたか?\n\nB さん:違いましたね。\n\n後藤:クラス担任、学年によって違うと思うんですけど、大体クリスチャンじゃなかった?\n\nB さん:大体違いましたね。中高はそんなにいなかったと思います。・・・礼拝も外部の方を呼んだりすることも多かったので。\n\n後藤:えっ、そうなんですか?\n\nB さん:はい、他の礼拝、教会の方が来てくださって、みたいな話が多かったので。\n\n後藤:なるほど。そうすると聖書を教える先生以外は、あまりキリスト教徒かどうかは別として、キリスト教に詳しくはないと。キリスト教徒でも詳しくない人もいるけどね。\n\nB さん:あまり、はい」。\n\n要するに、小学校の教師はクリスチャン率が高かったと推測されるし、たとえそうではなくてもキリスト教についての知識が多かった27 が、同じ系列であるのに中学になると、そういう教師が減ったという。進学実績の重要性の低い小学校では情操教育の比重が大きいので、キリスト教に親和的な教員を配し、一方、受験等の進路が評価に影響する中高ではそういう教員が減っているという実態が窺われる。つまりキリスト教主義学校の維持・発展が自己目的化するという側面を考察すると、キリスト教主義学校の設置目的の推測として「キリスト教の信者を増やすこと」よりも若干ではあるが、「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」が多い点を、先ほどはミッション・布教の土着化（いいかえると「徳育としてのキリスト教」、使命としてもミッションといったトリプルな信仰に通じる面での定着も布教と考えること）のレベルで捉えたが、そのことの妥当性は、競争率の高さの維持等、より実務的な要因も含めて吟味の余地があることになる。信徒を得る、シンパを確保する等の、直接的なミッションではない、与えられた使命の自覚、徳育としてのキリスト教を含めた広い意味でのミッションでさえ、副次的になる可能性も大いにあるからである。\n\nマートンの順機能、逆機能の用語は用いないが、本節で述べたような状況を井上順孝も捉えている。井上は当該著書出版の時期の 1997 年「現在」の宗教系の学校の宗教教育の現状を次のように記していた。「さて、宗教系の学校が宗教教育をどのように位置づけているかという点からすると、現在は大きく二つのタイプを見いだすことができる。一つは、宗教教育の実践を大きな目標のひとつとして教育がなされる場合であり、もう一つは単に教育事業に進出するというだけで、宗教教育の実を上げることはほとんど目標としていないものである。極端に言えば、後者においては、教育が一つの事業として成り立てばいいのであり、学業やスポーツ、その他の活動において、優秀な学生を集め、その学校の知名度が上がることが、むしろ大きな目的である。前者のタイプの宗教教育は、戦前に多かったが、戦後はしだいに後者のタイプが主流になっている」（井上ほか 1997 p.20）。そして「現在」宗教系の学校の率の高いのが高等教育であるとも付言する。「一九九三年における、全国の小学校、中学校、高校、短大、大学の総数（分校、夜間部を含む）、うち私立の数、それに一九九三年度における宗教教団関連の学校数を検討してみると、教育機関に占める私学の比率は、とくに高等教育において顕著である。その中で宗教系の学校の占める比率はきわめて高い」（井上ほか 1997 p.20~21）。この井上編集の著書から 25 年経ている現在、ますます「後者のタイプ」、すなわち「優秀な学生を集め、その学校の知名度が上がることが、むしろ大きな目的」である宗教系学校の増えていることは想像に難くない。\n\nなお、コロナ禍の影響もあって、我々はキリスト教主義学校の幹部や聖書・キリスト教学の教員にまだインタビューできていない。またカトリックとプロテスタントの学校の差はウェブモニターの中で多数派ではないキリスト教主義学校通学経験者をさらに分割することになるので、まだ捉えられていない。さらに先述の「日本社会の「トリプル信者」を生み出す要素はいろいろあるだろうが、重要なのは中学校・高等学校の教育ではないかと思う」（井上ほか 2018 p.139）という先行研究での発言に本研究もまずは依拠したので、大学のみキリスト教主義学校に在籍した者と下から上がってきた者との違いも充分に調査できてはいない。これらの側面からの吟味は今後の課題としたい。\n\n\n6. 本稿全体の結論\n\n本稿は科研費挑戦的萌芽研究「キリスト教主義学校から見る日本人の寛容と洋化-ステークホルダーらの期待と文化資本」（2018~2020年度）（課題番号:18K18590）のウェブモニター調査の一部（なおかつ最重要と我々が思う部分）の変数のクロス集計結果の分析報告であり、それの結果報告の具体的に肉付けに必要な限りでのインタビューでの発言やウェブ情報、文献資料からの引用を加えたものである。\n\nこの研究全体の研究目的は 1.2「本研究の目的」に記したように、日本のクリスチャンの数は日本の人口比で 1.6% 前後しかいないにもかかわらず、キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多いのはなぜかを究明することである。\n\nこの全体の研究目的に即しつつウェブモニター調査の作業仮説に移行するための作業仮説として、1.2 では、「キリスト教主義学校は何を目的として設立されているか」ということの解明が、《「日本の人口比で 1.6% 前後しかいない」という事実が、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況と矛盾する状況》の解明に役立つ作業仮説であると考え、これを作業仮説1とした。\n\nまたもう一つの、この全体の研究目的に即しつつウェブモニター調査の作業仮説に移行するための作業仮説としては、これも 1.2 で既述のことではあるが、この全体の研究目的の《「日本の人口比で 1.6% 前後しかいない」という事実は、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況と矛盾する状況》である点に着目している。この人気の理由の解明には、キリスト教主義学校の生徒・学生イメージを、人びとがどのように捉えているかを見ることである。よってこれもこの全体の研究目的に適う作業仮説であると考えられる。これを作業仮説2とした。\n\nここで挙げた作業仮説1に対応させて、我々のウェブモニター調査では、キリスト教主義学校設立目的の推測を問う設問Q34を設けた。分析の際は、この設問の各選択肢ごとにその選択肢を MA ベースにおいて選んだ人を 1、選ばなかった人を 0 とする離散変数を作った。このそれぞれの選択肢による離散変数を従属変数として、キリスト教主義学校当事者性に関わる Q12 の組変数 Q12G を主な独立変数として、クロス集計を行った。この結果は 3.1 で概ね記した。\n\n4.「調査結果のまとめと結論」で記したことをこの段落ではほぼ繰り返すが、Q34 の分析結果として、キリスト教主義学校に通った者等、キリスト教主義学校への関与度の高い者の方が、関与度の低い者に較べて、キリスト教主義学校の設置目的として信者の獲得を挙げる者の比率は、有意差がない（p値=0.13）ものの、少ない傾向にある。なお、有意差がなかった理由を本稿 2.1 では検出力不足（データ数の不足）によるものと考察した。\n\n他方、「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校に通ったことのある者の方に多い（カイ二乗<0.01）ことも分かった。また「信者の子弟の教育要求への対応」、「教会以外の、キリスト教に触れる敷居の低い舞台としての役割を果たすこと」を選ぶ者も、キリスト教主義学校に通った者等、キリスト教主義学校への関与度の高い者の方に多いことが分かった。なお、この「「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校に通ったことのある者の方に多い（カイ二乗<0.01）こと」については、後述する作業仮説 3 のトリプルな信仰にも関連する。\n\n作業仮説 1 についてここで小括するが、作業仮説1は「キリスト教主義学校は何を目的として設立されているか」ということの解明が、《「日本の人口比で 1.6% 前後しかいない」という事実が、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況と矛盾する状況》の解明に役立つ作業仮説であるというものであった。これとこの前段落の結果と照らし合わせると、布教という目的を日本のキリスト教主義学校は放棄しているとまではいえないまでも、ほとんど強く有していないから、「日本の人口比で1.6%前後しかいない」という事実と、「一定程度以上の定員比率を誇り、また人気を集める学校も多い」という事実とが矛盾しないことになっているといえる。\n\nつぎにここで挙げた作業仮説2に対応させて、我々のウェブモニター調査では、キリスト教主義学校の生徒・学生イメージについて Q29 で聞いた。分析方法は、Q34 と基本的に同様で、この設問の各選択肢ごとにその選択肢を MA ベースにおいて選んだ人を 1、選ばなかった人を 0 とする離散変数を作り、このそれぞれの選択肢による離散変数を従属変数として、キリスト教主義学校当事者性に関わる Q12 の組変数Q12G を主な独立変数として、クロス集計を行うことにした。この結果は 3.2 で記した。\n\nこれも作業仮説 1 の場合と同様、4.「調査結果のまとめと結論」で記したことと、この段落とこの段落の次の段落はほぼ重複するが、井上ほか (2018) はキリスト教主義学校の女子は「きれい、金持ち、キリスト」の 3K であるということを強調していて、佐藤 (2006) でも同様の記載はあったので、その点をまず吟味した。金持ちであるイメージについてはある程度妥当した。しかしきれいという点はまったく妥当しないことが分かった。3Kのうちキリストは同義反復的であるので措くとすると、1 勝 1 敗である。また井上ほか (2018) ではキリスト教主義学校では愛と奉仕の精神を教育目標に掲げるところが多いと、実際の教育効果の検証なしに、ウェブページの分析をして述べていた。したがって「他人を思いやる心が豊かである」を Q29 の選択肢に入れた。これは Q12G とクロス集計すると、キリスト教主義学校当事者の方が有意にこれを選ぶ者が多かった。しかし実際に通ったことのある者よりも当事者性の低い、緩やかな当事者の方がこれを選ぶ者が多かった。その点で、これは「他人を思いやる心」を涵養させうるだろうという家族等周囲の期待がその数字に表れていると考えられる。またこの Q29 で「該当なし /答えたくない」を除き最も選ばれたのは「外国語が得意である」である。女性皇族のキリスト教主義学校関与の是非を問う Q11G とクロス集計すると、キリスト教主義学校への関与を是とする者ほど、「外国語が得意である」を選んでいることが分かった。また Q12G とクロス集計すると、キリスト教主義学校関与度と関連はあったが、通学経験者よりは緩やかな当事者の方に「外国語が得意である」を選ぶ人が多かった。また「話し上手である」を選んだ人選ばなかった人をクロス集計すると、やはり通学経験者よりは緩やかな当事者の方にこの選択肢を選ぶ人が多かった。\n\nまたこの Q29 でその次に選ばれたのは、「教養が豊かである」で、これも女性皇族のキリスト教主義学校関与の是非を問う Q11G とクロス集計すると女性皇族のキリスト教主義学校への関与を「望ましい」と考える者は、「教養が豊かである」を選ぶ人が多かった。またこれを Q12G とクロス集計すると、キリスト教主義学校にかかわりの高い者ほど、キリスト教主義学校出身者を「教養が豊かである」と自認していることも分かった。\n\nこれらの結果から、女性皇族のキリスト教主義学校関与を是と捉える層からの皇室外交への期待がうかがわれる。また「外国語が得意である」と「教養が豊かである」が、Q29 で「該当なし /答えたくない」を除き一番目、二番目に選ばれ、「他人を思いやる心が豊かである」が六番目であることを考察すると、キリスト教主義学校の意義を、宗教教育をはじめとする情操の涵養のレベルで捉えるよりも、外国語や教養などのより実利レベルで捉える人の方が多いということになる。\n\nそこで作業仮説2に関する小括であるが、作業仮説 2 は「キリスト教主義学校の生徒・学生のイメージを、キリスト教主義学校の当事者を含めた世間の人びとはどのように考えているか」ということの解明が、《「日本の人口比で 1.6% 前後しかいない」が、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況》の解明に役立つ作業仮説であるというものであった。これとこの前々段落と前段落の結果と照らし合わせると、少なくとも東京・神奈川在住の若年層においてキリスト教主義学校が、井上ほか (2018) のいうような3Kだというブランドイメージが少なくも「きれい」ということについて定着しているわけではない可能性が分かった（有意差なしは証明が難しいが「きれい」については実数そのものさえも少ない）。しかし「外国語が得意である」と「教養が豊かである」が皇族女子のキリスト教主義学校への関与を是とする人から選ばれていることから、やんごとなきところに親和性があるという意味でのブランドイメージはあるといえよう。また「外国語が得意である」「他人を思いやる心が豊かである」「話し上手である」という選択肢は、キリスト教主義学校関与度と有意な関連はあったものの、通学経験者よりは緩やかな当事者の方にこれを選ぶ者が多かった。このことはキリスト教主義学校の生徒・学生イメージのうちの肯定的なもののいくつかは、通学経験者よりは緩やかな当事者の方に支持されているイメージであるといえるであろう。したがって作業仮説に合わせて考えると、キリスト教主義学校の肯定的イメージが《「日本の人口比で 1.6% 前後しかいない」が、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多い」という状況》をもたらしているといえるが、それら肯定的イメージのいくつかは、キリスト教主義学校の通学経験者よりは緩やかな当事者から支持されているということが分かった。つまり現実よりはイメージ先行の部分もあるということである。\n\nまた「外国語が得意である」と「教養が豊かである」が、Q29 で「該当なし /答えたくない」を除き一番目、二番目に選ばれ、「他人を思いやる心が豊かである」が六番目であることを考察すると、キリスト教主義学校の意義を、宗教教育をはじめとする情操の涵養のレベルで捉えるよりも、外国語や教養などのより実利レベルで捉える人が多いということになる。その点でも作業仮説1での分析結果で申し上げた、布教という目的を日本のキリスト教主義学校はほとんど強く有していないから、「日本の人口比で 1.6% 前後しかいない」という事実と、「一定程度以上の定員比率を誇り、また人気を集める学校も多い」という事実とが矛盾しないということを、補強的に裏付ける結果であるともいえる。\n\nただ Q34 について先にふれたように、「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校に通ったことのある者の方に多い。この問題はトリプルな信仰に関する作業仮説 3 に通じる。\n\n1.4 で論じたように、作業仮説 1 や作業仮説2での議論は、「「日本のクリスチャンの数は日本の人口比で1.6% 前後しかいないにもかかわらず、キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多いのはなぜかを究明すること」という命題の後半部分、「キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多いのはなぜかを究明すること」に焦点を当てていた。他方、もしもトリプルな信仰が成立するとすれば、「日本のクリスチャンの数は日本の人口比で 1.6% 前後しかいないにもかかわらず」という部分を吟味することになる。よってトリプルな信仰についての意識がキリスト教主義学校通学経験者の方が、それ以外よりも多いかどうかを、作業仮説3とし、主にこれは本稿 3.3 で論じた。\n\nキリスト教主義学校の通学経験者はそうでない者より、原家族と共にキリスト教以外の仏教や神道の宗教的行事に参加してきた。この結果は井上ほか (2000) でカトリック系の高校生が、神道系高校の生徒以上に、初詣をするという過去の知見も我々と同様の方向性を示しているといえる。したがってキリスト教主義学校の通学経験者は、キリスト教にまつわる行事・習俗であるか否かを問わず宗教あるいは宗教にまつわる習俗を尊ぶ環境に育ってきた者が多いといえる。そのことはトリプルな信仰ということをある意味で裏づける。「ある意味」と留保をつけたのは、習俗が形式のみであるという批判もありうるからである。ただし井上ほか (2018) の佐藤 (2006) への言及部分に関連づけて1.3.2にて申し上げたように、習俗、形式が内面化すると信仰として精神化しうる。したがってトリプルな信仰に結びつきうる。確かにキリスト教主義学校の教育効果としてトリプルな信仰があるかどうかはここでは分からないが、トリプルな信仰への期待、いいかえると宗教教育を通じての情操の情操の涵養への期待のなかで、キリスト教主義学校の通学経験を有する回答者自身が、両親等の保証人から、キリスト教主義学校に送り込まれた可能性を示す結果といえる。つまり同志社でいわれたとCさんがいう、「徳育としてのキリスト教」という発言とも整合する。また作業仮説1の箇所で申し上げた、Q34 の「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校に通ったことのある者の方に多いということは、単に情操教育の対象として以上に、トリプルな信仰に通じる場として実際にキリスト教主義学校が機能していることを裏づける。\n\nさらにそのことを裏づけるものが Q49 の結果である、Q49 は「以下の宗教において、入信していなくても教えを守ることによって、救済やご利益（ごりやく）が得られると思いますか。当てはまるものをそれぞれ選んでください」という設問で神道と世界三大普遍宗教合わせて 4 宗教について同じ尺度で聞いている。キリスト教主義学校通学経験者の方がキリスト教について「そう思う」人が多い。\n\n以上の結果から作業仮説3は裏付けられた。\n\n井上ほか (2018) における郭の議論からすると、既成仏教や神社神道においては、入信というプロセスを経ないでも、それらの宗教の信仰者といえる。それと同様、キリスト教も、一般の日本人の意識からすると、もしも洗礼というプロセスが不要であれば、仏教や神道と同様、三つ目の信仰の対象としうるということになる。要するに、その場合には「1.6%」というのは見かけ上の数値に過ぎないということになる。洗礼というプロセスは経ないもののキリスト教主義学校通学経験者の方がトリプルな信仰に親和的なことが分かった。\n\nこれによって科研費研究全体の研究目的「「日本のクリスチャンの数は日本の人口比で 1.6% 前後しかいないにもかかわらず、キリスト教主義学校が日本の私立学校のなかで一定程度以上の定員比率を誇り、また人気を集める学校も多いのはなぜかを究明すること」という命題のうち「日本のクリスチャンの数は日本の人口比で 1.6% 前後しかいない」部分が、じつは見かけ上低い数値であるにすぎない可能性が示唆される。\n\nさらにこのことと関連してキリスト教主義学校のウェブ調査等の検討から、キリスト教主義学校の教育目的を布教という狭い意味のミッションよりは、神に与えられた使命としてのミッションを果たすという広い意味で、当事者たちから理解されていることが分かった。またこの「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校に通ったことのある者の方に多いということは、洗礼と非洗礼で一線を画し、首尾一貫性を重んじるという、従来いだかれてきた日本のキリスト教像とは異なった見方を、キリスト教主義学校当事者たちがとっているということにもなる。これは先行研究の井上ほか (2018) のいう「トリプルな信仰」としてのキリスト教という考え方に照応する結果であるともいえる。もちろん教育基本法の縛りの影響も勘案する必要があり、今回そこにあまり言及出来てはいない。今後、キリスト教主義学校の経営陣へのインタビューをする中で、その面も含めて、より明確にしていきたい。\n\nICU元副学長で東京女子大学学長を現在務める森本あんりは、『不寛容論―アメリカが生んだ「共存」の哲学』でロジャー・ウィリアムズ (1603~1683) の寛容論を高く評価し、彼について多くの章を割いて分析・紹介する。裕福な家庭のお抱え牧師だったウィリアムズは、先住民の権利等、当時としては過激な主張をしてマサチューセッツ植民地から追放され、今のロードアイランド州に赴き、そこの先住民を対等な立場の人間として扱い、『アメリカ語理解の鍵』という本も著した。ここでいう「アメリカ語」とは先住民の言語の意味である（森本 2020 p.129）。そこで扱われている例文は先住民の生活ぶりを内側から観察して得た内容になっている。このように非常に先住民からの信頼の厚かったウィリアムズではあったが、形だけの宣教を彼は拒否する。「自分がその気になれば、何千という先住民を、いや彼らの国全体を、キリスト教へと回心させることだってできる、と彼は豪語している。先住民の間で彼が得ていた信頼の厚さを考えれば、それも「から威張り」とは言えないだろう。けれども彼は、洗礼という「儀式」で外面的なキリスト教徒を作ることには反対なのである。むしろそれは、反キリストの業である。外から仕向けられた回心は、真の信仰を伴わない。ウィリアムズにとって、伝道とは「回心者の獲得」ではない。先住民であろうとイギリス人であろうと、ただ神の恵みによって生まれ変わることを求めるだけである。信仰は内心の自由からのみ生まれるからである」（森本 2020 p.134~135）。\n\nこのようなウィリアムズの宣教と洗礼についての議論と、Q34 の「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」を選ぶ者が、キリスト教主義学校に通ったことのある者の方に多いということとは照応するのではないだろうかという思いを胸に、本稿を閉じることとしたい。\n\n\nデータ可用性\n\nFigshare:キリスト教主義学校に関する調査_ローデータ20200324.xlsx. https://doi.org/10.6084/m9.figshare.21366417.v1 (Goto, 2022a)\n\n上記プロジェクトには下記の基礎データが含まれている。\n\n・キリスト教主義学校に関する調査_ローデータ20200324.xlsx（ウェブモニター調査の回答）\n\nFigshare:キリスト教主義学校に関する調査　調査票画面.pdf. https://doi.org/10.6084/m9.figshare.21367356.v1 (Goto, 2022b)\n\n上記プロジェクトには下記の拡張データが含まれている。\n\n・キリスト教主義学校に関する調査　調査票画面.pdf（本論文で扱われたウェブモニター調査の調査票）\n\nFigshare:キリスト教主義学校調査票画面についての補足説明.txt. https://doi.org/10.6084/m9.figshare.21367632.v1 (Goto, 2022c)\n\n上記プロジェクトには下記の拡張データが含まれている。\n\n・キリスト教主義学校調査票画面についての補足説明.txt（調査票の補足説明）\n\nFigshare:キリスト教主義学校　インタビュー調査　調査依頼書.docx. https://doi.org/10.6084/m9.figshare.21367776.v1 (Goto, 2022d)\n\n上記プロジェクトには下記の拡張データが含まれている。\n\nキリスト教主義学校　インタビュー調査　調査依頼書.docx（インタビュー調査の依頼書）\n\n以上のデータは、クリエイティブ・コモンズ表示 4.0 国際 Creative Commons Attribution 4.0 International license (CC-BY 4.0) の条件下で利用することができる。\n\nなお、本論文のインタビューの回答は、本論文に含まれている部分を除いて、公開することができない。インタビューの内容が調査対象者の私的な情報や対象者を特定できる情報、ライフストーリーや思想・信条上の問題など対象者に不利益を生じうる情報を多く含むために、データを非公開とすることが倫理審査に基づく同意書に書かれていることが、その理由である。同分野の研究者や査読者がデータの閲覧を希望する場合には、データの利用目的と方法を明記の上、責任著者後藤宛て (ygoto@slis.tsukuba.ac.jp) に連絡して頂きたい。調査対象者と逐次相談の上、開示可能な箇所を開示する。\n\n\n謝辞\n\n髙井啓介先生（関東学院大学）には、調査票のキリスト教にかかわる部分を事前にご校閲いただく等、諸般にわたりご教示賜りました。ここに記して深く感謝の意を伝えたいと思います。また、本稿にそのインタビュー記録の一部を引用させていただいた A さん、B さん、C さんはじめ、我々のインタビューに応じて下さり、貴重な話を提供してくださった方々にも、感謝いたします。", "appendix": "参考文献\n\n一般社団法人キリスト教学校教育同盟:設置校数表: 2022年10月9日最終閲覧日.Reference Source\n\n井上順孝, et al.:宗教と教育―日本の宗教教育の歴史と現状.弘文堂:國學院大學日本文化研究所編;1997.\n\n井上順孝, et al.:科研費報告書「現代日本における宗教教育の実証的研究」(1998~1999)報告書.國學院大學;2000.\n\n井上章一, et al.:ミッションスクールになぜ美人が多いのか.朝日新聞社;2018.\n\n苧阪良二:明治から昭和初期にいたる実験心理学の形成過程.心理学評論. 1998; 41 (3): 333–358.\n\n内村鑑三:余は如何にして基督信徒となりし乎. 鈴木俊郎（翻訳）. 岩波書店. 1958.\n\n学校法人雙葉学園:教育理念.最終閲覧日2022年10月9日.Reference Source\n\n加藤周一, 木下順一, 丸山真男, et al.:日本文化の隠れた形.岩波書店; 2004.\n\nクリーグ波奈:キリスト教主義学校の役割とその教育的意義.東京大学教育学研究科紀要;2017; 56. : 377–387.\n\nGoto Y:キリスト教主義学校に関する調査_ローデータ20200324.xlsx. figshare. [Dataset].2022a. Publisher Full Text\n\nGoto Y:キリスト教主義学校調査票画面についての補足説明.txt. figshare. [Dataset].2022b. Publisher Full Text\n\nGoto Y:キリスト教主義学校調査票画面についての補足説明.txt. figshare. [Dataset].2022c. Publisher Full Text\n\nGoto Y:キリスト教主義学校　インタビュー調査　調査依頼書.docx. figshare. [Dataset].2022d. Publisher Full Text\n\n佐藤八寿子:ミッション・スクール―あこがれの園.中央公論社;2006.\n\nChristian Today:少子化、信徒教員の減少、迫る教育改革　カトリック学校の現状と将来(品田典子(講演者)(2018年11月29日23時05分). 2022年10月9日最終閲覧日.Reference Source\n\n島本千也:鎌倉別荘物語.岩森書店;1994.\n\n白百合学園中学校・高等学校:宗教教育: 最終閲覧日2022年10月9日.Reference Source\n\n武田清子:背教者の系譜.岩波書店;1973.\n\n武田清子:未来をきり拓く大学―国際基督教大学五十年の理念と軌跡―.国際基督教大学出版局;2000.\n\n竹中正夫:日本組合基督教会の歴史と課題:その百年にあたって.基督教研究.1987; 48(2):128–172.\n\n田中裕:「解説」西田幾多郎講演集.西田幾多郎著. 岩波書店、所収;2020.\n\n玉川学園:「波多野精一―小原國芳の師、玉川の丘へ」:2014.03.18. 最終売閲覧日2022年12月4日.Reference Source\n\n長野清泉女学院:よくある質問.最終閲覧日2022年10月9日.Reference Source\n\n日本学術会議社会学委員会Web調査の課題に関する検討分科会:提言　Web 調査の有効な学術的活用を目指して(2020).最終閲覧日2022年10月9日.Reference Source\n\n橋本満:近代日本における「宗教」の発見，あるいは宗教社会学の起点.日本語・日本学研究.東京外大;2015; 133–144.\n\n波多野精一:基督教の起源　他一篇.岩波書店;1979.\n\n濱田陽:「無宗教」への「対話」一チャペル・ウェディングと、日本のキリスト教一.宗教と社会;2001; 7. : 23–46.\n\n廣瀬つぎ子:雙葉小学校創立三十年記念誌　小百合 (1941). のびのび会\n\nフェリス女学院中学校・高等学校:キリスト教活動:　最終閲覧日2022年10月9日.Reference Source\n\nベラー・ロバート:1991.心の習慣―アメリカ個人主義のゆくえ(原著1985=邦訳1991). 薗進ほか訳.みすず書房;\n\n堀孝:「内村鑑三」と出会って.勁草書房;1996.\n\nマートンロバート・キング:社会理論と社会構造(原著1949=邦訳1962). 森東吾ほか訳.みすず書房;1962.\n\n丸山真男:日本の思想.岩波書店;1961.\n\n森孝彦:シカゴ万国宗教会議:1893年.同志社アメリカ研究1990; 26. : 1–21.\n\n森本あんり:不寛容論―アメリカが生んだ「共存」の哲学.新潮社;2020.\n\n森本あんり:「日本人は多神教だから寛容」通説は本当なのか　統計調査をもとに考察する「無寛容の正体」(2021年2月8日). 東洋経済オンライン. 2022年10月9日最終閲覧日.Reference Source\n\n立教女学院中学校・高等学校:立教女学院のキリスト教教育: 最終閲覧日2022年10月9日. 立教女学院中学校・高等学校:立教女学院のキリスト教教育: よくある質問. 最終閲覧日2022年10月9日.Reference SourceReference Source\n\n六十年史編集委員会編:六〇年の歩み. 立教女学院小学校.1997.\n\n若松英輔:内村鑑三.岩波書店;2018.\n\n和辻哲郎:和辻哲郎全集第6巻.岩波書店;1962.\n\n\nFootnotes\n\n1 栄光学園中学・高等学校は以前から神父を兼ねる教諭が同じイエズス会の上智大学に転出するケースはあった。例えばヨゼフ・ピタウ神父（のちの大司教）は若い頃栄光学園中学の教諭を務め、そのあとハーバードで学位を取り上智大学で教え、学長も務めた。もっとも 2016 年に学校法人栄光学園が同じイエズス会の六甲学院、広島学院、上智福岡中学校・高等学校と共に上智学院と法人統合したので、現在では上智大学と同一学校法人の下にある中学・高校となっている。\n\n2 例えば我々の調査では Q31 で「あなたの日本のキリスト教主義学校のイメージを形づくった学校の種類」を SA で性別と課程を組み合わせた 9 選択肢から一択で聞いたが、「特にどれとはっきりイメージしていない」を除き、最も多いのは「中学校高校女子校」 17.8% であり、大学ではない。他方、「中学校高校男子校」 4.4% 、「中学校高校共学校」 11.3% ゆえ、中学校高校の限りではイメージとしての女子バイアスはある。しかし大学については、現代日本で男子大学はないという現実の非対称性の問題もあるが、「女子大学」 8.0% に対して「大学・共学」 14.7% で、ここでは女性バイアスもない。\n\n3 本科研費研究交付年の 2018 年時点では「現代日本における宗教教育の実証的研究」 (1998~1999；研究代表者井上順孝）で行われた研究会やシンポジウムについてのウェブ情報が多くあって、本研究のウェブ調査の調査票を作るに際して大いに参考にした。ここもその一つである。ただし 2022 年度現在、この 1998~1999 の共同研究参加者の多くが退職されたためか、それら当時閲覧したウェブサイトの大部分が閉じられてしまっている。よってこの研究の報告書（井上順孝（研究代表者）科研費報告書『「現代日本における宗教教育の実証的研究」 (1998~1999) 報告書』 (2000、國學院大學）を 1. で記したように借り出し閲覧したが、ウェブに載っていた多くの情報はここには載っていない。閲覧時にログを保管しそこなった本稿筆頭筆者の怠慢のなせる業である。したがって、ここでは研究名のみに言及し、頁数や URL には言及しない。\n\n4 ただこの現象は日本だけというのも早計である。WHAM! の Last Christmas 等、すでにスタンダードと化している洋楽の多くもいわゆるアガペーではなくエロースを扱っている。\n\n5 佐藤卓己は京大教育学部に転出する前、日文研助教授として井上の後輩同僚であった。\n\n6 ヒンズー教の牛、イスラム教の豚、仏教の僧侶の肉類全般のように、食のタブーは多くの宗教につきものである。\n\n7 もっとも以下の記事公開時点で ICU 教授で副学長も務めた牧師の森本あんり（現、東京女子大学学長）は、日本人の寛容性は限定付きのものであるという。そもそも無宗教的で、宗教に無関心故、他者の宗教に寛容ではある。他人事である限り寛容であるが、「寛容の問いが自分自身に及び、深刻な利害が身の回りにひた寄せてくると」、容易に不寛容に転化しうる、という（森本あんり「「日本人は多神教だから寛容」通説は本当なのか　統計調査をもとに考する「無寛容の正体」」. 東洋経済オンライン）。このような森本の指摘にも、本研究の限界として、留意が必要である。このネット記事の元となったと称する著書で森本は次のように言う。「日本人は、クリスマスとお正月を一緒に祝い、生まれたときにはお宮参りをし、結婚式を教会で挙げ、葬式は寺に依頼する。だから宗教に寛容だ、というのが通説だが、こうして見る限り、どうやらそれはうわべだけの話のようである」（森本 2020 p.6）。一方この著書で森本は「本書が「中世的」と表現してきた寛容理解は、ごく大ざっぱに言うと、寛容論の分野で「伝統的な寛容」と呼ばれるものにあたる。この意味での寛容は、相手をしぶしぶ認めることである。相手を是認せず、その思想や行為に否定的であり続け、できれば禁止したり抑圧したりしたりしたいが、そうもいかないので、しかたなくその存在を認める、という態度である」（森本 2020 p.278）。それに対して最近の学説は、相手を承認する「肯定的寛容」「強い寛容」「認知としての寛容」「水平的寛容」であるという。しかし森本は「それらの主張もよくわかるのだが、ふりかえって自分の足元を見れば、それが常に可能というわけではないだろう」（森本 2020 p.279）とも述べて、「肯定的寛容」等の現代的な寛容がかえって反動としての「リベラル疲れ」をもたらす懸念も表明する。さらに現代的な寛容は「自分の信念への妥協を強いる」（森本 2020 p.280~281）可能性があるともいう。「相手にどういう態度で接するかだけでなく、内心で相手をどう評価するかまで寛容であれと要求するのは、潜在的にはとても不寛容である」（森本 2020 p.275）。\n\n8 しかも南原の師である内村はまだ禁教が解けて日の浅かった日本の既存のプロテスタント教会からさえ、不敬事件、再臨運動、それぞれの折に、厳しく批判されてきた（若松 2018）。\n\n9 読者モデルの掲載される誌面は私服紹介のコーナーが多く、2000 年代のファッション誌は今と違いプチプラコーデの記事が稀であったことからも、たとえ金持ちではなくても、基本的に少なくとも金持ちを装うことも、きれいと同時に必要とされたはずである。\n\n10 注 (3) と同様。なお、「現代日本における宗教教育の実証的研究」（1998~1999；研究代表者井上順孝）より前の成果ではあるが、井上ほか (1997) において、田口めぐみが「中等教育における宗教教育の意義」という論文で、宗教系の中等教育機関で行われる宗教教育は、宗教への関心を高める等、宗教教育としての効果はあるが、その学校の宗教を信じさせるという宗派教育としては有効ではないと述べた上で、次のように書く。「ただし、自分が通った学校がもつ宗教には、多少の親近感をもつようだ。仏教系の学校に通った学生は、日本人の宗教は仏教であると答えた者が他の宗教系に通った学生より若干多かった。キリスト教系の学校に通った学生は、「キリスト教をテーマにした小説」には他の宗教系の学校に通った学生よりも高い関心を寄せている。」（井上ほか 1997 p.225）。これらの記述はある意味、その宗教のシンパを形成していると見ることはできる。ただし、それを目的としているというよりは結果的にそうなっているということではあるが。\n\n11 ここも注 (3) と同様。一方、井上ほか (1997) において井上順孝は教育基本法の国公立学校での宗教教育の制約を指摘したうえで、「他方、宗教に関する寛容の態度及び宗教の社会生活上における地位は尊重され、また、私立学校においては、軍国主義的教説以外であれば、すべての宗教教育を自由に行えることとなった」（井上ほか 1997 p.11）と記す。さらに「宗教教育は、通常、三つのカテゴリーに分けて考えられている。第一は、特定の立場にたった宗教教育、第二は、宗教知識教育、第三は宗教的情操教育である。私立学校においては、児童生徒の信教の自由を認めた上でなら、この三つとも可能である」（井上ほか 1997 p.19）とも記される。\n\n12 ただキリスト教主義学校の教職員で自分自身や家族の通学歴等ない者をこの Q12 では漏らしてしまった点は、今後の検討課題である。\n\n13 属性項目なので SA で聞いたが、トリプルな信仰の有無という本研究のテーマの性格上、MA と SA の併用もありえた点は、調査票作成上の反省点である。\n\n14 本論では、キリスト教主義学校としてカトリックとプロテスタントを分けずに扱っているが、我々の共著者の一人は、近日まで、カトリックの中高一貫校とプロテスタントの中高一貫校にそれぞれ別の娘が通っていた。その共著者から、自分のような弱い当事者の視点からは、カトリック学校とプロテスタント学校には、「ミッション」のとらえ方、その「ミッション」を達成するための方策には違いがあると見受けられる、との意見が示された。プロテスタントとカトリックの「ミッション」概念の違いを視野に入れることは、今後の課題になる。\n\n15 キリスト教主義学校以外も含めた学校法人全般においては、そういう疑念をもたれる実例が絶対にないとはいえないが、そうすると理事長には背任の疑いがもたれ、学校法人自体も私学助成金の大幅減額等、文部科学省から大きなペナルティを受ける可能性を伴うことは 2021 年新聞報道等を賑わせた事例からも明らかである。\n\n16 なおプロテスタントの学校は佐藤のいうようにキリスト教伝道団体から独立しているが、カトリックの場合、法律上宗教法人と学校法人は別法人であるという点は措いて、それぞれの修道会が事実上系列の学校法人を監督している。したがってプロテスタントの場合ほどにはミッション・スクールでは「ない」ということを強調するモチベーションは、カトリックの場合は存在しないと推察できる。\n\n17 ここの本文で「等」というのは井上ほか (2018) で直接言及はされていないが、キリスト教主義学校卒業生以上に、一高校長（兼東大教授）新渡戸とその友人内村が、一高、東大に進む政財官学界の有力者の卵をプロテスタントに改宗させた部分も大きいはずだからである。新渡戸自身、南部藩の上級藩士の家柄で札幌農学校入学前から階層の高い人間である。内村も士族である。\n\n18 なお佐藤 (2006) は、ミッション・スクール的なリベラル・アーツは、それを学ぶことによって人を自由にするもの（佐藤 2006 p.147）で、他方、竹内洋が対象とするような旧制高校の修養主義的な教養主義はむしろその対極にある（佐藤 2006 p.156）と論じているが、新渡戸自身、一高というトップの旧制高校の校長と東大教授を務めた人物でもあり、なおかつ代表作の一つに『武士道』という著書もあるので、対比する部分と同時に連続する部分もあるはずで、その点の検討は今後の課題となる。しかし竹内のいう教養主義はデカンショマルクスというドイツ観念論と唯物論を中心にしたもので、英米の教養を身につけた新渡戸とは異質な部分も多い。新渡戸における教養とは何かを問うことは、リベラル・アーツ的な教養と修養主義的な教養との連続性と異質性を見る手がかりになると考えられる。\n\n19 15~29歳を対象とした調査のため、結婚による階層再生産戦略が検出できていない可能性がある。結婚による階層再生産戦略については、より高い年齢層を対象とした調査が必要だと考える。\n\n20 先述のように、井上はファッション誌の読モ（読者モデル）にキリスト教主義学校出身が多いことを数え上げていたが、読モ起用率の高さが若年層向き女性ファッション誌の売れ行き不振や休刊を招いた一因ともされる。読モレベルの容姿の女性の着こなしなら今では無料で SNS で閲覧できるからである。それどころか最近は SNS のインフルエンサーが女性ファッション誌の誌面に登場したり、専属モデルに抜擢されたりすることも多いし、男性向けのグラビア誌でもインフルエンサーの起用率は高い。SNS で人気を博するのに、通常、通学している学校は関係ないので、キリスト教主義学校の通学者が特にそこで目立つわけではない。現代の若い世代は、そのような情報環境に生きているので、このような結果になっている可能性もあると推測される。\n\n21 波多野は『基督教の起源』にてまずユダヤ教の特質を律法にみる。「ユダヤ教の基礎は「律法（おきて）」である。ユダヤ人は「律法」において神の聖なる意志を見た。真の宗教と偽の宗教と、また神の選民と他のすべての民族（異邦人）とを区別するは律法と考えられた」（波多野 1979 p.12）。よって「もと無意識的に行われた風俗習慣が神の命令として意識的に守られるべきものとなり、その煩瑣な規定を加うるに至っては、律法の宗教的意義は問題とならざるを得ない」（波多野 1979 p.15）。というのも国語さえ失ったユダヤ人にとって、国民の特色を最も表わすものは風俗習慣であるからである。「道徳は異邦人とてもっている。これに反して、風俗習慣において国民の特色は最も目立って現れている」（波多野 1979 p.15）。したがって民族に固有の風俗習慣を重んじるユダヤ教は、ユダヤ人にとっての宗教という党派性を脱し得なかった（波多野 1979 p.18~19）。そのようなユダヤ教にとって「神の国」の実現とはユダヤ人国家の再現という地上的なものであった。一方、イエスの「神の国」という言葉には、こういうユダヤ的地上的な部分もあったが、排他的ではなくより普遍的なものであった。「彼は政治より宗教を、ユダヤ人より人間を、イスラエルよりも神を重んじた」（波多野 1979 p.64）。このイエスの普遍的な側面を強調したのがパウロである。「十字架とメシアとの全く相容れぬ両端は今や相結び附いて、ユダヤ人的感情の誤れると律法の宗教上に全く価値なきとを証明した。それ故にユダヤ人たるはもはや宗教上必要の条件ではない。・・・かくしてパウロは、始めてキリスト教を、ユダヤ教を超越した人間的世界的なる新しい宗教として経験した」（波多野 1979 p.134）。また「イエスの神は愛の神であった」（波多野 1979 p.100）。然るにユダヤ教においては神は王であり、主であった。「神は主、人は僕（しもべ）―これはユダヤ人に最も普遍的なる観念であった。神の万能は何事も勝手気儘になすを得。ユダヤ人は、この近づき難き親しみ難き専制君主に服従するを、神を愛すると名づけた」（波多野 1979 p.17）。すなわち「パウロによれば、イエスは律法のなし得ぬ人類の救いをなし遂げんために世に送られたメシヤ（キリスト）即ち（特別の意味における）神の子である」（波多野 1979 p.168）。「パウロにとっては「恵みによってのみ義とせらる」と「信仰によってのみ義とせらる」とは全く同義であった。信仰とは何ぞ。そは人類を救わんとする神意に子供のごとく信頼するの謂である。吾人自身は罪人、何らほこるべき功もない、ただ謙遜と感謝とをもって神の恵みを受くべきのみである。この態度こそ信仰である」（波多野 1979 p.172）。このようにパウロは律法ではなく、信仰こそが救いに通じると解した。\n\n22 そもそも背教するにしても有島武郎のようにそのこと自体を、大きな思想的宿命、自分の背負っていく枷として考えていた人も一定数いたと考えられるが、そのイメージばかりが従来先行していて、佐藤 (2006) の指摘する老婦人のような人の存在にはあまり世間の意識が向かなかった可能性も考えられる。\n\n23 プロテスタントだとプロテスタントのなかでの宗派自体は親と違うことは割とあるだけでなく、ロバート・ベラーにいわせると合衆国ではそれが子どもの自立、親離れの証として推奨されることもある(ベラー1985=1991 p.72)という。いずれにせよ多くの人はキリスト教の枠内には収まっている・あるいは近年では無宗教が増えているにせよ「キリスト教か無宗教(理神論等も含めて)」の枠内にはある。\n\n24 日欧の受洗率の違いといっても、向こうの数字は、原則、幼児洗礼の数を加えているのであろうから、また幼児洗礼したものに洗礼のための受講は課さないのであろうから、じつは差は見かけほど多くないということもありうる。あと欧米に受洗前の講義のない分、埋め合わせとして身体化した風習や普段の日曜学校があるとすれば、日本の習俗のみの受容（仮に「のみ」に留まると仮定して）も身体化というレベルで欧米とのある程度の相同性がありうるともいえる。\n\n25 このような津城のいう「自由キリスト教」の影響は、桜美林や玉川のみならず、同志社でもある程度想定されうる。竹中正夫 (1987) はアメリカン・ボードの背景にある思想は、理性主義的なユニテリアンではなく、人間の自由意志を批判的に論じたジョナタン・エドワーズにまで遡れるという（竹中 1987 p.131）。その上で日本組合基督教会の特徴として、自治の精神、自給の精神、自由の精神の三つを挙げる。これら三つはあとに挙げたものほど時系列的にも、後になる。新島襄自身が関与したのは一つ目のみである。三つ目の自由の精神についてはこの論文ではその端緒を 1898 年第3回日本組合基督教会での宮川経輝の講演に求める。よって新島の没年 1890 年より後になる。ここで宮川は 1. 精神を先にして形骸を後にする2.日本人の伝道は日本人が当たる 3. 寛容、の三つを唱えた（竹中 1987 p.136）。その上でこの論文では日本組合基督教会におけるこの自由の精神の代表格を、海老名弾正であるとする。海老名は三位一体説の解釈を巡って 1902 年福音同盟会から除名された。海老名の解釈は独自ではあったが決してユニテリアン的ではなかったと断った上で、竹中は海老名のキリスト像がキリストの神聖を強調する「高いキリスト論」ではなく、その人間性を強調する「低いキリスト論」であったとする。その上で竹中は「組合教会の中においても、キリスト論に巾があった」（竹中 1987 p.138）とし、組合教会は「高いキリスト論」も「低いキリスト論」も併存することを容認する寛容性があったと論じる。このような「低いキリスト論」の代表格であった海老名は同志社の第8代総長 (1920~1928) にもなった。また日本組合基督教会の三元老は上述の宮川、海老名と共に小崎弘道であるとするが、小崎に米国への伝道を勧める松山常次郎に小崎が「自分は同志社社長の時代に宣教師から異端と目されているから北米伝道はできない」と答えた言葉を紹介する（竹中 1987 p.143）。同志社「社長」は現在では「総長」に呼びかえられ、同志社の公式ウェブで第2代総長として小崎の名前が出ている。小崎は万国宗教大会で「日本のキリスト教の特徴について語ったが、宣教師との関係については、日本の教会においては宣教師は補助者であり、神学においても教会政治においても主導権は日本のキリスト者が握っている点を紹介した。特に神学について宣教師は正統主義神学を教えているが、日本のキリスト者の中では自由主義神学 (Liberal Theology) が主流である点を指摘した」（森 1990 p.16）。このようにユニテリアンではないものの、広義の「自由キリスト教」にその比較的初期の総長からして親和的であったということは言えようである。また戦後ではあるが 14 代総長は住谷悦治である。住谷は本稿筆頭筆者の研究対象中井正一の、戦前における人民戦線運動の庇護者のような存在であり、叔父の住谷天来から洗礼を受けたクリスチャンであり、マルクス主義者でもあった。\n\n26 京大哲学科は東大哲学科時代にケーベルの影響を強く受けた人物が多かった。西田もケーベルの思い出を書いているし、本稿筆頭著者の研究対象中井正一の恩師深田康算は、漱石も寺田寅彦もケーベルの寓居を訪問するといつも深田がいたと記すほどの愛弟子であったし、波多野精一は、波多野の弟子の小原国芳の設立した玉川学園の公式ウェブによると、ケーベルが「おれの弟子は波多野一人だ。外にはニヒツ（無い）」と語っていたというほど優秀な弟子であった (https://www.tamagawa.jp/introduction/history/detail_6750.html) （最終閲覧日 2022 年 12 月 4 日）。和辻哲郎はケーベルに読んでもらうため英文で卒論を書き、ケーベルについての著書も弘文堂のアテネ文庫から出している。ケーベルの「キリスト教的信念は実に確固としていたのである。（波多野）博士は先生の個性におけるこのギリシア的自由とキリスト教的敬虔との総合に強く打たれた」（和辻 1962 p.27）。それに反して、東大哲学科を引っ張っていた井上哲次郎は内村の不敬事件を厳しく糾弾するなど、反キリスト教の立場を前面に出していた。反キリスト教的な井上よりもキリスト教的敬虔を示すケーベルに親愛の情を示したスタッフが京大哲学科には多かったといえるが、これは御大西田の意図による部分が大きいのか他の意思（例えば漱石と懇意な京都帝国大学文科大学初代学長狩野亨吉の意向）が強く働いているのかは、今後調べていきたい。なお西田のジェームズ開眼を元良の影響と、本稿本文では書いたが、万国宗教会議に参加した釈宗演の推薦によって米国に長期滞在していた大拙によるものとの説もある（橋本 2015 p.137）。なお元良も西田、大拙、漱石同様、円覚寺に参禅し釈宗演の指導を受けている。\n\n27 例えば井上順孝は井上ほか (1997) 所収の「大学生における宗教教育への関心と宗教意識」において宗教系中高卒業生（宗教系の中高の双方あるいはいずれかを卒業し、通っている大学は宗教系・非宗教系を問わない）と宗教系大学在学生卒業生を主に分析する。宗教系中高卒業生は「宗教に関係した学校に通った事で、その宗教を信じようと思ったことがありますか」という設問に対して「信じるようになった」 21.5% 、「興味をもったが信じない」 39% 、「信じない」 30.9% 、「反発を感じた」 4.5% であった（井上ほか 1997 p.149）。それらについて井上は次のように記す。「信じるようになった者が約二割、興味を抱いたのが約四割で、約六割に宗教系の学校に通ったことの効果が見られる。（中略）また、とくに信じようと思ったことがない者も約三割いるが、かえって反発を感じた者となると、五%弱である」（井上ほか 1997 p.149~150）。このように宗教系中学校高校の宗派教育の効果を一定程度認める記述をする。他方、宗教系大学生の分析に際しては同じ設問を比較していないのであるが、「今通っている大学に宗教的色彩がある」と感じている 66.9% に、どんな点に「宗教的色彩」を感じたかを自由回答で聞いた回答を紹介し、分析する。「肯定的、批判的、中立的な意見のうち、とりわけ目立って多いというものはなかった。ただ特徴的なことを一つ挙げれば、授業、行事の双方とも、強制された形態のものに対しては、強い反発があるということである。（中略）一般的に言って、中学や高校の宗教教育への反応に比べて、大学における宗教的色彩に対しては否定的反応が増える傾向にあると考えていいだろう」（井上ほか 1997 p.157）。井上はその理由を「強制への反感」にあると推察する。「やはり、かなりの学生は大学を「自由な学問の府」として捉えていると思われ、これと齟齬するような宗教的な授業や行事は、抵抗が大きいと推測される」（井上ほか 1997 p.157）。井上は「自由な学問の府」という部分を強調するが、ここの本稿本文のインタビューにあるように、小中高と上がるにつれて、宗教教育を担える人材が減っているという要因も、中高と大学の宗教教育の効果の差の原因にあるという可能性を我々は考えている。ただこれについても今後事例を積み重ねないといけないことは言うを俟たない。なお同じ著書の中で田口めぐみ「中等教育における宗教教育の意義」は上記の「宗教に関係した学校に通った事で、その宗教を信じようと思ったことがありますか」という設問についてもう少し細かい分析をしている。それによると「男子よりも女子の方が」そこの学校の「宗教に対し好感をもっている」（井上ほか 1997 p.215）とし、また「この結果の中で一番興味深いのは、宗教系の学校に中学校三年間または高校三年間しか通っていない学生と六年間通った学生との差である。ここでは、宗教教育が長期にわたってなされれば、その分影響も多いということが明確である。逆に反発を抱く学生の割合も多い」（井上ほか 1997 p.215）とも分析する。" }	[ { "id": "270385", "date": "07 Jun 2024", "name": "Shuhei Fujii", "expertise": [ "Reviewer Expertise 宗教学　宗教心理学　宗教認知科学" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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論文の要約　本論文は、日本のキリスト教徒は人口の約1.6％しか存在しないにもかかわらず、キリスト教主義学校が日本で人気を集めている理由を究明するために、東京都・神奈川県在住の 15～29 歳を対象としたウェブモニター調査を実施し、その結果の分析を行っている。　その際の作業仮説として、「（1）キリスト教主義学校は何を目的として設立されているか」「（2）キリスト教主義学校の生徒・学生イメージを、人びとがどのように捉えているか」を明らかにすることによって、日本のキリスト教徒は人口の1.6％しか存在しないにもかかわらず、キリスト教主義学校が日本で人気を集めている理由が解明されると想定されている。また、日本人は仏教と神道に加え、キリスト教も緩やかに信仰しているのではないかという「トリプルな信仰」説を踏まえ、（3）そのような信仰の有無を明らかにすることもまた、第3の作業仮説として設定されている。　質問票における質問項目は多岐にわたっているが、本論文では主に3つの作業仮説と関連する質問の結果が分析されている。作業仮説1については、キリスト教主義学校への関与度が低い回答者は布教や資金集めをキリスト教主義学校の目的と考えやすかったが、関与度が高い回答者はその割合が少なく、「信者にならなくても、キリスト教の教えを守ることによって、救われる可能性のある人を増やすこと」の回答が多かった。こうした点から、結果は日本にキリスト教徒が少ないにもかかわらずキリスト教主義学校が人気を集めている状況と矛盾しないと述べられている。　作業仮説2については、キリスト教主義学校に通う生徒が「みめうるわしい」というイメージはあまり抱かれておらず、一方で「外国語が得意である」「教養が豊かである」「品がよい」などの肯定的イメージが目立ち、これがキリスト教主義学校の人気の理由とみなしうるとしている。　作業仮説3については、キリスト教主義学校の通学経験者は仏教や神道の宗教的行事にもより参加している傾向があり、必ずしも信者にならないとしても、キリスト教の教えを守ったり、宗教的情操を涵養したりすることがキリスト教主義学校に期待されていることから、この仮説も裏付けられたとしている。　この結果を踏まえ、そのような複合的な信仰の形態がありうることを考慮すると、日本のキリスト教徒が1.6％だということは、見かけ上低い数値であるにすぎないという可能性を指摘している。\n本論文の評価　本論文は、キリスト教主義学校の目的やその生徒について人々が抱いている意識を実証的に明らかにしている点だけでなく、「トリプルな信仰」という視点から、特定宗教を信仰する／信仰しないという従来の二者択一的な宗教観を見直し、明確に信仰せずとも、教えに共感をもつ、宗教的情操を涵養するといった形での宗教への関与のあり方を提示している点もまた有意義である。この視点は、現代の宗教の状況についての他の研究とも整合的である。米国では「Believing without Belonging」という言葉がしばしば用いられている。たとえばタナカ（2010）は米国には300万人の仏教徒がいるだけではなく、250万人ほどの仏教同調者もおり、さらに約2500万人が仏教から「重要な影響」を受けているとしている。　同時に、宗教組織への明確な所属を伴わない「スピリチュアル」な活動に関心を持つ人も増加しており、アメリカ心理学会の第36部門は2010年に「宗教とスピリチュアリティ学会」へと改名しているほか、日本でもパワースポット巡りや占い、ヒーリングなどが人気を集めている。　そうした状況において、日本のキリスト教についても同調者という形で信者数以上の関係者が存在しており、その同調者を増やすためにキリスト教主義学校が寄与しているという仮定は妥当なものだといえ、本研究結果でもその仮定が裏付けられている。　他方で、本論文にはいくらか不足していると思われる点が見受けられる。それらについて以下に指摘していきたい。\n先行研究の扱いについて　全体として、本論文が参照している先行研究は不十分である。金児（2011）が指摘しているように、戦後の宗教心理学的研究においてはキリスト教系学校を対象とした質問紙調査が多数行われており、決して不足しているわけではない。　例として、寺川（1983）はキリスト教を含む宗教系学校に質問紙を送り、教育理念や実践について尋ねている。真野（1984）はプロテスタントのクリスチャン・スクールに宗教教育の目的・現状・成果などを尋ねている。生徒を対象としたものとしては、真野（1986）がキリスト教主義高等学校の女子生徒に対し、本論文でも扱っているような自校のイメージについて尋ねた例がある。　より新しい研究としては、現在進行中のものであるが、清泉女子大学の森本真由美氏が上智大学教育イノベーションプロジェクトとして、カトリック大学の伝統をどのように学生に継承していくかという課題から、面接調査を実施している。その成果の一部は、日本心理学会第87回大会の公募シンポジウム「神学・宗学は実証的宗教心理学と連携・協働することができるのか――実証的宗教心理学の挑戦（2）」として発表されている。　確かに、一般の回答者に対してキリスト教主義学校のイメージを尋ねた研究は管見の限りでは見当たらないが、キリスト教系高校出身者と非キリスト教系高校出身者のキリスト教への態度の差などを調査した中村（1976）などもあり、本論文の目的からして十分参照に値するものである。　また、「2. ウェブモニター調査の概要」ではキリスト教徒の割合の比較先として、井上らの2000年の文献が参照されているが、より新しい文献を用いるべきだろう。ISSPの結果を用いている小林（2019）ではキリスト教を信仰していると答えた割合は1.2％、國學院大學研究開発推進機構日本文化研究所（2022）の結果はキリスト教系大学生を多く含むものにもかかわらず、約1.4％となっている。総じて近年になるほど数値は減少する傾向にあるので、これらと比較すれば本論文の3.0％という数値はさらに際立つといえる。　さらに、前述の小林（2019）に加え庭野平和財団（2020）はキリスト教を含む宗教への肯定感やイメージについて尋ねており、こちらの結果も本論文にとって有意義なものとなるだろう。\n結果の取り扱いについて　それぞれの結果の分析は概ね妥当と思われるが、事前の仮説に反する結果の取り扱いにやや不十分な点が感じられる。たとえば、Q34のキリスト教主義学校に対する設置母体のメリットとして、「教団組織の資金集め」が最も多く選ばれている。これは現実とは乖離しているということであるが、それにもかかわらずなぜそのようなイメージが持たれているかは検討の余地があるのではないだろうか。そしてこの回答は、決してキリスト教主義学校に対してよいイメージをもっていることを示すものではないだろう。このようなネガティブな選択肢がもっと含まれていた場合、いかなる結果になったのかは気になる点である。　同様に、Q29のキリスト教主義学校の学生のイメージとして、「外国語が得意である」が最多となった点もより重視するべきであるように思われる。井上ほか（2018）においても、「女子アナが多い」など、実利的な面が大いに強調されているし、実際に有名なキリスト教系大学は、英語教育などに力を入れていることは確かであろう。そのため人格面に加え、「就職に有利である」「高収入である」などの実利的な質問を増やし、人格面とどちらが重視されるかを検討することも可能と思われる。　こうした点は問題点というよりは、今後の課題とみなしうるものだろう。一足飛びに仮説が検証されたとみなすのではなく、予期していなかった結果に目を向けることで、ますます多くの事実を明らかにすることができるように思われる。\n\n改善の提案　本論文の改善点として提案できることは、第一に先行研究の参照を増やすことである。先行するキリスト教主義学校の調査や世論調査を十分に参照することによって、本論文がいかに新しい知見をもたらしたかという点をより明確にすることができるだろう。　第二の点は、議論を整理することである。本論文は分量が非常に多く、途中にインタビューの記述なども差し挟まれ、把握しづらいものとなっている。「3. 調査結果とその分析」においても、それぞれの節が3つの作業仮説といかに対応しているかがわかりづらい。それぞれの節の冒頭にナビゲーションとなる解説を付加し、個々の質問項目の検討はいかなる疑問を明らかにするために行っているのかを明確にし、取り上げる質問項目を取捨選択することによって、この点は改善されるであろう。　また「5. 考察」においても、キリスト教の布教のあり方やキリスト教主義学校の目的はどのようであるべきかといった点に議論が迂回しているように思われる。もしこの主題を論じることが元来の目的であるならば、その点を「1. 先行研究と問題の所在」に明記すべきである。\n参考文献井上章一ほか（2018）．ミッションスクールになぜ美人が多いのか――日本女子とキリスト教　朝日新聞出版金児暁嗣（監修）松島公望・河野由美・杉山幸子・西脇良（編）（2011）．宗教心理学概論　ナカニシヤ出版小林利行（2019）．日本人の宗教的意識や行動はどう変わったか　放送研究と調査， 69(4), 52-72. 國學院大學研究開発推進機構日本文化研究所（編）（2022）．第13回学生宗教意識調査報告―改訂増補版―　國學院大學研究開発推進機構日本文化研究所真野一隆（1984）．私立学校の中・高一貫教育の原理と内容に関する理論的並びに実践的研究――プロテスタントのクリスチャン・スクールに焦点をあてて　日本私学教育研究所紀要， 20(1), 89-134. 真野一隆（1986）．私立学校の女子教育の歴史と現実――プロテスタントのクリスチャン・スクールに焦点をあてて　日本私学教育研究所紀要， 22(1), 37-72. 中村陽三（1976）．青年期における宗教意義に関する実証的比較研究――公立校生徒とキリスト教学校生徒との比較日本私学教育研究所紀要， 11(1), 157-173. 庭野平和財団（2020）．日本人の宗教団体への関与・認知・評価に関する世論調査報告書　庭野平和財団　https://www.npf.or.jp/pdf/2020_research.pdf タナカケネス（2010）．アメリカ仏教――仏教も変わる、アメリカも変わる　武蔵野大学出版会寺川幽芳（1983）．私立大学における宗教教育の現状をめぐって仏教文化研究所研究紀要， 13, 13-55.\n\n本研究は明確かつ正確に提示されたものであり、最新の文献を引用していますか。一部該当\n\n研究設計は適切で学術的価値がありますか。はい\n\n方法と分析について第三者による再現が可能となるよう十分な詳細が提示されていますか。はい\n\n（該当する場合は要回答）統計分析および解釈は適切ですか。はい\n\n結果の基礎となるソースデータはすべて入手可能で再現性を十全に保証していますか。はい\n\n結論は結果により妥当な裏付けを得ていますか。一部該当", "responses": [] }, { "id": "207591", "date": "04 Jul 2024", "name": "Anri Morimoto", "expertise": [ "Reviewer Expertise 神学・宗教学" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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本論文の要約\n\n本論文の課題は、「日本のクリスチャンの数は日本の人口比で1.6%前後しかいないにもかかわらず、キリスト教主義学校が日本の私立学校の中で一定程度の定員比率を誇り、また人気を集める学校も多いのはなぜか」を究明することである。著者たちによると、この課題には二つの論点が含まれている。一つは前半部分「日本のクリスチャンの数が日本の人口比で1.6%前後しかいない」であり、もう一つは後半部分「キリスト教主義学校が日本の私立学校の中で一定程度の定員比率を誇り、また人気を集める学校も多い」である。前半と後半は「にもかかわらず」という相反の関係と捉えられており、本論文の課題はその関係に合理的な説明を与えることにある。　本論文の結論は、前半部分については「宗派に帰属しない日本人のトリプル信仰」からすると、「キリスト教に洗礼という手続きが不要であればキリスト教を含めトリプルな信仰をする人は多かったはず」であると推測し、1.6%という数字が「見かけ上の数値にすぎない」可能性を示唆する。　後半部分については、キリスト教主義学校のイメージが「教養が豊かである」「外国語が得意である」「他人を思いやる心が豊かである」「話し上手である」などの点において肯定的であること、また「布教という目的を日本のキリスト教主義学校はほとんど強く有していない」ことの二点を挙げ、それらが前半の事実と「矛盾しない」と結論づけている。\n評価\n\n本論文の著者たちは、課題究明のためにコロナ禍の下でウェブモニター調査を行い、キリスト教主義学校に関する世間一般のイメージという把捉の難しい対象を統計的に数値化する方法を編み出している。特にその対象者を卒業生や在校生などの「強い当事者」、無関係の「部外者」、そして両者の間の中間的カテゴリーとして家族などの「緩やかな当事者」という三つのコホートに分けて設定し、それぞれにクロス集計をかけることで調査の精密度や信頼度を高めている点は、高く評価することができる。さらに、数値結果に具体的な肉付けをするためのインタビューを行い、ウェブや文献の資料も参看するなど、調査には独自で入念な手順が踏まれている。筆頭著者は思想・哲学系の社会学者であるが、対象領域は宗教学、風俗論、教育社会学、さらには日本思想史など複数の分野に関わっており、課題の設定や手法の選択に伴う困難と限界も十分に自覚されている。　調査の副産物として、本論文が一般社会にありがちな偏見の所在を示し、それらに根拠がないことを統計的に明らかにしている点も評価に値する。キリスト教主義学校に通う女子が「きれい」「容姿端麗」であるというイメージは、今日の倫理感覚からすれば調査項目とすること自体がはばかられるが、それをあえて項目化したことで明示的に否定する結果を導くことができている。キリスト教主義学校が「教団組織の資金集め」を目的としている、という印象も、著者たちが「現実にはありえない」選択肢と認識しつつ項目化したことで、それが無根據であることが示される結果となっている。　他方、改善が可能な点もある。まず、質問の項目や対象者などの偏差に注意が必要である。実際の質問項目を見ると、プロテスタントとカトリック、女子校・男子校・共学校の違いについては著者たちも留意しつつ調査を進めていることが認められる。しかし、仏教系や神道系の学校は質問項目には含まれているものの、詳細な比較検討がなされていない。たとえば「他者を思いやる心」の涵養などは、仏教系学校でも十分に高い数値となることが予想されるし、他宗教の行事に参加することへの寛容度の比較も分析可能なはずである。日本のキリスト教徒を人口比で1.6%（ないし1.0%）とすることの出典や根拠、東京都と神奈川県の数字を「日本」の数字へと外挿することの是非などにも説明が望ましい。　これらの空隙を補填するかのようにいくつかのインタビュー会話が記載されているが、いずれも個的で多義的なエピソードで、客観的なデータを補完するとまでは言えない。文献資料の参看となると、唐突に現代でなく明治時代の内村鑑三や海老名弾正や波多野精一が引用され、あるいはトマス・アクィナスの修道会入会理由が引用されており、本来の文脈を顧慮しない恣意性が散見される。　本論文の課題設定そのものの評価については、改善提案に記しておく。\n先行研究\n\n前半の課題は、これまで「日本のキリスト教受容」「キリスト教の土着化・文脈化」という問題群で扱われてきたやや古典的な問いである。先行文献も豊富だが、本論文ではわずかに武田清子と丸山眞男が引用されているのみである。本論文で「トリプル信仰」として引用されている信仰の多重性や複相性についても、国内外で議論が重ねられてきた。多くのアジア的な宗教にとり、宗教は意識的な決断の事柄であるよりも儀礼や習慣などの生活様式に密着した事柄である。入信儀礼の位置づけも多様であり得るため、ある宗教の理解を他の宗教の理解にあてはめることには慎重でなければならない。「トリプル信仰」は仏教や神道の視点から論じられたものであるため、キリスト教の視点から論じられた同種の議論を参看することは有益と思われる。森本あんり（2004年）は、特にキリスト教のアジア的な文脈化を類型論とともに論じており、宗教信仰の重層化とりわけ二重信仰のあり方に光を当てている。同書には、ビーヴァンスやシュライターなどこの問題群を扱うのに不可欠と思われる先行研究も紹介されている。他に、南山宗教文化研究所にも豊富な文献の蓄積がある。　他方、キリスト教に洗礼が必要か否か、救済に洗礼が不可欠か否かという問題群も、キリスト教神学の歴史において繰り返し問われ続けてきた。著者たちが論文末尾で引用しているロジャー・ウィリアムズはその論者の一人だが、古代中世を通して交わされてきた議論を概観するには、森本あんり（2018年）と同書に付された文献一覧が便利である。　後半の問いは、本研究にも引用されている「キリスト教学校教育同盟」に照会すれば、過去の事例研究やデータ資料が得られるはずである。\n改善提案\n\n本研究課題前半部の問いについては、「キリスト教に洗礼という手続きが不要であればキリスト教を含めトリプルな信仰をする人は多かったはず」という仮定に基づいた推測をもって結論とされているが、その推測を裏付けるためにはやや異なった質問項目が必要かもしれない。著者たちも一部自覚しているとおり、「関心がある」と「入信する」とは違うし、「内心で信じている」のと「それを公言する」のも別である。実際の質問項目でも「文化資源としての側面」と「純粋な宗教としての側面」という二面が区別されているが、これらの区別は連続的なので数値化するには工夫が求められる。　上記推測が正しいかどうかを確認するには、「もし洗礼という手続きが不要だったなら、あなたは自分のことをキリスト教徒だと思いますか」といった趣旨の設問がもっとも直接的だろう。だが、その前提をなす仮定「もし洗礼が不要だったら」は、「キリスト教徒であること」の内容にあらかじめ大きな変更を加えており、設問自体を無効化しかねない。それは「もし野球にバットが不要だったなら、あなたは野球選手になりますか」と尋ねることに似る。　宗教にはそれぞれ固有の準拠枠があるので、これを大きく改変することを前提とした上での設問には限界が伴う。日本の仏教や神道では明確な儀式を経ずにも信徒であることが可能であることから、他宗教でもそのような儀式は省略可能だ、と想定されているとすれば、そこには調査者の宗教観が無自覚に前提され作用していることになろう。　文中には内村の「無教会主義」も触れられている。無教会では洗礼という儀式が不要なので、著者たちの想定に従うならば、ここには多くの日本人キリスト教徒が集うはずだが、現実には内村の時代から今日に至るまで、無教会の信徒は極端な少数派に留まっている。武田清子の引用によれば、無教会主義はすでに日本で正統の一部と見なされているので、極端な少数派であることの理由を無教会主義への異端視に求めることはできない。とすると、本研究前半部の結論的推測には別の理由づけが必要となるように見える。　後半部の結論についてはおおむね分析の結果を信頼できるが、日本のキリスト教主義学校に多くの生徒・学生が集まることは、それらの学校の一般的なイメージが肯定的であることや学校が「布教」を主目的としていないことと「矛盾しない」、という判定だけではやや漠然としている。これも著者たちが自覚的に記していることであるが、キリスト教主義学校を志望する動機には、「進学・就職実績」など教育理念とは無関係の世俗的な利点が作用することが知られている。前半と後半が「矛盾しない」という判定を越えて、より積極的な因果関係ないし相関関係を見出すことに焦点が当てられるならば、本研究はいっそう大きな意義をもつことと思われる。\n参考文献\n森本あんり『アジア神学講義』、創文社、2004年。森本あんり『異端の時代』、岩波書店、2018年。 Bevans, Stephen B. Models of Contextual Theology, Revised and Expanded Edition. Maryknoll, New York: Orbis Books, 2002. Hesselgrave, David J. and Edward Rommen. Contextualization: Meaning, Methods, and Models. Grand Rapids, Michigan: Baker Book House, 1989. Morimoto, Anri. \"Two Types of Religion,\" Humanities: Christianity and Culture 33 (2002): 19-26. Schreiter, Robert J. Constructing Local Theologies. Maryknoll, New York: Orbis Books, 1985. van der Veer, Peter. \"Syncretism, Multiculturalism and the Discourse of Tolerance.\" Charles Stewart and Rosalind Shaw, eds., Syncretism/Anti-Syncretism: The Politics of Religious Synthesis, 196-122. London and New York: Routledge, 1994. Yung, Hwa. Mangoes or Bananas? The Quest for an Authentic Asian Christian Theology. Oxford: Regnum Books International, 1997.\n\n本研究は明確かつ正確に提示されたものであり、最新の文献を引用していますか。一部該当\n\n研究設計は適切で学術的価値がありますか。一部該当\n\n方法と分析について第三者による再現が可能となるよう十分な詳細が提示されていますか。はい\n\n（該当する場合は要回答）統計分析および解釈は適切ですか。一部該当\n\n結果の基礎となるソースデータはすべて入手可能で再現性を十全に保証していますか。はい\n\n結論は結果により妥当な裏付けを得ていますか。一部該当", "responses": [] } ]	1	https://f1000research.com/articles/12-973
https://f1000research.com/articles/11-1164/v1	11 Oct 22	{ "type": "Brief Report", "title": "Potential of in vivo stress reporter models to reduce animal use and provide mechanistic insights in toxicity studies", "authors": [ "Francisco Iñesta Vaquera", "Febe Ferro", "Michael McMahon", "Colin J. Henderson", "C. Roland Wolf", "Francisco Iñesta Vaquera", "Febe Ferro", "Michael McMahon", "Colin J. Henderson" ], "abstract": "Chemical risk assessment ensures protection from the toxic effects of drugs and manmade chemicals. To comply with regulatory guidance, studies in complex organisms are required, as well as mechanistic studies to establish the relevance of any toxicities observed to man. Although in vitro toxicity models are improving, in vivo studies remain central to this process. Such studies are invariably time-consuming and often involve large numbers of animals. New regulatory frameworks recommend the implementation of “smart” in vivo approaches to toxicity testing that can effectively assess safety for humans and comply with societal expectations for reduction in animal use. A major obstacle in reducing the animals required is the time-consuming and complexity of the pathological endpoints used as markers of toxicity. Such endpoints are prone to inter-animal variability, subjectivity and require harmonisation between testing sites. As a consequence, large numbers of animals per experimental group are required. To address this issue, we propose the implementation of sophisticated stress response reporter mice that we have developed. These reporter models provide early biomarkers of toxic potential in a highly reproducible manner at single-cell resolution, which can also be measured non-invasively and have been extensively validated in academic research as early biomarkers of stress responses for a wide range of chemicals at human-relevant exposures. In this report, we describe a new and previously generated models in our lab, provide the methodology required for their use and discuss how they have been used to inform on toxic risk. We propose our in vivo approach is more informative (refinement) and reduces the animal use (reduction) compared to traditional toxicity testing. These models could be incorporated into tiered toxicity testing and used in combination with in vitro assays to generate quantitative adverse outcome pathways and inform on toxic potential.", "keywords": [ "Risk assessment", "alternative in vivo methods", "chemical toxicity", "early biomarkers", "in vivo toxicology", "mechanistic toxicology", "oxidative stress", "DNA damage." ], "content": "Abbreviations\n\nAhR: aryl hydrocarbon receptor\n\nhCG: human chorionic gonadotropin\n\niAs: inorganic arsenic\n\nNBF: neutral-buffered formalin\n\nOCDE: Organisation for Economic Co-operation and Development\n\nPBS: phosphate-buffered saline\n\nPFA: para-formaldehyde\n\nTCDD: Tetrachlorodibenzo-p-dioxin\n\n2-MCPD: 2-Monochloropropane-1,3-diol\n\n3-MCPD: 3-monochloropropane-1,2-diol\n\n\nIntroduction\n\nDevelopment of new manmade chemicals has underpinned an enormous rise in quality of life in areas such as pharmaceuticals, technology, or food production.1 However, humans and the environment need to be protected from potential short/long-term toxic consequences of exposure to these agents and their derivatives. Regulatory procedures ensure appropriate risk assessments are conducted on novel agents before approval for commercial use. In the absence of human data, studies with multi-cellular experimental models are required to detect and estimate toxic potential. In vivo models, although with limitations, provide the closest estimation of recommended levels of safe exposure in a physiological context. The implementation of such tests must be appropriate, responsible, and humane, by reducing the numbers of animals in a study, refinement of measurements and implementation of less invasive endpoints.2 This is achieved by continuous application of the 3Rs principles, particularly when vertebrate models such as rodents are being used and is reflected in OCDE guidelines updates. For example, in acute inhalation studies, death as an endpoint was substituted for clinical signs of toxicity such as hypoactivity, bodyweight loss, and irregular respiration.3 However, clinical signs often reflect major disturbances of homeostasis, which are very unlikely to occur at relevant human exposure levels and do not inform on the mechanisms involved. Additionally, these tests are prone to subjectivity, inter-animal variability, require harmonisation between testing sites and need large numbers of mice for prolonged periods. For example, the OCDE-451 guideline for carcinogenic analysis recommends studies lasting for two years and groups of up to 50 animals for each testing condition.4\n\nNew approaches are being proposed to reduce reliance on animal testing for regulatory or research purposes. Although progress has been made, most models still have significant limitations, including the inability to recapitulate the cellular complexity of physiological structures of mammals, or the biochemical and physiological differences between simple organisms and the pathways which define chemical toxicity. Despite promising efforts to develop organoids, a harmonized model has been difficult to establish due to issues such as marked cell variability, aberrant activation of signalling pathways and the lack of physiological complexity.5 For example, metabolic activation of some compounds can initiate toxicity in a different distal tissue. Also, invertebrates do not provide an alternative to higher organisms because of differences in the pathways that define the mode of action as well as the sensitivity of target cells to chemicals. For example, vertebrates are sensitive to the effects of non-genotoxic carcinogens such as TCDD. However, invertebrates are insensitive, consistent with vertebrates possessing an AhR that binds TCDD-like chemicals, whereas invertebrate AhR homologs do not.6 Consequently, the use of complex organisms such as rodents remains a recommended approach for toxicity testing.\n\nTo overcome these challenges, the introduction of new methodologies to conduct “smart” in vivo studies with the same level of risk confidence has been proposed.7 Consistent with this paradigm, in this communication we propose the incorporation of toxic stress reporter mice into such smart in vivo studies (Table 1). We describe how stress reporter mice can overcome some of the issues surrounding traditional in vivo toxicity studies, accelerate toxicity testing and aid in risk assessment while simultaneously reducing animal use. The rationale is that cells are protected from toxic stress through the induction of specific cytoprotective genes (Figure 1A). Activation of these reporters provides an early biomarker of toxic potential and provides mechanistic information with minimal inter-animal variability. Such models also provide a refined, highly sensitive method of relating dose to toxic effects, a prerequisite for risk assessment. At the molecular level, reporter mice generate easily measurable endpoints in a manner reflecting transcriptional activation of different endogenous genes (Figure 1B). For example, the p21 gene is rapidly activated in response to DNA damage. Therefore, p21 transcriptional activation provides excellent biomarkers of DNA toxic stress. These models carry multiple complementary reporters, including luciferase (non-invasive imaging) as well as β-galactosidase (post-mortem, fine-scale spatial mapping of stressed cells by histochemistry). Moreover, the Hmox1 model expresses hCG, a secreted marker that can be detected in blood. Molecular details for the generation of these lines are described elsewhere (Table 1). We also present a new stress reporter model, HMOX1 stress shadowing mice, where cells exposed to stress are permanently marked and their subsequent fate is tracked (Figure 1C).\n\nReferences to the original publications and subsequent use in toxicologic studies are provided.\n\n\nMethodology\n\nAll animals used in our studies were bred and maintained at the University of Dundee. Mice were housed in open-top cages in temperature-controlled rooms at 21°C, with 45-65% relative humidity and 12 h/12 h light/dark cycle. Mice had ad libitum access to food and water. Animals were subjected to standard health and welfare monitoring (twice daily). Environmental enrichment was provided. All animal work was performed under appropriate ASPEL licencing and approved by the Welfare and Ethical use of Animals Committee of the University of Dundee. Experimental design was undertaken following the 3Rs principles and according to the ARRIVE guidelines.\n\nData was obtained using either male or female mice, unless specific sex sensitivity was detected. Mice were 10–18 weeks old, heterozygous for the reporter allele, littermates or age-matched (within three weeks). Animals were randomly assigned to treatment groups. The number of mice per group was determined using the G*power calculator. For bioluminescence and hCG expression, previous work confirmed normal distribution of data using a Kolmogorov-Smirnoff test with standard deviation of 0.35. Sample size required to detect a two-fold difference between groups was n=4, using a standard power of 80% and a type I error of 5%. To compare the expression levels between two different groups a t-test was run. Scientists were aware of the nature of the compounds been administered. Analysts can be blinded to sample identity, as was done in our study.\n\nTissues were rapidly harvested post mortem and fixed for 2 h in 4% PFA (brain, small intestine, skin), 3 h in 10% NBF (liver) or for 18 h in Mirsky’s fixative (other tissues). Tissues were dehydrated for at least 24 h in 30% (w/v) sucrose/PBS buffer at 4°C. Organs were later embedded in Shandon M-1 Embedding Matrix (Thermo Fisher Scientific) in a dry ice-isopentane bath. Sectioning was performed on an OFT5000 cryostat (Bright Instrument Co.). Except for lung (14 μm) and brain (20 μm), all sections were cut at 10 μm thickness (all reagents from SIGMA Aldrich [Merck], unless otherwise indicated).\n\nSections were rehydrated in PBS at room temperature (15 min) before incubation overnight at 37°C in X-gal staining solution: phosphate buffer solution (PBS) (pH 7.4) containing 2 mM MgCl2, 0.01% (w/v) sodium deoxycholate, 0.02% (v/v) Igepal-CA630, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1mg/ml 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (Thermo Fisher Scientific). The next day, slides were washed in PBS, counterstained in Nuclear FastRed (Vector Laboratories) (4 min), washed twice in distilled water (2 min) and dehydrated through 70% and 95% ethanol (4.5 and 1 min respectively) before incubation in Histoclear (VWR) (3 min), air-dried and mounted in DPX mountant (all reagents from Sigma Aldrich, except otherwise indicated).\n\nIn vivo luciferase imaging and hCG measurement were performed as described before.8\n\n\nResults\n\nWe exemplify the utility of our reporter models to address different toxicology scenarios using previously published studies. We describe for first time the HMOX1 “stress shadowing” model, that can be applied to the study of chemical exposure toxicity during development and effects in later life.\n\niAs) is present in drinking water and polluted air exposing millions of people.9 Epidemiological studies have linked iAs exposure to the development of numerous diseases including cognitive impairment, cardiovascular failure and cancer. One limitation in the study of iAs toxicity is that the concentrations required to identify potential toxic effects on mouse physiology exceed realistic environmental exposure levels.10 The Hmox1 reporter model provides a robust in vivo approach to detect iAs-associated toxicity in a cell- and tissue-specific manner, and, importantly from an NC3Rs point of view, prior to any overt toxic phenotype. In a series of dose- and time-response studies we identified tissue-specific HOTT reporter activation at environmentally relevant concentrations in studies that lasted a maximum of four weeks. Moreover, we demonstrated that oxidative stress is the major in vivo mechanism of iAs toxicity using pharmacological (antioxidant N-acetylcysteine) and in vivo genetic approaches (Figure 2A). This demonstrates how the models can also be used to rapidly test treatments of disease.11 Similarly, we have used these reporter models to understand the toxicity of additional environmental chemicals, including cadmium (a nephro- and hepato-toxin found in cigarette smoke), “forever” chemicals and diesel emissions (Inesta-Vaquera, manuscripts in preparation). The information generated can be used in risk assessments and inform policy for health interventions.\n\nA. NRF2_Hmox1 reporter mice.11 iAs 10 mg/kg, heart tissue 20×. B. Hmox1 reporter model.8 APAP (Acetaminophen), 300 mg/kg; NAC (N-acetylcysteine), 300 mg/kg; LacZ staining in liver tissue (10×). C. p21 reporter model, Cisplatin 10 mg/kg.17 LacZ staining in liver tissue (10×).\n\n2-MCPD and its isomer 3-MCPD are formed during the refinement of vegetable oils in processed foods. 3-MCPD has been classified as non-genotoxic, and possibly carcinogenic, to humans (category 2B; IARC, 2012). In a conventional two-year long feeding study in rats (group size=50), 3-MCPD-induced nephrotoxicity and testicular toxicity. Moreover, an increased incidence for Leydig cell tumours and kidney tubular adenomas was observed.12 The mechanism of 3-MCPD toxicity was not determined, although studies suggested that induction of oxidative stress was involved.13 A shorter (28-day) study using the HOTT model (only five animals per group), reported nephrotoxicity, testicular and liver toxicity. Strikingly, these studies identified a previously unreported induction of oxidative stress in the brain.14,15 Importantly, the effects of 2-MCPD, which has not been studied in carcinogenicity studies, were less pronounced, suggesting that the molecular toxicity mechanisms of 2-MCPD and 3-MCPD are potentially different.14,15 A similar approach was used to demonstrate the capacity of the food preservative ethoxyquin to induce liver toxicity, using a combination of our hGSTP1 and NRF2-nulled reporter mice.16\n\nThe safety of new therapeutics is paramount in the drug development process. Many drugs targeting the DNA damage response currently developed as anti-cancer therapies have the potential to increase toxicity to normal tissues. The use of reporter mice can accelerate the drug development process by determining which agents (and/or their combinations) possess a stronger toxic potential (Figure 2B and C). We have determined the capacity of several DNA-damaging agents (ionizing radiation, cisplatin, etoposide) to induce DNA damage in tissues using reporter mice.17 We also illustrated the power of this approach to evaluate the safety of new therapeutic regimens involving combination therapies. We demonstrated that a single exposure to olaparib, a PARP inhibitor licenced for the treatment of a number of cancers, caused DNA damage in mucosal cells lining the mouse large intestine, which was exacerbated in this organ, and the kidney by co-administration of ionizing radiation, suggesting that olaparib has carcinogenic potential.\n\nDrug metabolism is a major step in drug development and pharmacology. Humanised models of drug disposition are described in an accompanying report (see Henderson et al.16). The reporter technology can also be applied to identify activators of transcription factors responsible for induction of hepatic drug metabolising enzymes. For example, the constitutive androstane receptor (CAR) activates genes involved in drug metabolism, lipid homeostasis, and cell proliferation. We have developed a humanised rodent model of hCAR/hPXR containing a CYP2B6-LacZ transgene that allows the screening of large numbers of compounds, and studied the capacity of phenobarbital to activate this pathway in vivo.18\n\nWe report a novel Hmox1 stress-shadowing model. Stress-shadowing is a fate-mapping approach where Cre is driven from environmentally responsive rather than lineage-restricted genes. We engineered a new ‘driver’ mouse strain in which Cre is expressed from the Hmox1 locus (Hmox1Cre). We also generated a new reporter/tracer strain ROSA26LSL-LacZ-T2A-Luc in which a floxed silencing element (Lox-STOP-Lox, LSL) prevents expression of a dual reporter element harbouring both LacZ and luciferase. By crossing these ‘driver’ and ‘tracer’ strains, we generated the HMOX1 stress-shadowing mouse. Stress-induced expression of Hmox1 lead to Cre expression and, consequently, excision of the floxed silencing element. This permanently switches on the reporter, providing an enduring molecular mark that can be read at any subsequent time point (Figure 1C). We present a study confirming a low expression of the reporter in tissues of untreated young mice (six weeks old). Interestingly, the reporter becomes activated at older ages in untreated animals, especially in long-lived cells such as cardiomyocytes, neurons, and smooth-muscle cells (Figure 3). We have confirmed that activation is dependent on Cre and there is no ‘leakiness’ of reporter expression in the ROSA26LSL-LacZ-T2A-Luc line (data not shown, but available on request). Therefore, this line can track post-natally the fate of cells exposed during gestation to chemicals that induce Hmox1. This approach was successfully validated in another of our lines where Cre recombinase is driven from the endogenous Cyp1a1 locus, an AhR-responsive gene.19\n\nLacZ staining performed in indicated tissues.\n\n\nDiscussion\n\nWe propose the use of stress reporter models to reduce and refine the use of rodents in toxicity testing. We have demonstrated that reporter expression is a reproducible approach to quantify the potential toxic effects of a test substance, avoiding the variability of clinical scoring in conventional studies. Groups of only four animals are required to detect robust changes in luciferase/hCG expression differences between two treatments. Moreover, in our experience, groups of just three mice are sufficient to obtain reproducible results when measuring ß-galactosidase reporter expression as a qualitative endpoint (absence or presence of signal). This is achieved due to stable and specific reporter expression, which can also capture transient metabolic changes difficult to observe by other methods such as transcriptional profiling, as is the case with HO1.\n\nThe reporter approach offers a higher sensitivity in detecting homeostasis disturbance compared to traditional clinical signs of toxicity, paving the way to extensive study refinement. For example, test compound concentrations can be at relevant environmental/clinical exposure scenarios, below doses inducing overall toxicity. Similarly, study durations can be significantly reduced. These in vivo reporters can therefore be used as early toxicity biomarkers for quantitative risk assessment on test substances and provide valuable information on toxicity-associated mechanisms. Moreover, the ubiquitous distribution of these reporters ensures that relevant adverse effects in tissues not directly exposed to the test substance are not missed. Importantly, the assays required to measure reporter expression are inexpensive, fast, reliable and obviate the need for sophisticated laboratory equipment.\n\nThe application of reporter mice could provide critical information about potential toxicity in the drug discovery process to avoid unnecessary traditional toxicity studies, and/or initiate studies to establish whether such changes may be of clinical relevance. However, the interpretation of cellular responses observed using these reporters need careful considerations. For example, cisplatin can activate the DNA-damage reporter in liver, but it does not cause hepatotoxicity. The nature of the study will decide the next step in toxicity studies and will still depend on what murine parameters are present. We propose that the generation of stress reporter models on a humanized genetic background will help to overcome some of the potential caveats related to species differences in drug and foreign compound metabolism. For example, in drug toxicity, testing the incorporation of stress reporters into models humanized for metabolic enzymes (e.g. P450s) may further increase the predictive power of in vivo studies in relation to risk to man.\n\n\nData availability\n\nfigshare: F1000R-NC3R Publication - Potential of in vivo stress reporter models to reduce animal use and provide mechanistic insights in toxicity studies, https://doi.org/10.6084/m9.figshare.c.6049598.v2.21\n\nfigshare: ARRIVE Essential 10 Checklist for “Potential of in vivo stress reporter models to reduce animal use and provide mechanistic insights in toxicity studies”, https://doi.org/10.6084/m9.figshare.20528352.v1.22\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Bibliography\n\nOeppen J, Vaupel JW: Demography. Broken limits to life expectancy. Science. 2002; 296(5570): 1029–1031. PubMed Abstract \| Publisher Full Text\n\nFischer I, Milton C, Wallace H: Toxicity testing is evolving! Toxicol Res (Camb). 2020; 9(2): 67–80. Publisher Full Text\n\nSewell F, et al.: A global initiative to refine acute inhalation studies through the use of ‘evident toxicity’ as an endpoint: Towards adoption of the fixed concentration procedure. Regul. Toxicol. Pharmacol. 2015; 73(3): 770–779. Publisher Full Text\n\nFelter SP, et al.: Hazard identification, classification, and risk assessment of carcinogens: too much or too little? - Report of an ECETOC workshop. Crit. Rev. Toxicol. 2020; 50(1): 72–95. PubMed Abstract \| Publisher Full Text\n\nParish ST, et al.: An evaluation framework for new approach methodologies (NAMs) for human health safety assessment. Regul. Toxicol. Pharmacol. 2020; 112: 104592. Publisher Full Text\n\nAnkley GT, et al.: Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. Environ. Toxicol. Chem. 2010; 29(3): 730–741. PubMed Abstract \| Publisher Full Text\n\nBall N, et al.: A framework for chemical safety assessment incorporating new approach methodologies within REACH. Arch. Toxicol. 2022; 96(3): 743–766. PubMed Abstract \| Publisher Full Text\n\nMcMahon M, et al.: Measuring in vivo responses to endogenous and exogenous oxidative stress using a novel haem oxygenase 1 reporter mouse. J. Physiol. 2018; 596(1): 105–127. PubMed Abstract \| Publisher Full Text\n\nStraif K, et al.: A review of human carcinogens--Part C: metals, arsenic, dusts, and fibres. Lancet Oncol. 2009; 10(5): 453–454. PubMed Abstract \| Publisher Full Text\n\nTokar EJ, et al.: Cancer in experimental animals exposed to arsenic and arsenic compounds. Crit. Rev. Toxicol. 2010; 40(10): 912–927. PubMed Abstract \| Publisher Full Text\n\nInesta-Vaquera F, et al.: Application of the in vivo oxidative stress reporter Hmox1 as mechanistic biomarker of arsenic toxicity. Environ. Pollut. 2021; 270: 116053. PubMed Abstract \| Publisher Full Text\n\nCho WS, et al.: Carcinogenicity study of 3-monochloropropane-1,2-diol in Sprague-Dawley rats. Food Chem. Toxicol. 2008; 46(9): 3172–3177. PubMed Abstract \| Publisher Full Text\n\nBuhrke T, et al.: Oxidative inactivation of the endogenous antioxidant protein DJ-1 by the food contaminants 3-MCPD and 2-MCPD. Arch. Toxicol. 2018; 92(1): 289–299. PubMed Abstract \| Publisher Full Text\n\nSchultrich K, et al.: Effects of 2-MCPD on oxidative stress in different organs of male mice. Food Chem. Toxicol. 2020; 142: 111459. PubMed Abstract \| Publisher Full Text\n\nSchultrich K, et al.: Correlation between 3-MCPD-induced organ toxicity and oxidative stress response in male mice. Food Chem. Toxicol. 2020; 136: 110957. PubMed Abstract \| Publisher Full Text\n\nHenderson CJ, McLaren AW, Wolf CR: In vivo regulation of human glutathione transferase GSTP by chemopreventive agents. Cancer Res. 2014; 74(16): 4378–4387. PubMed Abstract \| Publisher Full Text\n\nMcMahon M, et al.: Olaparib, Monotherapy or with Ionizing Radiation, Exacerbates DNA Damage in Normal Tissues: Insights from a New p21 Reporter Mouse. Mol. Cancer Res. 2016; 14(12): 1195–1203. PubMed Abstract \| Publisher Full Text\n\nMcMahon M, et al.: Constitutive androstane receptor 1 is constitutively bound to chromatin and ‘primed’ for transactivation in hepatocytes. Mol. Pharmacol. 2019; 95(1): 97–105. PubMed Abstract \| Publisher Full Text\n\nSchiering C, et al.: Feedback control of AHR signalling regulates intestinal immunity. Nature. 2017; 542(7640): 242–245. PubMed Abstract \| Publisher Full Text\n\nVilla M, et al.: Aryl hydrocarbon receptor is required for optimal B-cell proliferation. EMBO J. 2017; 36(1): 116–128. PubMed Abstract \| Publisher Full Text\n\nInesta F: F1000R-NC3R Publication - Potential of in vivo stress reporter models to reduce animal use and provide mechanistic insights in toxicity studies. figshare. [Dataset]. Collection.2022. Publisher Full Text\n\nInesta F: ARRIVE guidelines checklist - F1000 report. figshare. Dataset.2022. Publisher Full Text" }	[ { "id": "174523", "date": "06 Jun 2023", "name": "Mark Miller", "expertise": [ "Reviewer Expertise in vivo rodent models", "air pollution", "cardiovascular disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a short review with original data outlining the use of the investigators’ mouse models with reporters of stress pathways. Data is shown demonstrating the presence of the reporter in histological sections from mice treated with various xenobiotics or aged animals for pathways that include oxidative stress and DNA damage, with prevention by anti-oxidants or genetic knockout. The authors conclude that these models could be used to detect activation of key stress pathways at more environmentally relevant exposures of stimulants without inducing overt toxicity, while offering the potential to reduce the length of exposure and number of animals per group. The paper is clearly written and the conclusions are reasonable for the examples shown in the paper.\nMinor comments:\nPlease state the source of the SD=0.35 used for the power calculations. I accept that the biological variability of the reporter expression is very low, however, n=4/group is very low for statistical analysis and risk of type I and II errors. The authors should comment on this. Similarly, the authors may also note that the presence of the reporter will likely be one variable measured in any detailed study using these mice but inevitably other functional and pathological parameters will be measured as well – these will still require higher n-numbers. Nonetheless, experiments with n=4 with reporter expression alone would still have great value as a screening and dose-finding tool.\n\nIt would be useful to show some quantitative data as well as the qualitative images. In particular, this could be used to demonstrate the variability of the reporter parameter or that more environmentally-relevant doses of stressors can be used, i.e. a dose response curve to a stimulant required to induce the reporter in these mice compared to a typical parameter used assess toxicity in normal mice to demonstrate.\n\nIf space permits, I would like to see more data from the examples outlined in the narrative text. The authors mention that these models can be used for non-invasive imaging – this data in particular would be good to show.\n\nPlease add a little further discussion on the physiological relevance of these pathways. For example, playing devil’s advocate, a dose of xenobiotic/drug may stimulate the reporter, however, this could be functionally harmless and it is only from greater/longer exposure that other biological responses become impaired causing a detrimental effect on health. In which case the reporter may give a false impression of the dose of exposure that is harmful, and potentially cause a drug to fail screening despite being promising in terms of risk-benefit in moderated doses.\n\nIs there any impact on breeding or known non-specific effect of the reporter compared to the wild-type mice?\n\nEnsure that figure legends are fully explained, e.g. describe why APAP and cisplantin are used – many readers will not be familiar with the type of stress/toxicity caused by these agents.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9976", "date": "29 Aug 2023", "name": "Francisco Inesta-Vaquera", "role": "Author Response", "response": "1. Please state the source of the SD=0.35 used for the power calculations. I accept that the biological variability of the reporter expression is very low, however, n=4/group is very low for statistical analysis and risk of type I and II errors. The authors should comment on this. Similarly, the authors may also note that the presence of the reporter will likely be one variable measured in any detailed study using these mice but inevitably other functional and pathological parameters will be measured as well – these will still require higher n-numbers. Nonetheless, experiments with n=4 with reporter expression alone would still have great value as a screening and dose-finding tool. The SD=0.35 is based on previous experience on using reporter animals (references 8, 17). We have extensively characterized the basal and inducible reporter activation across sex, age and tissues of the mentioned reporter mice. A sentence has been added to clarify this point. “The changes in reporter activation are highly reproducible, and therefore we are confident 4 animals/group is sufficient to avoid Type I and II errors. We acknowledge that where additional end points are to be measured simultaneously, this may be required to use a higher number of mice.” 2. It would be useful to show some quantitative data as well as the qualitative images. In particular, this could be used to demonstrate the variability of the reporter parameter or that more environmentally-relevant doses of stressors can be used, i.e. a dose response curve to a stimulant required to induce the reporter in these mice compared to a typical parameter used assess toxicity in normal mice to demonstrate. Table 1 provides multiple examples of where quantitative luciferase reporter activity (quantitative data) was measured after chemical exposure (references 8 and 17). For example, when mice were exposed to 30 mg/kg of haemin, a 10-fold induction of bioluminescence due to Hmox1 reporter luciferase activity was observed (ref. 8). 3. If space permits, I would like to see more data from the examples outlined in the narrative text. The authors mention that these models can be used for non-invasive imaging – this data in particular would be good to show. Due to space limitations we have summarized data on non-invasive imaging in table 1, including references where the images are shown. 4. Please add a little further discussion on the physiological relevance of these pathways. For example, playing devil’s advocate, a dose of xenobiotic/drug may stimulate the reporter, however, this could be functionally harmless and it is only from greater/longer exposure that other biological responses become impaired causing a detrimental effect on health. In which case the reporter may give a false impression of the dose of exposure that is harmful, and potentially cause a drug to fail screening despite being promising in terms of risk-benefit in moderated doses. We now include the following sentence in the discussion of the manuscript: “Cellular responses to toxic agents activate adaptive responses that protect cells from cell death. However, when these pathways are overcome toxicity results. Therefore, the activation of these pathways does not necessarily correlate with toxicity, but are indicative of toxic insults”. 5. Is there any impact on breeding or known non-specific effect of the reporter compared to the wild-type mice? No, both heterozygous and homozygous reporter mice are viable and fertile. 6. Ensure that figure legends are fully explained, e.g. describe why APAP and cisplantin are used – many readers will not be familiar with the type of stress/toxicity caused by these agents. Figure legend 2 now reads: “Figure 2. Demonstration of the utility of stress reporter mice to define mechanisms of toxicity. A. NRF2_Hmox1 reporter mice [11]. iAs, a naturally occurring metalloid, was dosed at 10mg/kg, p.o. and 24h heart tissue (20x) was processed for LacZ staining. B. Hmox1 reporter model [8]. Acetaminophen (APAP; in excess, such as an overdose, results in rapid depletion of glutathione within the liver), 300mg/kg p.o.; NAC (N-acetylcysteine), 300mg/kg; LacZ staining in liver tissue (10x). C. p21 reporter model was tested with the anti-cancer drug Cisplatin 10mg/kg, which causes damage to nuclear and mitochondrial DNA [17]. LacZ staining in liver tissue (10x).”" } ] }, { "id": "153304", "date": "07 Jul 2023", "name": "Heather M. Wallace", "expertise": [ "Reviewer Expertise Toxicology and risk assessment particularly of contaminants" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and well written overview addressing the the use of in vivo reporter models and how these might provide mechanistic detail not available via other methods. The paper discusses toxic risk and the text would benefit from this being clearly defined. The use of \"smart\" approaches is good but is this being used in the well known sense of smart goals? The authors argue that n = 4 is sufficient for the in vivo studies. It would be good to provide the evidence supporting this statement. It is stated that risk assessment is qualified by biomarkers but this statement needs support and explanation.\n\nA minor correction OCDE should be OECD through out.\n\nIs the work 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": "9977", "date": "29 Aug 2023", "name": "Francisco Inesta-Vaquera", "role": "Author Response", "response": "1.- The paper discusses toxic risk and the text would benefit from this being clearly defined. We now define “Toxic risk” as the “likelihood of a chemical causing an adverse health effect”. 2.- The use of \"smart\" approaches is good but is this being used in the well-known sense of smart goals? The term “smart approaches” has been defined as “tier 3 non-conventional in vivo toxicity testing systems” (7). 3.- The authors argue that n = 4 is sufficient for the in vivo studies. It would be good to provide the evidence supporting this statement. As per above reviewer one’s comment 1. 4.- It is stated that risk assessment is qualified by biomarkers but this statement needs support and explanation. A new sentence has been added to the manuscript: “These in vivo reporters can therefore be used as early biomarkers of toxicity for quantitative risk assessment by detecting adverse effects at low levels of exposure.” 5.- A minor correction OCDE should be OECD through out. This has been corrected." } ] } ]	1	https://f1000research.com/articles/11-1164
https://f1000research.com/articles/10-1148/v1	12 Nov 21	{ "type": "Systematic Review", "title": "Enterprise resource planning implementation within science and technology park (STP) organisations: an avenue for future research. A systematic review.", "authors": [ "Sharbani Harun", "Magiswary Dorasamy", "Abdul Aziz Bin Ahmad", "Ching Seng Yap", "Saida Harguem", "Abdul Aziz Bin Ahmad", "Ching Seng Yap", "Saida Harguem" ], "abstract": "Background: Enterprise resource planning (ERP) is critical to enhancing the ability to control commercial activities and results in a competitive advantage when combined with an organisation's existing competitive advantages. However, our practise review reveals that end users resist ERP implementation because the resulting changes will alter the current status quo. The implementation of an ERP system in an organisation is complex as it affects multiple areas of the business. Resistance to change is cited as a factor of ERP failure. Methods: In this study, we conducted a systematic literature review using Transfield’s five stages and established a conceptual framework for ERP system implementation in science and technology parks (STPs). Articles collected from Emerald, Science Direct, ProQuest and Scopus databases between 1st June 2021 and 15th June 2021. Two authors were assigned to check the suitability of the articles in order to avoid risk of bias. Articles were analysed based on components of a research paper and the data was tabulated using MS Excel. Results: Only eight papers (0.011% of all the papers) appeared when we searched for papers related to ERP with a focus on post ERP Implementation, end-user behaviours, organisational performance, and the accelerated SAP (system application and product) methodology. We found that there are hardly any articles on ERP post implementations in STP context particularly based on the evaluation part of accelerated SAP.\n\nConclusions: Results indicate the lack of studies in this field, particularly those addressing issues related to STP. This study attempted to broaden the understanding of the ERP's effectiveness, particularly in terms of an organisation's operational performance.", "keywords": [ "Enterprise Resource Planning (ERP)", "Organisational Performance", "Accelerated ASAP", "Science & Technology Park (STP)", "Post implementation", "Malaysia", "End User Behaviour", "Systematic Literature Review" ], "content": "Introduction\n\nOrganisations must improve their own business practices and procedures to survive in a rapidly changing business environment. Enterprise resource planning (ERP) systems are the most significant development incorporating information technology use and are quickly becoming the backbone of organisations. The challenges and high failure rates associated with ERP implementation have been extensively discussed in the literature. Companies typically experience business problems after investing a significant amount of money in an ERP system but are inevitably left with an ERP investment for which they received nothing in return.1 In terms of ERP implementation, one of Malaysia's selected science and technology parks (STPs) is in a similar situation. End-users frequently object to ERP implementations due to the inevitable changes in their current work environment.2 According to a study, ERP implementations fail because employees cannot articulate and meet their changing expectations.3 The acceptance or rejection of technological change has long been recognised as a factor in a person's ability to enhance performance and productivity, while low productivity and dissatisfied customers may result from the unfavourable attitude of employees towards ERP implementation. In addition, excessive organisational spending can lead to a negative view of the ERP system among stakeholders. The selected STP organisation for this study decided to continue implementing ERP based on an assessment of the benefits and challenges of the implementation because it is an important component of their digital transformation initiative. The non-financial and financial benefits were used to justify the organisation's decision to implement ERP. After years of implementing the ERP system in the selected STP in Malaysia, it is still unsure whether the anticipated benefits, such as improved operational performance and the value-added through the integration of business processes with best practices, have been realised as no evaluation of the ERP system's post-implementation benefits has been conducted. From the preliminary interviews with end-users in the STP organisation, mixed reactions to the use of ERP, both positive and negative, can be reasonably assumed.\n\nThe most difficult phase of an ERP project is the implementation.4 Most leading ERP providers, such as SAP (system application and product), have their own implementation methodologies. The accelerated SAP (ASAP) methodology was created by SAP. ASAP5 is a methodology for planning, managing, and delivering SAP efficiently and cost-effectively. However, as the ERP lifecycle has no evaluation component, the benefits of implementing and using ERP for the selected STP could not be evaluated. Thus, after completing the ERP implementation project, these evaluation elements may be incorporated into ASAP, demonstrating the benefits of ERP. This study will eventually lead to the development of a framework that incorporates evaluation elements into the post ERP implementation phases of the ASAP methodology. Therefore, this study aims to analyse the factors that contribute to user resistance and provide necessary information for decision-makers to complete the actions required to achieve success in the case of STP.\n\nERPs are critical tools for acquiring the capability to control commercial activities and result in a competitive advantage when combined with a firm’s existing competitive advantages.6 A study recommended several strategies for maximising the ERP system’s benefits and thus improving the overall organisational performance.7 Businesses primarily implement ERP to streamline operations, improve performance and create value by integrating business processes with best practices, management functions, real-time reporting, and the ability to study data. An ERP system enables a business to holistically manage all aspects of its operations to remain competitive in the current business environment.8\n\nERP systems enable companies to handle their operations effectively with possible benefits, including increased process efficiency, improved data analysis, increased consistency in decision-making, lean inventory, improved communication across the supply chain, and strong customer experience.9 Furthermore, ERP has resulted in substantial cost savings, increased return on investment, and improved access to information, ultimately improving consumer decision-making.10 In addition, ERP increases productivity, decreases downtime, reduces lead times, decreases the order cycle time, minimises inventory, reduces the system installation time, reduces quality issues, decreases the scrap rate, decreases the rework rate, and increases the overall company productivity and pay.11 According to studies, ERP systems can also help an organisation increase its capacity and flexibility in response to changing client needs.12\n\nCompanies usually have business problems after investing a significant amount of money in an ERP system but end up with an ERP investment for which they have not received anything in return.13 In terms of ERP implementation, the STP in Malaysia selected for this study is in a similar situation. Following the implementation of ERP in the selected STP, the lack of end-user support in the system’s use and the absence of organisation performance evaluation were observed. Understanding critical issues will aid organisations in the successful carrying out of ERP systems in the future. Managers and those responsible for information must also understand and quantify the benefits of the ERP system to justify ongoing costs and the impacts to the organisation.14 Generally, the impacts and benefits of implementing an ERP system are not realised until the system has been completed and is fully operational.\n\nAccording to the literature, several theories can shed light on the challenges mentioned above for STP context in the Table 1. These theories were obtained from preliminary reading prior to the systematic review.\n\nERP = enterprise resource planning.\n\nThe objectives of this proposal are as follows:\n\na) To analyse the research gaps on the implementation of ERP in the STP organisation in Malaysia\n\nb) To propose a conceptual framework for the STP based on the literature review\n\n\nMethods\n\nThis study was approved by the Research Ethical Committee (REC) of Multimedia University (EA2722021). This article is reported in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.30\n\nThis is a systematic literature review paper that highlights the challenges faced by STPs regarding ERP. Thus, we aim to present a preliminary conceptual framework that the body of knowledge in the field of ERP can adopt in STPs. To bridge the research gap in this field, we searched the current research landscape using Tranfield’s five stages.21 Tranfield's method is a well-known and highly cited method for carrying out systematic reviews of the literature. Our literature review was based on the following five stages:\n\na) Planning the review\n\nb) Identifying and evaluating studies\n\nc) Extracting and synthesising data\n\nd) Reporting descriptive findings\n\ne) Utilising the findings to inform research and practice\n\nThe primary objective of this review is to ascertain the nature and scope of the research conducted on the selected STP’s ERP implementation and subsequent organisational performance. We intend to provide researchers with a comprehensive review of post ERP implementation in STP, with particular emphasis on end-users who oppose ERP implementation due to the disruption of the current status quo. The objective is to provide information on the hidden causes of such behavioural change and inform the knowledge community about potential research directions for the field.\n\nThis study aims to identify solutions that will enable managers to make more informed decisions, mitigate the impact of technological change on the organisation’s social system, significantly reduce the implementation time, increase manager training and balance the effectiveness of ERP in manufacturing firms.22 The knowledge gained from this research might have broad implications for raising managers’ and employees’ awareness of the critical nature of preparation before implementing ERP systems and technical change.23\n\nThis study aims to examine the organisational benefits of ERP after implementation and their impact on the organisational efficiency and operational performance of a Malaysian STP. This study's objectives include determining the ERP system's post-implementation benefits and its effective use in Malaysia’s STP. The researchers believe that understanding all the shortfalls is critical to the full comprehension of the consequences of ERP implementation in the Malaysian STP. Additionally, this research aims to develop an ERP framework for future use in Malaysian STP organisations to maximise the benefits of post-ERP implementation.\n\nInclusion and exclusion criteria\n\nArticles were collected from database inception to 15 June 2021. Table 2 shows the inclusion and exclusion criteria of the articles. The search was conducted between 1 June 2021 – 15 June 2021.\n\nSearch strategy\n\nWe searched four major online databases (Emerald, Scopus, Pro Quest, Science Direct) as they are largest databases for social science journal articles. The following keywords were used: (1) enterprise resource planning (ERP), (2) ERP AND post-implementation, (3) ERP AND post-implementation AND end-user behaviours, and (4) ERP AND post-implementation AND end-user behaviours AND organisational performance AND Accelerated SAP (ASAP) methodology. The keywords set used in this study is provided in Table 3.\n\nTwo authors collected the papers together collaboratively. We collected data related to findings and themes. Stages 4 and 5 of Tranfield are combined and presented in the following sections.\n\n\nResults\n\nA PRISMA flow diagram as shown in Figure 1 illustrates the flow of paper extractions based on the inclusion and exclusion criteria. The number of papers included in this review are presented in Table 3.\n\nThe number of articles found per database and search strategy is presented in Table 3. The Scopus database recorded the highest number of ERP papers. When the search was executed with many keywords combined, Science Direct showed more related papers than the other online databases. We found only eight papers when the search for the final combination of ERP, post-implementation, end-user behaviours, organisational performance, and ASAP was executed. The fraction of papers are shown in Table 4. We further analysed the eight papers to gain some insights. Two of the papers were found to be unrelated papers. Hence, only six papers were included in this analysis, as shown in Table 5.\n\nCSF=critical success factor.\n\n\n\n• This methodological approach has been put to the test by running a validation experiment with the company partner of the research project.\n\n• Analyzing results showed that the methodology was an effective way of determining the capabilities that firms should have in order to determine the most appropriate implementation strategy.\n\n\n\n• The findings of this study show that organisational culture functions as a moderator and moderates the relationship between CSF and ERP project implementation success in India.\n\n\n\n• This article emphasised five factors that influence ICT projects: the likelihood of success, the duration of the project, the complexity of the project, the types of projects, and the methods by which projects are completed.\n\n• The dimensions were investigated in order to identify the critical constructs and elements that needed to be considered. The various dimensions were plotted using a multidimensional model.\n\n\n\n• Despite the fact that each adopting organisation has its own set of goals for its systems project, we discovered many similarities in motivations, concerns, and strategies across organisations.\n\n• This study identifies several critical issues in ERP project management.\n\n\n\n• The findings show that there is an association between organisational culture and ERP implementation problems, but there is no direct evidence that there is an association between national culture and implementation problems. Furthermore, the findings show that these various implementation issues can be caused by a misalignment between a small set of core values indicative of a customer's organisational culture.\n\n• At the end of the paper, we review our predictions, draw conclusions about them and the work of the key authors of national and organisational culture, and discuss future work.\n\n\n\n• ERP systems are related to business engineering (BE), which places emphasis on decades of rigorous research that seeks to establish the most efficient business practises.\n\n• As a part of the overview of ERP strategic applications for industrial marketing, there is also a description of the various cases used for applications. For instance, an effective streamlined sales order process is showcased, and the managerial implications are also identified.\n\nTable 4 shows the results based on all the sources and groups of keywords mentioned above. When the ‘ERP’ was used, 72635 articles were discovered. This figure dropped to 3,189 when we used ‘ERP + post implementation’. When we used the keywords ‘ERP + post-implementation + end-user behaviours + organisational performance’, 524 articles were produced. Following the careful selection based on the inclusion and exclusion criteria described in the following section, eight papers about ‘ERP + post-implementation + end-user behaviours + organisational performance + Accelerated SAP (ASAP)’ were identified.\n\nThis study identifies several critical issues in the ERP project as illustrated in Table 5. The research reveals that the organisation’s motivations, concerns, and strategies shared many similarities. The findings indicate that organisational culture has an impact on the relationship between the critical success factor (CSF) and the ERP project’s success. Nonetheless, no direct connection seems to exist between national culture and implementation issues.\n\n\nDiscussion\n\nThere are several gaps identified in this study. There are limited studies on post ERP implementation success in the context of STP focusing on end-user behaviour and organisational performance using a balance scorecard approach. This limitation is exhibited in terms of lack of evidence and study on STP particular case studies. The case studies are crucial for STPs to understand and adopt any possible recommendations. Implication of this study contributes to the decision of STP to invest in ERP in order to fully harvest the benefits.\n\nA limitation of this study is the number of keywords selected. Keyword selections are based on the research focus. However, it is possible to obtain more articles if the keywords are expanded to a field of study that is not specific in nature, such as STP. This could possibly lead to publication biases.\n\nGap 1: The result presents a clear research gap in post-ERP implementation success in the context of the STP that focuses on organisational performance using the balanced scorecard approach.\n\nGap 2: A research gap in post ERP implementation success exists in the context of the STP that focuses on end-user behaviour to prevent resistance to change.\n\nGap 3: A research gap exists in terms of the post ERP implementation component in the ASAP methodology, i.e. evaluating the post-implementation is crucial to ERP success.\n\nWe propose a conceptual framework in Figure 2 for studies to improve the post-implementation success by focusing on the ASAP methodology to focus on the evaluation stage using three main theories to understand three different dimensions:\n\n1. End-user behaviour: Sociotechnical system theory\n\n2. ERP implementation: Unified theory of acceptance and use of technology model\n\n3. Organisational performance: Balanced scorecard (BSC)\n\nERP=enterprise resource planning; ASAP=Accelerated SAP (System Application and Product).\n\nResearchers involved in examining the relationship between post ERP implementation success and STP could consider the main findings of the current study to explore options for future work. The two major themes for future research consideration include the following:\n\n• Theme 1: Post ERP implementation evaluation using BSC to evaluate organisation performance\n\n• Theme 2: ERP end-user behaviour towards resistance to changes\n\n\nConclusions\n\nThis study aims to inform the ERP for STP knowledge community about the research gaps in the application of theories in post ERP implementation success and organisation performance based on the published literature. In this study, we applied the five-stage methodology of Tranfield et al. (2003) in writing papers based on the comprehensive review of the literature in a given area. This methodology was used to understand the extent and nature of post ERP implementation in STP research in organisational performance. From the extensive search of 72,635 papers in the ERP domain, our search list was narrowed to eight papers (0.011%) that examined the post ERP implementation involving STPs, end-user behaviour, organisational performance, and the ASAP methodology. Our review of the eight papers suggests that the scope for more significant research on the two major areas is present. More empirical work is required to improve the understanding of the determinants of post ERP implementation success factors in the context of STPs. The implication for organisations is that they will have an improved plan in place to harvest their ERP investment in the future.\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 ‘Enterprise resource planning implementation within science and technology park (STP) organisations: an avenue for future research. A systematic review’. https://doi.org/10.6084/m9.figshare.16821982.30\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank the Multimedia University, Malaysia for the support and opportunity to publish this paper.\n\n\nReferences\n\nIsmail A, Harun S: The Effect of Enterprise Resource Planning (ERP) Systems Implementation on Organizational Performance.\n\nLiere-Netheler K, Vogelsang K, Hoppe U, et al.: Towards the User: Extending the Job Characteristics Model to Measure Job Satisfaction for ERP Based Workplaces-A Qualitative Approach. CONF-IRM. 2017; (p. 37).\n\nBento F, Costa CJ, Aparicio M:ERP Conceptual Ecology. World Conference on Information Systems and Technologies. Cham:Springer;2019, April; (pp. 351–360).\n\nMueller SK, Mendling J, Bernroider EW: The roles of social identity and dynamic salient group formations for ERP program management success in a postmerger context. Inf. Syst. J. 2019; 29(3): 609–640. Publisher Full Text\n\nSupriyono S, Sutiah S: Improvement of Project Management Using Accelerated SAP Method in the Odoo ERP.2020.\n\nAlomari IA, Amir AM, Aziz KA, et al.: Effect of enterprise resource planning systems and forms of management control on firm’s competitive advantage. Asian Journal of Accounting and Governance. 2018; 9: 87–98. Publisher Full Text\n\nSolomon F: Challenges And Effect of Implementing Enterprise Resource Planning (ERP) On Organisational Performance (A Case Study at Ethiopian Agricultural Transformation Agency). Addis Ababa University;2019.(Doctoral dissertation.\n\nUllah A, Baharun RB, Nor K, et al.: Enterprise resource planning (ERP) systems and user performance (UP). International Journal of Applied Decision Sciences. 2018; 377–390.\n\nAli M, Miller L: ERP system implementation in large enterprises–a systematic literature review. J. Enterp. Inf. Manag. 2017; 30: 666–692. Publisher Full Text\n\nSeyal AH, Rahman MNA:Investigating Impact of Inter-Organizational Factors in Measuring ERP Systems Success: Bruneian Perspectives. Enterprise Information Systems and the Digitalization of Business Functions. IGI Global;2017; (pp. 178–204). Publisher Full Text\n\nAboAbdo S, Aldhoiena A, Al-Amrib H: Implementing enterprise resource planning ERP system in a large construction company in KSA. Procedia Computer Science. 2019; 164: 463–470. Publisher Full Text\n\nYi W, Mei S, Li Q, et al.: How choice influences risk processing: an ERP study. Biol. Psychol. 2018; 138: 223–230. PubMed Abstract \| Publisher Full Text\n\nAbd Elmonem MA, Nasr ES, Gheith MH:Automating Requirements Elicitation of Cloud-Based ERPs. International Conference on Advanced Intelligent Systems and Informatics. Cham:Springer;2017, September; (pp. 171–180).\n\nBerić D, Stefanović D, Lalić B, et al.: The implementation of ERP and MES Systems as a support to industrial management systems. Int. J. Ind. Eng. Manag. 2018; 9(2): 77–86.\n\nThomassen OJ, Heggen K, Strand R: Applying principles of sociotechnical systems onto working environment research.2017.\n\nDemyanova OV, Andreeva EV, Sibgatullina DR, et al.: Evaluation of effectiveness of information systems implementation in organization (by example of ERP-systems). J. Phys. Conf. Ser. 2018, May; (Vol. 1015(No. 4): p. 042009). IOP Publishing.Publisher Full Text\n\nPARTO, A.: The Impact of Enterprise Resource Planning System on Iranian Firms Performance. Universiti Teknologi Malaysia;2017. (Doctoral dissertation).\n\nSislian L, Jaegler A: ERP implementation effects on sustainable maritime balanced scorecard: evidence from major European ports. Supply Chain Forum: An International Journal. 2020, October; Vol. 21(No. 4): pp. 237–245. Taylor & Francis.Publisher Full Text\n\nSemenoff J: How and why? explaining the factors that influence ERP system usage from the end-users perspective: a literature review.2020.\n\nChakraborty M, Al Rashdi S:Venkatesh et al.'s Unified Theory of Acceptance and Use of Technology (UTAUT) (2003). Technology Adoption and Social Issues: Concepts, Methodologies, Tools, and Applications. IGI Global;2018; (pp. 1657–1674).\n\nBecheikh N, Ziam S, Idrissi O, et al.: How to improve knowledge transfer strategies and practices in education? Answers from a systematic literature review. Res. High. Educ. J. 2010; 7: 1.\n\nOseni T, Foster SV, Mahbubur R, et al.: A framework for ERP post-implementation amendments: A literature analysis. Australasian. J. Inf. Syst. 2017; 21. Publisher Full Text\n\nKabir MR: Impact of ERP Implementation on Productivity and Profitability: An Empirical Study on the Largest Bangladeshi Steels Manufacturer. Int. J. Entrepreneurial Res. 2020; 3(4): 88–94. Publisher Full Text\n\nCapaldo G, Rippa P: A planned-oriented approach for EPR implementation strategy selection. J. Enterp. Inf. Manag. 2009; 22: 642–659. Publisher Full Text\n\nAnnamalai C, Ramayah T: Does an implementation stage act as a moderator in enterprise resource planning (ERP) projects in India? An empirical study. Asian Journal of Research in Banking and Finance. 2012; 2(2): 200–227.\n\nJoseph N: Conceptualising a multidimensional model of information communication and technology project complexity. South African Journal of Information Management. 2017; 19(1): 1–14. Publisher Full Text\n\nKumar V, Maheshwari B, Kumar U: An investigation of critical management issues in ERP implementation: empirical evidence from Canadian organizations. Technovation. 2003; 23(10): 793–807. Publisher Full Text\n\nKrumbholz M, Maiden N: The implementation of enterprise resource planning packages in different organisational and national cultures. Inf. Syst. 2001; 26(3): 185–204. Publisher Full Text\n\nGardiner SC, Hanna JB, LaTour MS: ERP and the reengineering of industrial marketing processes: A prescriptive overview for the new-age marketing manager. Ind. Mark. Manag. 2002; 31(4): 357–365. Publisher Full Text\n\nDorasamy M, Harun S: Prisma Flow Diagram and Checklist. figshare. Figure. 2021. Publisher Full Text" }	[ { "id": "121593", "date": "08 Feb 2022", "name": "Amizawati Mohd Amir", "expertise": [ "Reviewer Expertise Management Accounting and Control System" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe aim of the study is to understand that ERP adoption in STP organisations is a current research topic. However, the justification for the motivation of the study is unclear. Given that the ERP has a long development history starting back in the 1990s, what aspect of ERP is the study focusing on? The ERP concept itself has a very broad term and definition, coming from various perspectives and fields of knowledge, hence the focus of ERP itself needs to be clarified. There are various success factors related to ERP adoption, such as technology challenges, human factors, and financial constraints. Thus, a systematic review of prior research is pertinent to highlight the significance of the proposed study.\nThe paper states high failure reported on ERP implementation, critical discussion on past evidence contributing to the failure may highlight the challenges which could be investigated further. Despite the very limited study done in STP organisations, substantial work published related to ERP adoption need to be reviewed to strengthen and clarify the research focus. Additionally, the study foresees the ERP as a tool for enhancing business competitive advantage. Yet, technology cannot stand alone to promise success. It facilitates organisations to create capability and ability to succeed. Hence, it is suggested that the conceptual framework be revised. A revisit on the proposed theories is also recommended as the Balanced Scorecard is not a theory.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-1148
https://f1000research.com/articles/12-341/v1	28 Mar 23	{ "type": "Research Article", "title": "Central composite design driven optimization of sustainable stability indicating HPLC method for the determination of Tigecycline and greenness assessment", "authors": [ "Hani Mohammed Hafez", "Sona Soliman Barghash", "Marwa M. Soliman", "Moustafa K. Soltan", "Mohamed Abd Elrahman", "Noha Salah Katamesh", "Sona Soliman Barghash", "Marwa M. Soliman", "Moustafa K. Soltan", "Mohamed Abd Elrahman", "Noha Salah Katamesh" ], "abstract": "Background: Tigecycline (TGC) is a recently developed antibiotic to battle resistant bacteria. The procedures outlined in the literature for analyzing TGC involve chemical solvents that could be hazardous. Therefore, this study aimed to create a sustainable and stable HPLC technique for quantifying Tigecycline in lyophilized powder. The powerful chemometric tool, experimental design (ED), will be applied to analyze the variables' interaction and impact on the selected analytical target profiles. Response surface methodology provides a tutorial on using the central composite design with three levels of variables and quadratic programming to optimize the design space of the developed method. Methods: The New HPLC method consisted of an aqueous buffer and ethanol as a green mobile phase run on a reversed-phase symmetry C18 column. A full resolution between the Tigecycline and its degradation product peaks was achieved in a short analytical runtime. Results: Further, the specificity, accuracy, precision, robustness and stability indicating power of the proposed approach were verified through stress degrading testing. Conclusions: Finally, the analytical eco-scale and the green Analytical Procedure Index (GAPI) were utilized to determine how environmentally friendly the recommended method was compared to other published approaches.", "keywords": [ "Tigecycline", "Green liquid chromatography", "GAPI", "Stability indicating", "HPLC", "experimental design" ], "content": "Introduction\n\nTigecycline (TGC) is a commonly used antibiotic for treating bacteria resistant to other antibiotics. TGC was the first antibacterial agent of the glycylcycline family, and it has shown some promise as a treatment for those afflicted with infections that are resistant to other methods.1,2 It was created due to the rising prevalence of antibiotic-resistant pathogens, including S. aureus, Acinetobacter, and E. coli. Due to its structural modifications, its therapeutic action has been widened to encompass both +ve and –ve gram bacteria and types resistant to several drugs.3\n\nAdditionally, it has previously been examined by research participants in clinical studies as a single-drug therapy for treating bacterial infections that are difficult to treat. Indeed, its pharmacodynamics and pharmacokinetics aspects all play a role in this. Studies have indicated that TGC is more effective in treating complicated intra-abdominal and severe skin infections. TGC can only be obtained through intravenous administration.4 TGC is a chemical molecule that is partially synthesized and obtained through fermentation. Because it possesses polar groups such as hydroxyl, amide, and amine groups, it is a tetracyclic molecule with a high degree of polarity, as shown in Figure 1 (which depicts the molecule's chemical structure). The chemical formula C29H39N5O8 has a molecular weight of 585.65 (Figure 1). The reported solubility of TGC was found in water.5–7\n\nBefore a pharmaceutical product may be created and marketed, the chemical stability of an active pharmaceutical substance must be proven. The International Conference on Harmonization mandates that recent pharmaceutical components and dosage forms undergo stability testing.8–10 Investigation is required to understand the intrinsic stability qualities of the active substance in which hydrolysis is a potential issue. Acidic and basic hydrolyses are the most commonly used tests.9,10 TGC was previously determined using spectrophotometric methods,11 fluorescence determination,12–14 and liquid chromatography coupled with ultraviolet and mass spectrometry detection in human plasma15–26 and pharmaceutical preparations.27–32 Other methods, such as stability-indicating,33–39 have also been used to determine the drug's parenteral dosage form. The bulk of these processes take significant time, are harmful to the surrounding ecosystem, and require using acetonitrile as a mobile phase.\n\nGreen analytical chemistry (GAC) has recently become a focus of attention in the scientific community. GAC is a subfield of analytical chemistry that aims to eliminate harmful materials' application in analytical techniques and reduce pollution. The amount of energy used and the amount of waste produced can be reduced40,41 without impacting the analytical performance of the method. Consequently, this study focused on substituting less dangerous and environmentally friendly solvents for existing mobile phases to provide eco-friendly solutions first. TGC determination may be accomplished using simple and rapid chromatographic techniques that do not need sample extraction, pre-treatment filtration, or derivatization. The (GAPI), the Green Analytical Procedure Index, and the Eco-scale analytical tool were used to assess the chromatographic method's environmental friendliness.42,43 In addition, tests of drug products' chemical stability are subjected to artificially induced degradation to evaluate the results of these tests.\n\nHPLC is a dynamic separation technology with many applications, but the procedure is ultra-critical because so many parameters must be modified before each run. Therefore, these methods need more understanding. For example, the experimental design (ED) approach was utilized to save the time and effort required to reduce the experiment's number, reagent consumption, and laboratory work. ED identifies and quantifies links between factors and responses. Furthermore, ED explores how several factors affect response levels.44,45 All significant variables and levels were examined in a response surface methodology (RSM) and central composite design (CCD). This design comprises a central core of a two-level factorial design (2n). This core, in its role, consists of 2n outer points and one center point. The application of ANOVA allows for the computation of the significance of the models' coefficients of studied variables.45,46\n\nFinally, to accomplish this goal, researchers have developed a stability-indicating HPLC approach that is environmentally friendly, easy, conventional, and sensitive, utilizing the advantages of ED methodology. This method is used to measure degradation products in TGC dosage form simultaneously. The approach was validated by adhering to the International Conference on Harmonization (ICH) standards.47,48\n\n\nMethods\n\nTGC was donated by the QPS laboratory (Egypt). Ammonium acetate, triethylamine (TEA), ammonium hydroxide, ethylene diamine tetra-acetic acid disodium salt dihydrate (EDTA), and acetic acid were obtained from Merck (Germany). Without additional purification, HPLC-grade ethanol from Merck (Germany) was utilized. A Milli-pore analytical deionization system collected deionized water (Bedford, MA). Tygacil® (Pfizer, U.S.A.) lyophilized vial containing fifty milligrams of TGC was purchased from wholesale suppliers during the product's shelf life.\n\nA quaternary pump and a solvent chamber with an auto-sampler injection system were both parts of the Alliance HPLC instrumentation (Waters, U.S.A.) used in this study. A conventional flow cell was coupled to a Waters photodiode array for detection. The data was gathered with the help of the Empower 3 program. All results were calculated in Design Expert 13 software from Stat-Ease Inc. (Minneapolis, U.S.A.) and Microsoft Excel 2016 (Microsoft Corporation, U.S.A.). Lab pH meter model AD1030 from ADWA in Romania was used to adjust the mobile phase's pH to the desired level. The accurate weighing was accomplished using an analytical balance of SA 210 D (Scientech, U.S.A.).\n\nPreparing an ammonium acetate pH 6.0 buffer\n\nTransfer 3.85 g ammonium acetate (50 mM), 5.82 g EDTA (20 mM), and 0.2 mL TEA(0.2% v:v) to 1000 mL HPLC grade water, sonicated to dissolve, and then adjust to pH 6.25with glacial acetic acid.\n\nOptimized chromatographic conditions\n\nThe assessment of TGC in pharmaceutical drug products and degradation investigations was performed on a reversed-phase symmetry C18 column (10* 0.46 centimetre, 3.5 micrometres), Waters (Ireland). For the liquid chromatography, a mobile phase of 85 volumes of buffer solution (50 mM ammonium acetate, 20 mM disodium edetate, 0.2% triethylamine) and 15 volume ethanol was used at a temperature of 40 degrees Celsius. It was filtered with a membrane filter from a Millipore of 0.45-micrometre pore size. The analysis was carried out at a flow rate of 1.0 millilitres per minute utilizing UV detection at 275 nanometers. The reference material and the samples were injected with a volume of 40 μL, which was the same.\n\nWater is considered the most suitable solvent for TGC because of its high solubility in water.5,6 The standard stock solution was produced by accurately weighing 25 mg of the TGC reference substance, moving it to a twenty-five millilitres flask, and diluting it with distilled water until it reached a TGC concentration of 1000 μg mL-1. The freshly prepared stock standard solution was then diluted with water to the desired concentration before being filtered through a membrane filter with a pore size of 0.45 micrometres (Millipore).\n\nAliquots were taken from TGC standard stock solutions and transferred into appropriate volumetric flasks to prepare concentrations of 8-60 μg mL-1 for TGC to complete each flask, and distilled water was utilized. The calibration graphs were created by graphing the area under the peak versus TGC concentration in μg mL-1 and calculating the regression equations.\n\nThe stock solution should be diluted for the precision test to produce solutions containing 32, 40, and 48 μg mL-1. These solutions will be evaluated using three separate measurements of TGC samples on the same day (intra-day). Then, it was performed over two more days to assess the investigation's intermediate precision than initially planned (inter-day). For the objective of the accuracy test, exactly known quantities of TGC were successively transferred to a placebo solution to generate solutions with concentration levels of 32, 40, and 48 μg mL-1, equivalent to 80, 100, and 120 percent of the actual analytical level.\n\nTo make the sample solution, weighing and mixing Tygacil® vials with 50 milligrams of TGC in each twenty-five millilitres flask was necessary. A suitable volume of the solution was added and mixed to the volume with distilled water (40 μg mL-1) and injected.\n\nAn analytical method that successfully evaluates and assesses the active constituents without interfering with degradation products, process contaminants, inactive ingredients, or any other possible impurities is known as a stability-indicating procedure.5,10,47 A standard reference solution and pharmaceutical preparations of 1 mg mL-1 concentration were submitted to rapid forced degradation under acidic, basic, neutral, oxidative, and photolytic conditions to study the interference in the measurement of TGC. The ICH guidelines Q1A (R2) and Q1B were used for these degradation investigations, including solid and solution phases.5,10\n\nAcidic forced degradation\n\nTen milliliters of the TGC stock solution were treated with 100 mM HCl to hasten acidic degradation. Subsequently, the solution was examined at 25 and 60°C for an hour.5,10\n\nAlkaline forced degradation\n\nTo initiate alkaline degradation, a portion of ten milliliters of TGC stock solution was exposed to ten milliliters of 100 mM sodium hydroxide at 25°C for two hours.5,10\n\nOxidative forced degradation\n\nTo commence the oxidative degradation, a portion of ten milliliters of TGC stock solution was maintained at 25°C for two hours, shielded from illumination, with ten milliliters of 5 percent H2O2.5,10\n\nThermal forced degradation\n\nUnder neutral conditions, both TGC powder and solution were thermally degraded. TGC powder was maintained in a 60°C oven. A portion of the treated material was weighed and diluted in a 25-millilitre flask. A TGC stock solution was mixed with distilled water and heated to 60°C.5,10\n\nPhotolytic forced degradation\n\nTGC powder and solution were tested for photolytic degeneration after twenty-four hours of exposure to near-ultraviolet radiation. There was enough TGC powder weighed and tested under ultraviolet radiation. In addition, an amount of TGC stock solution equal to ten milliliters of distilled water was treated with ultraviolet light for testing.5,10,47 Therefore, every sample was taken at the intervals that were specified above. If necessary, it was treated before being injected to stop the degradation, and then it was diluted with distilled water to the target concentration (40 μg mL-1).\n\nInitially, a trial and error approach was used to learn about the method's efficacy and to identify critical, independent parameters and their impacts on dependent responses or parameters. Establishing a separation between TGC and its degradants with a resolution of more than 2.0 is the primary goal of the RP-HPLC method development, in addition to other important parameters that affect the accuracy and precision of the method. For example, the ED of the proposed method was set, and the central composite design (CCD) with the response surface method (RSM) was used.46\n\nThe ED initially begins with the pre-determination of the principle parameter crucial to the method efficacy; therefore, it should be measured during the ED (critical quality attributes (CQA)). The next step was determining the acceptable value of the CQA according to universal pharmacopoeias such as USP and BP, defined as analytical target profile (ATP). The optimum resolution (Rs) between peaks is considered the principle CQA. The peak symmetry, capacity factor, and other CQAs were also investigated (Table 1). The proposed ATPs had to be determined (Table 1) to achieve good reliability of the results. The next step is to define the main variables of the proposed chromatographic method that affect the CQAs and ATPs values which are defined as critical method parameters (CMPs) (Table 1).44\n\nFurthermore, the RSM with a three-level CCD was selected for the ED. In other words, the design comprises 3 important factors independently acting to build the design. The independent factors investigated were the ratio of green solvent (% Ethanol), the pH value of aqueous mobile phase (pH), and the concentration of additives EDTA and TEA.\n\nTwenty runs, each with a unique condition, made up the CCD (Table 2). The aqueous portion of the mobile phase, 100 mL in volume, was made for each run. Once the pH was adjusted, the mobile phase was eluted at a rate of 1.0 mL min-1 in each run. TGC and its degradation products were measured at 275 nm. After collecting and analyzing all responses, models were created in design expert 13 to determine the interactive effects between the CQAs and the CMPs. The optimization process was designed to find the optimal Rs between the peaks of TGC and its degradation products without surrendering the other parameters that eventually determine the system's efficacy. Throughout this process, optimization was carried out both numerically and graphically.\n\n\nResults\n\nPreliminary tests for screening\n\nThe HPLC approach was selected to separate and quantify TGC and its degradation products. To establish a method for monitoring the stability of a product, the proposed chromatographic conditions must be developed and optimized. The impact of the relevant factors was screened using the preliminary experiments. TGC has a molecular weight of 585.65, log P values of -3.86, and pKa values of 3.19, 6.4, 7.54, and 9.14, respectively.6,49 For example, water was selected as the appropriate solvent for TGC due to its high water solubility.5,6 In addition, all stock solutions in water were refrigerated for optimal drug stability in amber containers.\n\nFurthermore, an ultraviolet spectrophotometer scans 10 μg mL-1 TGC solution to find appropriate wavelengths. The wavelength of the measurement must be 20 nanometers longer than the ultra-violet cutoff of the solvent.50 The ultraviolet cutoff of ammonium formate containing EDTA and TEA mixed with ethanol (which were used in reversed-phase liquid chromatography trials) was 210 nm.50 Consequently, the UV detector can be set at 275 nm for TGC detection. According to Figure 2, the latter wavelength would show acceptable sensitivity for the TGC peak with good peak shape and baseline in the HPLC chromatogram compared to a wavelength of 245 nm, so it was chosen for ED examinations (Figure 2).\n\nTwo columns were employed to perform performance investigations. These columns were a VDSpher C18-E column (250.46 cm, 5 μm) from Nouryon (Sweden) and symmetry C18 Columns (100.46 cm, 3.5 μm) from Waters (Ireland). TGC is an antibiotic that resembles the chemical properties of tetracycline. TGC causes tailing peaks in a reversed-phase column by forming chelate complexes with metal ions and binding to the silanol group (Si-O-R). Experiments demonstrated that the column, symmetry C18 Column, was more suitable because it had excellent chromatographic performance, ideal resolution, sensitivity, and peak symmetry while maintaining a shorter run time. A preliminary trial showed that mobile phase pH greater than 4 improves the peak shape of TGC but still needs improvement. As a result, some additives for mobile phases were tested for improving TGC peak shape and the resolution with its degradation products.\n\nHigher pH values (4 or higher) combined with different concentrations of ethanol, EDTA, or TEA led to greater interference between the peaks of TGC and degradation products and a faster elution. These methods were developed to prevent adsorption on reversed-phase columns and chelate complexes formation. Therefore, the variables' minimum and maximum values were set at (10 and 20%), (5 and 7.5 mM), (10 and 40 mM), and (0.1 and 0.3%) for the percent of ethanol, mobile phase pH, EDTA molar concentration and percent of TEA, respectively. These values were utilized to figure out the possible range of the variables.\n\nRSM and CCD study\n\nDesign Expert 13 software was used to make the ED, and RSM was used to explore the influence of the variables.51 The model uses a CCD design, combining the independent factors in 20 different and five-centric runs. Our optimization study focused on the following factors: ethanol concentration, mobile phase pH, EDTA molar concentration, and percent of TEA. This study aimed to explore the sweet spot for these four factors. In addition, three distinct degrees of evaluation were carried out for each variable: -α, 0, and +α in Table 2.\n\nAs a result, the quadratic model can determine which parameters are most crucial. CCD is used for response modelling and optimization because it can comprehend the interactions between multiple variables.44–46 the rotatable CCD was chosen because of its high consistency and relatively low variability. This experiment consisted of 20 randomized iterations that were used to assess the factor effects using (-α), (-1), (0), (+1), and (+α) values of examined variables.\n\nResponse analysis and optimization\n\nThe five selected responses were evaluated based on the criteria (ATP). Coded prediction models or equations allow one to compute the importance of various factors in light of the data at hand.45 For instance, an artificial model selection approach used the Akaike information criterion (AIC) to improve accuracy. The P value is used in statistical analysis to determine the degree of significance (Table 3). Compared to the threshold for statistical significance set at (P value = 0.05), all proposed models have minor P values (0.0001), indicating their significance. However, higher P values for the lack of fit indicate that they are not statistically significant (<0.05) (Table 3). A positive coefficient in Table 3 positively impacts the corresponding response, whereas a negative coefficient indicates the opposite.51 Therefore, to ensure model validity, other CCD aspects must be investigated. Finally, strong linearity between the adjusted and expected regression coefficient (R2) was proved if the difference was smaller than 0.2 (Table 3).\n\n* A=%Ethanol, B=Buffer pH, C=EDTA Conc, and D=%TEA.\n\nThere was also the signal-to-noise ratio, which had to be high and was approved if a value greater than 4 indicated appropriate precision. Based on previously existing data, it was possible to determine the relevance of the examined factors to the selected CQAs. The adequate precision allows for evaluating the signal-to-noise ratio, which should be greater than 4.0. Consequently, as shown in Table 3, the findings demonstrate that this model can explore the design space.\n\nIn Figure 3 and Figure 4, contour and 3D surface plots depict the optimal responses against numerous combinations of essential variables. Finally, optimization was performed using both graphical and numerical approaches. In this stage, solutions are developed for the ideal chromatography settings with the best levels of desire by utilizing the limitations of the variables and their responses, as shown in Figure 5.\n\nThe primary objective of the optimizations was to attain a reasonable value for Resolution (>2). Other parameters, including peak symmetry, theoretical plates, capacity factor, and the shortness of analysis time (run time), were also considered. With graphical optimization, it's possible to put these limitations into practice. Finding the “sweet spot” (optimal value) between design space and robustness requires adjusting factor values (yellow area) (Figure 5). However, results that fall into the gray area are generally undesirable. The pH varied between 6.0 and 6.4, the molar concentration of EDTA was between 10 and 40 mM, and the quantity of TEA was between 0.1 and 0.3 percent. The acceptable ranges are illustrated in Figure 5. When using the post-analysis procedure, the computed mean was inside the 95% confidence interval (CI) of the possible values.\n\nThe method was validated by establishing its specificity, linearity, precision, accuracy, and robustness. The validation was done following the ICH requirements.47\n\nSpecificity\n\nTesting for excipient interference was performed by injecting a placebo (house-made solution of lyophilized powder excipient), and a known TGC concentration (40 μg mL-1) was added to the placebo solution. The ability of the method to identify degradation products from the TGC peak is shown to have been successfully demonstrated by the excellent separation of the peaks, as shown in Figure 6.\n\nLinearity and range\n\nThe linearity was confirmed by creating three different analytical graphs based on water, each with six different TGC levels in the 8–60 μg mL-1. Before the injection of the solutions, the column was first allowed to become equilibrated for at least twenty minutes while the mobile phase was being passed via the system. As a point of reference, the area of peaks of the chromatograms and the concentrations of TGC were plotted on the analytical graph to construct the calibration graphs of TGC. Table 4 and Figure 7 state that the results, calibration equation, and determination coefficient were calculated using regression analysis with the least-squares method.\n\nPrecision and accuracy\n\nThe precision of the approach was assessed based on its repeatability and intermediate precision. Six determinations were performed on the same day and under the same conditions to test the repeatability (intra-day) using the same TGC concentration sample. Analyses performed on three separate days (inter-day) and by other analysts in the same laboratory helped determine the method's intermediate precision (between analysts). The accuracy of the proposed method was assessed by adding 32, 40, and 48 μg mL-1 to a sample solution equivalent to 80, 100, and 120 percent of the actual analytical level. As can be seen in Table 5, the percentage of sample recovery was utilized to calculate the percent bias (% error) between the recorded average concentrations and those that were added.\n\nLOD and LOQ\n\nThe LOQ and LOD values were calculated using ICH,47 and the results are presented in Table 4. Calculating LOQ and LOD was accomplished by determining the standard deviation of the intercept and slope ratio over three different analytical graphs. These values were obtained using a linear regression model and multiplying the latter percentage by their respective detection and quantitation limit factors of 3.3 and 10. In addition, the LOQ was tested in an experiment.\n\nRobustness\n\nFor routine analysis, the robustness of the suggested procedure can be judged by its capacity to withstand modest and purposeful changes in method parameters. For example, using the same samples (40 μg mL-1 TGC) under five different analytical parameters, the robustness assessment of the chromatographic procedure for TGC quantification is illustrated in Table 6.\n\n* Proposed method is composed of a mobile phase containing ammonium acetate (0.05 M), disodium edetate (0.02 M), and triethylamine (0.2% v:v) (pH 6.25) - ethanol (85:15, v/v) running on a Symmetry C18 column at a flow rate = 1 mL min-1. Detection was set at 275 nm, and the column temperature was kept at 40°C.\n\nSystem suitability test\n\nA system suitability test was also performed to verify the system's repeatability and resolution for the analysis. This test consisted of 6 replicate injections of a standard solution containing 40 μg mL-1 TGC. Measurements were taken of peak symmetry, theoretical plates, and capacity factor. Observe and record the relative standard deviation of the peak area of injected standard samples and the retention times of all injections.\n\nMeasurement of TGC in drug products\n\nAfter diluting the lyophilized TGC vial with water (40 μg mL-1), filtering, and injecting in triplicate, the percent recoveries of the TGC against the pure compound were determined.\n\n\nDiscussion\n\nICH Q1A (R1) and Q1B recommendations were applied further to study the degradation of TGC under various stress conditions.10,48\n\nWhen no information regarding potential degradation products was available, stress degradation tests were employed to test the method's stability-indicating aspects. In addition, all stress degradation investigations generated probable degradation products. It was found that TGC was labile to all forced degradation conditions, including acidic, alkaline oxidative, thermal hydrolysis, and UV degradation. TGC showed significant decreases in the area under all conditions, ranging from 30 to 70% (Table 7). Furthermore, each type of forced degradation scenario produced a distinct pattern of degradation products corresponding to specific degradation pathways. The TGC reference material and Tygacil had nearly identical findings and practices.\n\nRetention time of TGC is 4.1 min.\n\ndeg stands for “degradation product”.\n\nTo determine whether the proposed method was environmentally friendly, we used the analytical eco-scale to calculate the penalty points gained by each analysis step. A grade of over 75 suggests a special green assessment, while a degree of over 50 shows an acceptable green evaluation, and a grade of 50 indicates inadequate green assessment.40 Analytical procedures can be evaluated using the Green Analytical Procedure Index (GAPI), a new tool that assesses the environmental friendliness of the entire process. Using a unique symbol with five pentagrams, it is possible to use GAPI to analyze and quantify the ecological impact of each phase of an analytical technique. The colors green, yellow, plus red in the GAPI pentagram each stand for a different level of impact: low, middle, as well as high, respectively.\n\nThe number of Globally Harmonized System (GHS) of Classification and Labeling of Chemicals hazard pictograms was multiplied by the degree of hazard warning, which was multiplied by 1, and danger, which was multiplied by 2, to calculate the number of penalty counts that should be assigned to each reagent. Because the GHS risk pictograms are printed on the bottles of the reagents, it is easy to determine the level of danger connected with using the chemicals.41–43,52 The interpretation of the GAPI pentagrams for the proposed chromatographic approach and its comparison with the reported methods33–35 are described in Table 8 and illustrated in Figure 8. The recommended procedure was more environmentally friendly than the reported methods. As a result, it can be used for routine analysis without causing harm to the environment.\n\n\nConclusion\n\nIn this research work, an ED approach for analytical method development consists of (i) developing a full grasp of the intended purpose, (ii) developing forecasting solutions, (iii) constructing an insightful system suitability solution that helps to identify modes of failure, and (iv) follows a design of experiments approach to method development. A central composite design was used to determine the impact of four chromatographic parameters on the chosen CQAs based on the risk assessment. The optimum analytical conditions were projected by a numerical optimization method. These conditions were located by flagging all parameters in an overlay plot. To study the main effects and interactions among different CMPs with the CAAs, 2D-contour plots and 3D-response surface plots were drawn.\n\nThe proposed method estimated the average amount of TGC in pharmaceutical products without interfering with the excipients. The process separates TGC from its degradation products rapidly. Eco-Scale and GAPI also demonstrated the method's greenness, which makes it more suitable for daily use.", "appendix": "Data availability\n\nFigshare: Tig QbD chromatograms, https://doi.org/10.6084/m9.figshare.22153613.v1. 53\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors would like to express their gratitude to quality for pharmaceutical services laboratories (QPS), Tenth of Ramadan, Egypt.\n\n\nReferences\n\nBrust K, Evans A, Plemmons R: Tigecycline in treatment of multidrug-resistant gram-negative bacillus urinary tract infections: a systematic review. J. 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Publisher Full Text\n\nBahgat EA, Hafez HM, El-Sayed HM: Kabil, NA, Development of a solvent-free micellar HPLC method for determination of five antidiabetic drugs using response surface methodology. Microchem. J. 2022; 179: 107446. Publisher Full Text\n\nInternational Conference on Harmonization (ICH): Technical Requirements for the Registration of Pharmaceuticals for Human Use. Validation of analytical procedures, Text and methodology.2005; Q2(R1). (Last accessed October 28, 2022).\n\nInternational Conference on Harmonization (ICH): Technical Requirements for the Registration of Pharmaceuticals for Human use. Stability testing: photostability testing of new drug substances and products Q1B.1996. (Last accessed October 28, 2022).\n\nLast accessed October 28, 2022. Reference Source\n\nLast accessed October 28, 2022. Reference Source\n\nHafez HM, El Deeb S, Naji EAA, et al.: Design of an Experimental Study for the Simultaneous Determination of Cefepime, Piperacillin and Tazobactam Using Micellar Organic Solvent-Free HPLC. Separations. 2022; 9: 215. Publisher Full Text\n\nSaid NS, Nasr ZA, Abdel-Razeq SA: Green stability indicating UV- spectrophotometric techniques for estimation of tenofovir alafenamide in bulk form and dosage forms. Azhar Int. J. Pharm. Med. Sci. 2021; 1: 102–110. Publisher Full Text\n\nHafez H: Tig QbD chromatograms. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "175969", "date": "08 Jun 2023", "name": "Eman I. El-Kimary", "expertise": [ "Reviewer Expertise Pharmaceutical Analytical Chemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes a green stability indicating assay method for the determination of Tegicycline antibiotic in lyophilized powder with a full greenness assessment of the developed method. Response surface methodology using central composite design was applied for the optimization of experimental conditions. The method was fully validated according to the ICH guidelines. I have the following comments:\n\nIn the abstract, what is meant by (stable HPLC)? The whole abstract needs to be revised. In the results part of the abstract, please mention some analytical figures of merit. The conclusion part of the abstract also needs to be edited.\n\nPlease update the introduction part with previously published stability indicating chromatographic assays. Also, in the discussion part, compare the degradation results in the proposed method with those obtained in previously published stability indicating assays.\n\nIn page 3 2nd paragraph, please remove (which depicts the molecule's chemical structure). Also, please revise the sentence (The reported solubility of TGC was found in water) as it is not clear.\n\nIn materials and chemicals, please add the full name of QPS.\n\nUnder (Optimized chromatographic conditions), remove (which was the same.) at the end of this paragraph. Also, please write (centimetre, micrometres, degrees Celsius, etc) in symbols not in words. In general, please revise the symbols of units in the whole manuscript to be in accordance to the metric system.\n\nSome titles need to be more concise. e.g. (Construction of calibration graphs for the linearity test) could be (Construction of calibration graphs), (Preparation of solution for accuracy and precision tests) could be (Accuracy and precision).\n\nUnder thermal forced degradation, Please clarify whether TGC was subjected to thermal degradation in the solid state, followed by thermal degradation of its solution in water or not as it is not clear.\n\nPlease write the full name of QMTP.\n\nUnder section Results, Optimization, Preliminary tests for screening, 2nd paragraph, please correct ammonium formate to ammonium acetate. Also, in this paragraph the choice of detection wavelength is not clear. Please discuss briefly the absorption characteristics of TGC and its effect on choosing the detection wavelength.\n\nWhy EDTA is added to the mobile phase?\n\nThe quality of figure 6 needs to be improved.\n\nIn table 5 and 6, please write %Recovery not R% and %RSD not RSD.\n\nIs the work 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": "9877", "date": "10 Aug 2023", "name": "Hani Hafez", "role": "Author Response", "response": "First reviewer: (Prof. Dr. Eman I. El-Kimary) The manuscript describes a green stability-indicating assay method for determining Tigecycline antibiotic in lyophilized powder with a full greenness assessment of the developed method. Response surface methodology using a central composite design was applied to optimize experimental conditions. The method was fully validated according to the ICH guidelines. I have the following comments: In the abstract, what is meant by (stable HPLC)? The whole abstract needs to be revised. In the results part of the abstract, please mention some analytical figures of merit. The conclusion part of the abstract also needs to be edited. Answer 1: Thank you for our valuable comment, (stable HPLC) has been changed to stability, indicating the HPLC method within the abstract. The results and conclusions parts have been edited to fulfill the statement to clarify the significance of the proposed study. Please update the introduction part with previously published stability indicating chromatographic assays. Also, in the discussion part, compare the degradation results in the proposed method with those obtained in previously published stability-indicating assays. Answer 2: Thank you for our valuable comment; the introduction was updated by adding new reported methods (Ref 40). The discussion part was also updated to include the required comparison. On page 3, 2nd paragraph, please remove (which depicts the molecule's chemical structure). Also, please revise the sentence (The reported solubility of TGC was found in water) as it is unclear. Answer 3: Thank you, the first sentence has been removed, and the second one has been edited and transferred to the discussion part. In materials and chemicals, please add the full name of QPS. Answer 4: Thank you, the full name of QPS has been added. Under (Optimized chromatographic conditions), remove (which was the same.) at the end of this paragraph. Also, please write (centimetre, micrometres, degrees Celsius, etc) in symbols, not words. In general, please revise the symbols of units in the whole manuscript to be in accordance with the metric system. Answer 5: Thank you, the first sentence (which was the same) has been removed, and the other modifications were addressed. Some titles need to be more concise. e.g. (Construction of calibration graphs for the linearity test) could be (Construction of calibration graphs), (Preparation of solution for accuracy and precision tests) could be (Accuracy and precision). Answer 6: Thank you, these titles were edited. Under thermal forced degradation, Please clarify whether TGC was subjected to thermal degradation in the solid state, followed by thermal degradation of its solution in water, as it is unclear. Answer 7: Thank you, the paragraph was edited to describe only the degradation in the solid state. Please write the full name of QMTP. Answer 8: Thank you, the QMTP was changed to ATP, as described above in Table 1 Under section Results, Optimization, Preliminary tests for screening, 2nd paragraph, please correct ammonium formate to ammonium acetate. Also, in this paragraph, the choice of detection wavelength is not clear. Please discuss briefly the absorption characteristics of TGC and its effect on choosing the detection wavelength. Answer 9: Thank you, the ammonium formate was corrected to ammonium acetate. The paragraph was also edited to present the merit of choosing 275 nm over 245 nm wavelength. Why is EDTA added to the mobile phase? Answer 10: Thank you, the following sentence was added under (EDTA was added to the mobile phase to prevent forming of these chelate complexes as metals favor complexing with EDTA than TGC.). It was added under Results, Optimization, Preliminary tests for screening, 3rd paragraph. The quality of figure 6 needs to be improved. Answer 11: The quality of Figure 6 was improved. In table 5 and 6, please write %Recovery not R% and %RSD not RSD. Answer 12: Thank you, %Recovery and %RSD were added" } ] }, { "id": "175432", "date": "14 Jun 2023", "name": "Ali Fouad", "expertise": [ "Reviewer Expertise Medicinal chemistry", "Pharmaceutical analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic of this manuscript is interesting. However, the text is contains some inaccurate expressions as from the technical/scientific, as well as from the linguistic points of view.\nBelow just a number of small changes to the article.\nIn introduction, paragraph three, LC determinations should be differentiated to HPLC And LC/MS/MS methods (ref. 15-26).\n\nThe chemical structure of TGC needs to be adjusted by chem-draw.\n\nIn page 4, optimized chromatographic conditions mobile phase might be written as 85:15 (v:v). Also, nm is more common than nanometre.\n\nIn page 5, Forced degradation tests of TGC, (neutral) should be removed.\n\nIn page 5, Acidic forced degradation was examined at 25 and 60°C while Alkaline forced degradation was examined at 25°C only. In table 7, the results were for acidic and alkaline at 25 and 60°C for each.\n\nIn table 1, what is QMTP? It is quality method target profile, it's used only abbreviated, and it should be illustrated.\n\nCCD, ED, CQA, ATPs, CMPs and QMTP approaches were used in this manuscript, so, some details about each should be added in the introduction or in methodology.\n\nAuthors should go through the manuscript regarding English grammar and make some edits.\nOverall, the article is useful, albeit there are some statements which are scientifically not supported by proper reference and some statements which need to be rephrased.\n\nIs the work 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": "9878", "date": "10 Aug 2023", "name": "Hani Hafez", "role": "Author Response", "response": "Second reviewer: (Prof. Dr. Ali Fouad) The topic of this manuscript is interesting. However, the text contains some inaccurate expressions from the technical/scientific, as well as from the linguistic points of view. Below are just several small changes to the article. In paragraph three's introduction, LC determinations should be differentiated into HPLC and LC/MS/MS methods (ref. 15-26). Answer 1: Thank you, the paragraph was edited. The chemical structure of TGC needs to be adjusted by chem-draw. Answer 2: Thank you, the chemical structure of TGC was adjusted by chem-draw In page 4, optimized chromatographic conditions mobile phase might be written as 85:15 (v:v). Also, nm is more common than nanometre. Answer 3: Thank you, the required modifications were done In page 5, Forced degradation tests of TGC, (neutral) should be removed. Answer 4: Thank you, (neutral) was changed to thermal. On page 5, Acidic forced degradation was examined at 25 and 60°C while Alkaline forced degradation was examined at 25°C only. In table 7, the results were for acidic and alkaline at 25 and 60°C for each. Answer 5: Thank you, Table 7 was corrected In table 1, what is QMTP? It is quality method target profile, it's used only abbreviated, and it should be illustrated. Answer 6: Thank you, Table 1 was corrected and the QMTP was changed to ATP, as described above in Table 1. CCD, ED, CQA, ATPs, CMPs and QMTP approaches were used in this manuscript, so, some details about each should be added in the introduction or in methodology. Answer 7: Thank you, the paragraph was edited to describe only the degradation in the solid state. Authors should go through the manuscript regarding English grammar and make some edits. Answer 8: Thank you, the manuscript was revised and edited. Overall, the article is useful, albeit there are some statements which are scientifically not supported by proper reference and some statements which need to be rephrased. Answer 9: the article was grammarly re-checked and re-phrased" } ] }, { "id": "175968", "date": "15 Jun 2023", "name": "Hala E. Zaazaa", "expertise": [ "Reviewer Expertise Pharmaceutical analytical Chemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGreenness assessment and QbD are essential aspects during method development. There is some interest in publishing the manuscript which, however, it also has a number of significant shortcomings that should be addressed before the manuscript is acceptable for indexing:\nWhile authors summarize a comparison between their suggested method and the previously published ones, they missed this article1 that was published in International Journal of Applied Pharmaceutics 2021. I ask the authors to add it illustrating the difference between their work and the aforementioned article.\n\nIt was valuable that the authors summarize the difference between their suggested method and the previously published ones in a tabulated form and show the greenness assessment of each.\n\nIt will be worthy to add other greenness assessment tools such as Eco-scale and NEMI.\n\nPlease add a reference for the buffer preparation.\n\nPreparing reference substance solutions should not contain any results information so the statement \"Water is considered the most suitable solvent for TGC because of its high solubility in water\" should be omitted and transferred into the discussion section. The same for \"An analytical method that successfully evaluates and assesses the active constituents without interfering with degradation products, process contaminants, inactive ingredients, or any other possible impurities is known as a stability-indicating procedure.5,10,47\" under Forced degradation tests of TGC.\n\nLinearity and range are separate items according to ICH guidelines. Also Precision and accuracy.\n\nNo need for Figure 7.\n\nThe precision should be expressed as repeatability and intermediate precision not (intra-day) and (inter-day) in Table 5.\n\nAccording to ICH guidelines, robustness should be tested by system suitability parameters especially resolution not only recover %.\n\nWhere are the results of the System suitability test?\n\nReference number of the proposed method (d) is missing in Table 8.\n\nNo statistical analysis was illustrated by the authors.\n\nIs the work 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": [ { "c_id": "9879", "date": "10 Aug 2023", "name": "Hani Hafez", "role": "Author Response", "response": "Third reviewer: (Prof. Dr. Hala E. Zaazaa) Greenness assessment and QbD are essential aspects during method development. There is some interest in publishing the manuscript which; however, it also has a number of significant shortcomings that should be addressed before the manuscript is acceptable for indexing: While authors summarize a comparison between their suggested method and the previously published ones, they missed this article1 that was published in International Journal of Applied Pharmaceutics 2021. I ask the authors to add it illustrating the difference between their work and the aforementioned article. Answer 1: Thank you, the article was added. The comparison was created from 2 points: the degradation percentage (Table 7) and the greenness assessment (Tables 8 and 9). It was valuable that the authors summarize the difference between their suggested method and the previously published ones in a tabulated form and show the greenness assessment of each. Answer 2: Thank you, the comparison was created from 2 points: the degradation percentage (Table 7) and the greenness assessment (Tables 8 and 9). It will be worthy to add other greenness assessment tools such as Eco-scale and NEMI. Answer 3: Thank you, the eco-scale and NEMI were added. Please add a reference for the buffer preparation. Answer 4: Thank you, the trials in the development stage obtained the buffer preparation. Preparing reference substance solutions should not contain any results information so the statement \"Water is considered the most suitable solvent for TGC because of its high solubility in water\" should be omitted and transferred into the discussion section. The same for \"An analytical method that successfully evaluates and assesses the active constituents without interfering with degradation products, process contaminants, inactive ingredients, or any other possible impurities is known as a stability-indicating procedure.5,10,47\" under Forced degradation tests of TGC. Answer 5: Thank you, the sentences were edited and transferred. Linearity and range are separate items according to ICH guidelines. Also precision and accuracy. Answer 6: Thank you for your valuable comment, It is correct, as you mentioned, but the linearity should be described in a certain range according to the Q2R1 (The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample). So that we merge linearity and range in the same paragraph. Precision and accuracy were edited and separated No need for Figure 7. Answer 7: Thank you, it was removed The precision should be expressed as repeatability and intermediate precision, not (intra-day) and (inter-day) in Table 5. Answer 8: Thank you, it was done According to ICH guidelines, robustness should be tested by system suitability parameters, especially resolution, not only recover %. Answer 9: Thank you for your valuable comment; the robustness test was performed on authentic material without degradation products, but the resolution was considered in the development stage and it was a critical quality attribute (CQA) with targeted ATP is NLT 2 in quality by design approach to select the appropriate design space. Where are the results of the System suitability test? Answer 10: Thank you, the results of the System suitability test were added in table 1 Reference number of the proposed method (d) is missing in Table 8. Answer 11: Thank you, the proposed method (d) is the new method of this paper. No statistical analysis was illustrated by the authors. Answer 12: Thank you," } ] } ]	1	https://f1000research.com/articles/12-341
https://f1000research.com/articles/12-72/v1	18 Jan 23	{ "type": "Systematic Review", "title": "Impact of the COVID-19 pandemic on the incidence and clinical outcomes of diabetic ketoacidosis among male and female children with type 1 diabetes: systematic review and meta-analysis", "authors": [ "Edinson Dante Meregildo-Rodriguez", "Franco Ernesto León-Jiménez", "Brenda Aurora Dolores Tafur-Hoyos", "Gustavo Adolfo Vásquez-Tirado", "Franco Ernesto León-Jiménez", "Brenda Aurora Dolores Tafur-Hoyos", "Gustavo Adolfo Vásquez-Tirado" ], "abstract": "Background: Some studies suggest that the SARS-CoV-2 pandemic increased the incidence of type 1 diabetes mellitus (T1DM) and diabetic ketoacidosis (DKA). However, the impact of this pandemic on pediatric T1DM is still mostly unknown. Therefore, we aimed to assess the effect of the COVID-19 pandemic on clinical outcomes in children with T1DM. Methods: We systematically searched for six databases up to 31 August 2022. We included 46 observational studies, 159,505 children of both sexes with T1DM, and 17,547 DKA events. Results: The COVID-19 pandemic significantly increased, in both sexes, the incidence of 1) DKA (OR 1.68; 95% CI 1.44–1.96), 2) severe DKA (OR 1.84; 95% CI 1.59–2.12), 3) DKA in newly diagnosed T1DM (OR 1.75; 95% CI 1.51–2.03), and 4) ICU admissions (OR 1.90; 95% CI 1.60–2.26). However, we did not find a significant association between this pandemic and 1) the incidence of T1DM, 2) the incidence of DKA in established T1DM, 3) the incidence of KDA complications, 4) the length of hospitalization stay, and 5) mortality. Subgroup analysis showed that the study design and the continent of origin accounted for the heterogeneity. Conclusions: The pandemic SARS-CoV-2 raised, in both sexes, the risk of DKA, severe DKA, DKA de novo, and ICU admissions.", "keywords": [ "COVID-19", "SARS-CoV-2", "type 1 diabetes mellitus", "diabetic ketoacidosis", "pediatrics", "child", "systematic review", "meta-analysis" ], "content": "Introduction\n\nType 1 diabetes (T1DM) is an autoimmune disease traditionally associated with viral infections.1,2 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus shows a great affinity for the angiotensin-converting enzyme (ACE) 2 receptor and other receptors present in the islets of Langerhans in the pancreas. Therefore, the SARS-CoV-2 virus could induce insulin resistance, hyperglycemia, and diabetes mellitus (DM) decompensation. On the contrary, hyperglycemia could worsen the prognosis of the COVID-19 disease.3,4 There seems to be, in fact, a bidirectional relation between COVID-19 and DM.5,6\n\nSome studies suggest that during the COVID-19 pandemic, the risk of T1DM has increased.7–9 Similarly, three systematic reviews and meta-analyses have shown an increase in the risk of developing DKA and severe DKA during the COVID-19 pandemic compared to the pre-pandemic era.10–12 Besides, people with DM are disproportionately affected by COVID-19. For example, they are more susceptible to be admitted to an intensive care unit (ICU) than those non-diabetic patients.13 In addition, patients with T1DM have 3.5 times higher mortality rates from COVID-19 than those without T1DM.14 However, data are still controversial. Some studies found no differences in the percentage of newly diagnosed T1DM complicating with DKA in COVID-19 and non-COVID-19 periods.15,16 Therefore, it is maybe due to more difficult access to healthcare systems.15 In fact, despite the advantages of telemedicine during the pandemic, several reports have shown that a considerable quantity of T1DM patients has presented complications, probably associated with fear and delay in seeking medical help.17 Another factor that could increase the incidence of T1DM, diabetic ketoacidosis (DKA), and severe DKA is the widespread use of steroids during the pandemic due to their ability to induce insulin resistance, hyperglycemia, and de novo DM.18,19\n\nAn international multicenter study in Europe and the USA aimed to examine the impact of the COVID-19 pandemic on the prevalence of DKA in pediatric type 1 diabetes. The researchers noted that the DKA prevalence at T1DM diagnosis during the pandemic years was 39%, significantly higher than the estimated prevalence of 33% for the two previous years. However, they did not find significant differences by sex or age.20\n\nAlthough the evidence suggests that the SARS-CoV-2 pandemic has raised the incidence of T1DM, DKA, and severe DKA, information about the impact of COVID-19 on other clinical outcomes is scarce. Thus, we aimed to determine the impact of this pandemic on the probability of developing pediatric T1DM, DKA, severe DKA, DKA in newly diagnosed and established T1DM, ICU admissions, DKA complications, length of hospitalization stay, and mortality due to DKA.\n\n\nMethods\n\nWe conducted this systematic review and meta-analysis following the recommendations of the Cochrane Handbook,21 the PRISMA,22 and the AMSTAR 223 guidelines. We previously registered the protocol in PROSPERO (CRD42021278821). We searched for observational (cohort, case-control, and cross-sectional) studies and randomized control trials published until 31 August 2022, in Medline (PubMed), Google Scholar, Scopus, ScienceDirect, EMBASE, and Web of Science. We combined different keywords, controlled vocabulary terms (e.g., MeSH and Emtree), and free terms following a predefined PECO framework (population: “children with type 1 diabetes mellitus”; exposure: “COVID-19” OR “SARS-CoV-2”; comparator: “NOT COVID-19” OR “NOT SARS-CoV-2”; outcome: “diabetic ketoacidosis” OR “DKA” OR “incidence” OR “hospital stay” OR “intensive care unit admission” OR “mortality” (Extended data). We did not limit searches by date or language.\n\nWe excluded case reports, case series, duplicated publications, and papers in which most patients were >18 years old or had other types of diabetes mellitus. Four independent reviewers examined articles, and a fifth researcher resolved discrepancies. We screened references from retrieved documents for additional articles. We reviewed the papers found and verified the compliance of the components of the PECO framework and the inclusion and exclusion criteria. In addition, we extracted and recorded the essential information from each article in a spreadsheet: authors' names, year and country of publication, type of study, number of patients, sex of the patients, number of events, the measure of association, and adjusted confounders if reported.\n\nInitially, we planned to perform subgroup analyses according to DKA severity, mortality, length of hospitalization, and sex. However, as the protocol stipulated, these subgroup analyses would be executed if feasible regarding information and data. In addition, in concordance with the SAGER guidelines,24 we defined sex as the biological attributes associated with physical and physiological features separated as mutually exclusive and complementary categories (male or female); that is, the sum of both is equal to the total number of cases.\n\nWe pooled the number of patients and events of interest in the quantitative synthesis and calculated odds ratios (ORs) with 95% confidence intervals (95% CIs) using the Mantel-Haenszel method. We considered the risk ratio (RR) equivalent to OR if the incidence of the event evaluated was fewer than 10 percent.25 We used forest plots to represent the quantitative synthesis and assessed heterogeneity among studies with Cochran’s Q test and Higgins I2 statistic. We predefined that if heterogeneity was not significant (p > 0.05, I2 statistics < 40%), we would use a fixed-effects model. We carried out sensitivity and subgroup analyses and assessed the risk of bias with the Newcastle–Ottawa Scale (NOS) tool.26 Finally, we examined the publication bias using a funnel plot.\n\nIn addition, we compare the incidence per 105 children/year of T1DM between the pre-pandemic and pandemic period using medians and interquartile ranges (IQRs) and the Mann–Whitney test.\n\n\nResults\n\nWe collected 112 studies, 98 in the primary screening and 14 in the secondary examination. Following the removal of duplicated articles, there were 87 articles left that we examined in title and abstract. Subsequently, we found and analyzed 46 papers in full text. We considered these 46 papers for qualitative and quantitative synthesis (Figure 1).\n\nOf the 46 studies included in this review, six studies were cross-sectional studies (CSS), two papers were case-control studies (CCS), and thirty-eight documents were prospective or retrospective cohort studies (PCS, RCS). This review includes a total of 159,505 children with T1DM, 17,547 events of DKA—5,792 episodes of severe DKA, 15,600 episodes of DKA in de novo T1DM, and 521 episodes of DKA in established T1DM, 791 ICU admissions, 822 DKA related complications, and one death (Table 1).\n\nT1DM: type 1 diabetes mellitus, DKA: diabetic ketoacidosis, RCS: Retrospective cohort study, PCS: Prospective cohort study, CSS: Cross-sectional study, ISPAD: International Society for Pediatric and Adolescent Diabetes, ADA: American Diabetes Association, DCCP: Diabetes Canada Clinical Practice, WHO: World Health Organization, ICU: admission to the intensive care unit, UC: unicenter, MC: multicenter, ND: not described.\n\nFollowing the approach of most studies, we analyzed outcomes comparing the pre-pandemic and the pandemic periods—regardless of their COVID-19 status (positive or negative)—instead of reporting events in children with COVID-19 positive or negative. Consequently, we only included papers that reported both groups of children (a pre-pandemic and a pandemic cohort). The lack of a pre-pandemic group was the leading cause of the exclusion of most studies (Extended data).\n\nWe analyzed nine outcomes (pre and during the COVID-19 pandemic) in children: 1) the incidence of T1DM, 2) the incidence of DKA in T1DM, 3) the incidence of severe DKA in T1DM, 4) the incidence of DKA in de novo T1DM, 5) the incidence of DKA in established T1DM, 6) the incidence of ICU admissions due to DKA in T1DM, 7) the incidence of DKA complications in T1DM, 8) the length of hospitalization stay, and 9) the risk of mortality due to DKA.\n\nT1DM: type 1 diabetes mellitus, DKA: diabetic ketoacidosis, RCS: Retrospective cohort study, PCS: Prospective cohort study, CSS: Cross-sectional study, ISPAD: International Society for Pediatric and Adolescent Diabetes, ADA: American Diabetes Association, DCCP: Diabetes Canada Clinical Practice, WHO: World Health Organization, ICU: admission to the intensive care unit, UC: unicenter, MC: multicenter, ND: not described.\n\nDuring the pre-COVID-19 era, the median incidence of DKA among children with T1DM was 17.28 per 105 patients/year (IQR 10.87–26.9), and during the COVID-19 era, the incidence of DKA among children with T1DM was 19.35 per 105 patients/year (IQR 14.65–34.7). However, this difference was not statistically significantly (p = 0.41, Mann–Whitney test).\n\nCompared to the pre-COVID-19 era, the COVID-19 era increased the odds of DKA by 68% (OR 1.68; 95% CI 1.44–1.96) (Figure 2a) among children with T1DM.\n\nCompared to the pre-COVID-19 era, the COVID-19 era increased the odds of severe DKA by 84% (OR 1.84; 95% CI 1.59–2.12) (Figure 2b) among children with T1DM.\n\nCompared with the pre-COVID-19 era, the COVID-19 era increased the odds of DKA by 75% (OR 1.75; 95% CI 1.51–2.03) (Figure 2c) among children with newly diagnosed T1DM.\n\nCompared to the pre-COVID-19 era, the COVID-19 era did not significantly increase the odds of developing DKA (OR 0.98; 95% CI 0.79–1.21) (Figure 2d) among children with established T1DM.\n\nCompared with the pre-COVID-19 era, the COVID-19 era increased the odds of ICU admission due to DKA by 90% (OR 1.90; 95% CI 1.60–2.26) (Figure 2e) among children with T1DM.\n\nCompared to the pre-COVID-19 era, the COVID-19 era did not significantly increase the odds of DKA complications (OR 1.39; 95% CI 0.81–2.38) (Figure 2f) among T1DM pediatric patients. Authors reported different definitions for “DKA complications”, including acute kidney injury,42 pulmonary edema,36 stroke,54 brain edema,36,39 altered mental status,32,42,45,54 treatment with hypertonic saline or mannitol,45 hypokalemia,36,56 hypocalcemia, or hypophosphatemia.36 On the other hand, a study did not define “DKA complication.”\n\nCompared with the pre-COVID-19 era, the COVID-19 era did not significantly affect the duration of hospital stay due to DKA (MD 0.18; 95% CI -0.11–0.46) (Figure 2g) among children with T1DM.\n\nWe found two studies33,39 evaluating this outcome. Unfortunately, only one39 of these two studies reported events during the pandemic. Consequently, we decided not to conduct a meta-analysis for this clinical outcome.\n\nOf the 46 studies included, 40 had a low risk of bias and six had a high risk of bias according to assessment with the Newcastle-Ottawa Scale (NOS) tool (Table 2).\n\nThe funnel plot suggested publication bias (Figure 3).\n\n\nDiscussion\n\nTo our knowledge, this is the first systematic review and meta-analysis that asses nine outcomes associated with the effect of COVID-19 on pediatric T1DM and DKA. According to our results, the COVID-19 pandemic significantly increased the incidence of DKA (OR 1.68; 95% CI 1.44–1.96), severe DKA (OR 1.84; 95% CI 1.59–2.12), DKA in newly diagnosed T1DM (OR 1.75; 95% CI 1.51–2.03), and ICU admissions (OR 1.90; 95% CI 1.60–2.26). Conversely, we found no association between the COVID-19 pandemic and the incidence of T1DM, DKA in established T1DM, DKA complications, the length of hospitalization stay, and mortality (Figure 2a-g). These findings are in agreement with other meta-analyses.\n\nNassar M et al.72 conducted a systematic review to identify the prevalence, clinical presentation, and outcomes of T1DM in patients with COVID-19. They searched for observational studies in four databases. The results evaluated were the duration of hospital stay, general ward admission, ICU admission, frequency of DKA, serious hypoglycemia, and mortality. They included 15 papers in the qualitative analysis. They had reports that included information from both children and adults with COVID-19. The frequency of T1DM among patients with COVID-19 varied between 0 and 30%, while the prevalence of COVID-19 among patients with T1DM varied between 0 and 17%. The assessed outcomes ranged widely among the studies. Furthermore, the study’s duration of hospital stay, general ward admission, ICU admission, frequency of DKA, and serious hypoglycemia varied significantly among the included studies.\n\nThe systematic review by Nassar M et al.72 has several limitations reported by the authors. First, they could not perform a meta-analysis because of the lack of studies with appropriate information. Besides, the characteristics of the participants differed widely among the studies. Therefore, in our meta-analysis, we excluded 13 of the 15 studies of the paper by Nassar M et al. because these studies combined adults with pediatric patients or did not have a control (pre-pandemic) group.\n\nRahmati M et al.12 systematically explored the occurrence of de novo T1DM in children and its complications, such as diabetic ketoacidosis, previously and in the times of the pandemic of COVID-19. First, they carried out a systematic search of four databases. Then, they performed a quantitative synthesis comparing the probabilities of developing T1DM and diabetic ketoacidosis in children with T1DM before (the year 2019) and during (the year 2020) the pandemic. They also examined glycemic and glycated hemoglobin levels in pediatric participants with de novo T1DM previously and at the time of this pandemic. They found that the overall incidence ratio of T1DM in 2019 was 19.73 per 105 children and 32.39 per 105 in 2020. During 2020 the cases of de novo T1DM, diabetic ketoacidosis, and serious diabetic ketoacidosis raised significantly. Similarly, in 2020, the median glycemic and glycated hemoglobin levels in pediatric participants with de novo T1DM during the COVID-19 era increased notably. They concluded that the pandemic raised the likelihood of developing de novo T1DM, diabetic ketoacidosis, and serious diabetic ketoacidosis in children.\n\nRahmati M et al.12 conducted heterogeneity and sensitivity analysis and assessed the possibility of publication bias. However, their paper only included studies covering the first wave of the SARS-CoV-2 pandemic. Conversely, our study includes more recent studies that covered subsequent waves. In addition, we excluded two studies of the review by Rahmati M et al., one because it did not report DKA cases nor a numerator to calculate an incidence ratio, and the other because there was no control (pre-pandemic) group.\n\nAlfayez OM et al.10 conducted a systematic review aiming to study the characteristics of DKA before and during the pandemic of SARS-CoV-2 in children with T1DM. First, they searched for observational and found 20 documents on DKA. Then, they performed a random model analysis and reported that the pandemic, compared to the period before, significantly raised the probability of developing DKA and serious DKA. Similarly, pediatric patients with de novo T1DM presented a substantially greater risk of developing DKA during the pandemic than those patients during the pre-COVID-19 era. However, the heterogeneity was significant in all of these estimates (I2 = 44%–71%). Two papers mentioned the likelihood of DKA among children with previously diagnosed T1DM, and this probability was not statistically increased during the COVID-19 era. The authors concluded that their research evidenced that DKA likelihood, particularly the chance of developing serious DKA, raised significantly during the COVID-19 period.\n\nAlfayez OM et al.10 reported subgroup and sensitivity analysis, and explored the possibility of publication bias. Although their systematic review collected fewer than half as many studies as ours, all included studies had an adequate control group. Consequently, their conclusions are closer to ours. Nevertheless, we highlight that of the studies included by these authors, one is a case-control study,35 and four follow a cross-sectional design.36,46,53,60 In studies of cross-sectional and case-control design, we only are able to assess odds, not risk. Therefore, a better measure of the effect size would have been to report odds ratios instead of risk ratios,73,74 as the authors estimated.\n\nElgenidy A et al.11 conducted a meta-analysis to investigate the increase of DKA in pediatrics at the time of the SARS-CoV-2 pandemic. In three databases, they looked for papers evaluating the frequency of diabetic ketoacidosis. The researchers reported 24 studies, including 124,597 pediatric patients with T1DM. Their main finding was that the pandemic raised statistically significantly the likelihood of developing DKA in children with de novo T1DM (RR 1.41; 95% CI 1.19–1.67; p < 0.01), particularly of those with serious DKA (RR 1.66: 95% CI 1.30–2.11) compared with the pre-COVID-19 era. Statistical heterogeneity was substantial (I2 = 86% and 59%, respectively). They found no important rise in the probability of developing DKA during the pandemic in pre-existing T1DM or combined—de novo and pre-existing T1DM children compared with the pre-COVID-19 era. They concluded that the likelihood of DKA in children with de novo T1DM had risen in the time of the SARS-CoV-2 pandemic and tended to present in more serious forms.\n\nElgenidy A et al. performed subgroup and sensitivity analysis and evaluated the risk of publication bias. However, in our meta-analysis, we excluded 5 of the 17 studies quantitatively analyzed by Elgenidy A et al. because they did not report any of the events of interest or the lack of a control group. Moreover, these authors also included two case-control studies35,55 and cross-sectional studies36,46,53,60 and reported relative risks instead of odds ratios. Therefore, the same considerations previously mentioned for the meta-analysis of Alfayez OM et al. should apply.\n\nThe heterogeneity was significant in this systematic review and meta-analysis (I2 > 90%, p < 0.05). According to the subgroup examination, the type of study design and the provenance region of the studies explained this lack of homogeneity among studies (test for subgroups difference I2 = 83.2%, p = 0.003; I2 > 49.4%, p = 0.11; respectively). Sensitivity analysis did not alter the global size estimate, showing good consistency. Because of the limited data among studies, we decided not to carry out subgroup analysis and meta-regression according to other variables. Unlike the study by Elgenidy A et al., because most studies do not provide complete information, we did not perform subgroup according to the type of onset of diabetes (de novo, established, or combined—de novo and established—T1DM) or the degree of diabetic ketoacidosis (serious, moderate, or mild). On the contrary, following Rahmati M et al. and Alfayez OM et al., we analyzed the diabetes onset and the degree of DKA as an independent outcome.\n\nWe highlight several strengths in our meta-analysis: 1) the strategy search was comprehensive and compiled a more significant number of papers than any other previous systematic review or meta-analysis, 2) all the studies that we included involved a control (pre-pandemic) group, 3) all the papers that we included examined clinical—not surrogate—outcomes, and 4) we carried out sensitivity and subgroup analysis and examined for possible publication bias. Then, our conclusions are stronger than those previously reported by any other meta-analysis.\n\nThis study has important limitations: 1) heterogeneity was significant, 2) we were not able to carry out subgroup analyses regarding other essential factors such as age or sex, 3) it is possible that there exists a publication bias, as was suggested by our funnel plot, and finally, 4) we could not establish definite conclusions on other important outcomes such as the likelihood of T1DM, DKA complications, the duration of hospitalization stay, and mortality due to DKA. In addition, although we initially planned to perform subgroup analyzes according to sex, due to the scarcity of data (most studies combined information for both sexes), it was not possible to achieve this sub-analysis. In fact, none of the previously cited systematic reviews could perform a subgroup analysis according to sex.\n\n\nConclusions\n\nOur systematic review shows that the SARS-CoV-2 pandemic significantly impacted T1DM and DKA outcomes in pediatric patients. This pandemic increased 1) the risk of DKA, 2) the risk of serious DKA, 3) the risk of DKA in children with de novo T1DM, and 4) ICU admissions due to DKA. Conversely, the relation of the SARS-CoV-2 pandemic with other outcomes such as 1) the incidence of pediatric T1DM, 2) the incidence of DKA in established pediatric T1DM, 3) the incidence of complications due to DKA, 4) the length of hospitalization stay, and 5) the risk of mortality due to DKA, were not statistically significant. Nonetheless, clinicians should interpret these findings with caution due to several limitations. Consequently, more research is still necessary to improve knowledge of the relationship between SARS-CoV-2 and diabetic ketoacidosis. Nevertheless, our results imply that healthcare systems should be alert and prepared for a potential rise in diabetic ketoacidosis cases, especially severe DKA cases, in future waves of viral respiratory pandemics.\n\n\nAuthor roles\n\nEDM-R: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Visualization, Writing, Original Draft Preparation; FEL-J: Data Curation, Formal Analysis, Investigation, Writing – Review & Editing; BADT-H: Investigation, Writing – Review & Editing; GAV-T: Conceptualization, Investigation, Supervision, Validation, Writing – Review & Editing", "appendix": "Data availability\n\nFigshare: Figshare3: Raw data, https://doi.org/10.6084/m9.figshare.21644723. 75\n\nThis project contains the following underlying data:\n\n- Figshare3-Raw data.xlsx (Search strategy, Table S1; Included primary studies, Table S2; Excluded primary studies)\n\nFigshare: Figshare4: Extended data, https://doi.org/10.6084/m9.figshare.21644786. 76\n\nThis project contains the following extended data:\n\n- Figshare4-Supplementary materials.docx (Search strategy, Table S2- Excluded primary studies)\n\nFigshare: PRISMA checklist for “Impact of the Covid-19 pandemic on the incidence and clinical outcomes of diabetic ketoacidosis among children with type 1 diabetes: systematic review and meta-analysis”, https://doi.org/10.6084/m9.figshare.21625790. 77\n\nFigshare: Figshare2: PRISMA Flowchart, https://doi.org/10.6084/m9.figshare.21625775. 78\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nHyöty H: Viruses in type 1 diabetes. 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An Overview of Reported Statistical Methods and Appropriateness of Interpretations in Cross-sectional Stud-ies with Dichotomous Outcomes in Veterinary Medicine. Front Vet. Sci. 2017; 10: 193.\n\nAlavi M, Hunt GE, Visentin DC, et al.: Using risk and odds ratios to assess effect size for meta-analysis outcome measures. J. Adv. Nurs. 2020; 76: 3231–3234. Publisher Full Text\n\nMeregildo-Rodriguez ED:Figshare3: Raw data. [data set]. figshare. 2022. Publisher Full Text\n\nMeregildo-Rodriguez ED: Figshare4: Supplementary materials. figshare. Online resource.2022. Publisher Full Text\n\nMeregildo-Rodriguez ED: Figshare2: PRISMA checklist. figshare. Online resource.2022. Publisher Full Text\n\nMeregildo-Rodriguez ED: Figshare1: PRISMA Flowchart. figshare. Online resource.2022. Publisher Full Text" }	[ { "id": "172554", "date": "25 Jul 2023", "name": "Carlos J. Toro-Huamanchumo", "expertise": [ "Reviewer Expertise Evidence-Based Medicine", "Epidemiology & Public Health", "Obesity & Metabolism" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have carefully gone through your manuscript titled \"Impact of the COVID-19 pandemic on the incidence and clinical outcomes of diabetic ketoacidosis among male and female children with type 1 diabetes: systematic review and meta-analysis\". Below are my comments and suggestions for improving your submission:\nThe objective of evaluating the incidence of T1D is not clearly stated, especially since the title mentions that your population is children with T1D. It would seem this is not a variable as all subjects already have this diagnosis. Could you clarify this further? It is not clear how clinical trials were considered as potential studies to be selected. Could you elaborate on the criteria used for this selection? When you state \"We excluded case reports, case series, duplicated publications, and papers in which most patients were >18 years old or had other types of diabetes mellitus\", could you specify what you mean by \"most\"? Does this refer to >50%? >75%? This point needs to be explicitly clarified. The process of how four independent reviewers examined the articles and a fifth investigator resolved discrepancies is unclear, especially given that there are only four authors listed in total. Please note that only three authors are listed on PROSPERO. The eligibility criteria need to be more precisely defined. The PECO question seems more oriented towards assessing whether having COVID-19 is associated with any of those outcomes, and not whether the incidence of each of the outcomes increased during the pandemic vs. pre-pandemic. In this sense, rather than formulating a PECO, it may be more useful to describe the inclusion criteria for studies in detail (as some previous systematic reviews have done). Please clarify why you preferred to use OR instead of RR. The statistical procedure for obtaining MD for the duration of hospital stay is not mentioned. Could you provide more detail on this? I strongly recommend presenting the complete results of the NOS, not just the final outcome.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "10037", "date": "16 Nov 2023", "name": "Edinson Dante Meregildo-Rodriguez", "role": "Author Response", "response": "The authors appreciate the reviewer's comments, which will enhance this article's quality and methodological rigor. The objective of evaluating the incidence of T1D is not clearly stated, especially since the title mentions that your population is children with T1D. It would seem this is not a variable as all subjects already have this diagnosis. Could you clarify this further? Replay: Thank you for this valuable comment. As we stated in the last paragraph of the Introduction, we aimed to determine the impact of this pandemic on the risk (probability) of developing pediatric T1DM, DKA, severe DKA, DKA in newly diagnosed and established T1DM, etc. So, we collected studies with participants, children with and without T1DM, DKA, severe DKA, DKA in newly diagnosed and established T1DM, etc. However, we must recognize that our PECO strategy did not fully reflect this intention. Therefore, in this new version of the manuscript, we have slightly modified the structure of the PECO question, and we have described with more detail the inclusion criteria following the example of previous similar systematic reviews. It is not clear how clinical trials were considered as potential studies to be selected. Could you elaborate on the criteria used for this selection? Replay: Thank you for this insightful observation. We included RCTs in our strategy search to carry out a broad and sensitive search. However, from the methodological and ethical point of view, and considering the exposure was \"COVID-19\" OR \"SARS-CoV-2\", it was improbable to find any published RCT, which was confirmed by our results. All the studies were observational. When you state \"We excluded case reports, case series, duplicated publications, and papers in which most patients were >18 years old or had other types of diabetes mellitus\", could you specify what you mean by \"most\"? Does this refer to >50%? >75%? This point needs to be explicitly clarified. Replay: Thank you for this comment. According to The Cochrane Handbook, the criteria for considering the population included in studies in a systematic review should be sufficiently broad to encompass the likely diversity of studies and the likely scenarios in which the interventions will be used, but narrow enough to ensure that a meaningful answer can be obtained when studies are considered together. A cutoff of more than 50% ensures that most participants in the included studies are children, which increases the likelihood that the results are relevant to children. Then, In the new version of the manuscript, we have included a small paragraph indicating that we considered a >50% cutoff for excluding studies. The process of how four independent reviewers examined the articles and a fifth investigator resolved discrepancies is unclear, especially given that there are only four authors listed in total. Please note that only three authors are listed on PROSPERO. Replay: Thank you for this insightful comment. You are right. Consequently, we have made the pertinent corrections in this revised manuscript version. Furthermore, we have updated the protocol in PROSPERO, including the four authors. The eligibility criteria need to be more precisely defined. The PECO question seems more oriented towards assessing whether having COVID-19 is associated with any of those outcomes, and not whether the incidence of each of the outcomes increased during the pandemic vs. pre-pandemic. In this sense, rather than formulating a PECO, it may be more useful to describe the inclusion criteria for studies in detail (as some previous systematic reviews have done). Replay: Thank you for this accurate comment. We have made the following modifications in our manuscript: 1) we have modified the Population component of our PECO question, and 2) described with more detail the inclusion criteria following the example of previous similar systematic reviews. Please clarify why you preferred to use OR instead of RR. Replay: Thank you for this insightful comment. Of the 46 studies included in this review, six studies were cross-sectional studies (CSS), two papers were case-control studies (CCS), and thirty-eight documents were prospective or retrospective cohort studies (PCS, RCS). The only measure of association that these study designs have is the OR. Consequently, we used OR, not RR. The statistical procedure for obtaining MD for the duration of hospital stay is not mentioned. Could you provide more detail on this? Replay: Thank you for this valuable comment. In this revised version of our manuscript, we have modified the respective paragraph detailing the statistical method used for obtaining MD and SD. Furthermore, we have included the corresponding references that support these statements. I strongly recommend presenting the complete results of the NOS, not just the final outcome. Replay: Thank you for this important. In this revised version of our manuscript, we have modified \"Table 2. Bias assessment of the included studies,\" presenting the complete Newcastle-Ottawa Scale (NOS) tool." } ] } ]	1	https://f1000research.com/articles/12-72
https://f1000research.com/articles/11-1184/v1	17 Oct 22	{ "type": "Research Article", "title": "Seroprevalence of exposure to SARS-CoV-2 in domestic dogs and cats and its relationship with COVID-19 cases in the city of Villavicencio, Colombia", "authors": [ "Dumar Alexander Jaramillo Hernández", "María Clara Chacón", "María Alejandra Velásquez", "Adolfo Vásquez-Trujillo", "Ana Patricia Sánchez", "Luis Fabian Salazar Garces", "Gina Lorena García", "Yohana María Velasco-Santamaría", "Luz Natalia Pedraza", "Lida Carolina Lesmes-Rodríguez", "María Clara Chacón", "María Alejandra Velásquez", "Adolfo Vásquez-Trujillo", "Ana Patricia Sánchez", "Gina Lorena García", "Yohana María Velasco-Santamaría", "Luz Natalia Pedraza", "Lida Carolina Lesmes-Rodríguez" ], "abstract": "Background: Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, different animal species have been implicated as possible intermediate hosts that could facilitate the transmission of the virus between species. The detection of these hosts has intensified, reporting wild, zoo, farm, and pet animals. The goal of this study was to determine the seroprevalence of anti-SARS-CoV-2 immunoglobulins (IgG) in domestic dogs and cats and its epidemiological association with the frequency of coronavirus disease 2019 (COVID-19) patients in Villavicencio, Colombia. Methods: 300 dogs and 135 cats were randomly selected in a two-stage distribution by clusters according to COVID-19 cases (positive RT-qPCR for SARS-CoV-2) within the human population distributed within the eight communes of Villavicencio. Indirect enzyme-linked immunosorbent assay (ELISA) technique was applied in order to determine anti-SARS-CoV-2 IgG in sera samples. Kernel density estimation was used to compare the prevalence of COVID-19 cases with the seropositivity of dogs and cats. Results: The overall seroprevalence of anti-SARS-CoV-2 IgG was 4.6% (95% CI=3.2-7.4). In canines, 3.67% (95% CI=2.1-6.4) and felines 6.67% (95% CI=3.6-12.18). Kernel density estimation indicated that seropositive cases were concentrated in the southwest region of the city. There was a positive association between SARS-CoV-2 seropositivity in pet animals and their habitat in Commune 2 (adjusted OR=5.84; 95% CI=1.1-30.88). Spearman's correlation coefficients were weakly positive (p=0.32) between the ratio of COVID-19 cases in November 2020 and the results for domestic dogs and cats from the eight communes of Villavicencio. Conclusions: In the present research cats were more susceptible to SARS-CoV-2 infection than dogs. This study provides the first positive results of anti-SARS-CoV-2 ELISA serological tests in domestic dogs and cats in Colombia with information about the virus transmission dynamics in Latin America during the COVID-19 pandemic.", "keywords": [ "anthropozoonosis", "coronavirus", "immunoassay", "public health" ], "content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) emerged in the Huanan Seafood Wholesale Market in Wuhan, China, in December 2019.1 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the causal agent of this disease, which was declared as pandemic by the World Health Organisation (WHO) on 11th March 2020.2 The zoonotic origin of COVID-19 has been evidenced thanks to the high genomic similarity of SARS-CoV-2 with coronaviruses (CoV) recovered from bats3 and pangolins, the latter considered potential intermediate hosts of the virus.4 Betacoronaviruses such as SARS-CoV-2 belong to the family Coronaviridae. They exhibit linear single-stranded RNA of positive polarity5 and cause respiratory and gastrointestinal diseases in mammals.6 Even though humans are the most frequent route of transmission, it has been reported that cats (Felis catus) and dogs (Canis lupus familiaris) are susceptible to SARS-CoV-2 infection.7 This way, reverse zoonosis (anthropozoonosis) is viable through close contact with owners during acute infections.8\n\nSince the beginning of the SARS-CoV-2 outbreak, different animal species have been implicated as possible intermediate hosts that could facilitate the virus transmission between species.9 This is the reason why the determination of these hosts has intensified, evidencing a number of reports involving wild, zoo, farm and pet animals.10 The zoonotic nature from which the transmission hypothesis has started, determines the importance of investigating animal species considered natural reservoirs of SARS-CoV-2.11 However, concern for the control and reduction of the spread of the virus has led to more vigorous investigation of the role that pet animals, such as dogs and cats, play in the spread of the disease. Although a cat-to-human transmission case was reported,12 it has been clearly defined that domestic canines and felines do not play a relevant role in the virus transmission to humans.8,13\n\nOn the other hand, human-animal transmission has been widely reported.14,15 This fact generates the need to investigate the implications for public and animal health, taking into account that animals are an epidemiological part of this pandemic.16 Various epidemiological and experimental studies, through serological detection of antibodies against SARS-CoV-2, neutralising antibodies, and detection of viral genome by reverse transcriptase polymerase chain reaction (RT-qPCR), have confirmed SARS-CoV-2 in pet animals around the world.17 Likewise, the occurrence of emerging variants has been described, as well as their influence on animals,18 for example, the Alpha variant (B.1.1.7) in dogs and cats with clinical signs of myocarditis,19 and the Delta variant (B.1.617.2) in dogs with clinical digestive and respiratory symptoms.20 Regarding the Omicron variant (B.1.1.529), concluded that the SARS-CoV-2 virus accumulated mutations within host cells in mice, giving rise to the Omicron variant that was transmitted to humans, indicating a ‘ping-pong’ (spillover and spillback) evolutionary trajectory between species.21 Transmission of SARS-CoV-2 Delta variant (AY.127) from hamsters to humans22 and animal-to-human transmission of SARS-CoV-2 within mink farms23 have also been reported.\n\nSusceptibility to SARS-CoV-2 is determined by the affinity between the receptor-binding domain (RBD) of the viral spike (S) glycoprotein and the angiotensin-converting enzyme 2 (ACE2) of the host cell. Therefore, since vertebrates have conserved domains of ACE2, transmission of the virus between species becomes possible.24 Canines have lower susceptibility to SARS-CoV-2 infection in contrast to felines8,25 that exhibit greater respiratory pathology and efficient transmission of the virus to other felines through aerosols.26\n\nIn the context of the rapid evolutionary trajectory between species that SARS-CoV-2 has been developing, and taking into account the Report No. 13 of the World Organisation for Animal Health (OIE) of 31st May 2022, which reported 676 outbreaks in animals affecting 23 different species in 35 countries,27 the need for seroepidemiological monitoring in pet, wild and synanthropic animals becomes essential in order to broadly understand the adaptation, evolution and transmission of SARS-CoV-2.7\n\nIn Colombia, in December 2021, a lion exhibited symptoms of infection days after being in contact with a COVID-19 positive keeper.28 However, seroepidemiological studies of exposure to SARS-CoV-2 by pet animals have not been reported to date in the country. The goal of the present study was to determine the seroprevalence of anti-SARS-CoV-2 immunoglobulins (Ig) class G (IgG) in domestic dogs and cats and its epidemiological association with the frequency of COVID-19 patients in Villavicencio city, Colombia.\n\n\nMethods\n\nThis research was endorsed by the Bioethics Committee of the Universidad de los Llanos, according to Minute 02 by consensus of April 6, 2021. In addition, all the owners of the dogs and cats involved in this study signed the respective informed consent.\n\nThis is a cross-sectional epidemiological study conducted in Villavicencio, Colombia. It consisted of applying a characterisation survey and taking blood samples from domestic canines and felines. The sample was estimated using the formula for size by proportions in finite populations, using the results obtained by Patterson et al.29 as a reference of p, with SARS-CoV-2 seroprevalence of 9.1% (3.3% in canines and 5.8% in felines) in pet animals in Italy. The population assessed in the present study corresponded to 68,651 domestic canines and felines in Villavicencio (47,573 canines and 21,078 felines), according to estimates from the report on anti-rabies vaccination of dogs and cats in Colombia.30 The confidence interval (CI) considered was 95% and the ‘Z’ value was 1.96 (1-α). The absolute precision considered was 0.15% (d = 0.0051).\n\nThe participants were selected based on their mandatory participation in the 2021 rabies vaccination campaign, carried out in Villavicencio (Meta State) by the health secretary. A probabilistic sampling was conducted by randomly selected two-stage clusters of domestic dogs and cats from the eight communes that compose the urban area of Villavicencio (Figure 1), which consisted of the random and proportional selection of individual dogs and cats, the sampling proportion of each cluster was determined according to the frequency of COVID-19 cases (RT-qPCR testing) in each commune31 (according to Table 1); for this, the EpiInfo v. 3.0 software, from the US Centers for Disease Control and Prevention (CDC) was used (https://www.cdc.gov/epiinfo/esp/es_index.html). The inclusion criteria considered domestic dogs and cats that had lived constantly in their homes for a minimum of two months before starting the present study. The animals that had consumed immunomodulatory medication (e.g., corticosteroid-type immunosuppressants) one week before the sampling were not included in the study.\n\nThis figure is an original figure produced by the authors for this article.\n\nActive human cases (RT-qPCR) in November 2020; data from the Villavicencio Municipal Health Secretary (2021).\n\nA total of 435 blood samples were taken (300 domestic canines and 135 domestic felines). For this purpose, the authors of this study collected the blood from the jugular or cephalic vein, previous disinfection of the area with alcohol using a 21-gauge needle or vacutainer. Haemostasis was facilitated by applying pressure with sterile gauze in the sampling site for approximately 30 sec. The samples were centrifuged at 2000 g (Centrifuga Eppendorf 5424R) within three hours after being taken, and the sera were stored at -20 °C until analysis in a freezer (ABBA CVANF502B1). Table 1 shows the representative distribution of ‘n’ by commune (235 neighbourhoods) in Villavicencio.\n\nPet animals characterization was performed through a survey applied to the owners, following the model of a SARS-CoV-2 study that involved dogs and cats with COVID-19 patients in a metropolitan area.32 The characteristics of each pet recorded were: name; sex; age; species; breed; and owners’ names. This survey also inquired whether the individuals that cohabited with the pet animals (spontaneous communication) had histories of positive or negative RT-qPCR testing for COVID-19, and whether there were histories of clinical signs of the animals, such as signs in the upper or lower respiratory tract, or non-specific digestive signs (e.g., vomiting, diarrhoea, among others). Coordinates of the houses where the pets lived, were also recorded. The survey can be found as Extended data.49\n\nIgG antibodies against the nucleocapsid protein (N) of SARS-CoV-2 in the sera of domestic dogs and cats were qualitatively determined using the indirect enzyme-linked immunosorbent assay (ELISA) (ID Screen® SARS-CoV-2, double antigen multi-species [IDvet, Grabels, France]) according to the manufacturer's instructions. Each plate contained 96 microwells sensitised with recombinant antigen of purified N protein of SARS-CoV-2, to which the following items were added: two negative controls (NC); two positive controls (PC); and 92 problem sera previously homogenised by vortexing. The optical density (OD) reading was performed using the Cytation 3 multimodal microplate reader (BioTek Instruments, Inc. Winooski, VT, USA) with a wavelength of 450 nm. In total, 435 problem sera samples and 25 pre-pandemic canine sera previously stored at -20 °C were analysed. Using the OD data of each well, the sample/positive control (S/P) ratio was calculated, which was expressed as a percentage using the following formula:\n\nThe test was validated when the mean OD value of the PC was greater than 0.350, and the ratio of the mean OD values of the PC and NC was greater than three. The samples were considered positive if the S/P ratio was greater than or equal to 60%, doubtful samples or samples in the gray zone had S/P ratios between 50% and 60%, and samples with S/P ratio less than or equal to 50% were considered negative.\n\nThe step-by-step protocol of this trial has been deposited under the title: Immunoassay of SARS-CoV-2 in dogs and cats V.1, DOI: dx.doi.org/10.17504/protocols.io.5qpvorn29v4o/v1 in protocolos.io (https://www.protocols.io/view/immunoassay-of-sars-cov-2-in-dogs-and-cats-v-1-5qpvorn29v4o/v1).\n\nThe frequencies of the data obtained in the survey and transformation of quantitative variables into categories for their subsequent analysis were estimated. The punctual seroprevalence (P) of SARS-CoV-2 in pet animals in Villavicencio was expressed as a proportion using the following formula, considering 95% CI:\n\nThe risk association measure odds ratio (OR), calculated by the binomial logistic regression model with 95% CI; was used in order to determine whether the frequency of active COVID-19 cases in humans by commune was related to SARS-CoV-2 seropositivity (exposure) of pet animals. Likewise, Spearman correlation was used to establish a possible relationship in the increase of cases in domestic animals at homes with COVID-19. Finally, using Kernel density analysis, the prevalence of COVID-19 in humans by commune was compared to anti-SARS-CoV-2 IgG seropositivity in domestic dogs and cats. A confidence level of 95% was used for all statistical calculations. Statistical estimates were made using the R 4.2 software, and the maps using the QGIS 3.10 software.\n\n\nResults\n\nThe overall seroprevalence of anti-SARS-CoV-2 IgG was 4.60% (95% CI = 3.2-7.4).49 Specifically, in canines the results indicated 3.67% (95% CI = 2.1-6.4), and in felines 6.67% (95% CI = 3.6-12.18) (Table 2). Twenty seropositive individuals (11 canines and 9 domestic felines) were detected through the enzyme immunoassay. In general, 22 animals with a history of respiratory signs (e.g., cough, runny nose, among others) were detected, of which 9.10% (95% CI = 2.53-27.81) were seropositive for SARS-CoV-2 (Table 3). Additionally, seven immunoassay results were classified as doubtful (gray area). Likewise, all 25 canine pre-pandemic sera were negative (Figure 2).\n\nThe following variables were taken into account: species, age, city communes and owners with positive or negative RT-qPCR testing.\n\n* p <0.05.\n\nIg = immunoglobulin class G; OD = optical density; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.\n\nRegarding the eight communes, there was a general seroprevalence of 0, 13.63%, 6.67%, 8.06%, 1.02%, 4.76%, 4.82% and 2.5%, respectively (Table 2). In the map obtained through Kernel density analysis (Figure 3), it is observed that the density of cases was concentrated mainly in the west of the city. Communes with higher densities of SARS-CoV-2 seropositive animals were 2 and 4 in comparison to COVID-19 cases in humans, with a greater number of positive cases in communes 5, 7 and 8. Other visible sites of concentrations of seropositive animals, though with lower density, corresponded to communes 7 and 8. Finally, communes 1, 3, 5 and 6 had densities ranging from low to zero.\n\nThe darker shade represents the highest density of seropositive animals, and the clearest represents the sites with the lowest densities. This figure is an original figure produced by the authors for this article. COVID-19 = coronavirus disease 2019; IgG = immunoglobulin class G.\n\nRegarding the SARS-CoV-2 exposure and the risk factors analysed, a statistically significant association between SARS-CoV-2 seropositivity and Commune 2 was found (adjusted OR = 5.84; CI 95% = 1.1-30.88). On the other hand, no significant statistical association was found (p >0.05) between anti-SARS-CoV-2 IgG seropositivity and the other items assessed (Table 2). Additionally, among the twenty seropositive animals, only one owner spontaneously confirmed to have positive RT-qPCR result for COVID-19.\n\nAdditionally, a Spearman correlation of p = 0.32 was found between the ratio of COVID-19 positive cases (RT-qPCR testing) of humans in November 2020 and domestic dogs and cats from the eight Villavicencio communes, result classified as a positive weak correlation. Finally, a strong positive correlation of 0.81 was found between the feline species and their SARS-CoV-2 seropositivity, as well as a positive correlation of 0.68 between the canine species and their SARS-CoV-2 seropositivity.\n\n\nDiscussion\n\nIn the present study, the seroprevalence of SARS-CoV-2 in canines was 3.67% (11/300) and in felines 6.67% (9/135). Felines had more risk of becoming infected with SARS-CoV-2 that dog (adjusted OR = 2.07; 95% CI = 0.78-5.46) (Table 2) this tendency was no statistically different. In similar studies, Barroso et al.7 found SARS-CoV-2 seroprevalence of 4.7% in dogs and 21.7% in cats in Portugal, determining that, among seropositive animals, 50% had been possibly infected by human-animal transmission. On the other hand, 33.3% of seropositive cats had possibly been infected via the cat-cat route. Colitti et al.33 found a SARS-CoV-2 seroprevalence of 2.3% in dogs and 16.2% in cats in Italy, and Fritz et al.13 found a SARS-CoV-2 seroprevalence of 15.4% in dogs and 23.5% in cats from France. In all the studies mentioned, SARS-CoV-2 prevalence was higher in cats and its transmission was mostly related to exposure to humans when they were more seropositive and more susceptible to infection.27,33,34\n\nAs a result of the present study, a positive relationship between seropositivity and the age of the animals was observed.The older animals between 11 and 15 years exhibited this tendency predisposition, but it was not statistically different (adjusted OR = 1.61; 95% CI = 0.16-15.93) (Table 2). In this sense, a significant trend was found in the fatality and mortality rates of COVID-19 with advanced age in humans,35 given that there is a weakened immune system, underlying chronic diseases, multiple drug therapies, lack of attention and self-care, poor environmental hygiene, loneliness, and lack of adequate support from other family members in this population.36 These reasons could be considered with equal value in the case of animals, especially pet animals.37 On the other hand, Shi et al.25 reported that three-month-old canines exhibited low susceptibility to experimental infection, contrary to the results obtained in cats, since animals aged less than 100 days and up to nine months were highly susceptible to SARS-CoV-2 infection.\n\nIn the present study, no significant differences were found for respiratory and digestive symptoms of the animals sampled according to their SARS-CoV-2 seropositivity (X2 = 0.8206; p = 0.365) (Table 3). These results are similar to those reported by Pagani et al.38 and Shi et al.25 i.e., cats infected with SARS-CoV-2 were asymptomatic or highly susceptible to subclinical infections. Contrarily, in Germany, Keller et al.39 reported animals with mainly respiratory symptoms, describing the case of a cat with unresolved pneumonia, which was associated to the owner positive test for COVID-19. SARS-CoV-2-specific nucleic acid analysis was performed, revealing the complete genome and the presence of infection in that patient.\n\nIn both canines and felines, the highest seropositivity occurred in Commune 2 (13.63% [6/44]) (Table 2), which is located in the southwest of Villavicencio. Despite the fact that it is a commune with a low population (19,491 inhabitants),40 it has been reported with the highest number of inhabitants per house (6 inhabitants) in comparison to the other communes,41 suggesting that having more than one individual infected with SARS-CoV-2 in the same household increased the risk of infection in these pet animals.33 Likewise, this commune presented a positive association between the seropositivity of the animals sampled (adjusted OR = 5.84; 95% CI = 1.1-30.88) (Table 2) and the seropositivity of the owners, similarly, Colitti et al.33 found a positive association between COVID-19 positive owners and their felines’ SARS-CoV-2 seropositivity (OR = 2.5; 95% CI = 1.3-5.2), which may be related to the duration of the pets’ exposure to the infected owners, and the close contact of the felines with their owners, suggesting the development of antibodies in domestic animals as a consequence of viral transmission from owners.33,34,42 In the present study, the association between positive COVID-19 cases (RT-qPCR testing) in humans versus seropositivity in canines and felines from the eight communes of Villavicencio was weakly positive (Spearman's correlation of p = 0.32). On the other hand, the study conducted by Van Aart et al.43 showed that none of the felines had been infected with SARS-CoV-2 despite the fact that these were living with their positive COVID-19 owners. Therefore, these associations between species should be analysed considering different factors.\n\nAnimals and humans are susceptible to a large number of different coronaviruses, in fact, it has been shown that all pathogenic human coronaviruses have their origin in animals, which is why studies should focus on their role in the transmission of SARS-CoV-2.44 In the present study, a human-animal transmission was considered based on the results of Smith et al.45 in the United Kingdom, who ruled out that dogs and cats were reservoirs of infection for humans. However, we cannot be sure about the trasmission direction, which will only be confirmed through further studies. Ultimately, successful elimination of SARS-CoV-2 will only be possible by assessing and controlling transmission in all susceptible animal species, a one health approach that could prevent the re-emergence of the virus in the future.46,47\n\n\nConclusion\n\nThe present study provides the first positive results of anti-SARS-CoV-2 serological tests (ELISA) in domestic dogs and cats in Colombia, with information about the dynamics of virus transmission in Latin America and the world during the COVID-19 pandemic. As mentioned above, cats were more susceptible to natural SARS-CoV-2 infection than dogs, following similar dynamics described in other studies.7,33,48 The present study does not provide evidence that domestic canines and felines are sources of infection for humans; however, further studies focused on one health should not be ruled out47 in order to improve our knowledge about transmission, epidemiology and dynamics of SARS-CoV-2 and promote a better response to possible future pandemics.\n\n\nData availability\n\nFigshare: Seroprevalence of exposure to SARS-CoV-2 in domestic dogs and cats and its relationship with COVID-19 cases in the city of Villavicencio, Colombia. https://doi.org/10.6084/m9.figshare.21271137.49\n\nThis project contains the following underlying data:\n\n- Dataset.xlsx (data on OD IgG Anti-SARS-CoV-2, positive control sample ratio (Indirect ELISA), data on domestic dogs and cats that participated in the project.)\n\nFigshare: Seroprevalence of exposure to SARS-CoV-2 in domestic dogs and cats and its relationship with COVID-19 cases in the city of Villavicencio, Colombia. https://doi.org/10.6084/m9.figshare.21271137.49\n\nThis project contains the following extended data:\n\n- Consent form.docx\n\n- Questionnaire.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors are grateful to the support of the General Research Division of Universidad de los Llanos. Likewise, to professionals and other employees of the Villavicencio Municipal Health Secretary involved in the rabies vaccination program for domestic dogs and cats. An earlier version of this article can be found on SSRN: https://ssrn.com/abstract=4156064 or doi: http://dx.doi.org/10.2139/ssrn.4156064.\n\n\nReferences\n\nWorobey M: Dissecting the early COVID-19 cases in Wuhan. Science. 2021; 374: 1202–1204. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization (WHO): WHO Director-General’s opening remarks at the media briefing on COVID-19-11 March 2020.2020 [cited 2022 March 6].Reference Source\n\nZhou P, Yang XL, Wang XG, et al.: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579: 270–273. PubMed Abstract \| Publisher Full Text Publisher Full Text\n\nZhang T, Qunfu W, Zhang Z: Probable Pangolin origin of SARS-CoV-2 associated with the COVID-19 Outbreak. Curr. Biol. 2020; 30(7): 1346–1351.e2. 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PubMed Abstract \| Publisher Full Text\n\nYen H, Sit T, Brackman C, et al.: Transmission of SARS-CoV-2 delta variant (AY.127) from pet hamsters to humans, leading to onward human-to-human transmission: a case study. Lancet. 2022; 399: 1070–1078. PubMed Abstract \| Publisher Full Text\n\nMunnink B, Sikkema R, Nieuwenhuijse D, et al.: Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans. Science. 2021; 371(6525): 172–177. PubMed Abstract \| Publisher Full Text\n\nChen Y, Guo Y, Pan Y, et al.: Structure analysis of the receptor binding of 2019-nCoV. Biochem. Biophys. Res. Commun. 2020; 525: 135–140. PubMed Abstract \| Publisher Full Text\n\nShi J, Wen Z, Zhong G, et al.: Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2. Science. 2020; 368(368): 1016–1020. PubMed Abstract \| Publisher Full Text\n\nGaudreault N, Trujillo JD, Carossino M, et al.: SARS-CoV-2 infection, disease and transmission in domestic cats. Emerg. Microbes Infect. 2020; 9: 2322–2332. PubMed Abstract \| Publisher Full Text\n\nOrganización Internacional de Sanidad Animal (OIE), Mapa de registro de casos de animales infectados por el SARS-CoV-2. Reporte 13.2022 [cited 2022 July 3].Reference Source\n\nOrganización Internacional de Sanidad Animal (OIE), Mapa de registro de casos de animales infectados por el SARS-CoV-2. Reporte 8.2022 [cited 2022 July 3].Reference Source\n\nPatterson EI, Elia G, Grassi A, et al.: Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy. Nat. Commun. 2020; 11(6231): 6231. PubMed Abstract \| Publisher Full Text\n\nMinisterio de Salud y Protección Social (MINSALUD): Reporte de Vacunación Antirrábica de perros y gatos, Colombia año 2017.2017 [cited 2022 March 7].Reference Source\n\nSecretaria de Salud Municipal de Villavicencio: La situación de los barrios, comunas y corregiminetos de Villavicencio frente al COVID-19.2021 [cited 2022 March 7].Reference Source\n\nCalvet GA, Pereira SA, Ogrzewalska M, et al.: Investigation of SARS-CoV-2 infection in dogs and cats of humans diagnosed with COVID-19 in Rio de Janeiro, Brazil. PLoS One. 2021; 16(4): e0250853. Publisher Full Text\n\nColitti B, Bertolotti L, Mannelli A, et al.: Cross-Sectional serosurvey of companion animals housed with SARS-CoV-2–infected owners, Italy. Emerg. Infect. Dis. 2021; 27(7): 1919–1922. PubMed Abstract \| Publisher Full Text\n\nDileepan M, Di D, Huang Q, et al.: Seroprevalence of SARS-CoV-2 (COVID-19) exposure in pet cats and dogs in Minnesota, USA. Virulence. 2021; 12(1): 1597–1609. PubMed Abstract \| Publisher Full Text\n\nEsmaeili DE, Fajari A, Naguili B, et al.: Case fatality and mortality rates, socio-demographic profile, and clinical features of COVID-19 in the elderly population: A population-based registry study in Iran. J. Med. Virol. 2022; 94: 2126–2132. PubMed Abstract \| Publisher Full Text\n\nLiu K, Chen Y, Lin R, et al.: Clinical features of COVID-19 in elderly patients: A comparison with young and middle-aged patients. J. Infect. 2020; 80: e14–e18. PubMed Abstract \| Publisher Full Text\n\nPati S, Panda SK, Acharya AP, et al.: Evaluation of geriatric changes in dogs. Veterinary world. 2015; 8(3): 273–278. PubMed Abstract \| Publisher Full Text\n\nPagani G, Lai A, Bergna A, et al.: Human-to-Cat SARS-CoV-2 transmission: Case report and full-genome sequencing from an infected pet and its owner in northern Italy. Pathogens. 2021; 10(252). PubMed Abstract \| Publisher Full Text\n\nKeller M, Hagag TI, Balzer J, et al.: Detection of SARS-CoV-2 variant B.1.1.7 in a cat in Germany. Res. Vet. Sci. 2021; 140: 229–232. PubMed Abstract \| Publisher Full Text\n\nDepartamento Administrativo Nacional de Estadística (DANE): Censo Nacional de Población y Vivienda - CNPV – 2018.2018; [cited 2022 March 15].Reference Source\n\nUniversidad Santo Tómas: Los procesos de configuración territorial en Villavicencio a partir de la migración histórica en el municipio.2018 [cited 2022 March 15].Reference Source\n\nHamer SA, Pauvolid-Corrêa A, Zecca IB: SARS-CoV-2 Infections and viral isolations among serially tested cats and dogs in households with infected owners in Texas, USA. Viruses. 2021; 13(5): 938. PubMed Abstract \| Publisher Full Text\n\nVan Aart A, Velkers AE, Fischer FC, et al.: SARS-CoV-2 infection in cats and dogs in infected mink farms. Transbound. Emerg. Dis. 2021; 69: 3001–3007. PubMed Abstract \| Publisher Full Text\n\nCui J, Li F, Li Z: Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 2019; 17(3): 181–192. PubMed Abstract \| Publisher Full Text\n\nSmith S, Anderson ER, Cansado-Utrilla C, et al.: SARS-CoV-2 neutralizing antibodies in dogs and cats in the United Kingdom. Current Research in Virological Science. 2021; 2: 100011. PubMed Abstract \| Publisher Full Text\n\nSharun K, Tiwari R, Rahman, et al.: SARS-CoV-2 vaccine for domestic and captive animals: An effort to counter COVID-19 pandemic at the human-animal interface. Vaccine. 2021; 39(41): 7119–7122. PubMed Abstract \| Publisher Full Text\n\nHedman H, Krawczyk E, Helmy Y, et al.: Host diversity and potential transmission pathways of SARS-CoV-2 at the human-animal interface. Pathogens. 2021; 10(2): 180. PubMed Abstract \| Publisher Full Text\n\nStevanovic V, Vilibic T, Cavlek I, et al.: Seroprevalence of SARS-CoV-2 infection among pet animals in Croatia and potential public health impact. Transbound. Emerg. Dis. 2020; 68(4): 1767–1773. PubMed Abstract \| Publisher Full Text\n\nGarcía C, Clara M, Jaramillo D, et al.: Seroprevalence of exposure to SARS-CoV-2 in domestic dogs and cats and its relationship with COVID-19 cases in the city of Villavicencio, Colombia. figshare. [Dataset].2022. Publisher Full Text" }	[ { "id": "159713", "date": "26 Jan 2023", "name": "Diego A. Forero", "expertise": [ "Reviewer Expertise Human health", "molecular biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes an analysis of seroprevalence of anti-SARS-CoV-2 IgG in a sample of 300 dogs and 135 cats from a city in Colombia. Several major and minor issues should be addressed:\nA better description of the rationale of the main aim of the study is needed - why are you studying only seroprevalence?\n\n\"SARS-CoV-2 seroprevalence of 9.1% (3.3% in canines and 5.8% in felines)\" is not clear (it is not clear that it is additive).\n\nIn \"Villavicencio (Meta State)\". \"state\" is not clear, in the context of the organization of Colombia.\n\nA better description of the recruitment strategy for the current study would be useful.\n\nIn Methods, a better description of \"commune\" is needed.\n\nIt is not clear whether the authors examined directly some of the animals, for key clinical signs.\n\nKey parameters, such as specificity and sensitivity, are not described for the ELISA assay used. In addition, in Methods it would be important to include citations to key previous papers using the same kit.\n\nIn Statistical analysis, a description of the specific R packages used is needed.\n\nTable 2 is incomplete (the header for a group of rows is missing) and the statistical analysis in table 2 is not clear.\n\nFigure 2 might be presented as supplementary information.\n\nFigure 3 is not clear - it is not clear what is the signal for humans and what is the signal for animals; in addition authors might use colors in this figure.\n\nData for correlation analyses should be presented.\n\nIn the Discussion section, authors must keep into account the lack of statistical significance of some findings.\n\nPossible cross-reactivity of the ELISA assay is not discussed in the manuscript.\n\nLimitations of the current study and recommendations for future studies are needed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9329", "date": "21 Mar 2023", "name": "Dumar Alexander Jaramillo Hernández", "role": "Author Response", "response": "1. A better description of the rationale of the main aim of the study is needed - why are you studying only seroprevalence? R/ One of the main limitations of our study is the lack of economic support. We could have done more advanced studies such as seroincidence, detection of neutralizing antibodies, or isolation of viral genomic material. Nevertheless, we just study seroprevalence in order to include an important and representative sample size of cats and dogs in Villavicencio. 2. \"SARS-CoV-2 seroprevalence of 9.1% (3.3% in canines and 5.8% in felines)\" is not clear (it is not clear that it is additive). R/ Change accepted: Methods Type of study and sample size paragraph Line 2 “The sample was estimated using the formula for size by proportions in finite populations, using the results obtained by Patterson et al.29 as a reference of p, with SARS-CoV-2 seroprevalences of 3.3% in dogs and 5.8% in cats from Italy”. 3. In \"Villavicencio (Meta State)\". \"state\" is not clear, in the context of the organization of Colombia. R/ Colombia is divided into 32 departments and one capital district (Bogotá). Each Department has a capital city. In the case of the Meta Department, the capital city is Villavicencio. Change accepted: The word “State” will be changed for Department on each occasion. E.g. Methods Sampling and inclusion criteria paragraph Line 1 “The participants were selected based on their mandatory participation in the 2021 rabies vaccination campaign, carried out in Villavicencio (capital of Meta department) by the health secretary”. However, in some publications the word “state” means “departamento”. 4. A better description of the recruitment strategy for the current study would be useful. R/ The inclusion and exclusion criteria used in order to estimate the sample size are mentioned under the subheading “Sampling and inclusion criteria”. Change accepted: The subheading will change to “Sampling, inclusion, and exclusion criteria” The sentence “The animals that had consumed immunomodulatory medication (e.g., corticosteroid-type immunosuppressants) one week before the sampling were not included in the study.” Will be changed for: “We determined as exclusion criterion, the animals that had consumed immunomodulatory medication (e.g., corticosteroid-type immunosuppressants) one week before the sampling.” 5. In Methods, a better description of \"commune\" is needed. R/ In Colombia, Departments are subdivided into municipalities which are in turn subdivided into “corregimientos” in rural areas and into “comunas” (communes) in urban areas. Each commune, in turn is divided into neighborhoods. Change accepted: Methods Sampling and inclusion criteria paragraph Line 2 “A probabilistic sampling was conducted by randomly selected two-stage clusters of domestic dogs and cats from the eight communes (subdivisions) that compose the urban area of Villavicencio (Figure 1), …”. 6. It is not clear whether the authors examined directly some of the animals, for key clinical signs. R/ No, animals were randomly selected among those who participated in the rabies vaccination campaign. Once these were selected, a survey was applied, and one of the questions was related to the clinical signs. 7. Key parameters, such as specificity and sensitivity, are not described for the ELISA assay used. In addition, in Methods it would be important to include citations to key previous papers using the same kit. R/ Change accepted: Methods Immunoassay paragraph Line 1. IgG antibodies against the nucleocapsid protein (N) of SARS-CoV-2 in the sera of domestic dogs and cats were qualitatively determined using the indirect enzyme-linked immunosorbent assay (ELISA) (ID Screen® SARS-CoV-2, double antigen multi-species [IDvet, Grabels, France]) according to the manufacturer's instructions. For cats, the kit presents 63% of sensitivity and 96% of specificity. For dogs, the kit has 36% of sensitivity and 85% of specificity. Previous papers used the ID Screen® kit in their studies (48-51). References 48. Bessière P, Vergne T, Battini M, Brun J, Averso J, et al.: SARS-CoV-2 Infection in Companion Animals: Prospective Serological Survey and Risk Factor Analysis in France. Viruses. 2022; 14(1178). PubMed Abstract I Publisher Full Text 49. Diezma-Díaz C, Álvarez-García G, Regidor-Cerrillo J, Miró G, Villanueva-Saz S, et al.: A comparative study of eight serological methods shows that spike protein-based ELISAs are the most accurate tests for serodiagnosing SARS-CoV-2 infections in cats and dogs. Front. Vet. 2023. Sci. 10(1121935). Publisher Full Text 50. Barua S, Hoque M, Adekanmbi F, Kelly P, Jenkins-Moore M, et al.: Antibodies to SARS-CoV-2 in dogs and cats, USA. Emerg Microbes Infect. 2021. 10(1):1669-1674. PubMed Abstract I Publisher Full Text 51. Neira V, Brito B, Agüero B, Berrios F, Valdés V, et al.: A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners. Emerg Microbes Infect. 2021. 10(1):376-383. PubMed Abstract I Publisher Full Text 8. In Statistical analysis, a description of the specific R packages used is needed. R/ Change accepted: Methods Statistical analysis paragraph Line 9. Statistical estimates were made using the R 4.2 software with the packages dplyr, MASS, corrplot and epiDispaly, and the maps using the QGIS 3.10 software. 9. Table 2 is incomplete (the header for a group of rows is missing) and the statistical analysis in table 2 is not clear. R/ In theTable 2, the header of the row 1, 2, 3, 4 … 8: is “Communes”. This header is included in the original manuscript. On the other hand, statistical analysis included in table 2 are described in the fourth paragraph of results: Line 1 “Regarding the SARS-CoV-2 exposure and the risk factors analysed, a statistically significant association between SARS-CoV-2 seropositivity and Commune 2 was found (adjusted OR = 5.84; CI 95% = 1.1-30.88).” 10. Figure 2 might be presented as supplementary information. R/ Change not accepted, it is important to show the OD distribution among communes, showing the positive results and grey area results. 11. Figure 3 is not clear - it is not clear what is the signal for humans and what is the signal for animals; in addition authors might use colors in this figure. R/ Change accepted: Results Figure 3 will be change by the colored one. 12. Data for correlation analyses should be presented. R/ Change not accepted, here we present a table with the data for the correlation analyses about the association between positive COVID-19 cases (RT-qPCR testing) in humans versus seropositivity in pets. Nevertheless, it is not necessary to show the table in the paper, since we mention the important data in the discussion (paragraph 4, line 10). Human Dogs Cats Pets Dogs 0,01 1 -- -- Cats 0,33 0,17 1 Pets 0,32 0,68 0,81* 1 *p-value = 0.01467 13. In the Discussion section, authors must keep into account the lack of statistical significance of some findings. R/ In the results and discussion sections, we mentioned and pointed out that results were not significant (just the results associated with commune 2 were statistically significant); however, there were trends that correlate with other studies and we mentioned them explaining that these were not significant (Eg. Discussion paragraph 2, line 1): “As a result of the present study, a positive relationship between seropositivity and the age of the animals was observed. The older animals between 11 and 15 years exhibited this tendency predisposition, but it was not statistically different”. 14. Possible cross-reactivity of the ELISA assay is not discussed in the manuscript. R/ Change accepted: The following paragraph will be added to the discussion section. Discussion Although the 20 canine pre-pandemic sera reacted negatively to the immunodiagnosis, cross-reactions with ancestral coronaviruses in canines and felines are possible, but in low probability when are compared to commercial ELISAs with neutralizing antibody tests (54, 55). 54. Perera R, Ko R, Tsang O, Hui D, Kwan M et al.: Evaluation of a SARS-CoV-2 surrogate virus neutralization test for detection of antibody in human, canine, cat, and hamster sera. J Clin Microbiol. 2021;59(2):e02504–20. PubMed Abstract I Publisher Full Text 55. Hughes EC, Amat JAR, Haney J, Parr J, Logan N, et al.: SARS-CoV-2 serosurveillance in a patient population reveals differences in virus exposure and antibody-mediated immunity according to host demography and healthcare setting. J Infect Dis. 2021;223(6):971-980. PubMed Abstract I Publisher Full Text 15. Limitations of the current study and recommendations for future studies are needed. R/ Change accepted: The following paragraph will be added to the discussion section. Discussion The possible cross-reactivity and the need to verify if the reactive antibodies are neutralizing for SARS-CoV-2 are the main limitations of our study; likewise, it is advisable to carry out studies of SARS-CoV-2 neutralization from the serum bank obtained, since this is the gold-standard test for the Centers for Disease Control and Prevention (CDC) (56). 56. Centers for Disease Control and Prevention. Coronavirus disease 2019 (COVID-19) 2020 interim case definition, approved April 5, 2020. 2023 [cited 2023 February 9]. Reference Source" } ] } ]	1	https://f1000research.com/articles/11-1184
https://f1000research.com/articles/9-1481/v1	18 Dec 20	{ "type": "Research Article", "title": "Factors affecting elective cesarean section in women with multiple pregnancy in Caruban, Indonesia", "authors": [ "Muhammad Pradhiki Mahindra", "Mahendra Tri Arif Sampurna", "Muhammad Pradhika Mapindra", "Apriska Mega Sutowo Putri", "Aries Krisbiyantoro", "Rozi Aditya Aryananda", "Muhammad Pradhiki Mahindra", "Muhammad Pradhika Mapindra", "Apriska Mega Sutowo Putri", "Aries Krisbiyantoro", "Rozi Aditya Aryananda" ], "abstract": "Background: Caesarean sections have become the most popular method for delivering twin babies because of the safety concerns associated with a natural birth. This study aims to identify the maternal characteristics and obstetric parameters that serve as risk factors influencing caesarean delivery in twin pregnancies by comparing women delivering via caesarean section and vaginal birth. Methods: A retrospective chart review design was used to analyse 47 women with multiple pregnancies from the medical records at a primary referral hospital in East Java, Indonesia. Women delivering vaginally were then compared with women who underwent a caesarean section to identify any differences between the groups. Results: More women delivered by caesarean section than by vaginal birth. In this study, women were more likely to undergo a caesarean section if they had a previous history of undergoing a caesarean section or if there were no foetal malpresentations during their pregnancy. There was a significant difference in maternal age between groups. We found that foetal malpresentation did not significantly increase the likelihood of caesarean delivery in women with multiple pregnancies. Conclusions: The percentage of multiple pregnancies delivered via caesarean section is quite high. Similar to previous studies, we identified a few factors including foetal malpresentation and labour augmentation that did not significantly increase the risk of caesarean delivery. However, we suggest that there is more than one reason responsible for the increase in caesarean sections in multiple pregnancies.", "keywords": [ "cesarean section", "complication", "multiple pregnancy", "vaginal birth" ], "content": "Introduction\n\nMultiple pregnancies are currently much more common than in the past as a result of older maternal age and pregnancies resulting from assisted reproductive technologies1. Multiple pregnancy accounts for 1% to 4% of total births, with a prevalence ranging from 0.9% to 1.4% in England and Wales and 0.9% to 2.4% in Brazil2. The lowest incidence rate of multiple pregnancy is in East Asia (fewer than eight twin births in 1000)3. Multiple pregnancy is associated with higher maternal and perinatal risks than singleton pregnancy, including pre-eclampsia, hypertension, hyperemesis, postpartum haemorrhage, premature rupture of the membranes and anaemia2,4. The rate of perinatal mortality is 2–3 times higher in multiple pregnancy than in singleton newborns. Multiple pregnancy can increase preterm birth, foetal growth restriction, low birth weight and intrapartum anoxia5.\n\nA case complicated by multiple pregnancy has the same delivery options as a singleton pregnancy: caesarean or vaginal delivery. This choice depends on multiple factors and remains controversial in numerous studies. Many important factors are involved when determining the delivery method, including women’s parity, foetal weight, foetal presentation, gestational weight and maternal clinical conditions6. Some studies have explained that if both foetuses are in a cephalic presentation, the vaginal mode is preferred7. Vaginal delivery is possible if the foetus weighs more than 1500 grams. Caesarean delivery is the best choice when the first foetus is non-cephalic. Another large retrospective population study found that compared with caesarean delivery, increased neonatal morbidity and mortality were observed for the second twin when vaginal delivery was performed. That study also indicated that elective caesarean delivery is safer than vaginal delivery and results in the best maternal and perinatal outcomes8. However, although evidence for the preference of an elective caesarean section for multiple pregnancy is lacking, the rates of elective caesarean section for multiple pregnancy have increased throughout the world9. The main objective of this study was to evaluate and provide reliable information on the contributing risk factors such as maternal characteristics, obstetric histories and recorded pregnancy complications that influenced the decision to undertake a caesarean section or vaginal birth in multiple pregnancy cases.\n\n\nMethods\n\nThis retrospective chart review investigated secondary data that were obtained from written medical records at Caruban General Hospital in Caruban, East Java, which has become one of the main primary referral hospitals that receives many high-risk pregnancy cases from the primary health centres and antenatal clinics in the region of Madiun.\n\nThis analysis used a purposive sampling method from the records of pregnant women with multiple pregnancies in the hospital’s data registry from 1 December 2014, to 15 December 2019. We identified 95 medical records of pregnant women based on several criteria. We obtained the data of women who had experienced multiple pregnancy and undergone elective caesarean section or vaginal delivery in the Caruban and Madiun regions of East Java, Indonesia. The inclusion criteria were as follows: women older than 18 years without mental disorders and who were medically stable (oxygen level > 93%, heart rate 60–100 bpm, blood pressure less than 80/60 mmHg). The exclusion criteria were as follows: emergency caesarean section, medically unstable (oxygen level < 93%, heart rate below 60 or above 100 bpm, women with altered mental status), death of the newborn and death of the mother. However, there were no cases of neonatal or maternal death or injury. Medical records with incomplete and unclear information on maternal characteristics were not included. The final sample consisted of 47 women.\n\nUsing a data extraction form (see extended data10), information on maternal characteristics including age, educational background and occupational status and information on obstetric histories such as previous history of caesarean section and parity status, were obtained. We decided to categorise educational background into graduates and unqualified degree. Occupational status was subcategorised into unemployed, civil servant, private employee and entrepreneur. Parity status was classified into nulliparous, primiparous and multiparous. Data on perinatal events including foetal malpresentation, labour augmentation, prenatal haemoglobin levels and gestational hypertension were also recorded (see underlying data for the full dataset11).\n\nThe analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 16 for Windows. Quantitative data are presented as odds ratios (ORs) with confidence intervals (CIs) and means with the standard deviation. Chi-square tests were used to compare the caesarean and vaginal birth groups, and normal probability plots were generated to evaluate the data distribution. We then used an ANOVA for the normally-distributed data and the Kruskal–Wallis test for the non-normally-distributed data. A P-value of <0.05 was considered significant.\n\nThis analysis was approved by the ethics committee of Caruban General Hospital (No. 800/ 4532/ 402.102.110/ 2020). The requirement to obtain informed consent was waived by the ethics committee of Caruban General Hospital before the study was conducted. The study was conducted according to the relevant guidelines. The patient data records were coded and anonymised. The information recorded is confidential and was used only for the study purpose.\n\n\nResults\n\nAs shown in Table 111, among the 47 mothers who met our inclusion criteria, more than half were between 21 and 35 years old, and most had a low educational status and worked as private employees. Additionally, 12 women underwent a vaginal delivery whereas the other 35 women birthed their babies abdominally. Thus, more women underwent a caesarean section than gave birth vaginally. Figure 1 indicates that the main indication for a caesarean section in this study was a caesarean scar, followed by malpresentation and foetal distress. Perinatal complications were principally dominated by malposition of the foetuses, which were not in the vertex position. During the labour process, 18 of the 47 women suffered prolonged labour. Six women came to the hospital with a history of premature membrane rupture without uterine contractions. Gestational hypertension was also reported in 12 women and 11 women had Hb levels below 10 g/dL in their last recorded Hb examinations in late pregnancy.\n\nBased on the analysis, the average age at delivery differed significantly between women who underwent caesarean and vaginal delivery (p = 0.00). Other maternal characteristics including primiparous as the most common parity status (p = 0.73), educational status (p = 0.46) and being unemployed (p = 1.00) did not differ significantly between the two groups of women stratified by delivery method.\n\nAdditionally, we evaluated the obstetric history of the included mothers. In total, 21 women had a history of previous caesarean section, which was found to significantly increase the likelihood of undergoing another caesarean section (OR = 16.5, 95% CI = 1.91 to 142.29, p = 0.02). Women who suffered prolonged labour during their delivery were significantly less likely to undergo a caesarean delivery (OR = 0.20, 95% CI = 0.49 to 0.81, p = 0.03). Women who did not experience malpresentation of their foetuses during pregnancy were more likely to undergo a caesarean delivery (OR = 8.25, 95% CI = 0.95, 71.09, p = 0.03). However, in this study, premature rupture of the membrane, gestational hypertension and gestational anaemia did not significantly contribute to an increased likelihood of a caesarean section. Pregnancy term did not differ significantly between the women undergoing caesarean section and vaginal birth. Neonatal outcomes including 1-minute APGAR (appearance, pulse, grimace, activity, and respiration) scores and babies’ birth weights did not differ significantly for both the first and second babies between both groups of delivery methods (Table 211). When comparing caesarean section and vaginal delivery, both first babies (p = 0.82) and second babies (p = 0.38) had APGAR scores ≥ 7 (Table 211). The first babies delivered after caesarean section vs vaginal birth did not differ significantly (2114 grams ± 469.37 vs 1938.33 grams ± 562.36, p = 0.29) (Table 211). Similarly, the second babies following caesarean section vs vaginal birth also did not differ significantly (2038 ± 478.75 vs 1878.33 ± 417.45, p = 0.31) (Table 211).\n\nAPGAR: appearance, pulse, grimace, activity, and respiration\n\n\nDiscussion\n\nBased on the study results, a planned caesarean section appears preferable to a vaginal birth for women with multiple pregnancies when the mothers have a history of previous caesarean section and in cases of malpresentation of the foetus. The neonatal birth weight and 1-minute APGAR scores of the twin babies born by both methods of delivery in this study were similar. Adverse perinatal outcomes including perinatal mortality and transient tachypnea of the newborn, were not significantly different between groups. However, the second twin had a higher risk of suffer perinatal outcomes than the first following vaginal delivery12. Combined vaginal/caesarean delivery is another option. However, it may be complicated by a longer interval of prolonged rupture of membranes, leading to endometritis and sepsis in the second twin.13. A large nationwide study of multiple pregnancy cases identified a greater risk of uterine rupture in women who delivered their babies via natural birth14. However, previous studies reported that there was no difference in the perinatal outcomes between caesarean section and vaginal birth in multiple pregnancy15,16. Conversely, maternal aspects are an important consideration when choosing the delivery mode in women with a twin pregnancy. However, some studies have reported that the use of forceps or vacuum extractions in vaginal birth could contribute to birth lacerations that may lead to gynaecologic morbidities17–20.\n\nIn this comparison study between planned caesarean section and vaginal delivery, we found that there was a significant difference in the maternal age between the two groups. This finding was supported by a previous study that showed a significantly increased trend of caesarean delivery with increased maternal age21. However, a study from Korea found there was no significant difference caesarean section rates in women with multiple pregnancies according to maternal age22. The other difference we identified was that women undergoing caesarean section were more likely to have undergone a previous caesarean section. A similar study comparing women having caesarean sections and vaginal births reported that caesarean section was significantly more likely to occur in women with previous caesarean histories23. Another report revealed that caesarean sections must not be considered mandatory for multiple pregnancies24. However, a study from Myles (2009) demonstrated that caesarean sections had a higher success rate and a lower probability of developing uterine rupture than vaginal delivery25.\n\nThis study found that women who underwent labour augmentation by using misoprostol or oxytocin did not have a higher probability of caesarean section. Supporting our finding, a study by Arulkumaran showed that approximately 78% of women with a caesarean scar who were administered labour augmentation had safe vaginal births whereas the other women who had undergone second caesarean sections were suffering from cephalo-pelvic disproportion26. Furthermore, a prospective cohort study on 153 women with caesarean scars in Saudi Arabia suggested that labour induction to promote vaginal delivery did not contribute to the increased likelihood of a second elective caesarean section or adverse effects on neonatal outcomes27. By contrast, a study in Sweden reported the increased likelihood of caesarean delivery after performing labour induction using oxytocin and cervical ripening in women with multiple pregnancy28. Another study also explained that labour induction or augmentation was safe for women to promote successful vaginal delivery in multiple pregnancy cases29.\n\nIn our study, we found that non-cephalic presentations did not significantly increase the likelihood of undergoing a caesarean section. Our results supported previous studies that reported relatively similar outcomes for women with non-cephalic presentations who delivered their babies abdominally and vaginally30,31. Furthermore, a caesarean section in multiple pregnancies with non-cephalic presentations is likely to occur following external cephalic attempts11. A report from France suggested that the type of presentation must not be considered the main consideration for caesarean section in multiple pregnancies10.\n\nOur findings demonstrate that women with multiple pregnancies had a higher tendency to deliver their babies abdominally via caesarean section. Maternal age and previous history of a caesarean section should be considered when determining whether to perform a caesarean delivery in women with multiple pregnancies. However, the supporting findings are still limited. This study also noted that malpresentation and labour augmentation do not increase the likelihood of a caesarean section compared with vaginal delivery. However, this finding was rather weak due to the small size of the study population, and thus choosing between a caesarean section and vaginal delivery must be made after considering the patient’s preference and the risks and benefits.\n\n\nData availability\n\nFigshare: Underlying Data - Factors Affecting Elective Cesarean Section in Women with Multiple Pregnancy at a Primary Referral Hospital in Indonesia. https://doi.org/10.6084/m9.figshare.13166735.v111\n\nThis project contains the following the underlying data:\n\n- Cesarean Section VS Vaginal Birth (Maternal Factors).sav\n\n- Cesarean Section VS Vaginal Birth (Neonatal Factors).sav\n\nFigshare: Data Extraction Form - Factors Affecting Elective Cesarean Section in Women with Multiple Pregnancy at a Primary Referral Hospital in Indonesia. https://doi.org/10.6084/m9.figshare.13238534.v110\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgement\n\nWe would like to thank the director of Caruban Hospital, Dr. Djoko Santoso, for his encouragement in permitting us to collect the data for this study.\n\n\nReferences\n\nLindroos L, Elfvin A, Ladfors L, et al.: The effect of twin-to-twin delivery time intervals on neonatal outcome for second twins. BMC Pregnancy Childbirth. 2018; 18(1): 36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSantana DS, Surita FG, Cecatti JG, et al.: Multiple Pregnancy: Epidemiology and Association with Maternal and Perinatal Morbidity. Rev Bras Ginecol Obstet. 2018; 40(9): 554–562. PubMed Abstract \| Publisher Full Text\n\nSmits J, Monden C: Twinning across the developing world. PLoS One. 2011; 6(9): e25239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaine K, Murzakanova G, Sole KB, et al.: Prevalence and risk of pre-eclampsia and gestational hypertension in twin pregnancies: A population-based register study. BMJ Open. 2019; 9(7): e029908. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nObiechina NJ, Okolie VE, Eleje GU, et al.: Twin versus singleton pregnancies: The incidence, pregnancy complications, and obstetric outcomes in a Nigerian tertiary hospital. Int J Womens Health. 2011; 3(1): 227–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchachter-Safrai N, Karavani G, Haj-Yahya R, et al.: Risk factors for cesarean delivery and adverse neonatal outcome in twin pregnancies attempting vaginal delivery. Acta Obstet Gynecol Scand. 2018; 97(7): 845–851. PubMed Abstract \| Publisher Full Text\n\nFelix Martins Santana E, Melo Correa V, Bottura I, et al.: Time and Mode of Delivery in Twin Pregnancies. Mult Pregnancy - New Challenges. 2019. Publisher Full Text\n\nSchmitz T, Azria E, Cabrol D, et al.: L’accouchement par voie basse des grossesses gemellaires est-il encore une option raisonnable? Analyse des donnees de la litterature. J Gynecol Obstet Biol Reprod. 2009; 38(5): 367–376. Publisher Full Text\n\nBarrett JFR, Hannah ME, Hutton EK, et al.: A randomized trial of planned cesarean or vaginal delivery for twin pregnancy. N Engl J Med. 2013; 369(14): 1295–305. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahindra MP, Sampurna MTA, Mapindra MP, et al.: Data Extraction Form - Factors Affecting Elective Cesarean Section in Women with Multiple Pregnancy at a Primary Referral Hospital in Indonesia. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13238534.v1\n\nMahindra MP, Sampurna MTA, Mapindra MP, et al.: Underlying Data - Factors Affecting Elective Cesarean Section in Women with Multiple Pregnancy at a Primary Referral Hospital in Indonesia. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13166735.v1\n\nHofmeyr GJ, Barrett JF, Crowther CA, et al.: Planned caesarean section for women with a twin pregnancy. Cochrane Database Syst Rev. 2011; (12): CD006553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlexander JM, Leveno KJ, Rouse D, et al.: Cesarean Delivery for the Second Twin. Obstet Anesth Dig. 2009; 29(3): 120–121. Publisher Full Text\n\nFord AAD, Bateman BT, Simpson LL, et al.: Vaginal birth after cesarean delivery in twin gestations: A large, nationwide sample of deliveries. Am J Obstet Gynecol. 2006; 195(4): 1138–42. PubMed Abstract \| Publisher Full Text\n\nHenriksen TB, Sperling L, Hedegaard M, et al.: Cesarean section in twin pregnancies in two Danish counties with different cesarean section rates. Acta Obstet Gynecol Scand. 1994; 73(2): 123–8. PubMed Abstract \| Publisher Full Text\n\nVarner MW, Leindecker S, Spong CY, et al.: The Maternal-Fetal Medicine Unit cesarean registry: Trial of labor with a twin gestation. Am J Obstet Gynecol. 2005; 193(1): 135–40. PubMed Abstract \| Publisher Full Text\n\nMeyer S, Schreyer A, De Grandi P, et al.: The effects of birth on urinary continence mechanisms and other pelvic-floor characteristics. Obstet Gynecol. 1998; 92(4 pt 1): 613–8. PubMed Abstract \| Publisher Full Text\n\nZetterstrom JP, López A, Anzén B, et al.: Anal incontinence after vaginal delivery: A prospective study in primiparous women. Br J Obstet Gynaecol. 1999; 106(4): 324–30. PubMed Abstract \| Publisher Full Text\n\nWilson PD, Herbison RM, Herbison GP: Obstetric practice and the prevalence of urinary incontinence three months after delivery. Br J Obstet Gynaecol. 1996; 103(2): 154–61. PubMed Abstract \| Publisher Full Text\n\nFathallah N, Spindler L, Zeitoun JD, et al.: Anal incontinence after childbirth. Colon and Rectum. 2018; 12(4): 235–42.\n\nLondero AP, Rossetti E, Pittini C, et al.: Maternal age and the risk of adverse pregnancy outcomes : a retrospective cohort study. BMC Pregancy Childbirth. 2019; 19(1): 261. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee YJ, Kim MN, Kim YM, et al.: Perinatal outcome of twin pregnancies according to maternal age. Obstet Gynecol Sci. 2019; 62(2): 93–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSansregret A, Bujold E, Gauthier RJ: Twin delivery after a previous caesarean: a twelve-year experience. J Obstet Gynaecol Can. 2003; 25(4): 294–8. PubMed Abstract \| Publisher Full Text\n\nAl-Obaidly S, Al-Ibrahim A, Khaldi HM, et al.: The impact of previous one caesarean section on obstetric decision in twin gestation. Eur J Obstet Gynecol Reprod Biols. 2016; 206: e5–e6. Publisher Full Text\n\nArulkumaran S, Ingemarsson I, Ratnam SS: Oxytocin augmentation in dysfunctional labour after previous caesarean section. Br J Obstet Gynaecol. 1989; 96(8): 939–41. PubMed Abstract \| Publisher Full Text\n\nAlsayegh AK , Roshdy S, Akef AH, et al.: Induction of Labor with Prostaglandin E2 in Women with Previous Cesarean Section and Unfavorable Cervix. Int J Health Sci (Qassim). 2007; 1(2): 211–6. PubMed Abstract \| Free Full Text\n\nJonsson M: Induction of twin pregnancy and the risk of caesarean delivery: a cohort study. BMC Pregnancy Childbirth. 2015; 15(1): 136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFausett MB, Barth WH, Yoder BA, et al.: Oxytocin labor stimulation of twin gestations: Effective and efficient. Obstet Gynecol. 1997; 90(2): 202–4. PubMed Abstract \| Publisher Full Text\n\nBisschop CNS, Vogelvang TE, May AM, et al.: Mode of delivery in non-cephalic presenting twins: A systematic review. Arch Gynecol Obstet. 2012; 286(1): 237–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHofmeyr GJ, Barrett JF, Crowther CA: Planned caesarean section for women with a twin pregnancy. Cochrane Database Syst Rev. 2011; (12): CD006553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGocke SE, Nageotte MP, Garite T, et al.: Management of the nonvertex second twin: Primary cesarean section, external version, or primary breech extraction. Am J Obstet Gynecol. 1989; 161(1): 111–4. PubMed Abstract \| Publisher Full Text" }	[ { "id": "76524", "date": "02 Feb 2021", "name": "Kian Djien Liem", "expertise": [ "Reviewer Expertise Neonatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comment:\nThis is a retrospective study which has the intention to identify the contributing risk factors for the decision of performing caesarean section in twin pregnancies. It is very difficult to get a reliable insight from this retrospective study about the contributing risk factors for caesarian section in multiple pregnancy. There are many reasons of the obstetricians for choosing caesarean section, which are not always necessary correlated with some real risk factors for the mother or the foetus. Possibly that some of these reasons are subjectively determined. Therefore it is not surprising to find that fetal malpresentation is not more common in the caesarean section group as compared to the group of vaginal delivery. Nevertheless, the result of this retrospective study can be used as a mirror for the obstetricians in order to think about the clear objective indications for performing of caesarean section in twin pregnancy. The authors should make an obstetrical recommendation for a reliable protocol of caesarean section indication in twin pregnancy.\n\nSpecific comments:\nAbstract:\nNo comment.\n\nIntroduction:\nLine 6 from the second paragraph: what is meant by gestational weight? Is this maternal weight or estimated fetal weight? The authors must clarify this.\n\nMethods:\nNo comment.\n\nResults:\nTable 1 - This part of the table is incorrect: Under vaginal birth is 1 of 12 never 66.7% and 11 of 12 is never 42.9%. The total percentage is more than 100%. Probably the OR and p-value has to be recalculated again.\n\nAlso in the text under the 3rd paragraph, the OR an p-value has to be checked also. I am wondering why fetal malpresentation didn’t result in more cesarean section. Especially when some fetal malpresentation will result in difficult either impossible vaginal delivery.\n\nDiscussion:\nIn the first line the authors make the following statement: “Based on the study results, a planned caesarean section appears preferable to vaginal birth for women with multiple pregnancies when the mothers have a history of previous caesarean section and in cases of malpresentation of the fetus”. But this seems in contradiction with their results that women who did not experience malpresentation of their fetuses during pregnancy were more likely to undergo a caesarean delivery. The authors should clarify this contradiction.\n\nIn the first paragraph the authors write: “However the second twin had a higher risk of suffer perinatal outcomes than the first following vaginal delivery”. But this statement is not supported by the data of their results.\n\nIn this chapter a discussion about the limitation of this study is missed. The authors should make a reflection about the limitations of their study. See also general comment.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1481
https://f1000research.com/articles/12-963/v1	10 Aug 23	{ "type": "Policy Brief", "title": "Multi-drug resistant Salmonella typhi: why should African countries move quickly toward adopting typhoid conjugate vaccine", "authors": [ "Adriano Lubanga", "Akim Nelson Bwanali", "Akim Nelson Bwanali" ], "abstract": "Africa has a high burden of typhoid fever causing significant morbidity and mortality especially among children. Over the years there has been increased development of Multi-drug and Extensively drug resistance typhoid strains in the continent which threatens typhoid treatment. This is compounded by a background that most African countries have limited diagnostic capacity, leading to high empiric use of antibiotics, and worsening of antimicrobial resistance. Access to safe drinking water, hygiene and sanitation facilities also remains a significant challenge for most countries. In this policy brief, we encourage African countries to consider quick adoption of highly efficacious and cost-effective available typhoid conjugate vaccines.", "keywords": [ "Typhoid Fever", "Multi-drug Resistance typhoid", "Typhoid Congugate Vaccine", "Extensive Drug Resistance Typhoid" ], "content": "Introduction\n\nTyphoid fever remains a significant public health burden resulting in high morbidity and mortality among millions of individuals in resource-constrained settings.1 Typhoid fever is caused by the bacterium Salmonella Typhi and is spread mainly through the ingestion of contaminated food or water. While the disease has been largely eliminated in higher-income countries with modern sanitary facilities and safe drinking water, it still persists as a significant public health issue in many low- and middle-income countries (LMICs) worldwide.2,3 This public health threat is further compounded by the emergent multi-drug resistant strains (H58 Salmonella). These multi-drug resistant strains have been on the rise especially in sub-Saharan Africa (SSA) and leads to prolonged illness and severe complications.4\n\nS. Typhi has reportedly been cultured from patients in 42 of 57 African countries until 2018. However, this could result from limited diagnostic capacities and underreporting in the remaining 15 countries.5 Many studies in Africa have reported an incidence rate that is similar to or even higher than that reported in South/Southeast Asia.5,6 A 2019 comprehensive review estimated the African incidence of typhoid fever from previous results and presented an initial pooled estimate of 112.1/100,000 person-years, with a 95% confidence interval (CI) of 46.7–203.5.7 This high incidence may likely be compounded by the impact of climate change in some countries in Southern parts of Africa.8 With such a huge impact of the disease on households, and health care systems, more innovative preventive efforts are required to curb the spread of the disease. Introduction of Typhoid conjugate vaccine (TCV) offers a new hope in addressing the enduring problem of typhoid fever as well as the evolving problem of multi-drug resistance in affected regions.\n\n\nTyphoid fever; current treatment options, development of multi-drug resistance, and economic impact of the disease\n\nAntibiotics have offered the most effective way to treat typhoid fever since 1940’s. Chloramphenicol, co-trimoxazole and ampicillin were the first recommended antibiotic treatment options for typhoid fever.9 Even though the antibiotics were highly effective during inception, the bacterium has evolved quite quickly, rendering most of them ineffective. Multi-drug resistant (MDR) typhoid, (resistance to chloramphenicol, ampicillin, and co-trimoxazole) first appeared in the 1970’s and has since spread globally.9 In the last decade, several regions in Africa have reported typhoid outbreaks, and these have been closely linked with MDR phenotypes, H58.S.9 This strain has been isolated in many parts of the world including SSA. Owing to the rapid rise of MDR strains, fluoroquinolones, azithromycin and third-generation cephalosporins were eventually recommended as second line and third line treatment options respectively. However, since 2016, extensive drug resistant strains (resistant to chloramphenicol, ampicillin, co-trimoxazole, streptomycin, fluoroquinolones, and third-generation cephalosporins) have been reported in Pakistan.10 This renders azithromycin as the only effective oral treatment option for typhoid. The emergence of these extensively drug resistant strains with a high potential to spread to other regions poses significant concerns to treatment of typhoid as treatment options are limited. Furthermore, most African countries have limited diagnostic capacity leading to increase in empiric treatment of infections, and eventually overuse of antibiotics.11 This will likely worsen the development of resistant strains and render all available treatment options ineffective.\n\nIntravenous antibiotics required in advanced, severe typhoid disease are more expensive and less readily accessible in most health care centers/facilities.12 In most developing countries, these antibiotics are mostly found in referral hospitals. This poses a significant economic burden on households as they must travel long distance to access care. Drug resistant cases leads to prolonged hospitalization, high case fatality and morbidity leading to increased pressure on health care systems.12\n\n\nTyphoid conjugate vaccine: A new hope for Africa\n\nIncrease in MDR and emergence of extensively resistant strains highlight the need for urgent escalation of preventive efforts such as adoption of Typhoid Conjugate Vaccine (TCV) and improvement in Water, Sanitation and Hygiene (WASH) facilities. However, the former offers a better option in most African countries where hope for improving water supply systems and hygiene practices has greatly staggered for a century. Approximately, 418 million people still lack even a basic level of drinking water service, 779 million lack basic sanitation services (including 208 million who still practice open defecation) and 839 million still lack basic hygiene services.13 With such threatening figures on inequitable access to safe drinking water and sanitation facilities, perhaps two more decades would elapse to alleviate the challenge. The problem is more likely to be compounded by the effects of Climate change, which further leads to disruption of WASH facilities. The cases of water borne diseases are likely to be on the increase, and typhoid is not an exception.\n\nThe TCV is highly effective and efficacious. The vaccine was prequalified for use for control of typhoid fever by WHO in 2018. The Vaccine is administered in a single dose and approved for children six months of age and older. Large Phase 3 efficacy studies conducted in Bangladesh, Malawi, and Nepal show that Typbar TCV is safe and highly effective, preventing 85%, 84%, and 79% of typhoid cases in vaccinated children, respectively.14 A recent phase 3 trial report from Malawi has also demonstrated the longer durability of protection, with little decrease in efficacy for up to 48 months. An age stratified analysis from the same study also demonstrated that the vaccine is efficacious in all age groups, including children <2 years old.4\n\nDespite being highly efficacious and prequalified by the WHO nearly four years ago, Most African countries haven’t adopted the TCV into their routine immunization programs.\n\n\nPolicy outcomes and implications\n\nEven though improving WASH facilities offers a longer term and most effective option for controlling typhoid fever, most African countries are far from achieving that. Adopting the TCV is not only the most effective approach, but also a cost-effective immediate solution for Africa. TCV can be administered as reactive campaign or as part of national preventive efforts. Studies have demonstrated reactive vaccination to be cost-effective if delays in vaccine deployment are minimal; otherwise, introduction of preventive routine immunisation with a catch-up campaign is the preferred strategy.15 Countries such as Zimbabwe, Liberia, and Malawi in partnership with international organizations have moved and adopted TCV into a routine immunization program. TCV has also shown to have a major role not only in reducing the burden of typhoid but also preventing the spread of drug-resistant typhoid strains. A recent modelling analysis projected that TCV introduction with catch-up campaigns could drastically reduce the number of drug-resistant typhoid cases and deaths.16 This analysis further predicts that two-thirds of cases and deaths due to fluoroquinolone resistant- and MDR typhoid could be averted through TCV introduction and the proportion of typhoid cases that are drug resistant could decrease by more than 16% in Gavi, the Vaccine Alliance-eligible countries over ten years. With the weakening pipeline in development of new antibiotics, TCV offers a new hope to most developing countries in Africa.\n\nFurthermore, studies done in Malawi indicate that typhoid can be economically catastrophic for families, despite accessible free medical care.17 The disease is also costly for government healthcare provision. With the high economic burden of the disease on family and increased pressure on health care systems, studies have demonstrated that introduction of routine immunization with TCV along with a catch-up campaign in children aged less than 15 years could be a cost-effective solution to combat the burden of typhoid fever, especially in countries with high typhoid incidence receiving Gavi support.\n\n\nActionable recommendations\n\n\n\n1. African countries should enhance cross-cutting typhoid research to establish determinants of the diseases and identify delivery approaches that optimize the effectiveness of TCV. This could ensure implementation of interventions in line with local contexts, thereby enhancing the odds of achieving desired effectiveness.\n\n2. Additionally, African countries should assess the economic impact and local feasibility of introducing TCV and move quickly toward adopting TCV, especially in typhoid endemic regions to control the disease and reduce spread of drug resistant strains. Due to potential low supplies of the vaccine, vaccination should be prioritized towards at risk populations in endemic countries that include children, adolescents and laboratory workers. This could be achieved by setting up and empowering taskforces that coordinate responses to MDR typhoid fever and suggest effective approaches for introducing the TCV.\n\n3. Strengthen disease surveillance and improve diagnostic capability for timely accurate detection and initiation of appropriate treatment for Enteric infections. This requires adequate equipment and human resource which remains a huge challenge in Africa. Therefore, this calls for sufficient financing that is driven by strong political commitment towards typhoid disease prevention and control. Additional funding may need to be secured from local and international health stakeholders.\n\n4. Improve access to WASH facilities as part of long-term preventive efforts for typhoid fever. Basic WASH packages along with health education on WASH utilization should be conducted in communities. This requires involvement of multiple stakeholders including district health management teams, community-based healthcare workers and community leader. WASH interventions together with vaccination form a vital integrated and comprehensive approach that is of utmost significance because typhoid transmission and development of drug resistance is multifaceted and dynamic.\n\n5. Ensure sustainability of intervention programmes. Due to the ever-evolving nature of MDR typhoid fever, vaccination programmes should be sustainable and fully include the communities. This could be done by integrating TCV into routine immunisation and conducting regular outreach clinics to ensure access of the vaccine for hard-to-reach communities.\n\n\nConclusions\n\nThe burden of typhoid fever is worsened by growing concerns about drug resistance. In view of the MDR typhoid in Africa highlighted in this article, it is imperative that countries adopt and adapt global policies for contextually appropriate use of TCV. Countries need to identify typhoid research priorities and set up typhoid national action plans (NAP) to oversee all collaborative efforts against MDR typhoid strains. The NAPs should outline a comprehensive prevention approach that should involve multiple stakeholders from the designing stage up to implementation.", "appendix": "Data availability\n\nThe data analysed during the development of this article is available and can be provided upon a reasonable request made to the corresponding author.\n\n\nReferences\n\nBasnyat B, Qamar FN, Rupali P, et al.: Clinical update: Enteric Fever. BMJ. 2021; 372: n437. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCutler D, Miller G: The role of public health improvements in health advances: the twentieth-century United States. Demography. 2005; 42(1): 1–22. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Typhoid and other invasive salmonellosis. Vaccine-preventable diseases, surveillance standards.2018. Accessed May 6, 2021. Reference Source\n\nPatel PD, Patel P, Liang Y, et al.: Safety and Efficacy of a Typhoid Conjugate Vaccine in Malawian Children. N. Engl. J. Med. 2021 Sep 16; 385(12): 1104–1115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim J-H, Im J, Parajulee P, et al.: A systematic review of typhoid fever occurrence in Africa. Clin. Infect. Dis. 2019; 69(Suppl 6): S492–S498. PubMed Abstract \| Publisher Full Text \| Free Full Text Google Scholar\n\nStanaway JD, Reiner RC, Blacker BF, et al.: The global burden of typhoid and paratyphoid fevers: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Infect. Dis. 2019; 19(4): 369–381. PubMed Abstract \| Publisher Full Text \| Free Full Text Google Scholar\n\nMarchello CS, Hong CY, Crump JA: Global typhoid fever incidence: a systematic review and meta-analysis. Clin. Infect. Dis. 2019; 68(Suppl 2): S105–S116. PubMed Abstract \| Publisher Full Text \| Free Full Text Google Scholar\n\nThe GAVI: Amidst crises, Malawi showcases resilience with major vaccination push. http\n\nBritto CD, Wong VK, Dougan G, et al.: A systematic review of antimicrobial resistance in Salmonella enterica serovar Typhi, the etiological agent of typhoid. PLoS Negl. Trop. Dis. 2018 Oct 11; 12(10): e0006779. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRasheed F, Saeed M, Alikhan NF, et al.: Emergence of Resistance to Fluoroquinolones and Third-Generation Cephalosporins in Salmonella Typhi in Lahore, Pakistan. Microorganisms. 2020 Sep 1; 8(9): 1336. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSteele AD, Hay Burgess DC, Diaz Z, et al.: Challenges and Opportunities for Typhoid Fever Control: A Call for Coordinated Action. Clin. Infect. Dis. 2016 Mar 15; 62(Suppl 1): S4–S8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim CL, Cruz Espinoza LM, Vannice KS, et al.: The Burden of Typhoid Fever in Sub-Saharan Africa: A Perspective. Res. Rep. Trop. Med. 2022 Mar 14; 13: 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUNICEF: Africa to drastically accelerate progress on water, sanitation and hygiene – report.Reference Source\n\nDr. Atuhebwe P ; WHO AFRO: Take on Typhoid; Now we know: typhoid conjugate vaccines are highly effective in African children.Reference Source\n\nPhillips MT, Antillon M, Bilcke J, et al.: Cost-effectiveness analysis of typhoid conjugate vaccines in an outbreak setting: a modeling study. BMC Infect. Dis. 2023; 23: 143. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBirger R, Antillón M, Bilcke J, et al.: Estimating the effect of vaccination on antimicrobial-resistant typhoid fever in 73 countries supported by Gavi: a mathematical modelling study. Lancet Infect. Dis. 2022 May; 22(5): 679–691. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLimani F, Smith C, Wachepa R, et al.: Estimating the economic burden of typhoid in children and adults in Blantyre, Malawi: A costing cohort study. PLoS One. 2022; 17(11): e0277419. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "239402", "date": "29 Aug 2024", "name": "Priscilla Rupali", "expertise": [ "Reviewer Expertise Infectious Diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe arguments are presented adequately. However, epidemiology of typhoid in Africa could have presented better. Changing epidemiology from SETA and TSAP could be presented. Economic burden needs a little bit of detailed expansion and suitable references. Treatment options that are being considered for XDR and MDR typhoid could be elaborated upon. There is data from other African countries now not just Malawi and that should be referenced.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Partly\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Yes\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-963
https://f1000research.com/articles/11-487/v1	03 May 22	{ "type": "Case Report", "title": "Case Report: Buprenorphine-precipitated fentanyl withdrawal treated with high-dose buprenorphine", "authors": [ "Nicholas L. Bormann", "Antony Gout", "Vicki Kijewski", "Alison Lynch", "Antony Gout", "Vicki Kijewski", "Alison Lynch" ], "abstract": "Background: Buprenorphine, a partial agonist of the mu-opioid receptor, is an increasingly prescribed medication for maintenance treatment of opioid use disorder. When this medication is taken in the context of active opioid use, precipitated withdrawal can occur, leading to acute onset of opioid withdrawal symptoms. Fentanyl complicates use of buprenorphine, as it slowly releases from body stores and can lead to higher risk of precipitated withdrawal. Objectives: Describe the successful management of buprenorphine precipitated opioid withdrawal from fentanyl with high doses of buprenorphine. We seek to highlight how no adverse effects occurred in this patient and illustrate his stable transition to outpatient treatment. Case report: We present the case of a patient with severe opioid use disorder who presented in moderately severe opioid withdrawal after taking non-prescribed buprenorphine-naloxone which precipitated opioid withdrawal from daily fentanyl use. He was treated with high doses of buprenorphine, 148 mg over the first 48 hours, averaging 63 mg per day over four days. The patient reported rapid improvement in withdrawal symptoms without noted side effects and was able to successfully taper to 16 mg twice daily by discharge. Conclusions: This case demonstrates the safety and effectiveness of buprenorphine at high doses for treatment of precipitated withdrawal. While other options include symptomatic withdrawal management, initiating methadone or less researched options like ketamine, utilizing buprenorphine can preserve or re-establish confidence in this life-saving medication. This case also increases the previously documented upper boundary on buprenorphine dosing for withdrawal and should provide additional confidence in its use.", "keywords": [ "Buprenorphine", "fentanyl", "Opioid-Related Disorders", "case report" ], "content": "Introduction\n\nBuprenorphine, a partial mu-opioid receptor agonist, has become the most prescribed treatment for opioid use disorder (OUD).1 With greater availability, non-prescribed use has also increased.2 When buprenorphine is taken after recent use of a full-agonist opioid, buprenorphine displaces the lower affinity molecule, causing a precipitated withdrawal.3 This rapid onset of opioid withdrawal symptoms including bone and muscle pain, diarrhea, insomnia, dysphoria, and anxiety, causes significant patient discomfort.\n\nThe rise of synthetic opioids such as fentanyl has complicated the treatment of precipitated withdrawal. Fentanyl is more potent than heroin, and has high lipophilicity leading to rapid uptake into body tissues and subsequent slow release.4,5 While use of ketamine has been suggested,6 conventional wisdom has been to utilize additional buprenorphine.7 Recent cases in the literature have shown safety and effectiveness of up to 40 mg of buprenorphine early in the withdrawal period.7–11 Herein, we describe the case of a patient who required 148 mg of buprenorphine over 48 hours for successful treatment of buprenorphine-precipitated withdrawal from fentanyl. Consent was obtained from the patient for his case to be used in the academic literature and is available upon request. As such, the University of Iowa Institutional Review Board has deemed it exempt.\n\n\nCase description\n\nA 21-year-old partnered, unemployed Caucasian male with no known past medical history, a psychiatric history of attention deficit hyperactivity disorder and unspecified anxiety, with a pertinent family history of an opioid use disorder in his brother and both his mother and maternal aunt having unspecified addiction to pills per his father, who lived in an apartment with a roommate however was being evicted due to late rent payments, presented with his father to our medication for addiction treatment (MAT) walk-in clinic located at a primary care outreach clinic for assistance discontinuing daily fentanyl use. Initial opioid exposure was through purchasing prescription opioids for six months, before transitioning to use of fentanyl after he had purchased it unknowingly. He endorsed daily fentanyl use of an unknown amount for six months, with an escalation in patient-estimated amount over the preceding two months. His typical method of use was insufflation or vaping. He had attempted to stop multiple times by tapering use with the goal of abstinence, however, was unsuccessful after occurrence of withdrawal symptoms led to eventual return to daily use of the previous amount. Other substance use consisted of non-prescribed alprazolam 1 mg daily that he had started taking in the previous weeks for anxiety symptoms. He denied other active substance use.\n\nAfter making plans to present to the MAT clinic, he abstained from using fentanyl to prepare for buprenorphine induction. His father drove him to the MAT clinic sixteen hours after last fentanyl use. While traveling to the clinic, he took non-prescribed buprenorphine-naloxone 8-2 mg, which immediately precipitated withdrawal. On initial evaluation in clinic, he had a clinical opioid withdrawal scale12 (COWS) score of 27, with diffuse pain, nausea, emesis, diarrhea, rhinorrhea, chills, yawning, anxiety, and restlessness. He also was intermittently agitated and having visual hallucinations. Due to lack of readily available medications in the outreach clinic, he was taken to the emergency department (ED) at the main hospital.\n\nAt the ED, he was given buprenorphine monoproduct (referred to herein as buprenorphine) 8 mg, which lowered COWS to 19 and provided approximately 45 minutes of relief. Screening labs in the ED consisted of a complete metabolic panel, complete blood count with differential, urinalysis, blood alcohol level, acetaminophen drug level, urine drug screen and electrocardiogram (ECG). This standard screening panel was largely within normal limits, with positive findings of presumptive positive for urine benzodiazepines, a minor elevation in neutrophils (count of 7,850 with normal range of 2,188 – 7,800/mm3), and ECG QTc interval of 463 millisecond (normal less than 430 millisecond). He received an additional 8 mg of buprenorphine 3 hours later which lowered COWS to 9. He subsequently was administered doses of 4 mg three times over the next 8 hours, which he did not feel provided as much relief as higher doses. This was increased to 16 mg doses of buprenorphine, which he tolerated without significant changes in vital signs, and provided symptomatic relief for 2 hours at a time. Medication administration record for buprenorphine can be seen in Table 1.\n\nOver the first 24 hours, he received 68 mg of buprenorphine and was routinely assessed by nursing. He became physically restless with increasing anxiety, however these improved with repeat dosing of buprenorphine. He was seen by a psychiatrist in the ED, who recommended admission to the medical-psychiatry unit for on-going management of the high-doses of buprenorphine. The patient’s goal was long-term abstinence from fentanyl use, and continued treatment with buprenorphine was felt to be the most direct method to accomplish that.\n\nOver the second 24-hour period from initial precipitation of withdrawal, he received an additional 80 mg of buprenorphine in 16 mg doses. His respiratory rate measured consistently between 16 and 18 breaths per minute (normal is typically considered 12 to 16) during this time. His daily buprenorphine dose requirement peaked on day two, with the goal for discharge of 16 mg twice daily, the maximum daily amount his insurance would cover. He tolerated this reduction over three days without exacerbation of symptoms or cravings.\n\nThe COWS was scheduled every 4 hours, and his scoring remained low with continued treatment. In addition to buprenorphine, he received gabapentin 300 mg thrice daily titrated to 600 mg thrice daily for physical discomfort and anxiety. His anxiety was worse earlier in his course, and he received lorazepam while on the unit, averaging a daily dose of 2 mg by mouth. He was discharged on 1 mg daily of clonazepam. He is now seen in the MAT outpatient clinic and has a severe opioid use disorder in early remission on buprenorphine-naloxone 24-6 mg once daily. His clonazepam use has been tapered, now taking less than 0.5 mg daily. He reports increased stability in his life, and relayed appreciation for the care and assistance he received in transitioning to MAT. He is followed by the senior author, who also saw him at both the walk-in clinic and main hospital. VK was the attending on the inpatient unit during his stay.\n\n\nDiscussion\n\nThis case builds upon existing literature by extending the upper extreme of known buprenorphine dosing for treatment of buprenorphine precipitated withdrawal. Previous case reports have used between 16 and 40 mg daily.7–11 In this case the range was doubled without significant adverse effect. A separate trial showed evidence for tolerability of one-time dosing of up to 96 mg of buprenorphine with goal of craving reduction.13 This however, to our knowledge, is the first report of a patient tolerating repeated days of high dose buprenorphine, averaging 63 mg per day over the first four days, with maximum 24-hour dose of 80 mg.\n\nThe US Food and & Drug Administration (FDA) has approved use of buprenorphine up to 32 mg daily. At this dose, the mu-opioid receptor nears saturation.14 Buprenorphine’s duration of action however is only 6–8 hours,15 which may partially explain the effectiveness of repeated high doses in this and other cases. Further research is needed in this area, such as incorporation of imaging techniques to quantify changes in receptor occupancy with re-dosing of buprenorphine.\n\nOpioid withdrawal is a strong negative reinforcer for patients, and the fear of withdrawal may prompt behavior changes intended to avoid such misery.16 Withdrawal specifically precipitated by buprenorphine is felt by some to be particularly uncomfortable, due to the abrupt displacement of opioid agonist by the high affinity buprenorphine molecule.3 It is reasonable that an experience with buprenorphine leading to withdrawal would limit one’s willingness to continue taking it or to utilize it for long-term maintenance therapy.17 However, with only three FDA approved medications for treatment of OUD and buprenorphine being the most readily accessible, attempts should be made to reassure the patient, optimize the initial exposure to buprenorphine even if use prompted a visit to the ED, and preserve or reestablish the patient’s confidence that this medication can provide benefit.\n\nAlong with administering additional buprenorphine, alternative recommendations for the treatment of acute buprenorphine precipitated withdrawal are discontinuation with symptomatic treatment (such as clonidine, ondansetron, loperamide and gabapentin) or utilization of full-opioid agonist such as methadone.7 In the moment, discontinuing the medication that caused acute withdrawal while providing symptomatic treatment may feel like the safest option, but this strategy misses an opportunity to initiate a potentially life-saving medication. Obtaining a start date for methadone maintenance therapy at an opioid treatment program out of the ED is also a large barrier to care and unrealistic for most parts of the United States. Ketamine has also been suggested, specifically in the ED.6 A noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, ketamine has the potential to suppress physiologic symptoms of withdrawal.18 In this patient, continued use of buprenorphine throughout his hospital stay helped the patient develop confidence in the medication and incorporate taking it into his daily routine.\n\nA commonly voiced concern for escalating doses of buprenorphine is respiratory depression. Buprenorphine’s effect on ventilation has been shown to plateau with a ceiling effect, unlike fentanyl, which can lead to apnea with increasing dose.19 While this case along with others7–11 have shown no issues with respiratory depression, it remains a practical concern. Scheduling the COWS can help mitigate this risk by utilizing symptom triggered dosing to inform daily requirement. Contamination of the drug supply, particularly with fentanyl, may also contribute to risk of respiratory depression. The occurrence of hallucinations in the case may be due to opioid withdrawal, which is a documented but less frequent symptom,20 or from a contaminant.\n\nTo conclude, this case provides additional evidence for the tolerability of high-dose buprenorphine and how the medication can be successfully tapered to a safe outpatient dose by discharge. There were no identifiable side effects to this total dosing of buprenorphine. As these doses exceeded the FDA approved limit, the use we describe is off label. This report builds upon existing literature and should provide additional confidence for providers in the emergency setting to opt for treatment of precipitated withdrawal with high-dose buprenorphine.\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\nReporting guidelines\n\nOpen Science Framework: CARE checklist for ‘Buprenorphine-precipitated fentanyl withdrawal treated with high-dose buprenorphine: a case report’. http://doi.org/10.17605/OSF.IO/9M468.21\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.", "appendix": "Acknowledgements\n\nThe authors extend their gratitude to the patient, who consented to publication to allow his case to help educate providers and improve care of future patients.\n\n\nReferences\n\nMorgan JR, Schackman BR, Leff JA, et al.: Injectable naltrexone, oral naltrexone, and buprenorphine utilization and discontinuation among individuals treated for opioid use disorder in a United States commercially insured population. J. Subst. Abus. Treat. 2018; 85: 90–96. Publisher Full Text\n\nLofwall MR, Walsh SL: A review of buprenorphine diversion and misuse: the current evidence base and experiences from around the world. J. Addict. Med. 8(5): 315–326. PubMed Abstract \| Publisher Full Text\n\nVarshneya NB, Thakrar AP, Hobelmann JG, et al.: Evidence of Buprenorphine-precipitated Withdrawal in Persons Who Use Fentanyl. J. Addict. Med. November 2021. Publish Ahead of Print. Publisher Full Text\n\nMcClain DA, Hug CC: Intravenous fentanyl kinetics. Clin. Pharmacol. Ther. 1980; 28(1): 106–114. Publisher Full Text\n\nArmenian P, Vo KT, Barr-Walker J, et al.: Fentanyl, fentanyl analogs and novel synthetic opioids: A comprehensive review. Neuropharmacology. 2018; 134(Pt A): 121–132. PubMed Abstract \| Publisher Full Text\n\nHailozian C, Luftig J, Liang A, et al.: Synergistic Effect of Ketamine and Buprenorphine Observed in the Treatment of Buprenorphine Precipitated Opioid Withdrawal in a Patient With Fentanyl Use. J. Addict. Med. November 2021. PubMed Abstract \| Publisher Full Text\n\nOakley B, Wilson H, Hayes V, et al.: Managing opioid withdrawal precipitated by buprenorphine with buprenorphine. Drug Alcohol Rev. 2021; 40(4): 567–571. Publisher Full Text\n\nDanilewitz M, McLean M: High-dose buprenorphine for treatment of high potency opioid use disorder. Drug Alcohol Rev. 2020; 39(2): 135–137. PubMed Abstract \| Publisher Full Text\n\nQuattlebaum THN, Kiyokawa M, Murata KA: A case of buprenorphine-precipitated withdrawal managed with high-dose buprenorphine. Fam. Pract. June 2021; 39: 292–294. Publisher Full Text\n\nHerring AA, Vosooghi AA, Luftig J, et al.: High-Dose Buprenorphine Induction in the Emergency Department for Treatment of Opioid Use Disorder. JAMA Netw. Open. 2021; 4(7): e2117128. PubMed Abstract \| Publisher Full Text\n\nJutras-Aswad D, Widlitz M, Scimeca MM: Treatment of buprenorphine precipitated withdrawal: A case report. Am. J. Addict. 2012; 21(5): 492–493. PubMed Abstract \| Publisher Full Text\n\nWesson DR, Ling W: The Clinical Opiate Withdrawal Scale (COWS). J. Psychoactive Drugs. 35(2): 253–259. Publisher Full Text\n\nAhmadi J, Jahromi MS, Ghahremani D, et al.: Single high-dose buprenorphine for opioid craving during withdrawal. Trials. 2018; 19(1): 675. PubMed Abstract \| Publisher Full Text\n\nGreenwald MK, Johanson C-E, Moody DE, et al.: Effects of buprenorphine maintenance dose on mu-opioid receptor availability, plasma concentrations, and antagonist blockade in heroin-dependent volunteers. Neuropsychopharmacology. 2003; 28(11): 2000–2009. PubMed Abstract \| Publisher Full Text\n\nFoster B, Twycross R, Mihalyo M, et al.: Buprenorphine. J. Pain Symptom Manag. 2013; 45(5): 939–949. Publisher Full Text\n\nKoob GF: Neurobiology of Opioid Addiction: Opponent Process, Hyperkatifeia, and Negative Reinforcement. Biol. Psychiatry. 2020; 87(1): 44–53. PubMed Abstract \| Publisher Full Text\n\nWhitley SD, Sohler NL, Kunins HV, et al.: Factors associated with complicated buprenorphine inductions. J. Subst. Abus. Treat. 2010; 39(1): 51–57. PubMed Abstract \| Publisher Full Text\n\nRasmussen K: The role of the locus coeruleus and N-methyl-D-aspartic acid (NMDA) and AMPA receptors in opiate withdrawal. Neuropsychopharmacology. 1995; 13(4): 295–300. PubMed Abstract \| Publisher Full Text\n\nDahan A: Opioid-induced respiratory effects: new data on buprenorphine. Palliat. Med. 2006; 20 Suppl 1: s3–s8. PubMed Abstract \| Publisher Full Text\n\nKumor KM, Grochow LB, Hausheer F: Unusual opioid withdrawal syndrome. A case-report. Lancet (London, England). 1987; 1(8535): 720–721. PubMed Abstract \| Publisher Full Text\n\nBormann NL: High dose buprenorphine case report. OSF. April 2022. Publisher Full Text" }	[ { "id": "184916", "date": "03 Aug 2023", "name": "Neil B. Varshneya", "expertise": [ "Reviewer Expertise Pharmacology (fentanyl)" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBormann et al. describe the successful management of buprenorphine-precipitated fentanyl withdrawal with high-dose buprenorphine and provide valuable insights into the management of opioid use disorder and the challenges associated with fentanyl exposure. This case report underscores the importance of individualized medication management in patients with opioid use disorder. The patient in this case had a history of opioid use disorder, including repeated fentanyl self-administration (with daily fentanyl use and dose escalation for 6 months), and developed severe withdrawal symptoms following non-medical, unsupervised self-treatment with buprenorphine/naloxone. The patient was subsequently managed in a medical setting with high doses of buprenorphine, which provided symptomatic relief in two hours intervals. The patient was eventually stabilized on a lower dose of buprenorphine and was discharged without exacerbation of symptoms or cravings. The report highlights the need for careful monitoring and tailored treatment plans for patients with opioid use disorder, especially in cases of fentanyl exposure. The report also underscores the importance of proper medication management for the acute onset of opioid withdrawal symptoms.\nOverall, this is a well-written an interesting manuscript and is timely given the role of fentanyl and its analogs in the ongoing opioid crisis. In general, the data are clearly presented. However, there are a few minor issues that deserve attention:\n\nThe authors write: “When buprenorphine is taken after recent use of a full-agonist opioid, buprenorphine displaces the lower affinity molecule, causing a precipitated withdrawal.” I think there are instances in which buprenorphine, with high enough doses, would still precipitate withdrawal by reducing receptor activation when competing with mu-opioid receptor ligands of higher affinity and efficacy. You should consider rewriting this sentence as follows: “Buprenorphine can precipitate withdrawal by reducing receptor activation through competitive displacement of higher efficacy mu-opioid receptor ligands”. Thank you for citing my work here.\n\nThe authors write: “Further research is needed in this area, such as incorporation of imaging techniques to quantify changes in receptor occupancy with re-dosing of buprenorphine.” You should consider including additional research strategies from the following article and cite it here:\nVolkow, N. D., & Blanco, C. (2023, Jun 1). Fentanyl and Other Opioid Use Disorders: Treatment and Research Needs. Am J Psychiatry, 180(6), 410-417. https://doi.org/10.1176/appi.ajp.20230273\n\nThe authors write: “However, with only three FDA approved medications for treatment of OUD and buprenorphine being the most readily accessible, attempts should be made to reassure the patient, optimize the initial exposure to buprenorphine even if use prompted a visit to the ED, and preserve or reestablish the patient’s confidence that this medication can provide benefit.” Please list the three FDA approved medications in parentheses following “only three FDA approved medications for treatment of OUD”.\n\nThe authors write: “A commonly voiced concern for escalating doses of buprenorphine is respiratory depression. Buprenorphine’s effect on ventilation has been shown to plateau with a ceiling effect, unlike fentanyl, which can lead to apnea with increasing dose.” You might consider citing the following study showing demonstrating the respiratory-depressant ceiling of buprenorphine vs fentanyl in animal models:\nVarshneya, N. B., Hassanien, S. H., Holt, M. C., Stevens, D. L., Layle, N. K., Bassman, J. R., Iula, D. M., & Beardsley, P. M. (2022, Jan). Respiratory depressant effects of fentanyl analogs are opioid receptor-mediated. Biochem Pharmacol, 195, 114805. https://doi.org/10.1016/j.bcp.2021.114805\n\nThe authors write: “This report builds upon existing literature and should provide additional confidence for providers in the emergency setting to opt for treatment of precipitated withdrawal with high-dose buprenorphine.” I wouldn’t say that N = 1 exactly inspires confidence, so I suggest using different language here to state that further research is needed.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "189490", "date": "03 Aug 2023", "name": "Ethan O Bryson", "expertise": [ "Reviewer Expertise Addiction", "Anesthesiology", "Electroconvulsive 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 case report describes the use of high-dose buprenorphine in the treatment of buprenorphine-precipitated fentanyl withdrawal. Given the considerable increase in fentanyl-adulterated opioids and non-opioids as well as an increase in persons with opioid use disorder specifically seeking out fentanyl this report is timely and appropriate.\nThere are some grammatical errors that need to be addressed prior to indexing, however, specifically:\n\"While use of ketamine has been suggested,6 conventional wisdom has been to utilize additional buprenorphine.7\" Please specify that this is for withdrawal treatment to make this a complete thought.\n\"As such, the University of Iowa Institutional Review Board has deemed it exempt.\" please specify what is exempt from what.\n\"A 21-year-old partnered, unemployed Caucasian male with no known past medical history, a psychiatric history of attention deficit hyperactivity disorder and unspecified anxiety, with a pertinent family history of an opioid use disorder in his brother and both his mother and maternal aunt having unspecified addiction to pills per his father, who lived in an apartment with a roommate however was being evicted due to late rent payments, presented with his father to our medication for addiction treatment (MAT) walk-in clinic located at a primary care outreach clinic for assistance. discontinuing daily fentanyl use.\"\nThis sentence is too long, and the reader may wonder who (the father? the aunt? or the patient) was late on their rent. Only include details like this if they are relevant. Also, please add \"with opioid use disorder\" in the patients' history or, if not previously diagnosed, something to that effect as this is more relevant than the other information which is included.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-487
https://f1000research.com/articles/12-962/v1	10 Aug 23	{ "type": "Research Article", "title": "Antioxidant and apoptotic activities of sitagliptin against hepatocellular carcinoma: An in vitro study", "authors": [ "Ruqaya Alameen", "Ahsan Bairam", "Maryam Al-Haddad", "Ahsan Bairam", "Maryam Al-Haddad" ], "abstract": "Background: Hepatocellular carcinoma (HCC) is the most common and aggressive type of liver cancer. Most chemotherapeutic medications nowadays imply oxidative stress leading to toxicity, which causes the necessity to find agents with better safety profiles against normal cells in addition to their anticancer activity. Sitagliptin has been shown to possess antioxidant as well as apoptotic properties by the specific suppression of dipeptidyl-peptidase 4, a glycoprotein produced in many tissues that have been thought to promote tumorigenesis and metastasis. Methods: Five groups of cell lines were included: Control (untreated HepG2 cells); cisplatin treatment HepG2 cells; sitagliptin treated HepG2 cells; combination of different concentrations of cisplatin plus sitagliptin (250 μg/mL) treated HepG2 cells, and finally, combination of different concentrations of sitagliptin plus cisplatin (25 μg/mL)-treated HepG2 cells. After an incubation period for 48 hours, the supernatants were collected to quantify the level malondialdehyde (MDA) and B-cell lymphoma-2 (BCL-2) by ELISA assay kits. Data were finally gathered and analyzed statistically. Results: Our findings indicated that sitagliptin significantly decreased the oxidative stress, particularly at high concentrations, through decreasing the MDA level. In addition, sitagliptin exhibited significant apoptotic activity against HepG2 cells through decreasing BCL-2 level. In combination with cisplatin, sitagliptin significantly potentiated the apoptotic effect and reduced the oxidative stress parameters. Conclusions: Sitagliptin showed apoptotic and antioxidant activity against HCC which may potentiate chemotherapeutic agents like cisplatin, in addition to reducing the oxidative stress against normal cells.", "keywords": [ "Apoptotic", "antioxidant", "cisplatin", "HepG2", "sitagliptin", "HCC", "control group", "level" ], "content": "Introduction\n\nHepatocellular carcinoma (HCC) is the most common tumor type of the liver and is highly aggressive. Approximately 90% of people with a cirrhosis diagnosis also have HCC. Causes of HCC have been identified, including viral infections, alcohol consumption, usage of aflatoxins, and genetics (Ioannou et al., 2007).\n\nAbout 75% of all cases of liver cancer occur due to HCC, making it the seventh most prevalent cancer and the second largest cause of mortality (Bray et al., 2018). There were predicted to be 713 new cases and 686 fatalities in Iraq in 2020 (IARC, 2020). According to GLOBOCAN 2020, liver cancer rates have recently increased sharply in many nations. Included in this set of nations are the countries of Iraq, Iran, Nepal, Afghanistan, Azerbaijan, and Qatar (Zhang et al., 2022). The world’s highest rates are found in Asia and Africa (McGlynn et al., 2021).\n\nTreatment for HCC typically consists of one or more of the following: surgical procedures, ablation, trans-arterial chemoembolization, and chemotherapies like cisplatin.\n\nCisplatin is activated upon entry into a cell. Cisplatin’s cytoplasmic chloride atoms are exchanged for water molecules. This hydrolyzed molecule is a highly powerful electrophile that may react with a wide variety of nucleophiles found in nucleic acids. Cisplatin inhibits tumor cell proliferation and induces apoptotic cell death by binding to the N7 on purine residues, thereby degrading DNA (Fraval et al., 1978).\n\nIn biology systems, anticancer drugs cause oxidative stress, which causes lipid peroxidation and the production of many electrophilic aldehydes. These oxidative stress byproducts may impede the effectiveness of anticancer treatments by slowing the progression of the cancer cells’ cell cycle (Conklin, 2004). Therefore, combining cisplatin with other drugs with antioxidant and apoptotic characteristics reduces concentration to avoid drug resistance and toxicity.\n\nRecently, significant anticancer effects of dipeptidyl peptidase-4 (DPP-4) inhibitors in cancer cells have been observed. In particular, the anti-diabetic medication sitagliptin was approved by the USA Federal Drug Agency (FDA) in 2006 as an inhibitor of DPP-4 (Aschner et al., 2006). Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones deactivated by the enzyme DPP-4. The hormone incretin increases insulin secretion from beta cells while decreasing glucagon secretion from beta cells (Scott, 2017). Since sitagliptin blocks DPP-4 activity, it also extends the effects of incretin hormones. Consequently, significant postprandial augmentation of insulin secretion and reduction of glucagon secretion is observed (Amritha et al., 2015).\n\nSitagliptin has been proven to improve the prognosis of several different types of cancer, including breast (Tseng, 2017), kidney (Kabel et al., 2018), and colon (Amritha et al., 2015), and modify the oxidative stress equilibrium during chemotherapy (Salama et al., 2022). Based on the above, our study attempted to assess the antioxidant and apoptotic activity of the sitagliptin in HepG2 liver cancer cell-line.\n\n\nMethods\n\nThe cells were cultured into a 96-well plate with Roswell Park-Memorial Institute-1640 (RPMI-1640) liquid medium that supplied by Gibco/UK and then incubated for 24 hours to promote the formation of a cellular monolayer (80% growth phase). Later, the old medium was discarded, and 200 μL of medium containing the test medicines was added (Meerloo et al., 2011). Five primary groups were utilized including the control group, which were: Control group (untreated HepG2 cells), cisplatin-treated HepG2 group, sitagliptin-treated HepG2 group, cisplatin plus sitagliptin-treated group which received a combination of different concentrations of cisplatin plus fixed concentration of sitagliptin (250 μg/mL), and the last group treated with a combination of different concentrations of sitagliptin plus fixed concentration of cisplatin (25 μg/mL). The plates were then kept in an incubator for 48 hours. The supernatant from each well was collected at the end of the exposure time and placed in a 1.5 mL sterile Eppendorf tube, and subsequently frozen at -20°C until measured by specific MDA and BCL-2 ELISA assay kits.\n\nBCL-2 concentration measurement\n\nHuman BCL-2 ELISA kit purchased from (BT LAB, Shanghai, China) was evaluated. The assay procedure was performed according to BT LAB protocol. Briefly, the standard stock solution was diluted by 1:2 to 1:16. A 50 μL of the standard was added to the standard well, 40 μL of the sample to the sample wells, and 10 μL of anti-Bcl2 antibody and 50 μL of streptavidin-HRP to the sample and standard wells. A sealant was used to close off the plate, allowing it to sit at 37°C for an hour. Repeatedly, the plate was washed in the wash buffer. Each wash should last between 30 seconds and 1 minute, and use 300 μm of wash buffer. 50 μL of substrate solution A, then repeated with substrate solution B was added to each well. After adding the substrate solutions, a color appears directly proportional to the concentration of Human BCl-2. Absorbance was taken at 450 nm when the process was stopped with an acidic stop solution. Then, a microplate reader that had a wavelength of 450 nm was used to measure each well’s absorbance value.\n\nMDA concentration measurement\n\nHuman MDA ELISA kit purchased from (BT LAB, Shanghai, China) was evaluated. The assay procedure was performed according to (BT LAB protocol). Briefly, the standard stock solution was diluted by 1:2 to 1:16. A 50 μL of the standard was added to the standard well, 40 μL of the sample to the sample wells, and 10 μL of anti-Bcl2 antibody and 50 μL of streptavidin-HRP to the sample and standard wells. A sealant was used to close off the plate, allowing it to sit at 37°C for an hour. Repeatedly, the plate was washed in the wash buffer. Each wash should last between 30 seconds and 1 minute, and use 300 μm of wash buffer. 50 μL of substrate solution A, then repeated with substrate solution B was added to each well. After adding the substrate solution, a color appears directly proportional to the concentration of human MDA. Absorbance was taken at 450 nm when the process was stopped with an acidic stop solution. Then, a microplate reader with a wavelength of 450 nm was used to measure each well’s absorbance value.\n\nThe data were gathered and analyzed using Microsoft Office Excel 2019 and IBM SPSS Statistics version 20. Means were compared using one-way ANOVA (Post hoc Tukey), and a p-value of 0.05 or lower was regarded to indicate a statistically significant difference.\n\n\nResults\n\nThe effect of cisplatin on BCL-2 level\n\nAfter incubating the HepG2 cells with cisplatin alone, a significant reduction in BCL-2 level (p≤0.001) was seen after exposure to all utilized concentrations of cisplatin in comparison with the control group, as shown in Figure 1.\n\n* p≤0.001.\n\nThe effect of sitagliptin on BCL-2 level\n\nAfter incubating HepG2 cells with sitagliptin, a significant decrement was noticed in BCL-2 level at all sitagliptin concentrations applied in comparison with the control group, as illustrated in Figure 2. Statistical calculations revealed that at a sitagliptin concentration of 31.25 μg/mL, BCL-2 level was decreased significantly at p≤0.01, while at higher concentrations, a reduction in BCL-2 level was highly significant (p≤0.001).\n\n p≤0.01, * p≤0.001.\n\nComparison between the effect of cisplatin alone versus combination of cisplatin plus sitagliptin on BCL-2 level\n\nComparing the apoptotic effect of cisplatin alone versus combination of cisplatin plus sitagliptin showed significantly lower BCL-2 level (p≤0.001) after exposure to combination than that obtained with cisplatin alone, as displayed in Figure 3.\n\n* p≤0.001.\n\nComparison between the effect of sitagliptin alone versus combination of sitagliptin plus cisplatin on BCL-2 level\n\nAs described in Figure 4, evaluating the apoptotic effect of sitagliptin alone versus combination of sitagliptin plus cisplatin demonstrated a highly significant decrement in BCL-2 level (p≤0.001) after exposure to combination versus sitagliptin alone.\n\n* p≤0.001.\n\nThe effect of cisplatin on MDA level\n\nThe results showed a significant increase in MDA level at p≤0.001 after exposure to all used concentrations of cisplatin in comparison with the control group, as shown in Figure 5.\n\n* p≤0.001.\n\nThe effect of sitagliptin on MDA level\n\nThe results showed a significant decrease in MDA level at p≤0.05 and p≤0.01 for the concentrations 500 μg/mL and 1000 μg/mL, respectively. However, the other concentrations of sitagliptin (31.25 μg/mL, 62.5 μg/mL, 125 μg/mL and 250 μg/mL) did not show significant changes in MDA level in comparison with the control group, as shown in Figure 6.\n\n* p≤0.05, p≤0.01.\n\nComparison between the effect of cisplatin alone versus combination of cisplatin plus sitagliptin on MDA level\n\nComparing the MDA level after exposing the HepG2 cells for cisplatin alone versus combination of cisplatin plus sitagliptin showed significantly lower MDA level after exposure to combination than that obtained with cisplatin alone, as demonstrated in Figure 7.\n\n p≤0.01, * p≤0.001.\n\nComparison between the effect of sitagliptin alone versus combination of sitagliptin plus cisplatin on MDA level\n\nFinally, exposing HepG2 cells to sitagliptin alone or combination of sitagliptin plus cisplatin further confirmed the antioxidant capability of sitagliptin. Indeed, as the concentration of sitagliptin increase, lower MDA levels were detected in both groups. In addition, significantly higher MDA levels (p≤0.001) were seen in cell treated with combination of sitagliptin plus cisplatin as expected, as shown in Figure 8.\n\n* p≤0.001.\n\n\nDiscussion\n\nIt is believed that inducing apoptosis is an effective method for treating cancer, given that the number of cancer cells depends on their development and death rate. On the other hand, cancer cells have developed various methods to resist death caused by apoptosis (Nachmias et al., 2004). One of these methods is the excessive production of anti-apoptotic proteins belonging to the BCl-2 family, leading to death resistance and reducing therapeutic efficacy (Letai, 2005). However, a potent cytotoxic response can be achieved by downregulating these proteins. The apoptotic pathway that begins inside mitochondria is strongly controlled by proteins of the BCl-2 family (Gross et al., 1999; Cheng et al., 2001).\n\nOur study indicated that the apoptotic effect of cisplatin on the HepG2 cell-line is explained by decreased BCl-2 level. Consistently with these findings, Xu et al. (2007) found that cisplatin and/or phenol decreased BCL-2 expression and increased Bax expression in human hepatoma cell lines, especially in the combination group. Moreover, compared to the control group, our finding showed a significant decrease in BCL-2 level in the HepG2-treated sitagliptin group, suggesting the apoptotic effect of sitagliptin on these corresponding cancer cells. Similarly, other study showed that sitagliptin suppressed cell growth and caused apoptosis in immortalized and primary glioblastoma cells (You et al., 2023). Based on the findings of Salama et al. (2022) in the Ehrlich solid carcinoma experimental model, sitagliptin showed anti-tumor activity through multiple mechanisms involving inhibition of cell proliferation and induction of tumor apoptosis. Interestingly, sitagliptin reduced apoptosis in normal cells as previously reported (Marques et al., 2014; Abo-Haded et al., 2017). Furthermore, the combination of sitagliptin and cisplatin demonstrated a synergistic apoptotic effect on cancer cells by significantly lowering BCL-2 levels. Yet, no previous data were reported concerning the effect of sitagliptin plus cisplatin combination on BCL-2 level.\n\nOverproduction of reactive oxygen species (ROS) leads to oxidative stress, which triggers mitochondrial malfunction and death (Liu et al., 2022). Tumor survival, growth, spread, and angiogenesis have all been linked to oxidative stress (Reuter et al., 2010). Multiple studies have indicated an increase in mitochondrial ROS as the mechanism of cisplatin toxicity (Martins et al., 2008). MDA, a marker of lipid peroxidation, was evaluated to determine the level of oxidative stress. The results of our study showed a significant increase in the MDA level of HepG2 cells when treated with cisplatin alone, which highlights the oxidative stress inducing capability of cisplatin. Studies on cisplatin cytotoxicity have indicated lowered hepatic GSH and elevated hepatic MDA, supporting the hypothesis that cisplatin cytotoxicity results from elevated oxidative stress in hepatic tissue (Aboraya et al., 2022). Moreover, liver tissue from cisplatin-injected animals showed increased oxidative stress (increased MDA and decreased GSH) (Taghizadeh et al., 2021). A recent study used A549 and DU145 cell lines found that exposure to cisplatin significantly increased intracellular ROS levels, and it was suggested that mitochondria are the origin of the ROS response induced by cisplatin in cancer cells (Marullo et al., 2013). On the other hand, the MDA level in the normal renal cells of mice treated with cisplatin elevated by an average of 1.5-fold, which triggered renal cell death due to ROS-dependent kidney damage (Soni et al., 2018).\n\nCombining cisplatin with other compounds containing antioxidant characteristics has been considered to reduce its toxicity. For example, the ability of sitagliptin to increase the activity of nuclear factor erythroid 2-related factor 2 (Nrf2), an inducer of various antioxidant enzymes, may be responsible for the antioxidant action (Li et al., 2019). Our result indicated that only higher concentrations (1000 μg/mL and 500 μg/mL) of sitagliptin showed a significant decrease in the MDA level of the HepG2 cell line in comparison to the control group. These findings are supported by several other studies which demonstrated the possible antioxidant impact of sitagliptin in various tissues and conditions, such as diabetic nephropathy (Marques et al., 2019) and Alzheimer’s disease (Li et al., 2019). In addition, the effect of a combination of sitagliptin plus cisplatin on MDA levels further demonstrated the oxidative stress-reducing ability of sitagliptin. This antioxidant effect clearly improved as the concentration of sitagliptin increased because the concentration of cisplatin was constant.\n\nThe antioxidant protective effect of sitagliptin may reduce cisplatin cytotoxicity in addition to potentiating its apoptotic activity. Importantly, the antioxidant beneficial effect of sitagliptin in combination with chemotherapeutic agents was also mentioned by Salama et al. (2022), who demonstrated that when sitagliptin combined with doxorubicin, the level of MDA was significantly decreased compared to both the control and doxorubicin-treated groups, suggesting partial protection from the oxidative stress against normal cells.\n\n\nConclusions\n\nSitagliptin could have antioxidant effect in higher concentrations. Moreover, sitagliptin revealed apoptotic activity against HepG2 cell-line based on BCL-2 measurement. The apoptotic effect of sitagliptin clearly enhanced the apoptotic activity of cisplatin, which may help in reducing the dose and subsequently the side effects of cisplatin in cancer treatment protocols.", "appendix": "Data availability\n\nFigshare: ELISA 2.xlsx, https://doi.org/10.6084/m9.figshare.23616819.v1 (Alameen et al., 2023).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAbo-Haded HM, Elkablawy MA, Al-Johani Z, et al.: Hepatoprotective effect of sitagliptin against methotrexate induced liver toxicity. PLoS One. 2017; 12(3): e0174295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAboraya DM, El Baz A, Risha EF, et al.: Hesperidin ameliorates cisplatin induced hepatotoxicity and attenuates oxidative damage, cell apoptosis, and inflammation in rats. Saudi J. Biol. Sci. 2022; 29(5): 3157–3166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlameen R, Ahsan FB, Al-Haddad M, et al.: ELISA 2.xlsx. Dataset. figshare. 2023. Publisher Full Text\n\nAmritha CA, Kumaravelu P, Chellathai DD: Evaluation of Anti Cancer Effects of DPP-4 Inhibitors in Colon Cancer- An Invitro Study. J. Clin. Diagn. Res. 2015; 9(12): FC14–FC16.\n\nAschner P, Kipnes MS, Lunceford JK, et al.: Effect of the dipeptidyl peptidase-4 inhibitor sitagliptin as monotherapy on glycemic control in patients with type 2 diabetes. Diabetes Care. 2006; 29(12): 2632–2637. 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\nCheng EH, Wei MC, Weiler S, et al.: BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell. 2001; 8(3): 705–711. PubMed Abstract \| Publisher Full Text\n\nConklin KA: Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integr. Cancer Ther. 2004; 3(4): 294–300. PubMed Abstract \| Publisher Full Text\n\nFraval HN, Rawlings CJ, Roberts JJ: Increased sensitivity of UV-repair-deficient human cells to DNA bound platinum products which unlike thymine dimers are not recognized by an endonuclease extracted from Micrococcus luteus. Mutat. Res. 1978; 51(1): 121–132. PubMed Abstract \| Publisher Full Text\n\nGross A, McDonnell JM, Korsmeyer SJ: BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 1999; 13(15): 1899–1911. Publisher Full Text\n\nIARC: (International Agency for Research on Cancer): Iraq fact sheets, Globocan.2020.\n\nIoannou GN, Splan MF, Weiss NS, et al.: Incidence and predictors of hepatocellular carcinoma in patients with cirrhosis. Clinical Gastroenterology and Hepatology: The Official Clinical Practice Journal of the American Gastroenterological Association. 2007; 5(8): 938–945.e4. Publisher Full Text\n\nKabel AM, Atef A, Estfanous RS: Ameliorative potential of sitagliptin and/or resveratrol on experimentally-induced clear cell renal cell carcinoma. Biomed. Pharmacother. 2018; 97: 667–674. PubMed Abstract \| Publisher Full Text\n\nLetai A: Pharmacological manipulation of Bcl-2 family members to control cell death. J. Clin. Invest. 2005; 115(10): 2648–2655. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Tian Q, Li Z, et al.: Activation of Nrf2 signaling by sitagliptin and quercetin combination against β-amyloid induced Alzheimer’s disease in rats. Drug Dev. Res. 2019; 80(6): 837–845. PubMed Abstract \| Publisher Full Text\n\nLiu Q, Wu D, Ma Y, et al.: Intracellular reactive oxygen species trigger mitochondrial dysfunction and apoptosis in cadmium telluride quantum dots-induced liver damage. NanoImpact. 2022; 25: 100392. PubMed Abstract \| Publisher Full Text\n\nMarques C, Gonçalves A, Pereira PMR, et al.: The dipeptidyl peptidase 4 inhibitor sitagliptin improves oxidative stress and ameliorates glomerular lesions in a rat model of type 1 diabetes. Life Sci. 2019; 234: 116738. PubMed Abstract \| Publisher Full Text\n\nMarques C, Mega C, Gonçalves A, et al.: Sitagliptin prevents inflammation and apoptotic cell death in the kidney of type 2 diabetic animals. Mediat. Inflamm. 2014; 2014: 1–15. Publisher Full Text\n\nMartins NM, Santos NA, Curti C, et al.: Cisplatin induces mitochondrial oxidative stress with resultant energetic metabolism impairment, membrane rigidification and apoptosis in rat liver. J. Appl. Toxicol. 2008; 28(3): 337–344. PubMed Abstract \| Publisher Full Text\n\nMarullo R, Werner E, Degtyareva N, et al.: Cisplatin induces a mitochondrial-ROS response that contributes to cytotoxicity depending on mitochondrial redox status and bioenergetic functions. PLoS One. 2013; 8(11): e81162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGlynn KA, Petrick JL, El-Serag HB: Epidemiology of Hepatocellular Carcinoma. Hepatology (Baltimore, Md.). 2021; 73 Suppl 1(Suppl 1): 4–13PubMed Abstract \| Publisher Full Text\n\nMeerloo JV, Kaspers GJ, Cloos J: Cell Sensitivity Assays: The MTT Assay. In: Cree IA, editor. Cancer cell culture: methods and protocols. (Methods in molecular biology, volume;731) London: Human Press; Second ed.2011; pp. 237–244.\n\nNachmias B, Ashhab Y, Ben-Yehuda D: The inhibitor of apoptosis protein family (IAPs): an emerging therapeutic target in cancer. Semin. Cancer Biol. 2004; 14(4): 231–243. PubMed Abstract \| Publisher Full Text\n\nReuter S, Gupta SC, Chaturvedi MM, et al.: Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 2010; 49(11): 1603–1616. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalama MM, Zaghloul RA, Khalil RM, et al.: Sitagliptin Potentiates the Anti-Neoplastic Activity of Doxorubicin in Experimentally-Induced Mammary Adenocarcinoma in Mice: Implication of Oxidative Stress, Inflammation, Angiogenesis, and Apoptosis. Sci. Pharm. 2022; 90(3): 42. Publisher Full Text\n\nScott LJ: Sitagliptin: A Review in Type 2 Diabetes. Drugs. 2017; 77(2): 209–224. Publisher Full Text\n\nSoni H, Kaminski D, Gangaraju R, et al.: Cisplatin-induced oxidative stress stimulates renal Fas ligand shedding. Ren. Fail. 2018; 40(1): 314–322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaghizadeh F, Hosseinimehr SJ, Zargari M, et al.: Alleviation of cisplatin-induced hepatotoxicity by gliclazide: Involvement of oxidative stress and caspase-3 activity. Pharmacol. Res. Perspect. 2021; 9(3): e00788. PubMed Abstract \| Publisher Full Text\n\nTseng CH: Sitagliptin May Reduce Breast Cancer Risk in Women With Type 2 Diabetes. Clin. Breast Cancer. 2017; 17(3): 211–218. PubMed Abstract \| Publisher Full Text\n\nXu SP, Sun GP, Shen YX, et al.: Synergistic effect of combining paeonol and cisplatin on apoptotic induction of human hepatoma cell lines. Acta Pharmacol. Sin. 2007; 28(6): 869–878. PubMed Abstract \| Publisher Full Text\n\nYou F, Li C, Zhang S, et al.: Sitagliptin inhibits the survival, stemness and autophagy of glioma cells, and enhances temozolomide cytotoxicity. Biomed. Pharmacother. 2023; 162: 114555. PubMed Abstract \| Publisher Full Text\n\nZhang CH, Cheng Y, Zhang S, et al.: Changing epidemiology of hepatocellular carcinoma in Asia. Liver Int. 2022; 42(9): 2029–2041. Publisher Full Text" }	[ { "id": "225056", "date": "30 Nov 2023", "name": "Ke Su", "expertise": [ "Reviewer Expertise Clinical and basic research on hepatocellular carcinoma" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFive groups of cell lines were included: Control (untreated HepG2 cells); cisplatin treatment HepG2 cells; sitagliptin treated HepG2 cells; combination of different concentrations of cisplatin plus sitagliptin (250 μg/mL) treated HepG2 cells, and finally, combination of different concentrations of sitagliptin plus cisplatin (25 μg/mL)-treated HepG2 cells. After an incubation period for 48 hours, the supernatants were collected to quantify the level malondialdehyde (MDA) and B-cell lymphoma-2 (BCL-2) by ELISA assay kits. Data were finally gathered and analyzed statistically.\n\nAlthough this is an interesting study, there are still some issues that must to be addressed. Prior to acceptance, the author must make corrections:\nThe author can supplement with animal experiments.\n\nDiscussion must be more focused on recent advances in the treatment of HCC, such as external beam radiation therapy (PMID:37818373), radiotherapy combined with sorafenib (PMID:36495367), PD-1 Inhibitors Combined with Antiangiogenic Therapy with TACE (37538403).\n\nMore biomarkers must be discussed by the author, such as ALP (PMID: 36685113), ALBI (37100384), and ALR Score (PMID: 37405321).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-962
https://f1000research.com/articles/12-961/v1	10 Aug 23	{ "type": "Research Article", "title": "Digital twin maturity levels: a theoretical framework for defining capabilities and goals in the life and environmental sciences", "authors": [ "Brett Metcalfe", "Hendriek C. Boshuizen", "Jandirk Bulens", "Jasper J. Koehorst", "Hendriek C. Boshuizen", "Jandirk Bulens", "Jasper J. Koehorst" ], "abstract": "Background: Digital twins (DT) are the coupling of a real-world physical asset to a virtual representation to provide insight and actionable knowledge. The benefits of DT are considered to include improvements in reproducibility, reliability of interventions, increased productivity, as well as increased time for innovation. For instance, a DT could be used to boost agricultural productivity whilst also meeting various targets (e.g., biodiversity, sustainability). Or a DT could be used to monitor a cell culture, predict interactions, and make subtle adjustments to maintain the environment allowing researchers to conduct other work. Yet in developing DT two fundamental questions emerge: ‘What will the DT capabilities be?’ (i.e., the range of features and possible actions) and ‘What will the DT do?’ (i.e., which capabilities will it utilise). Methods: Here we discuss a theoretical framework for DTs developed during Wageningen University & Research’s Investment Programme on DTs that aims to answer these questions. Focusing on the Life and Environmental Sciences to help developers and stakeholders to agree on the capabilities, purpose, and goal of a DT. As well as identifying iterative design stages that may help set interim development goals such as a minimum viable product. Results: This framework defines a DT as sitting at one of five maturity, or capability, levels associated with specific types of DT: a status, an informative, a predictive, an optimisation, and an autonomous twin. Conclusions: The aim of DTs is to make better, data-driven, decisions yet there can be a gulf between expectations of what a Digital Twin will do and the reality. The five maturity levels outlined here can be used to first identify and communicate about the type of Digital Twin required for a particular project prior to DT development. Bridging the gap between what project leads, developers, and stakeholders envision the end-product will be.", "keywords": [ "Digital Twins", "Maturity levels", "Capabilities", "Digital Twins for Life Sciences" ], "content": "Introduction\n\nWith the advent of concepts such as the Internet of Things (IoT) computers can now more readily ‘sense’ their environment in (near) real-time using a variety of embedded sensors all networked together.1,2 Many of these IoT devices are everyday items,1 others are small increasing their portability and ensuring that where there is the possibility of a connection to the internet, or another network, observations can be made.2 One application of such interconnected sensors is the acquisition of data from an asset that maybe too big (e.g., a city), remote (e.g., streetlights), or inaccessible (e.g., inside an air conditioning system) to study in detail with traditional data collection methods. Significantly, applying these interconnected sensors to a problem allows for the collection or generation of a continuous stream of data independent of analysts allowing for higher resolution, continuous datasets to be acquired. These developments are considered to have significantly furthered our progress to the next, fourth, iteration in our industrial development.3,4\n\nThe fourth industrial revolution (Industry 4.0 or 4IR) should see the transition from the digital revolution4 - represented by the switch from mechanical and analogue systems to automated digital electronics - to industrial processes dominated by cyber-physical systems, smart sensors, and cognitive computing. Conceptually the fourth industrial revolution is built upon expanding the digital technology revolution5,6 that occurred during the third industrial revolution through increased interaction and integration7,8 between the virtual and physical (i.e., “real world”) environments. Such interaction and integration can take many forms (Figure 1), such as (digital) models that simulate processes that occur in the physical world; digital shadows or digital threads that collect, store, and provide information about a physical object; or digital generators, devices that construct physical objects (e.g., 3D printers).\n\nVariants of digital technologies characterized by how information flows between the real (blue) and virtual (green) worlds. Examples of these varieties include 3D printers and barcodes here referred to as a digital generator and digital shadow respectively. Solid black lines denote an automated flow of information, whereas grey lines denote a ‘manual’ flow of information. Based upon Refs. 7, 8.\n\nAssociated with the coupling of the virtual and physical into cyber-physical systems is the potential formation of digital twins9–11 with commercial12–15 and academic16,17 interest piqued regarding the possible application of digital twins to various complex processes and systems (e.g., Ref. 18). Digital twins can be differentiated from other ‘digital’ variants (Figure 1) by the connection between the virtual and physical realms having a continuous automated two-way flow of information16 (Figure 2). Through this communication, a virtual representation simulates how a process or physical asset operates throughout its lifecycle19–21 from which decisions can be made that control the real-world process. Such simulations can be simple, for example keeping up to date readings of sensors that register the status of the physical asset. Or the virtual simulations can be anticipatory, using prior knowledge, time-series, and/or physics-based models to determine the likely future outcome based upon the current status of the physical asset. The feasibility of creating digital twins has existed for a time in the manufacturing and engineering sectors, where the intricacies of components and their complex processes are known in detail or can be intuited from blueprints.\n\n(A) Conceptual components of a digital twin, having a (1) physical asset; (2) virtual representation; (3) two-way communication; and (4) feedback or actionable knowledge. (B) In reality the various components maybe more complex. The terms used in (A) to describe the various stages, i.e., ’Collects’, ’Processes’, ’Discovers’, and ’Manages’, are from Ref. 12.\n\nFrom such information it is possible to place sensors that can generate data of sufficient quality and resolution to allow for the development of Digital twins that can monitor, control, and optimize processes. Or remove geographic and temporal constraints, so that operators can be trained remotely or decision maker can view historic, current and future states of the physical asset whilst not needing to be ‘on location’ or proximal to it.22,23\n\nBeyond the factory floor the digital revolution is taking place in industries related to the Life and Environmental Sciences.17 Such as Utilities,18 the Agricultural sector,24–28 Horticulture,29 Apiculture,30 Smart farming,24 Animal farming,31 as well as the Healthcare sector,32,33 and the Pharmaceutical Industries34 amongst others. Other twins aim to provide information on the built-environment (e.g., planning, environmental impact, air quality, etc.), advice on sustainable water management [1], or to help protect and restore (European) biodiversity [2]. More ambitious digital twins aim to provide a digital twin of the Oceans [3] and our Planet [4] to assist in the green transition.35\n\nSuch an interest in the possible application of Digital Twins to the Life and Environmental sciences has been ignited by the emergence of new techniques and new technology that can circumvent the complexity of real-world phenomenon. New techniques include those that can derive patterns and connections without the explicit requirement to know the process behind such patterns (e.g., machine learning) or those that enable a model to be kept synchronised with observations (e.g., data assimilation). Alongside the development36 of smaller more portable interconnected technology and devices,37 loaded with sensors38–40 that can acquire sufficient data to feed into the virtual half of the twin.\n\nYet given their integration into various aspects of our daily lives’ digital twins for the life and environmental sciences may have the potential to cause considerable socioeconomic upheaval. Such upheaval may occur through changing our interaction with ourselves with the development of personalised healthcare38,40–42 and (personal) environmental monitoring. Or via adapting environments (e.g., greenhouses) and organisms to meet our needs e.g., optimisation of agricultural practices. However, for all the possible benefits and opportunities of utilising digital twins for life science applications there are many barriers to success such as the ‘blueprints’ of a natural process or phenomenon being only partially known, too complex, technically unfeasible or unethical to be obtained. There is also the potential for such socioeconomic upheaval to exacerbate rather than curtail existing problems, such as those relating to social injustices or healthcare43–46 or ensuring continuation of poor practices (e.g., in agriculture47).\n\n\nAims and objectives\n\nGiven the socioeconomic opportunities and consequences applying of digital twins to life and environmental science questions17 it is important to consider a framework that is communicable to a range of stakeholders that not only conveys what a digital twin is but what it should do. Ill-defined definitions can lead to ambiguous projects goals; be counterproductive for product development; create too high expectations; and limit stakeholder participation. Defining the capabilities of the digital twin and which of these capabilities will be used in each situation will be part of the design process for developing digital twins. Clarification over what a digital twin will do, ultimately defines the test of whether it is feasible, prudent or necessary to develop a digital twin for a given problem.16,48,49 Likewise, a well-defined definition can help develop short-term goals to make a project achievable. Either by providing sufficient scope to a project to prevent scope-creep or through curtailing over ambition by allowing a project to be divided into a series of distinct iterations. Iterative design allows for a period of reflectance between design stages to assess the projects continued needs, requirements, direction, and goals. Furthermore, additional benefits includes having different iterations ready to apply to different problems. Here such a framework is outlined. First by discussing the definition of a digital twin (Section ‘Definition and maturity’), then the conceptual framework behind maturity levels that represent the functionality and capabilities of a digital twin (Sections ‘Definition and maturity’ and ‘Maturity levels’). Before outlining the maturity framework (Section ‘Maturity levels’) and its associated Digital Twin types (Section ‘Types of digital twin’) that is applicable to a range of applications in the life and environmental sciences.\n\n\nDefinition and maturity\n\nThere is no explicit formal definition of a digital twin leading to some divergence between various (scientific) disciplines (e.g., Ref. 50) about what a digital twin actually is. To further exacerbate the problem studies have shown that in some papers what is referred to as a digital twin has not always been clarified51 leading others to attempt to catalogue the variation in descriptions3,7,16,24,52–54 including identifying what can or cannot be considered a digital twin.7,8\n\nFrom a practical perspective here, it is considered that a definition of a digital twin is valid if it meets the following criteria of being a virtual representation, replica, or simulation of an actual, or possible, physical asset in (near) continuous two-way communication with its counterpart to play an active role in planning, decision, and control processes through providing feedback. The digital twin will be primarily data-driven and capable of, or have the capacity for, predicting future trends or developments over a period of time. Here feedback need not be an automated process but simply incorporation of the results of the twin in a decision-making process. There are four commonalities between the various types of digital twins: (1) a physical asset; (2) a virtual representation; (3) two-way communication; and (4) some form of feedback (Figure 2). In the following, let us expand upon these various components.\n\nPhysical asset\n\nA digital twin must be comprised of a physical asset in physical space and a virtual replica, copy, analogy, or representation in virtual space. There is no exclusivity with respect to twinning, so the same physical asset can be twinned by multiple digital twins.55 Likewise, the same sensors could be used for multiple twins. In some descriptions of digital twins whether they are a part or the whole asset has been differentiated by adding the following terms ‘component’, ‘asset’, ‘system or unit’, or ‘process’ to the term digital twins.15,56 Where a ‘component’ is part of an asset and a ‘system’, ‘unit’, and ‘process’ refer to a greater and greater collection of assets. Therefore, in digital twinning the physical asset need not be the ‘whole’ instead it can be a singular component of a larger system (e.g., a motor in a factory; the digestive tract of an animal; etc) or alternatively a group of individuals (e.g., a fleet of delivery vehicles; a herd of cows; a field; etc.) as long as it can conceivably or does physically exist. Similarly, digital twins that describe a prototype of a physical asset, explicitly one that can exist, have been referred to as a Digital Twin Prototype to differentiate it from those with a real-world physical asset. In the life sciences the digital twin prototype can possibly occur in the biotech industry representing organisms in a natural state or as genetically modified organisms, where the cells may be modelled first prior to their creation.\n\nVirtual representation\n\nIn the virtual environment, the physical asset is twinned by a virtual representation that should in theory provide the user with relevant information they could obtain were they to go out and physically inspect the asset in the real-world. Assuming that measurements, time, money, and distance are not limiting factors. Of course, in reality the twin utilises a subset of measurement components that either directly, or indirectly (e.g., proxies) capture various states of the measured system. The virtual representation can be broadly split into a visualisation component (what the user sees) and a processing component (how the data is transformed, used, or incorporated into models). Some definitions of digital twins implicitly state that in order to have maximum fidelity with the real-world asset by necessity the visualisation component of virtual representation must be a 3D model of the asset. Here, however, the requirement of the digital twin to have a 3D visualisation is omitted as ultimately the visualisation should depend upon the (i) type of data; (ii) the goal of the visualisation; and (iii) the needs, wants, and requirements of the user. Hence, the visualisation components can range from simple iconography, a dashboard, graphs and graphics, a 3D model, augmented reality or virtual reality. Whereas the processing components can range from just the data, a data-model, physics-based model, artificial intelligence, or some combination of all of these. Ultimately the form the virtual representation takes will be dependent upon the knowledge of the targeted user group and the purpose of the digital twin.\n\nTwo-way communication\n\nBetween the virtual-physical and physical-virtual spaces two-way communication should occur (Figure 1). Of course, the nature and method of this two-way communication will vary. Communication between the physical and virtual worlds may be via networked sensors, data upload, or manual data-entry (e.g., patient results). Communication between the virtual and physical worlds could be in the form a simple visualisation (e.g., icon forms or composite interactive dashboard) or more complex feedbacks and actions (e.g., automation).\n\nLikewise, the frequency of communication will also vary being dependent upon the use case. For instance, high frequency communication may be required where rapid changes occur but just as equally when trying to pinpoint subtle variations. It may be prudent to first identify the physical assets’ own unique trends and rhythms prior to defining the communication interval to ensure the right frequency of communication occurs. For example, if one was aiming to twin a plant then consideration must be made regarding the frequency of communication so as to sufficiently capture the various internal and external natural trends and rhythms (e.g., diurnal or seasonal cycles). In other instances, there may be variables or components of the Digital Twin that are updated only infrequently. For example, when representing a complete city, the volume of traffic might be updated continuously whereas road layout itself is semi-rigid - being fixed but requiring updates at a vastly different time scale and often performed manually by cartographers.\n\nActionable knowledge\n\nFinally, there must be an initiation of a decision-making process and where required feedback; with the Digital Twin providing the source of actionable knowledge to aid in the decision-making process. Actionable knowledge refers to the coupling of Science and Management.57 That is using directed action to bring about intended consequences (i.e., management) whose actions and consequences are defined by the generalisations provided by empirical research to describe or explain a particular phenomenon (i.e., science). Actionable knowledge will be knowledge that can provide either the specific conditions or actions necessary to ensure or lead to a particular set of intended consequences beyond the settings in which it was first created, and which can be applied to both individuals as well as to groups.\n\nDigital twin\n\nIn summary, a digital twin is composed of a virtual representation or replica of an actual, or possible, physical asset with these two counterparts in continuous communication so as to play an active role in planning, decision, and control processes. Following coupling between the physical and virtual twins both twins are collectively referred to as a Digital Twin Instance.11,16\n\nAs can be seen from the proceeding section a definition does not inform the reader of what a digital twin will do for a given problem.58 A concept, or definition, of Digital Twins will only provide a ‘foundation level’ knowledge, i.e., knowing the definition of a digital twin serves to differentiate it from other types of digital technology (Figure 1) and what components are key (Figure 2A) but not much else. This is because for each criterion (i.e., virtual asset; two-way communication; feedback; and a decision-making process) there can be a range of potential outcomes (Figure 2B) giving an equally wide variety of possible digital twin end products, or more aptly ‘phenotypes’ (i.e., observable characteristics or traits). How the various components of a Digital Twin are expressed and formulated must be discussed during the development phase. The resultant Digital Twin will depend upon the end goal of the digital twin, as well as any legal and technological practicalities, and the various decisions made in the design phase.\n\nSolving the question of what a particularly digital twin will do and its capabilities necessitates communication between developers, stakeholders, and users. Yet, communication between stakeholders requires a common frame of reference. Hence, in order to solve the issue of what a digital twin will do, several initiatives have placed digital twins onto a hierarchical framework (Figure 3), describing various types, capabilities, and the evolution59–63 or developmental stage26 of digital twins.64 These have been referred to as digital twin maturity levels,59,65,66 a series of digital twin integration levels,7 a continuum,60 a spectrum,65,67 archetypes,68 and/or a classification62 or taxonomy of digital twins,69 all of which seek to define the functionality of the various digital twin levels (Figure 3).\n\nComparison of the various levels of maturity59–63,65,70,71 placed upon a relative scale where the sizes of the boxes reflects the coarseness of the granularity. Blue boxes represent levels that do not represent a Digital Twin. Whereas, dashed unfilled boxes represent levels not specifically defined by the schema. References with a * are from online (business) blogs, ** from technical reports, and others from academic journals.\n\nPrevious work\n\nAs such investigating previous work is a useful starting point for defining a digital twin maturity model that is applicable to Life Science problems. However, several previous studies have expanded their models to incorporate other digital instances (Figure 1). For example, Kritzinger et al.’s7 digital twin integration levels put digital twins on a continuum involving digital models and digital shadows (Figure 1). For brevity only previous work differentiating between different types of digital twins will be discussed here. Where the different types of digital twin reflect differences in functionality of the digital twin. As opposed to the type of asset being twinned (i.e., Refs.15, 56).\n\nA quick review of this subset of the literature (Figure 3) suggests that the types of digital twin are generally differentiated by the level of insight that the twin provides about a problem or toward a goal.71 A digital twin having better insight can be seen as having increased capabilities alongside an increased capacity to perform the actions itself. It is our opinion that Digital twins can be broadly categorised into those that provide ‘Data about’, ‘Knowledge of’, ‘Suggestions for’ and ‘Control of’ a physical component, asset, process, or phenomenon. These can be split into data collection, analysis, prediction, optimisation, and autonomy (Figure 3). Differences between previous studies largely reflects the ‘coarseness’ of the various digital twin sub-divisions. For instance, ‘twins with business models’ reflecting a stage where the digital twin focuses upon optimising a process59 can itself be sub-divided into various substages: an initial substage coupling the twin to business operations, a subsequent substage recommending various decisions,60 and finally making those decisions autonomously.63 Yet despite differences between the assorted maturity models there is a general consensus that to reach the foundational level of a digital twin requires an availability of data71 and/or a connection between the physical and virtual worlds.65,70 The lowest level of a digital twin is generally reserved for a twin that simply relays the collected data, or real-time status, and the highest level when the twin has gained autonomy to control.72 With many prior maturity models referencing automobile automation or the SAE’s levels of driving automation for self-driving cars73 as a comparable frame of reference (e.g., Ref. 59). Between these two end members (data collection and automation) there are levels devoted to analysis (including modelling, simulating), prediction, and optimising (Figure 3).\n\nConceptual framework\n\nFor the framework outlined here we continue the use of the term maturity59,65,66 given how it denotes both a natural, and sequential, progression toward more complex states (e.g., see the data-driven manufacturing maturity model74). Although it is entirely feasible to also refer to it in terms of functionality or capability. Likewise, our maturity levels are also based on the presumption that the ultimate goal of digital twins is to streamline decision making processes through the automation of sequential tasks. Whether or not the ultimate goal of an autonomous twin is obtainable is another matter (see, Discussion) and there is some debate regarding digital twins and cyber-physical systems (e.g., Ref. 53). In our framework the various maturity levels can be conceptualised as a series of progressive stages74 that add functionality towards the goal of automation.73 At low digital twin maturity levels, the user or operator of the digital twin carries out more of the data capture, decision making, process and control operations. As a digital twin matures processes once controlled by human operators become subsumed by the virtual twin, until the process is fully automated. These levels focus upon providing insight upon a particular aspect of the object under study, with aspects that address the what’s, the how’s, and the why’s. What questions relate to conveying the basic research elements, (raw) data and information (‘what is the time’, ‘what value does X have’). How questions relate to understanding the relations of these basic research elements, e.g., through models, from which predictions can be inferred. Why questions relate to identifying cause and effect as well as whether something can or should be done. Why can be sub-divided into ‘appreciation of why’ and ‘why’. This sub-division is based upon the idea that there are instances where the reasoning or knowledge behind why something occurs may not be known (’why’) but an answer or explanation can still be formulated (‘appreciation of why’).\n\nTo explain the conceptualisation let us consider a simple example, that of traffic in a city. If we want to know the amount of traffic on the road, we can begin by asking a simple question, “What is the number of vehicles on the road?”. Such a question relates solely to the measurement of vehicles on the road (i.e., “6 vehicles” or “5 cars, 1 bus, …”) and it is up to the receiver of that data to provide the context. By changing our question to “What is the current volume of traffic?” context and meaning are added to the answer (i.e., “low”, “high”). But it might be prudent to know “How will traffic change over the course of a day, week, or month?” and therefore, understanding the variables necessary for prediction and modelling becomes necessary. Knowing the amount of traffic can be used to provide insight for a practical use, for example selecting the optimum route: “What routes are there from location to A to destination B?”. Finally, such knowledge could be used to address “What is the best route and best time to leave from A to get to B?”\n\nThe basis of this conceptualisation is the knowledge pyramid (or knowledge hierarchy; information hierarchy; knowledge management pyramid),75–77 a common feature in information and knowledge management.77 The knowledge pyramid is represented by a hierarchy commonly presented as a pyramid proceeding from the base of data to information, knowledge, understanding, and ultimately the pinnacle representing wisdom. To transition from data to information requires understanding relations; information to knowledge requires understanding patterns; and knowledge to wisdom requires understanding principles. The stages of the pyramid hierarchy can be mapped to the conceptualisation’s different questions, the data and information relating to ‘knowing what’, knowledge to ‘knowing how’, and wisdom to ‘knowing why’. Understanding is linked to ‘appreciating why’, the why might not be fully understood but cause and effect might be. The mapping between the hierarchy and these concepts has been argued by Zeleny76 who stated that to “manage wisely implies knowing why to do something; to manage effectively implies knowing what to do; to manage efficiently implies knowing how to do it (and to ‘muddle through’ implies nothing and having ‘lots of data’ around)” [5].\n\n\nMaturity levels\n\nFrom low to high the five maturity levels outlined here deal with ‘knowing what’ (data); ‘knowing what’ (information); ‘knowing how’ (knowledge); ‘appreciating why’ (understanding split into: understanding and insight); and ‘knowing why’ (wisdom).\n\nAt the data level the digital twin focuses upon data collection and generation of values that represent the various properties of the physical asset, presenting the user with its current state. The information level focuses on structuring data into a coherent and organised format, adding context, and ensuring that current and historic data are presented to the user. Knowledge involves the processing of this structured data using previous experience, either through identifying underlying pattern and trends or physics-based process models. Understanding is the exploration of ‘if/then’ situations through scenario testing and interactions, understanding is further divided into insight which focuses upon optimisation and developing new relationships. Finally, the wisdom level is reached, at this point the digital twin is fully autonomous providing feedback and control through understanding.\n\nThe maturity levels can be visualised as a matrix (Figure 4) in which the columns represent the various types of digital twin and the rows the concept, these can be represented as questions and aspects of the knowledge pyramid. The cells of the matrix are divided into tasks performed by a digital twin (green boxes in Figure 4C) and not by a digital twin (blue boxes in Figure 4C).\n\nThe maturity index is composed of five levels that address the level category of a twin. The maturity levels can be visualised as a matrix in which the columns represent the various types of digital twin, and the rows are the concepts which can be represented as either (A) questions or (B) aspects of the knowledge pyramid. (C) The cells of the matrix are divided into tasks performed by (Green) and not by (Blue) the digital twin.\n\n\nTypes of digital twin\n\nThese maturity levels (ML) can be represented by distinct types of digital twin (e.g., Ref. 59) which can be referred to from low to high as: status, informative, predictive, optimisation, and autonomous digital twins. The five types (Figure 4) of digital twin are defined as follows:\n\n• Digital twin maturity level 0 (ML0): No Twin. Measurements are disparate or non-existent, sensors are either not present or if they are, they lack any (inter) connected networking. The process maybe described by a process or physics model however, these are not linked to the real-world data and/or apply fixed boundary conditions.\n\n• Digital twin maturity level 1 (ML1): Status. Focus upon real-time data capture, collection and visualization of information.\n\n• Digital twin maturity level 2 (ML2): Informative. Real-time data capture and collection and incorporation of historical data or normative data (benchmarks) and visualization of the information.\n\n• Digital twin maturity level 3 (ML3): Predictive. Real-time data capture and collection with historic data coupled to a machine learning or physical process-based model.\n\n• Digital twin maturity level 4 (ML4): Optimisation. Real-time data capture and collection with historic data coupled to a machine learning or physical process-based model. The Virtual Twin allows for scenarios of different ‘if then/what if’ scenarios to be explored. Helping the operator to deduce the outcome of particular decisions.\n\n• Digital twin maturity level 5 (ML5): Autonomous. Real-time data capture and collection with historic data coupled to a machine learning or physical process-based model. The Virtual Twin deduces the optimal scenario and enacts controls that lead to it occurring.\n\n\nDiscussion\n\nDigital twins are being developed in a variety of disciplines both commercially and academically.17,18,24–34 Therefore, these projects may encompass a wide range of use cases and stakeholders who may not even have a cursory knowledge of what a digital twin is, let alone an understanding of what it will do. Likewise, there won’t be a ‘one-size-fits-all’ approach to developing digital twins (as outlined in the ‘Digital twin definition’ and ‘Going beyond a definition’ sections above). Therefore, as previously noted, development of a digital twin boils down to two fundamental methodological questions. The first question relates to the full range of the digital twins features and possible actions, i.e., ‘What will the digital twins capabilities be?’. The second aims to address which of the capabilities identified in the initial question the digital twin will it utilise, i.e. ‘What will the digital twin do?’. The answer to these questions can be considerably broad, especially depending upon the intricacies and peculiarities of the use case (e.g. note the range of expressions in Ref. 17). Critically it is important that in the process of describing what a digital twin will do that we end up with less of a wish list and more of a list of requirements for development. The levels of digital twin maturity proposed here aims to serve as a means for effective communication between project leaders, project owners, project participants, developers, and stakeholders about the intention of the project. Allowing developers to outline to interested parties what the goal and purpose of the digital twin will be. The maturity model should also act as a springboard in defining what is feasible with current resources; and, to know where a digital twin is in its development phase.\n\nAs a simple example, let us consider how the maturity levels could be applied to twinning something simple such as a house plant (Figure 2). The first step in digital twin development will be to perform an initial scoping exercise that will preliminary determine what type of digital twin (Figure 4) suits the intended objective.59 This exercise should identify the asset under study; an intended goal; prior knowledge; measurable and required variables; define processes; determine the expected values of key variables; define the intended users and stakeholders; and consider if and what output and (anticipated) feedback the digital twin will produce. For a house plant (our object) there are several parameters that can be directly affected by an individual (our stakeholder) to promote growth (our goal). These include providing sufficient light, water, and temperature, all of which can be measured to a greater or less degree of accuracy by commercially available sensors. Once the initial scoping exercise has been performed, a digital twin developer should use the answers obtained to sketch out the rough divisions between the various maturity levels. Using coarser divisions such as data collection and communication (ML 1-2); analytics, models, and predictive tools (ML3); and ‘use cases’ (ML 4-5) can help make preliminary design decisions. Following which, it is prudent to next identify previous or preliminary work that already fulfils or can feed into one or more of the maturity levels. Applying the maturity model follows an iterative design methodology with each level, or iteration, being a cycle of development, testing, analysis, and reflection. The result of each learning cycle can then be used in subsequent design iterations, either another iteration at the same maturity level or progression onward.\n\nAssuming that the process starts from the initial maturity level (ML1), design choices should address what sensors will be used and how these sensors will communicate their data to the virtual twin. For example, questions to be addressed include ‘What is the nature of the communication?’ e.g., WiFi, LoRa, (etc.), ‘How frequent will these sensors communicate?’ or ‘Will data arrive as a stream or be sent in batches?’ amongst others. This level adds low level knowledge of a process, such as insight into range and distribution of obtainable values and/or how such variables change spatially or temporally. Such knowledge may help develop understanding of a process. Returning to our example, a digital twin for a house plant at ML1 provides real-time data capture of the amount of light, the soil moisture, and the room temperature. Values can be collected by an IoT sensor and presented on a display next to the plant, or as values on a simple web server.\n\nHaving obtained and communicated data in ML1, at the next maturity level, ML2, such data will be given context. Here ‘context’ is conveying known information about the subject matter, either from prior knowledge or previous data. Defining which context will be added requires communicating with experts, users, or a combination of both to identify key parameters. For instance, for our house plant, context could be added through a knowledge rule that translates values, such as the light into ‘low’, the soil moisture into ‘dry’, or the temperature into ‘cold’. One could colour code the values on the display or add simple iconography to indicate (un)suitable conditions for the plant, allowing someone to identify problems and come up with their own actions. Such as moving the plant to a sunnier or shadier position, to water it, or to adjust the thermostat.\n\nProgressing to the next level requires the twin to shift from solely focusing on past and present values to predicting future values. Hence, ML3, involves adding predictive capacity. For our example that could be knowing the future value of soil moisture and when it will drop below a specific level that requires watering. Generally, predictions will be made via a model, though the exact type (e.g., mechanistic, process, physics based, machine learning, etc.) will depend the nature of the digital twin (e.g., the use case; availability of data; etc.). Though developers can utilise the previous levels to identify required inputs and outputs as well as those variables that are of importance, those that vary, and which could be conceivably parameterised. For instance, for our house plant the digital twin could incorporate a mechanistic model that calculates the length of day and the angle of the Sun by giving the latitude, date, and time which can then be used to constrain the upper or lower light levels depending on weather. Whereas, predicting soil moisture and/or temperature could make use of a time series machine learning algorithm to make predictions without direct inference of the cause of the variation. However, crucially the model(s) must allow for the updating of various states. Techniques such as data assimilation can be used to combine new observations and model forecasts to update the prediction.\n\nThe final levels (ML4 and ML5) transition from relaying knowledge (ML1 to ML3) to creating and using new knowledge, with these levels being split between optimisation (ML4) and automation (ML5). How either of these levels will be ultimately implemented by a developer will be highly dependent upon the needs and requirements of users, experts, and stakeholders as well as taking into account various practicalities. The distinction between ML3 and ML4 is the addition of potential corrective actions and their outcomes. In our house plant example, optimisation could focus on resolving questions such as: ‘When is the optimal moment to water to maintain constant soil moisture levels?’ or’Is it better to water when the room is cooler?’. How those questions and their answers are presented will again depend on project specifics, for example they could be embedded in a user interface or developers could allow a user to test them out in a sandbox environment. Once it is possible to optimise a process, then a natural progression is the possible addition of some degree of automation, i.e., bypassing the decision maker to quickly and consistently provide a corrective action. Therefore, at the final maturity level, ML5, the process is autonomous. For our example, as the moisture levels drops the digital twin would react by watering the plant to sustain an optimal amount of soil moisture.\n\nWhilst our example discusses the full spectrum of maturity levels projects developing digital twins should be aiming for and settling upon the right maturity level for a particular use case rather than reaching for higher maturity levels. This might seem far from aspirational however, the goal should be to have the best fit of digital twin to a particular goal or problem. Nor is it the intention of the maturity levels presented here is to produce a ranking system. As it is more than likely that specific digital twin use cases will have an upper maturity level limit that reflects the legal and ethical considerations as well as technological feasibility. For instance, for a given problem the highest level of a Digital Twin is dependent upon consensus on the expected values a set of variables should have and the certainty of relevant knowledge (Figure 5 based upon concepts in Refs. 59, 78–83). In situations where there is a lack of both knowledge and certainty of the values, a Status Digital Twin could provide the necessary foundational knowledge. Where there is consensus on the values and high degree of certainty regarding our knowledge of a process, then an Autonomous Digital Twin might be more prudent.\n\nWhich twin is for you? Comparing certainty of knowledge verses consensus of values. Deciding on which digital twin is required for a given problem also necessitates a critical look upon known knowns, known unknowns and unknown unknowns. In this example the initial digital twin is dependent upon two variables, whether consensus is reached on the values a variable should have allowing for knowledge rules to be produced and the certainty of relevant knowledge. Based upon ideas and concepts relating to digital twins and uncertainty communication described elsewhere.59,78–83\n\nOur house plant example shows that the capabilities of a digital twin will however, be constrained by the availability and feasibility of technology. Automating the watering of a house plant is far more feasible than automating the movement of a plant to track light. Therefore, in this case the more automated levels will focus upon solutions that are feasible. However, it should be noted that the maturity levels outlined here do not directly refer to the technological readiness of Digital Twins or their constituent components. Technological readiness being defined by the technical progress or maturity of a technology under evaluation, a technology is ranked along a technology readiness level (TRL) scaled from research is beginning (= 1) to full scale deployment (= 9). For digital twins the feasibility of obtaining each of the maturity levels defined here will depend on the individual project, its intended goal, and thus the technology associated with it. In some instances, it is conceivable that the technology associated with higher maturity levels (e.g., machine learning models, control systems, etc.) are more ready than the technology associated with lower maturity levels (e.g., automated data capture and ingestion). Where automated or networked sensors do not yet exist for example, lower maturity levels that rely on data collection may not be obtainable but higher maturity levels such as modelling (using other or legacy datasets) may not be so constrained. Therefore, a mapping between maturity and technological readiness remains highly dependent on the topic of the digital twin. Although, it is the authors opinion that the maturity levels outlined here and the TRL are complimentary. The TRL will reflect the state of technical innovation7,54,84 and key enabling technologies.85,86 By using the maturity index to first identify and communicate about the type of digital twin required for a project and its goals, developers can use the TRL at the next phase of project development to identify technological feasibility and/or bottlenecks.\n\n\nConclusion\n\nAs part of Industry 4.0, physical processes, assets, or phenomenon will be in the future linked to a virtual representation referred to as digital twins. Digital twins are physical assets linked by a two-way flow of information to a virtual representation that is intended to provide actionable knowledge. These characteristics can be expressed in different ways making knowing the type, format, and capabilities of a digital twin somewhat ambiguous. To ensure fruitful dialogue between stakeholders and digital twin developers here we presented a model of the capabilities along a path to a fully autonomous digital twin. This maturity index is composed of five levels that address ‘knowing what’ (data); ‘knowing what’ (information); ‘knowing how’ (knowledge); ‘appreciating why’ (understanding and insight); and ‘knowing why’ (wisdom). Each represented by a distinct type of digital twin: status; informative; predictive; optimisation; autonomous. These types of digital twin share commonalities and the natural progression between the levels can be used to define the human and computer tasks. At low maturity levels, such as the status digital twin, the operator will perform many of the complex tasks. Proceeding from this low maturity level denoted by low complexity and few autonomous attributes the types of digital twin become increasingly complex and automated.\n\n\nAuthor contributions\n\nAll authors contributed to the conceptualisation and writing.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nIBM: What is the internet of things? IBM. Reference Source\n\nHoldowsky J, Mahto M, Raynor M, et al.: Inside the Internet of Things (IoT) A primer on the technologies building the IoT. A primer on the technologies building the IoT. Deloitte University Press; 2015.\n\nNegri E, Fumagalli L, Macchi M: A Review of the Roles of Digital Twin in CPS-based Production Systems. Procedia Manuf. 2017; 11: 939–948. 2351-9789. Publisher Full Text\n\nMaynard AD: Navigating the fourth industrial revolution. Nature Nanotech. 2015; 10(12): 1005–1006. Number: 12 Publisher: Nature Publishing Group. 1748-3395. 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January 2021; 46(1): 191–196. 1740-634X. Number: 1 Publisher: Nature Publishing Group. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFagherazzi G: Deep Digital Phenotyping and Digital Twins for Precision Health: Time to Dig Deeper. J. Med. Internet Res. 2020; 22(3): e16770. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLydon-Staley DM, Barnett I, Satterthwaite TD, et al.: Digital phenotyping for psychiatry: accommodating data and theory with network science methodologies. Curr. Opin. Biomed. Eng. March 2019; 9: 8–13. 2468-4511. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilbanks JT, Topol EJ: Stop the privatization of health data. Nature News. 2016; 535(7612): 345–348. Section: Comment. Publisher Full Text\n\nZarocostas J: How to fight an infodemic. Lancet. 2020; 395(10225): 676. 0140-6736, 1474-547X. Publisher: Elsevier. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEditorial: More accountability for big-data algorithms. Nature News. September 2016; 537(7621): 449. Section: Editorial. PubMed Abstract \| Publisher Full Text\n\nBruynseels K, Santoni de Sio F, van den Hoven J : Digital Twins in Health Care: Ethical Implications of an Emerging Engineering Paradigm. Front. Genet. 2018; 9: 1664-8021. Publisher: Frontiers. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBronson K: A digital “revolution” in agriculture?: Critically viewing digital innovations through a regenerative food systems lens.Duncan J, Carolan M, Wiskerke JSC, editors. Routledge Handbook of Sustainable and Regenerative Food Systems. Routledge; August 2020; pp. 336–349. 978-0-429-46682-3. Publication Title: Routledge Handbook of Sustainable and Regenerative Food Systems. Publisher Full Text\n\nMuse ED, Topol EJ: Digital orthodoxy of human data collection. Lancet. 2019; 394(10198): 556. 0140-6736, 1474-547X. Publisher: Elsevier. Publisher Full Text\n\nHuckvale K, Venkatesh S, Christensen H: Toward clinical digital phenotyping: a timely opportunity to consider purpose, quality, and safety. NPJ Digit. Med. September 2019; 2(1): 11–88. 2398-6352. Number: 1 Publisher: Nature Publishing Group. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorkamani A, Andersen KG, Steinhubl SR, et al.: High-definition medicine. Cell. 2017; 170(5): 828–843. 0092-8674, 1097-4172. Publisher: Elsevier. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarricelli BR, Casiraghi E, Fogli D: A Survey on Digital Twin: Definitions, Characteristics, Applications, and Design Implications. IEEE Access. 2019; 7: 167653–167671. 2169-3536. Publisher Full Text\n\nWright L, Davidson S: How to tell the difference between a model and a digital twin. Adv. Model. Simul. Eng. Sci. March 2020; 7(1): 13. 2213-7467. Publisher Full Text\n\nBoyes H, Watson T: Digital twins: An analysis framework and open issues. Comput. Ind. 2022; 143: 103763. 0166-3615. Publisher Full Text\n\nLiu M, Fang S, Dong H, Xu C: Review of digital twin about concepts, technologies, and industrial applications. J. Manuf. Syst. 2021; 58: 346–361. 0278-6125. Publisher Full Text\n\nWalsh J, Tolle D: ASSESS-Theme-Positioning-Paper-Digital-Twins-1.10.pdf. ASSESS Theme Positioning Paper Version 1-10, ASSESS Initiative.2020. Reference Source\n\nManca L, Grugni R, Mirzazadeh R: Digital twin: A digital copy of reality that enables you to simulate and find answers in a risk free and secure environment. Technical report, Engineering (eng.it).2019. Reference Source\n\nArgyris C: Actionable Knowledge: Design Causality in the Service of Consequential Theory. J. Appl. Behav. Sci. December 1996; 32(4): 390–406. 0021-8863. Publisher: SAGE Publications Inc. Publisher Full Text\n\nBroo DG, Schooling J: Digital twins in infrastructure: definitions, current practices, challenges and strategies. Int. J. Constr. Manag. August 2021; 23: pp. 1254–1263. 1562-3599, 2331-2327. Publisher Full Text\n\nKalwani S: The Evolution of Digital Twins for Asset Operators.2017. Reference Source\n\nSocha D: The Digital Twin Maturity Continuum.September 2018. Reference Source\n\nMadni A, Madni C, Lucero S: Leveraging Digital Twin Technology in Model-Based Systems Engineering. Systems. January 2019; 7(1): 7. 2079-8954. Publisher Full Text\n\nWilking F, Schleich B, Wartzack S: Digital twins - definitions, classes and business scenarios for different industry sectors. Proceedings of the Design Society. 2021; 1: 1293–1302. 2732-527X. Publisher Full Text\n\nRao AS: Evolution of digital twins open data science conference.May 2022. Reference Source\n\nSingh M, Fuenmayor E, Hinchy EP, et al.: Digital twin: Origin to future. Applied System Innovation. 2021; 4(2): 36. 2571-5577. Number: 2 Publisher: Multidisciplinary Digital Publishing Institute. Publisher Full Text\n\nEvans S, Savian C, Burns A, et al.: Digital twins for the built environment: An introduction to the opportunities, benefits, challenges and risks. Technical report. Hertfordshire, UK: The Institution of Engineering and Technology (IET); 2019. Reference Source\n\nKorovin G: Digital twins in the industry: Maturity, functions, effects.Kumar V, Leng J, Akberdina V, et al., editors. Digital Transformation in Industry, Lecture Notes in Information Systems and Organisation. Cham: Springer International Publishing; 2022; pp. 1–12. 978-3-030-94617-3. Publisher Full Text\n\nBolton A, Butler L, Dabson I, et al.: The Gemini Principles: Guiding values for the national digital twin and information management framework. Technical Report CDBB_REP_006. Cambridge, UK: Centre for Digital Built Britain; 2018. Publisher Full Text\n\nvan der Valk H , Haße H, Möller F, et al.: Archetypes of digital twins. Bus. Inf. Syst. Eng. 2022; 64(3): 375–391. 1867-0202. Publisher Full Text\n\nPai M, Parle D, Smith C: A Digital Twin Taxonomy for Industry Implementations. Technical report. Bangalore, India: Wipro Limited; December 2019. Reference Source\n\nGriffith C, Truelove M: Framework for spatially enabled digital twins: Information paper. Information Paper, CSIRO Data 61, Australia.2021.\n\nKharche V: Digital Twin Maturity.January 2022. Reference Source\n\nAssad Neto A, Ribeiro da Silva E, Deschamps F, et al.: Digital twins in manufacturing: An assessment of key features. Procedia CIRP. 2021; 97: 178–183. 2212-8271. Publisher Full Text\n\nOn-Road Automated Driving (ORAD) committee: Taxonomy and definitions for terms related to driving automation systems for on-road motor vehicles. Ground Vehicle Standard J3016_202104. USA: SAE International; 2021. Publisher Full Text\n\nWeber C, Königsberger J, Kassner L, et al.: M2ddm – a maturity model for data-driven manufacturing. Procedia CIRP. 2017; 63: 173–178. 2212-8271. Publisher Full Text\n\nAckoff RL: From Data to Wisdom Presidential Address to ISGSR, June 1988. J. Appl. Syst. Anal. 1989; 16: 3–9.\n\nZeleny M: Management support systems: Towards integrated knowledge management. Hum. Syst. Manag. 1987; 7(1): 59–70. 0167-2533. Publisher: IOS Press. Publisher Full Text\n\nRowley J: The wisdom hierarchy: representations of the DIKW hierarchy. J. Inf. Sci. April 2007; 33(2): 163–180. 0165-5515. Publisher: SAGE Publications Ltd. Publisher Full Text\n\nBergsma P: Besluiten onder onzekerheid Hoe gebruiken bestuurders van de Nederlandse waterschappen statistische informatie over onzekerheid bij beslissingen over wateroverlast? Master’s thesis, Wageningen University and Research, Wageningen, The Netherlands.2016. Reference Source\n\nWehrens R, ten Broeke G , Stigter H, et al.: Digital Twins for decision making; The Uncertain half of the Twin. Wageningen Environmental Research WENR 3071. Wageningen, The Netherlands: Wageningen Environmental Research; 2021. Publisher Full Text\n\nBoogerd A: Van Droge Kennis naar Natte NatuurDe Interactie tussen Natuurwetenschap en Beleid over Verdroging. Utrecht: Uitgeverij Eburon; 2005. 9059720768/9789059720763. Reference Source\n\nTurnhout E, Hisschemöller M, Eijsackers H: Science in Wadden Sea policy: from accommodation to advocacy. Environ. Sci. Pol. 2008; 11(3): 227–239. 1462-9011. Publisher Full Text\n\nPetersen AC: Simulating Nature: A philosophical study of computer-simulation uncertainties and their role in climate science and policy advice. 1st ed.Apeldoorn – Antwerpen: VU University Amsterdam PhD Proefschrift. Het Spinhuis; 2006. 978 90 5589 280 8. Reference Source\n\nHuijs S: Het vertellen van verhalen: een politieke manier van omgaan met onzekerheid.van Asselt M , Petersen A, editors. Niet bang voor onzekerheid. Vol. 1. . Den Haag, NL: Boom Juridische uitgevers; 2003; pp. 105–118. 978-90-5931-287-6.\n\nReimsbach-Kounatze C: Benefits and challenges of digitalising production.OECD, editor. The Next Production Revolution: Implications for Governments and Business. Paris: OECD Publishing; 2017; pp. 73–117. 978-92-64-27115-9. Reference Source\n\nCommission of the European Communities: Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee and the Committee of the Regions: “Preparing for our future: developing a common strategy for key enabling technologies in the EU” - {COM(2009) 512}. Commission Staff Working Document SEC(2009)1257. Brussels: Commission of the European Communities; 2009.\n\nButter M, Fischer N, Gijsbers G, et al.: Horizon 2020: Key enabling technologies (kets), booster for european leadership in the manufacturing sector. Study for the ITRE Committee IP/A/ITRE/2013-01 PE 536.282, European Parliament. Directorate-General for Internal Policies. Brussels: Policy Department A: Economic and Scientific Policy; 2014.\n\n\nFootnotes\n\n1 https://www.wur.nl/nl/onderzoek-resultaten/onderzoeksinstituten/environmental-research/show-wenr/digital-twin-voor-waterbeheer.htm\n\n2 https://biodt.eu/news/biodt-digital-twin-prototype-help-protect-and-restore-biodiversity\n\n3 https://www.ocean-twin.eu/\n\n4 https://digital-strategy.ec.europa.eu/en/policies/destination-earth\n\n5 Note that since publication of Zeleney’s 76 work the levels in the knowledge pyramid that relate to ‘what’ and ‘how’ have switched. At the time data was how and knowledge was what, now data is what and knowledge is how. Here the quote is used in full as per the original unmodified." }	[ { "id": "198894", "date": "08 Sep 2023", "name": "Koen Smit", "expertise": [ "Reviewer Expertise Digital Twin Technology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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,\nThank you for proposing your contribution titled “Digital twin maturity levels: a theoretical framework for defining capabilities and goals in the life and environmental sciences” for publication in F1000 Research.\nThe paper aims to answer two questions about which capabilities are tied to a DT and how the capabilities will be utilized by the DT. The paper proposes a theoretical view on five maturity stages for DT capabilities. The specific focus of the contribution is interesting as the life sciences domain can potentially benefit greatly from digital twin technology and the body of knowledge should converge on practices to help structure the design of this complex technology to be used successfully in practice. Based on my analysis, I have the following considerations for the authors to improve the contribution:\n\nAbstract/background should focus more on life sciences as the claim that DT capabilities must be explored is not true for, e.g., the manufacturing industry, where DT’s are used for quite some time now. Consider pointing out the relevance for the specific domain better.\n\nI have some difficulty with the concept of ‘new sensors’ on top of sensing (in blue) in figure 2 on page 5. While I like the notion that on a detailed level, digital twins are more complex due to all concepts mentioned in figure 2b, the orange part should be explained better or left out. Adding to this, consider adding to figure 2 that, depending on the level of required capabilities, complexity increases as it now presents a situation in which all DTs should be enhanced with AI/ML, which is not always required.\n\nOne of the main statements in the contribution about why such a framework should be researched is as follows: “Ill-defined definitions can lead to ambiguous projects goals; be counterproductive\n\nfor product development; create too high expectations; and limit stakeholder participation.” Please consider adding strong sources for this as there are many contributions that focus on these issues and readers should be informed about this knowledge.\n\nAnother point that could be added is that big bang approaches in developing DT solutions often fail because of complexity and therefore fail to meet expectations. Iterative development is mentioned in the contribution, so I agree that this helps in expectation management and has a higher chance of successful implementation in practice. Again, there are, in the context of DT, but surely outside of that as well (general IS-research), a lot of contributions that point towards this universal problem and helps make the point of avoiding big bang approaches. Consider discussing this, accompanied with appropriate sources, in the aims and objectives-section of the paper.\n\nThe contribution presents multiple other works from the DT body of knowledge that focus on (the quantification of) maturity levels and models. How this maturity model adds to the already available knowledge should be discussed more extensively in the beginning of the contribution.\n\nI find figure 3 very comprehensive and must complement the authors on the informative value it brings to the paper, though consider enlarging the figure a bit to improve readability.\n\nAre the results of the subset of contributions in the DT body of knowledge complete? The authors do not explain how these papers were selected. For example, why is the following reference, which is very relevant in the context of this study, not included (disclaimer: I am not a (co)-author of this specific reference):\nUhlenkamp, J. F., Hauge, J. B., Broda, E., Lütjen, M., Freitag, M., & Thoben, K. D. (2022). Digital twins: A maturity model for their classification and evaluation. IEEE Access, 10, 69605-69635.\n\nMy previous comment also points out a larger problem with the contribution, regarding its methodological quality. While the contribution is theoretically scoped, I would expect details about how the selection/filtering in the DT body of knowledge is performed to derive the presented levels and characteristics in figure 3, 4 and 5. This is a major flaw in the contribution that should be improved upon.\n\nFigure 3s most left column is categorized as ‘no twin’. While I know that multiple papers refer to this lowest stage of maturity in a similar manner, consider delving into what it can be referred to as, because the current label is hardly informative for practitioners. The textual information presented in this column does not really help in informing the reader. Later, the focus of the discussion seems to point towards more disconnected sensors and models of both the physical and virtual world, so disconnected or non-linked. If that is the largest caveat for this level, why not refer to it as such in the model?\n\nThe example presented in page 12 contains a paragraph in which a ‘knowledge rule’ is used to explain logic that is required to translate data into contextual information or categories. The term knowledge rule is new to me, as far as I am aware such logic is often referred to as business rules. Consider replacing the concept of knowledge rules or explain more specifically what they are (if different from business rules/logic).\n\nThe authors state the their opinion is that the Technological Readiness Level is important to take into account in the context of utilizing DT technology and establishing maturity levels. The levels presented in the contribution are complementary to the TRL, but how that translates to the model exactly is kept in the dark, unfortunately. Consider illustrating or describing how they complement each other as it is stated that TRL should be taken into account. At least provide an example, e.g., in conjunction with the example provided earlier in the sections.\n\nEven if the contribution is theoretically scoped, I would expect the authors to discuss possible limitations of the study and its results in a dedicated discussion section. Unfortunately, such a section is not present. Also, future research directions in this (and other) domain(s) are important notions to base further research upon. However, these are not presented at the end of the paper. Consider adding these sections as they contribute towards proper academic discussion and follow-up.\nMinor spelling/grammar:\nPage 3: real-world phenomenon should be: phenomena (plural)\n\nPlease consider carefully checking the entire paper for punctuation accuracy as, for example, I found some comma’s to be incorrectly used in sentences.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "208820", "date": "16 Oct 2023", "name": "Mezzour Ghita", "expertise": [ "Reviewer Expertise Digital Twins", "Smart manufacturing", "Advanced Driver Assistance Systems" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper proposes a five maturity level framework that discuss all the aspects for proper integration of digital twins especially for life science application. The authors draw an initial macroscopic view what it can be defined as a digital twin and gives all relevant description of the element constituting the context for digital twins. The presented work describes several contributions that can help in the practical development and implementation of DT in the field. However some modifications are needed to enhance technical aspects of the work.\nThe authors in their introduction claims that the framework is intended to help integration of DT for life and environmental sciences this cannot be apprehended through out the paper except for the use case that gives just an overview of how the framework can be exploited. It would be relevant to highlight what industry 4.0 technologies are already in use in the field and where they fail including for digital twins. Authors can get inspiration from some patent developed in the agricultural field and also from work on industry 5.0 that deals same obejctives. The aim would be to define a proper field related problematic that value the proposed framework in this context. Some interesting papers are referred by the authors and patents can give some additional value to this.\n\"There is no explicit formal definition of a digital twin\" actually there is work in progress for the definition of a digital twin standard by ISO including formal definition of the context. Its true that in the field of interest discussed here it was not defined yet formally but in general the norm would serve as a reference in this case.\nThe framework presents maturity levels through a knowledge pyramid perspective but at the end no explicit process is defined of how this levels can be apprehend its left to the user to decide what questions, measures, criteria can help to achieve this evaluation. The use case give some insights but no explicit procedure is defined which at the end makes it difficult for technical evaluation of the framework and its feasibility. To deal with this the authors proposed to include TRL but again no explicit description is provided for replication. TRL can also help to include some additional relevant points that hinder efficient and large scale inclusion of DT in some fields as instance communication, costs and all project specific management aspects. This point can be highlighted as a following research direction of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "208826", "date": "16 Oct 2023", "name": "Pratik Maheshwari", "expertise": [ "Reviewer Expertise Supply chain Digital Twin" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 an intriguing topic, \"Digital twin maturity levels: a theoretical framework for defining capabilities and goals in the life and environmental sciences.\" However, the paper's implications and contributions to the present state of the art are somewhat limited. Several observations and recommendations are outlined below:\nThe introduction provides a clear overview of the research area. However, it would be beneficial to more explicitly emphasize the significance and relevance of digital twin maturity levels in the context of life and environmental sciences.\n\nThe literature review is comprehensive and well-organized. It effectively establishes the groundwork for the proposed framework. However, to enhance the paper's currency, consider incorporating more recent research findings, especially those published within the last two years.\n\nThe research objectives and framework are well-defined and aligned with the research problem. Nevertheless, it would be helpful to provide clarification on how this framework differentiates itself from existing ones, where applicable.\n\nWhile the paper briefly outlines the methodology employed in developing the framework, enhancing its credibility could be achieved by providing more comprehensive details regarding research methods and data sources.\n\nThe proposed digital twin maturity levels framework is presented clearly and logically. Nonetheless, supplementing it with practical examples or case studies that illustrate its application within the life and environmental sciences would enhance its comprehensibility.\n\nThe discussion section offers valuable insights into the implications of different maturity levels. However, a more detailed analysis of potential challenges and limitations in implementing these levels would provide greater depth to the paper.\n\nIt is advisable to highlight practical applications and real-world scenarios where this framework can prove beneficial in the life and environmental sciences. Additionally, discussing how it can address specific challenges within these fields would be insightful.\n\nThe paper's writing requires improvement; there are areas where language could be clarified to enhance understanding. Consider rephrasing sentences or providing additional explanations as needed.\n\nThe figures and tables are indeed helpful, but ensuring they are labeled and explained with utmost clarity is essential. If feasible, consider incorporating additional visual aids to further enhance reader comprehension.\n\nThe conclusion effectively summarizes the paper's key findings and contributions. However, it would be advantageous to suggest potential directions for future research or extensions of the framework, thereby providing a more forward-looking perspective.\n\nIt is recommended to meticulously review the references to ensure completeness and accuracy in citations. Additionally, verify that they conform to the chosen citation style consistently.\n\nLastly, emphasize the paper's contribution to the digital twin field and its potential impact on the life and environmental sciences. A concise summary of these aspects can enhance the paper's overall significance\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-961
https://f1000research.com/articles/12-960/v1	10 Aug 23	{ "type": "Clinical Practice Article", "title": "Repair of ischial tuberosity pressure ulcer with a perigluteal muscle flap", "authors": [ "Guan Lifeng", "Fazal Rahim", "Muhammad Awais Khan", "Guo Tao", "Fang Chao", "Wang Xiaoni", "Muhammad Saad", "Fazal Rahim", "Muhammad Awais Khan", "Guo Tao", "Fang Chao", "Wang Xiaoni", "Muhammad Saad" ], "abstract": "Background: Pressure ulcers are caused by long-term pressure on tissues. Hip pressure ulcers are the most common type. Pressure ulcers in the buttocks are classified into three types: sacrococcygeal, ischial tuberosity, and femoral tuberosity.\nMethods: We used perigluteal muscle tissue flaps to treat nine cases of grade IV pressure ulcers from January 2016 to August 2018. A retrospective study was conducted on all patients who had an ischial tuberosity pressure ulcer repaired with a perigluteal muscle flap. The gluteus maximus myocutaneous flap was combined with the gluteal fasciocutaneous flap, the biceps femoris myocutaneous flap was combined with the gluteal fasciocutaneous flap, the gracilis myocutaneous flap, the biceps femoris myocutaneous flap, and the semitendinosus and semimembranosus myocutaneous flap. Wounds of different sizes and depths were encountered, the details of which are discussed in treatment methods. Data from patients, including age, gender, cause of injury, wound dimensions, the timing of flap coverage, and complications, were analyzed.\nResults: The appearance of the skin flap was complete after muscular tissue flap repair, with no visible scar hyperplasia. However, after six to 12 months of follow-up, the flap was soft and left a small linear scar.\nConclusions: In the repair of an ischial tuberosity pressure ulcer, a peri gluteal muscle flap provides both aesthetic and functional results. Before using muscle tissue flap transplantation, it is recommended to perform multiple debridement + VSD to improve the wound foundation.", "keywords": [ "Surgical Flaps", "Ischial tuberosity", "Pressure Ulcers" ], "content": "Introduction\n\nLong-term pressure on body tissues, blood circulation problems, necrosis, and skin and soft tissue defects cause pressure ulcers and are most commonly found at bony protrusions. Hip pressure ulcers have the highest clinical incidence. Pressure ulcers in the buttocks are mostly concentrated in three areas: sacrococcygeal, ischial tuberosity, and femoral tuberosity. Because of the presence of a bursa in the Ischial tuberosity, when a pressure sore occurs, the entire bursa is easily affected, resulting in a sore that is larger than the skin necrosis area “the entire bursa is easily affected when a pressure sore occurs”.1 Furthermore, it is close to the perineum, which is easily infected and aggravates the sore, making clinical repair difficult. From January 2016 to August 2018, we successfully used the perigluteal muscle tissue flap to treat 11 cases of grade IV pressure ulcers at the ischial tuberosity.\n\n\nCase series\n\nA spinal cord paralysis caused by a traumatic vertebral fracture affected nine males and two females aged 19 to 52. One patient was bilaterally paralysed, while the others were unilateral paralysed. The average time between recurrent non-healing skin ulcers was 14 months, and all patients had grade IV pressure ulcers of the ischial tuberosity. A wound secretion culture was performed on all patients.\n\nAfter admission, patients underwent routine examinations and received symptomatic treatment for comorbidities such as anemia, hypoproteinemia, and electrolyte disturbances. We changed the dressing on sore wounds two to three times per day, and the first debridement was performed five to seven days after admission. During the operation, as much necrotic tissue, chronic inflammatory tissue, and infected tissue as possible were removed.\n\nSome of the sores even reached the ischium—bone, causing periosteum and cortical bone degeneration and necrosis. After thorough debridement of the wound, the negative pressure material was placed on it and covered with a film. five to seven days after the operation, the negative pressure material was opened and one or more debridements were performed depending on the condition of the wound base, and VSD treatment was continued. After each debridement, the negative pressure was maintained at -11.97-9.31 kPa, and continuous saline irrigation was administered. The ulcer base was significantly improved after one to three debridement + VSD treatment in 9 patients, and fresh granulation tissue was formed at the bottom of the incubation cavity. The key to surgery is to pack and seal the deep submerged hole because the sore is a deep cavity wound with a narrow opening and a wide base. We opted for a musculocutaneous flap in conjunction with a facial flap or a simple musculocutaneous flap. Four cases were repaired with a gluteus maximus myocutaneous flap combined with a gluteal fasciocutaneous flap, two cases with a biceps femoris myocutaneous flap combined with a gluteal fasciocutaneous flap, two cases with a gracilis myocutaneous flap, one case with a biceps femoris myocutaneous flap, and two cases with a semitendinosus and semimembranosus myocutaneous flap combined with the gluteal fasciocutaneous flap (the size of the flap was 11 cm × 9 cm ~ 28 cm × 18 cm). All 9 patients were sutured directly in the donor area. Anti-infective treatment after surgery was increased and sensitive antibiotics to patients who had precise bacterial culture results were given, for symptomatic treatment.\n\nExample 1\n\nThe patient was a 52-year-old Chinese male with paraplegia for eight years and a pressure sore at the left ischial tuberosity caused by a car accident for 13 months. The patient was typically mobile in a wheelchair. The patient’s overall nutritional status was good, and routine examinations revealed no significant abnormalities. The sore on the left ischial tuberosity was 7 cm × 4 cm in diameter and 10 cm deep, with some secretion and a mild odor. On the third day of admission, the first debridement was performed. From shallow to profound, chronic inflammatory tissue and clear necrotic tissue were removed layer by layer. The base was deep up to the ischial cortex, and the sore’s deep cavity was 13 cm × 8 cm. After debridement, negative pressure dressing was applied on the wound surface and the sore was sealed, vacuum sealing drainage (VSD) treatment after the operation was continued; the negative pressure value was kept at -10.64 kPa, and was flushed with normal saline continuously. Every five to seven days, the negative pressure dressing was replaced and the wound was properly debrided. Two such negative pressure treatments were performed. On the 16th day of admission, we performed a repair surgery. The intraoperative design was a 15 cm × 9 cm ipsilateral gluteus maximus muscle flap that was rotated counterclockwise to fill the residual cavity. The flap incision was 15 cm long and extended outward and upward to form a facial flap and fascial skin. To seal the wound, the flap was rotated counterclockwise and covered the muscle flap. The muscle and skin flaps were all alive and well after the operation, and the appearance was good. See Figure 1. During the six- to 12-month follow-up period, the flap remained soft and left a small linear scar.\n\nA. Pressure ulcer on the day of admission; B. Ulcer base during the first debridement; C. VSD treatment after the first debridement; D. Ulcer bottom during the second debridement; E. Hip repair during surgery; F. The gluteus Maximus muscle flap rotates counterclockwise to fill the deep residual cavity at the Ischial tuberosity; G. The hip fascia flap rotates counterclockwise to cover and seal the pressure sore; H. The calf fascia flap was rotated counterclockwise to cover and seal the pressure sore. The skin flap was well-shaped three months after surgery, leaving only a small linear scar.\n\nA 36-year-old Chinese man presented with 5 years of paraplegia caused by a traffic accident, as well as 2 months of pressure sores on the right Ischial tubercle. The sores had a typical cystic granulomatous type of grade IV pressure sores, and the skin defect was 5 cm × 4 cm and 3.5 cm deep. On the bottom, there was aging granulation tissue. There was chronic inflammatory skin and soft tissue 1.5-2 cm wide around the wound’s skin, and the injury had little secretion. In the first debridement, the unstable inflammatory tissue surrounding the ulcer was removed. The cyst wall was removed entirely as well. The granulation tissue was removed, the base was enlarged to up to 7 cm × 6 cm, and the bottom was deepened to the superficial muscle layer. Following debridement, the wound was filled with negative pressure material, sealed, and treated with continuous VSD. The negative pressure was -10.64 kPa and was washed continuously with normal saline. One week later, the injury had healed and the medial gracilis muscle flap had been designed. The muscle flap was created within 10 cm of the pubic tubercle-medial semitendinosus muscle connection. The flap was 8 cm × 6 cm in size, and the pedicle was 7 cm × 5 cm in length. The first incision was made at the distal end of the flap to find the gracilis, the myocutaneous flap was separated from the far end and near to 10 cm below the midpoint of the inguinal ligament, and the sore joint was cut to form an open channel; the flap was rotated counterclockwise to cover the wound. We sutured the incision directly on the donor site of the medial thigh. After the operation, all of the flaps survived, and their appearance was satisfactory. Figure 2 depicts this. During the six- to 12-month follow-up, the texture was soft, with only a faint linear pattern.\n\nA. Pressure sore situation on admission day; B. During the first debridement, the ulcer base appears; C. The development of a gracilis muscle flap during the repair procedure; D. To cover the wound, the gracilis muscle flap rotated counterclockwise. E. Closed sore, donor area sutured directly; F. Finally, 3 months after the operation, the skin flap appears to be in good condition, with only a small linear scar.\n\nTreatment outcomes\n\nAfter debridement + VSD + muscle flap combined with fasciocutaneous flap or musculocutaneous flap repair, all patients’ ulcers were healed. Patients in our department were hospitalized for 28 to 81 days. Bacterial cultures were positive in seven of the eleven patients with wound secretions. The appearance of the skin flap was complete after muscular tissue flap repair, with no visible scar hyperplasia. However, after 6 to 12 months of follow-up, the flap was soft and left a small linear scar.\n\n\nDiscussion\n\nPressure ulcers are more common in people who have limb dysfunction, are unconscious, or are in forced postures. They were previously known as “bed sores” but they are now more commonly known as pressure ulcers, and they mostly occur in protruding parts of human bones. The buttocks are the most commonly affected area of the body when it comes to pressure ulcers. Pressure ulcers in the buttocks are most common in the sacrococcyx, ischial tuberosity, and femoral tuberosity. The sacrococcygeal region has the highest incidence, which mostly occurs in patients who have been supine for an extended period of time. However, pressure ulcers at the ischial tuberosity are more common in patients who have paralyzed lower limbs and have been in wheelchairs for a long time. Repairing a severe pressure ulcer is complicated, the treatment cycle is lengthy, and the cost is exorbitant. As a result, for people at high risk of pressure ulcers, prevention is preferable to treatment, and the proper family care model is advocated.2 If a pressure ulcer develops, the wound will quickly deepen and expand if not treated properly. Early and effective treatment is critical: 1. Avoid long-term pressure and rotate frequently. 2. Maintain clean and dry skin. 3. Use decompression devices like an air bed and a decompression mat. Early-stage I to II pressure ulcers are mostly treated with protection and conservative dressing changes.\n\nLong-term vertical pressure is the leading cause of pressure ulcers. The normal capillary pressure ranges between 12 and 30 mmHg. When the local pressure exceeds 16 mmHg, capillary tissue perfusion can be blocked, resulting in pressure sores. When the local pressure exceeds 30-35mmHg and lasts for two hours, it can result in a pressure ulcer. Necrotic tissues are classified into ulcer type, full-thickness tissue necrosis type, sinus type, and cyst wall granuloma type based on the depth and clinical manifestations of pressure ulcers.3 Pressure ulcers at the ischial tuberosity have distinct characteristics when compared to the other two parts. The skin of the ischial tubercle is located at the buttock fold, which has a lot of skin and soft tissues. In patients who usually sit for a long time, their soft skin tissues move horizontally repeatedly on the seat. A shearing force is produced by the combination of horizontal friction and vertical pressure. This shearing force causes the adjacent two layers of tissue to slide in the opposite direction, resulting in skin and soft tissue displacement and rupture. Under these conditions, a latent cavity or sinus is formed, which exacerbates the destruction of deep tissues. As a result, ischial tuberosity pressure ulcers have a small-scale skin defect with deep enlarged penetrating ischial tuberosity necrosis.4 Furthermore, the ischial tuberosity is close to the perineum, the surrounding environment is moist, and urine and stool contamination are likely to worsen the infection. In severe secondary infections, there will be a foul smell or a large amount of purulent secretions, which can easily penetrate deep tissues and cause tendons and periosteum to be inflamed, thickened, hardened, and destroying their bones and joints. As a result, it complicates repair treatment. Pressure sores in the ischial tuberosity are difficult to heal in a chronic wound; deep-tissue necrosis is common, and residual bacteria are persistent. As a result, thorough debridement is required before repair, and VSD is a novel type of closed drainage technology. It has been widely used in the treatment of various types of wounds with positive results.5 In addition to the effect of continuous drainage, VSD treatment can hasten necrotic tissue shedding, hasten residual cavity sealing, improve local blood supply, promote vascularization, and prevent microbial invasion and infection in the external environment.6 Pressure sore mouth after VSD treatment can lay the groundwork for future repair. In this group of patients, one or more negative pressure treatments effectively controlled the infection symptoms, and the basal tissue of the latent cavity became fresh. The main goal of surgical pressure sore repair in the ischial tuberosity is to fill the residual cavity, eliminate the sinus tract, and seal the skin and soft tissue defects. It is critical to pack the residual cavity. Muscle tissue has an abundant blood supply and a powerful anti-infection ability. It is ideal for filling a large residual cavity. To repair deep pressure sores at the ischial tuberosity, we used muscle flap packing in conjunction with fasciocutaneous flap covering or a simple muscle flap alone. We chose the lower part of the buttock, the medial thigh, and the posterior thigh as the donor sites for the muscle tissue flap based on the principle of “obtaining materials from nearby.” The gluteus Maximus muscle tissue is densely packed with blood vessels. The gluteus maximus muscle flap is one of the preferred packing tissues for the large residual cavity. The gluteus maximus muscle flap is lifted from the distal end in the direction of the gluteus Maximus muscle bundle. The inferior gluteal artery perforating branches are located in the middle of the gluteal region, 5 cm above the lateral third of the gluteal fold. Depending on the size of the residual cavity, either full-thickness or part-to-section cutting can be performed. We proceeded with caution in order to protect the main inferior gluteal artery during the procedure. The amount of muscle tissue required to fill the ischial tuberosity’s deep residual cavity was obtained. Pressure ulcers frequently consume far more skin and soft tissue than is required to seal the wound. As a result, when selecting the gluteus maximus muscle flap, we usually use only the muscle flap and the fascia flap. Skin soft tissue flaps are typically formed by extending and incising along the buttock folds to inform and transfer. They can be transferred counterclockwise after formation, reducing the fasciocutaneous flap’s tissue volume. The gracilis musculocutaneous flap is located on the inner thigh and is concealed. This muscle is an auxiliary muscle of the inner thigh muscle group, and its removal has little effect on function. The main nutrient vessel is the gracilis branch of the deep femoral artery, and its entry point is about 9 cm below the inguinal ligament midpoint.7 Because the gracilis muscle is thin, musculocutaneous flaps are frequently used to transfer the wound and cover it directly. It is appropriate for wound repair with a shallow wound base. If the amount of tissue filled by the musculocutaneous flap is insufficient, the flap can be appropriately enlarged. To fill the residual cavity, the peripheral part of the tissue is removed from the epidermis to form part of the subcutaneous tissue flap.8 Posterior thigh muscles include the biceps femoris, semitendinosus, and semimembranosus, which all originate from the ischial tubercle. Their blood supply is given to them in stages. The main blood supply to the posterior femoral side comes from the first perforating branch of the deep femoral artery, located 8 cm below the ischial tubercle.9 Clinically, depending on the size and depth of the pressure sore, either a simple muscle flap packing or a muscle tissue pedicle myocutaneous flap directly covering can be used.\n\nThe biceps femoris tissue, on the other hand, is extensive and suitable for formation. The large residual cavity is filled with a single muscle flap. Simultaneously, semitendinosus and semimembranosus are small and thin, making them more suitable for the direct formation of myocutaneous flaps for transfer and repair.\n\nThe IV-degree pressure sore of the ischium tubercle takes a long time to heal. The blood circulation of the tissue flap chosen above, for example, is extremely reliable. In the cases, there no occurrence of blood supply disturbance of the tissue flap itself, and the factor that affects the healing time is often that the transplanted tissue flap cannot be adhered to the wound base completely. In summary, the main reasons are as follows: 1. Anemia, hypoproteinemia, and electrolyte imbalances are examples of systemic nutritional status deviations. 2. Bacteria that remain in deep tissues can easily infect. 3. The expansion is insufficiently thorough, as is the basement’s remaining ecological organization. 4. Patients with lower-limb paralysis have poor neutrophil function. 5. Long-term postoperative prone position, patients with more difficult body posture. 6. Turning over activities that can easily cause the ischial tuberosity joint traction. In response to the aforementioned preoperative and postoperative issues, it is recommended to perform multiple expansions + VSD to improve the foundation of the wound before using muscle tissue flap transplantation, in addition to the symptomatic adjustment of systemic nutrition, the use of sensitive antibiotics to fight infection, and strengthening of postural care. As soon as possible after transplantation, it should be ensured that the muscle tissue flap heals and closes the injury.\n\n\nEthical considerations\n\nThe study’s participants were treated in accordance with the values outlined in the Helsinki Declaration.\n\n\nPatient consent\n\nEach patient provided informed consent for the publication and the patient’s privacy was not infringed without informed consent.", "appendix": "Data availability\n\nAll data are available as part of the article and no additional data sources are required.\n\n\nAcknowledgments\n\nWe appreciate the assistance and effort of the authors included in this study. We also acknowledge the support of the Natural Science Foundation of Ningxia and the National Science Foundation of China. Fazal Rahim is the first co-author of this article.\n\n\nReferences\n\nHenglin H, Chuan’an S, Jiake C, et al.: Biceps femoris long head muscle flap combined with semi-V-shaped posterior femoral fascia skin flap in repair of ischial tuberosity pressure ulcers. Chinese Journal of Burns. 2012; 28(1): 57–59.\n\nGuanghua L, Xiao Z, Ying L, et al.: Analysis of risk factors for pressure ulcers in patients at home. Chinese Journal of Burns. 2014; 30(3): 278–280.\n\nXuanping Z, Huarong H, Shurong Z, et al.: Experience of recurrence and re-repair after repair of capsular bag granulomatous pressure ulcer at the sciatic tubercle. Chinese Journal of Injury and Repair (Electronic Edition). 2017; 12(3): 197–199.\n\nJian L, Chunlin H, Heping Z: Treatment of ischial tuberous pressure ulcers with bilateral adipose fascia flaps. Chinese Journal of Plastic Surgery. 2014; 30(6): 421–423.\n\nCoban YK: Combination of negative pressure wound therapy and hyalomatrix application for soft tissue defect of the great toe. The International Journal of Lower Extremity Wounds. 2012; 11(3): 155–156. PubMed Abstract \| Publisher Full Text\n\nBranch of Burn Surgery, Chinese Medical Association, Editorial Board of “Chinese Journal of Burns”: National expert consensus on the application of negative pressure closed drainage technology in burn surgery (2017 edition). Chinese Journal of Burns. 2017; 33(3): 129–135.\n\nYanjin W, Zhenyu C, Yiqing X, et al.: Clinical application of gracilis flap to repair perineal defect. China Journal of Aesthetic Plastic Surgery. 2012; 23(9): 539–541.\n\nChu Guoping L, Guozhong ZY, et al.: Partially exfoliated posterior femoral bilobed skin flap for repair of 15 cases of ischial tubercle pressure ulcer. Chinese Journal of Burns. 2018; 34(8): 559–561.\n\nChunlin H, Yudong G: Flap Surgery (Second Edition) [M]. Shanghai: Science and Technology Press; 2013; 558–560." }	[ { "id": "254295", "date": "03 Apr 2024", "name": "Zairong Wei", "expertise": [ "Reviewer Expertise I mainly focus on several research areas", "including the design", "acquisition and clinical application of skin flaps", "skin and nerve regeneration", "wound repair", "and lymphedema." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “Repair of ischial tuberosity pressure ulcer with a perigluteal muscle flap” by Lifeng G et al. introduces the repair of ischial tuberosity pressure ulcer with perigluteal tissue flap. As known, the skin flap for repairing pressure ulcer needs to have the advantages of rich blood supply, good wear resistance, sufficient thickness, and strong anti-infection ability. Perigluteal tissue flap is undoubtedly one of the ideal choices. The manuscript is somewhat innovative and has reference value, but I have a few suggestions to make: 1. In “Abstract and Introduction”, explain the significance of choosing \"perigluteal muscle tissue flaps\". 2. In the Introduction section, the author mentions the relationship between pressure ulcers and bursa. Please have the author carefully check the grammar. 3. Please verify the data, 9 cases in “Abstract and Treatment method”, and 11 cases in “Introduction and Clinical data”. 4. Provide detailed data for all patients, such as age, gender, culture results, nutritional status, duration of paralysis, duration of pressure ulcers, depth of pressure ulcers, debridement and VSD usage status, and comorbidities, presented in the form of tables. 5. Based on the application of the flap in the manuscript, I believe that naming it \"gluteal tissue flap\" would be more appropriate. Moreover, some terminologies are used improperly, such as “facial flap”. 6. The authors should integrate clinical data and describe it in the appropriate part of , at least positive bacterial cultures are not indicative of treatment outcomes. 7. The discussion should focus on the research content and the latest studies, with an emphasis on the advantages, indications, contraindications, precautions of choosing the perigluteal tissue flap and the authors’ experience. 8. Discussion section, the last paragraph is difficult to understand. Is the author trying to express the reasons why pressure ulcers are difficult to heal or flaps are easy to loss? If that's the case, it may be more reasonable to explain why choosing a gluteal tissue flap is more appropriate in the first paragraph.\n\nIs the background of the cases’ history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? 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 conclusion balanced and justified on the basis of the findings? Partly", "responses": [] }, { "id": "254314", "date": "16 Apr 2024", "name": "Aydın Turan", "expertise": [ "Reviewer Expertise reconstructive surgery", "maxillofacial surgery", "microsurgery", "oncological surgery and hand surgery." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1-Pressure ulcers in the buttocks are mostly concentrated in three areas: sacrococcygeal, ischial tuberosity, and femoral tuberosity. It can be changed in shape. Because in the literature, they are generally defined as sacral, ischial and trochanteric pressure sores. 2-It should be explained why osteotomy is not performed to expand the bone surface area in ischial pressure sores. 3- Anterolateral thigh myocutaneous flap is a known and reliable flap option for ischial pressure sores. Information on this subject can be given in the introduction and discussion sections. 4-Editing the study in terms of English grammar and structure by a professional editing organization or person will increase its understandability. 5-The number of cases differs in the method and Case series sections. It should be corrected. 6-Literature research and evaluations regarding treatment options are insufficient.\nThe result of the review: The Study may be re-evaluated once the above-mentioned deficiencies have been completed.\n\nIs the background of the cases’ history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the conclusion balanced and justified on the basis of the findings? Yes", "responses": [] }, { "id": "233630", "date": "28 Aug 2024", "name": "Reto Wettstein", "expertise": [ "Reviewer Expertise Wound healing", "tissue engineering", "pressure injury reconstruction", "body contouring" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a series of 11 cases of pressure injury reconstruction. It seems as if the preparation before flap surgery involves several debridements and negative pressure wound therapy. The actual closure of the defect (stage IV PIs) is performed with a \"double\" flap: muscle and fasciocutaneous (not facial) or a myocutaneous flap. Whereas the authors seem to have good short term results - as far as it is described in the treatment outcome, the rational to use this sort of flap is not presented. In the discussion the authors describe generalities of pressure injuries that are not relevant to the aim of the article (description of the perigluteal muscle flap?). Also it seems as if there are several different perigluteal flaps. The authors fail to address the question why a muscle flap is needed in a location that is anatomically not or only partially covered by muscle tissue - that is less resistant to pressure than a fasciocutaneous flap and a comparison of their results with the literature is not possible due to the small number of cases.\n\nIs the background of the cases’ history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the conclusion balanced and justified on the basis of the findings? No", "responses": [] }, { "id": "241106", "date": "08 Oct 2024", "name": "Luca Negosanti", "expertise": [ "Reviewer Expertise plastic surgeon specilized in pressore sores reconstruction in spinal cord injury patients." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nthe article describes the reconstruction of ischial ulcers with different types of flaps. the language is not entirely correct (example: femoral tuberosity is the greater trochanter?). the technique used for the reconstruction is adequate but alternatives such as local fasciocutaneous flaps which are less invasive and allow different solutions in case of recurrences are not discussed. In case of recurrence, what approach could you use? recurrence should always be taken into consideration for pressure ulcers. in our experience the reconstruction is well performed with local fasciocutaneous flaps, we consider muscle flaps only for trochanteric sores. the two stage procedure is necessary in this cases?\n\nIs the background of the cases’ history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the conclusion balanced and justified on the basis of the findings? No", "responses": [] } ]	1	https://f1000research.com/articles/12-960
https://f1000research.com/articles/11-794/v1	18 Jul 22	{ "type": "Research Article", "title": "Modern approaches to address the concept of territorial division in Islamic jurisprudence", "authors": [ "Hajed A. Alotaibi" ], "abstract": "Background: This article examines how current jurists can deal with a juristic division of the world into abode of \"Islam” and “infidelity\". The success with which jurists re-evaluate this dichotomy will have major impacts on how an Islamic state interacts with non-Muslim governments on the one hand, and Muslims residing temporarily or permanently in a non-Muslim territory on the other. Methods: Here, an attempt made to review the concepts of Dar Al-Islam and Dar Al-Harb from two perspectives: that of the dialectical limits and liberalization, as discussed in secondary material such as books, research articles, and reports. Results: this paper has explored and discussed the criteria for identifying Dar Al-Islam and Dar Al-Harb in Islamic jurisprudence. Conclusions: This article analyzes the various arguments put forward to establish new ways of peaceful coexisting and maintaining healthy international connections.", "keywords": [ "Modern Approaches", "Territorial Division", "Islamic jurisprudence" ], "content": "Introduction\n\nThe terms “Dar Al-Islam” (land of Islam or peace) and “Dar Al-Harb” (land of war or blasphemy) have become vital to modern-day Islamic juristic discourse and some Muslim scholars, especially those of the Hanafi schools such as Alkasani (2010) and Alsarkhasi (2001), who divide the entire world into Dar Al-Islam and Dar Al-Harb. In addition, others (e.g. Alzuhaili, 1981) have added a third term, “Dar Al-Ahd” (house of truce or treaty), to the existing Islamic division of the world between Dar Al-Islam and Dar Al-Harb. Arguably, the introduction and use of this third term is a response to the many circumstances and events that have emerged which challenge the old Islamic binary discourse. For example, there have been political developments in matters of state sovereignty in the public arenas of international law, social change, and geographical movements and affiliations. Ongoing immigration has caused a crucial reconsideration of the Dar Al-Islam/Dar Al-Harb divide because, for example, Muslim immigrants have been required to adapt themselves to new and different realities (i.e. new countries, new peoples, multiple religions, etc.) in order to apply for citizenship or for a passport from that country and so on. There has been considerable focus on immigrants’ children and the extent to which they adapt themselves to such challenges or conserve their original culture, religion, and languages within their new communities.\n\nThe issue of immigration has led to some other crucial questions arising that have required a further critical review of the old juristic division between Dar Al-Islam and Dar Al-Harb. For example, can the concept of Dar Al-Islam still exist while the state of Islam is divided into multiple countries (i.e. it is not a single state/empire as in the past)? Can the non-Muslim world be described as a place of war, while Muslims find more protection and security in the West than their Islamic countries? Is it possible to invest some of the juristic opinions into developing a broader concept of homeland, thereby facilitating the idea of coexistence, and creating an understanding between those who differ in religion and residency?\n\nAlshirazi (2018) claims that the relationships that once existed and prevailed between countries, regarding the concepts of sovereignty, the minority, and the majority, are no longer the same in this era in which treaties and agreements have a prominent impact on the international reshaping of the United Nations. Additionally, advanced and advancing information technology have impacted on the reshaping of international relations, with technology also having a strong impact on the spread of information, and transmitting ideas and beliefs to people around the globe regardless of objections, barriers, or issues pertaining to rights/permission.\n\nIn contemporary times, interactions between the countries of the world are largely based on mutual commercial benefits, political and military collaborations, as well as mutually agreed uses of social spaces, all of which are enacted independently of belief and ideology. Given these contemporary international transformations and formations, it is arguably not tenable for contemporary jurists to posit a juristic division of the world based solely on the dichotomy of “the house of Islam” and “the house of infidelity”. It follows, given this, that there is a question as to whether they will adhere to this division or respond to the changes of their era. The response has serious ramifications for the nature of Islamic countries’ relationships with non-Muslim governments on the one hand, and Muslims living temporarily or permanently in non-Muslim territories on the other.\n\n\nMethods\n\nIn order to assess the data suitability, the author applied certain criteria. For instance, I searched using the keywords mentioned above, to find out in Google Scholar and in Saudi Digital Library SDL, which has access via our institutions to major research databases. Also, access to more than 169 Databases including ProQuest, Emerald, Web of Science, Scopus, Taylor & Francis and many others. Additionally, SDL has access to more than 50,000 journals around the globe and more than 5,000,000 postgraduates’ theses and dissertations. Therefore, after specifying those secondary research data, the author has prepared the required discussion using qualitative approach, dividing them into sections based on thematic titles as shown throughout the paper. For example, breaking the discussion into its appropriate places of the paper, trying to look at both sides of an argument so as to overcome the alleged bias, if any, in the paper. Moreover, based on secondary data such as books, research articles, and reports, this paper reviews the concepts of Dar Al-Islam and Dar Al-Harb from two perspectives: those of dialectical constraints, and liberalization. It additionally examines the Islamic division of the world through the old Islamic juristic discourse by examining the criteria for defining Dar Al-Islam and Dar Al-Harb in Islamic jurisprudence. The material from this preliminary discussion is then used to suggest new ways of peaceful coexistence as well as the development of good international relationships.\n\n\nResults\n\nSergeevich (2020) traces the evolution of the concepts of Dar Al-Islam/Dar Al-Harb in Arabic dictionaries as well as Islamic jurisprudence in order to examine their development. He concludes that the concept of a world divided into two geo-religious territories emerged in the early centuries of Islam, that the terms represent the distinction between the Muslim and non-Muslim worlds, and that they are the standard used to decide who belongs to both of those realms. It is also the case, however, that the terms also possess resonances of cultural symbols and values. Huwaidi (1990) observes that some Muslim jurists, in the past and present, have divided the world into two major parts. Others such as Alzuhaili (1981) have added two additional divisions, “Dar Al-Ahd” (land of the covenant or treaty) and “Dar Al-Hyad” (land of neutrality). As Huwaidi (1990) points out, dividing the world into two or three named sectors is not just the invention of Muslims, even if Muslim jurists adopted and developed it. The Romans, for example, divided people into Patriots, Latins, and Foreigners, with “foreigners” originally being called “enemies”, “others” or “barbarians”. Huwaidi argues that the Romans came to adopt this latter way of thinking due to the rise of Islam at that time. Indeed, the Romans began to use the parallel religious concepts of “Christian homes” and “homes of disbelief”.\n\nIn Islamic juristic discourse, however, more complex criteria are applied to those lands. Indeed, there is no single basis that can be considered to define the lands of Islam, of war, or of covenant, even though a search will reveal various definitions of those terms. Alzuhaili (1981) argues that there is no such thing as a “Dar Al-Hyad”(land of neutrality) in Islam since it is included in “Dar Al-Ahd” (the land of the covenant). This is because the concept of the land of the covenant has been expanded to cover all non-Muslim countries in the world today, as long as infallibility remains and fighting does not exist. Peace is the foundation of Islamic international relations. Ahmad (2008) additionally cites Alzuhaili’s argument that the legal repercussions of an act done in such a state (i.e. Dar Al-Hyad or land of neutrality) are rarely discussed in Islamic jurisprudence. This may be because such a state did not exist in the immediate vicinity of the jurists in question or, more crucially, because the inhabitants of such a state did not have the legal protection of the Islamic state due to the absence of a treaty. For a variety of reasons, the old Islamic juristic texts have defined the lands of Islam, Harb, and Ahd in various ways. However, from the following definitions, it is evident that Muslim jurists differ in their definitions of the lands due to their differences of opinion about the standards and controls set out for them. Definitions are largely based on the following criteria:\n\n1) The criterion of worship (the ability to demonstrate the two testimonies – that Allah is the one God and that Mohammed is his prophet – and the Pillars of Islam, such as prayer, zakat, and so on).\n\n2) The criterion of authority and governance (the rule of Islamic law or lack thereof).\n\n3) The criterion of military power (i.e. whether the military power supports a benign Islamic government or is at the disposal of other militant groups).\n\n4) The criterion of the size of the population, whether large or small.\n\nAs a result, Muslim jurists have given “Dar Al-Islam” several definitions, and these include that of Alsarkhasi (2001, p1253) who notes that Dar Al-Islam is a name for the place that is in the hands of Muslims, a sign of which is that Muslims feel secure in it. Meanwhile Al-Kasani (2010, p131), of the Hanafi school, has written that: “What is meant by adding the houses of Islam and disbelief is not Islam or disbelief in themselves. Rather, what these terms describe are security and fear, respectively. Consequently, it means that if there is safety for Muslims at all times, then it is the abode of Islam. Otherwise, it is the abode of unbelief”.\n\nAbu Mansur Al-Baghdadi of the Shafie school (1980, p270) argues that: “Dar Al-Islam is every house (territory) in which the call of Islam has arisen from among its people without a guard, judge or tribute”. He further observes that, “Muslim law covers the people of the dhimma (non-Muslims with protected status) if the people of the heresy did not fight against the people of Sunnah, for it is the abode of Islam”. He concludes, “If the matter is against what we have mentioned, then it is the abode of unbelief”. Meanwhile, Ibn Muflih of the Hanbali School (2003, p190) defines the differences between Dar Al-Islam and Al-Harb as follows: “Every land that has been conquered by Muslim armies and that has a Muslim government is the land of Islam, but if the rule of the infidels prevails, according to Islamic teachings, then the land is the land of Al-Harb. There are no other additional houses”.\n\nAccording to the Zaydi sect of Shia Muslims, “Dar Al-Islam is where the two testimonies and prayer appear, and where there is, additionally, no blasphemy, except in the eyes of Muslims” (Alshawkani, 2004, p57). He argues that when Jews and Christians practice their religion in Muslim countries this is not deliberately anti-Islamic. It is simply that such religious practices offend Muslims because they deviate from Islam.\n\nThe definitions of contemporaries who cling to the old juristic division of the lands do not deviate from the terminology of the ancients. For instance, such scholars see that Dar Al-Islam is every country in which Islamic law prevails in legislation and implementation, and that power and pride are with Muslims, whether the majority of the population are Muslim or not. As for the house of war, this encompasses all lands in which there is no sovereignty or protected status for Muslims, where Islamic judgments in law are not applied, and where Muslims are not able to live under an actively Islamic government (Abu Zahra, 1995, p277; Afifi, 1986, p128; Alzuhaili, 1981, p175).\n\nFrom the discussion above, it becomes clear that Muslim jurists differ in their definitions of Dar Al-Islam, Dar Al-Harb and other terms used to describe the relationship between Muslim and non-Muslim lands, territories, and governments. However, Ahmad (2008) argues that the central distinction between Al-Islam and Al-Harb has been widely misinterpreted in recent years. It is commonly conceived that the partition of the universe into two domains is inextricably linked to the belief that the usual relationship between Dar Al-Islam and Dar Al-Harb is uniformly one of enmity. In light of such comments, it is frequently asserted that one must either embrace or reject the following two assertions:\n\n• That the globe can be divided into a “Land of Islam” and a “Land of War” and\n\n• That the relationship between Muslim and non-Muslim countries is necessarily one of hostility.\n\nThose scholars who see the two assertions as inextricably linked argue that the divide envisioned by Muslim jurists is permanent and unchangeable. This may, in turn, lead to violent thinking. However, according to Bonzatto and Ortunes (2015), fundamentalism is a recent but not always violent movement. Commenting further, it should be noted that Bonzatto and Ortunes (2015) examine the concept of a house of peace and a house of war, and note various interpretations. Fundamentalism, according to Bonzatto and Ortunes, “presents the threads coming from a period of conflict and reform in Islam.” As a result, each fundamentalist group has its own interpretation of the Qur’an, as well as its own political and religious goals. However, for a better knowledge of the subject, it is necessary to rely on some books by authors who hold opposing viewpoints, such as Bernard Lewis and Edward Said.\n\nBadar and Nagata (2017) look at how the terms Dar Al-Islam and Dar Al-Harb have evolved throughout Islamic history, and note how extremist groups have misinterpreted them, and what their current legal standing is. Badar and Nagata concluded that modern extremist groups have resurrected the use of some older Islamic doctrines and intentionally misconstrued them to achieve their goals. They opine that the most prominent example of such a group is Daesh (the Islamic State of Iraq and Syria). Daesh has issued takfir (excommunication) pronouncements against Muslim governments, and claimed that only Daesh-controlled territory is Dar Al-Islam, while all other Muslim countries are Dar Al-kufr (having a Muslim population but not governed by Islam). Meanwhile, to explore why that Daesh is the only Dar Al-Islam, scholars such as Bernard Lewis (2004, p73) wrote that the universality of the Muslim revelation is the foundation of the requirement for jihad. It is the obligation of those who have accepted God’s word and message to struggle unceasingly to convert or, at the very least, subjugate those who have not. This responsibility has no time or space constraints. It must continue until the entire globe has either joined Islam or succumbed to the Islamic state’s sovereignty. Until then, the world is divided into two houses: the house of Islam (Dar Al-Islam), where Muslims govern and Islamic law reigns supreme, and the house of war (Dar Al-Harb), where the law of war – or the potential for Muslim conquest – reigns supreme.\n\nThose who do not agree that the typical international relationship between Muslim and non-Muslim political entities is one of conflict claim that Muslim jurists drew this distinction based on the conditions of their time, and that it is unsustainable in present times. As contemporary thinker Tariq Ramadan (1999) argues, Dar Al-Islam and Dar Al-Harb are notions that are not found in either the Qur’an or the Sunnah, whose ideas are global and eternal, and transcend all geographical boundaries. In contrast, during the first three centuries of Islam, Islamic scholars evaluated and classified the many places in and around them at that time. For example, they looked at the geographical divisions, the powers in place through religious affiliation and influence, and the ever-changing nature of alliances.\n\nThe practice of dividing the world into two domains (Islam and Harb) has been retained primarily by the Hanafi School; a majority of Muslim schools such as the Maliki, Shafie, and Hanbali have rejected this division. Therefore, the question arises as to what motivated Hanafi scholars to establish such a division and why most other Muslim scholars have rejected it. According to the Hanafi School, Muslims who live in Dar Al-Islam are protected (ma’soum) by Islamic law. For Muslims living outside of Dar Al-Islam, protection merely means that those who abuse their rights will be held accountable in the Next World, i.e. in the hereafter, by God’s court. This is known as “Ismah bi’l Islam,” or protection based on one’s association with Islam (Alsarkhasi, 2001).\n\nAccording to Hanafi jurists, this means that the rights of Muslims living outside Dar Al-Islam’s territorial bounds will not be enforced by the Islamic state’s courts, as these courts can only exercise jurisdiction over the area under the effective control of Dar Al-Islam’s Imam (i.e. the ruler). In other words, Islamic courts are unable to take recognition of a judicial action that occurs outside of their jurisdiction. The Islamic state’s courts do not have jurisdiction over wrongs committed outside the borders of the state unless it has a contract or treaty with the body that has suzerainty over that territory. In contrast, the Islamic state is obliged to preserve the rights of all those who dwell permanently, or even temporarily, inside its borders, whether Muslim or non-Muslim. All people living within an Islamic state’s geographical boundaries are assured of legal protection, whether Muslim or non-Muslim. This is known as the “protection of one’s rights as a result of residing in an Islamic country” (Alsarkhasi, 2001).\n\nThe bottom line of the Hanafi School’s position is that “Ismah” (used here according to its core meaning of “protection”) guaranteed solely to those who live inside the geographic bounds of the Islamic state, regardless of whether they are Muslims or non-Muslims, according to what we may call, the municipal law of Islam. Furthermore, Islamic law differentiates between various non-Muslim political bodies based on their attitude toward Islam and Muslims. If a non-Muslim country is actively at war with Dar Al-Islam, it is referred to as Dar Al-Harb whilst if it signs a peace treaty with Dar Al-Islam, it is known as Dar Al-Ahd. However, people living in Dar Al-Harb do not enjoy ‘Ismah’ (legal protection) from the standpoint of the Islamic state’s territorial authority and its courts (Alkasani, 2010). In instance of Dar Al-Ahd, however, and because the Islamic state has signed a treaty with that entity, certain jurisdiction may be ceded to the Islamic state. If this is the case, some sort of “Ismah” may be established for individuals who live there. The provisions of the treaty that has been signed will determine the status of the previously hostile country and its inhabitants.\n\nIn contrast to the Hanafi School, the majority of Shafie, Maliki, and Hanbali Muslims believe that Islamic law has no territorial boundaries. As a result, if a Muslim disobeys a Shariah norm, he will be punished not only in the Hereafter but also in this life by the Islamic state’s courts. Similarly, if a non-Muslim resident of a non-Muslim state abuses the rights of any Muslim, the wrongdoer will be punished by the Islamic state’s courts if he is captured by Muslims or enters the Islamic state. It follows, that “Ismah,” or legal protection, is conferred because of a person’s religious affiliation (Islam) rather than his or her domicility in a politically determined land (Dar). Concerning non-Muslim citizens of an Islamic State (Ahl Al-Dhimmah), it should be noted that it is believed that they are protected as long as Muslims have signed a Dhimmah treaty with them. A majority of Shafie, Maliki, and Hanbali Muslims thus compare non-Muslims to alien non-Muslims (musta’minn) in Dar Al-Islam; protected by Muslims under an Aman (reciprocal safety) contract (Ibn Qudamah, 1999; Ibn Alqayim, 2014).\n\nTariq Ramadan’s (1999) assertion that the division of the world into Dar Al-Islam and Dar Al-Harb does not exist in the Quran or Sunnah is open to challenge, especially about Sunnah literature. His un-nuanced assertion may have led to confusion about this matter. The next section of this paper discusses some Islamic juristic rulings including some from the basic sources of Islamic law, on the Islam/Harb distinction, from a territorial (as opposed to purely religious) perspective.\n\nAhmad (2008) argues that though we cannot always deduce directly the teachings of the Islamic texts (i.e. the Quran and the Sunnah), certain things may be inferred. Thus, instead of interpreting the texts literally, Hanafi scholars such as Alkasani and Alsarkhasi strive to extract general principles from these texts and then apply them to the entire field of law. If a ruling emerges from one of the most authoritative texts that is different from the literal interpretation given in a text of lesser authority, they interpret the secondary text in light of the general principle. This may result in them treating the rule mentioned in the secondary text as an exception to the general principle if compatibility between the two is impossible. Through this approach, the Hanafi preserve uniformity in the judicial system.\n\nPassages and traditions, ranging from inferred principles from the Quran to explicit directives in both the Quran and other kinds of Islamic literature, establish different laws for Muslims who live in and beyond Dar Al-Islam. For example, the Qur’an classifies some Muslims who immigrated from Makkah (at that time Dar Al-Harb) to Medinah (at that time Dar Al-Islam) as “fuqara” (poor; empty-handed) even though they owned property in Makkah (Qur’an 59: 8). According to Hanafi scholars, the reason for this interpretation was that they had lost the titles to their property in their native land because of their move to Dar Al-Islam. Another proof of their loss of ownership was the fact that the Prophet Muhammad did not return these assets to them after the conquest of Makkah (Hamidullah, 1987; Alsarkhasi, 2001).\n\nWhen the Muslims in Makkah were persecuted and forced to flee to the Islamic state of Medinah, those who did not migrate were denied the Islamic state’s protection (Quran 16: 106–110 and 4: 97–99). The Quran thus asserts that the Islamic state (Dar Al-Islam) has no legal responsibility to protect the rights of such people. However, if requests for assistance are received regarding “matters of faith,” the Islamic state is obliged to assist those people, even militarily, if necessary (Quran 8:72; Quran 4: 75–76). It is also the case that the Islamic state is compelled to act according to the conditions of any treaty they sign. Further comment on the territorial nature of the Islam/Harb divide is found in the case of Abu Busair and his associates. These people were not subject to the Islamic state’s control since they were Muslims who had immigrated to places other than Medinah. However, the Prophet did not feel legally obliged to hand them over to the non-Muslim rulers of Makka either. It is worth noting that, as a non-Hanafi scholar, Ibn Alqayim (2014), a well-known Hanbali jurist, sees grounds for a territorial jurisdiction in this case. He claims that by not putting a halt to these people’s activities, the Prophet did not break any treaty provisions because they were “out of his control”. In short, they were not under his jurisdiction.\n\nAdditional evidence of the Islam/Harb divide as a territorial matter includes the idea, at least according to Hanafi jurists, that non-Muslims can acquire ownership of property they capture from Muslims if they carry it to Dar Al-Harb. Several arguments have been advanced in support of this rule. It is important to note, however, that taking possession of property by force does not provide a justifiable reason for its ownership within Dar Al-Islam. According to the Hanafi school of thought, this is because the basis for “Ismah” (legal protection) is Hirz (safe custody), which is based on Dar (a land’s jurisdiction) rather than Din (religious belonging).\n\nHowever, what of the territorial Islam/Harb divide and monetary exchanges? According to Abu Hanifah and Muhammad Ibn Al-Hassan Al-Shaibani, if a Muslim exchanges one Dirham for two Dirhams in Dar Al-Harb, the transaction is valid, because the Muslim exchange partner took the property with his or her consent. The matter of a Muslim’s property in Dar Al-Harb is unlike that of the property of the musta’minn (Non-Muslims with a protection contract) in a territory of Islam. Musta’minn property becomes legally protected under the contract of Aman (reciprocal safety) (Alsarkhasi, 2001 p.104)\n\nMoreover, if a Muslim soldier commits adultery or fornication (Zina) in Dar Al-Harb, he will not receive the appropriate punishment because the cause of action occurred beyond the Islamic state’s jurisdiction. However, if he commits such an act within the Muslim army’s camp, additional punishment can be imposed. The same is true when it comes to imposing the Qisas (death) penalty (Alsarkhasi, 2001 p. 84, 115, 116)\n\nCorrespondingly, the courts of Dar Al-Islam are unable to resolve any musta’min issues (issues concerning non-Muslim temporary residents of the Dar Al-Islam) that emerged while they were still in Dar Al-Harb. The musta’min do not become residents of Dar Al-Islam just by having a contract of Amn. However, they also need to be in Dar Al-Islam territory at the time that the issue arises if legal redress is required.\n\nMuslims should still respect a person’s rights if he comes to Dar Al-Islam from Dar Al-Muwda‘ah (a territory of mutual peace) without a new Amn contract. Similarly, if he enters Dar Al-Harb and Muslims control the land thereafter, Muslims must maintain his rights even there, “his status is similar to that of a dhimmi who joins a Dar Al-Harb that is later controlled by Muslims.” (Alsarkhasi, 2001 p.98). The above examples are from Hanafi scholars’ points of view.\n\nIn contrast, other Muslim scholars, including the Maliki, Shafie, and Hanbali schools are of the view that Islamic law is applicable everywhere, especially, to Muslims as well as to those who are in contract with Dar Al-Islam, though this depends on the contents of their contracts. They argue that the legal protection “Ismah” of the faithful Muslim relates to a Dar Al-Islam based on the universal religion and not merely one of physical territory. According to this perspective – and with reference to the example previously given - those Muslim immigrants who lost the titles of their property in their native land as a result of their move to Dar Al-Islam, were entitled to have their property returned once Makkah was conquered by Muslims, as happened later on in history. Those Muslims who did not migrate from Dar Al-Harb were entitled to the Islamic state’s protection (Ibn Qudamah, 1999; Ibn Alqayim, 2014).\n\nMoreover, if the “location” of Dar Al-Islam and Islamic law can be anywhere where there are Muslims, non-Muslims cannot acquire the ownership of property that they may capture from Muslims if they carry it to Dar Al-Harb. What is more, if a Muslim exchanges one dirham for two dirhams in Dar Al-Harb, the transaction will be void because Muslims are bound by Islamic law everywhere. It is also the case that if a Muslim soldier commits adultery or fornication (i.e. Zina) in Dar Al-Harb, he must submit to the appropriate punishment. Similarly, the courts of Dar Al-Islam can oversee and resolve any musta’min issues that emerged whilst the plaintiffs were still in Dar Al-Harb but only once the issue has been raised in Dar Al-Islam. It remains the case, however, that the situation regarding Abu Busair and his associates is not applicable to the concept of Dar Al-Islam as a religious concept rather than one of geographical place. The Prophet Muhammad’s treaty with the Makkans included a provision requiring him to return anyone who migrated to Medinah, where he then lived. Abu Busair and his associates immigrated to a different place from Medinah (in other words, they moved outside the geographical bounds of the treaty). Thus, the Prophet did not break any treaty provisions Ahmad (2008).\n\nThe discussion above makes clear that both major views emanating from the Islamic schools of jurisprudence are inaccurate. This is because the division of the world into two parts, whether framed in religious or territorial terms, is juristic; hence, it lacks both legal and Sharia reasoning, although the division itself is arguably based on the divine texts of the Quran and Sunnah. Yet this division is purely derived from received wisdom rather that constant rational argument. Eqigg (2017) claims that continuing to adopt the old juristic view of the concepts of “house of Islam” and “house of war” can lead to unfortunate results. For instance, in the mentality of terrorist movements, it could provide justification for aggressive acts directed against non-Muslims in their lands, whether as combatants or civilians. In addition, a too simplistic division of the world into Islam and Harb may cause extremists to harm Muslims residing in non-Muslim countries. Furthermore, by advocating the juristic dictionary definitions of homelands, the jurists may encourage some Muslims in the West to abrogate the money and properties of non-Muslims by enabling them to argue that it is permissible to deceive the private and public facilities of some countries on the pretext that since their inhabitants live in a “house of war/unbelief”, the property is free for Muslims. In doing so, they make two grave errors:\n\nFirst, they are flouting the legal and Sharia rulings that urge the fulfillment of pledges and the keeping of trusts and charters - even where these have been entered into with people who profess a different religion. The second is that they substantiate the negative image of Muslims that may already exist in non-Muslim countries.\n\nContemporary Muslim jurists such as Aljudai (2007), Alhaj (2006) and Alfaitori (2006) approach the concept of an Islam/Harb divide from several angles in order to analyze and evaluate this heritage according to what is consistent with the Qur’anic vision of civilized living. In addition, Bork (2017) discusses modern views of the territorial division between the Lands of Islam and War in ancient Islamic legal theory, and in so doing focuses on views that challenge obsolete constructs of the world and advocates appropriate substitutes. He does this by utilizing the perspectives of two modern Muslim thinkers: Tariq Ramadan and Wahbah Al-Zuhail who have both attempted to establish new models which consider present realities, as well as offering a vision of Muslim-Non-Muslim cooperation based on Islamic law. The perspective of many contemporary jurists can be summarized as follows.\n\nFirst, they emphasize that the issue is generally perceived through the lens of custom and usage rather than according to binding legislation. Moreover, based on the criteria referred to above, contemporary juristic writings unanimously state that the division of the world into “Dar Al-Islam”, “Dar Al-Harb” and “Dar Al-Ahd” is the work of the jurists in the second century of the Prophet Mohammed. Alhaj (2006) observes that such a division arose from, “the necessities and conditions imposed by the special societal circumstances at that time”. This juristic development of the original sources is not based on significant legal evidence but rather, on, well-intended interpretation. It should not, therefore, be taken as a binding religion, but rather as a guide. Moreover, Ian argues that Dar Al-Harb, in particular, could have been “civilized” in the same way that the Byzantine Empire had been, but that it had not bowed to God’s will as defined by Islamic theology and jurisprudence. It is also worth noting that this terminology is fluid and has evolved over time, with varying degrees of consensus among scholars and rulers about what it actually indicated in terms of geographical region.\n\nSome contemporary researchers such as Alali (2006) have attempted to establish this juristic division by citing a group of hadith texts in which the terms “Dar Al-Islam” or “Dar Al-Hijrah” are mentioned, and their opposites, “house of polytheism” or “house of infidelity”. They argue that such texts confirm that this division was common in the words of the Companions. However, whilst these references can possibly be interpreted as describing a divided world, they are not definitive. Meanwhile, the context in which the terms “Dar Al-Hijrah”, “house of the faithful” and “house of immigrants” mentioned are in the context of a doctrinal and faith dimension, which has nothing to do with the political significance that later jurists added to it. Whatever the motives and purposes that led those jurists, either from Hanafi or from other Islamic schools, to make this division; they biased their interpretation of it more towards faith and belief than towards the politics of a geographically conceived Islamic nation (Alhaj, 2006). Furthermore, their interpretation of the concept does not describe either an original or a permanent basis for the geography of the land of the Islamic caliphate, which represents a starting point for all perceptions about the nature of internal and external relations, particularly, for the Muslim community. Secondly, contemporary jurists such as Al-Qaradawi (2010) and Topolyak (1997) have employed a new concept of “safety” to emphasize that the “house of Islam” at present is no longer confined to the historical boundaries of the old “house of Islam”. Rather, they suggest, it has expanded greatly to include every spot in which there is a Muslim who acknowledges to his Lord the two testimonies and establishes his laws in himself according to his ease and ability. Badr (1982) claimed that historical Islam’s international connections have gone through three periods of varying duration: the ages of expansion, interaction, and coexistence. Any attempt to define Islamic international relations regulation must keep this historical context in mind.\n\nSuleiman Muhammed Topolyak, a modern Islamic researcher, records in his discussion of the ruling on the political asylum of a Muslim in a Non-Muslim country that the concept of ‘abode of infidelity’ has changed today from what it meant in the past. For instance, we can see that a foreigner in a non-Muslim country enjoys legal and judicial protection, which are the same as a citizen, regardless of a religion. Yet, in many cases, these immigrant Muslims are allowed to practice their religious rituals, establish mosques and Islamic centers without objection (Topolyak, 1997). In the same way, Al-Qaradawi (2010) states that a Muslim may reside in any country in which the environment of religious, political, and civil freedom prevails, even if it is a secular country. Especially, if immigration or residence in those countries is for legitimate purposes such as seeking sustenance, knowledge or asking for security.\n\nBy referring to the old juristic tradition, we find such words among Alshafie (2001), where he says: “the Messenger of God said: the imposition of emigration can only be on those who afford it, but those who have been tempted in their religion in a country where they embraced Islam. Because the Messenger of God authorized people in Makkah to stay there in Makkah after their conversion to Islam such as Al-Abbas bin Abdul-Muttalib because they did not fear sedition\". Ibn Taymiyyah (2005) believes that the criterion of either residing in the abode of infidelity or emigration is based on the extent to which a Muslim can obey God in that abode or not. Therefore, there is no doubt that this can only be achieved with the presence of security and safety. If a Muslim find this in a place where he lives, then there is no need to emigrate. This is the all-encompassing principle\".\n\nTo explain more, the command to emigrate in Islamic law is related to the existence of safety elements in religion, self, and accommodation. However, with the conquest of Makkah, the necessity to emigrate was no longer necessary as it was a base of Islam and, additionally, safety exists there. Therefore, emigration is to a place that achieves for a Muslim the purpose of emigration, which is to enable him to establish his religion and protect himself from harm. This indicates that staying among non-Muslims is not self-blameworthy. Rather, it depends upon the extent of what is achieved from it in the interests of the Muslim. When contextualizing some Hadiths in this regard, confirming the legitimacy of the residency of a Muslim in other than the abode of Islam, including the survival of a group of companions who immigrated to Abyssinia and did not join the Messenger of God in the house of Islam, i.e. Medinah, during the seventh year of the Hijra. The Prophet did not blame them for immigrating to the abode of Islam nor did he issue anything that offended their stay among non-Muslims in Abyssinia (Eqigg, 2017). It was previously stated in the early juristic definitions of Dar Al-Islam that the Hanafis make a criterion of distinguishing Dar Al-Islam from the other houses by “achieving security and safety for Muslims”. This safety can be achieved according to Zaydi jurists by the ability of just showing the two Shahada and some rituals of Islam (Alshawkani, 2004, p575; Alsarkhasi, 2001).\n\nThird, the specific political and social data of the world has changed. The juristic classification of homelands, as previously mentioned, is a discretionary classification dictated by the conditions of the Islamic nation and the nature of international relations existent at the given time. However, the situation has changed, and the existence of recognized international treaties, the criminalization of wars that have not arisen from a response to aggression and resistance to occupation, and the emergence of states of citizenship, as well as the rights of religions, races, and colors necessitate that that the whole world has become a space for tolerance and peaceful coexistence between and among all human beings. Henkel (2004) conducted an empirical study in Germany and concluded that many religious Muslims have recently undergone a major shift to a more “inclusive” position. To understand this transition, Henkel studied the transnational experience of Turkish Muslims in Germany, especially the experience of the “second generation” of Turkish immigrants, as well as the recent development of Turkey itself, where Islam has accelerated its integration into modern society. As Henkel notes, for many religious Muslims of the traditional Turkish Islamic tradition, a free society has become a social background conducive to the practice of Islam.\n\nCommenting upon associated themes, it can be seen that some of the analysis of the Orientalist Bernard Lewis is based on the classic Islamic comparison between Dar Al-Islam and Dar Al-harb. In his reading, he shows the inherent hostility of Islam to non-Muslims. However, the conflict is only one aspect of the complex relationship that exists between Islam and “Western” societies. For most Muslims, Dar Al-Islam and Dar Al-Harb are no longer relevant categories that define their social relationships with non-Muslims. However, the conceptual opposition of Dar Al-Islam and Dar Al-Harb points to a question that remains important to religious Muslims and that has been answered in different ways at different times: in what kind of society can one live a Muslim’s life? According to Arangul (2017), millions of Muslims presently live under non-Muslim governance. This is a level of magnitude unmatched in the past. Muslims were unwilling to legitimize non-Muslim supremacy for a variety of reasons during periods when they had free and/or prosperous countries. However, when historical reality begins to turn against them politically and economically, immigrant Muslims have grown to believe that this is feasible if they follow certain Islamic teachings.\n\nMoreover, the “house of Islam”, whose standards were defined by old jurists, no longer exists except in historical and juristic works. The abode of Islam became distributed among many nations of more than fifty countries, and its names became linked to races and nationalities as well as, sometimes, the names of families and royals (i.e. not in the name of the religion itself, which was the basis for belongings). In addition, whereas Muslim people of the old abode of Islam did not leave unless they were merchants, involved in Jihad or as tourists or visitors to other places for limited periods, today, they are in many parts of the world in which Muslims permanently reside, either through long-standing affiliations to those regions or by action.\n\nGiven these new determinants, Mawlawi (1990) believes that the criteria that the jurists specified in the past to distinguish the house of Islam from the house of war are no longer valid for defining an accurate description of this house in this era. Accordingly, he poses the questions: What is the standard to the Sultan of Islam, what are the implementations of its provisions, and what is needed to establish its rituals? Is it the establishment of Islam completely? This means that most Muslim countries are no longer a “house of Islam” today (Haikal, 1996). Is it sufficient to apply the provisions of Islamic personal status to the exclusion of all other laws? This means that we exclude the “house of Islam” of ancient Islamic countries like Turkey and Tunisia. Is it sufficient for Muslims to freely perform the rituals of Islam such as prayer, fasting, Hajj and zakat to be considered a “house of Islam” based on the legacy of the past? There are Muslims and they practice their rituals more freely than in some Muslim countries Mawlawi (1990). As for the “house of war”, it is no longer clearly defined, given that Muslims are now linked with most of the countries of the world that are under the banner of the United Nations with treaties, charters, and alliances that cannot be revoked as long as everyone abides by their requirements per what is customary in “international legitimacy” Mawlawi (1990).\n\nIn addition to this, the “house of war” - according to Howeidi (1990) is no longer necessarily located in the square of others. Rather, most wars in this time take place in the homes of Muslims; a scenario that did not occur in the time of the ancient jurists, as they only envisioned war between the Muslims as a whole, and the non-Muslims as a whole. Then “our jurists who used the term Dar Al-Harb did not live the terrestrial unity that we live in today, but they lived in a world of separate islands which do not coexist, hence, it was the jurisprudence of war.\n\nAhmad (2008) wrote that the Quran and Sunnah texts do not lend themselves to such an extension of the abode of Islam and war because, like all past Messengers of God, the Prophet’s wars included an element of Divine punishment for his foes, and he had, therefore, conclusively demonstrated the reality of his mission beyond all doubt, at least as far as his immediate addressees in seventh-century Arabia were concerned. Other than the Prophet’s closest addressees, non-Muslims cannot face any worldly punishment for their rejection of Islam. This is because it would violate the Quranic prohibition on compulsion in questions of religion (Qur’an 2: 256). This part of the Prophet’s mission was also acknowledged by the fuqaha, who used it as the foundation for various regulations. In any case, Jihad does not advocate for the coercive conversion of non-Muslims to Islam. Rather, Muslims must oppose those who seek to disrupt the Divine plan by harassing those who seek to force them to accept or reject a certain faith.\n\nTherefore, what we need today is the “jurisprudence of peaceful coexistence”; a reality that is different in quantity and quality. Mawlawi (1990) also claimed that the West is no longer a house of battle, but rather a house of call because the premise on earth is that it is for the call, based on the Almighty’s statement that “We did not send you but a mercy for the worlds”. Moreover, jurisprudence councils in Europe, America, and India have unanimously agreed that the land of Muslim minorities is a place of covenant and call, not a place of conflict (Al-Awa, 2006). It is critical, therefore, to be cautious when words from old Islamic international law such as “Dar Al-Islam,” “house of infidelity,” “house of war,” and “house of covenant,” to the realities of today’s world and individual countries as a consequence of the changed nature of international relationships, their complexities, and the nature of overlapping attributes of the same.\n\n\nConclusion\n\n“Dar Al-Islam” and “Dar Al-Harb,” have become dangerous phrases in Islamic jurisprudence. However, several circumstances have emerged that put into doubt Dar Al-Islam and Dar Al-Harb in classic Islamic discourse. State sovereignty, immigration, geographical mobility, and citizenship are all effective examples of such ongoing issues. As a result, Muslim jurists have been compelled to adjust to a new and different reality (new countries, people, religion, language, and so on). As a result, new approaches to the old Islamic jurisprudence’s concept of geographical partition have, and are, being developed. It follows that some legal opinions could be invested to expand the concept of homeland, promote peaceful coexistence, and resolve the issue of religious and residency differences. Issues pertaining to how an Islamic state interacts with non-Muslim governments on the one hand, and how Muslims reside temporarily or permanently in non-Muslim territory, have been influenced by this debate. The concept of Dar Al-Islam and Dar Al-Harb, as well as the criteria for identifying Dar Al-Islam and Dar Al-Harb in Islamic jurisprudence have been examined in this work and new path for the furtherance of peaceful coexistence and healthy international relationships identified.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgments\n\nThe author is very thankful to all the associated personnel in any reference that contributed to/for this research. The author would additionally like to thank the Deanship of Scientific Research at Majmaah University for supporting this work under Project number R-2022-169.\n\n\nReferences\n\nAbu Zahra M: International Relations in Islam. Beirut:Dar Alfikr Alarabi;1995.\n\nAfifi M: Islam and International Relations. Beirut:Dar Al-Raed Al-Arabi;1986.\n\nAhmad M: The Notions of Dār al-Ḥarb and Dār al-Islām in Islamic Jurisprudence with Special Reference to the Ḥanafī School. Islamic Studies. 2008; 47(1): 5–37.\n\nAlali Y: Juristic Geography of the World. Islamic Magazine Al-Ma’rifah. 2006; (45): 103.\n\nAl-Awa M: Islamic Jurisprudence in the Way of Renewal. Cairo:Safir International Publishing;2006.\n\nAl-Baghdadi A: Fundamentals of Religion. Beirut:Dar Al-Kutub Al-Ilmiyya;1980.\n\nAlfaitori A: The Purposes of Difference of Two Worlds. Islamic Magazine Al-Ma’rifah. 2006; (45): 125.\n\nAlhaj A: Doctrinal Perspective and Quranic division of the globe. Islamic Magazine Al-Ma’rifah. 2006; (45): 69.\n\nAljudai A: Division of the globe in Islamic jurisprudence and its impact on reality. Dublin:European Council for Fatwa and Research;2007.\n\nAlkasani AM: Bada’ Alsana’i. 3rd ed.Beirut:Scientific Books House;2010.\n\nAl-Qaradawi Y: Homeland and Citizenship in the Light of Doctrinal Origins and Legal Objectives. Cairo:Dar Al-Shorouk;2010.\n\nAlsarkhasi M: Sharh Alsiyar Alkabir. Cairo:Eastern Company for Advertising;2001.\n\nAlshafie M: Alumm. Cairo:Darul Wafaa;2001.\n\nAlshawkani M: Alsail Aljarrar. Riyadh:Daru Ibn Hazm;2004.\n\nAlShirazi M: The duality of the juristic division of the world into “House of Islam and House of Infidelity” a critical approach. 2018. accessed on 1st of May 2021.Reference Source\n\nAlzuhaili W: Athar Alharb Fi Al-Islam. Damascus:Dar Alfikr;1981.\n\nArangul M: On the Possibility of Living under Non-Muslim Rule From the Perspective of Islamic Law. 2017.\n\nBadar M, Nagata M: Modern Extremist Groups and the Division of the World: A Critique from an Islamic Perspective. 2017.Publisher Full Text\n\nBadr M: A Survey of Islamic International Law. Proceedings of the Annual Meeting (American Society of International Law). Cambridge University Press;1982; Vol. 76 (APRIL 22-24, 1982): pp. 56–61 (6 pages).\n\nBonzatto EA, Ortunes L: Fundamentalisms: Among the House of Peace and the House of War.2015. Publisher Full Text\n\nBork J: Models of coexistence: approaches for overcoming the classical legal division between the ‘Land of Islam’ and the ‘Land of War’ by contemporary Islamic scholars. 2017. Publisher Full Text\n\nEqigg M: The doctrinal division of the world between narrowing and liberation of the concept of homeland. Morocco, Fiqh and Law Journal. 2017.Reference Source\n\nHaikal M: Jihad and fighting in legal politics. Riyadh:Dar Ibn Hazm;1996.\n\nHamidullah M: Collection of political documents in the time of the Prophet. Beirut:Alnafais House;1987.\n\nHenkel H: rethinking the Dar Al-harb: Social change and changing perceptions of the West in Turkish Islam. 2004. Publisher Full Text\n\nHoweidi F: Citizens not dhimmis. The position of non-Muslims in the Muslim community. Cairo:Dar Al-Shrouq;1990.\n\nIbn Alqayim M: Alturuq Alhukmiah. Riyadh:Dar Altadmoriyah;2014.\n\nIbn Muflih M: Alfurou. Riyadh:Al-Risalah publishing;2003.\n\nIbn Qudamah A: Almughni. Beirut:the World of Books;1999.\n\nIbn Taymiyyah A: Majmoo ’Al-Fatwas. Mansoura:Dar Al-Wafaa;2005.\n\nLewis B: The Political Language of Islam. Karachi:Oxford University Press;2004.\n\nMawlawi F: The Sharia foundations of relations between Muslims and non-Muslims. Beirut:Dar Al-Rahad Al-Islamiyah;1990.\n\nRamadan T: To be A European Muslim: A study of Islamic Sources in the European Context. Leicester:The Islamic Foundation;1999.\n\nSergeevich V: Dar Al-Islam/Dar Al-Harb: Categories of Space in Medieval Islam.2020. Publisher Full Text\n\nTopolyak M: Political rulings for Muslim minorities in Islamic jurisprudence. Amman, Jordan:Dar Al-Nafaes;1997." }	[ { "id": "144597", "date": "08 Aug 2022", "name": "Chris PreJean", "expertise": [ "Reviewer Expertise My expertise is premodern Hanbali jurisprudence. My research discusses the relations between Muslim and non-Muslims", "including the rules of war and jihad", "territorial boundaries and jurisdiction." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEditorial Note: This report was updated on [23/09/22] to include references to works cited by the reviewer.\nIn his “Modern approached to address the concept of territorial division in Islamic jurisprudence,” H.A. Alotaibi outlines some of the contours of Muslim juristic discourses of territoriality and its association with migration. In premodern discourse, territoriality is often divided into the Abode of Islam, meaning a territory ruled by a Muslim power, and the Abode of War, meaning a territory ruled by a non-Muslim power. Alotaibi shows that the discourse of premodern jurists and modern reformers is not always so neatly divided into a binary system. What’s more, modern reformers reformulate the premodern discourse to fit an international world order governed by larger bodies such as The United Nations. After a review of the sources, the author attempts to identify a new path forward for “peaceful coexistence” and “good international relations.”\nIs the work clearly and accurately presented and does it cite the current literature?\nThe author makes it clear that some premodern and modern authors divide the Islamic world into two parts. This was well said by Khaled Abou El Fadl nearly three decades ago:\n“Premodern Muslims discussed many different types of abodes, depending on the particular objective being considered, whether territorial, theological, jurisdictional, or legal. But in all cases, it is a gross oversimplification to claim that in Islam, the world is divided into the abode of Islam the abode of infidels or war' (p190).1\nThe author also mentions some of the complexity of premodern discourse, but lacking from engagement is mention of just how complex the situation in premodern legal works was. A look at Abou El Fadl’s works would greatly enhance this paper.2 Assumptions of a binary world divided into two territories is, as the author hints at, a rehash of orientalist views of Islam’s dichotomous worldview in which Muslims viewed Islam as in a constant state of war. But the author might have also mentioned that many modern Muslims have adopted this view without questioning its assumptions, which would help contextualize the essay and the causes of misunderstandings of the concepts of territoriality and jurisdiction.\nThe article’s organization could have benefited from streamlining a thesis. By the end of the article, I was left without an idea of what contention was being made. Was there supposed to be an argument about a new model for coexistence, and if so, why was this dealt with in the short penultimate paragraph? What the article seems to be is a review of some of the issues associated with territoriality and jurisdiction. An organizational structure fitting this purpose might have been more beneficial to the reader. For example, the author might have organized around the rulings emanating from one’s of religion or the jurisdiction.\n\nThe paper engages the well-known and foundational texts of the major schools of Islamic law in the premodern period. There is some engagement with the texts, but many of the citations are translations without enough context or are unclear translations. For example: “Muslim law covers the people of the dhimma (non-Muslims with protected status) if the people of the heresy did not fight against the people of Sunnah, for it is the abode of Islam” (p4). The same is to be said of the translation and citation – which also includes an incomplete sentence – of Shafi’i in paragraph 2, p.9: “When contextualizing some Hadiths in this regard…” Throughout the paper, the phrase “those who…” is used abstractly without clear referents. See two examples at the beginnings of paragraphs on p.5. There are many awkward or hard transitions with unclear referents that make it difficult to follow the rationale of the statements, for example, on p.4: “The definitions of contemporaries who…”. The flow of content does not always logically follow due to a lack of signposts or organization. See for example p.6 where the author transitions to Ramadan’s assertions that appeared many paragraphs prior.\nThe paper would be much improved if terms were used consistently. In the introduction, Dar al-Islam is referred to as the “land of Islam or peace” and the Dar al-Harb as the “land of war or blasphemy”. But at the end of the introduction, they are called the “house of Islam” and the “house of infidelity,” respectively; later in the translation of al-Kasani’s text the term “abode” is used. And there are more examples of inconsistency. It should also be noted that the translation of Dar al-Harb as the land of “blasphemy” does not seem to equate to the general sense of the meaning of premodern jurists. The alternate to Dar al-Harb is typically – though there are more – Dar al-Kufr or Dar al-Shirk, both meaning something like the abode of unbelief or association (with other gods), neither of which mean blasphemy. Some other better word choices might be made. Instead of “ancient” and “old” to refer to juristic activity before the modern period, “premodern” or “classical” might be used instead. For clarity, the translation of ma’sum and ‘isma as “protection” might be termed “inviolability (of person and property).” This would be helpful to distinguish ‘isma from hirz. Lastly, in my knowledge, the more common term used in juristic discourse for a pact of safety is Aman, as in ‘ahd al-aman, rather than Amn.\nThe list below notes some of the factual errors, unclear statements, and citation issues.\nP.1: “In addition, others (e.g. Alzuhaili, 1981) have added a third term, ‘Dar Al-Ahd’ (house of truce or treaty), to the existing Islamic division of the world between Dar Al-Islam and Dar Al-Harb.”\n\nThe Dar al-Ahd was spoken of since at least the 10th century, and the way the sentence is written makes it seem as if the modern reformer created the category.\n\nP.8: “Secondly, contemporary jurists such as Al-Qaradawi (2010) and Topolyak (1997) have employed a new concept of “safety” to emphasize that the “house of Islam” at present is no longer confined to the historical boundaries of the old “house of Islam”. Rather, they suggest, it has expanded greatly to include every spot in which there is a Muslim who acknowledges to his Lord the two testimonies and establishes his laws in himself according to his ease and ability.\"\n\nThis “new” view is the same one espoused by premodern jurists who also thought that Islam is wherever a Muslim brought it, which is acknowledged in the paper (p. 7). But the paper does not cite Mawardi who made this claim. It would be helpful to show or engage the sources to clarify what is new about the view or perhaps why it mattered to the researcher or jurist being cited. It is unclear what this view adds to the development of the discourse on territoriality and jurisdiction from the premodern to the modern period.\n\nP9: “This is a level of magnitude unmatched in the past”.\nThis claim proceeds a statement that millions of Muslims live under non-Muslim governments. But Muslims lived at least partially under non-Muslim governance in many colonial territories, not to mention the Mongol invasion of Islamic lands in the 13th century or the large populations of Muslims living in Sicily in the 11th, to mention just a couple examples. Some nuance would be helpful here, especially since the abode of Islam might still be considered as such even after being conquered by a non-Muslim ruler such as a Mongol. For instance, according to some in the Hanafi school, wherever Islamic law is applied, the territory is to be considered the Dar al-Islam.\n\nP. 9-10: “In addition, whereas Muslim people of the old abode of Islam did not leave unless they were merchants, involved in Jihad or as tourists or visitors to other places for limited periods, today, they are in many parts of the world in which Muslims permanently reside, either through long-standing affiliations to those regions or by action.\"\n\nWhat is the basis for this contention? Is the theory of juristic discourse conflated with the fact that they were? Some proof of this contention is necessary to establish its veracity. Or alternatively it might be said that the jurists held positions that might theoretically be followed by a believer. I might suggest a review of migration studies or social history of the premodern period for explicit examples.\n\nP.10: “Rather, most wars in this time take place in the homes of Muslims; a scenario that did not occur in the time of the ancient jurists, as they only envisioned war between the Muslims as a whole, and the non-Muslims as a whole.”\n\nThis is difficult to understand based on other claims in the article that Orientalists such as Bernard Lewis (and Schacht might also be included here) associate a dichotomous view of the world as ones always at enmity with one another. I am not sure that the envisioning of war between Muslims and non-Muslims was of religious origin, but one of medieval societies. In the medieval period all nations were seen in conflict unless some agreement had been made. Something else to consider is whether jurists ever discuss intra-Muslim conflict, rebellion (Kharijites for example), or living under an unjust Muslim ruler.\n\nAre sufficient details of methods and analysis provided to allow replication by others?\nWhile the article outlines some of the issues associated with premodern juristic discourse, a deeper, precise, and more substantive dive into them would have been welcome. How did the premodern law schools and their rulings on territoriality develop over time? The same could be said about the discussion of the Muslim reformers. There is mention of Abu Zahra, the twentieth-century Egyptian reformer, and al-Zuhayli, a Syrian jurist, but little to no mention is made of other well-known modern reformers (Shaltut, for example) or how they relate. Nor is there mention of the impacts of colonialism on reformers and its significance to the development of ideas of territorial boundaries in Islamic international law. Also unclear to me is the lack of specificity of the “changed nature” of international relations that resulted in reinterpretations or abrogation of premodern juristic discourse, though the author does well to mention the United Nations. Likewise, there is no discussion of premodern juristic discourse on the abrogation of the necessity for all Muslims to migrate from foreign territory to Islamic territory. All of these left me with methodological questions: why were certain modern jurists or researchers chosen as representatives of new models of juristic discourse territorial division, and how do they fit into their contexts? What were some of the contradictions in the sources that they were working out?\nAre all the source data underlying the results available to ensure full reproducibility?\nThere are references to Islamic magazines or journals that are not easily locatable and cannot therefore be accessed and analyzed by a reviewer (Alshirazi is one: the link leads to the home page). Likewise, one source citation (Eqigg) links to an unpublished PDF or produced by an unknown source that might become irretrievable. The author cites Alfaitori but does not engage his ideas. It is unclear whether Bork is a Muslim jurist, as seems evident from the article. Volume and page numbers of books should be included for reproducibility. For example, Ibn Qudama and Ibn Qayyim al-Jawziyya are cited without reference to volumes or page numbers.\nDoes he fulfill his purpose?\nThe article promised to identify a new path forward for peaceful coexistence and better international relations, but they are only emphasized in a final paragraph. No specific path is identified, outlined, or examined. Instead, the article is a review of different juristic discourses, which can be found in many academic articles and books published over the last three decades or more. The article might better foregrounded a path of peaceful coexistence by relating it to the history of juristic discourse. In the end, I did not leave with a better understanding of the overlap of migration with territoriality and jurisdiction, nor of how new theories of territoriality by Muslim jurists helps the author identify a better path forward between Muslim and non-Muslim communities.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "144595", "date": "06 Sep 2022", "name": "Che Zarrina Sa’ari", "expertise": [ "Reviewer Expertise Islamic thought" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 should further explain that this juristic division of the world is used/practiced in political matters or dividing borders in the contemporary world and give evidence to support this claim. Specify the states or countries that practice this division especially from Hanafi jurists.\n\nIf the author cannot prove that this juristic division of the world is used/practiced to show the borders or states, and that it has only been discussed in the jurists' books, what is the importance of this issue being discussed in this article? Please provide convincing arguments.\n\nAdd more current sources (within 5 to 10 years back) to support your data.\n\nThe author cited many quotations from many scholars/books that show his weakness in dealing with and analyzing collected data. Thus, he could not show his own understanding of the issues discussed.\n\nThe terms used are not consistent, please relook to the translation of the term Dar al-Islam, Dar al-Harb and Dar al-Ahd.\n\nThis article aims to identify and introduce new approaches to expand the concept of homeland, promote peaceful coexistence, and resolve the issue of religious and residency differences as well as develop a new path forward for peaceful coexistence and better International relations. However, the author only highlighted them in the conclusion without identifying, examining, outlining, and discussing specific new paths.\n\nThis article can be said to be written in the form of a review paper and very little highlights its own ideas in building a new idea to face the juristic division of the world.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-794
https://f1000research.com/articles/12-958/v1	10 Aug 23	{ "type": "Research Article", "title": "Low back pain functional disability and associated factors: A cross-sectional study in Peruvian amateur athletes", "authors": [ "Jaime Rosales-Rimache", "Yaritza Chavez-León", "Yaritza Chavez-León" ], "abstract": "Background: Low Back Pain Functional Disability is one of the most critical physical limitations found in athletes, even leading to their temporary or permanent retirement. In this sense, this research aimed to evaluate the frequency of lumbar functional disability and its associated factors. Methods: We designed transversal research and evaluated athletes from the Peruvian Sports Institute (IPD) of Ica in athletics, basketball, and table tennis. We administered a file to obtain demographic and epidemiological data. We identified low back pain with the DN4 instrument (Douleur Neuropathique 4 Questions) and lumbar functional disability with the athlete's disability index. Results: We evaluated 130 amateur athletes (table tennis=30, basketball=50, and athletics=50), with an average age of 23.9±8.2 years, and constituted 63.9% of men. 30% (CI95: 22.3-38.7%) of the population presented lumbar functional disability (moderate 56.4%, severe 30.8%, and very high 12.8%). Factors associated with lumbar functional disability were age (PR: 1.05, CI95: 1.01-1.09), female sex (PR: 2.21, CI95: 1.08-4.50) and neuropathic pain (PR: 0.35, CI95: 0.16-0.80). Conclusions: The frequency of lumbar functional disability is high among athletes who practice table tennis, basketball, and athletics. It is crucial to strengthen fitness programs accompanied by a periodic medical evaluation that allows the timely identification of injuries and potential functional disability due to low back pain.", "keywords": [ "Sport", "Athletics", "Basketball", "Table tennis", "Low Back Pain Functional Disability" ], "content": "Introduction\n\nLow Back Pain Functional Disability is an alteration generated by a musculoskeletal problem at the lumbar level that can manifest with pain.1 Globally, there are no figures estimated by official institutions such as the World Health Organization; however, there are authors who affirm that the disability caused by low back pain reaches figures between 5.45 and 6.73 million people affected between the years 1990 and 2016.2 Other international studies have shown that this condition is present in a higher prevalence rate among athletes, compared to the general population, even doubling said rate.3 In the case of America, the evidence at the Latin American level is quite limited; however, in the United States, low back pain and its functional disability is the second most crucial health problem in the loss of years of healthy life.4 In Peru, musculoskeletal diseases occupy the sixth place of disease burden. In the specific case of low back pain, there are no national registries that show its prevalence or incidence in the general population; however, various studies show that it is a common reason for the loss of workdays,5 calculating losses of 149 million work days per year due to low back pain.6 However, low back pain and the functional disability generated are more prevalent in certain risk groups, such as those who handle heavy loads, workers with non-ergonomic positions, and people dedicated to sports activities.7\n\nApproximately it is estimated that 80% of people experience at least one episode of back pain during their lives.8 This rate varies between 1% and 30% among people dedicated to sports activities depending on age, training program and the type of sport practiced.9–11 Sports are classified according to static-dynamic activity in 3 levels: Low (e.g., golf, cricket, and bowling), medium (American football, rugby, jumping), and high (cycling, triathlon, boxing, and canoeing).12 Among highly competitive athletes, low back pain is one of the most common causes of game loss and lifelong disability.3 Disability assessment is one of the most critical components of the health care service, and self-reported measures of disability are currently the most common way to assess patients with back pain.13 In this sense, various specific indices have been developed to assess the functional status of the lower back in the population at risk.14 The most popular are the Roland-Morris Disability Questionnaire15 and the Oswestry Low Back Pain Disability Index.16 However, these scales were designed to assess back pain in the general population with low physical and functional activity performance.17 These tools may have some limitations in assessing the specific physical demands, deficits, and temporary disabilities of athletes and their specific exercise and activity levels; Furthermore, the expectations of athletes regarding their functional status and therapeutic results differ significantly from those of the general population.18 However, despite the limitations above, there are instruments such as the Micheli Functional Scale (MFS) to assess pain and functional levels in young athletes18 and the Athlete Disability Index (ADI) to assess functional disability of the lower back in athletes.3\n\nIn Peru, the most formally practiced sports are soccer and volleyball. However, sports such as tennis, basketball, and athletics are being promoted by the Peruvian Sports Institute. Tennis includes movements that involve muscle tension, rotation, and stretch-shortening cycles with the participation of different muscle groups in the legs, shoulders, hips, and back, among others.19 The ballistic and repetitive trunk movements required in tennis have been associated with radiological abnormalities in the lumbar spine20 and with evidence of low back pain in elite young tennis players.21 It has even been observed that excessive mechanical force can injure the vertebrae in the lower back.22 Basketball is characterized as a fast and intense sport involving accelerations, sudden stops or landings from a jump, body contacts, and handling of the ball during fast movements.23 These movements can increase the risk of low back pain. However, little scientific evidence associates low back pain exclusively with basketball. The prevalence of low back pain in basketball players is very high. Close to 50% have been reported in players who have practiced the sport for 12 consecutive months.24 Low back pain is known to be the third most frequent ailment among basketball players, preceded by ankle and knee injuries.25 Athletics is made up of many sports that involve movements such as the role of internal rotation of the upper arm in striking and the influence of elastic energy and muscle pre-stretch on stretching and shortening actions,26 which are subject to dysfunctional movements if they are not supervised.27 This scenario increases the risk of low back pain. It can be exacerbated by characteristics attributed to the athlete, such as flexibility, lower limb length discrepancy, sacroiliac movement dysfunction, and preseason training intensity.28\n\nOur research aimed to determine the frequency of low back pain functional disability and the identification of associated factors. We believe that early identification of low back pain and functional disability associated with the practice of sports in amateur athletes can improve primary prevention strategies in amateur athletes, reducing the risk of recurrent musculoskeletal injuries in their professional sports life.\n\n\nMethods\n\nWe designed a cross-sectional study with an analytical component to evaluate amateur athletes who practiced table tennis, basketball, and athletics and are part of the Peruvian Sports Institute of the district of Ica. Between October and November 2018, we evaluated 130 athletes selected for convenience, in continuous activity, and who practice sports such as tennis, basketball, and athletics. On the other hand, we exclude athletes under 12 years of age and over 50 years of age, with special abilities, and with spinal surgery.\n\nWe performed the post-estimation of power in a logistic regression model.29 We considered a proportion of low back pain (as a proxy measure for functional disability due to low back pain) of 0.156 for athletes who play basketball and 0.077 for those who play tennis, according to what was reported by Ansari S. et al.30 Likewise, we use a confidence level of 95% and an adjustment coefficient R2 of 0.25, obtaining a power of 81.2%.\n\nData collection form: we prepared a form that allowed us to obtain demographic data (age, sex), and anthropometry (weight and height, which were measured with the Weighing Scale With Height & Weight [Great Medic, China] with a precision of 100 grams and 0.5 cm), risk factors (practice time (number of hours per week for the development of sports activity), handling of heavy loads (manual handling of weights over 25 kg (men) or 15 kg (women)) as part of work activity), cigarette consumption (continuously in the last two months), alcohol consumption (continuously in the last three months) and obesity (BMI greater than 29.99 kg/m2).\n\nAssessment of low back pain: we used the DN4 scale (Douleur Neuropathique 4 Questions) validated in Spanish,31 whose purpose was the differential diagnosis between neuropathic pain and non-neuropathic pain of a somatic nature. The Assessment of the presence of neuropathic pain was made from 10 items focused on the description and signs of pain perceived by the patient. The answers were evaluated dichotomously (No/Yes). The maximum score was 10 points, and a total score greater than or equal to four was classified as neuropathic pain, while scores less than four were classified as non-neuropathic pain.\n\nAthlete’s disability index: we applied a questionnaire made up of 12 questions, of which each question had four possible options and was scored from 0 to 3 with higher values indicating a more severe condition (no pain/restrictions = 0 points; no restrictions some pain = 1 point; some restrictions to limit pain = 2 points; and restricted due to pain = 3 points). The sum of the scores for the 12 questions makes the total score between 0 and 36. To calculate the athlete’s level of functional disability due to low back pain, indicated as a percentage, the total score was divided by 36 and multiplied by 100. This instrument has been validated by Zamani et al.3 Those with scores between 0% and 20% were considered athletes without a functional disability due to low back pain; and with disabilities greater than 20% and subclassified into moderate (21%-40%), severe (41%-60%), very high (61%-80%), and sports retirement (81%-100%) disabilities.\n\nWe describe the characteristics of athletes with absolute and relative frequencies. We compared Low back pain functional disability by the independent variables using the chi-square test. We also compared the total scores according to the type of sport with the nonparametric Kruskal-Wallis test. The factors associated with functional disability due to low back pain were identified using a generalized linear model with a Poisson family and Log link. We calculated the prevalence ratio as a measure of association. We consider p<0.05 as a significant value in all hypothesis tests. We performed calculations using Stata Corp (Stata College Station, TX, USA), version 17.\n\nThe study was authorized by the Ica Regional Sports Council with Official Letter N° 344-2018-IPD-CDR-Ica, issued on August 13, 2018. Since people were evaluated and data was obtained by applying various instruments indicated in the investigation, we obtained written informed consent with the presence of a witness (it was the coach on duty who provided evidence that the objectives of the study, benefits, and risks were explained to the potential participant). The information generated was encrypted and stored with a security key and exclusive access to the principal investigator.\n\n\nResults\n\nTable 1 shows the descriptive characteristics of 130 amateur athletes from the Peruvian Sports Institute at the Ica campus, of which 63.9% were male. The mean age was 23.9 ± 8.2 years. Regarding the anthropometric characteristics, the average weight and height were 64.8 ± 10.8 kg and 1.65 ± 0.08 m, respectively. The body mass index had a mean of 23.8 ± 3.9 kg/m2 (min: 16.7 and max: 36.3). The presence of social habits such as cigarette and alcoholic beverage consumption was low. The frequency of athletes in table tennis, basketball, and athletics was 23.0%, 38.5%, and 38.5%, respectively.\n\nTable 2 shows the characteristics of pain and functional disability due to low back pain. We found that all the athletes reported low back pain, and depending on the type, it was of neuropathic and non-neuropathic origin in equal proportions. The mean perceived pain score was 3.5 ± 1.8 (min: 0, max: 7 points) according to the DN4 instrument. 61.5% reported low back pain lasting approximately 1 to 3 continuous months. Functional disability due to low back pain presented a mean percentage score of 17.3 ± 18.3% (min: 0.0% - max: 69.4%). Functional disability due to low back pain occurred in 30% (CI95: 22.3-38.7%), of which 56.4% had a moderate disability, and 30.8% presented severe disability. No athlete with a disability that generated sports withdrawal was observed.\n\nTable 3 shows the results of the association with functional disability due to low back pain in bivariate analysis. We identified that gender, age, obesity, duration of low back pain, and pain were factors associated with functional disability due to low back pain. We observed that functional disability was higher among women and older athletes. Likewise, those who manifested pain for up to 12 months represented almost all athletes with low back pain functional disability. In addition, athletes with neuropathic pain had a higher frequency of functional disability than those with non-neuropathic pain. The type of sport practiced was not a determining factor in functional disability due to low back pain. Additionally, we compared the athlete’s disability index scores for table tennis, basketball, and athletics with medians of 6.9, 9.7, and 13.8, finding no significant differences (p = 0.470, Kruskal-Wallis test).\n\na Median and interquartile range.\n\nb Mann-Whitney nonparametric test.\n\nc Fisher's exact test.\n\nd Yates-corrected chi-square.\n\nTable 4 identifies the factors associated with low back pain functional disability in multivariate analysis. We evidenced that, for each increased year of life, the risk of occurrence of functional disability due to low back pain increases significantly by 5% (PR: 1.05, CI95: 1.01-1.09). Female athletes have approximately twice the risk of low back pain functional disability compared to male athletes (PR: 2.21, CI95: 1.08-4.50). Athletes with non-neuropathic pain had a 65% lower risk of functional disability due to low back pain than those with neuropathic pain (PR: 0.35, CI95: 0.16-0.80). No type of sport was associated with low back pain functional disability.\n\na Generalized linear model with Poisson family and Log link.\n\n\nDiscussion\n\nOur results show that sports such as table tennis, basketball, and athletics are not significantly associated with the occurrence of low back pain functional disability; however, other factors do promote its condition, including age, female sex, and perceived pain in the lumbar region. Although our study population consisted of amateur athletes between the ages of approximately 16 and 35, it is essential to consider that there are structural differences in the spine of an adolescent compared to an adult. The spine of a child or adolescent at the level of the nucleus pulposus is moderately hydrophilic and more significant than that of an adult. It allows more effective absorption of force and its central distribution to the adjacent vertebrae.32 With adulthood, the composition of the nucleus pulposus begins to change and generates a distribution of force to more peripheral areas of the disc.33 Therefore, age is a determining factor in spinal injury, especially at the lumbar level. Injuries and fractures are more common in the adolescent spine, with numbers as high as 47% of young athletes, and are believed to be caused by incomplete bone maturation present in the neural arch.34,35\n\nWe found that 30% of athletes have low back pain functional disability at different levels (moderate, severe, and very high). This figure is much lower than that reported in other investigations. Noormohammadpour et al. reported that close to 90% of the athletes had mild lumbar functional disability using the Oswestry and Roland Morris scale,36 not finding categories of severe and very high disability. Reiss et al. also found higher scores for lumbar functional disability among Jiu-Jitsu athletes (10 points) compared to control subjects (6 points), using the Quebec scale.37 As observed in previous research, the disability rate is very high compared to our findings. This situation could explain by the instruments used (e.g., Oswestry, Roland-Morri’s questionnaire, and Quebec scale), which are oriented towards evaluation. Of low back pain functional disability in the general population. These instruments are beneficial in situations related to clinical conditions in the patient,38 and they have not been designed to evaluate athletes.36 The etiopathogenesis of pain is different in athletes compared to the general population. Therefore, the functional capacity is also different. This situation could lead to overvaluation and incorrect classification of lumbar functional disability among athletes.\n\nAccording to our results, all athletes, without exception, presented low back pain at different levels of pain and origin, which denotes a severe problem with pain control and management. Other investigations have shown a very high prevalence of low back pain among athletes; for example, Fett et al. reported a prevalence of low back pain of 88.5% in elite athletes.39 In general, the rate of low back pain among athletes is very high, according to international evidence, compared to the general population; however, from the point of view of sports medicine, pain is a manifestation attributed to sports practice that is reduced and eliminated by various pain control programs. However, the constant manifestation of pain and its level leads to the loss of functionality of the lumbar region in the athlete; this, in turn, generates limitations in sports practice and may even end in sports retirement.32\n\nThe prevalence of low back pain and its functional disability may vary depending on the sport being practiced. For example, it has been described that up to 11% of gymnasts and 50% of soccer players presented low back pain. Recurrence of the type of injury that causes the pain again may be sport-specific. For example, herniated lumbar discs are more common in soccer players and weightlifters, disc degeneration and spondylolysis are more common in gymnasts, and traumatic lumbar spine injuries are more common in wrestlers and hockey players.40 However, there are sports that have a lower risk of low back pain and functional disability. Table tennis is a sport that was taken as the reference group in our study, considering that the risk of low back pain is low, even in highly elite athletes.21 However, the frequency of low back functional disability among table tennis players was the lowest compared to the other two sports evaluated. In fact, due to the movements involved in basketball and athletics, the risk level of low back pain is much higher, especially in those who also handle heavy loads and have low back flexibility problems.41\n\nPeople involved in impact sports appear to have risk factors for specific spinal pathologies that correlate with the load and repetition demands of specific activities. For example, elite athletes who engage in longer, more intense training have a higher incidence of the degenerative disc disease and spondylolysis than athletes who do not. On the other hand, the data suggest that amateur athletes can be protected from low back injuries prior to physical conditioning.41 Athletes are often well conditioned with greater flexibility and higher pain thresholds than the general population. These characteristics can serve as protective factors; however, athletes with high demands on the lumbar spine usually do not tolerate functional limitations in their activities. Low back pain among athletes is approximately 30%, which can even be higher in elite athletes, such as Olympic competitors. Low back pain can even be attributed to lumbar disc degeneration.42\n\nThe study had some limitations, including the need for temporality, typical of a cross-sectional design, and the number of athletes evaluated. Although our study seeks to identify factors associated with low back functional disability, in order not to bias our results, we used a generalized linear model including potential confounders to adjust the association measures. We could not include instruments for the biomechanical study of athletes, which would have been necessary to understand better the etiopathogenesis of low back pain and functional disability.\n\nTimely identification of low back pain and functional disability among athletes makes it possible to improve primary prevention programs aimed at reducing sports injuries. In the case of amateur athletes starting a sports season, they must undergo a physical and medical evaluation, which allows the identification of specific risk factors, such as previous injuries that have not been completely rehabilitated or muscle weakness and inflexibility. It is recommended that physical conditioning be carried out over several weeks, with a gradual increase in the frequency and intensity of training, to achieve a safe adaptation according to the demands of the sport. The growth process in the athlete is a crucial factor that must be considered in the training stage since young athletes are prone to lose muscle flexibility and imbalance, which predisposes them to injury.43 In this sense, the medical staff, evaluators, and even the coach must be aware of the changes in the spinal development of the young athlete to help diagnose and treat spinal injuries in a timely manner.\n\n\nConclusions\n\nAmateur athletes who practice table tennis, basketball, and athletics have a high frequency of low back pain functional disability. Likewise, low back pain is present in all athletes, with significant differences by type of sport practiced. Female athletes are more prone to low back pain functional disability, and the risk of this increases significantly with age. In this sense, it is essential to identify the main risk factors for injuries among athletes.", "appendix": "Data availability\n\nFigshare: Dataset peruvian athletes.csv, https://doi.org/10.6084/m9.figshare.23101466.v2. 44\n\nThis project contains the following underlying data:\n\n- Data.csv\n\n- Codebook_Data.csv\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe appreciate the support of the coaches of the Peruvian Institute of Sport in Ica.\n\n\nReferences\n\nHartvigsen J, Hancock MJ, Kongsted A, et al.: What low back pain is and why we need to pay attention. Lancet. 2018; 391(10137): 2356–2367. PubMed Abstract \| Publisher Full Text\n\nWu A, Dong W, Liu S, et al.: The prevalence and years lived with disability caused by low back pain in China, 1990 to 2016: findings from the global burden of disease study 2016. Pain. 2019; 160(1): 237–245. 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PubMed Abstract \| Publisher Full Text\n\nFritz JM, Irrgang JJ: A comparison of a modified Oswestry Low Back Pain Disability Questionnaire and the Quebec Back Pain Disability Scale. Phys. Ther. 2001; 81(2): 776–788. PubMed Abstract \| Publisher Full Text\n\nFett D, Trompeter K, Platen P: Back pain in elite sports: A cross-sectional study on 1114 athletes. PLoS One. 2017; 12(6): e0180130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNagashima M, Abe H, Amaya K, et al.: Risk Factors for Lumbar Disc Degeneration in High School American Football Players: A Prospective 2-Year Follow-up Study. Am. J. Sports Med. 2013; 41(9): 2059–2064. PubMed Abstract \| Publisher Full Text\n\nLawrence JP, Greene HS, Grauer JN: Back pain in athletes. J. Am. Acad. Orthop. Surg. 2006; 14(13): 726–735. Publisher Full Text\n\nAttal N, Ayache SS, Ciampi De Andrade D, et al.: Repetitive transcranial magnetic stimulation and transcranial direct-current stimulation in neuropathic pain due to radiculopathy: a randomized sham-controlled comparative study. Pain. 2016; 157(6): 1224–1231. PubMed Abstract \| Publisher Full Text\n\nPatel DR, Kinsella E: Evaluation and management of lower back pain in young athletes. Transl. Pediatr. 2017; 6(3): 225–235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosales J: Dataset peruvian athletes.csv. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "228444", "date": "19 Dec 2023", "name": "Gopal Nambi", "expertise": [ "Reviewer Expertise Musculoskeletal and Sports physiotherapy" ], "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 title of the study is not clear and self-explanatory. Abstract:\nMention a brief background of the study – it is not clear. Mention the study design, study duration and study setting. Mention the character of the study participants. The outcome measures used in the study are not sufficient. Mention the statistical tests used for the study. Present the reports with 95%CI with upper and lower limits for all outcome variables. The conclusion of the study is not clear; it should be drawn based on study reports.\nManuscript:\nPlease summarize the introduction part as it is not scientifically written. The need for the study is not mentioned clearly in recent references. Mention the gaps monitored by the researcher in the previous studies. Include the clinical significance of the study for the researchers, clinicians and patients. Mention the study design, study duration and study setting. Mention the character of the study participants. Include the diagnostic criteria of the disease and its ICD classification. Mention who has diagnosed the participants and their qualifications and experience. Mention the outcome variables with their reliability and validity. Include the sample size calculation with a suitable reference. The samples included in the study are not sufficient to generalize the results. The statistical tests included are not apt for this study. Present the reports with 95%CI with upper and lower limits for all outcome variables. Summarize the discussion part. Include the mechanism behind the association between these variables. The conclusion of the study is not clear; it should be drawn based on study reports. Include the real-time limitations faced by the researcher. Include the future recommendations of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "260770", "date": "24 Apr 2024", "name": "Zarina Zahari", "expertise": [ "Reviewer Expertise Musculoskeletal Physiotherapy" ], "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\nTitle- not satisfactory. should the population be specific, eg, Functional disability among Peruvian amateur athletes with neuropathic LBP OR the association between functional disability and demographic factors among ...... abstract - insufficient details in obj of the study, the method- demographic & epidemiological data? lack of details in design.. cross-sectional?, statistical analysis result: unable to relate with obj of the study conclusion: does not relate to findings\nText introduction - satisfactory but most of the refs are old >5 years. The problem statement is not clear. The associated factors should be spelled out clearly in the last paragraph method: the subheading of study area and participants was not synchronise with the text. Lack of sample size calculation & its justification. Pls write a clear research instruments with their reliability & validity values & cut-off point (if any). Statistical analysis was unclear and not synchronize with the study obj. The use of DN4 is to identify radiculopathy among LBP sufferers and not to detect LBP. May need to use other instrument. results: the descriptive findings of demographic, anthropometric & functional disability are fine but I would suggest if they can be merged into 1 table. I'm not sure of table 3 & 4. They are quite confused to match with the obj. Why need 2 different association analysis? discussion: satisfactory & sound conclusion: I'm not clear of \"frequency\" of LBP functional disability. is it referring to prevalence or number of incidence?\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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-958
https://f1000research.com/articles/11-1180/v1	17 Oct 22	{ "type": "Review", "title": "Integration of single-cell RNA sequencing and spatial transcriptomics to reveal the glioblastoma heterogeneity", "authors": [ "Adrian Perdyan", "Urszula Lawrynowicz", "Monika Horbacz", "Bozena Kaminska", "Jakub Mieczkowski", "Adrian Perdyan", "Urszula Lawrynowicz", "Monika Horbacz", "Bozena Kaminska" ], "abstract": "Glioblastoma (GBM), a deadly brain tumor, is still one of the few lasting challenges of contemporary oncology. Current therapies fail to significantly improve patient survival due to GBM’s tremendous genetic, transcriptomic, immunological, and sex-dependent heterogeneity. Over the years, clinical differences between males and females were characterized. For instance, higher incidence of GBM in males or distinct responses to cancer chemotherapy and immunotherapy between males and females have been noted. However, despite the introduction of single-cell RNA sequencing and spatial transcriptomics, these differences were not further investigated as studies were focused only on exposing the general picture of GBM heterogeneity. Hence, in this study, we summarized the current state of knowledge on GBM heterogeneity exposed by single-cell RNA sequencing and spatial transcriptomics with regard to genetics, immunology, and sex-dependent differences. Additionally, we highlighted future research directions which would fill the gap of knowledge on the impact of patient’s sex on the disease outcome.", "keywords": [ "single-cell RNA sequencing", "spatial transcriptomics", "glioblastoma", "heterogeneity", "immunology", "sex" ], "content": "Introduction\n\nGlioblastoma (GBM) is one of the deadliest human tumors, with a 14-month median survival length and five-year overall survival (OS) of approximately 6.8% (https://seer.cancer.gov/). Despite intensive research and the introduction of novel therapeutic regimens, the survival rate has not been improved in the last decades.1 The disappointing results are mainly due to ineffective surgical resection and rapid local progression.2 Moreover, there are no useful biomarkers to detect the emergence of GBM, and the early course of the disease is often asymptomatic.3 Nowadays, GBM is treated with surgery, temozolomide-based (TMZ) chemotherapy, and radiotherapy.2 The failure of conventional and targeted therapies is most likely due to intratumoral heterogeneity, intrinsic mechanisms of cell death resistance (due to a high frequency of PT53 and PTEN mutations) and redundant prosurvival signaling pathways.4 Additionally, in accordance with recent reports, patient sex may have a major impact on GBM therapeutic outcomes and prognosis. The response rate to conventional therapies is higher in females, whereas immunotherapy works better in males due to higher molecular and cellular heterogeneity of glioma cells.5 Moreover, a mutation in the isocitrate dehydrogenase 1 encoding gene, IDH1, contributes to better chemotherapy outcomes and prolonged OS in males only.6–8 On the other hand, hypermethylation of the promoter of the MGMT gene coding for O6-methylguanine-DNA methyltransferase enhances the effect of TMZ chemotherapy and prolongs OS in females.8,9 On the other side, there are several negative prognostic biomarkers which are listed in the Figure 1.8,10–13\n\nLegend: Btg2 – B cell translocation gene 2; CDK – cyclin-dependent kinase; FZD7 – frizzled class receptor; Hmg2 – high mobility group box 2; IDH1 – isocitrate dehydrogenase 1; IL – interleukin; MGMT – O6-methylguanine-DNA methyltransferase; MHC – major histocompatibility complex; p63 – transformation-related protein 63; RB1 – retinoblastoma protein 1; Shh – sonic hedgehog human; TNF-α – tumor necrosis factor alpha.\n\nOver ten years ago, the first GBM transcriptional subtypes were identified with a partial enrichment of PDGFRA or EGFR alterations.14,15 However, with the recent introduction of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST), new cellular states were exposed. Interestingly, distinct states may co-exist within the tumor with variate frequency and are associated with genetic alterations.4,16 Moreover, GBM heterogeneity manifests in unique developmental states of GBM cells in the tumor. GBM mimics mechanisms of neural development, thus it contains GBM stem cells (GSCs) which are thought to play a major role in possessing tumor-propagating potential and exhibiting preferential resistance to radiotherapy and chemotherapy.17,18 Finally, GBM has a heterogeneous and highly immunosuppressive TME composed of normal brain residents such as neurons, astrocytes, oligodendrocytes, and microglia, immune system infiltrating cells including mostly monocytes/macrophages, as well as endothelial and mesenchymal cells.19,20 Glioma-associated microglia and macrophages (GAM) are the most abundant population of immune cells, constituting up to 30% of the tumor mass.21–23 Despite the high content of glioma-associated microglia and macrophages (GAM), GBM is considered immunologically “cold” due to relatively poor infiltration of activated T cells and anergic state of those present in TME.24 Moreover, the broad range of cell-to-cell interactions between cancer cells and components of the TME affects the biological status of the tumor with an increase in its evasion capacity and resistance to treatment.25\n\nIn summary, a better understanding of multilayer GBM heterogeneity, including genetics, epigenetics, developmental stages, TME, and immunology, is required to establish effective therapies.16 Thus, the purpose of this study was to discuss recent advances in exposing the GBM heterogeneity and highlight future research directions.\n\nAlthough scRNA-seq should expose a detailed functional characterization of transcriptomics of a single cell and allows integration of in vivo states with in vitro models, in principle, it provides an indirect inference of cellular interactions.26 However, with the recent introduction of spatial transcriptomics (ST), it is believed that spatial and functional organization are strictly related, especially in the context of a neuronal tissue.27 ST is a revolutionary method that enables the characterization of cellular interactions and spatial organization of examined tissues.28 Given the fact that ST is not yet at single-cell resolution, methods for the integration of scRNA-seq and ST are vital to understand the heterogeneity of GBM (Figure 2). With the rapid development of complex algorithms and machine learning technologies, a variety of tools to integrate scRNA-seq and ST were recently developed, which enabled a precise characterization of GBM and its TME (Table 1).29–35\n\nSingle-cell RNA sequencing provides high-throughput and high-resolution profiling of gene expression. However, it lacks spatial information due to tissue dissociation. Conversely, ST offers a spatial context without single-cell resolution. Currently, the spot diameter of ST and Visium platforms is 100 μm and 55 μm, respectively, capturing from one to 30 cells. Thus, to gain a spatial single-cell resolution, it is necessary to integrate both methods.\n\nOver the years, with the constant introduction of more advanced sequencing methods and the launching of The Cancer Genome Atlas (TCGA) in 2008, a few layers of transcriptional GBM heterogeneity were discovered (Figure 3) providing a classification of GBM subtypes.14,16,26 In 2010, Verhaak et al., using bulk-RNA sequencing data of 200 GBM patients from TCGA, identified four distinct transcriptional subtypes of GBM: 1) classical, 2) mesenchymal, 3) proneural, and 4) neural. The results were further validated in a combined cohort of 260 GBM patients from previous studies and confirmed the association of each subtype with certain genetic events.37–40 The classical subtype was highly associated with chromosome 7 amplification paired with chromosome 10 loss, EGFR gene alterations, and disruption of RB, Notch, and Sonic hedgehog signaling. The mesenchymal subtype was characterized by alterations in NF1 and PTEN genes affecting the AKT pathway and expression of mesenchymal markers and tumor necrosis factor superfamily and NF-κB proteins. On the other side, the proneural subtype was associated with various gene alterations, including PDGFRA, IDH1, TP53, PIK3CA/PIK3R1, and higher expression of OLIG2, SOX, DCX, DLL3, ASCL1, and TCF4. Finally, the neural subtype was associated with the expression of neuron markers NEFL, GABRA1, SYT1, and SLC12A5. However, it was later confirmed that it is not a tumor-specific subtype of GBM with a lack of gene abnormalities but resected fragments with a high contribution of normal tissues.15 Next, identified subtypes were assigned to neural cell types using transcriptomic data gene sets.41 The classical, mesenchymal, and proneural subtypes were associated with murine astrocytic, astroglia, and oligodendrocytic signatures, respectively. Interestingly, the frequency of each subtype can vary within the same tumor as multiple subtypes can co-exist or change over time and as a response to therapy.4,42 Interestingly, the prevalence of mesenchymal, neural, and proneural subtypes is higher in males than females, while the classical subtype occurs with the same frequency.10,43\n\nLegend: AC-like – astrocyte-like; CDK4 – cyclin-dependent kinase 4; EGFR – epidermal growth factor receptor gene; MES-like – mesenchymal-like; NF-1 – neurofibromatosis type 1 gene; NPC-like – neural progenitor-like; OPC-like – oligodendrocyte-progenitor-like; PDFGRA – platelet-derived growth factor receptor A gene; scRNA-seq – single-cell RNA sequencing; ST – spatial transcriptomics; TCGA – The Cancer Genome Atlas.\n\nIn 2019, Neftel et al. showed the relationships between genetic subtypes and cellular states by deconvolution of scRNA-seq and TCGA bulk data on GBMs with lineage tracing in GBM murine models.16 The researchers depicted four cellular states: 1) neural progenitor-like (NPC-like), 2) oligodendrocyte-progenitor-like (OPC-like), 3) astrocyte-like (AC-like), and 4) mesenchymal-like (MES-like) corresponding to previously established TCGA signatures (Figure 3).14 As previously mentioned, these states may co-exist within the same tumor with different frequencies influenced by genetic alterations in CDK4, PDGFRA, EGFR, and NF1, which favor a particular state, respectively. Based on distinct gene expression patterns signatures can be divided into mesenchymal (MES1-like [hypoxia-independent], MES2-like [hypoxia-dependent]) and neuro-developmental (NPC1-like, NPC2-like, OPC-like, AC-like) states.44,45 In general, GBM cells correspond primarily to one of the four states, however, each of the tumors contains at least two cellular states, with most tumors containing all four states. The most frequent hybrid states are AC-like/MES-like, NPC-like/OPC-like, and AC-like/OPC-like.\n\nOn the other side, scRNA-seq provided novel insights into GBM immunology (Figure 4), especially on the localization of GAMs within the tumor. Microglia tend to reside in the tumor periphery with the adjacent brain parenchyma, while tissue-invading monocyte-derived macrophages (MDM) are most abundant within the tumor core.46–48 Moreover, the expression of immune checkpoint receptor ligands differs in myeloid cells between tumor core and peritumoral tissue.46 Comparison of microglial cells from human IDH wild-type GBM and age-matched controls revealed substantially downregulated expression of microglia core genes and upregulated expression of inflammatory- (IFI27, IFITM3), metabolic- (LPL, APOE, TREM2), and hypoxia-associated (HIF1A, VEGFA) genes.49 In terms of sex-dependent differences, Ochocka et al. showed that microglia MHC class II-associated genes were significantly upregulated and more reactive in males than in females.50 Furthermore, Pombo et al. investigated the evolution of functional GAM profiles across disease stages by sequencing samples from newly diagnosed and recurrent GBMs. Microglia-derived GAMs were predominant in newly diagnosed tumors, but were surpassed by more heterogeneous MDMs in the recurrent ones, especially in the hypoxic tumor environment.51 Regarding T cells, Mathewson et al. showed that the inhibitory natural killer (NK) cells receptor CD161 is expressed in tumor-infiltrating lymphocytes, but absent in T regulatory cells (Tregs) or patient-matched peripheral blood mononuclear cells (PBMCs). Moreover, CLEC2D (CD161 ligand) was primarily expressed by malignant and myeloid cells, revealing similarities with the PD-1/PDL-1 (programmed death-1/programmed death-1 ligand) system.\n\nNK cells, natural killer cells, Tregs, T regulatory cells, APC, antigen presenting cells. A) Expression of CD161 receptor by NK cells and its ligand CLEC2D by malignant and myeloid cells. B) T-cell exhaustion mediated by TME cells releasing interleukin-10. C) Expression of immune checkpoints on tumor and peritumoral myeloid cells.\n\nLocal cellular interactions between tumor and cells located in TME play a major role in the adaptation of GBM and facilitate growth, infiltration, and therapy resistance, contributing to unique spatial signatures in GBM.52–54 In 2022, Ravi et al. published an atlas of spatially resolved transcriptomics of 28 specimens (20 patients) and complemented it with spatially resolved metabolomics and proteomics.26 The researchers described five spatially distinct transcriptional programs of GBM: 1) radial glia, 2) reactive-immune, 3) neural development, 4) spatial OPC, and 5) reactive-hypoxia (Figure 3). The first two were associated with high expression of astrocyte -related genes (GFAP, AQP4, VIM, CD44). Specifically, radial glia program had an increased expression of radial-glia-associated genes (HOPX, PTPRZ1) and reactive-immune program had a functional enrichment of inflammation-associated genes (HLA-DRA, C3, CCL4, CCL3) and interferon-γsignaling. The next two programs were associated with neuronal lineages (neurons or oligodendrocytes) and were named accordingly. The last program was associated with hypoxia-response (VEGFR, HMOX1, GAPDH) and glycolytic (LDHA, PGK1) genes. In order to integrate novel programs with already established bulk and single-cell classifications, spatial-weighted regression and bilateral integration of the top-scoring gene signatures were carried out, confirming an overlap between radial glia, spatial OPC, neuronal, reactive-hypoxia, and AC-, OPC-, NPC-, and MES2-like (hypoxia-dependent) states, respectively.\n\nReactive-immune program\n\nThe hybrid meta-modules described by Neftel et al. were associated with reactive-immune program suggesting a close functional relationship between AC- and MES-like states.16 In further analysis, an imaging mass cytometry-based single-cell profiling showed significant enrichment of myeloid and lymphoid cells across hybrid regions. A substantial enrichment of tumor-associated myeloid cells and T cells was found among the reactive immune program. Moreover, the mean PD-1 protein level on T cells was increased, suggesting locally enhanced immunosuppression. These findings were supported by the enrichment of CD163+ myeloid cells, which support phagocytosis and immunosuppression. Furthermore, a previous study by Ravi et al. in 2022 focused on T-cell dysfunction and indicated that exhausted T cells are preferentially located within regions with mesenchymal transcriptional programs. The study revealed that the spatial and functional interaction between the myeloid and lymphoid compartment leads to an interleukin-10 mediated T cell exhaustion.55 This was further confirmed that the enrichment of memory and exhausted T cells occur in the reactive-immune and reactive-hypoxia areas.26\n\nReactive hypoxia program\n\nThe reactive hypoxia program is associated with histologically confirmed areas of necrosis and a high prevalence of copy-number alterations (CNAs), including focal amplification of oncogenes or losses of tumor suppressors. Spatially resolved metabolomics of reactive-hypoxia regions revealed a significant enrichment of the pentose phosphate pathway, phosphoadenylate metabolism, glycolysis, and amino sugar metabolism.26 Ravi et al. showed that hypoxia and oxidative stress highly contribute to genomic instabilities, including various chromosomal alterations, that are driving forces in GBM resistance to therapies. Moreover, the reactive-hypoxia program is prevalent among non-cycling cells resulting in a S-phase arrest contributing to the genomic instability.56 Finally, based on migratory gene-expression signatures, the effect of oxidative stress on cellular migration was explored, revealing the opposing drivers of genomic diversity, resulting in clonal evolution in GBM.\n\n\nConclusions\n\nIn this mini-review, we showed a comprehensive overview of the GBM heterogeneity which was revealed by scRNA-seq and ST, and the urgent need to integrate both methods in future research. We discussed distinct layers of heterogeneity with regard to GBM genetics, transcriptomics, immunology, and patients’ sex. Worryingly, despite a growing number of clinical reports about the impact of patients’ sex on GBM prevalence, therapeutic outcomes, and prognosis, there are a few studies considering patients’ sex while using scRNA-seq or ST. Hence, in the future, it is warrant to distinguish patients’ sex in order to characterize potential differences which could have a major impact on the development of therapeutic agents and overcoming GBM treatment resistance.\n\n\nData availability\n\nThere are no underlying data associated with this article.", "appendix": "References\n\nDelgado-López PD, Corrales-García EM: Survival in glioblastoma: a review on the impact of treatment modalities. Clin. Transl. Oncol. 2016; 18(11): 1062–1071. 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PubMed Abstract \| Publisher Full Text\n\nPombo Antunes AR, Scheyltjens I, Lodi F, et al.: Single-cell profiling of myeloid cells in glioblastoma across species and disease stage reveals macrophage competition and specialization. Nat. Neurosci. 2021; 24(4): 595–610. PubMed Abstract \| Publisher Full Text\n\nBhandari A, Koppen J, Agzarian M: Convolutional neural networks for brain tumour segmentation. Insights Imaging. 2020; 11(1): 77. PubMed Abstract \| Publisher Full Text\n\nHeiland DH, Gaebelein A, Borries M, et al.: Microenvironment-Derived Regulation of HIF Signaling Drives Transcriptional Heterogeneity in Glioblastoma Multiforme. Mol. Cancer Res. 2018; 16(4): 655–668. PubMed Abstract \| Publisher Full Text\n\nGrimes DR, Jansen M, Macauley RJ, et al.: Evidence for hypoxia increasing the tempo of evolution in glioblastoma. Br. J. Cancer. 2020; 123(10): 1562–1569. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "153463", "date": "24 Oct 2022", "name": "Dieter Henrik Heiland", "expertise": [ "Reviewer Expertise Single cell omics", "spatially resolved multiomics", "computational biology", "software development", "mathematical cancer-models" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 enjoyable overview of the various layers of omic knowledge that have been gathered over the last few years. The article is well-written and provides the information expected from a mini-review. The illustrations are well laid out and clear.\nIn Fig. 2, the correct nomenclature should be taken into account. It says \"reactive glia\", which is incorrect and should be called \"radial glia\". After minimal adjustments support the indexing.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "9593", "date": "09 Aug 2023", "name": "Jakub Mieczkowski", "role": "Author Response", "response": "In Fig. 3 the wording “reactive glia” was used because it is an original wording used by the authors of the primary publication. The purpose of the article was to show and point out used nomenclature and provide a good background for further studies." } ] }, { "id": "161164", "date": "17 Feb 2023", "name": "Theo Mantamadiotis", "expertise": [ "Reviewer Expertise Cancer biology", "brain cancer", "molecular biology", "gene expression", "animal models", "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\nThe mini-review explores the very interesting observations that male patient with GBM exhibit a higher incidence and fair worse than female patients with GBM. The authors discuss many valid biological phenomena related to the tumor microenvironment and sex-specific differences among tumor infiltrating immune cells and other cell types found in GBM.\nAlthough the sex-specific biology is discussed, the review is divided into sections based on technologies, rather than biology. This approach distracts from the interesting biology.\nTable 1 seems more appropriate for a technical or bioinformatic focused review, unless the authors can demonstrate that each of these approaches has discovered something relevant to the biology of sex-specific differences.\nOther parts of the manuscript which require improvement are Figure 1 legend text. The diagram and legend could be better presented. A clear explanation of what Figure 1 shows is required.\nFigure 3 is difficult to understand with all the layers of information. I don’t understand what “Chronological..” means in the figure title. Some inner dark blue sections indicate biological information (hypoxia/development), while others indicate cell types and words like “Spatial-OPC”. Can this figure be edited to fix these inconsistencies? The figure legend needs more text to explain what is shown.\nThe manuscript needs editing to correct language errors. For example, in the Abstract, the words “However, despite..” are used together. “However” is redundant here, so should be deleted. The word ”exposed” as used in the abstract and with the body text, is wrong – the authors mean “revealed”. Another example is in the Conclusions section. Near the end of Conclusions, “it is warrant” is used but it's unclear what the context is.\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? No\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "9594", "date": "09 Aug 2023", "name": "Jakub Mieczkowski", "role": "Author Response", "response": "Table 1 seems more appropriate for a technical or bioinformatic focused review, unless the authors can demonstrate that each of these approaches has discovered something relevant to the biology of sex-specific differences. Table 1 was removed from the main text. There were no sex-specific differences mentioned in these articles. Other parts of the manuscript which require improvement are Figure 1 legend text. The diagram and legend could be better presented. A clear explanation of what Figure 1 shows is required.[AP1] Figure 1 was changed accordingly. A detailed description of the information present in Figure 1 is present in the introduction section. The title of Figure 1 was corrected. Figure 3 is difficult to understand with all the layers of information. I don’t understand what “Chronological..” means in the figure title. Some inner dark blue sections indicate biological information (hypoxia/development), while others indicate cell types and words like “Spatial-OPC”. Can this figure be edited to fix these inconsistencies? The figure legend needs more text to explain what is shown. The title and description of the figure were changed accordingly. Names of specific subtypes were cited from primary publications. The goal of this article was to show and point out these differences which should be unified in future studies. The manuscript needs editing to correct language errors. For example, in the Abstract, the words “However, despite..” are used together. “However” is redundant here, so should be deleted. The word ”exposed” as used in the abstract and with the body text, is wrong – the authors mean “revealed”. Another example is in the Conclusions section. Near the end of Conclusions, “it is warrant” is used but it's unclear what the context is. The main manuscript text was edited accordingly." } ] }, { "id": "163667", "date": "01 Mar 2023", "name": "Georges Nemer", "expertise": [ "Reviewer Expertise Transcriptomics", "exome sequencing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review by Perdyan A et al. recapitulates the historical classification of different types of glioblastomas. Overall, the mini-review captures all 4 pillars of heterogeneity: genetics, transcriptomics, immunogenomics, and sex.\n\nIn the form, there are a couple of typos and grammar mistakes as well as redundant \"words\" that should be corrected like \"on the other side\" on pages 3 and 6 and the repetition of \"on the other hand\" in different contexts.\n\nThe authors should also correct the label \"in this study\" on page 4, as this is a mini-review.\n\nFor the content, all is well-covered but it would be better if sex is integrated into the other 3 pillars of heterogeneity.\n\nThe mini-review can be Approved after these minor corrections.\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? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "9595", "date": "09 Aug 2023", "name": "Jakub Mieczkowski", "role": "Author Response", "response": "In the form, there are a couple of typos and grammar mistakes as well as redundant \"words\" that should be corrected like \"on the other side\" on pages 3 and 6 and the repetition of \"on the other hand\" in different contexts. The authors should also correct the label \"in this study\" on page 4, as this is a mini-review. The main manuscript text was edited accordingly. For the content, all is well-covered but it would be better if sex is integrated into the other 3 pillars of heterogeneity. We integrated sex into one of the three pillars of heterogeneity. Unfortunately, there are only a few studies investigating sex-dependent differences. Studies on which the heterogeneity classifications were made did not take into account this matter. Hence, the purpose of this mini-review was to point out and address this gap in the field." } ] } ]	1	https://f1000research.com/articles/11-1180
https://f1000research.com/articles/12-447/v1	26 Apr 23	{ "type": "Research Article", "title": "Enhanced human periodontal ligament stem cell viability and osteogenic differentiation on two implant materials: An experimental in vitro study", "authors": [ "Sara F. El Shafei", "Shereen N. Raafat", "Engy A. Farag", "Shereen N. Raafat", "Engy A. Farag" ], "abstract": "Background: Surface roughness of dental implants impacts the survival of adult periodontal stem cells and rate of differentiation. This research was conducted to test how human periodontal ligament stem cells behaved on yttria stabilized tetragonal zirconia polycrystals and polyetheretherketone (PEEK) discs with different surface topographies. Methods: Discs roughening was prepared by sandblasting. Stem cells were cultivated on zirconia discs with a polished surface, PEEK discs with a polished surface, sandblasted zirconia discs and sandblasted PEEK discs. Cells viability was assessed after 24, 48, 72 hours. Scanning electron microscopy was used to examine the adherence and attachment of cells. Osteoblastic differentiation capacity was studied by checking the mineralization clusters development through alizarin red S staining and alkaline phosphatase assay. ANOVA and the Tukey post hoc test were used for the statistical analysis. Results: Polished PEEK discs showed lower cell viability, whereas roughened sandblasted zirconia and PEEK discs showed the highest proliferation rates and cell viability percent. The osteogenic differentiation was enhanced for rough surfaces in comparison to polished surfaces. Sandblasted zirconia and PEEK discs showed a markedly increased mineralized nodule development and ALP enzyme activity compared to the polished surface and control. Conclusions: Micro- topographies creation on the PEEK implant surface enhances stem cell attachment, viability, and osteogenic differentiation.", "keywords": [ "PEEK", "PLDSCs", "Sandblasting", "Zirconia" ], "content": "Introduction\n\nThe biological interface between a dental implant and the host tissue in which it is embedded is a critical determinant of clinical outcome.1 To enable osteointegration of the implant, osteoconductive material and coatings are usually used.2 However, in case of significant loss of bone volume (and the unsuitability of bone autografts), the use of osteoconductive materials only will not suffice. The use of osteoinductive material should provide an improved clinical outcome. These materials should stimulate ontogenesis, increase vascularization and hence improve mechanical stability of the implant.3\n\nHuman periodontal ligament stem cells (hPDLS) have mesenchymal stem cells characteristics, may regenerate, and develop into a variety of cells, including osteoblasts. Furthermore, their angiogenic potential has been demonstrated via the release of vascular endothelial growth factor (VEGF), indicating that they play an important role in implant osseointegration.4\n\nThe biocompatibility of titanium with bone tissue makes it a popular material for bone implants. Unfortunately, due to its dark color, titanium implants may become obvious through the gingiva.5,6 Given their color, tetragonal zirconia polycrystals (TZP) on the other hand show outstanding esthetic performance. Additionally, due to their biocompatibility and favorable mechanical properties, TZP has become a pillar material for dental restorations.7 However, from an osteointegration perspective, titanium still shows superiority.8\n\nPEEK is a synthetic polymer which shows potential as an aesthetic dental implant material given its tooth-like color. It possesses exceptional chemical tolerance and biomechanical characteristics making it well-suited for biomedical applications. Notwithstanding, PEEK's bioactivity and osseointegration are also debatable.9\n\nSurface topography modification by lithography, pattern transfer, sandblasting, acid-etching, or plasma immersion ion implantation has been shown to promote cellular adhesion and proliferation. Micro-topographical modifications also create areas in which the bone infiltrates and grows, enhancing implant fixation and stability into bone tissue.10\n\nSandblasting is one of the most versatile techniques used for implant surface modification. Increased surface area of implants to promote osseointegration is its main benefit. The rougher surfaces also improve osteoblast adhesion and proliferation.11,12\n\nIn comparison to titanium implants, the significance of surface alterations in cell proliferation and the development of osteogenic tissue on zirconia and PEEK implants is much less understood. This study aimed to ascertain the effect of implant material (TZP and PEEK) and surface characteristics (rough versus smooth) on human periodontal ligament stem cell growth and osteogenic differentiation.\n\n\nMethods\n\nThis study was conducted from 1st of February 2022 to 1st of November 2022. This study is an experimental in vitro study investigating hPDLS behavior using different implant materials with different topographies.\n\nYttria stabilized tetragonal zirconia polycrystals (ZrO2 balanced, Y2O3< 5.15 mass %, HfO3< 3 mass%, Al2O3 < 0.5 mass%, SiO2 <0.02 mass %, Fe2O3 <0.01, Na2O <0.04) (Bruxzir Shaded, Glidewell) and polyether ether ketone (PEEK) disks 1 mm thick and 10 mm in diameter were utilized. Four experimental groups of discs were formed according to the type of material and surface treatment: experimental zirconia discs with polished surface (Z); experimental zirconia discs with a sandblasted surface (ZS); experimental PEEK discs with smooth surface (P) and experimental PEEK discs with a sandblasted surface. Using a polishing apparatus (Ecomet 3, Buehler), polished TZP samples were first ground on the two sides with diamond discs (70 m, then 45 m). They were then polished with a polishing cloth, diamond particles measuring 3 and 9 microns, and colloidal silica measuring 0.6 microns.13 PEEK polished samples, however, were produced by first polishing the PEEK disc with 1000 and 2000 grit silicon carbide sandpapers, then finishing with 8000 grit alumina lapping film to offer a smooth surface.14 At 50 mm perpendicular distance on all sides, particles of 110 m alumina (Korox 110, BEGO) were used to sandblast specimens of TZP and PEEK at 5 bar air pressure using a sandblaster (Basic eco, Renfert). All discs were sandblasted for 10 seconds.15 All samples underwent a 10 minutes ultrasonic cleaning procedure using acetone and distilled water before being used in cell culture experiments, followed by a half an hour UV disinfection lamp application on each side of each disc.\n\nPeriodontal ligament (PDL) cells isolation and culture\n\nUnder the guidance of the British University in Egypt's Ethical Committee (approval # 22-003) and with patients' (n=3) full agreement and signing an informed consent, three human permanent teeth were gathered from healthy donors at the oral and maxillofacial dental department. The teeth had been extracted for orthodontic purposes. The patients were informed about the study privately and the patient’s data confidentiality was ensured. The patients’ inclusion criteria were; not suffering from any chronic diseases, and average age between 20–40 years. The patients were not further included in the study and were considered as teeth donors only. The selected teeth were free from any carious lesions.\n\nThe extracted teeth were kept in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO), which received antibiotic doses (300 U/ml penicillin and 300 mg/ml streptomycin; Sigma). Within the first 24 hours from teeth extraction, primary cell culture was performed. Sterile phosphate-buffered saline (PBS, Sigma) was used to disinfect the PDL tissues removed from healthy third molars root surfaces. PBS was then added with antibiotics at escalating doses to provide further protection. Stem cells were isolated from these PDL tissues by using the outgrowth method.16 Smaller pieces of PDL were put onto 35 mm plates (NUNC, Roskilde) with 1 mL of culture media, which was composed of Dulbecco's modified Eagle's medium/nutrient combination F-12 Ham medium (DMEM/F12, Sigma) with 10% fetal bovine serum (FBS, Gibco) added as a supplement and 1% penicillin/streptomycin. 37°C incubation was conducted with 5% CO2 in a humid atmosphere. An inverted microscope was used to monitor cell growth and morphology. The cells were trypsinized and sub-cultured once they had reached 80% confluence. Every three days, the medium was changed. All tests were carried out on cells acquired during the fourth passage (Figure 1a,b).\n\n(a) Cell aggregates at passage 0; (b) Confluent cells at passage 4 (Mag. 40×).\n\nIdentification of isolated PDL cells as mesenchymal stem cells (MSCs)\n\nCell surface marker identification. The expression of specific surface markers on the extracted PDLSCs was identified by fluorescence-activated cell sorting (FACS). The cells were collected, moved, and then fixed for 15 minutes in 4% paraformaldehyde. First, 3% bovine serum albumin (BSA; Sigma-Aldrich) was used to incubate the cells before being incubated for one hour with primary antibodies (eBioscience, Thermo Fisher Scientific), produced against CD105, CD90, CD73, CD34, CD45, and HLA-DR for 1 hour. The secondary antibody was applied and left on the cells for 45 minutes at room temperature after being rinsed with wash buffer. After three rounds of washing, a flow cytometer (FACSCalibur, BD Biosciences) was used to evaluate the cells.17\n\nMultilineage differentiation. To evaluate the in vitro multipotential differentiation capacity of hPDLSCs, 5×104 cells/ml from the fourth passage were cultured in 24-well plate with OsteoDiff media, AdipoDiff media, and CondroDiff media (Human mesenchymal stem cell functional identification kit, R&D Systems) for three weeks to achieve osteogenic, adipogenic, and chondrogenic differentiation, respectively. Alizarin red staining (Sigma-Aldrich) was used to assess mineralization, which demonstrates osteogenesis. Oil red O solution (Sigma-Aldrich) was utilized to find lipid droplet aggregation, which demonstrates adipogenesis. Alcan blue staining (Sigma-Aldrich) was used to identify glycosaminoglycans verifying chondrogenic differentiation.\n\nCell viability. Six discs of each group (Z, ZS, P and PS) were put at the bottom of a 6-well plate. Cells were then put to the wells at a density of 30×104 cell for each well. The culturing of cells was on the discs for the following 24, 48, and 72 hours in which cellular viability was evaluated, utilizing methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay (Sigma Aldrich), relative to control cells being cultured in 6-well plates without discs. Absorbance was recorded using a benchtop microplate reader (Thermo Scientific Multiscan).18 The findings were presented as mean viability % relative to controls cultured in the absence of disks.\n\nCell morphology. Cells were seeded in 6-well plates on Z, ZS, P and PS discs as detailed earlier and 72 hours were spent cultivating. Subsequently, 4% glutaraldehyde was used to fix the cells for 2 hours at 4°C.19For critical point drying, samples dehydration was done using increasing ethanol concentrations from 25%,50%, 75%, 95%, to 100%, 5 minutes in each concentration. Samples were subjected to sputter-coating with gold using 15 mA for 4 min (HUMMER 8.0, ANATECH). Discs without cells and those with cultured cells were analyzed by scanning electron microscopy (SEM, Leo Supra 55).\n\nOsteogenic differentiation assay. For osteogenic differentiation assay, cells were implanted in a 6-well plate at a density of (30 × 104 cells/well) on disks. The medium was changed the following day to osteogenic differentiation medium, which contains MEM-Alpha, 10% FBS, dexamethasone 100 nm (Sigma Aldrich, Steinheim), ascorbic acid-2-phosphate 200 uM (Sigma Aldrich), and beta glycerophosphate 10 mM (Merck, Darmstadt).20 Differentiation was performed for 14 days. A positive control was achieved using cells cultivated in osteogenic media without discs, whereas a negative control used cells cultured in normal medium (DMEM). Both osteogenic induction medium and basal culture medium were altered twice per week. After 14 days of cultivation, the osteogenic distinction capacity was assessed by the calcium deposition mineralization assay using alizarin red staining and alkaline phosphatase (ALP) activity.\n\nAlizarin red S assay. Once calcified, mineralized nodules were observed after 14 days of induction, alizarin red S staining was conducted. In more detail, the medium was aspirated and the cells were fixed with 10% formaldehyde (Sigma, v/v) at room temperature for 15 minutes. After three PBS washes to remove non-attached cells, the plates were stained for 30 minutes at room temperature with 40 mm alizarin red S (Sigma, pH 4.2). To eliminate excess stain, cells were washed 3 more times with PBS before being examined using a light microscope. Additionally, at 405 nm, the absorbance was measured after staining was solubilized in a 10% glacial acetic acid (Sigma-Aldrich) solution.21\n\nAlkaline phosphatase (ALP) activity assay. ALP activity of PDLSCs on every specimen was assayed after differentiation for 14 days. Briefly, monolayers were cleaned twice with PBS and then once with 1 mL alkaline phosphatase buffer (ALPB). One mL of ALPB was added to each well and equal volumes of para-Nitrophenylphosphate (p-NPP) (Sigma Aldrich) equilibrated to 4°C were added. Immediately, 50 ul were removed and mixed with same volume of NaOH to stop the reaction. The previous step was repeated every minute for each well till 10 minutes had passed. The rate of accumulation of p-nitrophenolate was plotted for each well and the initial rate of the reaction, a way to express the rate of the reaction, was calculated by obtaining the gradient at the linear phase for each group. Monolayers were then cleaned in PBS and kept at -20 o C for total protein quantification.22 Total protein amounts were detected utilizing enhanced BCA protein assay kit (Pierce) based on the manufacturer’s procedures.23 The absorbance at 405 nm was detected by spectrophotometry. The initial rate of the reaction in each group (slope) was divided by protein concentrations (mg).\n\nEach in-vitro assay was carried out in triplicate and analyzed across three distinct studies. Graph-Pad Prism v8.1.0 (RRID:SCR 002798) was used to conduct ANOVA test (RRID:SCR_002798), followed by a pair-wise Tukey's post hoc test, after validating the homogeneity of variance and normal distribution of the data.\n\n\nResults\n\nAccording to flow cytometry findings, isolated PLDSCs were CD44 (92.11%), CD73 (98.76 %), CD90 (95.67%), and CD105 (95.52%) positive; and less than 5% for CD34 (4.44%), CD45 (4.48%), and HLA-DR (2.66%) (Figure 2a). The rates are provided in respect to the presence of mesenchymal stem cell surface markers. Additionally, hPDLSCs were found to be capable of differentiating into osteoblasts, chondroblasts, and adipocyte-like cells (Figure 2b).\n\nAn MTT assay was utilized to calculate the viability amount of the hPLDSCs (Figure 3). Data are presented after 24,48, and 72 hours of exposure of the PLDSCs to discs and were normalized to the control (cells + medium). Polished PEEK surface discs showed a notably reduced cell viability % in comparison to the three other groups at 24,48 and 72 hours (p<0.05). The sandblasted roughened zirconia and PEEK discs showed increased cell viability % throughout the whole experiment, the roughened PEEK surface possessed significant higher cell viability % compared to the same respective group of smooth polished surface (p<0.001), and the roughened Zirconia discs also showed higher cell viability % compared to the same respective group of smooth polished surface (p<0.05). However, the percentage of cell viability was not significantly different between the roughened zirconia and PEEK discs.\n\nThe data are presented as absorbance values (570 nm) at 24, 48, and 72 h of the PLDSCs being exposed to discs. p significant difference of the respective group in comparison to the Z group, #p significant difference of the respective group in comparison to the P group, p values <0.05 are statistically significant. For each material's surface properties, each experimental condition was run in triplicate.\n\nThe discs’ surface microstructure and stem cell morphology are shown in Figure 4. Cells with extending cytoplasmic processes were frequent and well-observed in smooth polished zirconia discs while no well-defined cells were seen attached to the polished PEEK discs. Cell adhesion and structure of hPLDSCs on roughened sandblasted specimen surfaces revealed the presence of numerous well-attached-type cells with extending stretched cytoplasmic processes.\n\nOn day 14, extracellular matrix rich in calcium was assessed as a byproduct of the osteogenic differentiation process when stained with alizarin red S (Figure 5). There were notable variations in between the groups examined; the negative control (cells+normal medium) displayed fewest mineralized nodules amounts; the positive control (osteogenic medium only) exhibited higher amount of calcium deposition (p<0.001) than polished zirconia and PEEK groups (Figure 5). When compared to the other experimental groups, roughened sandblasted zirconia and PEEK groups possessed greatest grades of mineralized nodules (p<0.001).\n\n(Mag. 40×). (a) Control (b) Osteogenic medium (c) Smooth zirconia surface (d) Smooth PEEK surface (e) Roughened zirconia surface (f) Roughened PEEK surface. Every experimental condition was carried out in triplicate for every material surface characteristics. (g) Alizarin red staining data from three independent experiments were statistically analyzed and given as mean standard deviation, p significant difference of the respective group compared to the control, #p significant difference of the respective group in comparison to the osteo group. $p significant difference of the respective group in comparison to the Z group. p values <0.05 is statistically significant.\n\nThe ALP assay kinetic profile demonstrated the aggregation of the yellow p-nitrophenolate (p-NP) as a growing product among the various groups (shown in Figure 6a). The control group possessed the lowest rate of accumulation, the polished zirconia and PEEK showed lower rate of p-NP accumulation than the roughened sandblasted disks of same respective groups. Statistical analysis was done by calculating the slope of each curve and divided by the total amount of protein in each well. The results showed that the ALP activity at day 14 in cells cultured on the polished zirconia and PEEK discs was significantly lower than the positive control (p<0.05); polished PEEK discs possessed the lowest ALP activity compared to the polished zirconia discs and positive control (p<0.001). Roughened sandblasted Zirconia and PEEK discs indicated most ALP activity in comparison to the polished discs (p<0.001) (Figure 6b). Regarding ALP activity, no discernible change was found in cells cultured on the roughened sandblasted zirconia discs, PEEK discs and the positive control group, suggesting that roughened sandblasted surfaces can stimulate the osteogenic potential of PDLSCs.\n\na) An illustration of an ALP assay's kinetic curve showing how different groups over time accumulated the yellow p-nitrophenolate product. b) The initial rate of the reaction in each group by calculating the slope, values were scaled back to the quantity of total protein. The values revealed as mean±SD of three independent experiments, *p significant difference of the respective group in comparison to the control (cells in normal medium), #p significant difference of the respective group in comparison to the osteo group (osteogenic medium), $p significant difference of the respective group compared to the Z group. p values <0.05 is statistically significant.\n\n\nDiscussion\n\nTitanium and zirconia are commonly used as dental implant materials, whereas PEEK is widely used in orthopedic research. PEEK, on the other hand, has little biological activity when utilized as an implant, making it unable to successfully osseointegrate with adjacent bone tissue. These flaws significantly restrict the osteogenic properties of PEEK, which is a crucial factor for implants long-term stability. As a result, recent years have seen a surge in research into various methods of modification that might improve PEEK's biological activities, such as cell compatibility, osteogenic activity, and antibacterial capability.24\n\nBy enhancing bone-to-implant connection and peri-implant bone density, implant surface modification aimed to accelerate the early phases of osteointegration.25 The surface pattern can be altered by acid etching and airborne particle abrasion, or the surface's physiochemical characteristics can be altered through coating application to enhance the surface's bioactive and osteoconductive qualities.26\n\nMeanwhile, MSCs are capable of self-renewal and cell lineage differentiation into a wide range of cell types, including osteoblasts, chondrocytes, adipocytes, neuroblasts, and myoblasts. hPDLSCs have been used in this study for the advantages of MSCs as self-renewal, multipotency and immunomodulation, in addition to being a cell population that is conveniently accessible and has the required properties for future clinical use in human studies.27\n\nThis study assessed the in vitro behavior of hPLDSCs on TZP and PEEK with varying surface topography. The sandblast method was used to micro-roughen the surface; this approach has been utilized for metal implants and is straightforward and affordable.28 To assess the behavior of hPLDSCs on different substrates, we employed different in vitro assays including cell viability, morphology and osteogenic potential. Our results showed that polished TZP had better hPLDSCs cell attachment and viability than polished PEEK, whereas sandblasted TZP and PEEK had the highest significant hPLDSCs viability and osteogenic differentiation.\n\nCell viability was higher on the rough TZP surface than on the smooth surface. Different surface topography presumably modifies the shape and arrangement of the cells' cytoskeletons influencing cell attachment proteins secretion, hence enhanced cell viability and proliferation.29 The viability of hPLDSCs on sandblasted PEEK was significantly higher than on smooth PEEK. Sandblasting produced peaks and valleys on PEEK surfaces resulting in increased PEEK's surface roughness and increased contact angle.30 Danen et al.31 proposed that cell adhesion proteins have an arginine-glycine-aspartate peptide sequence that serves as the integrin-binding domain, a protein known to play a role in the adhesion of osteoblasts to biomaterials, and is essential for initial cell attachment, morphology, and proliferation. Because roughened PEEK has a larger surface area, more proteins can be adsorbed on it, resulting in better initial cell attachment and proliferation. Our findings agree with those of Sunarso et al.,14 suggesting that roughened PEEK could achieve cell-cell interactions faster than mirror smooth PEEK.\n\nIn the current study, the smooth surface specimens of TZP had micro-topography. It displayed advanced cell attachment, a well-adhered cell morphology, and some fibres running between cells, indicating that cells aggregated together. This could be due to the presence of micro-scale granules in the smooth-surfaced TZP. The observed filamentous scaffolding was previously reported to be important in cell adhesion and spread.32\n\nThe SEM observations revealed that the cells in the roughened groups exhibited multiple aggregates of cells. Cell attachment samples revealed to be dependent on the finger-like cytoplasmic extensions formation, which was clearly observed on the roughened PEEK specimens in contrast to the polished smooth PEEK. These structures appeared to serve as anchors for the cells to the underlying surface.8 These findings imply that cell rearrangement might result from the sandblasted structure's conversion of the material's physical signals into intracellular biological signals.33\n\nThe alizarin red staining and ALP activity assays were used to detect the impact of the tested specimens on the osteogenic potential of hPLDSCs. Our findings revealed that calcium-rich extracellular matrix and ALP activity were significantly higher on sandblasted surfaces than on smooth specimens. All samples showed evidence of calcium nodule development, with differences in aggregation and dispersion based on the topographical type. The mineralization tendency was confirmed by the distribution and density of the nodules. These findings are consistent with Sima et al.34 who proved that increased surface granulation, particularly on sandblasted surfaces, indicates that roughened surfaces act as mineralization nucleation centres.\n\nThe enhanced osteogenic activity of hPLDSCs could be due to the synergistic effect of micro-topography which may provide a biomimetic surface and influences hPLDSC proliferation and osteogenic differentiation.2 A previous study by Taniguchi et al.35 examined how surface roughness affected cultured osteoblast-like cell morphology, proliferation, differentiation and reported that a roughened surface has a greater chance to speed up the calcification process and boost cells' osteogenic ability. Furthermore, the study showed that surface roughness promoted osseointegration of the peri-implant area after application on induced bone defects of rats’ tibiae.\n\nSandblasted zirconia and PEEK surfaces with microscale roughness may provide a suitable niche for hPLDSC proliferation and osteogenic differentiation. A study by Zhu B et al.36 discovered groove-ridge patterns on the surface of polystyrene that resembled collagen fibrillar architecture more closely in profile and size and that it could be used as a scaffold to provide directed physical cues for osteoblast-like cell orientation, spreading, and directional mineralization.\n\nSurface roughness has an impact on cell attachment, proliferation, differentiation, and mineralized matrix synthesis. Our results are in line with those of Lincks et al.,37 who found that roughness is an important variable in promoting osteogenic differentiation and appears to affect cell orientation. Furthermore, as surface roughness increases, so does bone development rates and the percentage of surface area in close interaction with bone. In addition, Schneider et al.38 studied the influence of implant surface microtopography on the expression of transcription factors regulating osteoblast differentiation. It was observed that there is a relation between cell attachment and surface architecture through gene expression regulation.\n\nTo summarize the current study findings, we found that roughened PEEK exhibited high PLDSCs attachment and viability, close to that of roughened zirconia and higher in comparison to the polished PEEK. In addition, higher level of mineralized matrix and ALP activity were observed on the roughened TZP and PEEK. Sandblasted PEEK showed improved osteogenic potential through elevation of the responses of hPLDSCs. A strategy that has the potential to improve PLDSC attachment is the development of micro-topographies on the PEEK surface, viability and osteogenic differentiation. Future efforts should be directed at detailed understanding of the development of more efficient and the study of PEEK modification techniques to enhance accelerated osteogenic differentiation.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: https://figshare.com/articles/dataset/Raw_data_xlsx/21724385.v1. 39\n\nThis project contains the following extended data\n\n‐ Supplementary raw data for and statistical analysis of all in vitro assays\n\n‐ Supplementary data of the control figures of flow-cytometry in stem cells characterization\n\n‐ All in vitro assays protocols and procedures were done in accordance to; Mesenchymal stem cells assays and applications. https://link.springer.com/protocol/10.1007/978-1-60761-999-4_17\n\nData are available under the terms of the creative commons attribution 4.0 international license CC BY 4.0.\n\n\nAcknowledgements\n\nThe authors would like to thank all members in the Stem cells and Tissue Culture Hub, Centre of Innovative Dental Sciences, Faculty of Dentistry, The British University in Egypt (BUE), for their assistance in the study.\n\n\nReferences\n\nParithimarkalaignan S, Padmanabhan TV: Osseointegration: An Update. J. Indian Prosthodont. Soc. 2013; 13(1): 2–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHirano T, Sasaki H, Honma S, et al.: Proliferation and osteogenic differentiation of human mesenchymal stem cells on zirconia and titanium with different surface topography. Dent. Mater. J. 2015; 34(6): 872–880. PubMed Abstract \| Publisher Full Text\n\nKazimierczak P, Przekora A: Osteoconductive and Osteoinductive Surface Modifications of Biomaterials for Bone Regeneration: A Concise Review. Coatings. 2020; 10(10): 971. Publisher Full Text\n\nMarconi GD, Fonticoli L, Della Rocca Y, et al.: Human Periodontal Ligament Stem Cells Response to Titanium Implant Surface: Extracellular Matrix Deposition. Biology (Basel). 2021; 10(9): 931. Publisher Full Text\n\nSicilia A, Cuesta S, Coma G, et al.: Titanium allergy in dental implant patients: a clinical study on 1500 consecutive patients. Clin. Oral Implants Res. 2008; 19(8): 823–835. 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PubMed Abstract \| Publisher Full Text\n\nHong L, Sultana H, Paulius K, et al.: Steroid regulation of proliferation and osteogenic differentiation of bone marrow stromal cells: a gender difference. J. Steroid Biochem. Mol. Biol. 2009; 114(3–5): 180–185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodríguez-Lozano FJ, López-García S, García-Bernal D, et al.: Cytocompatibility and bioactive properties of the new dual-curing resin-modified calcium silicate-based material for vital pulp therapy. Clin. Oral Investig. 2021; 25(8): 5009–5024. PubMed Abstract \| Publisher Full Text\n\nKrause U, Seckinger A, Gregory CA: Assays of osteogenic differentiation by cultured human mesenchymal stem cells. Methods Mol. Biol. 2011; 698: 215–230. Publisher Full Text\n\nLi J, Zhang F, Zhang N, et al.: Osteogenic capacity and cytotherapeutic potential of periodontal ligament cells for periodontal regeneration in vitro and in vivo. PeerJ. 2019; 7: e6589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa Z, Zhao X, Zhao J, et al.: Biologically Modified Polyether Ether Ketone as Dental Implant Material. Front. Bioeng. Biotechnol. 2020; 8: 620537. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Oirschot BAJA, Alghamdi HS, Närhi TO, et al.: In vivo evaluation of bioactive glass-based coatings on dental implants in a dog implantation model. Clin. Oral Implants Res. 2014; 25(1): 21–28. PubMed Abstract \| Publisher Full Text\n\nMajhi R, Majhi RK, Garhnayak L, et al.: Comparative evaluation of surface-modified zirconia for the growth of bone cells and early osseointegration. J. Prosthet. Dent. 2021; 126(1): 92.e1–92.e8. Publisher Full Text\n\nMarconi GD, Fonticoli L, Della RY, et al.: Enhanced Extracellular Matrix Deposition on Titanium Implant Surfaces: Cellular and Molecular Evidences. Biomedicines. 2021; 9(11): 1710. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe Guéhennec L, Soueidan A, Layrolle P, et al.: Surface treatments of titanium dental implants for rapid osseointegration. Dent. Mater. 2007; 23(7): 844–854. Publisher Full Text\n\nBoyan BD, Lohmann CH, Dean DD, et al.: Mechanisms involved in osteoblast response to implant surface morphology. Annu. Rev. Mater. Sci. 2001; 31: 357–371. Publisher Full Text\n\nOurahmoune R, Salvia M, Mathia TG, et al.: Surface morphology and wettability of sandblasted PEEK and its composites. Scanning. 2014; 36(1): 64–75. PubMed Abstract \| Publisher Full Text\n\nDanen EHJ, Yamada KM: Fibronectin, integrins, and growth control. J. Cell. Physiol. 2001; 189(1): 1–13. Publisher Full Text\n\nWei C, Gong T, Pow EHN, et al.: Adhesive and oxidative response of stem cell and pre-osteoblasts on titanium and zirconia surfaces in vitro. J. Investig. Clin. Dent. 2019; 10(3): e12407. PubMed Abstract \| Publisher Full Text\n\nSun Y, Sun J, Wu X, et al.: Mechanism of zirconia microgroove surface structure for osseointegration. Mater. Today Adv. 2021; 12: 100159. Publisher Full Text\n\nSima LE, Bonciu A, Baciu M, et al.: Bioinstructive Micro-Nanotextured Zirconia Ceramic Interfaces for Guiding and Stimulating an Osteogenic Response In Vitro. Nanomater. 2020; 10(12): 1–25.\n\nTaniguchi Y, Kakura K, Yamamoto K, et al.: Accelerated Osteogenic Differentiation and Bone Formation on Zirconia with Surface Grooves Created with Fiber Laser Irradiation. Clin. Implant. Dent. Relat. Res. 2016; 18(5): 883–894. PubMed Abstract \| Publisher Full Text\n\nZhu B, Lu Q, Yin J, et al.: Alignment of osteoblast-like cells and cell-produced collagen matrix induced by nanogrooves. Tissue Eng. 2005; 11(5–6): 825–834. PubMed Abstract \| Publisher Full Text\n\nLincks J, Boyan BD, Blanchard CR, et al.: Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition. Biomaterials. 1998; 19(23): 2219–2232. PubMed Abstract \| Publisher Full Text\n\nSchneider GB, Perinpanayagam H, Clegg M, et al.: Implant Surface Roughness Affects Osteoblast Gene Expression. J. Dent. Res. 2003; 82(5): 372–376. Publisher Full Text\n\nEl Shafei S, Raafat SN, Farag EA: Enhanced human periodontal ligament stem cell viability and osteogenic differentiation on two implant materials: An experimental in vitro study, Raw data. figshare. 2022. Publisher Full Text" }	[ { "id": "171166", "date": "14 Jul 2023", "name": "Samar Darwish", "expertise": [ "Reviewer Expertise In vivo studies", "inflammation", "hepatology", "endocrinology", "cancer", "oxidative stress" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 scientists examined and tested how human periodontal ligament stem cells responded to polyetheretherketone (PEEK) discs with various surface topographies and yttria-stabilized tetragonal zirconia polycrystals. The study clarifies how forming micro-topographies on the surface of PEEK implants improves stem cell adhesion, vitality, and osteogenic differentiation. However, there are few minor revisions that I advise the authors to take them in concern:\nAbstract section:\nIt is preferred not to write the keywords in their abbreviated forms, such as PEEK and PLDSCs.\n\nIntroduction section:\nIt is observed that all reference numbers are written after the full stops of the sentences in the whole manuscript. It is preferred to write the reference numbers before not after.\n\n3rd paragraph, 2nd Line: What is meant by “Given their color...”, please rephrase the sentence to give a clear idea.\n\n4th paragraph, 1st line: “PEEK is a synthetic polymer which shows potential...\", there is a missing word I think, please revise.\n\n4th paragraph, 3rd line: the authors mentioned that PEEK's bioactivity and osseointegration are also debatable, please write in more detail what you mean by debatable to clarify your idea.\n\nMethods section:\nPEEK abbreviation refers to which word? Polyetheretherketone or polyether-ether-ketone? Please check.\n\nIn 'Disc preparation section': please write more details about how yttria stabilized tetragonal zirconia polycrystals were prepared or utilized.\n\nIn 'Periodontal ligament (PDL) cells isolation and culture': the authors mentioned that \"The extracted teeth were kept in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO), which received antibiotic doses (300 U/ml penicillin and 300 mg/ml streptomycin; Sigma).\" Please mention how many doses were given and the dosing frequency.\n\nIn 'Statistical analysis': \"(RRID:SCR_002798)\" is repeated, mention only once.\n\nResults section:\nFigure 2a: if possible, it is preferred to increase the resolution to make the numbers more clear.\n\nFigure 2b: put labels on the 6 different pictures to identify them clearly.\n\nFigure 3: again the resolution needs to be increased.\n\nFigure 5: there are two rows of pictures, (a, b , c….). What is the difference? Is it the magnification? Please clarify and label them because it is only mentioned that \"(Mag. 40x)\".\n\nIn the comment on 'Mineralization assay calcium deposition' results, mention the percentage or folds difference calculated between the groups as you can.\n\nDiscussion: the discussion was well described and covered the study points in detail\nIn the last paragraph: what did the authors mean by this sentence, \"A strategy that has the potential to improve PLDSC attachment is the development of micro-topographies on the PEEK surface, viability and osteogenic differentiation\"? Please rephrase to clarify your idea.\n\nReference section:\nSome references are preferred to be renewed such as: no 8, 29, 31, 37\n\nIs the work 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": "10017", "date": "08 Aug 2023", "name": "Sara El Shafei", "role": "Author Response", "response": "Thank you for your efforts in reviewing our manuscript. All the comments have been addressed. Reviewer 1: Abstract section: It is preferred not to write the keywords in their abbreviated forms, such as PEEK and PLDSCs. -Corrected PEEK in abstract and keywords highlighted -Corrected hPLDSCs in keywords and highlighted Introduction section: 1-It is observed that all reference numbers are written after the full stops of the sentences in the whole manuscript. It is preferred to write the reference numbers before not after. -The followed reference numbering style in text is according to the journal submission regulations 2-3rd paragraph, 2nd Line: What is meant by “Given their color...”, please rephrase the sentence to give a clear idea. -Rephrased and highlighted 3-4th paragraph, 1st line: “PEEK is a synthetic polymer which shows potential...\", there is a missing word I think, please revise -Added missing word and highlighted 4-4th paragraph, 3rd line: the authors mentioned that PEEK's bioactivity and osseointegration are also debatable, please write in more detail what you mean by debatable to clarify your idea -Clarified the sentence and highlighted Methods section: 1-PEEK abbreviation refers to which word? Polyetheretherketone or polyether-ether-ketone? Please check. -Clarified the word and highlighted 2-In 'Disc preparation section': please write more details about how yttria stabilized tetragonal zirconia polycrystals were prepared or utilized -Rephrased to clarify the idea and highlighted 3- In 'Periodontal ligament (PDL) cells isolation and culture': the authors mentioned that \"The extracted teeth were kept in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO), which received antibiotic doses (300 U/ml penicillin and 300 mg/ml streptomycin; Sigma).\" Please mention how many doses were given and the dosing frequency. Antibiotic-antimycotic drug is added once in the culture medium bottle (500 ml) which is used in the whole study; changing medium for the cells is maximum 2-5 ml / well or flask twice per week. 4- In 'Statistical analysis': \"(RRID:SCR_002798)\" is repeated, mention only once. -Removed and mentioned only once. Results section: 1-Figure 2a: if possible, it is preferred to increase the resolution to make the numbers more clear. 2-Figure 2b: put labels on the 6 different pictures to identify them clearly -Figure 2a. Edited and added in the manuscript -Figure 2b. Edited and added in the manuscript 3-Figure 3: again the resolution needs to be increased -Edited and added in the manuscript Figure 5: there are two rows of pictures, (a, b , c….). What is the difference? Is it the magnification? Please clarify and label them because it is only mentioned that \"(Mag. 40x)\". -Done and labeled In the comment on 'Mineralization assay calcium deposition' results, mention the percentage or folds difference calculated between the groups as you can - Mean values of absorbance of the study group are added in the manuscript and highlighted. Discussion: 1-In the last paragraph: what did the authors mean by this sentence, \"A strategy that has the potential to improve PLDSC attachment is the development of micro-topographies on the PEEK surface, viability and osteogenic differentiation\"? Please rephrase to clarify your idea. -Rephrased and highlighted" } ] }, { "id": "171165", "date": "25 Jul 2023", "name": "Sahar El Refai", "expertise": [ "Reviewer Expertise Oral 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 article is clearly written covering all the details of the material and methods, I acknowledged the standardization in the used technique and the clarity of the results which mostly verified the aim despite figure 2b, (the most right image should have been titled chondrogenesis instead of the 2 images having the osteogenesis title.\nIn regards to the discussion, it was sequential and consistent, however would have preferred if the author highlighted the kind of cell/material binding being regulated by cell type and implant material.\nFinally, the addition of opposing opinions to the results addressed in the present research would have enriched the current study outcomes and elucidated all reported literature views.\n\nIs the work 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": "10018", "date": "08 Aug 2023", "name": "Sara El Shafei", "role": "Author Response", "response": "Thank you for your efforts in reviewing our manuscript. All the comments have been addressed. 1-despite figure 2b, (the most right image should have been titled chondrogenesis instead of the 2 images having the osteogenesis title -Done and corrected. 2- preferred if the author highlighted the kind of cell/material binding being regulated by cell type and implant material Added in the manuscript, discussion section and highlighted. 3- the addition of opposing opinions to the results addressed in the present research would have enriched the current study outcomes and elucidated all reported literature views Added in the manuscript, discussion section and highlighted." } ] } ]	1	https://f1000research.com/articles/12-447
https://f1000research.com/articles/12-952/v1	08 Aug 23	{ "type": "Study Protocol", "title": "The comparative study on axial and coronal CT images using maximum intensity projection for evaluating urinary calculus - a study protocol", "authors": [ "Syed Asrar ul haq Andrabi", "Suhas Tivaskar", "Anurag Luharia", "Albert Paul", "Shreya Naik", "Suhas Tivaskar", "Anurag Luharia", "Albert Paul", "Shreya Naik" ], "abstract": "Urinary calculus is a medical condition characterized by the formation of hard deposits in the urinary tract. It is also known as kidney stones and can cause severe pain and discomfort. The most common type of urinary calculus is composed of calcium, but it can be made of other substances such as uric acid or struvite. Risk factors for developing urinary calculus include dehydration, family history, certain medical conditions, and certain medications. Urinary calculi are a common condition that can cause significant discomfort and morbidity in affected individuals. CT imaging is the gold standard technique that helps diagnose urinary calculi. During the examination, axial and coronal images are commonly used. However, the optimal imaging approach has not been clearly defined. The study will include patients with suspected urinary calculi who undergo CT imaging at a single institution. Both axial and coronal images will be reconstructed using M.I.P. The primary outcome measure will be the sensitivity and specificity of the two imaging approaches for detecting urinary calculi. Secondary outcome measures will include the time required for image reconstruction, the radiation exposure associated with each approach, and the subjective preference of the radiologists for one approach over the other. The study is expected to provide valuable information on the optimal imaging approach for evaluating urinary calculi. If one approach is superior, it could lead to a more efficient and effective diagnosis and treatment of this common condition. Additionally, the study will provide data on the radiation exposure associated with each approach, which could inform clinical decision-making and improve patient safety. In this study, the protocol aims to compare the diagnostic performance of axial and coronal C.T. images using maximum intensity projection (MIP) for the evaluation of urinary calculi.", "keywords": [ "Maximum intensity projection (M.I.P.)", "Urinary calculus", "urinary tract", "dehydration", "image reconstruction", "Axial and coronal projections." ], "content": "Introduction\n\nThe Renal Calculi or kidney stones are crystal aggregates within the kidney. Nephrolithiasis is a Greek word. Nephrosis means “kidney”, and lithos means “stone”. It refers to kidneys having stones or calculi in the collecting system. Kidney stones (or) Nephrolithiasis exclusively refers to the presence of calculi (or) stones in the kidneys.1 Renal stones continue to be a prevalent issue, impacting around 10% of individuals at some stage. The occurrence of kidney stones has risen in recent years, although this could be partially attributed to enhanced identification methods.2\n\nFor patients who are complaining of acute flank pain and feeling of urolithiasis, unenhanced C.T. is the imaging modality of choice. However, not every patient in this situation has stone illness; others have a different diagnosis for their acute pain, such as diverticulitis, appendicitis, or even pancreatitis.3\n\nContrary to urolithiasis, contrast-enhanced C.T. (C.E.C.T.) is a better tool for evaluating these entities. It can be challenging to clinically distinguish between these conditions and stone disease, and some patients may need clarification on whether a C.T. scan should be done with or without contrast. Additionally, pyelonephritis and renal abscess, which could mimic or worsen symptomatic urolithiasis, are challenging to detect on unenhanced CT.4\n\nFew studies have specifically looked at the phenomena that have been Theorized to decrease the sensitivity of C.T. for identifying renal stones.\n\nMaximal-intensity projection (M.I.P.) techniques generate a two-dimensional image by projecting a line (or search ray) through a dataset in a given volume, highlighting the voxel with the highest attenuation value. M.I.P. is commonly used in C.T. angiography for evaluation and display purposes.5\n\nIn our study, we explored the effectiveness of coronal M.I.P. reformations in detecting and measuring the Hounsfield density of urinary tract stones. We hypothesized that the improved visibility of stones on M.I.P. images would enhance the detection of urinary tract stones compared to regular axial and coronal reformats. Additionally, since M.I.P.s eliminate partial volume-averaging effects, density measurements may be more precise.6\n\nThe purpose of the study is to identify which CT Projection (Axial, Coronal M.I.P.) is best for the Detection of Renal stones; the current study was to compare the detection rate and size measurements of Renal calculi on Axial And coronal MIP images.\n\n\nProtocol\n\nThe ethical statement indicates that the study protocol on the comparative evaluation of urinary calculus has received approval from the Institutional Ethics Committee at D.M.I.H.E.R. (DU), Warda. The study protocol has been assigned the IEC number DMIHER (DU)/IEC/2023/588, confirming its compliance with ethical standards, The study will include 120 patients, and all participants will be requested to provide written informed consent. The statement provides reassurance that the study will be conducted with due consideration for ethical principles.\n\nThe present study constitutes a prospective comparative investigation aimed at assessing the efficacy of Normal Coronal and axial projections vs coronal and axial projections using maximum intensity projection (M.I.P.) in the assessment of urinary calculus. One hundred twenty patients with established urinary calculus will be randomly selected. The primary objective of this investigation is to discern and compare the diagnostic accuracy of M.I.P. in the detection of urinary calculi using these two distinct imaging planes. Additionally, the study endeavours to evaluate the sensitivity of urinary calculus by determining its size, location, and composition.\n\nIn order to be considered for inclusion in the study, participants need to satisfy the following requirements:\n\n• The study will enrol patients referred to Acharya Vinoba Bhave Rural Hospital for urinary calculus evaluation\n\n• Only patients referred to the radiology department for C.T. Abdomen or KUB imaging will be considered for enrolment\n\n• Participants should be 18 years or older to be eligible for inclusion in the study\n\n• Written informed consent will be obtained from all prospective participants prior to enrolment, in adherence with ethical principles\n\nThe following criteria must be met by potential participants in order to be excluded from the study:\n\n• the presence of contraindications for C.T. imaging, such as pregnancy,\n\n• renal impairment,\n\n• inability to provide informed consent,\n\n• participation in other clinical trials within the last three months.\n\nRationale: This study examined whether axial or coronal maximum-intensity-projection (M.I.P.) reformations enhance renal stone identification in computer tomography by determining the sensitivity of thin axial and coronal M.I.P. images for the diagnosis of renal stones.\n\nAim: This study’s objective is to assess the efficacy of computed tomography in axial and coronal renal stone identification.\n\n\n\n1. To identify the morphological structure of stones’ size.\n\n2. To identify the external structure and features of the renal bladder\n\n3. To identify the Renal calculi in the urinary tract\n\n4. To identify the location of the renal stone in the abdomen\n\nOne hundred twenty participants (n=120) were enrolled in the study, consisting of individuals aged 18 years and above who were visiting the radiology department for Abdo-Pelvic CT scans.\n\nSampling procedure and formula used:\n\nZ2 is the level of Significance at 5%, i.e. 95%\n\nConfidence interval =1.96\n\nP = Expected prevalence or proportion = 2.8% = 0.028\n\nD=precision (if the precision is 3%, then d = 0.03)\n\nn = 120\n\nStudy Reference: Sajal Sayeed\n\nFormula reference: Daniels’s formula\n\nSoftware used: SPSS 27.0 version\n\n\nMethodology\n\nOver two years, we plan to recruit 120 patients over the age of 18 who have been referred to A.B.V.R.H. hospital. The study will focus on the visible anatomy and specific information regarding the size, shape, and location of the urinary stones and the techniques used to capture the images.\n\nDuring the study, we will compare the calculus size measurements in three planes: A.P. × TRANS × CRANIOCAUDAL between the C.T. Axial, Coronal, and CT M.I.P. Axial and Coronal images, with a focus on stones measuring 4 mm, 6 mm, and 8 mm with your innovative study, you hope to provide valuable insights into the efficacy of using maximum intensity projection for the evaluation of urinary calculus, potentially paving the way for improved diagnostic and treatment options for patients.\n\nDepartment of Radio Diagnosis, Acharya Vinoba Bhave Rural Hospital, Datta Meghe Institute of Higher Education & Research. Relevant dates, including periods of recruitment: October 2022 – October 2024.\n\nAll the patients presented to the Department of Radio Diagnosis, Acharya Vinoba Bhave Rural Hospital, Datta Meghe Institute of Higher Education & Research for a CT Abdomen and Pelvis scan will be considered.\n\nOutcome: Overall, the study protocol provides a structured approach for investigating the comparative efficacy of axial and coronal C.T. images using maximum intensity projection to evaluate urinary calculus. The study’s real findings may offer fresh perspectives on the identification and management of urinary calculi. They could aid in the creation of better imaging methods for this illness.\n\nData analysis: The study will be conducted once the final report has been finished.\n\nType of study: Closs sectional study\n\nDuration of study: 2 years\n\nPlace of study: Datta Maghe Institute of higher education and Research Sawangi Wardha\n\n\n\n1. The study will use a 16-slice S.O.M.A.T.O.M. Machine CT scanner to perform Abdomen/KUB scans.\n\n2. 120 patients who are 18 years or older and referred to A.B.V.R.H. hospital will be considered.\n\n3. The technique of reconstructed soft-tissue window imaging will be used for the C.T. scan.\n\n4. The reconstructed Axial, Coronal images, and Axial, Coronal M.I.P. images of the abdomen and KUB will be used to compare the findings.\n\n5. The study will describe visible anatomies, such as size, shape, and location, as well as specific information about identified stones and imaging techniques used to capture the images.\n\n6. The study will take place over a two-year period, during which the patients will receive counseling.\n\n7. The calculus size measurement will be compared in 3 planes (A.P. × TRANS × CRANIOCAUDAL) between C.T. Axial, Coronal, and CT M.I.P. Axial and Coronal images.\n\n8. The size of the calculi to be measured will be 4 mm, 6 mm, and 8 mm.\n\n\nDiscussion\n\nThe results of the study suggest that thin images obtained from contrast-enhanced C.T. (CECT) during the portal venous phase are highly effective in detecting renal stones measuring 2 mm or larger. However, for patients experiencing acute flank pain with suspected renal stone disease, unenhanced C.T. is still the recommended imaging test. Traditionally, C.E.C.T. has been considered inadequate for detecting renal stones and is not recommended in such cases. Flank pain can be caused by conditions other than urolithiasis, and various renal disorders can present similar symptoms.7 This poses a challenge in selecting the most suitable imaging protocol when evaluating patients suspected of having urolithiasis along with other conditions like appendicitis or diverticulitis.\n\nIf contrast material is used to optimize the evaluation, it is important to be aware of the sensitivity of C.E.C.T. in detecting renal stones.8 The reduced sensitivity of C.E.C.T. in this regard is attributed to the enhanced appearance of the kidneys, which diminishes the contrast between the stones and the renal parenchyma. Recent studies have reported varying sensitivities in detecting renal stones with C.E.C.T.9 One study found an overall sensitivity of 81% for detecting renal stones, with higher sensitivity for larger stones. Another study focusing on arterial phase CT detected 75% of renal calculi and all stones larger than 5 mm. The differences in sensitivities could be due to variations in slice thickness, as thinner slices have been shown to enhance renal stone detection on unenhanced CT.10\n\nThin images from portal venous phase C.E.C.T. demonstrate high sensitivity in detecting renal stones measuring 2 mm or larger. However, for patients with acute flank pain and suspected renal stone disease, unenhanced CT remains the recommended imaging test. C.E.C.T. has historically been deemed inadequate for detecting renal stones and is not recommended for this purpose.11 It is important to consider the challenges in accurately diagnosing renal stones, as other conditions can mimic urolithiasis symptoms. The choice of imaging protocol should be carefully considered, taking into account potential alternative conditions and the sensitivity of C.E.C.T. in detecting renal stones. Thinner image slices have shown improved detection of renal stones on unenhanced CT.12\n\nOngoing; expected to complete before October 2024", "appendix": "Data availability\n\nNo underlying data are associated with this article.\n\nNo extended data are associated with this article.\n\n\nReferences\n\nPrstojevic JK, Junuzovic D, Hasanbegovic M, et al.: Characteristics of Calculi in the Urinary Tract. Mater. Soc. 2014 Oct; 26(5): 297–302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCorwin MT, Lee JS, Fananapazir G, et al.: Detection of Renal Stones on Portal Venous Phase CT: Comparison of Thin Axial and Coronal Maximum-Intensity-Projection Images. Am. J. Roentgenol. 2016 Dec; 207(6): 1200–1204. PubMed Abstract \| Publisher Full Text\n\nChu G, Rosenfield AT, Anderson K, et al.: Sensitivity and value of digital CT scout radiography for detecting ureteral stones in patients with ureterolithiasis diagnosed on unenhanced CT. Am. J. Roentgenol. 1999 Aug; 173(2): 417–423. PubMed Abstract \| Publisher Full Text\n\nEisner BH, McQuaid JW, Hyams E, et al.: Nephrolithiasis: What Surgeons Need to Know. Am. J. Roentgenol. 2011 Jun; 196(6): 1274–1278. PubMed Abstract \| Publisher Full Text\n\nDym RJ, Duncan DR, Spektor M, et al.: Renal stones on portal venous phase contrast-enhanced CT: Does intravenous contrast interfere with detection? Abdom. Imaging. 2014 Jun; 39(3): 526–532. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCarthy CJ, Baliyan V, Kordbacheh H, et al.: Radiology of renal stone disease. Int. J. Surg. 2016 Dec 1; 36: 638–646. Publisher Full Text\n\nCorwin MT, Hsu M, McGahan JP, et al.: Unenhanced MDCT in suspected urolithiasis: improved stone detection and density measurements using coronal maximum-intensity-projection images. Am. J. Roentgenol. 2013 Nov; 201(5): 1036–1040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel U, Walkden RM, Ghani KR, et al.: Three-dimensional CT pyelography for planning of percutaneous nephrostolithotomy: accuracy of stone measurement, stone depiction and pelvicalyceal reconstruction. Eur. Radiol. 2009 May; 19: 1280–1288. PubMed Abstract \| Publisher Full Text\n\nGhani KR, Patel U, Anson K: Computed tomography for percutaneous renal access. J. Endourol. 2009 Oct 1; 23(10): 1633–1639. Publisher Full Text\n\nRydberg J, Kopecky KK, Tann M, et al.: Evaluation of prospective living renal donors for laparoscopic nephrectomy with multisection CT: the marriage of minimally invasive imaging with minimally invasive surgery. Radiographics. 2001 Oct; 21(suppl_1): S223–S236. PubMed Abstract \| Publisher Full Text\n\nDillman JR, Caoili EM, Cohan RH: Multi-detector CT urography: a one-stop renal and urinary tract imaging modality. Abdom. Imaging. 2007 Aug; 32: 519–529. PubMed Abstract \| Publisher Full Text\n\nOlcott EW, Sommer FG, Napel S: Accuracy of detection and measurement of renal calculi: in vitro comparison of three-dimensional spiral CT, radiography, and nephrotomography. Radiology. 1997 Jul; 204(1): 19–25. PubMed Abstract \| Publisher Full Text" }	[ { "id": "206211", "date": "05 Oct 2023", "name": "Elanchezhian Somasundaram", "expertise": [ "Reviewer Expertise CT", "AI", "Medical Physics" ], "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 writing is not clear and confusing to read.\n\nThe objectives are also not clear. What is the clinical benefit of evaluating a MIP image? How is this going to reduce the dose? There is no difference in dose or exam protocol for generating the coronal or axial series. It is a single scan from which both series are reconstructed, so I don't understand about the dose being different for both planes.\n\nAlso, clinicians are going to use a 3D volume to localize the kidney stones and measure them from the CT scan. I am not sure how you propose to localize the stones from a MIP image.\n\nWhat methodology are you going to use to create the MIP? No details about this are presented.\n\nNo feasibility study or sample images provided to even know if the MIP would shows the kidney stones clearly.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [ { "c_id": "10685", "date": "13 Apr 2024", "name": "syed asrar", "role": "Author Response", "response": "Sir, I hold your opinions in high regard, and I genuinely appreciate your insights. Sir, may I kindly inquire where you find aspects of my study confusing? Your guidance is invaluable to me, and I am eager to enhance my understanding. Additionally, I would be grateful for your suggestions regarding variables that could contribute to improving my study habits. Your esteemed advice will undoubtedly aid me in achieving greater academic success" } ] } ]	1	https://f1000research.com/articles/12-952
https://f1000research.com/articles/10-1116/v1	05 Nov 21	{ "type": "Research Article", "title": "Association between media use and poor sleep quality among senior high school students: a cross-sectional study", "authors": [ "Wisit Chaveepojnkamjorn", "Jutiporn Srikaew", "Pratana Satitvipawee", "Supachai Pitikultang", "Soontaree Khampeng", "Jutiporn Srikaew", "Pratana Satitvipawee", "Supachai Pitikultang", "Soontaree Khampeng" ], "abstract": "Background: Poor sleep quality (PSQ) is an increasing health problem among adolescents. Mobile phones and portable media devices have become a part of children’s lives and may affect their sleep duration and quality. This study aimed to explore the prevalence of PSQ and identify the association between media use and PSQ among adolescents studying in high school grades 10-12. Methods: This cross-sectional study was conducted in central Thailand. A multi-stage sampling technique was used to enroll 777 adolescents from eight schools from August to October 2016. The research instruments comprised factors of demographics and consumption behaviors and the Pittsburgh Sleep Quality Index questionnaire. Multivariable logistic regression was used to calculate adjusted odds ratios (ORadj) and 95% confidence intervals (CI). Results: Prevalence of PSQ was 56.24%. The study subjects were mostly 16-17 years old (67.82%) and female (70.39%). Multivariable logistic regression, after controlling for possible confounders, revealed an increased odds of PSQ among those who used a social media device (OR=1.34, 95%CI=0.97-1.87), and showed a higher proportion of social media use in the PSQ group. Conclusion: A surveillance system to detect media use and PSQ should be conducted accompanied by knowledge sharing on media use among parents, teachers and adolescents. To determine causal relationships, further longitudinal studies will be required to test the association between media users and PSQ. This study may also provide some implications for health promotion on sleep quality of senior high school students.", "keywords": [ "media use", "poor sleep quality", "senior high school students" ], "content": "Abbreviations\n\nCMB: the China Medical Board\n\nGPA: grade point average\n\nGSQ: Good Sleep Quality\n\nORadj: adjusted odds ratio\n\nORc: crude odds ratio\n\nPSQ: Poor Sleep Quality\n\nPSQI: the Pittsburgh Sleep Quality Index\n\n\nIntroduction\n\nSleep is an essential part of life and plays important roles in physical and mental health.1,2 Adolescents experience significant changes to the body and mind associated with sex hormones.3 Insufficient sleep has been one of the most important public health problems among adolescents. Concerning the aspect of sleep, a few studies have found that poor sleep quality (PSQ) was associated with the amount of daytime sleep, exhaustion, weight gain, obesity, impaired memory and motor vehicle accidents.4–6 PSQ is currently a widespread issue in most societies. The prevalence of PSQ among adolescents was reported to range from 32 to 62%7–11 reflecting a wider range of PSQ prevalence. In Thailand, the prevalence of PSQ among adolescents was reportedly 32 to 48%.7,8 Insufficient sleep not only impacts at a personal level, but also can cause major impact on a larger scale through a high burden of non-communicable diseases,12 many events such as motor vehicle crashes,13 workplace accidents, increased mortality and reduced quality of life.14 Media use such as watching TV and using electronic devices are activities that cause PSQ among children and adolescents. Especially among school age group, having a TV in the bedroom can disturb sleep resulting in decreased sleep duration and insufficient sleep. In addition, media use may increase the activity of physiological arousal, inadequate sleep hygiene practice and difficulty falling asleep.11 Some studies have shown the association of media use related to PSQ.15–17 Hence, the present study aimed to seek the prevalence of PSQ and determine its association with media use among senior high school students in Ratchaburi Province, Thailand. Provinces of Western Thailand which are composed of Kanchanaburi, Phetchaburi, Prachuap Khiri Khan, Tak and Ratchaburi, geographical region and academic area are similar so the authors selected Ratchaburi Province as the area of study because of its characteristics as a proxy of western provinces of Thailand. Ratchaburi is located on the bank of the Mae Klong river and one of the western provinces of Thailand with an area of about 5,196 square kilometer. It lies 80 km west of Bangkok, and borders Myanmar to the west with the Tenasserim Hills as a natural border containing a population of 871,714 and density of 170 km2 in 2017.18,19\n\n\nMethods\n\nA cross-sectional study was carried out between August and October 2016 to explore PSQ and identify the association between media use and PSQ occurrence among senior high school students in Ratchaburi Province, Thailand.\n\nThe sample size was calculated using a formula to estimate the population proportion with specified absolute precision20 according to the following assumption: 32% of PSQ among adolescents (P),7 with 95% confidence interval and 5% specified absolute precision (d). As a multistage sampling technique was employed to identify study subjects, a design effect of 2 was used. The calculated sample size totaled 709. Also, approximately 10% was added to adjust for nonresponses. Thus, the final sample size was at least 777.\n\nA multi-stage stratified sampling technique was used to identify study subjects from senior high schools in Ratchaburi Province. Schools were stratified by student numbers, namely, extra large (>2,500), large (1,500-2,499) and medium (500-1,499). We randomly selected at least one school from the list of three school categories: urban and rural public schools and private schools. The selection of schools was based on a list of schools obtained from the Provincial Education Office and willingness of school administrators to participate in the study. For each of the schools, the student sample size was calculated proportional to the size of the schools.\n\nThe study was conducted in accordance with the ethical principles in the Declaration of Helsinki, and the protocol was reviewed and approved by the Human Research Ethical Review Committee of the Faculty of Public Health, Mahidol University (COA. No. MUPH 2016-097). The purpose of this study was explained to school principals and teachers of the target schools. Permission was obtained from these schools and students; written informed consent was obtained from the student’s parents or legal guardians after informing them of the study details (the objectives of study, methods and protection of human rights). Parents or legal guardians were told that participating in the survey was voluntary and that the survey would remain anonymous. Confidentiality was maintained throughout the study using anonymous technique (schools and respondents were identified by code numbers to ensure confidentiality and the results were analyzed as a whole group).\n\nStudy population was senior high school students grades 10-12 during the educational year 2016 in Ratchaburi province.\n\n\n\na) Students who studied in grades 10-12.\n\nb) Students who studied in high schools that were under the control of the secondary education service area office 8, Ratchaburi province\n\nc) Students who were willing to participate in the study and provided the written informed consent.\n\nd) Students who provided the written informed consent signed by their parents or legal guardians.\n\n\n\na) Students who were absent from school on a period of data collection.\n\nb) Students who were chronically ill during the time of study.\n\nResearchers contacted the educational administrators and the teachers for data collection. The paper-based questionnaire was provided for the participants to fill data at the free time from studying at their school. Researcher and research assistants explained the details of questionnaire and answered the questions from participants. This process was approximately 40 minutes. Information was collected using a self-administered, anonymous questionnaire comprising three parts, namely, demographics, consumption behaviors relating to sleep quality, sleep quality assessment and media-used evaluation. A copy of the questionnaire can be found in the Extended data.35 Sleep quality was evaluated using the Pittsburgh Sleep Quality Index (PSQI) translated to Thai with a cutoff point of scores > 5 was classified as poor sleepers and ≤5 was classified as good sleepers.20–23 Reliability was tested revealing a Cronbach’s alpha of 0.86.\n\nThe data were encoded and processed for statistical analysis by using SPSS for Windows, Version 18). Categorical variables were given as frequency and percentage, crude odds ratio (ORc), 95 % CI of OR and p-value. Moreover, numerical variables were expressed as mean, median, minimum and maximum, standard deviation and quartile deviation. Univariate analysis was performed using univariable logistic regression to differentiate proportional exposures between poor and good sleepers for categorical variables. Adjusted odds ratio (ORadj) and 95% CI of OR were calculated from multivariable logistic regression to examine associations between media use and PSQ occurrence, adjusted for potential confounders. All statistics were performed using two-sided tests, and the criteria of p <0.05 was judged to be statistically significant.\n\n\nResults\n\nIn total, 777 students were selected for the present study. The majority were female (70.39%), aged 16 to 17 years (67.82%), studying in Grade 12 (35.39%), GPA 3.01 to 3.50 (40.14%), monthly family income ≤10,000 THB (44.67%), no smoking (98.33%) and no alcohol consumption (85.33%), as shown in Table 1.\n\nThe prevalence of PSQ was 56.24%. Using univariable logistic regression analysis, associated demographic factors of PSQ among adolescents included illness history during the last month, coffee and tea consumption, reading, annoyance, poor ventilation, stress, depression and sleep duration (p < 0.05), as shown in Table 2. In case of media use, we found an association between social media use and PSQ (OR = 1.53, 95%CI = 1.13-2.08), as shown in Table 3. Using multivariable logistic regression analysis, regarding association between social media use and PSQ among adolescents (adjusted for potential confounders), social media users were 1.34 times at risk compared with those of nonusers (OR = 1.34, 95%CI = 0.97-1.87) but without significance, as shown in Table 4. Comparing PSQ and good sleep quality (GSQ) groups, the most commonly activity before bedtime was social media (44.56%, 37.38%) and television watching (20.78%, 30.29%) respectively. Further, we found a higher proportion of social media use in the PSQ group, as shown in Table 5.\n\n* Statistically significant (p < 0.05).\n\n* Statistically significant (p < 0.05).\n\n\nDiscussion\n\nOur findings demonstrated that PSQ prevalence rate was about 56% higher than related studies conducted in Thailand.7,8 Evidence from related studies on PSQ among college students showed PSQ prevalence was approximately from 32 to 62%.7–11 The difference of PSQ occurrence might have stemmed from various factors, namely, environment, lifestyle, household characteristics, social media and activities, health behaviors etc. Univariable analysis showed that social media use played a critical role in the development of PSQ among adolescents (OR = 1.53, p = 0.006). However multivariable logistic regression analysis did not indicate significant differences (OR = 1.34, 95%CI = 0.97-1.87). Some studies indicated adolescents who used social media before bedtime had lower sleep efficiency.15–17,24–27 Mobile phone use among young students for daily calling, using e-mail, text messaging and social network services were associated with short sleep duration, PSQ, excessive daytime sleepiness and presenting insomnia symptoms.15,28,29 Higher frequency and volume of social media use had significantly greater odds of having sleep disturbance among young adults,24,26 while one study showed a better sleep quality among users.30 The present study showed the prevalence of social media use before bedtime in the PSQ group was approximately 44.56%. One half of social media users spent over 2 hours per day. The average time for social media use was 3.58 hours per day, and this might have affected sleep pattern. A related study showed users who spent 0.5 to 2 hours per day on social media were more likely to have poor sleep than those of spent less than 0.5 hours.31 In addition, the meta-analysis studies reported social media users before bed were more likely to have insufficient sleep and tended to have PSQ.27,32 Some related studies have suggested blue light emitted from smart phones might disturb sleep.33,34 Therefore, monitoring social media use among adolescents, and cooperating with parents, caregivers, teachers and the adolescents themselves is recommended to reduce PSQ problems.\n\nThis study encountered a few limitations that need to be addressed. First, cross-sectional surveys reduced the ability of the study to make direct causal inferences. Second, these data apply only to those aged 14-19 years as the study subjects; therefore, they could not represent all adolescents. Moreover, data collection might have excluded subjects absent from schools. Finally, all data were based using a self-report method subject to recall bias.\n\n\nConclusion\n\nPSQ surveillance systems should be established along with knowledge sharing programs regarding associated factors of PSQ among adolescents with their parents and teachers. We recommend that the use of media and the presence of media equipment in bedroom should be limited. This may be beneficial to sleep quality.\n\n\nData availability\n\nOSF: Association between media use and poor sleep quality among senior high school students: a cross-sectional study. https://doi.org/10.17605/OSF.IO/KV2BJ.35\n\nThis project includes the following underlying data.\n\n- SAV Dataset\n\nOSF: Association between media use and poor sleep quality among senior high school students: a cross-sectional study. https://doi.org/10.17605/OSF.IO/KV2BJ.35\n\nThis project includes the following extended data.\n\n- Appendix A (The certificate of ethical approval)\n\n- Appendix B (A copy of the questionnaire)\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\nAll authors would like to express their sincere thanks for the valuable contributions to the study provided by students and staff of participating high schools. We also thank those not mentioned for their kindness and encouragement. A previous version of this article is published on Research Square: https://www.researchsquare.com/article/rs-81395/v1\n\n\nReferences\n\nZhang Z, Chen T, Jin X, et al.: Sleep patterns, sleep problems and associations with reported sleep quality in Chinese school-aged children. Am J Public Health Res. 2013; 1(4): 93–100. Publisher Full Text\n\nMindell JA, Meltzer LJ: Behavioral sleep disorders in children and adolescents. Ann. Acad. Med. 2008; 37(8): 722–728.\n\nMcDermott B, Baigent M, Chanen A, et al.: Clinical practice guidelines: depression in adolescents and young adults. Melbourne (Australia): beyond blue: the national depression initiative. 2011.\n\nMalhotra S, Kushida CA: Primary hypersomnias of central origin. Continuum. 2013; 19(1): 67–85. Publisher Full Text\n\nGiannotti F, Cortesi F: Family and cultural influences on sleep development. Child Adolesc. Psychiatr. Clin. N. Am. 2009; 18(4): 849–861. 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PubMed Abstract \| Publisher Full Text\n\nChaveepojnkamjorn W, Srikaew J, Satitvipawee P, et al.: Association between media use and poor sleep quality among senior high school students: a cross-sectional study. OSF. 2021. Publisher Full Text" }	[ { "id": "99456", "date": "25 Nov 2021", "name": "Pongdech Sarakarn", "expertise": [ "Reviewer Expertise Statistical method for health modeling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript addresses a very important issue for solving the problem of the adolescent group. However, some components should be considered and added up for fulfilling the content to implication or implementation. I have the following comments:\nThe gap of knowledge for the association between media use and poor sleep quality (PSQ) among senior high school students should be identified clearly. For example, the magnitude of related factors for such an association, in previous studies, have not been justified based on some limitations (conditions). This study can add something more regarding detecting the precision or properly-related factors compared to previous studies\n\nMedia use is a key factor – I would like to know how it is associated with the PSQ in this study, but the authors didn’t mention the factor in the manuscript. Therefore, the media use should be identified clearly in the methods part.\n\nPotential confounders which are included in the multi-variable analysis step should also be identified in the methods section. According to such confounders, some studies (Fatima et al., 20161, Galland et al., 20172) pointed out that the PSQ had a higher prevalence in girls than in boys. From the results, what do you think about the gender influence in your study? Why didn’t you add the factor as confounder in the model? Or, can you expand more on the subject of gender variables?\n\nFor explicitly identifying the magnitude of estimation, the 95% CI of the PSQ prevalence should be added.\n\nIn Table 5, I am confused regarding PSQ and good sleep quality (GSQ) for each type of media use. Given that they are opposites, a table comparing them may be difficult to interpret and lead to misunderstanding. Also, the total of each row doesn’t add up to 100%, could you please explain how you want to want to present this table, and how you calculate the percentage of both (PSQ and GSQ) in each type of the media use? I suggest that the column of GSQ should be removed from the table, because the remainder of PSQ can be interpreted as GSQ. However, each row cannot be calculated total as 100%, so please describe more for this table.\n\nAccording to the results in Table 3, 4 and 5, data management should be mentioned and described in the part of data analysis, regarding how social media is considered and included into the final model.\n\nIssues with the statistical method issues should be mentioned, in particular the finding that is not statistically significant. For example, is the standard logistic regression appropriate for this data or not? As students from the same room may be correlated, while some students from the different room may be independent. Therefore, the statistical methods for correlating the outcome, like the generalised estimating equation (GEE) and logistic regression, should maybe be analysed based on identifying the cluster as with the room\n\nFinally, even if the data of this study was collected before the transmission of the COVID-19, the issue of media use can change, and affect the topic. I think the author should mention this point in the discussion or limitation part.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "7514", "date": "08 Aug 2023", "name": "Wisit Chaveepojnkamjorn", "role": "Author Response", "response": "User Comment: I am an author of this article. This manuscript addresses a very important issue for solving the problem of the adolescent group. However, some components should be considered and added up for fulfilling the content to implication or implementation. I have the following comments: To The gap of knowledge for the association between media use and poor sleep quality (PSQ) among senior high school students should be identified clearly. For example, the magnitude of related factors for such an association, in previous studies, have not been justified based on some limitations (conditions). This study can add something more regarding detecting the precision or properly-related factors compared to previous studies Response Thank you very much for your comments. The objectives of study aimed to seek the prevalence of PSQ and determine its association with media use among senior high school students. We justified the definition of PSQ by using the Pittsburgh Sleep Quality Index (PSQI) for poor and good sleepers. Media use is a key factor – I would like to know how it is associated with the PSQ in this study, but the authors didn’t mention the factor in the manuscript. Therefore, the media use should be identified clearly in the methods part. Response The authors made considerations about the media use, and made the questionnaire for collecting the media use such as watching TV and using electronic devices before bedtime. Potential confounders which are included in the multi-variable analysis step should also be identified in the methods section. According to such confounders, some studies (Fatima et al., 20161, Galland et al., 20172) pointed out that the PSQ had a higher prevalence in girls than in boys. From the results, what do you think about the gender influence in your study? Why didn’t you add the factor as confounder in the model? Or, can you expand more on the subject of gender variables? Response Demographic factors associated with PSQ (such as illness history during the last month and etc) were controlled by the multivariable logistic regression. It showed at the end of Table 4. The issue of gender, Table 2 sex variable didn’t show the association with PSQ and we didn’t add this one in the model. For explicitly identifying the magnitude of estimation, the 95% CI of the PSQ prevalence should be added. Response The PSQ was showed in the result section (PSQ and associated factors) and we are ready added the 95% CI of the PSQ (52.75-59.74). In Table 5, I am confused regarding PSQ and good sleep quality (GSQ) for each type of media use. Given that they are opposites, a table comparing them may be difficult to interpret and lead to misunderstanding. Also, the total of each row doesn’t add up to 100%, could you please explain how you want to want to present this table, and how you calculate the percentage of both (PSQ and GSQ) in each type of the media use? I suggest that the column of GSQ should be removed from the table, because the remainder of PSQ can be interpreted as GSQ. However, each row cannot be calculated total as 100%, so please describe more for this table. Response Table 5 showed the proportion of media use among the PSQ and the GSQ. We would like to seek the difference of the media use in both groups. According to the results in Table 3, 4 and 5, data management should be mentioned and described in the part of data analysis, regarding how social media is considered and included into the final model. Response We are ready added your recommendations. The data were verified, encoded and processed… We added at the end of potential confounders (enter method) Issues with the statistical method issues should be mentioned, in particular the finding that is not statistically significant. For example, is the standard logistic regression appropriate for this data or not? As students from the same room may be correlated, while some students from the different room may be independent. Therefore, the statistical methods for correlating the outcome, like the generalised estimating equation (GEE) and logistic regression, should maybe be analysed based on identifying the cluster as with the room Response We concerned and realized of the statistical use for analysis. Statistical methods such as logistic regression is a tool to prove the association between the exposure and the outcome. However, every statistics had its limitation and criteria for appropriate use. Finally, even if the data of this study was collected before the transmission of the COVID-19, the issue of media use can change, and affect the topic. I think the author should mention this point in the discussion or limitation part. Response We think the pandemic of COVID-19 changed the learning of everyone . The trend of social media use was rapidly increasing every generation groups . The further studies should be concerned after the COVID-19 pandemic." } ] }, { "id": "166408", "date": "18 Apr 2023", "name": "Kavita Batra", "expertise": [ "Reviewer Expertise Global health", "Health behaviors Research", "Maternal and Child Health", "Statistics", "Epidemiology", "COVID-19" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle: Please consider adding the place of the study in the title.\n\nIntroduction: The introduction section is not backed up by sufficient literature review. Relevant references can be added to strengthen the argument of sleep problems among countries with similar standing. Comparison with other developed countries is highly advised.\n\nData are too old. Several things have been changed now especially with the COVID-19 pandemic.\n\nSince authors translated the tool in another language, it becomes critical to assess the psychometric validity of the tool. Please describe.\n\nWhat estimates did the authors use to perform stratified sampling? After the stratification, how did the authors ensure random selection?\n\nPlease do the bivariate group comparisons via Chi-square first.\n\nExpand on the potential value of the 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? 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": "9589", "date": "21 Apr 2023", "name": "Wisit Chaveepojnkamjorn", "role": "Author Response", "response": "Respose to this report: We believe that the title is appropriate and concise. The place of study is showed in the methods. The introduction indicated burden of poor sleep quality and media use. The authors studied this study during August-October 2016 and submitted this article on 1st July 2021 for publication in the F1000Research. We believed in the tool PSQI version Thai, it is a good tool which we had reference 23. We used the multi stage stratified sampling technique (we added the design effect of 2 and added 10% for non-response). We used the univariable logistic regression for bivariate analysis." } ] } ]	1	https://f1000research.com/articles/10-1116
https://f1000research.com/articles/12-948/v1	07 Aug 23	{ "type": "Opinion Article", "title": "Whose Ear?: Proposal to conserve the name Auricularia auricula (L.) Underw. for Auricularia auricula-judae (Bull.) Quél.", "authors": [ "Kendra C. Autumn", "Bryn T. M. Dentinger", "Bryn T. M. Dentinger" ], "abstract": "Auricularia auricula-judae is a saprobic European jelly fungus with traditional culinary and medicinal significance, often said to resemble a human ear. It was originally named Tremella auricula by Linnaeus and has been moved to different genera since, but its specific epithet was also changed from auricula to auricula-judae by Bulliard in 1789, which is not normally a valid nomenclatural alteration. However, due to the practice of \"name sanctioning\" in the mycological nomenclatural code, this change has been accepted. This article outlines the nomenclatural and cultural history of the controversial name Auricularia auricula-judae and suggests its return to the original specific epithet auricula, as well as the designation of an epitype specimen.", "keywords": [ "mycology", "taxonomy", "nomenclature", "historical mycology", "Jew's ear", "jelly ear" ], "content": "Introduction\n\nThe jelly fungus Auricularia auricula-judae (Bull.) Quél. (Auriculariaceae, Auriculariales, Agaricomycetes, Basidiomycota) is a saprobic mushroom that grows on multiple hardwood species and has a long history of medicinal and culinary use (Kout and Wu 2022). Previously, various Auricularia specimens from Europe, North America, and Asia were regarded as belonging to a single species, A. auricula-judae, but molecular systematics have shown that European specimens form a distinct monophyletic clade (Wu et al. 2015, 2021). To reflect these findings, true A. auricula-judae are considered to occur solely in Europe, although similar Auricularia species have garnered interest both traditionally and in modern times as food and medicine, particularly in Asia.\n\nA. auricula-judae has several common names: Judas’ ear, Jew’s ear, wood ear, tree ear, or jelly ear. This profusion of common names may represent awkwardness around the specific epithet. David Arora’s popular foraging-focused book All That the Rain Promises and More… uses an invalid species name, Auricularia auricula, removing -judae from the epithet, and offers the common name “Wood Ear”, with “Tree Ear” and “Judas’ Ear” as alternatives (Arora 1991). Similarly, the 1975 A Field Guide to Western Mushrooms lists only Auricularia auricula (Smith 1975). It’s unclear whether this habit of shortening the epithet is simply a response to the bulky hyphenation, or of the resemblance of the term “judae” to “Judaism” and other words associated with the Jewish people and religion. This superficial resemblance may have resulted in the common name Jew’s ear, or a reference to Jews may have been purposeful from the creation of the current epithet.\n\n\nHistory\n\nThe binomial Auricularia auricula-judae emerged from a string of name changes during the 18th and 19th centuries. Linnaeus created the species Tremella auricula in 1753, as documented in Species Plantarum (Figure 1) (Linné 1753). In 1789, Bulliard appended “-judae” to the specific epithet in his publication Herbier de la France, functionally changing the name to Tremella auricula-judae, an invalid alteration in regard to taxonomic convention (Herbier de la France 1780-93). In 1822, Fries moved the species to the genus Exidia in the second volume of Systema mycologicum (Fries 1821), preserving Bulliard’s specific epithet and creating the name Exidia auricula-judae. The current name Auricularia auricula-judae was established by Quélet in 1886 and recorded in Enchiridion fungorum in Europa media et praesertim in Gallia vigentium (Figure 2) (Quélet 1886).\n\nAlthough Bulliard’s change to the specific epithet has no basis in the general rules of taxonomic naming, it was solidified by its acceptance by Fries, for whom there is a special provision in Article F.3 of the International Code of Nomenclature for algae, fungi, and plants (May et al. 2019). Because of this exception, the publication of Exidia auricula-judae in Systema mycologicum “sanctions” the epithet auricula-judae and excepts it from the nomenclatural principle that would dictate its return to the original auricula as designated by Linnaeus.\n\nA nomenclaturally inconsistent binomial due to name sanctioning isn’t the only issue with the species name as currently designated. When Bulliard established species in Herbier de la France, he didn’t designate physical type specimens, rather allowing skillfully rendered image plates to serve as iconotypes (Figure 3). This means that there is no one individual specimen representing the morphological and genetic qualities used to define A. auricula-judae, creating potential for taxonomic confusion. In our modern era, whole genome sequencing allows us to investigate and estimate species boundaries using an abundance of biological information. Without a biological specimen taxonomically tied to a species name, we risk imprecise application of genomic data.\n\nThe origins of the name A. auricula-judae are complicated, both in publication and in culture. Arguments have been made that “judae” was appended to the specific epithet as a reference to Judas Iscariot, justifying the common name Judas’ ear, from which Jew’s ear would have been bastardized (Harding 2008). This interpretation relies on several historical references to Judas hanging himself in an elder tree as penance for betraying Jesus (Knowles 2005) and the observation that A. auricula-judae sometimes grows on elder trees (Kout and Wu 2022). Indeed, Buillard refers to elder trees (Sambucus) in his description: “vient sur différentes sortes de bois mais plus ordinairement sur les vieux troncs de Sureau.”\n\nA dictionary of folk etymology published in 1882 states that the name “Jew’s ear”, describing an ear-like fungus, was a corruption of “Judas’ ear” (Palmer 1882). However, The Herball, printed in 1597, contains the usage of “Iewe’s eare” to describe a wood-dwelling mushroom, with no mention of Judas (Gerard 1597). The Regiment of Life, published even earlier in 1544, also references mushrooms called “iewes eares” (Goeurot and Phayer 2011). If Jew’s ear is a mere bastardization and the original intention was to associate the fungus with Judas, the corruption must have occurred quite early, persisting until the present. It’s also interesting to note that when Bulliard published the name Tremella auricula-judae in 1789, he lists a common name “LA TREMELLE OREILLE-DE-JUDA”—ear of “Juda”, but not Judas specifically.\n\nIn French, Judas is spelled exactly the same as it is in English, so did Buillard make an orthographic error with the common name, or was he referring to something else? The French “Juda” can be translated as “Judah.” One meaning of Judah is the Kingdom of Judah, an Israelite Kingdom in the Iron Age centered around Jerusalem and from which Jewish people are primarily descended. Thus “Judah” could be interpreted as interchangeable with “Jew.” However, the translation of the French “Juda” is not entirely clear. Interestingly, the definition for the heading “Juda” in a 1789 French dictionary (Dictionnaire de l’Académie françoise 1798) is “Ouverture pratiquée à un plancher, et communément fermée d’ une petite trappe amovible, pour voir ce qui se passe au-dessous.”, translated into English as “Opening made in a floor, and commonly closed with a small removable trapdoor, to see what is happening below.” Could Buillard have been simply referring to a peephole? Yet, dictionary entries under various headings from 1694 to 1932 also refer to “les Rois de Juda” or “la tribu de Juda,” clearly demonstrating the well-established use of the term “Juda” in direct reference to Jewish people. It seems likely that, given the prevalence of antisemitic attitudes throughout Europe in the 18th Century, Buillard’s emendment of the Linnaen epithet was intentionally referring to Jewish people and reflects a widely held prejudice of the time. Perhaps Buillard’s embellishment was inspired by his resistance to the emerging tolerance espoused by the revolutionaries that overthrew the French monarchy in the same year Herbier de la France was published.\n\nEven if we accept the premise that the added “judae” is a reference to Judas, not the Jewish people, the distinction obviously isn’t clear, judging by the many recorded instances of the name Jew’s ear over several centuries, as well as its use in guidebooks from the 20th century. Author Patrick Harding, who argues that a change to the common name would be the “result of political correctness where it is not necessary”, goes on to say: “The name Jew’s ear is a reminder of the folklore surrounding Judas (himself a Jew …”, revealing that, to Harding, even when the ear is Judas’, it’s still relevant that it’s the ear of a Jew.\n\nGoogle Scholar lists multiple papers with “Jew’s ear” in their titles published in 2020 and later focused on members of the genus Auricularia (Ekowati et al. 2020; Islam et al. 2021; Pumnuan et al. 2021; Vyshnavi and Pramod 2022; Xu et al. 2020). Whether or not one finds this common name offensive, it has become unusual to call a taxon by a name that references an ethnic group. For example, the International Ornithological Community World Bird List has updated several common names of South African birds from previous names that incorporated the term “Hottentot”, a derogatory name addressing the Khoikhoi indigenous group (Driver and Bond 2021). While this proposal does not recommend any kind of universal rules for avoiding future offensive scientific names or replacing existing ones, it’s an issue worth considering when creating or altering nomenclature. Social and scientific concerns may intersect: organisms with names that are associated with sensitive, controversial, or offensive subject matter might be avoided as research topics, biasing or impeding scientific progress.\n\nName conservation has been proposed for several fungal taxa to accommodate for discrepancies between the valid binomial and the most commonly used binomial for a species. This issue is most heightened in medicine, where confusion over the identity of organisms could have significant consequences to the ability to treat patients. The name Cryptococcus gattii (Vanbreus. and Takashio) Kwon-Chung and Boekhout was conserved against Cryptococcus hondurianus Castell. under Article 14 of the International Code of Nomenclature for algae, fungi, and plants after Cryptococcus neoformans var. gattii Vanbreus. and Takashio was raised to species level (Kwon-Chung et al. 2002). Although C. hondurianus had priority, the name was not in use within the research community, while C. gattii was already widely used and its adoption would minimize difficulties in scientific communication.\n\nSimilarly, the phytopathogens Balansia claviceps Speg. and Claviceps paspali F. Stevens & J.G. Hall and the entomopathogen Tolypocladium inflatum W. Gams were all proposed for name conservation by Rossman et al. (2017) under Article 14. For each of these taxa, the oldest epithet in the first genus named is valid, but a different epithet has come into common use and a return to the oldest epithet would be disruptive. Name conservation was especially pertinent for T. inflatum due to its medicinally important status as the natural source of the immunosuppressant drug cyclosporin. Article 14 of the Code deals with name conservation and allows for nomenclatural stability to take precedence over nomenclatural priority were approved by the General Committee.\n\nAlthough Auricularia auricula-judae is undeniably frequently used in scientific and popular mycological literature, A. auricula may be used more often, based on a Google Scholar search for “auricularia auricula-judae” returning ~5,050 results, whereas “auricularia auricula -judae” returns ~7,510 results (placing a hyphen, preceded by a space, in front of a term excludes it from Google search results). In our opinion, this unusual, hyphenated epithet, commonly informally or accidentally abbreviated, presents a similar case to name conservations described above.\n\n\nConclusions\n\nWe propose that Auricularia auricula-judae be returned to the original epithet designated by Linnaeus in Species Plantarum, creating the binomial Auricularia auricula. A. auricula is less controversial, less cumbersome, and already somewhat in use as an invalid synonym published in Mycologia by Bernard Lowy in 1952 (Lowy 1952). We also suggest designating a physical epitype in addition to the existing iconotype illustration created by Bulliard in Herbier de la France (Figure 3). Our recommendation is that an epitype specimen should be selected from the dataset of Wu et al. (2021), which delineated species boundaries using a multigene phylogeny.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nArora D: All that the rain promises and more: a hip pocket guide to western mushrooms. Ten Speed Press; 1991.\n\nDictionnaire de l’Académie françoise [Texte imprimé], revu, corrigé et augmenté par l’Académie elle-même: À Paris, chez J. J. Smits et Ce. imp.-lib.5th ed.1798.\n\nDriver RJ, Bond AL: Towards redressing inaccurate, offensive and inappropriate common bird names.2021.\n\nEkowati N, Maharning AR, Ratnaningtyas NI, et al.: Effects of Ethyl Acetate Extract of Jew’s Ear Mushrooms (Auricularia auricula) on Cytotoxic and Apoptosis of Cervical Cancer Cells (HeLa). IOP Conference Series: Earth and Environmental Science. IOP Publishing; 2020, November; (Vol. 593(1): p. 012011).\n\nFries E: Systema mycologicum: Sistens fungorum ordines, genera et species, huc usque cognitas, quas ad normam methodi naturalis determinavit. Lundae, Ex Officina Berlingiana; 1821; (Vol. 1. ). 1821-[1832]. Reference Source\n\nGerard J, Dodoens R, Priest R, et al.: The Herball, or, Generall historie of plantes. Imprinted at London. Iohn Norton; 1597. Reference Source\n\nGoeurot J, Phayer T: The regiment of life, whereunto is added a treatise of the pestilence, with the boke of children, newly corrected and enlarged by T. Phayre. Houssemaine, Nicolas de, d. 1523. Early English Books Online Text Creation Partnership.2011. accessed 30 March 2023. Reference Source\n\nHarding P: Collins Mushroom Miscellany. Collins; 2008. Reference Source\n\nHerbier de la France: ou, Collection complette des plantes indigenes de ce royaume; avec leurs proprie ́te ́s, et leurs usages en medecine, Paris, Chez l’auteur, Didot, Debure, Belin.1780-93. Reference Source\n\nIslam T, Ganesan K, Xu B: Insights into health-promoting effects of Jew’s ear (Auricularia auricula-judae). Trends Food Sci. Technol. 2021; 114: 552–569. Publisher Full Text\n\nKnowles E: The Oxford Dictionary of Phrase and Fable. Oxford University Press; 2005. Retrieved 1 May. 2023. Publisher Full Text\n\nKout J, Wu F: Revealing the Cryptic Diversity of Wood-Inhabiting Auricularia (Auriculariales, Basidiomycota) in Europe. Forests. 2022; 13(4). Article 4. Publisher Full Text\n\nKwon-Chung KJ, Boekhout T, Fell JW, et al.: (1557) Proposal to Conserve the Name Cryptococcus gattii against C. hondurianus and C. bacillisporus (Basidiomycota, Hymenomycetes, Tremellomycetidae). Taxon. 2002; 51(4): 804–806. Publisher Full Text\n\nvon Linné C : Species plantarum: Exhibentes plantas rite cognitas ad genera relatas, cum diferentiis specificis, nominibus trivialibus, synonymis selectis, locis natalibus, secundum systema sexuale digestas: Vol. t.1 (1753). Berlin, Junk, 1908.1753. Reference Source\n\nLowy B: The Genus Auricularia. Mycologia. 1952; 44(5): 656–692. Reference Source\n\nMay TW, Redhead SA, Bensch K, et al.: Chapter F of the International Code of Nomenclature for algae, fungi, and plants as approved by the 11th International Mycological Congress, San Juan, Puerto Rico, July 2018. IMA Fungus. 2019; 10: 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPalmer AS: Folk-etymology: A Dictionary of Verbal Corruptions Or Words Perverted in Form Or Meaning, by False Derivation Or Mistaken Analogy. G. Bell and Sons; 1882. Reference Source\n\nPumnuan J, Insung A, Phutphat S: Carbaryl Insecticide Decomposition after Application on Oyster and Jew’s Ear Mushrooms Cultivations. Science & Technology Asia. 2021; 169–175.\n\nQuélet L: Enchiridion fungorum in Europa media et praesertim in Gallia vigentium. O. Doin; 1886. Reference Source\n\nRossman AY, Allen WC, Castlebury LA, et al.: (2517–2519) Proposals to conserve the names Balansia claviceps against Ephelis mexicana, Claviceps paspali against Ustilagopsis deliquescens, and Tolypocladium inflatum against Cordyceps subsessilis (Ascomycota: Sordariomycetes: Hypocreales). Taxon. 2017; 66(3): 749–750. Publisher Full Text\n\nSmith AH: A Field Guide to Western Mushrooms. University of Michigan Press; 1975. Reference Source\n\nVyshnavi AS, Pramod R: Effect of supplements and growth regulators on the productivity of Jew’s ear mushroom (Auricularia auricula-judae). The Pharma Innovation Journal. 2022; 11(12): 4261–4265.\n\nWu F, Tohtirjap A, Fan LF, et al.: Global Diversity and Updated Phylogeny of Auricularia (Auriculariales, Basidiomycota). J. Fungi (Basel). 2021 Nov 3; 7(11): 933. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu F, Yuan Y, He S-H, et al.: Global diversity and taxonomy of the Auricularia auricula-judae complex (Auriculariales, Basidiomycota). Mycol. Prog. 2015; 14(10). Publisher Full Text\n\nXu Z, Meenu M, Xu B: Effects of UV-C treatment and ultrafine-grinding on the biotransformation of ergosterol to vitamin D2, physiochemical properties, and antioxidant properties of shiitake and Jew’s ear. Food Chem. 2020; 309: 125738. Publisher Full Text" }	[ { "id": "245333", "date": "23 Feb 2024", "name": "Rong-Ju Xu", "expertise": [ "Reviewer Expertise Phylogenetics", "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\nThis manuscript, titled \"Whose Ear?: Proposal to conserve the name Auricularia auricula (L.) Underw. for Auricularia auricula-judae (Bull.) Quél.\", explores the nomenclatural and cultural history of the fungus Auricularia auricula-judae, advocating for a return to its original specific epithet \"auricula\". The authors provide a thorough examination of the historical, cultural, and scientific aspects surrounding the naming of this species. They argue that the name change to \"auricula-judae\" does not conform to standard nomenclatural practices and that the original name \"auricula\" should be restored to reduce confusion and align with modern taxonomy principles. Additionally, they propose designating a physical epitype to provide clarity and stability to the species taxonomy.\n\nOverall, the paper presents a compelling argument for revisiting the nomenclature of Auricularia auricula-judae, blending scientific rigour with cultural and historical awareness.\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": "261262", "date": "11 Apr 2024", "name": "Nattapol Kraisitudomsook", "expertise": [ "Reviewer Expertise wood-decay fungi taxonomy", "evolution", "and 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\nMajor comments: The authors did an excellent job weaving a story about Auricularia auricula-judae and its problematic epithet. I applaud the authors initiative to take actions combating racism and antisemitism. And I am totally on board with having an actual fungarium specimen as epitype. I only have a few concerns and questions below.\nI think another possible reason for Bulliard to emend the name is to avoid taxonomic confusion. Has the authors looked to make sure that there were not two separate Tremella auricula species descriptions around 1789?\nAnd if Buillard really added -judae to refer to the Jews, then I wonder. What about this fungus made him think of the Jewish people? As a non-Western descent, I am only aware of the red curly hair and the hooked nose stereotypes. Did he think that Jews had venous ears or something similar to A. auricula? I think it would be a nice addition to this article if the authors researched and discussed this part.\n\nLastly, for me, the main reason to conserve a scientific name should be for clarity and usage. In this case, auricula-judae is also commonly used (5,000 results is a lot) to represent the wood ear mushroom. On one hand, it is preferred to have a consistent scientific name. On the other hand, the proposed conservation might be disruptive to researchers who are currently using 'auricularia-judae' to talk about this fungus. So usually I would be on the fence about this proposal. However, with added antisemitic connotation, I favor the conservation of the name Auricularia auricula.\n\nMinor comments: 1. For \"antisemitic attitudes throughout Europe in the 18th Century...and reflects a widely held prejudice of the time\" - this line might need a citation as it should be a historical fact. 2. For \"auricularia auricula -judae returns ~7,510 results\" - I understand what you did, but it might be confusing to an average reader because the two names look very similar. Why not just say \"auricularia auricula\" returns about 7,500 results. The authors don't have to go into a technical detail on how to exclude -judae in Google Scholar searches. 3. For \"lists only Auricularia auricula (Smith 1975).\" the name should be italicized.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-948
https://f1000research.com/articles/12-358/v1	31 Mar 23	{ "type": "Research Article", "title": "Relationship between anal swab PCR for SARS-CoV-2 with gastrointestinal clinical manifestations and severity of COVID-19 infection in Indonesia", "authors": [ "Virly Nanda Muzellina", "Murdani Abdullah", "Juferdy Kurniawan", "Aulia Rizka", "Virly Nanda Muzellina", "Juferdy Kurniawan", "Aulia Rizka" ], "abstract": "Introduction: Coronavirus disease 2019 (COVID-19) cases caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Indonesia remain high. The virus can bind with ACE2 receptor which is not only found in the lungs, but also in the digestive tract. Thus, it allows SARS-CoV-2 infection in the gastrointestinal tract, gastrointestinal manifestations, and detection of viral RNA on anal swab using polymerase chain reaction (PCR). There hasn’t been similar study about the role of anal swab in Indonesia yet. Therefore, this study aims to determine the relationship between SARS-COV-2 anal swab PCR with gastrointestinal clinical manifestations, and the severity of COVID-19 in Indonesia. Methods: This is an analytical study with cross-sectional design. Samples were obtained from hospitalized COVID-19 patients from July 2020 to January 2021. Demographic data, clinical manifestations, severity, and SARS-CoV-2 anal swabs PCR were collected using case report form. Results: A total of136 patients were analyzed. 52 patients (38.2%) had positive SARS-CoV-2 anal swabs PCR and 84 patients (61.8%) had negative results. The most common gastrointestinal clinical manifestations were nausea and vomiting in 69 patients (50.7%), anorexia in 62 patients (45.6%), and abdominal pain in 31 patients (22.8%). There were 114 patients (83,8%) classified as mild-moderate symptoms and 22 patients (16,2%) classified as severe-critical symptoms. There was a statistically significant relationship between the gastrointestinal tract SARS-CoV-2 infection and gastrointestinal clinical manifestations (P=0.031). There was no statistically significant relationship between the gastrointestinal SARS-CoV-2 infection and the severity of COVID-19 infection (P = 0.844). Conclusions: This study showed there is a significant relationship between SARS-CoV-2 anal swab PCR with gastrointestinal clinical manifestations. There is no significant relationship between anal swab PCR with the severity of COVID-19 infection. Further studies should aim to assess the relationship using larger samples while also assessing the possibility of fecal-oral transmission.", "keywords": [ "SARS-CoV-2", "COVID-19", "anal swab", "gastrointestinal clinical manifestations", "the severity of COVID-19" ], "content": "Introduction\n\nA virus with a new strain named coronavirus was discovered in the city of Wuhan, China on December 31st, 2019. On January 12, 2020, WHO (World Health Organization) announced this disease was named COVID-19 (coronavirus disease 2019) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1 Indonesia reported its first case in March, 2020. 2 The cases in Indonesia in March 2022 reached 5,974,646 with 154,062 cases of death and the Case Fatality Rate (CFR) of 2.58%. 3\n\nSARS-CoV-2 can encode and express a spike glycoprotein (S) that can bind to the Angiotensin Converting Enzyme 2 (ACE2) receptor for entry into human cells. 4 The ACE2 receptors are not only found in the lungs, but also in the gastrointestinal tract which allows infection of enterocytes, causing gastrointestinal clinical manifestations, and detecting viral RNA on anal swab examination. 4 A systematic review found that 1 in 5 patients with confirmed COVID-19 had gastrointestinal symptoms. 5 Another study that evaluated gastrointestinal symptoms reported the prevalence of gastrointestinal symptoms including anorexia (26.8%), nausea and vomiting (10.2%), diarrhea (12.5%), abdominal pain (9.2%), and gastrointestinal symptoms. other (17.6%). 6 – 8\n\nThe Center of Disease Control and Prevention (CDC) has determined that the standard test for the diagnosis of COVID-19 is to use Reverse Transcription Polymerase Chain Reaction (RT-PCR) from upper and/or lower respiratory tract specimens. 9 However, positive RT-PCR results were not only found from respiratory tract specimen but also from gastrointestinal tract specimens like feces (29% – 53.42% positivity rate), anal swab, and endoscopic biopsy specimens from the stomach, duodenum, ileum, and rectum. 10 – 13 The RT-PCR test using an anal swab has a good specificity for confirming COVID-19 cases, which is 93.8%. 12 The role of anal swab sampling during the COVID-19 pandemic is getting more attention from researchers for several reasons. Severe COVID-19 patients showed higher detectable levels of viral RNA, high viral load, and early positive detection in anal swabs that can affect disease progression. 14 Other studies around the world have shown different results and similar studies have not been found in Indonesia. 5 , 6 This study aims to determine the relationship between anal swab PCR for SARS-CoV-2 with gastrointestinal clinical manifestations and severity in COVID-19 patients in Indonesia.\n\n\nMethods\n\nThis study is a branch of the main research entitled “The Value of Anal Swab RT-PCR for Diagnosis of COVID-19 in Adults in Indonesia”. 15 This was an analytic study with a cross-sectional design. This research data in this manuscript is taken from the main research data. There were three potential source of bias, i.e. recruitment bias, allocation bias, and measurement bias. To anticipate the recruitment bias, the author was choosing a sampling method from the main research, which was taken with consecutive sampling. To anticipate the allocation bias, the author was examining anal PCR SARS-CoV-2 swabs at the Virology and Cancer Pathobiology Research Center RSCM-FKUI, which has been verified by the Ministry of Health of the Republic of Indonesia. To anticipate the measurement bias, the author was examining patients directly to collect the demographic data, clinical manifestations, severity, and anal swab PCR SARS-CoV-2 into the main study. The classification of the severity of COVID-19 infection is carried out according to the classification from the Ministry of Health of the Republic of Indonesia.\n\nThis research was an analytic study with a cross-sectional design using an etiological approach. The cross-sectional study design allows for an assessment of risk factors and their impact at the same time. Thus this study design excels in convenience, cost efficiency, and does not require re-evaluation for the patients included.\n\nThis study was approved by the Research Ethics Committee of Universitas Indonesia, Jakarta (Ethical Approval Number KET-639/UN2.F1/ETIK/PPM.00.02/2020), on June 22nd 2020. Data was collected between July 2020 until January 2021. Written informed consent was obtained from all patients to participate in this study and publication of the patients’ details.\n\nThe sample were adult confirmed COVID-19 patients with a positive nasopharyngeal swab PCR who were hospitalized at dr. Cipto Mangunkusumo (RSCM), Mitra Keluarga Hospital Depok, Mitra Keluarga Kelapa Gading Hospital, and Ciputra Hospital during the period July 2020 to January 2021. This time frame was chosen because of the COVID-19 cases in Indonesia were begin to increase nationally at that time point. Minimal respondents for this study is 96, based on formula for two independent samples. The target population is patients with COVID-19 infection above or equal to 18 years, with gastrointestinal manifestations who are hospitalized at selected hospitals for a specified period of time. This study used total sampling from the main research that were using consecutive sampling. The subjects were selected using consecutive sampling and subject inclusion criteria were patients admitted to a COVID-19 hospital who were confirmed positive for nasopharyngeal or nasal swabs using RT-PCR and aged over 18 years. Subject exclusion criteria were patients in critical condition at the time of initial hospital admission or patients who had hematochezia, melena, or other anatomical/physiological abnormalities that did not allow anal swab sampling.\n\nPatients who met the inclusion criteria were asked to participate in this study by signing the informed consent statement. The data collected included: the patient’s characteristics, clinical manifestations, disease severity, and results of the SARS-CoV-2 PCR anal swab using a case report form (CRF) which comes from both the patients directly through interview and their written medical records.\n\nThe collected data were processed and analyzed using SPSS software, version 25.0. Patient characteristics were reported using simple statistics such as n (number of patients), % (percentage of patients), and mean (SD). The relationship between anal SARS-CoV-2 swab PCR with gastrointestinal clinical manifestations and the severity of COVID-19 infection was assessed with a chi-square categorical comparative statistical test and the prevalence ratio (PR) with a 95% confidence interval. Other factors related to the dependent factor were assessed using multivariate logistic regression analysis and calculated odds ratio (OR) with 95% confidence interval.\n\n\nResults\n\nThe flow of patients recruited in this study were shown in this diagram:\n\n\n\nThere were 136 patients included in this study, consisting of 68 women (50%) and 68 men (50%) with an average age of 44.1 years ( Table 1 ). 41 There were 15 people (11.0%) who were active-smokers. The most common comorbidities found in patients were hypertension 17.6% (n=24), diabetes mellitus 15.4% (n= 21), followed by other lung diseases 6.6% (n=9). There were 52 patients (38.2%) who had positive SARS-CoV-2 anal PCR swabs and 84 patients (61.8%) had negative anal swabs. The most common gastrointestinal clinical manifestations were nausea and vomiting in 69 patients (50.7%), decreased appetite in 62 patients (45.6%), and abdominal pain in 31 patients (22.8%). There were 114 patients (83.8%) classified as mild-moderate and 22 patients (16.2%) classified as severe-critical.\n\nThere was a statistically significant relationship between the anal swab SARS-CoV-2 PCR and gastrointestinal clinical manifestations in the form of diarrhea or nausea and vomiting (P = 0.031, PR = 1.546) shown in Table 2. This was demonstrating that the SARS-CoV-2 anal swab PCR can be used as a sign or risk factor for gastrointestinal clinical manifestations in the form of diarrhea or nausea and vomiting.\n\nFactors that influence gastrointestinal clinical manifestation in COVID-19 patients were assessed with a multivariate logistic regression analysis test. The factors were anal swab, comorbidities, smoking, male gender, and age over 50 years old. The first stage was the selection of candidate variables that will enter the multivariate logistic regression model (Table 3). The variables anal swab, male gender, and age over 50 years old have P-value <0.25, so these variables deserved to be included in the multivariate model.\n\nThe results of statistical tests of multivariate logistic regression analysis to assess the factors that influence gastrointestinal clinical manifestations are described in Table 4. The anal swab has the greatest statically relationship with the severity of COVID-19 infection (P = 0.035) and an adjusted OR value of 2.184 (95% CI = 1.054-4.525). Another male gender variable has a P-value of 0.108 and an adjusted OR of 0.564 (95% CI = 0.281-1.134). The variable age over 50 years old has a P-value of 0.318 and an adjusted OR of 1.467 (95% CI = 0.691-3.112). Thus, the anal swab variable was an independent variable that affects the clinical manifestations of the gastrointestinal tract. In patients with the same gender and age, patients with positive anal swab were twice more likely to experience gastrointestinal clinical manifestations of diarrhea or nausea and vomiting, compared with negative anal swabs patients.\n\nThere was no statistically significant relationship between the SARS-CoV-2 anal swab PCR and the severity of COVID-19 infection (P = 0.844, PR = 0.984) shown in Table 5. This was demonstrating that the SARS-CoV-2 anal swab PCR cannot be used as a sign or risk factor for the severity of COVID-19 infection.\n\nFactors that influence the severity of COVID-19 infection were assessed with a multivariate logistic regression analysis test. The factors were anal swab, comorbidities, smoking, male gender, and age over 50 years old. The first stage was the selection of candidate variables that will enter the multivariate logistic regression model (Table 6). The variables “Comorbid” and “Smoking” had P-value <0.25, so these variables deserved to be included in the multivariate model. The variable “Anal swab” did not qualify for inclusion in the multivariate model because it had a P-value > 0.25, but was still included to assess the relationship of the anal swab in the multivariate model.\n\nThe results of statistical tests of multivariate logistic regression analysis to assess the factors that influence the severity of COVID-19 infection are described in Table 7. Smoking has the greatest statically relationship with the severity of COVID-19 infection (P = 0.003) and an adjusted OR value of 5.920 (95% = 1.839-19.061). The comorbid variable which has a P-value of 0.222 and adjusted OR 1.906 (95% CI = 0.676-5.372). The anal swab variable has a P-value of 0.940 and an adjusted OR of 0.962 (95% CI = 0.350-2.643). Thus, it demonstrates that the “Smoking” variable is an independent variable that affects the severity of COVID-19 infection. In patients with the same comorbidities and anal swab results, patients who smoked were almost 6 times more likely to have a more severe COVID-19 infection than patients who did not smoke.\n\n\nDiscussion\n\nVarious kinds of research around the world continue to be carried out about SARS-CoV-2 infection and the involvement of the gastrointestinal tract with the resulting clinical manifestations. The primary outcome of this study was to determine the relationship between anal swab PCR for SARS-CoV-2 with gastrointestinal clinical manifestations and the severity in COVID-19.\n\nIn this study, only 38.2% of the patients showed positive anal swab results to detect SARS-CoV-2 infection. A different proportion was found in the research by Gong-Qi et al. being as many as 53.6% of patients showed a positive result of SARS-CoV-2 anal swab. 16 The most common gastrointestinal manifestations in this study were nausea and vomiting (50.7%), decreased appetite (45.6%), and abdominal pain (22.8%). In the systematic review and meta-analysis of Ren Mao et al., the most common gastrointestinal manifestations were decreased appetite (21%), diarrhea (9%), and nausea and vomiting (6%). 17 The severity of COVID-19 in the study by Weiyin Lin et al. was 81.2% classified as mild-moderate and 19.8% as severe-critical. 7\n\nIn this study, there was a statistically significant relationship between the SARS-CoV-2 anal swab PCR variable and gastrointestinal clinical manifestations in the form of diarrhea or nausea and vomiting (P = 0.031). Similar findings were found in the study by Chaoqun Han et al. that concluded there was a statistically significant relationship between the detection of SARS-CoV-2 genetic material in feces and clinical manifestation of diarrhea or nausea and vomiting in patients (P = 0.033). 18 The opposite results were obtained in the study of Yong Zhang et al. The analysis results showed that the P-value was 0.0503, which means that there is no statistically significant relationship between the detection of SARS-CoV-2 genetic material in feces and clinical manifestation of diarrhea in patients. 19\n\nAt the time of writing this study, there is no definite reason or theory to explain the relationship between PCR SARS-CoV-2 anal swab and gastrointestinal clinical manifestations. Several hypotheses to support the relationship suggest a direct infection of SARS-CoV-2 to the gastrointestinal tract, direct invasion of the virus can damage the existing protective function of the gastrointestinal tract and increase permeability. 18 This will facilitate the invasion of pathogens in the gastrointestinal tract and cause clinical manifestations in the form of diarrhea, malabsorption of nutrients, and others. 18\n\nIn this study, it was found that there was no statistically significant relationship between the SARS-CoV-2 anal swab PCR and the degree of COVID-19 severity. Similar findings were obtained in the study of Weiyin Lin et al. that showed there was no statistically significant relationship between the SARS-CoV-2 anal swab PCR variable and the severity of both during hospital admission (P = 0.097) and during treatment (P = 0.229). 7 The opposite results were obtained in the Gong-Qi study. et al. that there was a statistically significant relationship between the positive anal swab results for SARS-CoV-2 RNA and the degree of severity (P = 0.029). 16\n\nSeveral studies showed results that were contrary to this study. The reasons underlying why other studies have concluded that there is a significant relationship between PCR SARS-CoV-2 anal swabs and the severity of COVID-19 infection are still largely unknown. Several hypotheses suggest that the high viral replication in the respiratory system allows the virus to actively penetrate the alveolus and endothelium of blood vessels to enter the bloodstream and infect the gastrointestinal epithelium. 7 Another hypothesis is that SARS-CoV-2 RNA is found in various locations of the gastrointestinal tract, starting from the esophagus, stomach, duodenum, and rectum. The wide availability of ACE2 receptors in the gastrointestinal tract can be a site of viral replication outside the lungs. Thus, the SARS-CoV-2 elimination from the respiratory system would be delayed, which can cause disease progression. 7 The different results between this study and other studies could also be due to the limited number of samples and the unbalanced proportion of samples classified as mild-moderate and severe-critical.\n\nSeveral factors were analyzed to assess their effect on gastrointestinal clinical manifestations, were anal swab, comorbidities (hypertension, diabetes mellitus, chronic obstructive pulmonary disease [COPD]), smoking, male gender, and age over 50 years old. Data analysis showed that the variables “Anal swab”, “Male gender”, and “Age over 50 years” had an influence on gastrointestinal clinical manifestations with “Anal swab” being the most dominant factor in influencing gastrointestinal clinical manifestations. ACE2 receptors in the gastrointestinal tract are the entry point for viruses to enter host cells. 6 , 8 , 11 , 20 Viruses can replicate and spread to various locations in the body. Viral infections will damage the intestinal mucosa and cause gastrointestinal manifestations in the form of diarrhea. 21 In certain individuals, a cytokine storm may occur due to the infection that will exacerbate the occurrence of this diarrhea. The body's response to the cytokine storm that occurs will cause intestinal hypoxia to tissue ischemia which also contributes to diarrhea. The existing inflammation affects the balance of microorganisms in the gastrointestinal tract which will lead to more prominent gastrointestinal symptoms. 20 , 22\n\nComorbid factors did not affect gastrointestinal clinical manifestations in this study. The opposite finding was found in the study by Uday C. et al. The study performed a multivariate analysis to assess factors associated with gastrointestinal manifestations. The analysis results showed that patients with comorbidities had an adjusted OR of 6.87 (95% CI = 1.22–38.79) and a P value of 0.027. 23\n\nIn this study, smoking did not affect gastrointestinal clinical manifestations. This finding was supported by the research of Ting Zhan et al. that showed there is no statistically significant relationship between smoking history and gastrointestinal manifestations (P = 0.501). 24\n\nMale gender had a correlation with gastrointestinal clinical manifestations in this study; men were more likely to be infected by bacteria and viruses than females. This may be because women have strong innate and adaptive immune responses. 25 However, an opposing study showed that gastrointestinal symptoms in the form of decreased appetite (p-value <0.001) and diarrhea (p-value 0.002) in COVID-19 infection were significantly more common in women compared to men. 26\n\nThe factor “Age over 50 years old” has an influence on gastrointestinal clinical manifestations in this study which is supported by other studies. A similar study with multivariate analysis showed gastrointestinal symptoms including diarrhea, nausea, vomiting, abdominal pain, and decreased appetite had a positive association with the patient’s age. Human aging is associated with innate and adaptive immunity decline, and will gradually lose the defense function to against infection. Thus, older adults are more vulnerable to COVID-19 infection. 27\n\nIn this study, several factors were assessed for their influence on the severity of COVID-19 infection. Those factors were anal swab, comorbidities (hypertension, diabetes mellitus, COPD), smoking, male gender, and age over 50 years old. Data analysis showed that the variables “Comorbid” and “Smoking” had an influence on the severity of COVID-19 infection with “Smoking” being the most dominant factor. A systematic review by J.E. Rod et al. processed data from 17 studies and collected up to 60 predictors or factors that influence the severity of COVID-19 infection. Factors tested using multivariate analysis, i.e. age, C-reactive protein, D-dimer, albumin, body temperature, Sequential Organ Failure Assessment (SOFA) score, and diabetes. The results of the analysis showed that diabetes is the most consistent comorbidity in predicting the severity of COVID-19 infection. Patients with diabetes mellitus may experience an over-inflammatory response and a hypercoagulable state that is associated with dysregulation of glucose metabolism. Both have an effect on the aggravation of the disease. 28 Patients with comorbid hypertension are known to be at higher risk for respiratory tract infections and an increased incidence of ICU admissions. 29 , 30 In patients with COPD, there has been the destruction of the lung parenchyma, chronic inflammation, airflow limitation, and the presence of infection could trigger COPD exacerbations. Thus, patients with COPD have a worse prognosis and more severe disease. Smoking behavior factors are known to cause disorders of the immune system in the lungs, prolonged lung structure damage, and increased ACE2 expression which underlies the degree of disease that occurs. 31 – 34 Thus, smoking patients can influence the COVID-19 severity.\n\nIn this study, the male gender was not included in the multivariate regression analysis because it did not have a statically significant influence on the severity of COVID-19. Several studies showed the relationship between gender and COVID-19 infection. Male gender is related to differences in behavior, social activities, roles in society, smoking and alcohol consumption. Comorbidities like hypertension, cardiovascular disease, and COPD is more likely in men which can influence the prognosis of COVID-19. In men, ACE2 expression is higher and the cytokine storms are increased compared to women. 35 – 37\n\nThe age over 50 years old variable did not affect the severity of COVID-19, thus it was not included in the multivariate regression analysis model in this study. Several studies had opposite results that showed aging is associated with an impaired mucociliary clearance which facilitates infection, weaker innate and adaptive immune systems, and coagulation abnormalities which can increase the risk of disseminated intravascular coagulation. Thus, leading to a more severe disease. 38 – 40\n\nAs far as our knowledge, this article is the first cross sectional study that has assessed the relationship between SARS-COV-2 anal swab PCR with gastrointestinal clinical manifestations and the severity of COVID-19 in Indonesia this far. Beside it has several limitations, i.e. the nature design of cross sectional study that cannot establish the long term causal effect of SARS-CoV 2 infection and the gastrointestinal symptoms, nor measuring the long term gastrointestinal outcome of this disease. Also, the SARS CoV-2 PCR only evaluate the presence of genetic materials in the specimen collected, but cannot establish the viability of the virus itself.\n\n\nConclusion\n\nThis study demonstrated that there is a statically significant relationship between PCR SARS-CoV-2 anal swab with gastrointestinal clinical manifestations (diarrhea or nausea and vomiting). Therefore, SARS-CoV-2 anal swab PCR can be used as a sign or risk factor for gastrointestinal clinical manifestations. There is no statistically significant relationship between the SARS-CoV-2 anal swab PCR and the severity of COVID-19 infection. Therefore, SARS-CoV-2 anal swab PCR cannot be used as a sign or risk factor for the severity of COVID-19 infection. Patients with gastrointestinal manifestations such as diarrhea or nausea and vomiting need to be more aware of the potential for COVID-19 infection, even though they do not experience respiratory manifestations. Patients with suspected COVID-19 infection with gastrointestinal manifestations (diarrhea, nausea, and vomiting) who show negative nasopharyngeal swab results are advised to have an anal swab examination. In this study, the number of samples analyzed had met the minimum requirement for a representative analysis, but analysis using a larger number of samples was needed to further assess the relationship between the anal swab SARS-CoV-2 PCR test results with gastrointestinal clinical manifestations and COVID-19 severity. The outcomes of the study can be considered to increase awareness of the possibility of fecal-oral transmission.", "appendix": "Data availability\n\nOpen Science Framework: Relationship between Anal Swab PCR for SARS-CoV-2 with Gastrointestinal Clinical Manifestations and Severity of COVID-19 Infection in Indonesia, https://doi.org/10.17605/OSF.IO/9E24G. 41\n\nThis project contains the following underlying data:\n\n- Raw data.xlsx (this file contains raw questionnaire and anal swab results)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nWang C, Horby PW, Hayden FG, et al.: A novel coronavirus outbreak of global health concern. Lancet. 2020; 395(10223): 470–473. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKesehatan K: Pedoman Pencegahan dan Pengendalian Corona Virus deases (Covid-19). 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhoshal UC, Ghoshal U, Mathur A, et al.: The Spectrum of Gastrointestinal Symptoms in Patients With Coronavirus Disease-19: Predictors, Relationship With Disease Severity, and Outcome. Clin. Transl. Gastroenterol. 2019; 11(12): E00259. Publisher Full Text\n\nZhan T, Tang Y, Han Z, et al.: Clinical characteristics of 195 cases of COVID-19 with gastrointestinal symptoms. Turk. J. Gastroenterol. 2021; 32(2): 148–154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJaillon S, Berthenet K, Garlanda C: Sexual Dimorphism in Innate Immunity. Clin. Rev. Allergy Immunol. 2019; 56(3): 308–321. Publisher Full Text\n\nSierpiński R, Pinkas J, Jankowski M, et al.: Sex differences in the frequency of gastrointestinal symptoms and olfactory or taste disorders in 1942 nonhospitalized patients with coronavirus disease 2019 (COVID-19). Pol. Arch. Intern. Med. 2020; 130(6): 501–505. PubMed Abstract \| Publisher Full Text\n\nRahman MM, Bhattacharjee B, Farhana Z, et al.: Age-related risk factors and severity of SARS-CoV-2 infection: A systematic review and meta-analysis. J. Prev. Med. Hyg. Pacini Editore S.p.A./AU-CNS. 2021; 62: E329–E371.\n\nRod JE, Oviedo-Trespalacios O, Cortes-Ramirez J: A brief-review of the risk factors for covid-19 severity. Rev. Saude Publica. 2020; 54: 60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaki N, Alashwal H, Ibrahim S: Association of hypertension, diabetes, stroke, cancer, kidney disease, and high-cholesterol with COVID-19 disease severity and fatality: A systematic review. Diabetes Metab. Syndr. Clin. Res. Rev. 2020; 14(5): 1133–1142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo W, Li M, Dong Y, et al.: Diabetes is a risk factor for the progression and prognosis of COVID-19. Diabetes Metab. Res. Rev. 2020; 36(7): e3319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao Q, Meng M, Kumar R, et al.: The impact of COPD and smoking history on the severity of COVID-19: A systemic review and meta-analysis. J. Med. Virol. 2020; 92(10): 1915–1921. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShastri MD, Shukla SD, Chong WC, et al.: Smoking and COVID-19: What we know so far. Respir. Med. 2021; 176: 106237. Publisher Full Text\n\nReddy RK, Charles WN, Sklavounos A, et al.: The effect of smoking on COVID-19 severity: A systematic review and meta-analysis. J. Med. Virol. 2021; 93(2): 1045–1056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGülsen A, Yigitbas BA, Uslu B, et al.: The Effect of Smoking on COVID-19 Symptom Severity: Systematic Review and Meta-Analysis. Pulm Med. 2020; 2020: 1–11. Publisher Full Text\n\nJun T, Nirenberg S, Weinberger T, et al.: Analysis of sex-specific risk factors and clinical outcomes in COVID-19. Commun. Med. 2021; 1(1). Publisher Full Text\n\nMohamed MS, Moulin TC, Schiöth HB: Sex differences in COVID-19: the role of androgens in disease severity and progression. Endocrine. 2021; 71(1): 3–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaitao T, Vermunt JV, Abeykoon J, et al.: COVID-19 and Sex Differences: Mechanisms and Biomarkers. Mayo Clin. Proc. 2020; 95(10): 2189–2203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhama K, Patel SK, Natesan S, et al.: COVID-19 in the elderly people and advances in vaccination approaches. Hum. Vaccin. Immunother. 2020; 16(12): 2938–2943. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBooth A, Reed AB, Ponzo S, et al.: Population risk factors for severe disease and mortality in COVID-19: A global systematic review and meta-analysis. PLoS One. 2021; 16(3 March): e0247461. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIzcovich A, Ragusa MA, Tortosa F, et al.: Prognostic factors for severity and mortality in patients infected with COVID-19: A systematic review. PLoS One. 2020; 15(11 November): e0241955. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuzellina VN, Kurniawan J, Rizka A, et al.: Relationship between Anal Swab PCR for SARS-CoV-2 with Gastrointestinal Clinical Manifestations and Severity of COVID-19 Infection in Indonesia. [Dataset]. 2023, March 1. Publisher Full Text" }	[ { "id": "168691", "date": "19 Jun 2023", "name": "Fauzi Yusuf", "expertise": [ "Reviewer Expertise Gastroenterology", "Hepatology", "Microbiota", "Short Chain Fatty Acid", "Helicobacter pylory", "Covid 19", "Colorectal cancer" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGastrointestinal symptoms occur in patients with COVID-19. Virus RNA can be detected in fecal samples even those collected after respiratory samples test negative.\nThere are many reports that Infection with SARS-CoV-2 virus, which causes COVID-19, results in respiratory as well as gastrointestinal symptoms and virus RNA has been detected in fecal samples.\nThis manuscript addresses enteric manifestations of SAR-CoV-2 not only as an interesting diagnostic challenge to clinicians when facing patients with COVID-19 symptoms on initial presentation but also signifies potential fecal transmission of this virus. The content of the abstract is already appropriate, structured, and informative.\nThe research’s background, problem formulation, objectives, literature review and novelty are relevant.\nThe content of the paper has consistent material, good theoretical framework, valid data, and appropriate information supported by clear tables and figures.\nIn the discussion part, all relevant matters are well-discussed.\nWith the increasing number of reported cases of COVID-19, there is a pressing need for more research on the enteric manifestations of COVID-19 and the temporal pattern of fecal shedding of the SARS-CoV-2 virus, particularly to gastroenterologists and endoscopists who may not be familiar with this disease.\nEven though the study design has potential bias, this analytic study with cross-sectional design is sufficient to allow application by others.\nI think the conclusions drawn adequately support the results, but still need more research about possible fecal oral transmission.\n\nIs the work 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": "172704", "date": "31 Jul 2023", "name": "Hery Djagat Purnomo", "expertise": [ "Reviewer Expertise Gastroentero Hepatologist", "Internist" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 cross-sectional study investigating the association between anal swab PCR for SARS-CoV-2 with gastrointestinal clinical manifestations and severity of COVID-19 infection. This study has many strengths including being a clear manuscript, uses some of new available datasets for analysis, and careful interpretation of the findings. However, some concerns remain as detailed below :\nIn the study design, research biases are more suitable to be included in the study limitations. There is repetition of the same sentence “this research was an analytic study with a cross-sectional design using an etiological approach”.\nFurthermore, the advantage of the study design that there is no need for re-evaluation should not be mentioned, because to determine the association between anal swab PCR with disease severity, it would be better if it’s done periodically/longitudinal.\n\nThe Inclusion and exclusion criteria did not mention the history of Gastrointestinal disease. Was there any influence to the GI symptoms of the subjects?\n\nRegarding comorbidities evaluation, the comorbidities were evaluated individually or as in a group of yes or no comorbidity?\n\nIn the results explanation of table 4.; it was written “The anal swab has the greatest statically relationship with the severity of COVID-19 infection (P = 0.035) and an adjusted OR value of 2.184 (95% CI = 1.054-4.525)”, while Table 4 is the result of multivariate regression factors that contribute to GI manifestations. Could you please correct?\n\nIn the sentence “Another male gender variable has a P-value of 0.108 and an adjusted OR of 0.564 (95% CI = 0.281-1.134)” ; it would be better using “moreover/furthermore/additionally”.\n\nIn the discussion, it has not been well explained the reason of the comorbidity analysis which is different from previous studies.\n\nGiven the limitation of the study, it is more appropriate to say that there is not enough evidence to support the association between Anal swab PCR and disease severity. It is due to study design that the evaluation was done once in the admission. Furthermore, there are others factors such as drugs, other comorbidities, virus viability that may influence the results.\n\nPlease consider adding suggestions for future research.\n\nThe conclusions are too long and inconclusive. Please consider to write in an concise sentence and show the impact of this research on clinical applications. Then “analysis using a larger number of samples was needed” should not be included in the conclusion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10027", "date": "09 Aug 2023", "name": "Murdani Abdullah", "role": "Author Response", "response": "1. In the study design, research biases are more suitable to be included in the study limitations. There is repetition of the same sentence “this research was an analytic study with a cross-sectional design using an etiological approach”.Furthermore, the advantage of the study design that there is no need for re-evaluation should not be mentioned, because to determine the association between anal swab PCR with disease severity, it would be better if it’s done periodically/longitudinal. Thank you for your input. We have moved the research biases in the study design to our study limitation. We also have deleted the same sentence in the article and not mentioned the need for re-evaluation. 2. The Inclusion and exclusion criteria did not mention the history of Gastrointestinal disease. Was there any influence to the GI symptoms of the subjects? Thank you for your input. We did not mention the history of gastrointestinal disease in the inclusion and exclusion criteria because there was no influence of the history of gastrointestinal disease with the gastrointestinal symptoms of the subjects. The gastrointestinal symptoms that are assessed in this study are the symptoms that appear in the present. 3. Regarding comorbidities evaluation, the comorbidities were evaluated individually or as in a group of yes or no comorbidity? Thank you for your input. The comorbidities were evaluated in a group of yes or no comorbidity 4. In the results explanation of table 4.; it was written “The anal swab has the greatest statically relationship with the severity of COVID-19 infection (P = 0.035) and an adjusted OR value of 2.184 (95% CI = 1.054-4.525)”, while Table 4 is the result of multivariate regression factors that contribute to GI manifestations. Could you please correct? Thank you for your input. We have already corrected the sentence into “The anal swab has the greatest statistically relationship that influence gastrointestinal clinical manifestations (P=0.035) with an adjusted OR value of 2.184 (95% CI = 1.054-4.525)”. 5. In the sentence “Another male gender variable has a P-value of 0.108 and an adjusted OR of 0.564 (95% CI = 0.281-1.134)” ; it would be better using “moreover/furthermore/additionally”. Thank you for your input. We have changed the word “another” into “additionally”. 6. In the discussion, it has not been well explained the reason of the comorbidity analysis which is different from previous studies. Thank you for your input. We have added the reason of the comorbidity analysis which is different from previous studies in the article. 7. Given the limitation of the study, it is more appropriate to say that there is not enough evidence to support the association between Anal swab PCR and disease severity. It is due to study design that the evaluation was done once in the admission. Furthermore, there are others factors such as drugs, other comorbidities, virus viability that may influence the results. Thank you for your input. We have combined the sentence you suggest into the article. 8. Please consider adding suggestions for future research. Thank you for your input. We have added the suggestions for future research in our article. 9. The conclusions are too long and inconclusive. Please consider to write in an concise sentence and show the impact of this research on clinical applications. Then “analysis using a larger number of samples was needed” should not be included in the conclusion. Thank you for your input. We have shortened the conclusion and the sentence “analysis using a larger number of samples was needed” has been deleted in the conclusion." } ] } ]	1	https://f1000research.com/articles/12-358
https://f1000research.com/articles/12-942/v1	07 Aug 23	{ "type": "Study Protocol", "title": "Role of specialized pro-resolving mediators on inflammation, cardiometabolic health, disease progression, and quality of life after omega-3 PUFA supplementation and aerobic exercise training in individuals with rheumatoid arthritis: a randomized 16-week, placebo-controlled interventional trial *", "authors": [ "Sebastián Jannas-Vela", "Alejandro A Candia", "Luis Peñailillo", "Paola Barrios-Troncoso", "Jeremy Zapata-Urzúa", "Joanny Rey-Puente", "Harold M Aukema", "David M Mutch", "Rodrigo Valenzuela", "Denisse Valladares-Ide", "Alejandro A Candia", "Luis Peñailillo", "Paola Barrios-Troncoso", "Jeremy Zapata-Urzúa", "Joanny Rey-Puente", "Harold M Aukema", "David M Mutch", "Rodrigo Valenzuela", "Denisse Valladares-Ide" ], "abstract": "Background: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by autoantibody production and synovial membrane damage. It significantly impairs overall function and quality of life. Consumption of omega-3 (n-3) polyunsaturated fatty acids (PUFAs) and regular aerobic exercise (AEx) training are reported to have positive effects on the progression of RA. However, the mechanisms behind these benefits are still inconclusive. This study protocol will investigate the effects of n-3 PUFA supplementation and AEx training on disease progression, cardiometabolic health, and quality of life, and their association with the plasma and synovial fluid levels of specialized pro-resolving mediators (SPMs) in subjects with RA. Methods: The study consists of a 16-week intervention period, during which participants will be randomly assigned in a double-blinded manner to one of four groups: placebo control (PLA), PLA+AEx, n-3, or n-3+AEx. The PLA groups will be given a gelatin-filled capsule, while the n-3 groups will be given n-3 PUFAs equivalent to 2.5 g/d of docosahexaenoic acid and 0.5 g/d of eicosapentaenoic acid. The AEx groups will perform exercise three times per week on a stationary electronically braked cycle ergometer at 60-70% of their VO2peak for 50-60 minutes. Before and after the intervention, participants will undergo RA-specific and functional measurements, peak aerobic capacity test, and a dietary and physical activity assessment. Venous blood and synovial fluid from the knee joint will be collected. Changes in disease progression, cardiometabolic health, and quality of life, as well as erythrocyte membrane composition to assess n-3 incorporation, SPM levels, inflammatory markers, and gene expression from blood and synovial fluid will be analyzed. Conclusions: The study aims to elucidate the SPMs that regulate the inflammatory gene expression pathways and associate them with the improvements in disease progression, cardiometabolic health, and quality of life after n-3 PUFA supplementation and AEx training. Registration: ClinicalTrials.gov #NCT05945693.", "keywords": [ "Omega-3", "exercise", "rheumatoid arthritis", "oxylipins", "specialized pro-resolving mediators", "inflammation", "gene expression." ], "content": "Introduction\n\nRheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease characterized by autoantibody production [rheumatoid factor (RF) and anti–citrullinated protein antibody] and damage of the synovial membrane of the joints, causing swelling, pain, stiffness, and fatigue, among other symptoms.1,2 The chronic inflammation around the joints leads to substantial damage to the cartilage, bones, tendons, and ligaments, thereby, significantly impairing overall function and quality of life.3 As a result, RA patients have a 50% greater chance of developing cardiometabolic diseases,4 such as hypertension, insulin resistance, obesity, and sarcopenia, thereby increasing the propensity of further disability and mortality.1,5–7 Worldwide, RA affects 0.2-1.0% of the total population, with a higher prevalence in women, being 3 to 8 times more frequent than in men.8,9\n\nTreatments for RA include the prescription of antirheumatic/anti-inflammatory pharmacological and biological medications such as nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and disease-modifying antirheumatic drugs (DMARDs). They lead to significant improvements in functionality (decreased stiffness) and quality of life; however, they result in increased healthcare costs. Importantly, these treatments have no effect or even augment the cardiometabolic risk factors associated with this disease, probably due to the sedentary lifestyle and poor dietary patterns of RA individuals.5–7 Furthermore, as these traditional treatments target and inhibit the signaling pathways that produce inflammatory cytokines, including tumor necrosis alpha (TNF-α) and interleukin-6 (IL-6), they lead to immunosuppression and other unwanted side effects.10 Interestingly, there is substantial evidence supporting that consumption or supplementation with omega-3 (n-3) polyunsaturated fatty acids (PUFAs) and regular aerobic exercise (AEx) training have positive effects on RA. For instance, several studies report significant benefits of n-3 PUFA on clinical and functional outcomes in RA individuals, usually when high doses were used (> 2g/d).11–13 In particular, regular consumption of fish oil rich in n-3 PUFAs leads to the incorporation of eicosapentaenoic acid (C20:5n-3, EPA) and docosahexaenoic acid (C22:6n-3, DHA) into blood and synovial fluid phospholipids.14 This has been shown to improve several disease-specific and functional outcomes of RA, such as morning stiffness,15 joint pain,16 number of swollen and tender joints,17 grip strength,18,19 decreased NSAID consumption,15,20 and health-related quality of life.13 In addition, regular consumption of n-3 PUFAs decreases the serum levels of the proinflammatory mediator thromboxane B2 (TXB2) and the production of prostaglandin E2 (PGE2) after 24 h incubation of blood with lipopolysaccharide (LPS).20 Moreover, n-3 PUFA supplementation reduces the production of several pro-inflammatory cytokines, including the blood levels of C-reactive protein (CRP),21 TNF-α22 and IL-1β.18 Surprisingly, the specific mechanisms behind the anti-inflammatory and positive health-related effects of n-3 PUFAs in RA individuals are still inconclusive.\n\nThe regular practice of physical activity/exercise is also an effective and multifactorial approach for improving several aspects of health in RA subjects, including cardiovascular, metabolic, musculoskeletal, and functional health.23 For instance, RA participants who performed 6 months of regular AEx [3 times per week at 70% of peak oxygen consumption (VO2peak)] improved their cardiorespiratory fitness (8% VO2peak) and cardiometabolic health.24 Furthermore, AEx training improved symptoms and specific clinical measures of RA, including the perception of fatigue, joint pain, physical function (decreased walk time and increased grip strength), quality of life [health assessment questionnaire HAQ)], and RA disease activity score (23%).6,23–26 These beneficial effects are most likely mediated by the anti-inflammatory effects of exercise; however, the mechanisms behind this effect are not yet known.\n\nBecause some of the putative mechanisms behind the effects of n-3 PUFA supplementation and AEx training overlap,27–29 the combination of these treatments in individuals with RA may further improve their health. However, to date, no study has examined the effects of n-3 PUFA supplementation and AEx training in individuals with RA. Further, the emergence of a novel class of lipid metabolites derived from n-3 PUFAs – specialized pro-resolving mediators (SPMs) – which activate pathways that reduce excessive inflammation, yet also enhance pathways necessary for tissue repair and regeneration,30,31 has initiated a promising new era of research that could explain the benefits observed after n-3 PUFA supplementation and AEx in individuals with RA. However, whether they are responsible for this adaptive response, also remains to be elucidated.\n\nResearch hypothesis: N-3 PUFA and AEx will have synergistic effects on disease progression, cardiometabolic health, and quality of life in individuals with RA, and these changes will be associated with SPM production in blood and synovial fluid.\n\nGeneral aim: To investigate the effects of n-3 PUFA supplementation and AEx training on disease progression, cardiometabolic health, and quality of life and their association with the plasma and synovial fluid levels of SPMs in individuals with RA.\n\nSpecific aims:\n\n(1) To compare the effects of n-3 PUFA supplementation, AEx training, and the combination of both on disease progression, cardiometabolic health, and quality of life in individuals with RA.\n\n(2) To compare the effects of n-3 PUFA supplementation, AEx, and the combination of both on the plasma and synovial fluid levels of the SPMs in individuals with RA.\n\n(3) To examine the relationship between the plasma levels of the n-3 PUFA derived SPMs with systemic disease progression, cardiometabolic health, and quality of life in individuals with RA\n\nTo achieve these aims we have designed a randomized, 16-week, placebo-controlled, blinded interventional trial.\n\n\nProtocol\n\nParticipants will be recruited from hospitals and private clinics in the central region of Chile, whereas all the interventions regarding AEx will be carried out at the Universidad de O’Higgins and the Hospital Regional de Rancagua. All institutions are in the Región del Libertador Bernardo O’Higgins, Chile.\n\nThe sample size calculation for this study is based on an α level of 0.05, a power (1-β) of 0.8, and effect size of 0.42 (morning stiffness).11 A minimum of 18 participants are necessary per group; however, 22 individuals will be recruited to account for a typical dropout rate of 20% in long-term intervention studies.\n\n88 individuals (18-65 y) with rheumatoid arthritis (RA) will be recruited via newspaper advertisements, posters, flyers, visits to local community centers, and referrals from associated centers and Rheumatologists. All participants will obtain medical clearance from their Rheumatologist before taking part in the study. Individuals with moderate disease activity, as defined by the Disease Activity Score 28 (DAS28) > 2.6 and < 5.1 will be included. Participants taking nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, or disease-modifying anti-rheumatic drugs (DMARDs) will be eligible; however, the dosages of these agents must be constant at least four weeks before, must remain within this limit throughout the study, and prednisone dose should not be higher than 7.5 mg/d.32 Individuals diagnosed with gastrointestinal or metabolic diseases, regular alcohol abuse, smokers, and dietary supplement intake (e.g., fish oil capsules) or consumption of fish > 2 times per week will be excluded. Further, individuals that perform regular aerobic exercise (> 150 min moderate intensity per week) or have any physical or biomechanical limitations to complete the exercise program will also be excluded. Finally, subjects will be excluded if they present blood levels of aspartate aminotransferase, alanine transaminase, or creatinine higher than 1.5 times the maximum normal limit and total bilirubin levels of more than 1.8 mg/dL.33,34 Participants will be encouraged to continue with their everyday normal activities. They will be fully informed of the nature and possible risks of the experimental procedures before providing their written informed consent. Participation in this study is voluntary.\n\nThis study consists of a 16-week intervention with n-3 PUFAs and/or aerobic exercise (AEx) training. The subjects will be randomly assigned in a double-blinded manner to one of four groups: placebo control (PLA), PLA+AEx, n-3, or n-3+AEx. A stratified randomized assignment (by block) process will be employed to ensure that the experimental groups are balanced for disease activity, pharmacological treatment, sex, and age. The principal investigator and pharmacy personal will be aware of patient allocation and will code the information. Patients and exercise intervention supervisors will be blinded for supplement intervention, while medical specialists and database analysis personal will be blinded for supplement treatment and exercise intervention. The study will be unblinded for patients and medical specialist if the participant worsens their condition according to the criteria described below or if they decide to drop out the study.\n\nThe training sessions will be conducted at the Laboratorio de Ciencias del Ejercicio en el Ciclo Vital (Lab-CERVITAL), Universidad de O’Higgins. The week before intervention the participants will perform RA specific tests (e.g., Disease Activity Score-28) and functional measurements (e.g., handgrip strength), peak aerobic capacity test, a dietary and physical activity assessment, and blood samples will be collected. Subsequently, participants will start their 16-week intervention (PLA, PLA+AEx, n-3, or n-3+AEx). At the end of the intervention the same initial measurements, questionnaires, and assessments will be collected. In a subgroup of participants (n=24 in total or n=6 per group) extraction of synovial fluid will be performed before and after the intervention.\n\nPrimary outcome: Early morning stiffness is one of the main outcomes significantly improved by n-3 PUFA supplementation in RA patients.11 Therefore, the difference in the duration (minutes) of early morning stiffness has been selected as the primary outcome to compare the intervention effect in each group after the intervention period.\n\nSecondary outcomes: Other relevant outcomes which allow the assessment of disease progression will be measured, such as the Disease Activity Score-28, DHA-SPMs, tender and swollen joint count, quality of life, grip strength, analgesic use, and TNF-α blood levels.\n\nThe participants will be supplemented with either 5 capsules per day of Omega UP (Newscience, Chile) equivalent to 2.5 g/d of DHA and 0.5 g/d of EPA or a placebo filled gelatin capsule. The current doses are within the limits recommended by the European Food Safety Authority and have been shown to be safe, to be incorporated into cell membranes, and to produce significant improvements in overall health in individuals with RA.35–38 Capsules will be placed into de-identified bottles by people not involved in the study and provided to the participants to ensure double blinding.\n\nPatients will be given 2-4 weeks of capsules at a time, with all capsules counted before and after this timeframe, and a small notepad will be provided for them to write down every time when the capsules are taken. Furthermore, patients will receive weekly phone calls and text messages to serve as reminders for treatment adherence. Capsule treatment will be discontinued if patients worsen their condition or if the patient decides to drop-out without questioning their decision.\n\nThe exercise intervention will be performed according to the recommendations from the European Alliance of Associations for Rheumatology (EULAR) consisting of aerobic type exercise training three times per week, on non-consecutive days, with a total time of 20-60 minutes.25 The AEx will be performed on a stationary electronically braked cycle ergometer starting at 40-50% of VO2peak for 20 minutes. The intensity and volume of cycling will then be gradually increased to target at least 60-70% of VO2peak for 50-60 minutes over the final 10 weeks. Heart rate, power output and rating of perceived exertion (RPE) will be monitored during training intervention.\n\nA brief talk about the exercise importance for their conditions will be made every first session of the week to create awareness about it. Also, sessions have been planned at moderate intensity to decrease the risk of discomfort and to improve adherence. The exercise intervention will be stopped if the patient presents any discomfort related to fatigue, consciousness, cardiac, respiratory, or muscular hassle. Furthermore, if at some point disease symptoms are an obstacle for exercise practice, the patient will be advised to drop out the study. Finally, all patients in the exercise group may drop out from the study at any point without justification.\n\nIn case of any question or inquiry during the follow-up, the patients will be given a phone number and email to contact study the personnel. Furthermore, during the phone calls made by the personnel a question regarding compliance with the study will be asked. If these measures are not enough for promoting participant retention and the patient decides to drop out, a question regarding their reason for discontinuation will be asked. This information will be analyzed to avoid future study dropouts.\n\nA data monitoring committee will not be needed in this study since it has minimal risks and will be a short period of intervention. Interim analyses will be made by the principal investigator and a selected team of personnel every two weeks, indicating the total number of reported adverse events and its severity, any other undesirable effect, and the number of dropouts.\n\nBody mass and height will be determined with a scale and wall-mounted stadiometer. The participant’s age, body mass index (BMI), educational status, socioeconomic level, years of diagnosis, smoking habits, comorbidities, and medications will be recorded.\n\nDuration of morning stiffness: This will be assessed by asking “How long is your morning stiffness from waking until maximum improvement?”.39 The duration of morning stiffness will be reported in minutes up to a maximum of 180 minutes.\n\nDisease Activity Score-28 (DAS28): Describes the severity of rheumatoid arthritis using clinical and laboratory data. It is composed of the number of swollen joints (total 28), the number of tender joints (total 28), the Westgren erythrocyte sedimentation rate (ESR), and patients’ global disease assessment through a visual analog scale (VAS). The DAS28 has been extensively validated for its use in clinical trials and it is required by the Chilean Ministry of Health.40\n\nHealth assessment questionnaire disability index (HAQDI): This tool evaluates the functional status of RA individuals. It consists of eight sections: dressing, arising, eating, walking, hygiene, reach, grip, and activities, with 2 or 3 questions for each section. Scoring is from 0 (without any difficulty) to 3 (unable to do). The 8 scores of the 8 sections are summed and divided by 8. This questionnaire has been validated to be used among Chilean RA patients.41\n\nQuality of life RA (QOL-RA) scale: This scale is an RA-specific health-related quality of life instrument consisting of an 8-item scale. Each item starts with the definition of an element to be considered in rating one’s quality of life, followed by a horizontal 10-point scale anchored with 1 (very poor) at one end and 10 (excellent) at the other end. The elements are physical ability, pain, interaction with family and friends, support from family and friends, mood, tension, arthritis, and health. The higher the QOL-RA Scale score, the higher the health-related quality of life. This scale has been validated to be used in the Spanish language.42\n\nAnalgesic use: Analgesic use will be measured using pill counts for paracetamol and NSAIDs.\n\nTimed Up and Go test: The participant starts in a seated position, stands up upon command, walks 3 meters, turns around, walks back to the chair, and sits down. The time stops when the subject is seated.\n\nShort Physical Performance Battery (SPPB): This battery of tests evaluates lower extremity functional performance using timed measures of standing balance, a 4-meter walk, and five repetitive chair stands.43\n\nHandgrip strength: Measures the maximum isometric strength of the hand and forearm muscles. The participant holds the dynamometer in the hand with the arm at a right angle and the elbow by the side of the body. When ready, the participant squeezes the dynamometer with maximum isometric effort for 3-5 seconds. Participants will be instructed to perform the test as fast and hard as possible and verbal encouragement will be provided simultaneously. The highest value out of three contractions will be used for analysis.\n\nThe VO2 peak will be determined during an incremental cycling test to exhaustion using a recumbent cycle ergometer following the recommendations of the American College of Sports Medicine,44 using a portable and validated gas analyzer.45 Blood pressure and heart rate will also be analyzed at rest and during the exercise test.\n\nThe participants will have to fill a dietary record using image-assisted method over three days (two consecutive weekdays and one weekend day) before and after the supplementation period for estimation of macronutrient intake.46 Similarly, participants will answer the Global Physical Activity Questionnaire (GPAQ) to assess their physical activity.47\n\nBlood samples will be collected after an overnight fast (12 h) using a needle and Vacutainer kit and will be immediately centrifuged to obtain plasma (1000 × g for 10 min at 4°C) and erythrocyte fractions (3000 × g for 10 min at 20°C). Both fractions will be frozen at −80°C until further analysis. A portion of venous blood will also be collected in LeukoLOCK™ Total RNA Isolation System for the capture and purification of intact RNA from white blood cells, which will be frozen and stored at –80°C until further processing.\n\nPlasma rheumatoid factor (RF) and lipid profile will be determined. Further, plasma interleukin-10, and TNF-α levels will be measured via commercial ELISA assay kits.\n\ncDNA will be prepared by reverse transcription of 1 ug of total RNA, using the SensiFASTTM cDNA Synthesis Kit (Bioline) according to manufacturer’s protocol. RNA quantity and quality will be assessed using a Nanodrop 8000 (ThermoScientific, Wilmington, DE, USA) and an Agilent 2100 BioAnalyzer (Agilent Technologies Inc., Santa Clara, California, USA), respectively. Real-time RT-qPCR will be carried out to quantify the changes in the expression of genes associated with inflammatory pathways and SPM synthesis, including TNF-α, interleukin-1β (IL-1β), IL-6, cyclooxygenase-2 (COX-2), lipoxygenase-5 (ALOX5), lipooxygenase-12 (ALOX12), lipooxygenase-15 (ALOX15), and calcium-independent phospholipase A2 VIA (iPLA2VIA). 18S ribosomal RNA (18SRNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) will be used as housekeeping genes. Data will be analyzed using the ΔΔCt method.\n\nAll individuals must obtain medical clearance from their Rheumatologist. RA subjects will attend the laboratory after an overnight fast, without having carried out any type of exercise in the previous 48 hours. Local anesthesia (1% Lidocaine) will be applied to the affected joint and synovial fluid will be obtained by a certified rheumatologist.48 Synovial fluid will be immediately placed on ice and aliquoted for specific analyses. All aliquots will be stored at −80°C until use.\n\nQuantitative extraction of total lipids from erythrocytes, plasma, and synovial fluid will be carried out according to Bligh and Dyer49 with the addition of BHT. Fatty acid methyl esters (FAMEs) from erythrocytes and plasma will be prepared according to Morrison and Smith.50 FAMEs will be extracted with 0.5 mL of hexane. FAMEs will be identified and quantified using a gas-liquid chromatograph (Agilent7890B, Santa Clara, CA, USA) equipped with a capillary column (Agilent HP-88, 100 m × 0.250 mm; I.D. 0.25 μm) and flame ionization detector (FID). Identification and quantification of FAMEs will be achieved by comparing the retention times and the peak area% values of unknown samples to those of a commercial lipid standard (Nu-Chek Prep Inc., Elysian, MN, USA). C23:0 will be used as internal standard (Nu-Chek Prep Inc., Elysian, MN, USA) and data will be processed using the Hewlett-Packard Chemstation software system.\n\nPlasma and synovial fluid samples (400 μL) will be analyzed in duplicate for oxylipins by HPLC/MS/MS, as previously described.51 Briefly, deuterated internal standards (Cayman Chemicals) will be added to plasma samples and adjusted to pH <3.0 prior to solid-phase extraction using Strata-X-SPE (Phenomenex) columns preconditioned with methanol, followed by pH 3.0 water. Samples will be loaded on the columns, washed, and free SPMs will be eluted with 100% methanol. The eluate will subsequently be dried and resuspended in solvent A (water/acetonitrile/formic acid, 70/30/0.02 v/v/v) for oxylipin analysis by HPLC/MS/MS, using a Luna 5-μm C18 column (100 Å, 250 × 2.0 mm, Phenomenex) on a Shimadzu Nexera XR HPLC, coupled to an ABSciex QTRAP 6500 MS operating in negative mode with electrospray ionization. Oxylipins will be eluted with the following gradient: 100% solvent A between 0 and 1 min, and then solvent B (acetonitrile/isopropyl alcohol, 50/50, v/v) is increased linearly to 25% at 3 min, to 45% at 11 min, to 60% at 13 min, to 75% at 18 min, and to 90% at 18.5 min. Solvent B will then be held at 90% until minute 20, dropped to 0% by 21 min, and held until 25 min. Quantification of oxylipins will be performed using a stable isotope dilution method.\n\nResults will be expressed as mean ± SD. A two-way ANOVA with repeated measures followed by Fisher’s least significant difference post-test for multiple comparisons between groups will be used. Linear regression models will be constructed to examine the association between SPMs and clinical, functional, and health parameters, accounting for participant age, sex, and BMI as covariates. A value of p<0.05 will be considered statistically significant.\n\nDissemination\n\nThe results will be communicated to the funding body through a formal report. Further, they will be communicated to the participants and the interested public at the Universidad de O’Higgins and will be disseminated through presentations at national and international scientific conferences. The study outcomes will be published through peer-reviewed journals, no matter the trial outcome. There is no limit to the publication of the trial results.\n\nStudy status\n\nThe recruitment of participants will begin during the winter (June-September) of 2023. The data collection of this study will be completed in December 2024.\n\n\nDiscussion\n\nRA is a chronic autoimmune and inflammatory disease characterized by inflammation around the joints leading to substantial damage of the cartilages, bones, tendons, and ligaments, significantly affecting overall function, quality of life, and increasing the propensity of mortality. The current treatments for patients with RA (DMARDs, NSAIDs, and anti-TNF biologics) intend to disrupt the inflammatory response and slow disease progression; however, their chronic consumption may lead to unwanted side effects (immunosuppression and infections) and prevent optimal resolution and tissue repair, which is imperative in RA. Further, the lack of robust and sensitive biomarkers to determine treatment responsiveness in RA has impeded proper tracing and development of novel individualized interventions. Therefore, elucidating the SPMs, as well as the underlying inflammatory and SPM synthesis transcriptional pathways, associated with the improvements in disease progression, cardiometabolic health, and quality of life after n-3 PUFA supplementation and AEx training could lead to the development of new markers and individualized treatment strategies that improve overall health and quality of life in RA individuals. Finally, these results could prompt the development of new pharmacological treatments that enhance the production of the SPMs observed after n-3 PUFA supplementation and AEx training to improve treatment outcomes in RA individuals.\n\nDue to ethical considerations (divergent inflamed joints between participants), we will only perform the synovial fluid procedure in a small subset of patients from each intervention group. We will ensure that all individuals receive the best medical attention before, during, and after the procedure. Before a participant is eligible, a full medical screening will be performed to assess any risks. The individuals will be followed up routinely 48 h after the procedure to check if any complication occurs, which is unlikely. If a complication occurs, the principal investigator of this study will take care of all associated medical procedure costs and will seek the best treatment for the participant.\n\nThis study received ethics approval from the Comité de Ética de Investigación en Seres Humanos #219-2021 (Universidad de Chile, Santiago, Chile). All participants must sign a consent form prior participation in the study.\n\nThis trial has been registered with ClinicalTrials.gov, #NCT05945693. The construction of this study protocol followed the Interventional Trials 2013 (SPIRIT) guidelines.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nWalsmith J, Roubenoff R: Cachexia in Rheumatoid Arthritis. Int. J. Cardiol. 2002; 85(1): 89–99. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "231475", "date": "14 Feb 2024", "name": "Łukasz Poniatowski", "expertise": [ "Reviewer Expertise Orthopaedics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe original article by Jannas-Vela et al \"Role of specialized pro-resolving mediators on inflammation, cardiometabolic health, disease progression, and quality of life after omega-3 PUFA supplementation and aerobic exercise training in individuals with rheumatoid arthritis: a randomized 16-week, placebo-controlled interventional trial\" covers a potentially interesting and emerging topic related to the rheumatoid arthritis. I regard the main point of this paper as highly attractive as well as the results are clearly presented. The text does not contain any major errors, therefore I have some minor comments and recommendations:\n1. There is a need to provide slightly more expanded introduction shortly mentioning/describing pathogenesis of RA and its impact of modern healthcare. 2. The figure summarizing and clarifying the results should be added. 3. Following references should be added and properly cited within the main text:\n- Turczyn P, Wojdasiewicz P, Poniatowski ŁA, Purrahman D, Maślińska M, Żurek G, Romanowska-Próchnicka K, Żuk B, Kwiatkowska B, Piechowski-Jóźwiak B, Szukiewicz D. Omega-3 fatty acids in the treatment of spinal cord injury: untapped potential for therapeutic intervention? Mol Biol Rep. 2022 Nov;49(11):10797-10809. doi: 1 0.1007/s11033-022-07762-x. - Wojdasiewicz P, Poniatowski ŁA, Turczyn P, Frasuńska J, Paradowska-Gorycka A, Tarnacka B. Significance of Omega-3 Fatty Acids in the Prophylaxis and Treatment after Spinal Cord Injury in Rodent Models. Mediators Inflamm. 2020 Jul 29;2020:3164260. doi: 10.1155/2020/3164260. PMID: 32801994; PMCID: PMC7411484.\n4. In some places the use of English could be improved on.\nCompleting this gaps will have an impact on the understanding the aim of the study and, from my point of view, is absolutely necessary.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "11608", "date": "25 Jun 2024", "name": "Sebastian Jannas Vela", "role": "Author Response", "response": "Thank you for your review and suggestions. Kind regards, Sebastián Jannas-Vela" } ] }, { "id": "276501", "date": "21 May 2024", "name": "Philip C Calder", "expertise": [ "Reviewer Expertise Omega-3 fatty acids", "Inflammation", "Lipid mediators", "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\nThis manuscript describes a study protocol which has already been peer reviewed and which already has ethical approval. It is well written and clear, the study design is good (2 x 2 factorial design), dosing level of omega-3 is good, combination of omega-3 and exercise a novelty, and clinical and lab outcomes are all relevant. I did not quite understand the rationale for why only a \"small subset of patients\" will be subject to synovial fluid isolation and it is unclear what \"a small subset\" means and how that might adversely impact the value of the data obtained.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11607", "date": "25 Jun 2024", "name": "Sebastian Jannas Vela", "role": "Author Response", "response": "Thank you for your positive review. We are using only a small subset of patients because we want to compare the levels of n-3 derived SPMs from plasma and synovial fluid of RA patients after the intervention, as these mediators increase almost twofold from baseline, no more than 5 participants are needed. Regards Sebastián Jannas-Vela" } ] }, { "id": "276499", "date": "08 Jun 2024", "name": "Chris Mcglory", "expertise": [ "Reviewer Expertise Skeletal muscle", "nutrition" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this interesting manuscript. After careful review we have highlighted some points/comments that we believe will improve the overall quality of the study and reporting of the final manuscript.\nIntroduction:\nAre there specific/published measures of function for RA? What are the key outcomes used to support this point? More info would benefit the connection between RA and development of metabolic disease. What is the burden of increased healthcare cost- is it on the individual or society? Specify meaningful clinical and functional outcomes Is the n-3 PUFA incorporated freely into the blood, or is it its incorporation into blood cells that have possible positive effects? Expand on the proposed function/mechanisms SPMs act through. Is there proposed interaction between the AEx and N-3 only groups? How might these compare to the combined group? Do you suspect and additive effect of the combined intervention?\nGeneral methods:\nWhat is the time frame of intervention vs. observation? Is this suitable to measure disease progression? Sample size: Why use morning stiffness for power calculation? Will this be the primary outcome? Is this the most influential factor clinically? What is the significance of this prednisone dose and why is it grounds for exclusion? Validation for excluding drinkers and smokers?\nGeneral design:\nIs there a specific time frame to recruit 88 participants? If there is more than 22% drop out, will you recruit more than 22 people per group? Can this study be considered double blind if the PI is aware of group allocation? What is the peak aerobic test used? What does the physical activity assessment consist of- is it self-reported? Would it be worthwhile to include more timepoints? Even just for blood and synovial fluid to see the time course of n-3 saturation? Why not try to recruit everyone for synovial fluid extraction? Is morning stiffness clinically meaningful? Does this measure alone influence quality of life or similar outcomes Why were these doses of DHA and EPA chosen specifically? Is there any concern that gelatin may confound results due to possible implications on joint health? Will the progression of exercise be standardized relative to time? Are the phone calls with compliance questions the same call/frequency as the adherence reminder? When is the participant asked to report this measure? On the phone, in person, or in writing? Will this involve active recall? Could this influence their reporting? Again, what is the clinical significance of morning stiffness? Will analgesic used be reported with respect to body mass or sex? Handgrip assessment should be across a standardized time\nDiscussion:\nWhat are some of the primary markers of inflammation for this disease?\nOther (pre reported RCT):\nStatus is listed as not recruiting, but is supposed to be done by Dec 2024? Omega-3 combined with exercise is not listed as a separate intervention Can masking be considered quadruple if the PI knows group allocation\n\nChris McGlory and Danielle Nyman.\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/12-942
https://f1000research.com/articles/12-941/v1	07 Aug 23	{ "type": "Research Article", "title": "Benchmarking graph representation learning algorithms for detecting modules in molecular networks", "authors": [ "Zhiwei Song", "Brittany Baur", "Sushmita Roy", "Zhiwei Song", "Brittany Baur" ], "abstract": "Background: A common task in molecular network analysis is the detection of community structures or modules. Such modules are frequently associated with shared biological functions and are often disrupted in disease. Detection of community structure entails clustering nodes in the graph, and many algorithms apply a clustering algorithm on an input node embedding. Graph representation learning offers a powerful framework to learn node embeddings to perform various downstream tasks such as clustering. Deep embedding methods based on graph neural networks can have substantially better performance on machine learning tasks on graphs, including module detection; however, existing studies have focused on social and citation networks. It is currently unclear if deep embedding methods offer any advantage over shallow embedding methods for detecting modules in molecular networks. Methods: Here, we investigated deep and shallow graph representation learning algorithms on synthetic and real cell-type specific gene interaction networks to detect gene modules and identify pathways affected by sequence nucleotide polymorphisms. We used multiple criteria to assess the quality of the clusters based on connectivity as well as overrepresentation of biological processes. Results: On synthetic networks, deep embedding based on a variational graph autoencoder had superior performance as measured by modularity metrics, followed closely by shallow methods, node2vec and Graph Laplacian embedding. However, the performance of the deep methods worsens when the overall connectivity between clusters increases. On real molecular networks, deep embedding methods did not have a clear advantage and the performance depended upon the properties of the graph and the metrics. Conclusions: Deep graph representation learning algorithms for module detection-based tasks can be beneficial for some biological networks, but the performance depends upon the metrics and graph properties. Across different network types, Graph Laplacian embedding followed by node2vec are the best performing algorithms.", "keywords": [ "network biology", "module detection", "graph clustering", "graph representation learning", "node embedding", "graph neural network", "machine learning" ], "content": "Introduction\n\nGraphs provide ubiquitous powerful representations of interacting components in many complex systems.1 In molecular biology, graphs are used to represent interactions between molecular components; for example, protein-protein interactions represent interactions between proteins, while gene regulatory networks describe interactions between transcription factor proteins and genes. A common property of graphs of complex systems, including molecular networks, is the presence of community structures or modules, defined by groups of densely interacting nodes. In protein-protein interaction networks, such modules often correspond to key biological processes needed for cells to accomplish different tasks.2 Disruptions to module connectivity have been implicated in many diseases.1 Therefore, the accurate detection of these modules is an important problem in the analysis of molecular networks.3 Graph representation learning (GRL),4 which aims to embed graph nodes in a d-dimensional space (Figure 1), offers a powerful framework for performing machine learning tasks on graphs, including clustering.5 Both deep and shallow graph representation algorithms exist; however, deep representation learning methods have been designed and tested on non-biological networks such as social networks, citation networks and recommendation systems6–9 or only tested on link prediction and node classification tasks.10 It is unclear if deep graph representation learning methods are beneficial for module detection of molecular networks.\n\nA GRL algorithm converts graph structure into node embeddings denoted as vectors for each node. A standard clustering method, such as K-means clustering, can be applied to embeddings to obtain group clusters.\n\nTo address this need, we benchmarked several state-of-the-art shallow and deep learning GRL frameworks for the task of module detection on both real molecular networks and synthetic benchmark networks. Our benchmark included three shallow GRL methods: Graph Laplacian embedding,11 DeepWalk,12 and node2vec13 and two deep GRL algorithms: Variational Graph Auto-Encoder (VGAE)7 and GraphSAGE.14 We evaluated the methods on one simulated and three real world molecular network datasets. Two of the three real network datasets captured the impact of single nucleotide polymorphisms (SNPs) identified from genome wide association studies (GWAS). We used multiple clustering metrics to evaluate the quality of the inferred clusters including modularity index, silhouette index, adjusted mutual information index (when cluster labels were available) and enrichment of gene ontology processes for modules inferred on the molecular networks. Our results show that on simulated benchmarks, deep GRL methods offered some advantages over shallow methods, however, on real networks we did not see a clear advantage of deep GRL methods over shallow GRL methods for detecting modules in biological networks.\n\n\nMethods\n\nWe applied five different representation learning algorithms to simulated and cell type-specific gene interactions that we previously generated.15\n\nLancichinetti-Fortunato-Radicchi (LFR) benchmark graphs\n\nWe evaluated all the algorithms on a synthetic network based on the LFR graph generator. We chose to use LFR benchmark graphs16 as they can support larger graphs and capture the distribution of node degree and community sizes of real-world networks, which often follow the power law distribution. We used the graph generator to obtain weighted undirected graphs with ground-truth non-overlapping communities.\n\nThe LFR benchmark has a number of parameters: (N,K,γ,β,μ,kmin,kmax,smin,smax). N is the number of nodes in the whole graph, K is the average degree of nodes, γ is the exponent value for the power-law distribution of node degrees, β is the exponent value for the power-law distribution of community sizes, and μ is the mixing the parameter controlling the percentage of the degree of each node interconnecting from its own community to another community. When the mixing parameter is low, there is a higher chance for an edge to stay within one community; while when it is high, there will be more interconnected edges between two communities than edges with two ends in the same community. The last four parameters kmin, kmax, smin, smax are the lower and upper bounds of node degree and community size when constructing synthetic networks.\n\nBy setting different parameters in the LFR package, we can generate networks of different topological properties and compare various node embedding techniques. Based on the statistics of the 55 cell-type specific networks, we set the number of nodes N=1000, the average degree of nodes K=102550100, the exponent for degree distribution γ=2, the exponent for community size distribution β=1, and the mixing parameter μ=0.10.5. We left the bounds of node degree and community size as default. For each parameter setting, we generated five sets of weighted synthetic networks. Since some of our GRL algorithms take unweighted graphs, we derived unweighted graphs by keeping edges with weights higher than the average weight. In total, there are eight groups of synthetic network datasets for each parameter setting with five networks each in total (Tables 1 and 2).\n\nThe unweighted networks comprise edges with weights larger or equal to the average edge weights in weighted ones.\n\nThese values can be used to compare with the optimal k value detected when applying K-Means in each algorithm.\n\nCell-type specific networks\n\nThe cell-type specific networks consist of 55 gene networks capturing protein-protein and transcription factor (TF) gene regulatory interactions in different cell types that were generated to link regulatory single nucleotide polymorphisms (SNPs) to genes from Baur et al.15 The cell types represent major cell and tissue types studied by the NIH Roadmap Epigenomics Project.17 To create these networks we obtained the protein-protein interactions from the STRING database,18 RRID:SCR_005223. The protein-protein network is not cell type specific and combined it with cell type-specific TF-gene regulatory interactions. The regulatory interactions were defined in a cell-type specific manner and included both promoter-proximal and distal regulatory interactions. To create the cell-type specific regulatory networks, we downloaded Roadmap DNase I data from NIH Roadmap Epigenomics Project for each cell type and sequence-specific motifs for transcription factors from JASPAR,19 RRID:SCR_003030. Accessible motif instances were identified by intersecting motif instances with DNase I peaks on gene promoters and distal regions that exhibited long-range interactions with genes.15 The proximal TF-target regulatory network captures interactions between TFs and gene promoters exhibiting a significant and accessible motif instance. The distal regulatory network captures the interactions between TFs and genes, where the TF’s motif instance is on a region that interacts with the gene. The proximal and distal regulatory networks and the protein-protein interaction networks were merged by concatenating edges into one unweighted network per cell type. The statistics of these networks are described in Table 3.\n\nDensity is calculated by dividing the maximum number of edges in a full network by the number of edges. The values presented in the table are in mean (std) format.\n\nPhenotype-specific diffused networks\n\nWe used the cell-type specific networks to identify parts of the network that were most affected by SNPs identified in different genome-wide association studies (GWAS,15). We refer to these as phenotype-specific networks. Briefly, for each of the 55 cell types, we scored genes in each network based on their propensity to interact with a non-coding SNP as assessed by the strength of the interaction between the region harboring a SNP and a gene promoter. The higher the score, the greater the strength of interaction between a gene and a region with the SNP. After assigning the node scores in the gene network, a two-step diffusion process was applied to first obtain scores for all other genes in the network using a regularized Laplacian kernel,20 followed by a second diffusion using a heat kernel to diffuse the signal among all genes. The output is a weighted adjacency matrix format where diagonal elements are node scores and off-diagonal elements are diffused edge weights. Since each cell type has a different network, we end up with 55 different nearly fully connected, weighted networks for each phenotype. We created these networks for SNPs from two phenotypes: Autism Spectrum Disorder (ASD) and Coronary Artery Disease (CAD). The statistics of these networks are described in Table 3.\n\n\nGraph representation learning algorithms\n\nGraph Laplacian embedding\n\nThe Graph Laplacian is a key operator for a given graph that has a large number of applications ranging from spectral clustering11 to using the Laplacian to smooth clusters on the graph.21–23 The normalized Graph Laplacian can be used to produce node embeddings as well and is computed as:\n\nHere I is the identity matrix and D is the degree matrix obtained by summing the edge weights associated with each node and adding the average degree to the diagonal of the degree matrix. The addition of the average degree transformation was shown to be useful for real-world graphs with non-uniform degree distributions.24 Next, we compute eigenvectors associated with the k smallest eigenvalues of the normalized Graph Laplacian matrix. Each node is then represented by corresponding entries in these k eigenvectors to produce a k-dimensional embedding of the node.\n\nDeepWalk\n\nThe DeepWalk12 algorithm is based on random walks, which uses the skip-gram model25 to learn a node representation. We note that the authors of DeepWalk refer to this algorithm as a deep learning algorithm; however, we use the classification provided by Hamilton et al.,6 which considers DeepWalk as a shallow representation learning algorithm. In DeepWalk, each node’s representation is obtained from a set of random walks of a fixed window size on the graph starting from that node. For each node v, the algorithm tries to find the embedding of node v that maximizes the log-likelihood of neighbors observed in the random walk. This yields the optimization objective function that maximizes PrukΦv, where ϕv, the embedding of node v and uk is a neighbor node. Naively optimizing this objective is computationally prohibitive, so DeepWalk uses a hierarchical softmax function where the neighbor nodes are stored as leaves on a binary tree and the above probability can be calculated by the product of the probability of observing nodes on the path from the root (vj):\n\nAs Deepwalk accepts unweighted graphs as input, we converted the fully connected weighted graph to an unweighted graph by keeping edges with the highest weights. Briefly, we computed the mean value of all edge weights in a network and used it as a threshold to remove the edges with weights lower than the threshold. Edges that remained in the graph were set to a weight of 1. After this step the networks for Autism Spectrum Disorder, the average node number reduces from 1358.45 to 1352.91 and the average edge number in the graphs reduces from 1,846,514.09 to 167,384.27 (approximately 1/10 of the edges in the original weighted networks), Table 3.\n\nnode2vec\n\nSimilar to the DeepWalk algorithm, node2vec13 also uses random walks and the skip-gram model on the graph to obtain node embeddings. However, in node2vec, biased random walks are used to better capture the node’s neighborhood information and are controlled by two parameters, p and q. The parameter p controls the probability of revisiting a node right after moving away from it, while q specifies the probability of visiting a node’s 1-hop neighbors.6 These parameters can be set to make the random walk behave more like a Breadth First Search algorithm (high p) versus Depth First Search (DFS, low p). As in DeepWalk, the embedding of a node is learned based on the probability of the neighbor set. This probability distribution is estimated efficiently by making the assumption that the probability of observing one node in a neighborhood is independent of another one and that a node u and its neighbor nodes should have a symmetric effect on each other’s feature space. Let Nsv denote the set of neighbors of v and ϕv denote the embedding of node v. The independence assumption allows the probability of the neighbor set to be written as:\n\nThe probability of node pairs is defined as a softmax function. We define the likelihood of node pairs as a softmax function of:\n\nnode2vec implements negative sampling strategies when optimizing the likelihood function and updating the node’s embedding features. This improves computation efficiency and becomes more scalable compared to the DeepWalk algorithm. We explored a range of p and q values to explore neighborhoods using a more BFS or DFS algorithm. In addition to the default setting of p=q=1, we also applied p, q with values pq∈1210.5210.51. In order to facilitate the comparison between various settings of p and q parameters, we used the default embedding dimension proposed in the algorithm implementation. The difference in performance under various p and q values is minor across all values of k, the number of clusters (Underlying data: Supplementary Figure 126,27). However, under the silhouette index, the default setting (p=1,q=1) always achieves top performance except when k=70. For consistency with DeepWalk, we set the embedding space dimension to be d∈64128256 (Table 4).\n\nGraphSAGE\n\nThe GraphSAGE framework generalizes an inductive Graph Convolutional Network (GCN) framework and estimates a function with learnable parameters that can generate node embeddings based on the node attributes and the attributes of its neighboring nodes.14 The function in turn is a deep neural network where each layer makes use of aggregation and propagation of information from a node’s neighborhood. Given a learned function, GraphSAGE uses a forward algorithm to generate node embeddings, that consist of message passing and aggregation functions. In the message-passing step, each node retrieves its neighboring nodes’ current embedding and passes it through an aggregation function. This is denoted as\n\nThe hyperparameters of GraphSAGE include the number of hidden layers and the dimension of each layer. We set the number of layers to 2 as recommended by6 and constrain the number of units in both the hidden and output layers to be d∈64128256 (Table 4). The objective function is optimized with batch gradient descent with a batch size of 256 and step size of 0.005. We also implement the negative sampling strategy by uniformly randomly selecting a target node to create an equal amount of negative links compared to positive ones. We use the mean aggregator function in the message passing step. As in the case of Deepwalk, we gave GraphSAGE unweighted versions of the phenotype-specific networks.\n\nVariational graph auto-encoders (VGAE)\n\nGraph auto-encoders (GAE)7 are commonly used unsupervised graph representation learning algorithms. They can be implemented either with or without node features. GAEs typically contain an encoder, which is often a Graph Convolutional Network (GCN)8 and a decoder, which uses inner products to predict edge presence. The loss function is based on the difference between the original adjacency matrix and the reconstructed one. Variational graph auto-encoder (VGAE)7 is an extension of GAEs that uses variational inference of model features. It assumes a prior distribution and encodes the node embeddings into a latent space via a GCN. In addition to the GAE loss function, its loss function minimizes the KL divergence between feature distributions and the standard normal distribution.\n\nWhen training the VGAE model, we set the number of layers to two so that it is consistent with GraphSAGE and the input node feature matrix as an identity matrix. The hidden layers and embedding space dimensions are also set to be 6464128128256256 (Table 4). The size of the embedding space does not significantly impact VGAE’s performance as assessed on the cell-type specific networks (Underlying data: Supplementary Figure 226,27). Since the encoder of VGAE, GCN, is also based on the aggregation of node embeddings from their neighbors, under similar reasoning as GraphSAGE, VGAE was applied to unweighted versions of the phenotype-specific graphs.\n\nTo define cell clusters we applied the K-means clustering algorithm to the node embeddings obtained from each GRL algorithm. We set the number of clusters, k, to vary from 10 to 100 with a step size of 10. To uniformly compare across algorithms, we had to pick a single k that was optimal for all algorithms. To this end, we obtained the k value for each method on each cell line that achieves the highest value based on the clustering evaluation metrics defined below. Then we select the mode value among all k values to be and use the clustering results from this k to compare across algorithms on different real and simulated networks. The optimal k values for the simulated dataset was k=30, for the cell type-specific networks was k=50, and was k=10 for both phenotype-specific networks. For selected cell lines, we additionally visualized the modularity and silhouette scores for each algorithm as a function of k (Underlying data: Supplementary Figures: 3–826,27). These values for individual cell lines are provided in Underlying data: Supplementary Tables 1–6.26,27\n\nFor the synthetic networks, we used three metrics to evaluate the quality of our clusters from each of the learned embeddings. Two of these, Modularity and Silhouette Index do not require any additional information to evaluate the quality of clusters. The third metric is Adjusted Mutual Information (AMI), which is used when there are ground truth labels for each cluster. AMI and Silhouette Index are widely used to assess the results from any clustering algorithm. Modularity is used specifically for graph clustering algorithms. All three metrics were used for the simulated networks, while SI and Modularity were used for the real networks.\n\nModularity\n\nModularity, proposed by Newman and Girvan,28 is the most widely used evaluation metric for the assessment on detected community structure. It represents the difference between the fraction of edges within one community and the fraction of edges in an equivalent network whose edges are randomly connected. Let i denote the cluster i, Ci denotes the nodes in cluster i, and E denotes the set of edges. Modularity is defined as follows:\n\nHere eii captures the proportion of edges within nodes of cluster i and ai is the expected number of edges for members in cluster i. The modularity score ranges from -1 to 1, with more positive values indicating greater community structure.\n\nSilhouette Index\n\nSilhouette Index29 measures how close a node is to its own cluster compared to its closest neighbor cluster and scores each node based on this schema. Each node’s overall average silhouette score is treated as a measurement of cluster quality. It is defined as:\n\nAdjusted Mutual Information Index (AMI)\n\nThe mutual information (MI) score is a similarity measurement between two sets of clusterings, U and V defined as:\n\nTo interpret the clusters identified on the cell-type and phenotype-specific networks, we used GO enrichment analysis (Release 106).30–32 GO enrichment analysis was done using a Hypergeometric test with FDR correction. We set the threshold of q-value to be 10−5 to define enriched terms. As GO enrichment can provide a large number of overlapping terms, we developed additional summary metrics to evaluate the GO enrichment: (a) the number of clusters enriched with any term, (b) the number of terms enriched in any cluster, (c) the distribution of the number of clusters a term is enriched in. This analysis was focused on the k=50 results to obtain the greatest resolution of the clusters, while being optimal for most methods. Finally, we used terms that were deemed specific to a cell line (defined as enriched in a small number of clusters), to bicluster the cell lines and the terms using Non-negative matrix factorization (NMF).33 We first filtered out the GO terms that are enriched in more than 20% of the cell lines and created a GO term count matrix, where the row of the matrix is a GO term and the column represents the 55 cell lines. The entry of the matrix is the number of times a term is enriched in the corresponding cell line (maxed at the number of clusters). This matrix was factorized with NMF to group GO terms into distinct term categories and cell lines into different categories with 10 factors. This enabled us to associate groups of terms with groups of cell lines. We reordered the GO terms and cell lines based on the group assignments and visualized the count matrix as heatmaps (Underlying data: Supplementary Figure 10 to Supplementary Figure 1226,27). The code for doing the NMF-based ontology summarization is available at.26\n\n\nResults\n\nWe compared our three shallow (DeepWalk, node2vec, Graph Laplacian) and two deep (GraphSAGE, VGAE) GRL methods on simulated networks with known ground truth and real cell type-specific molecular networks.\n\nWe first evaluated the different methods on the synthetic graphs from the LFR benchmark using three metrics, Modularity, AMI and Silhouette Index. The synthetic graphs were generated by setting the mixing parameter, α which controls the connectivity between clusters, to 0.1 or 0.5 (Methods). When α=0.1, 10 percent of edges connected to each node link to another node in a different cluster and the remaining edges stay in the node’s cluster. Thus α=0.1 is easier to cluster compared to α=0.5 which has more inter-cluster edges. For α=0.1, Graph Laplacian and node2vec embeddings performed overall the best for both modularity index (Figure 2A), followed closely by VGAE. VGAE achieved the highest score under silhouette index and AMI (Figure 2B & 2C). GraphSAGE outperformed others when evaluated by the modularity score, but had a relatively low score when considering the other metrics. This suggests that GraphSAGE is able to recover community structure better than other methods, even though the low dimensional embeddings of nodes within a cluster may not be as coherent as the other methods. These results also demonstrate the utility of assessing results using multiple metrics which can provide complementary information (e.g., modularity for GraphSAGE results). Deepwalk had the worst performance compared to other methods. Overall, VGAE, node2vec, and Graph Laplacian embedding had higher performance compared to GraphSAGE and Deepwalk.\n\n(A) The modularity scores with alpha = 0.1 and 0.5. (B) The Silhouette Index scores with alpha = 0.1 and 0.5. (C) The AMI scores with alpha = 0.1 and 0.5. Each algorithm is applied to simulated networks with the node degree [10, 25, 50, 100] and their scores form distributions that are summarized into box plots. For each data set, the optimal k is chosen based on the overall performance of all algorithms.\n\nFor α=0.5, which is more challenging from a clustering point of view, GraphSAGE and VGAE significantly dropped in performance under all three metrics. However, node2vec and Graph Laplacian were still robust for these graphs. GraphSAGE and Deepwalk were the two poorest-performing methods when aggregating the different metrics.\n\nWe next evaluated our five GRL methods for their ability to retrieve gene modules from 55 cell type-specific networks (Methods). These cell types corresponded to diverse cell lines and tissue types that were studied by the ENCODE ROADMAP consortium project.17 We first evaluated the quality of the modules based on Silhouette Index and Modularity, which did not require ground truth labels. The evaluation metrics were computed for each k (number of clusters), to determine the best number of clusters for each algorithm for each cell line (Underlying data: Supplementary Table 126,27). To compare across algorithms, we used k=50 as this was the best across all cell lines and methods when using either modularity or SI. Furthermore, the choice of k did not substantially affect the relative performance of algorithms (Underlying data: Supplementary Figure 326,27). Based on both modularity and SI for k=50, DeepWalk performed the best, followed by Graph Laplacian (Figure 3A & 3B). Node2vec had a better performance under the modularity index but achieved a worse silhouette score. The deep GRL method VGAE had a better SI score than node2vec, but was outperformed on modularity by node2vec. GraphSAGE had the worst performance using both metrics. Overall, DeepWalk and Graph Laplacian embedding had a consistently high performance using both metrics. Deep GRL methods had poor performance for Modularity, but we saw better silhouette scores for the VGAE algorithm.\n\n(A) The distribution of modularity across 55 cell-type specific networks. Each algorithm is applied to 55 cell line networks and their scores form distributions that are summarized into box plots. For each data set, the optimal k is chosen based on the overall performance of all algorithms. (B) The distribution of Silhouette Index scores across the 55 cell type-specific networks. As in Modularity, we picked the k that was best for each cell line. In general, Modularity and Silhouette Index picked a similar k. (C) tSNE visualizations of the embeddings learned by each method. The colors correspond to the different clusters. The colors are matched across methods so that the largest cluster is shown as cluster ID 1, followed by cluster 2, etc. (D) Number of clusters enriched with a GO term in 55 cell line networks. (E) Number of terms enriched in any cluster in 55 cell line networks. (F) Distribution of the number of cell lines a term is enriched in any cluster.\n\nTo further examine the inferred clusters, we used tSNE34 to visualize the node embedding of genes for each method in a two-dimensional space (Figure 3C) and color the nodes based on their cluster membership. To be consistent with the above analysis, we selected cluster assignments with k = 50 and plotted the 10 largest clusters. We selected the heart cell line since it had the highest Modularity across methods. For all methods, there was a clear visual separation between the members of each cluster (different colors). The tSNE visualization also showed that the Graph Laplacian embedding tended to identify less uniform-sized clusters, with two large clusters (2922-1448 genes) in comparison to the other methods.\n\nWe next interpreted the cluster assignments by performing GO enrichment analysis. Across 55 cell-type specific networks, we counted the number of clusters (out of a total of 50) that are enriched for a GO process term (Figure 3D). Across all methods, the majority of the clusters were enriched, with Graph Laplacian producing the largest number of enriched clusters across cell lines followed by DeepWalk and VGAE. Node2vec had the lowest average number of enriched clusters. This indicates that Graph Laplacian, DeepWalk and VGAE obtained the most biologically meaningful clusters for these networks. We next counted the total number of terms enriched in any cluster for each of the methods (Figure 3E). Across the 55 cell lines, we found that Graph Laplacian had the greatest number of terms on average, which was followed by node2vec and DeepWalk. Here, GraphSAGE had significantly fewer terms compared to any of the methods. We aimed to assess the overall specificity of the clusters for the GO terms by examining the distribution of the number of clusters a term is enriched in. We expect this distribution to be bimodal, with some general terms to be enriched in many clusters, while more specialized terms would be enriched in fewer clusters (Figure 3F). Here DeepWalk, VGAE and node2vec exhibited the clearest bimodal distributions. Graph Laplacian embedding tended to identify more generic terms (inferred in many clusters) and relatively fewer cluster-specific terms compared to other methods. In contrast, GraphSAGE had the opposite behavior with most terms enriched in a smaller number of clusters. In parallel, we examined the distribution of the number of cell lines a term was enriched in each of the GRL methods. Most methods with the exception of GraphSAGE exhibited a bimodal distribution, with most terms either being enriched in many of the cell lines (>50) or few cell lines (<4). Graph Laplacian was unique in that most of the terms were shared across cell lines. Finally, we used the specific GO terms for each method (defined by biclustering cell lines and the enriched GO terms using non-negative matrix factorization (Underlying data: Supplementary Figure 10,26,27 Methods). Both DeepWalk and VGAE grouped the fetal cells separately, providing more skewed cell line clusters, while the other methods provided more uniform groups of cell lines and their corresponding terms. The association of the GO terms with cell line clusters was consistent with the role of the cell lines for these methods. For example, in Graph Laplacian, we identified the immune cell lines associated with immune function, and developmental processes were associated with the embryonic stem cell lines. Taken together, these results show that shallow representation learning methods are able to recover biologically meaningful groupings of cell lines.\n\nNetwork-based approaches offer a powerful framework for interpreting sequence variants identified from GWAS by identifying pathways that might be jointly perturbed by a set of sequence variants.35,36 A common theme of these approaches is to map a set of sequence variants onto a network and interpret these variants based on subnetworks connected by these variants. As another application of GRL, we examined how the embedding affects the results for network-based GWAS interpretation. To this end, we considered two phenotypes, Autism Spectrum Disorder (ASD) and Coronary Artery Disease (CAD), which are among the most well-studied phenotypes in the NHGRI catalog.37 We focused specifically on non-coding variants, which are more challenging to interpret as they are often far away from genes. We used heat diffusion kernel to first construct weighted graphs for each phenotype (Methods), and then applied each GRL method to obtain an embedding followed by k-means clustering (Figure 4, Figure 5). Because GraphSAGE and DeepWalk require unweighted graphs, we first removed edges with weights less than the average edge weight per cell line and then gave the remaining edges as an unweighted graph as input to GraphSAGE and DeepWalk. For the VGAE algorithm, the encoder was based on Graph Convolutional Network that learns each node’s embeddings from its neighborhood. Since both ASD and CAD phenotype-specific networks were nearly fully-connected, this can produce noisy embeddings. Thus, we applied VGAE on the unweighted graphs as well.\n\n(A) The distribution of modularity across 55 cell line networks obtained with ASD. Each algorithm is applied to 55 cell line networks and the scores are summarized in a box plot. Shown are the results for k=10, which was chosen as the optimal k based on the overall performance of all algorithms. (B) The distribution of silhouette scores across the 55 networks when using k=10 clusters. (C) tSNE visualizations of the embeddings learned by each method. The colors correspond to the different clusters. The order of cluster numbers matches the decreasing order of cluster sizes. (D) Number of clusters enriched with a GO term in 55 cell line networks. (E) Number of terms enriched in any cluster of the 55 cell line networks. (F) Distribution of the number of cell lines a term is enriched in any cluster.\n\n(A) The distribution of modularity across 55 cell line networks obtained with CAD. Each algorithm is applied to 55 cell line networks and the scores are summarized in a box plot. Shown are the results for k=10, which was chosen as the optimal k based on the overall performance of all algorithms. (B) The distribution of silhouette scores across the 55 CAD phenotype-specific networks when using k=10 clusters. (C) tSNE visualizations of the embeddings learned by each method. The colors correspond to different clusters. The order of cluster numbers matches the decreasing order of cluster sizes. (D) Number of clusters enriched with a GO term in 55 cell line networks. (E) Number of terms enriched in any cluster in 55 cell line networks. (F) Distribution of the number of cell lines a term is enriched in any cluster.\n\nAs in the analysis of the cell-type specific networks, we chose the optimal k value based on the mode of the Modularity and Silhouette Index values across 55 cell lines and all 5 methods (Underlying data: Supplementary Table 3–6,26,27). The optimal k under these two metrics was 10. Based on the modularity metric, the top-performing method was Graph Laplacian followed by node2vec for the ASD phenotype network (Figure 4A). However, with the Silhouette Index, Graph Laplacian and node2vec had the worst performance (Figure 4B). Here, VGAE exhibited the best performance followed by GraphSAGE and node2vec. VGAE, GraphSAGE and DeepWalk accepted undirected weighted graphs and the opposite behavior of these algorithms for modularity and Silhouette Index could be due to that. These results demonstrate the benefit of deep learning frameworks for embedding graphs for GWAS analysis. We also visualized the clusters obtained for ASD phenotype networks in the heart cell line with t-SNE plots (Figure 4C). For all algorithms, the clustering structure was visually discernible with genes in the same cluster being positioned close together.\n\nWe next evaluated the different GRL methods based on GO enrichment of the inferred clusters. Here we considered k=50 to have a sufficient granularity of the clusters to capture specific biological processes. We examined the distribution of the number of clusters enriched with a GO process as well as the number of GO process terms that were enriched across the 55 cell lines. Graph Laplacian embedding produced the highest number of clusters enriched with biological functions, followed by node2vec (Figure 4D). DeepWalk and VGAE had a similar average number of clusters enriched and GraphSAGE had the fewest. GraphLaplacian and node2vec were equally good when considering the number of terms followed by DeepWalk (Figure 4E). Both GraphSAGE and VGAE had the fewest number of GO terms. Finally, we examined the distribution of the number of clusters for each term (Figure 4F). Only Graph Laplacian and node2vec exhibited a bimodal distribution, meaning that they can discover both generic and specific term GO terms. As we did previously, we selected the most specific terms (defined by their enrichment in any cluster of 20% of the cell lines) and applied NMF to find cell line and term clusters (Underlying data: Supplementary Figure 1126,27). Graph Laplacian and node2vec exhibited the clearest block diagonal structure with groups of cell lines associated with groups of GO terms. Several of these groupings were biologically meaningful, for example, the association of muscle and cardiac functions with a cell line cluster with heart (Graph Laplacian) and immune response associated with cd14 primary cells (node2vec).\n\nWe performed a similar analysis for the CAD phenotype network as well (Figure 5). We observed similar trends for the relative algorithm performance when using Modularity and Silhouette Index, with methods using undirected graphs exhibiting a higher Silhouette Index compared to methods using the weighted graphs (Graph Laplacian and node2vec) and an opposite trend when using Modularity (Figure 5A & 5B). Visualization of the tSNE plots for the heart cell line showed that while Graph Laplacian, GraphSAGE and VGAE retrieved layouts that were consistent with the clustering, DeepWalk and node2vec visualization were noisy (Figure 5C). A similar noisy layout was observed for other cell lines as well suggesting that the CAD networks were noisier than the ASD networks (Underlying data: Supplementary Figure 926,27). Based on the number of enriched clusters and terms enriched, Graph Laplacian performed best, followed by VGAE and GraphSAGE (Figure 5D). The low enrichment of the DeepWalk and node2vec algorithms is consistent with the lack of clustering structure in the tSNE results. The histogram of term specificity exhibited a bimodal distribution only with the GraphLaplacian embedding (Figure 5E); however, all three methods (Graph Laplacian, VGAE and GraphSAGE) produced well-separated cell line and biological process groups (Underlying data: Supplementary Figure 1226,27).\n\nOverall, our comparison of the GRL methods for the GWAS interpretation task showed that Graph Laplacian provided the most robust embedding under all metrics. Furthermore, the performance of the methods depended upon the metric and phenotype and deep GRL methods were less prone to noisy diffusion-based graphs compared to random-walk based methods (node2vec, DeepWalk).\n\n\nDiscussion\n\nGraph representation learning (GRL) has emerged as a powerful paradigm for applying machine learning algorithms on graphs. More recent deep graph representation learning algorithms have shown improved performance on numerous graph machine learning problems such as node classification and link prediction. Given the modular organization of molecular networks such as protein-protein and regulatory networks, a natural question is whether deep GRL methods have an advantage for detecting modules or clusters on graphs over shallow GRL methods. In this paper, we performed a comprehensive benchmarking study on synthetic networks and real molecular networks capturing protein-protein and regulatory interactions. On real networks, we considered two types of applications: detection of gene modules from cell type-specific networks and detection of candidate pathways affected by single nucleotide polymorphisms identified in GWAS. We observed some benefits of deep GRL algorithms on simulated networks as well as in situations where the graph may be noisy.\n\nOur experiments on simulated networks were helpful because of the availability of ground truth and a controlled experimental setting. In particular, the mixing parameter of 0.1, which results in sharper boundaries across clusters, all but DeepWalk perform well using Modularity. However, for a more challenging cluster structure, GraphSAGE dropped in performance. Between VGAE and GraphSAGE, which are both deep methods, VGAE was consistently better. It is likely that GraphSAGE is better for graphs where node attributes provide complementary information to the graph structure. When the networks are sparse, VGAE achieved equally good performance as node2vec and Graph Laplacian embeddings. However, shallow methods consistently outperformed deep learning frameworks for denser graphs where modules are harder to detect. Our experiments showed that deep GRL methods such as VGAE can outperform other algorithms but this depends upon the structure of the graph and the specific metric.\n\nOn real molecular networks, the relative performance of the algorithms depended upon the task. When defining gene modules across 55 cell line/type-specific networks, shallow GRL methods consistently outperformed both VGAE and GraphSAGE when using Modularity. However, when using the Silhouette Index, VGAE was comparable to shallow methods but GraphSAGE remained worse. Interestingly, despite low silhouette scores, all methods generated well-clustered tSNE visualizations and identified clusters that were enriched for gene ontology processes. When considering the task of GWAS interpretation, we found both GraphSAGE and VGAE and the shallow method, DeepWalk, to produce better silhouette scores, but this trend was reversed when using modularity. This observation was reproducible for the second phenotype as well. All three algorithms accepted filtered undirected graphs as input, while GraphLaplacian and node2vec accepted nearly fully connected weighted graphs. The pre-processing of graphs before providing them as input to graph embedding algorithms could be a major determinant of an algorithm’s performance. When compared to the ASD phenotype-specific graphs, we found DeepWalk and node2vec to produce noisy tSNE visualizations, which could be due to the greater noise in this graph and the susceptibility of random walk methods to noisy graphs. This was consistent with the relatively lower modularity score of node2vec compared to Graph Laplacian as well as a smaller number of enriched terms for these methods. Finally, across all network types, tasks and metrics, we found Graph Laplacian to provide consistently good embeddings, though it was not necessarily the best for some conditions. The only potential limitation of the Graph Laplacian is to infer unbalanced gene clusters for the cell-type specific networks. A direction of future work would be to consider alternative pre-processing of the graph or additional regularization to obtain more uniformly-sized clusters.\n\nOur study compared select deep GRL and shallow GRL algorithms on graphs that did not have any node attributes. One direction of future work is to expand the type of graphs to those with node attributes and accordingly increase the class of algorithms to be compared. Another direction of future work is to detect dynamic gene modules, for which there are shallow38 and deep learning algorithms.39\n\n\nConclusions\n\nIn conclusion, our benchmarking study shows that the performance of different embedding methods depends on the metrics used as well as network characteristics, such as sparsity and edge weight distribution. Deep GRL algorithms are not necessarily advantageous over shallow methods for module detection and this depends upon the algorithm, the type of graph and the specific module-based task. Between the two deep GRL algorithms, we found the VGAE algorithm to offer superior performance. Comparing across different graph types and module metrics, Graph Laplacian provided consistently robust and high-quality embeddings suggesting that it could be a good baseline algorithm to compare for newly developed representation learning algorithms.", "appendix": "Data availability\n\nZenodo: Underlying data for’Benchmarking graph representation learning algorithms for detecting modules in molecular networks’, https://doi.org/10.5281/zenodo.7876238 27\n\nThis project contains the following underlying data:\n\n• Cell Type Specific Networks.zip This includes all the cell type specific networks from the 55 cell lines.\n\n• ASD Phenotype Networks.zip This includes all the cell type-specific weighted networks, where the weights correspond to the output of diffusing signals on the graph using SNPs associated with ASD.\n\n• CAD Phenotype Networks.zip This includes all the cell type-specific weighted networks, where the weights correspond to the output of diffusing signals on the graph using SNPs associated with CAD.\n\n• Simulated Networks.zip This includes all the simulated networks used to evaluate the performance of different methods.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nAcknowledgements\n\nWe would like to thank Sara Knaack and Junha Shin for assistance with the generation and visualization of Gene Ontology enrichments.\n\n\nReferences\n\nBarabási A-L, Oltvai ZN: Network biology: understanding the cell’s functional organization. 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Res. 2008; 9(86): 2579–2605.\n\nBarrio-Hernandez I, Schwartzentruber J, Shrivastava A, et al.: Network expansion of genetic associations defines a pleiotropy map of human cell biology. Nat. Genet. March 2023; 55(3): 389–398. Number: 3 Publisher: Nature Publishing Group. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJia P, Zhao Z: Network-assisted analysis to prioritize GWAS results: principles, methods and perspectives. Hum. Genet. February 2014; 133(2): 125–138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSollis E, Mosaku A, Abid A, et al.: The NHGRI-EBI GWAS Catalog: knowledgebase and deposition resource. Nucleic Acids Res. January 2023; 51(D1): D977–D985. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee C, Wilkinson DJ: A review of stochastic block models and extensions for graph clustering. Appl. Netw. Sci. December 2019; 4(1): 1–50. Number: 1 Publisher: SpringerOpen. Publisher Full Text\n\nKazemi SM, Goel R, Jain K, et al.: Representation Learning for Dynamic Graphs: A Survey, arXiv:1905.11485 [cs, stat].April 2020." }	[ { "id": "201155", "date": "20 Sep 2023", "name": "Claude Pasquier", "expertise": [ "Reviewer Expertise Computer science", "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\nThis article \"Benchmarking graph representation learning algorithms for detecting modules in molecular networks\" investigates deep and shallow graph representation learning algorithms for gene module detection in molecular networks. The authors compare 5 graph representation learning (GRL) techniques on 4 different datasets, 1 synthetic and 3 real biological networks. The GRL techniques are divided into two categories, shallow and deep, containing 3 and 2 state-of-the-art methods respectively. The authors report that, while deep methods, especially Variational Graph Auto-Encoder (VGAE), perform well on synthetic networks, their performance varies on real networks, depending on the graph's characteristics and evaluation metrics. Shallow methods like Graph Laplacian embedding and node2vec often outperform deep methods in identifying gene modules. The paper concludes that the choice between deep and shallow graph representation learning methods for module detection tasks in biological networks depends on various factors, including the graph's structure and the evaluation metrics used.\nThe novelty of the article lies in the fact that the authors have specifically tackled the performance evaluation of graph representation learning techniques on biological networks, which is particularly interesting and differs from many other existing studies which focus on social and citation networks. Although the article provides valuable information on the comparison of deep and shallow graph representation learning methods for module detection we have identified some potential limitations or weaknesses of the study.\nMajor comments\n1. One significant issue with this paper is its heavy reliance on K-Means clustering as the sole evaluation metric for graph embeddings. The authors benchmark various graph representation learning algorithms and draw conclusions about embedding quality based on K-Means performance. However, it is well-known that clustering methods can be highly dataset-dependent, as is the choice of embedding method. As shown in Woma & Ngo (2019)1, the quality of clustering depends on both the embedding and clustering methods. The paper cannot assert, as it does, that examining K-Means results is a comprehensive measure of embedding quality. The authors should consider testing other clustering methods and discussing the results.\n2. Another important concern is the use by the authors of a network generator that preserves a certain community structure. Although the topology of the generated graphs may resemble social graphs to some extent, it is by no means certain that they accurately represent real biological networks. Some studies show that biological networks constitute a special case with very specific characteristics, particularly in the way the communities are formed (Gutiérrez-Bunster et al. 2014)2. This could perhaps explain why the results obtained on artificial and real biological graphs are different. It would be beneficial to discuss how this choice of synthetic data generation relates to the characteristics of real biological networks.\n3. Following on from the previous remark, it is regrettable that the metrics used are classic clustering metrics. Since the interest of this work is to compare LRM methods with graphs that have different properties from the graphs generally used in the literature, wouldn't it make sense to judge them with different metrics?\n4. The title of the article suggests that the authors are carrying out a study evaluating the performance of graph representation learning methods on molecular networks, but they focus their study on cell-type-specific networks. Although these networks are important in molecular biology, they do not cover the full diversity of biological networks, and the results may not be generalizable to other types of molecular networks. Here again, the article would benefit from addressing the limitations of the comparative analyses carried out and the difficulties of generalizing them.\n5. The authors tested different methods on artificial networks generated with Lancichinetti-Fortunato-Radicchi (LFR) graph generator, setting the number of nodes to 1000, the average degree of nodes K = 10, 25, 50 and 100, the exponent for degree distribution γ = 2, the exponent for community size distribution β = 1, and the mixing parameter μ = 0.1 and 0.5. So there are 8 different networks, listed in Table 1. Next, clustering results are presented for α (μ? see minor comments) equals to 0.1 and 0.5 (figure 2). It seems that the results obtained for each value of k have been averaged. This does not give a detailed view of the results for the different configurations. Since the authors present 8 test datasets, they should give the results for each one. If this is due to space constraints, they could include the detailed results as supplemental data.\n6. The authors are testing the methods on artificial networks of 1000 nodes, which is small for a biological network. The authors should add benchmarks on larger networks and discuss the performances of the algorithms.\n7. The description of the cell-type specific networks and phenotype-specific diffused networks is unclear. It is not evident whether the creation of these networks is part of the work described in this paper or if it was performed in the paper of Baur et al. The paper should clarify this point.\n8. The paper evaluates a relatively small set of deep and shallow graph representation learning algorithms. There are numerous other algorithms available in this field, and the selection may not represent the full spectrum of possibilities. The authors should either include a wider range of algorithms or explain how the selected algorithms are representative of existing methods.\n9. The evaluation using GO enrichment involves counting enriched terms but doesn't consider group size, which can impact the results. In the article, there's no data on the size of the clusters obtained; could the authors go into a little more detail, mentioning the size of the clusters and their distribution?\n10. Furthermore, the metrics used to indicate the relevance of clusters according to their GO annotation (the number of clusters enriched with a GO term, the number of terms enriched in any cluster and the distribution of the number of clusters in which a term is enriched) do not seem enough in our view. The authors need to provide a more detailed biological analysis.\nMinor comments\n1. The paper introduces the concepts of deep and shallow embedding quite late, on page 5, with a reference to Hamilton et al. This crucial terminology and distinction should be introduced earlier in the paper to enhance reader comprehension.\n2. The mixing parameter is referred to as μ on pages 3 and 4, similar to the notation used in Lancichinetti et al. However, it appears to be identified as α in the rest of the paper, including the tables. This inconsistency should be rectified for clarity.\n3. The variable k is first used to describe the average degree of the nodes before becoming the number of clusters used by the K-Means algorithm. This dual use of k can lead to confusion and should be clarified.\n4. This sentence doesn't seem to be well constructed: \"The protein-protein network is not cell type specific and combined it with cell type-specific TF-gene regulatory interactions.\"\n5. The objective function is optimized with batch gradient descent with a batch size of 256 and step size of 0.005. => The objective function is optimized with batch gradient descent with a batch size of 256 and a step size of 0.005.\n6. This yields the optimization objective function that maximizes Pr(uk∣∣Φ(v)), where ϕ(v), the embedding of node v and uk is a neighbor node => This yields the optimization objective function that maximizes Pr(uk∣∣Φ(v)), where ϕ(v) is the embedding of node v and uk is a neighbor node\n7. Zenodo: Underlying data for’Benchmarking [...]. => Zenodo: Underlying data for ’Benchmarking [...].\n\nIs the work 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": "201156", "date": "21 Sep 2023", "name": "Renming Liu", "expertise": [ "Reviewer Expertise Computational Biology", "Network Biology", "Graph Representation Learning", "Single Cell 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\nThis paper conducted a systematic study to compare two classes of approaches, shallow network embeddings, and graph neural network-based methods, for their usage in aiding the analysis of molecular networks, such as finding modules related to particular diseases or biological processes. The authors used well-developed graph embedding methods, including graph Laplacian eigenmaps, deepwalk, node2vec, GraphSAGE, and VGAE. After conducting systematic module detection evaluations using both synthetic and real molecular networks, the authors suggest that shallow graph embedding techniques, particularly the graph Laplacian eigenmaps, perform well in finding modular structures in the networks. GNNs, on the other hand, do not always provide benefits over shallow embedding methods despite their complexities.\nOverall, the paper is well written, and the claims made are well aligned with the results presented. However, I have a few questions and suggestions for the authors.\nPhenotype-specific diffused network construction: what is the purpose of the two-step diffusion process using regularized Laplacian and heat kernels? Could the authors provide some context or justification for this particular choice of processing instead of just using one diffusion kernel? Does this have any effect on the downstream evaluation?\n\nThe link prediction function used for GraphSAGE, and similarly VGAE, is unclear. Particularly, the authors mentioned that “for each noe, positive and negative links are generated via negative sampling and the parameters of the embedding function are learned via stochastic gradient descent”, but how the predictions for and edge is made from the model is unclear.\n\nIn equation (6), what is “q” in the summation? Should it be “1” instead?\n\nThe first results section title could be more specific. Deep GRL methods provide advantages in what general settings?\n\nIt would be beneficial to delve into a detailed discussion regarding the seemingly conflicting results observed in Fig 4.A and Fig.A, particularly concerning the disparities in rankings between VGAE and node2vec across the two metrics - modularity and SI. What are the potential reasons behind these discrepancies and the implications that future studies should consider?\n\nFor the network diffusion-based analysis, the authors applied a sparsifying threshold using the mean edge weight. This processing step seems to be one of the most crucial data processing for the embedding methods. Have the authors tried other thresholds besides the default edge weight and see if the overall major conclusion changes? The authors also mentioned that deep GRL methods perform less well when the networks are dense and noisy. Could increasing the sparsifying threshold be one of the solutions to further improve the GRL results and make them more on par with shallow embedding methods?\n\nAccording to the methods section, the GRL embeddings are trained by predicting the presence of edges in the network. Is the edge weight information also being used during the training process in the case of weighted 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": [] } ]	1	https://f1000research.com/articles/12-941
https://f1000research.com/articles/12-940/v1	07 Aug 23	{ "type": "Research Article", "title": "Morphological, structural, chemical, vibrational, thermal, pasting, and functional changes in pea starch during germination process", "authors": [ "Juan Carlos Lucas-Aguirre", "Víctor Dumar Quintero-Castaño", "Luisa Fernanda Castañeda-Cano", "Mario Enrique Rodríguez-García", "Víctor Dumar Quintero-Castaño", "Luisa Fernanda Castañeda-Cano", "Mario Enrique Rodríguez-García" ], "abstract": "Background: Changes in the structural and physicochemical properties of pea starch could be significantly affected by germination treatment, which can provide a theoretical basis for promoting the use of this starch in the food industry. Methods: This work aims to evaluate the effect of germination time on the structural, thermal, rheological and functional properties of pea starches to determine their potential in the production of fermented beverages. The physicochemical changes during the germination process of peas and native and germinated starch granules were evaluated. Results: For germination critical time was 0.985 days, with 95% of germinated grains. The starch grains did not undergo any morphological change during the germination process as seen in the scanning electron microscopy images, indicating the absence of the α and ß - amylases responsible for the starch splitting. The X-ray patterns revealed that the crystalline structures of pea starch with and without germination were unchanged and contained by hexagonal and orthorhombic glucopyranose crystals. The viscosity profiles of the starches do not show significant changes; the most representative change is the increase in the gelatinization onset temperature of the paste from germinated starches compared to native starch. The functional properties of starches showed generally low values, with statistically significant differences between water absorption index, water solubility index, and starch swelling power and germination time. Conclusions: In general terms, it can be concluded that lentil starch does not undergo significant changes in its physicochemical and functional integrity with respect to the grain germination process.", "keywords": [ "pea", "starch", "germination", "starch nanocrystals", "pasting." ], "content": "Introduction\n\nTo improve the nutritional value of legumes, preparation techniques have been developed to significantly increase the bioavailability of their nutrients. These techniques include germination, a complex metabolic process that activates and releases enzymes, responsible for changes in the chemical composition of the grain. Pea (Pisum sativum, L.) is a legume that has great nutritional potential due to its high protein content and has been proposed as an alternative protein source to soybeans in countries where soybeans are not native, or in situations where soybeans cannot be used due to allergic reactions or intolerances. The market has seen an increase in demand for gluten-free goods since, in certain people, gluten can result in autoimmune and immune-mediated health issues (Urbano et al., 2005; Xu et al., 2019). To satisfy the gluten-free market, the ideal scenario would be to select raw materials that can be produced at low cost and have excellent nutritional value, and are rich in protein, carbohydrates, vitamins and minerals. For instance, compared to cereal flours, which have a protein value of 3-7 g/100 g dry basis (db), legume flour has a protein concentration that ranges from 17-40 g/100 g (db). Legume flours have also been utilized to create dishes with a lower glycemic index since they have more dietary fiber than wheat flours. In addition, consumption of legume flours is associated with better human health, mainly due to the presence of bioactive components such as polyphenols (Urbano et al., 2005; Xu et al., 2019). However, the potential benefits may be limited by the presence of anti-nutritional factors, including trypsin inhibitory activity.\n\nGermination is responsible for changes in the chemical composition of the grain. Due to which lipids, carbohydrates (starch) and storage proteins within the seed are broken down to obtain the energy and amino acids necessary for the development of the pedicel, roots and at the end of the plant and to be more easily absorbed, which increase the healthy function of the grain (Urbano et al., 2005; Xu et al., 2019; Aguilar et al., 2020; Gao et al., 2022). Depending on temperature, humidity, light levels, and grain, the germination stage typically lasts between 4 and 5 days. The grains are rotated occasionally while regulating humidity and temperature. Enzymes like hemicelluloses, amylases, proteases, oxidases, among others, are engaged. In this stage metabolism is very active (Oseguera-Toledo et al., 2020). The functional properties of the starch, such as variations in relative viscosity, reducing sugars, apparent amylose, among others, change along with the germination process. As malting progresses so does the capability for water absorption (Hernández-Becerra et al., 2020).\n\nAccording to Esquivel-Fajardo et al. (2022), starch is a micron or submicron-sized particle made of amylose, amylopectin, water, minerals, salts, and nanocrystals with orthorhombic (A-type starch) or hexagonal crystalline structures (B-type starch). These elements have a significant impact on how the physicochemical properties of starch change during germination. This suggests that it is important to identify the modifications brought about by the germination process in each of these components.\n\nThe physicochemical properties of starch after germination can be effectively affected, depending on the type of crop and treatment conditions, but in peas such behavior is largely unknown. Gao et al. (2022) reported that the structural and physicochemical properties of pea starch could be significantly affected by germination treatment; these changes could provide a theoretical basis to support the use of this starch in the food industry. However, no information about the starch components affected by germination was reported.\n\nIn order to evaluate the potential use of pea flours and starches in food products, this work evaluated the impact of germination time on morphological, structural, thermal, vibrational, sticking and functional properties.\n\n\nMethods\n\nThe San Isidro pea variety, cultivated in Nariño state at the south of Colombia, was used, purchased at the central supply center of the city of Armenia-Quindio. Initially, 1500 g of peas were used, and a manual selection process was carried out to eliminate impurities and broken and/or irregularly shaped kernels. Then, 250 g were weighed for each day of germination for four days and subjected to a 12 hour soaking process in potable water (20°C) using twice the volume of water occupied by the dried peas, during which time rehydration of the peas was ensured. After the extra water was removed, the germination time started and was measured from time zero (0), until it reached 4 days (Figure 1), the period of time during which the hypocotyl had fully developed.\n\nEvery four hours during the four-day germination period, the peas were soaked and flipped over to ensure appropriate humidification and the excess water drained, to prevent the embryo from decaying and dying. Following the conclusion of each germination period, the starch was promptly extracted as explained below.\n\nThe germinated flours were produced once the germination process was complete by drying the germinated peas for 48 hours at 40°C in an oven with forced ventilation. To obtain flours, the dry germinated seed were milled using a coffee mill (Krups) and each one of the flours were then placed in zip-top bags and maintained in a controlled environment of temperature (20°C) and relative humidity (65%). The flour were named as follows: pea flour without germination (PF), the flour for the first day of germination (PF-1), second day (PF-2), third day (PF-3) and fourth day (PF-4). After drying, the flour was ground and sieved through 100 mesh (150 μm).\n\nMethodologies reported by AOAC (2005) were used for moisture (925.10), lipids (920.39), fiber (978.10), ash (942.05) and protein (920.15), where the moisture content was determined by the oven drying method (105°C, to constant mass); the total fat content was determined by the Soxhlet method; the fiber content by calcination of the dry residue remaining after acid and alkaline digestion of the sample; the ash content by incineration at 550°C; and the protein content was determined by the Kjeldahl method using 6.25 as a factor.\n\nThe isolated starch both without germination and germinated for each day was obtained following the methodology proposed by Contreras-Jiménez et al. (2018) and Londoño-Restrepo et al. (2018), where samples were ground with a blender (Krups) using distilled water, filtered on a 100 mesh, kept at 4°C for 24 h with distilled water, centrifuged 2270 x g (3000 rpm, Model TD5 Table Top Low Speed Centrifuge, Yingtai Instrument, Changsha, Hunan, China) at 25°C for 15 min, and the clean starch was dried at 45°C for 24 h. After drying, the starch was ground and sieved through 100 mesh (150 μm).\n\nThe amylose content of native and germinated pea starch was determined by the spectrophotometric method (620 nm) based on the formation of the amylose-iodine complex (Juliano et al., 1981), for which starch was dispersed in 1 M sodium hydroxide, brought to volume with distilled water (100 ml), the starch was gelatinized and the amylose concentration was determined using an iodine solution (2 g KI + 0.2 g I2) against a standard curve with known amylose concentration.\n\nThe number of grains that show the existence of a root is determined by obtaining three random samples of 100 grains. Every day, the development of the root was observed, and it was deemed to have germinated when the radicle ruptured the cell wall. When the pedicel was twice the size of the grain, germination had finished. A manual selection of the grains was done to eliminate broken and damaged grains in order to calculate this parameter (Hernández-Becerra et al., 2020).\n\nSEM images of the pea isolated starches (PIS) for different germination times were studied using a high vacuum scanning electron microscope (JEOL, JSM-6060LV at high vacuum; Jeol, Tokyo-Japan) with a resolution in HV mode. Each sample was fixed on a specimen holder with carbon tape and sputtered with gold. The analyses conditions used 20 kV electron acceleration voltage at 12–20 Pa pressure (Quintero-Castaño et al., 2020).\n\nParticle sizes were determined as percentiles D10, D50, and D90, using the Mastersizer 3000 (Malvern Instrument Ltd., Worcestershire, UK). The samples were dispersed in 500 mL of distilled water until a darkening value of 10±1% was obtained, considering the size distribution from Mie’s theory and using the refraction index of 1.52 (Lucas-Aguirre et al., 2019).\n\nX-ray diffraction patterns of isolated starch along germination process were carried out on a Rigaku-Ultima 4 diffractometer (Rigaku Miniflex, Texas, EE.UU). The operating conditions were 35 kV and 15 mA, with a CuKα radiation wavelength of λ=0.1540 nm, and the measurements were done from 5 to 35° on a 2θ scale. The patterns were carried out using High-Resolution X-ray diffraction with a step size of 0.01° to have a better intensity and resolution of each one of the possible diffracted peaks. The powder samples were packed into an aluminum pan (Quintero-Castaño et al., 2020).\n\nIsolated pea starches were characterized by FTIR spectrophotometer (Perkin Elmer, Spectrum Two) using ATR (attenuated total reflectance) to identify the main functional groups, and the transmittance was recorded from 600 to 4000 cm-1 (Contreras-Jiménez et al., 2018).\n\nThe gelatinization temperature and enthalpy of starches obtained from PSI, and isolated starches from each day of the germination process, were analyzed using a differential scanning calorimeter (Mettler Toledo, Greifensee, Switzerland). Calibration was performed using an Indium standard. The samples (2.0±0.1 mg) were prepared by adding deionized water to the samples until they reached 85% moisture (w/w). A similar ratio of starch/water was used for the viscosity measures. The pans were hermetically sealed using a press. Sample scanning was from 30 to 120°C at 7.5°C/min. Each measurement was conducted in duplicate (Contreras-Jiménez et al., 2018).\n\nApparent viscosity profiles of the PIS, and isolated starches obtained along the germination time, were obtained using a starch cell of an Anton Paar MCR 102 (St Albans, United Kingdom) rheometer following the methodology proposed by Rincón-Londoño et al. (2016). Each sample was heated from 50 to 92°C in 5.3 min and then held at a constant temperature of 92°C for another 5.3 min. After that, samples were cooled down to 50°C in 5.3 min and held at that temperature for 1 min. All the tests were carried out at a constant frequency of 193 rpm.\n\nThe water absorption index (WAI), the water solubility index (WSI) and the swelling power (SP) of the isolated starches were measured and determined using the methodology reported by Contreras-Jiménez et al. (2018), where a 2% W/V starch suspension was heated in a water bath at 90°C for 30 min. The starch samples were centrifuged at 2000 rpm for 20 min. The supernatant was removed and the sediment was weighed. Aliquots of supernatant were dried in an oven at 100°C to a constant weight, then the respective weights were taken and calculations were performed.\n\nAll the analysis were performed following a completely randomized design with three replications. An analysis of variance was performed (ANOVA) with 95% of confidence and Tukey test of mean comparison using Statgraphics Centurion XVIII software.\n\n\nResults and discussion\n\nThe morphological changes of the pea beans during germination are depicted in Figure 1(a) to (f). There are no noticeable morphological changes on the surface of the bean after soaking; instead, the bean swelled to nearly double its initial volume, due to hydration of the cotyledons and embryo (Figure 1(a) and (b)), while from the first day of germination, the hemicellulose enzymes were activated, the cell wall was broken, the radicle emerged (Figure 1(c)) and the tegument or testa began to separate from the grains. On days 2 and 3, the hypocotyl emerges, which initially remains curved (protected) (Figure 1(d) and (e)), and continues to grow up to three times the size of the grain. The teste continues to separate more easily and finally on day four, the hypocotyl straightens (Figure 1f), at which time germination stops and they are dried at 40°C for 48 h.\n\nThe proximate composition of the pea flours (PF) and flours obtained from day 1 to day 4 (PF-1 to PF-4) as a function of the germination day is shown in Table 1, presenting statistically significant differences in each of the evaluated variables (p<0.05). In fact, the germination process resulted in a large decrease in total fat content, equal to 74.52%, compared to its value on day 0. As a result of the energy-intensive metabolic activities required for this decrease in fat content, the respiratory rate increased (Aguilar et al., 2020; Hernández-Becerra et al., 2020; Oseguera-Toledo et al., 2020). Chemical composition is affected by genotype, agronomic conditions, growing conditions such as soil, moisture, temperature, pests, and diseases, and production management (Oseguera-Toledo et al., 2020).\n\nAsh content, which is correlated to the mineral content of the sample, also showed a decrease throughout the germination process, dropping as much as 50.0% by day 4, in comparison to its initial content. Several minerals are needed as coenzymes during germination in order to catalyze the transfer of proteins and carbohydrates to the radicle. These minerals can be lost during the hydration process and/or during germination and can be leached in water during soaking. Consequently, some of these minerals can be lost when the radicles are separated (debarking). The same behavior was seen during the malting of three quinoa varieties by Aguilar et al. (2020), and with two maize varieties by Hernández-Becerra et al. (2020).\n\nDuring germination, the protein and fiber contents of the peas increased proportionally by 107.84% and 36.46% respectively, with respect to the initial content. Some proteins can be anabolized from stored carbohydrates during germination, raising the protein content of the grain. All these differences depend on the metabolism of the grain; in addition, the permeability of the seed coat to water depends on its composition. For instance, the presence of phenols and flavonoids can lower permeability (Bewley et al., 2012; Aguilar et al., 2020). The previous behavior is contrary to that reported by Hernández-Becerra et al. (2020), with two maize varieties, where the protein content in the meals does not undergo changes throughout malting, which could be interpreted to mean that proteins can be degraded to polypeptides, peptides and amino acids that provide substances for de novo protein synthesis in the growing embryo, but all of them contribute to total nitrogen. Oseguera-Toledo et al. (2020) reports an increase in protein content in sorghum malt, indicating that enzymes can contribute to the increase in protein and that this process continues during fermentation, obtaining in both cases raw materials with higher nutritional value. This increase in protein content in pea would be important attribute that affects yeast nutrition, fermentation, foam stability and beer flavor, thinking about this possibility of use (Alfeo et al., 2018). Table 1 shows the changes on amylose content over the germination time. Germination is a kinematic process governed by enzymatic processes α and β-amylose hydrolyzed during of germination. In this table, the apparent amylose does not significantly change, but when amylopectin degrades, it transforms into amylose, so the values at the end are the same.\n\nPeas were once classified manually with broken, sick, or damaged beans being thrown away. Figure 2 shows the germination percentage as a function of time for the peas, showing that during the soaking time (12 h), the grains reach moisture values from 13.13±0.08% to values close to 40.53±0.52% moisture (wb) and an average of 5 grains germinate (Figure 1); during the first day of germination, germination percentages of over 95% are reached and over 98% during the second day (Figure 1).\n\nAccording to Contreras-Jiménez et al. (2018), who studied barley malting; Hernández-Becerra et al. (2020), who studied two types of maize; and Oseguera-Toledo et al. (2020), who studied sorghum malting, the behavior of germination % exhibits a sigmoidal and/or logistic shape curve. This kind of curve illustrates a second order kinetics in which the system passes through a localized transition between two saturation values. The crucial germination time, or tc, is a point at which the shift fundamentally takes place. For pea, tc is around 1 day, but for other products like maize, it is between 1.92 and 1.94 days and for barley, it is between 1.92 and 2.92 days.\n\nAs evidenced by the radicle shattering the cell wall on the second day of germination, peas reach their peak value before corn and barley, which germinated on the third and fourth days, respectively. This fact shows that extending of the period for peas boosts their uses when thinking about industrial processes. Other aspects of germination that need to be assessed include the activation of gibberellins, which are phytohormones that cause the interruption of the dormant stage of the seeds and cause them to germinate, and the beginning of latent enzymatic processes with the α and ß-amylases, which use starch and fats as energy sources. These include the grain’s level of hydration. This explains why germination suddenly increased by 60% about tc. For two days, gibberellins are created, therefore for the third day decreases (Gupta et al., 2010; Evans et al., 2009; Bradford et al., 2008).\n\nThe SEM images of the PIS and isolated starches obtained from day 1 to day 4 of germination (PIS-1 to PIS-4) are shown in Figure 3a to j, where Figure 3a and b represent the PIS, Figure 3c and d represent day1 (PIS-1), Figure 3e and f represent day2 (PIS-2), Figure 3g and h represent day 3 (PIS-3), and Figure 3i and j represent day 4 (PIS-4) of germination. No significant alterations to the starch surface were seen during pea germination. However, there is evidence of some protein and fat removal. As was reported for barley, α and β-amylase form micro-holes during this stage of germination (Contreras-Jiménez et al., 2018), for corn (Hernández-Becerra et al., 2020) and (Oseguera-Toledo et al., 2020).\n\nThe starch granules, which coincide with those described by Gao et al. (2022), have oval and kidney shapes with a few surface indentations. But with few changes during the germination process. Some starch granules presented only small changes on their surface, observing some indentations or rough surfaces, where an enzymatic attack could have occurred. All of this contrasts with what Gao et al. (2022), which states that after the first day of germination, the majority of the starch granules had notches on the surface, with obvious pores and more undulations on the surface of the germinated starch granules as germination progressed, changes that affect the other physical and thermal properties. These changes were produced by the enzymes responsible for amylolysis, in 2 varieties of pea.\n\nThe particle size distribution of starch granules D10, D50, and D90 indicate that 10, 50, and 90% of the starch granules have a smaller diameter than this value (Table 2). In general, D50 was used to describe the mean diameter. The D50 of the starch granules ranged from 53.72±4.65 μm to 56.8±3.41 μm.\n\nIn general, statistically significant differences were found in the average diameter in each of the percentiles (p<0.05), presenting a slight increase in the average diameter with advancing germination time, in percentiles D10 and D50, coinciding with what was reported by Gao et al. (2022), with starches from two pea varieties. In the D90 percentile, the average diameter of the starch decreased, which could be an effect of the enzymes in the germination process, which could penetrate inside the granules and hydrolyze from the helium region to the outside. This could result in the formation of surface pores, surface erosion, sponge-like erosion and degraded granules, for the grains where there is enzyme activity, which could be occurring superficially in the pea starch granules according to the SEM images obtained in this study, although no enzymatic activity is observed (Li et al., 2017).\n\nThe X-ray diffraction patterns for the PIS and starches obtained from peas subjected to germination for four days are shown in Figure 4. For indexing these patterns, tables provided by Rodríguez-García et al. (2021) were used. It was found that pea starches with and without germination contain a mix of hexagonal and orthorhombic glucopyranose crystals. Starches presenting this kind of mix are classified as C-type. These patterns revels that these crystalline structures do not suffer any damage during germination. Rodríguez-García et al. (2021) indexed the orthorhombic structure for A-type starch and hexagonal structure for B-Type B starch, and the dash lines in this figure correspond to the indexation of both structures. Gao et al. (2022) studied the structural transformation along with the germination process in two pea varieties. They results indicated that during germination the crystalline structures present in starch do not exhibit significant changes. However, they did not index the patterns to confirm that peas starch can be classified as C-Type. Liu et al. (2020) reported that different crystallinity degrees can indicate the differences in the chemical structure and composition of starches. However, from a crystallographic point of view the term chemical structure does make sense.\n\nThe variations in the relative crystalline percent as a function of germination day were shown in Figure 4b. The relative crystalline percent is around 17%, and this result showed that enzymatic attack during germination does not induce structural alterations. The tiny variations in this parameter may be related to the sample’s moisture content. While Rodríguez-García et al. (2021) assert that the crystallinity is directly related to the orthorhombic and hexagonal crystalline structures, Rupollo et al. (2011) noted that the relative crystallinity is typically related to the content and average chain length of amylopectin, the internal orientation and interaction degree of the double helix, and the moisture content of starch granules. Of course, the determinations of these patterns were performed at very low moisture (around 10%). This suggests that the out diffusion of bound water into these nanocrystalline formations altered their crystalline structure (Kim and Huber (2008, 2010) and Gong et al., 2016).\n\nFigure 5a displays the DSC thermograms of of native starch (PIS) and starches with different germination times (PIS-1 to PIS-4). This figure shows an endothermic thermal transition for PIS around 65.3°C, which corresponds to an ordered to disordered transition produced by the solvation of the orthorhombic nanocrystals and corresponds to the gelatinization. The gelatinization is a second order thermal event (irreversible transition).\n\nThe values correspond to average of enthalpy and standard deviation. Different letters in panel c show significant differences using α = 0.05.\n\nWhen employing the second derivative criterion presented in Figure 5b, it is evident that gelatinization corresponds to two thermal events even if a preliminary inspection of the endothermic transition does not indicate the presence of more than one transition. These events are directly related to the solvation of orthorhombic and hexagonal nanocrystals, which are what make up this starch according to Figure 4a, which demonstrated this. The first occurrence involves the solvation of the orthorhombic structure, which contains eight water molecules in its structure, and the second involves the solvation of the hexagonal structure, which has 32 water molecules in its crystalline structure (Esquivel-Fajardo et al., 2022).\n\nFor the nanocrystals solvation, a step known as gelatinization must occur. An ordered-disordered transition has been connected to starch gelatinization (Zobel et al., 1988). There is, however, no mention of the type of structure that is engaged in this transition. The endothermic event can be represented in two steps from a thermodynamic perspective. In the case of pea starch, the orthorhombic and hexagonal nanocrystals absorb energy to raise the temperature, the vibrational states reflect the energy, and the amplitude of the vibrations increases from To to Tp. From a quantum perspective, each vibrational state increases its amplitude, and each vibrational mode only reaches its maximum amplitude at Tp.\n\nThe endotherm peak returns to the initial level because each vibrational state is obliterated sequentially from Tp to Te. The ordered system is represented by the first stage of the endothermic transition, while the disordered system is represented by Tp. It is evident from a close examination of Figure 4a that neither orthorhombic nor hexagonal nanocrystals alter during germination.\n\nFigure 6 shows the changes in the vibrational spectrum of isolated native starch and germinated starches. In this section, attention was focused on the region in which the vibrational modes were changed due to the germination process. The description of the well-known bands is as follows: a germination split signal from day 1 to day 4 located at 1240, 1407 and 1456 cm-1 of the C-H group stretch corresponds to carbohydrates were observed.\n\nAccording to the results presented, the most important changes occurred during day 3 and day 4 of germination. At this time, the enzymatic process initiates the modification in the starch granules. No significant changes are shown after soaking. This can be explained because during soaking the seed hydrates and some starch granules can swell. At the same time, an internal enzymatic process in the pea is activated, although the granules remain intact. Germination modified the chemical bonds of the amylose and amylopectin to produce sugar chains that will be used by the grain as a source of energy and carbon for growth. Changes in the signal corresponding to –OH were found at 1275 cm-1, possibly showing the modifications of the glycosidic linkages in α(1–4) and α(1–6). The modification of the signal in the different stages of malting demonstrates the changes in the structure of the starch (Oseguera-Toledo et al., 2020).\n\nIn turn, the presence of characteristic vibrations for functional groups typical of starch could be evidenced (-CH-, -CH2-, C-OH, and groups -OH) to wavelengths to 3100, 2915, 2850, 1680, 998, and 855 cm-1 (Quintero-Castaño et al., 2020). The bands at 1640 and 1545 cm-1 can be attributed to amide vibrations. These bands are present due to the modification that the proteins undergo at the time of germination and that causes the vibration of the amino acids that are released (Contreras-Jiménez et al., 2018). Other authors have reported similar bands in the germination process of the pea. Xing et al. (2021) showed changes in the malting process of quinoa starch, mainly due to 1047 y 1022 cm-1 associated with the fingerprint region of the compound product of the vibration of bonds C-O-H. Chinma et al. (2022) reported significant changes to 2930 cm-1 for germinated pea fluor. These changes are associated with changes in the C-H vibrations typical of amylose and amylopectin chains.\n\nThe band at 2980 cm−1 (a) with absorber water (Chang et al., 2014). 2970 cm-1 is due to CH2 stretching vibration (b), 2930 cm-1 (c) to υ(C-H (Oniszczuk et al., 2019); the band at 2890 cm-1(d) to υst(C-H) or υ(C-H) in CH2/CH3 group, and the band at 2882 cm-1 (e) corresponds to υst(C-H) or υ(C-H) in CH2/CH3 group. The expression of these IR bands are an indicative of the amylose or amylopectin debranching produce more vibrational centers (quantum states).\n\nThe viscosity profiles of the PIS and germinated pea starches PIS-1, PIS-2, PIS-3, and PIS-4 are shown in Figure 7a, and the changes in maximum viscosity during germination are shown in Figure 7b. It was found that during germination time there were no significant changes in the viscosity profiles of the isolated starch, which agrees with the morphological analysis of the starches (SEM), where there were no changes on the grains surface. The behavior is in contrast to that observed in Puma and Palomero corn starches by Hernández-Becerra et al. (2020), malted barley by Contreras-Jiménez et al. (2018), and sorghum malting by Oseguera-Toledo et al. (2020), where it was discovered that during the germination and malting processes, peak and final viscosities decrease where starch is degraded. Indicating that the long chains of amylose and amylopectin were fractionated, producing an increase in reducing sugars and a decrease in apparent viscosity, due to the activation of α-amylases that degrade the amorphous component of starch, obtaining mono and disaccharides used as an energy source during malting, and therefore affecting viscosity and by the interaction of water with the starch crystals, which is corroborated by SEM images, where the starch grains are almost completely broken. X-ray analysis showed that the crystalline structures of the starch remains unchanged throughout the malting process (Contreras-Jiménez et al., 2014; Oseguera-Toledo et al., 2020). Londoño-Restrepo et al. (2018) showed that along with the pasting profile, the corn starch granules are disrupted, and at the end of the peak viscosity they showed that there are not any crystalline compounds, because of the X-ray pattern does not exhibit any crystalline phase. Recently, Esquivel-Fajardo et al. (2022) showed that at the end of the pasting profile of isolate avocado starch, some fraction of the crystalline components of the starch retains some grade of crystallinity.\n\nDifferent letters in panel b show significant differences using α = 0.05.\n\nThey also go against the findings of Gao et al. (2022), who found that as germination time increased, the parameters of the viscosity profiles of starches isolated from two native and germinated pea varieties revealed substantial statistical variations. When germination time was extended, the peak viscosity first rose and then fell, reaching its maximum values on days 3 for Xiwan variety 1 and day 1 for Xiwan variety 2, respectively. According to Xing et al. (2021), the peak viscosity of mung bean starch decreased when the germination time was extended to 72 h, while the peak viscosity of quinoa starch decreased as the germination time increased (Liu et al., 2020). The temperature at which the paste of germinated starches begins to gelatinize is likely to have increased when compared to native starch. According to some authors, this characteristic relates to the initial alteration of starch granules, which is the change from starch suspension in water to gel formation (Rincón-Londoño et al., 2016). Additionally, the thermal characteristics of starch are proportional to the pasting temperature. The same behavior was reported by Hernández-Becerra et al. (2020) for Puma and Palomero corn starches, which presented an increase in pasting temperature.\n\nAt the same time, native and germinated pea starches discovered in this study displayed higher peak and final viscosities than those obtained in malted Puma and Palomero corn (Hernández-Becerra et al., 2020); in malted barley (Contreras-Jiménez et al., 2018); and in two varieties of pea (Gao et al., 2022). According to the results of starch morphological analysis (SEM) and X-ray, higher viscosities are the result of less enzymatic modification of pea starches. These differences may be related to the amylose-amylopectin relationship, aggregation of starch grains, the fine structure of amylopectin, and the size of starch granules (Gao et al., 2022). At the same time, once native and germinated pea starch paste was cooled, their final viscosities often increased by a factor of two, from 10.000 cP to more than 20.000 cP.\n\nIt is important to highlight the possible presence of fat and protein residues in isolated pea starches at different germination times, which could influence the greater resistance of the starch granules to swelling and rupture, due to the formation of lipid-amylose and/or amylopectin complexes that increase the gelatinization onset temperature (Tg), increasing the integrity of the starch granule, where lipids act as adhesive material and contribute to the formation of granules that fuse to become compound granules.\n\nAdditionally, because of heating, protein residues are denatured and deformed, which encourages protein aggregation and the formation of disulfide bonds between proteins. These effects contribute to protein-protein and protein-starch interactions, which by binding exogenous proteins to the starch grains and preventing water from diffusing through it, affect Tg. Denatured proteins also reveal several hydrophilic groups (like -COOH, -NH2, -OH, and -SH) and hydrophobic amino acids (like Pro, Leu, Phe, Val, and Ile), which may all bind to the surfaces of starch granules by hydrogen bonding or hydrophobic interactions, increasing Tg. A similar effect was seen by Ribotta et al. (2012), who found that pea protein isolate (PPI) dramatically changed how corn and cassava starches pasted during the heating-cooling cycle, resulting in an increase in viscosity. In the samples of maize starch, the pasting temperature was lowered by the addition of PPI. Conversely, the inclusion of PPI enhanced the PV (peak viscosity), FV (final viscosity), BD (breakdown), and SB (setback) of both starch pastes during the heating-cooling process. The pasting temperature (PT) also increased marginally with protein isolates in cassava samples.\n\nTable 2 shows the results of the functional properties and the type of gel formed by pea isolated starch (PIS) and for day 1 to day 4 (PIS-1 to PIS-4) as a function of the germination (see Figure 7b), where it is initially observed that although in general there are statistically significant differences between WAI, WSI, and SP (p<0.05) and germination time, these are not large enough. At the same time, when comparing the functional properties, with respect to cassava starch, the report reference values are: WAI varies between 0.82 and 15.52 g gel/g sample; WSI: 0.27-12.32% and SP: 0.79 and 15.45% (Aristizábal and Sánchez, 2007; Wang et al., 2022). It can be concluded that pea starch has medium quality characteristics by developing medium WAI and SP values, and low values in WSI. Contreras-Jiménez et al. (2018), with isolated quinoa starch and Ozcan and Jackson (2005), with corn starch, reported values of 2.35±0.03 g gel/g starch and 1.77 g gel/g starch in WAI, respectively, low values with respect to pea starch (Table 2).\n\nDifferent letters in the same column show significant differences using α = 0.05.\n\nRegarding WSI, pea starch reported less than 2.63%, much lower than the figures reported by Contreras-Jiménez et al. (2018) for quinoa starch (4.56±0.0%). This finding indicates that pea starch presents an important number of soluble compounds, which can be included as fiber and other carbohydrates different from starch. In relation to the swelling power (SP) of pea starch, higher values were found with respect to quinoa starch 2.98±0.07% (Contreras-Jiménez et al., 2018); this last behavior may be related to viscosity, as the higher the SP values, the higher the viscosity value is expected, which is related to the type of gel (solid) obtained with pea starch.\n\n\nConclusions\n\nIn the kinematic process of germination, the timing of the distinct starch components is altered. The surface of the starch granules is not altered because of the enzymatic action during germination. While the protein content of the pea grain increased, this could be due to the anabolism of some proteins from reserve carbohydrates and/or enzymatic means. Fat was one of the components that decreased during germination from 2.08±0.06% to 0.53±0.05%, indicating that the fat is involved in several metabolic degradation processes that demand energy and facilitate the emergence of the radicle and hypocotyl in the pea grain.\n\nAmylose and amylopectin are fractionated during the early stages of germination by an enzymatic assault, although orthorhombic and hexagonal nanocrystals are unaltered. More in-depth research is required on this issue. For longer periods of time, these nano crystals serve as the primary energy source for the germination stage. Pea cannot be used for fermented beverages, as evidenced by the pasting behavior of the flours at rapid germination times.\n\n\nCRediT authorship contribution statement\n\nVíctor D. Quintero, Juan Carlos Lucas and Luisa F. Castañeda Formal analysis.: Conceptualization, Writing - review & editing. Mario E. Rodríguez, Juan C. Lucas. Investigation, Writing - review & editing. E. Rodríguez-García Mario: Resources, Writing - review & editing. Victor D. Quintero and Juan Carlos Lucas: Methodology, Resources, Writing - review & editing. Juan Carlos Lucas: Project administration.", "appendix": "Data availability\n\nfigshare: Data repository of the article titled “Morphological, structural, chemical, vibrational, thermal, pasting, and functional changes in pea starch during germination process”, https://doi.org/10.6084/m9.figshare.23605389.v1 (Lucas et al., 2023)\n\nThis project contains the following underlying data:\n\n- RVA PEA: RVA results.\n\n- Properties starch PEA: results of functional properties, amylose, proximal.\n\n- X-ray patterns: x-ray results.\n\n- IR stach: FTIR results native and germinated starches day 1-4.\n\n- DCS PEA: DSC results native and germinated starches day 1-4.\n\n- SEM Strach: native 0002-0013; Day 1_0001-0010; Day 2_0001-0004; Day 2_0001-0004; Day 4_0001-0009.\n\nData are available under the terms of the Creative Commons Zero ’No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgements\n\nEnglish edition by M.B.M. Carolina Tavares.\n\n\nReferences\n\nAguilar J, Miano AC, Obregón J, et al.: Malting process as an alternative to obtain high nutritional quality quinoa flour. J. Cereal Sci. 2020; 90: 102858.\n\nAlfeo V, Jaskula-Goiris B, Venora G, et al.: Screening of durum wheat landraces (Triticum turgidum subsp. durum) for the malting suitability. J. Cereal Sci. 2018; 83: 101–109. Publisher Full Text\n\nAOAC: Official Methods of Analysis. Gainthersburg, MD: Association of Official Analytical Chemists; 2005.\n\nAristizábal J, Sánchez T: Guía Técnica para la Producción y Análisis de Almidón de Yuca. Boletín de servicios agrícolas de la FAO. Roma, Italia: 2007; pp. 135. 1020-4334.\n\nBewley JD, Bradford K, Hilhorst H, et al.: Seeds: Physiology of development, germination and dormancy. New York Heidelberg Dordrecht London: Springer Science & Business Media. Springer; 2012. 978-1-4614-4692-7.\n\nBradford KJ, Benech-Arnold RL, Come D, et al.: Quantifying the sensitivity of barley seed germination to oxygen, abscisic acid, and gibberellin using a population-based threshold model. J. Exp. Bot. 2008; 59(2): 335–347. PubMed Abstract \| Publisher Full Text\n\nChang YJ, Choi HW, Kim HS, et al.: Physicochemical properties of granular and non-granular cationic starches prepared under ultra high pressure. Carbohydr. Polym. 2014; 99: 385–393. PubMed Abstract \| Publisher Full Text\n\nChinma CE, Abu JO, Adedeji OE, et al.: Nutritional composition, bioactivity, starch characteristics, thermal and microstructural properties of germinated pigeon pea flour. Food Biosci. 2022; 49: 101900. Publisher Full Text\n\nContreras-Jiménez BL, Figueroa-Cárdenas JD, Ávalos-Zúñiga R, et al.: Effect of steeping time and calcium hydroxide concentration on the water absorption and pasting profile of corn grits. J. Food Eng. 2014; 122: 72–77. Publisher Full Text\n\nContreras-Jiménez B, Del Real A, Millán-Malo BM, et al.: Physicochemical changes in barley starch during malting. J. Inst. Brew. 2018; 125: 10–17. Publisher Full Text\n\nEsquivel-Fajardo EA, Martínez-Ascencio EU, Oseguera-Toledo ME, et al.: Influence of physicochemical changes of the avocado starch throughout its pasting profile: Combined extraction. Carbohydr. Polym. 2022; 281: 119048. PubMed Abstract \| Publisher Full Text\n\nEvans ED, Li C, Harasymow S, et al.: Improved prediction of malt fermentability by measurement of the diastatic power enzymes β- amylase, α-amylase, and limit dextrinase: II. Impact of barley genetics, growing environment, and gibberellin on levels of α-amylase and limit dextrinase in malt. J. Am. Soc. Brew. Chem. 2009; 67(1): 14–22.\n\nGao L, Wu Y, Wan C, et al.: Structural and physicochemical properties of pea starch affected by germination treatment. Food Hydrocoll. 2022; 124: 107303. Publisher Full Text\n\nGong B, Liu W, Tan H, et al.: Understanding shape and morphology of unusual tubular starch nanocrystals. Carbohydr. Polym. 2016; 151: 666–675. PubMed Abstract \| Publisher Full Text\n\nGupta M, Abu-Ghannam N, Gallaghar E: Barley for brewing: Characteristic changes during malting, brewing and applications of its by-products. Compr. Rev. Food Sci. Food Saf. 2010; 9(3): 318–328. PubMed Abstract \| Publisher Full Text\n\nHernández-Becerra E, Contreras-Jiménez B, Vuelvas-Solorzano A, et al.: Physicochemical and morphological changes of corn grains and starch during the malting for Palomero and Puma varieties. Cereal Chem. 2020; 97: 404–415. Publisher Full Text\n\nJuliano BO, Perez CM, Blakeney AB, et al.: International Cooperative testing on the amylose content of milled rice. Starch – Stärke. 1981; 33: 157–162. Publisher Full Text\n\nKim H-S, Huber KC: Channels within soft wheat starch A- and B-type granules. J. Cereal Sci. 2008; 48(1): 159–172. Publisher Full Text\n\nKim H-S, Huber KC: Physicochemical properties and amylopectin fine structures of A- and B-type granules of waxy and normal soft wheat starch. J. Cereal Sci. 2010; 51(3): 256–264. Publisher Full Text\n\nLi C, Ohb SG, Lee DH, et al.: Effect of germination on the structures and physicochemical properties of starches from brown rice, oat, sorghum, and millet. Int. J. Biol. Macromol. 2017; 105: 931–939. PubMed Abstract \| Publisher Full Text\n\nLiu Y, Su C, Saleh ASM, et al.: Effect of germination duration on structural and physicochemical properties of mung bean starch. Int. J. Biol. Macromol. 2020; 154: 706–713. PubMed Abstract \| Publisher Full Text\n\nLondoño-Restrepo SM, Rincón-Londoño N, Contreras-Padilla M, et al.: Morphological, structural, thermal, compositional, vibrational, and pasting characterization of white, yellow, and purple Arracacha Lego-like starches and flours (Arracacia xanthorrhiza). Int. J. Biol. Macromol. 2018; 113: 1188–1197. PubMed Abstract \| Publisher Full Text\n\nLucas-Aguirre JC, Giraldo-Giraldo GA, Cortés-Rodríguez M: Stability during storage of coconut powder fortified with active components. Biotecnología En El Sector Agropecuario Y Agroindustrial. 2019; 17(2): 66–76.\n\nLucas J, Quintero-Castaño VD, Castañeda LF, et al.: Data repository of the article titled “Morphological, structural, chemical, vibrational, thermal, pasting, and functional changes in pea starch during germination process”, Version 2. Dataset. figshare. 2023. Publisher Full Text\n\nOniszczuk T, Combrzyński M, Matwijczuk A, et al.: Physical assessment, spectroscopic and chemometric analysis of starch-based foils with selected functional additives. PLoS One. 2019; 14(2): e0212070. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOseguera-Toledo ME, Contreras-Jiménez B, Hernández-Becerra E, et al.: Physicochemical changes of starch during malting process of sorghum grain. J. Cereal Sci. 2020; 95: 103069. Publisher Full Text\n\nOzcan S, Jackson D: Functionality behavior of raw and extruded corn starch mixtures. Cereal Chem. 2005; 82(2): 223–227. Publisher Full Text\n\nQuintero-Castaño VD, Castellanos-Galeano FJ, Álvarez-Barreto CI, et al.: Starch from two unripe plantains and esterified with octenyl succinic anhydride (OSA): Partial characterization. Food Chem. 2020; 315: 126241. PubMed Abstract \| Publisher Full Text\n\nRincón-Londoño N, Vega-Rojas LJ, Contreras-Padilla M, et al.: Analysis of the pasting profile in corn starch: structural, morphological, and thermal transformations, Part I. Int. J. Biol. Macromol. 2016; 91: 106–114. PubMed Abstract \| Publisher Full Text\n\nRodríguez-García ME, Hernández-Landaverde MA, Delgado JM, et al.: Crystalline structures of the main components of starch. Curr. Opin. Food Sci. 2021; 37: 107–111. Publisher Full Text\n\nRibotta PD, Colombo A, Rosell CM: Enzymatic modifications of pea protein and its application in protein–cassava and corn starch gels. Food Hydrocoll. 2012; 27(1): 185–190. Publisher Full Text\n\nRupollo G, Vanier NL, da Rosa Zavareze E , et al.: Pasting, morphological, thermal and crystallinity properties of starch isolated from beans stored under different atmospheric conditions. Carbohydr. Polym. 2011; 86(3): 1403–1409. Publisher Full Text\n\nUrbano G, Aranda P, Vílchez A, et al.: Effects of germination on the composition and nutritive value of proteins in Pisum sativum, L. Food Chem. 2005; 93: 671–679. Publisher Full Text\n\nWang Z, Mhaske P, Farahnaky A, et al.: Cassava starch: Chemical modification and its impact on functional properties and digestibility, a review. Food Hydrocoll. 2022; 129: 107542. Publisher Full Text\n\nXing B, Teng C, Sun M, et al.: Effect of germination treatment on the structural and physicochemical properties of quinoa starch. Food Hydrocoll. 2021; 115: 106604. Publisher Full Text\n\nXu M, Jin Z, Simsek S, et al.: Effect of germination on the chemical composition, thermal, pasting, and moisture sorption properties of flours from chickpea, lentil, and yellow pea. Food Chem. 2019; 295: 579–587. PubMed Abstract \| Publisher Full Text\n\nZobel HF, Young SN, Rocca LA: Starch gelatinization: An X-ray diffraction study. Cereal Chem. 1988; 65(6): 443–446." }	[ { "id": "213713", "date": "01 Nov 2023", "name": "Mária Herminia Ferrari Felisberto", "expertise": [ "Reviewer Expertise Cereal", "bakery products", "starch", "fiber" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Morphological, structural, chemical, vibrational, thermal, pasting, and functional changes in pea starch during germination process\" brings innovative and preliminary research into the behavior of starch, extracted from a non-conventional source, over different germination times. However, the work presents some points that need to be improved, or better described/explained, as highlighted below:\nAt the conclusion of the work summary, the authors mention lentil starch: I believe this was a typo, as it doesn't make sense in the context.\n\nIn the introduction to the work, when justifying the use of peas, the authors start by addressing soy and then enter the gluten-free category, which doesn't make much sense, since soy does not contain gluten. I suggest that the authors explore this argument further, following the line of unconventional or under-explored sources.\n\nStill in the introduction, the authors use the term \"humidity\", which I believe should be replaced by \"moisture content\".\n\nIn the material and methods, I suggest that the authors maintain the analyzes of germinated grains, following the germination process, maintaining a logical sequence of the steps carried out (go up to the Percentage of grain germination methodology).\n\nDiscussion of the results, in the topic \"morphological analysis of isolated stars\"... I believe there was an error in the elaboration of the last sentence, as the way it was used it doesn't make much sense.\n\nGrammatical error in line 8 of the topic \"structural characterization of isolated stars\". And the last sentence of this item was confusing with all the previous discussion... The evidence indicates or does not indicate type-C. What was the authors' conclusion? I suggest adding some reference that corroborates the conclusion obtained and presented in this sentence.\n\nThere is a grammatical error in the last sentence of the following paragraph.\n\nThere is a grammatical error (typing) in the first sentence of the topic \"thermal properties\".\n\nFigures 5 and 6: I suggest that the authors use color legends in the graphs, as the indication of the samples in them is confusing the reader.\n\nTopic \"vibrational analysis\" mentions some lengths that are not shown in the Figure. I suggest reevaluating the figure, or explaining the reason.\n\n\"amide vibrations\" Could this really be the expression? (second paragraph on page 12).\n\nPage 13, 4th paragraph: \"it is important....\" Would this really be a plausible explanation, given the very low protein and fat content that could be present in starch? I suggest adding references to this statement.\n\nConclusion topic mentions something about \"in-depth research....\" what do the authors mean? Longer germination time? Other analyses? Additional analyses?\n\nIs the work 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": "228140", "date": "05 Mar 2024", "name": "Lesław Juszczak", "expertise": [ "Reviewer Expertise food analysis", "carhohydrtes", "starch", "honey", "food rheology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 on manuscript: Morphological, structural, chemical, vibrational, thermal, pasting, and functional changes in pea starch during germination process by YJuan Carlos Lucas-Aguirre , Víctor Dumar Quintero-Castaño, Luisa Fernanda Castañeda-Cano, Mario Enrique Rodríguez-García submitted to F1000Research journal.\nIn the manuscript submitted for comments the authors studied the impact of germination time on morphological, structural, thermal and functional properties of starch. Generally, the manuscript is interesting but before acceptance should be corrected and supplemented in some points.\nDetailed recommendation: Title, page 3 – what do the authors understand by the vibrational properties of starch? page 3, lines 11-12 – literature sources should be added, page 3, line 18 – what does \"storage proteins\" mean? page 3, line 38 – properties of what? page 4, lines 1 and 15 – origin country should be added, page 4, line 20 – model, producer and origin country of spectrophotometer should be added, page 5, line 22 and page 11 – better will be: FTIR analysis, how was the sample prepared? page 5, line 32 – what parameters were determined? page 5, line 35 – Anton Paar is an Austrian company, page 5, line 38 – what parameters were determined? page 5, line 38 – rpm is not a unit of frequency, page 5, line 42 – instead of rpm, the centrifugal force should be provided, Table 2 – an explanation of the meaning of superscript letters should be provided in the legend, page 9, line 26 and figure 4b – in the methodology, the authors do not mention the calculation of relative crystallinity, Figure 7 – viscosity should be expressed in SI unit, page 13 – instead of cP, the SI unit should be used, page 14 – is this a legend for table 2 on page 9?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-940
https://f1000research.com/articles/12-939/v1	07 Aug 23	{ "type": "Study Protocol", "title": "Effect of Powerbreath Medic Plus versus Threshold IMT on maximal inspiratory pressure and functional capacity in post-operative ventricular septal defect closure: A protocol for a randomized controlled trial", "authors": [ "Chitrakshi A Choubisa", "Vishnu Vardhan", "Vishnu Vardhan" ], "abstract": "Background: Congenital heart disease is a general phrase that refers to any heart conditions that exist at birth, the most prevalent of which being ventricular septal defects (VSD) and foetal congenital malformations. VSD refers to the presence of aperture between the ventricles which leads to mixing of oxygenated and de-oxygenated blood. Although many VSDs close on their own, failing to do so can result in a number of issues and typically necessitates open heart surgery for closure of the defect. According to various studies, there is reduced functioning of diaphragm post cardiac surgeries which leads to occurrences of pulmonary complications such as lung collapse, fluid accumulation in pleura, pneumonia, acute respiratory distress syndrome and pneumothorax which can be debilitating and jeopardize recovery as well as increase hospital stay. There are several devices which are proven to improve the strength of inspiratory musculature and hence improve maximal inspiratory pressure and functional capacity post cardiac surgeries. Method: In this study we aimed to assess and evaluate the effectiveness of Powerbreath Medic Plus and Threshold Inspiratory Muscle Trainer (IMT) adjunct to conservative physiotherapy management on subjects who underwent VSD closure in a two arm parallel superiority randomized control trial on improving maximal inspiratory pressure and functional capacity. 60 individuals in total will be allocated equally into two groups, and both groups will be receiving treatment for four weeks with five sessions per week. After four weeks of therapy and two weeks after the intervention is through, the results will be assessed for follow-up. Predicted results: By increasing maximal inspiratory pressure and functional capacity, this technique may be used to treat complications following heart surgery if our study's premise is found to be significant. CTRI registration: CTRI/2023/03/051090", "keywords": [ "Ventricular septal defect closure", "congenital heart disease", "Powerbreath medic plus", "threshold IMT", "maximal inspiratory pressure", "functional capacity." ], "content": "Introduction\n\nCongenital heart diseases (CHDs), one of the most frequent foetal defects, can occur in as many as 13 out of every 1000 live births.1 CHDs are among the most prevalent foetal malformations. The most frequent acynotic congenital cardiac abnormalities in children are ventricular septal defects (VSD), which are the second most common congenital anomaly in adults.1,2 Shunt formation and inappropriate right-to-left ventricular communication are the primary causes of hemodynamic compromise in VSD due to presence of aperture between the ventricles.3,4 Many VSDs close on their own, however if they fail to, large defects can cause a number of problems, including pulmonary arterial hypertension (PAH), ventricular dysfunction, and a greater incidence of arrhythmias. If there are significant problems, the most common procedure in paediatric cardiac surgery is surgical closure of the ventricular septal defect.3,5–7\n\nTransient diaphragm dysfunction is reportedly frequent in the initial week following cardiac surgery, based on numerous studies.8,9 In patients receiving elective heart surgery, the prevalence of residual diaphragm dysfunction was 8%, and it was linked to poor respiratory outcomes.9,10 Several pulmonary complications, such as collapse of the lung, collection of fluid in pleura, pneumonia, cardiogenic pulmonary oedema, acute respiratory distress syndrome, pulmonary embolism, phrenic nerve injury, pneumothorax, sternal wound infection and inflamation as well as longer hospital stays and prolonged ventilator support, are caused by diaphragmatic dysfunction.8–12 Furthermore, cardiac procedures impair breathing mechanics, decrease lung volume, obstruct gas exchange, affect the ventilation-to-perfusion ratio, lung capacities, and promote physical inactivity, all of which lower functional capacity.13,14\n\nEarly ambulation and mobility, breathing exercises, coughing techniques, incentive spirometers, and strengthening of respiratory musculature are all cardiopulmonary physical therapy interventions that have been documented to be beneficial for lowering the occurrence of pulmonary problems.15 Patients having heart operations have shown possible positive effects from inspiratory muscle training (IMT).15 Numerous IMT devices are utilised to prevent diaphragmatic dysfunction and respiratory issues.15 Research on the application of Powerbreathe training and enhancing maximal inspiratory pressure in individuals who underwent VSD closure is scant. Data on physical therapy interventions and results in individuals who had VSD closure surgery are rare.\n\nIMT improves the capacity for functional activity as well as respiratory muscle strength. The two main categories of IMT equipment are devices with pressure- and volume-based loading.16,17 A pressure-based threshold device called Power Breathe (PwB) limits airflow during inspiration until it achieves a particular pressure. This inspiratory pressure is modifiable by adjusting the spring tension to match the patient’s maximal inspiratory pressure (MIP). The lineal resistance to the flow increment must be as low as possible when the pressure is surpassed and the valve is opened. Due to coupled action of respiratory musculature and the PwB device, higher lung charges of between 186 and 274 centimetres of water (cmH2O) pressure can be produced.18,19 The most popular inspiratory muscle trainer for improvising the power and endurance of the respiratory musculature is the Threshold IMT, a pressure-based loading apparatus. It can provide resistance up to 41 cm H2O and hence retraining the inspiratory muscles at different resistance levels.17\n\nThis article presents the protocol for the full study, whose objective is to assess and evaluate the effectiveness of the PowerBrethe medic plus device and the threshold IMT device in patients who underwent VSD closure in a two-arm parallel superiority/equivalence randomised control trial (RCT). The end point results will be compared on a marginal basis to determine effectiveness.\n\n\n\n1. To assess and evaluate the subjects who underwent VSD closure, intervened with Power Breathe Medic Plus adjunct to conservative physiotherapy management and its effect on improving maximal inspiratory pressure (Micro Respiratory Pressure Meter) and functional capacity (6 Minute Walk Distance Test) in the entire populace.\n\n2. To assess and evaluate the subjects who underwent VSD closure for the change in pulmonary function parameters (FEV1, FVC AND FEV1/FVC ratio) treated with Power Breathe Medic Plus adjunct to conservative physiotherapy management, to observe if it can improve pulmonary functioning in the entire populace.\n\n3. To analyse the efficacy over the treatment of Power Breathe Medic Plus along with conventional physiotherapy management and Threshold IMT along with conventional physiotherapy management for bringing on change in maximal inspiratory pressure and functional capacity for the patients who underwent VSD closure.\n\n\nProtocol\n\nThis protocol has been registered with CTRI (CTRI/2023/03/051090) on 27/03/2023.\n\nThis study will be conducted with written informed consent from all participants. Ethical approval was received from the institutional ethics committee (ref: DMIHER (DU)/IEC/2023/545) on 04/02/23.\n\nSingle centric, two arm parallel equivalence randomized controlled trial. Participants in the study will be split into two groups. Group-A (Powerbreath and conservative physiotherapy management) and Group-B (Threshold IMT and conservative physiotherapy management) by randomization for 1:1 allocation with intent to treat purpose.\n\nParticipants will be chosen from the Cardiovascular and Thoracic Surgery Unit at Acharya Vinobha Bhave Hospital Sawangi in Meghe, Wardha, Maharashtra, following approval from the institutional ethics committee of Datta Meghe Institute of Higher Education and Research. Potential participants will be identified through their medical records and evaluation. The participants will be screened as per inclusion and exclusion criteria followed by randomization using a computer-generated list. Allocation will be done by sequentially numbered opaque sealed envelopes. Allocation and participant enrolment will be done by the primary investigator. The therapist will be aware of the allocations. The inclusion and exclusion criteria for selection will be based on the cut-off values at baseline parameters when engaging participants. Throughout the six-month recruitment phase, a second source of recruitment will be used if more study participants are needed; this will be an additional local private hospital.\n\nTo compare improvements in maximal inspiratory pressure and functional capacity at the end point, the interventional group will receive Powerbreath and conservative physiotherapy management (Figure 1), and the control group will receive Threshold IMT and conservative physiotherapy management (Figure 1). Participants will be enrolled and evaluated at several intervals, including first visit and second visit for subject enrolment and screening respectively, baseline, 4 weeks, and 2 weeks after treatment for follow-up, when primary and secondary parameters will be measured. The study design is depicted in Figure 1.\n\n\n\n1. Patients clinically diagnosed with isolated VSD.\n\n2. Patients who have been operated on for VSD closure.\n\n3. Age group of 10-18 years.\n\n4. Patient’s guardian who will sign the consent form\n\n5. Both male and female.\n\n6. Patients with the ability to understand and follow instructions\n\n\n\n1. Patients with VSD associated with other congenital conditions such as Down’s syndrome, William’s syndrome, Noonan syndrome, and Turner syndrome.\n\n2. Patients on a post-operative mechanical ventilator.\n\nAfter screening and randomization, the experimental group will receive inspiratory muscle training with PowerBreathe along with conservative physiotherapy management (Table 1) which will start from post-operative day 3 (POD-3). The training will be delivered in-person at the hospital. Initially the postoperative training will begin with 40% of the preoperative MIP recorded, and weekly load increases of 5–10% will be made based on the patient’s tolerability. The patient will obtain postoperative training for 20 to 30 minutes per session, which will consist of six sets of five deep breathes against the trainer device, with only a brief rest period of 1 to 2 minutes in between each set. Training will be done twice a day, every day, for four weeks.20 The treatment will be discontinued or modified if patient is not willing to continue or faces any discomfort. To improve adherence to the treatment, counselling sessions will be arranged for patient and his/her family on how this device can be useful to improve lung function post-operatively along with various other medical interventions.\n\nAfter screening and randomization, the control group will receive inspiratory muscle training with Threshold IMT along with conservative physiotherapy management (Table 1) which will start from POD-3. The training will be delivered in-person at the hospital. Initially, the postoperative training will be 40% of the MIP that was observed pre-operatively, followed by an increase in load by 5–10% per week based on the tolerance of the patient. The patient will get postoperative training for 20 to 30 minutes per session, which will consist of six sets of five deep breaths against the trainer device, with only a brief rest period of 1 to 2 minutes in between each set. Training will be done twice a day, every day for four weeks.13 The treatment will be discontinued or modified if patient is not willing to continue or faces any discomfort. To improve adherence to the treatment, counselling sessions will be arranged for patient and his/her family on how this device can be useful to improve lung function post-operatively along with various other medical interventions.\n\nPrimary outcomes\n\n• Change in maximal inspiratory pressure and MicroRPM reading (respiratory pressure meter).\n\nMicroRPM demonstrated excellent inter-rater reliability for inspiratory pressure and good inter-rater reliability for expiratory pressure, as well as strong contemporaneous validity and test-retest reliability. In reference to concurrent validity for inspiratory and expiratory pressures, the intraclass correlation coefficientS (ICC) were 0.77 and 0.86, respectively. Inter-rater reliability revealed ICCs as 0.91 for inspiratory pressure and 0.84 for expiratory pressure, with test-retest reliability demonstrating an ICC of 0.87 for inspiratory pressure and 0.78 for expiratory pressure.21\n\n• Change in 6 MWD (Minute Walk distance) Test.\n\nThe 6MWD is a submaximal exercise test that is quick and easy to administer and is well-tolerated by the patient. With an ICC of 0.90, it is a standardized field test to assess functional capacity after exercise performance in people with a range of cardiac and pulmonary problems.22,23\n\nSecondary outcomes\n\n• Change in Pulmonary Function Test (PFT).\n\nThe simplest and most practical pulmonary function test (PFT) is spirometry, which measures air exhaled or inhaled during forceful movements. It is a quantifiable, repeatable, non-invasive, and comparatively easy technique for determining lung function. FEV1, FVC and FEV1/FVC ratio will be collected.24\n\nSafety outcomes\n\nAdverse events will be reported at each time. No adverse effects are anticipated.\n\nSample size calculation resulted at 5% level of significance considering both the sides at 5% error probability with total 10 % for Z(1-α) value =1.64 & (1- β) at power of 80 % = 0.84 measuring the mean difference (effect size) of δ= 10.3 & standard deviation (σ) = 15.75\n\nSample size calculation formula using mean difference:\n\nPrimary variable (maximum inspiratory pressure)\n\nMean ± SD (pre) result on maximum inspiratory pressure for conventional chest therapy (control group) = 103 ±15\n\nMean ± SD (post) result on maximum inspiratory pressure for conventional chest therapy (control group) = 113.3 ±16.5\n\nDifference in mean = 10.3\n\nAs per reference article.25\n\nConsidering 10% drop out = 2\n\nTotal samples required (n1 =n2 = 30 per Group)\n\nTotal sample size required (N) = 2*30 = 60\n\nAssumptions\n\nZα=1.64\n\nα=Type I errorat5%\n\nZβ=0.841−β=Powerat80%\n\nσ=Standard Deviation\n\nReference article: Inspiratory muscle training and functional capacity following coronary artery bypass grafting in high-risk patients: A pilot randomized and controlled trial.25\n\nData collection, management and analysis.\n\n\nAnalysis\n\nAll the results will be calculated using R studio software 4.3. Demographic variables as per the quantitative assessment will be nanalysed for the mean±SD and the frequency and percentage. Inferential statistic data with the variables will be tested for the normality using the Kolmogorov-Smirnov Test. A parametric test will be used if data follows normal distribution. The paired-t test will be used for pre- and post-analysis. Alternatively, the Wilcoxon Test will be used as a non-parametric test if the data are not normally distributed. Similarly, for the unpaired-t test, the Mann Whitney test will be used as an alternative. Association analysis for finding significance of cofounding parameters will be evaluated by using the Chi-squared test or Fisher’s exact test or by using multi-variant analysis. Sensitivity and specificity of the device will be tested over primary outcome (MIP). AUC (area under curve) will be calculated on the basis of observational values for finding accuracy of the device.\n\nInferential statistics will be utilized to compare the two groups for their mean change in primary variable (MicroRPM and 6MWD Test) between baseline, 4 weeks and follow up after 2 weeks using the linear mixed model. The two groups will be active treatment versus control treatment. The participants’ responses will be evaluated in light of the major variable’s variation from baseline to the timeline measured during the study (visit 1 and after the conclusion of 4 weeks, and follow-up after 2 weeks after the conclusion of the intervention). For research participants, random effects will be generalised, and treatment group and visit count will be taken into consideration while analysing fixed effects. With a corresponding 95% confidence interval (CI), the effect size over the mean change difference on the major variable from baseline to end line visit at 4 weeks and 2 weeks follow up will be measured.\n\nIn order to forecast the difference in impact size between the active and control groups, secondary outcomes (PFT) will be examined according to the aforementioned linear mixed model effect. If the data has a normal distribution, the t-test (unpaired) will be used to determine whether there is a significant difference between the means in comparison between the two groups. For non-normal distribution, the translation of the data to the normal distribution will be done using MATHEMATICAL algorithms. The alternative non-parametric tests (Chi square, Mann Whitney, and Wilcoxon tests) will be used if the data across the major variable still exhibit non-normal distribution.\n\n\nDiscussion\n\nCHDs, specifically VSD, are commonly encountered condition in children as well as adults which, if they fail to close on their own, require open heart surgery for the closure of the defect.1 Cardiac surgeries are used worldwide for treatment of patients with such heart conditions, and rates of post-operative complications are still significant, mainly involving pulmonary complications.3,5 This study’s objective is to assess and evaluate the effectiveness of the PowerBrethe medic plus device and the Threshold IMT device in patients who underwent VSD closure. The end point results will be compared on a marginal basis to determine effectiveness.\n\nIn their non-blinded randomised controlled experiment, Rhoia Neidenbach et al. (2023) reported that the effects of IMT on lung capacity and exercise capacity were assessed in a sizable cohort of 40 Fontan patients. They discovered a noticeably better oxygen saturation, which is a clinically meaningful improvement.26 In a randomised controlled trial conducted by Fatma A. Hegazy et al. in 2021, 100 patients who had undergone mitral valve replacement surgery participated. The researchers looked at the impact of postoperative high load, long duration inspiratory muscle training on pulmonary function and functional capacity using the Threshold IMT device. They discovered significant improvement in all measures (p 0.001) in the between-group study, and significant improvements in lung function, inspiratory pressure, and functional capacity (p 0.05) were found in the experimental group.13 In order to assess the effect of an inspiratory muscle strengthening programme using the PowerBreathe device on the ergogenic potential for respiratory and/or athletic performance, Diego Fernández-Lázaro et al. (2021) conducted a systemic review with meta-analysis. They came to the conclusion that the programme increased VO2 max, inspiratory muscle strength, pulmonary function, and sport performance.19 According to previous studies, training of inspiratory musculature is probably beneficial in increasing respiratory muscle strength, decreasing airway closure, and possibly even in enhancing breathing mechanics and lowering exercise-related dyspnea.27 These mechanistic alterations reflect the developing understanding of the function of inspiratory training in paediatric patients and can be used to explain improvements in symptomology and clinical outcomes.27\n\nWe are planning to present the study protocol and subsequent findings in conference proceedings.\n\nThe study has not yet started. Recruitment is expected to begin in July 2023.", "appendix": "Data availability\n\nNo data are associated with this protocol.\n\nSPIRIT checklist for ‘Effect of Powerbreath Medic Plus versus Threshold IMT on maximal inspiratory pressure and functional capacity in post-operative ventricular septal defect closure: A randomized controlled trial’, https://doi.org/10.5281/zenodo.7988644. 28\n\n\nAcknowledgements\n\nI would like to acknowledge Mr. Laxmikant Umate and Mr. Manoj Patil who have helped me in sample size calculation and data analysis planning.\n\n\nReferences\n\nAzhar AS, AlShammasi ZH, Higgi RE: The impact of congenital heart diseases on the quality of life of patients and their families in Saudi Arabia. Saudi Med. J. 2016 Apr; 37(4): 392–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXie D, Fang J, Liu Z, et al.: Epidemiology and major subtypes of congenital heart defects in Hunan Province, China. Medicine (Baltimore). 2018 Aug 3; 97(31): e11770. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDakkak W, Oliver TI: Ventricular Septal Defect. StatPearls. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2023 Jan 15]. Reference Source\n\nDixit R, Rai SK, Yadav AK, et al.: Epidemiology of Congenital Heart Disease in India. Congenit. Heart Dis. 2015; 10(5): 437–446. PubMed Abstract \| Publisher Full Text\n\nSchipper M, Slieker MG, Schoof PH, et al.: Surgical Repair of Ventricular Septal Defect; Contemporary Results and Risk Factors for a Complicated Course. Pediatr. Cardiol. 2017 Feb; 38(2): 264–270. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRao PS, Harris AD: Recent advances in managing septal defects: ventricular septal defects and atrioventricular septal defects. F1000Res. 2018; 7: F1000 Faculty Rev-498. Publisher Full Text\n\nRao PS: Consensus on timing of intervention for common congenital heart diseases: part I - acyanotic heart defects. Indian J. Pediatr. 2013 Jan; 80(1): 32–38. PubMed Abstract \| Publisher Full Text\n\nLaghlam D, Naudin C, Srour A, et al.: Persistent diaphragm dysfunction after cardiac surgery is associated with adverse respiratory outcomes: a prospective observational ultrasound study. Can. J. Anaesth. J. Can. Anesth. 2022 Dec 13; 70: 228–236. Publisher Full Text\n\nKodric M, Trevisan R, Torregiani C, et al.: Inspiratory muscle training for diaphragm dysfunction after cardiac surgery. J. Thorac. Cardiovasc. Surg. 2013 Mar; 145(3): 819–823. PubMed Abstract \| Publisher Full Text\n\nCordeiro ALL, de Melo TA , Neves D, et al.: Inspiratory Muscle Training and Functional Capacity in Patients Undergoing Cardiac Surgery. Braz. J. Cardiovasc. Surg. 2016; 31(2): 140–144. PubMed Abstract \| Publisher Full Text\n\nLaizo A, Delgado FE d F, Rocha GM: Complications that increase the time of Hospitalization at ICU of patients submitted to cardiac surgery. Rev. Bras. Cir. Cardiovasc. 2010; 25(2): 166–171. PubMed Abstract \| Publisher Full Text\n\nTanner TG, Colvin MO: Pulmonary Complications of Cardiac Surgery. Lung. 2020; 198(6): 889–896. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHegazy FA, Mohamed Kamel SM, Abdelhamid AS, et al.: Effect of postoperative high load long duration inspiratory muscle training on pulmonary function and functional capacity after mitral valve replacement surgery: A randomized controlled trial with follow-up. PLoS One. 2021 Aug 27; 16(8): e0256609. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShakouri SK, Salekzamani Y, Taghizadieh A, et al.: Effect of respiratory rehabilitation before open cardiac surgery on respiratory function: a randomized clinical trial. J. Cardiovasc. Thorac. Res. 2015; 7(1): 13–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGomes Neto M, Martinez BP, Reis HF, et al.: Pre- and postoperative inspiratory muscle training in patients undergoing cardiac surgery: systematic review and meta-analysis. Clin. Rehabil. 2017 Apr; 31(4): 454–464. PubMed Abstract \| Publisher Full Text\n\nShei RJ, Paris HL, Sogard AS, et al.: Time to Move Beyond a “One-Size Fits All” Approach to Inspiratory Muscle Training. Front. Physiol. 2022 Jan 10; 12: 766346. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaeotawee P, Udomittipong K, Nimmannit A, et al.: Effect of Threshold Inspiratory Muscle Training on Functional Fitness and Respiratory Muscle Strength Compared to Incentive Spirometry in Children and Adolescents With Obesity: A Randomized Controlled Trial. Front. Pediatr. 2022 Jul 7; 10: 942076. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMenzes: A review on respiratory muscle training devices - Google Scholar.[cited 2023 Jan 17]. Reference Source\n\nFernández-Lázaro D, Gallego-Gallego D, Corchete LA, et al.: Inspiratory Muscle Training Program Using the PowerBreath®: Does It Have Ergogenic Potential for Respiratory and/or Athletic Performance? A Systematic Review with Meta-Analysis. Int. J. Environ. Res. Public Health. 2021 Jul [cited 2023 Jan 16]; 18(13). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCargnin C, Karsten M, da Guaragna JCV , et al.: Inspiratory Muscle Training After Heart Valve Replacement Surgery Improves Inspiratory Muscle Strength, Lung Function, and Functional Capacity: A RANDOMIZED CONTROLLED TRIAL. J. Cardiopulm. Rehabil. Prev. 2019 Sep; 39(5): E1–7. PubMed Abstract \| Publisher Full Text\n\nSilveira BMF, Pereira MCB, Cardoso DR, et al.: New method for evaluating maximal respiratory pressures: Concurrent validity, test-retest, and inter-rater reliability. Braz. J. Phys. Ther. 2021 Dec; 25(6): 741–748. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUszko-Lencer NHMK, Mesquita R, Janssen E, et al.: Reliability, construct validity and determinants of 6-minute walk test performance in patients with chronic heart failure. Int. J. Cardiol. 2017 Aug 1; 240: 285–290. PubMed Abstract \| Publisher Full Text\n\nGiannitsi S, Bougiakli M, Bechlioulis A, et al.: 6-minute walking test: a useful tool in the management of heart failure patients. Ther. Adv. Cardiovasc. Dis. 2019 Aug 23; 13: 175394471987008. Publisher Full Text\n\nHuang YT, Lin YJ, Hung CH, et al.: The fully engaged inspiratory muscle training reduces postoperative pulmonary complications rate and increased respiratory muscle function in patients with upper abdominal surgery: a randomized controlled trial. Ann. Med. 54(1): 2222–2232. PubMed Abstract \| Publisher Full Text\n\nCordeiro ALL, Carvalho BSCD, Silva EGD, et al.: Inspiratory muscle training and functional capacity following coronary artery bypass grafting in high-risk patients: A pilot randomized and controlled trial. J. Clin. Transl. Res. 2022 Jun 17; 8(4): 266–271. PubMed Abstract\n\nNeidenbach R, Freilinger S, Stöcker F, et al.: Clinical aspects and targeted inspiratory muscle training in children and adolescents with Fontan circulation: a randomized controlled trial. Cardiovasc. Diagn. Ther. 2023 Feb 28; 13(1): 11–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhammar DM, Jones HN, Lang JE: Inspiratory Muscle Rehabilitation Training in Pediatrics: What Is the Evidence? Can. Respir. J. 2022 Aug 18; 2022: 1–18. Publisher Full Text\n\nChoubisa C: SPIRIT-checklist (Version v1). Zenodo. 2023. Publisher Full Text" }	[ { "id": "216838", "date": "08 Nov 2023", "name": "Shane Patman", "expertise": [ "Reviewer Expertise Critical care", "acute care", "rehabilitation", "physical therapy", "respiratory care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMany thanks for the opportunity to contribute to the peer review process for the study protocol within F1000Research entitled 'Effect of Powerbreath Medic Plus versus Threshold IMT on maximal inspiratory pressure and functional capacity in postoperative ventricular septal defect closure: A protocol for a randomized controlled trial'\nGeneral Comments\nSuggest rephrasing throughout submission to avoid starting sentences with abbreviations or numbers (e.g. such as: Abstract, Background, line 4; Abstract, Method, line 6; Introduction, paragraph 1, line 2; Introduction, paragraph 4, line 1)\nSuggest amending to depersonalize throughout submission (e.g. such as: Abstract, Method, line 1 ; Abstract, Predicted results, line 3)\nThe role of prehabilitation, some programs inclusive of the use of IMT, is a growing topic within contemporary literature within the adult major surgery cohorts (such as those listed below). It may be prudent for authors to acknowledge this topic and for them to comment as to why they are choosing to focus solely on the post-operative use of IMT in isolation for their project.\nYau, D. K. W., Underwood, M. J., Joynt, G. M., & Lee, A. (2021). Effect of preparative rehabilitation on recovery after cardiac surgery: A systematic review. Annals of physical and rehabilitation medicine, 64(2), 101391. https://doi.org/10.1016/j.rehab.2020.03.014\n\nSteinmetz, C., Bjarnason-Wehrens, B., Walther, T., Schaffland, T. F., & Walther, C. (2023). Efficacy of Prehabilitation Before Cardiac Surgery: A Systematic Review and Meta-analysis. American journal of physical medicine & rehabilitation, 102(4), 323–330. https://doi.org/10.1097/PHM.0000000000002097\n\nOlsen, D. B., Pedersen, P. U., & Noergaard, M. W. (2023). Prehabilitation before elective coronary artery bypass grafting surgery: a scoping review. JBI evidence synthesis, 21(6), 1190–1242. https://doi.org/10.11124/JBIES-22-00265\n\nAssouline, B., Cools, E., Schorer, R., Kayser, B., Elia, N., & Licker, M. (2021). Preoperative Exercise Training to Prevent Postoperative Pulmonary Complications in Adults Undergoing Major Surgery. A Systematic Review and Meta-analysis with Trial Sequential Analysis. Annals of the American Thoracic Society, 18(4), 678–688. https://doi.org/10.1513/AnnalsATS.202002-183OC\n\nGibbison, B., Murphy, G. J., Akowuah, E., Loubani, M., & Pufulete, M. (2023). Pre-operative and prehabilitation services in UK cardiac surgery centres. Anaesthesia, 78(3), 388–391. https://doi.org/10.1111/anae.15918\n\nKatsura, M., Kuriyama, A., Takeshima, T., Fukuhara, S., & Furukawa, T. A. (2015). Preoperative inspiratory muscle training for postoperative pulmonary complications in adults undergoing cardiac and major abdominal surgery. The Cochrane database of systematic reviews, 2015(10), CD010356. https://doi.org/10.1002/14651858.CD010356.pub2\n\nKendall, F., Oliveira, J., Peleteiro, B., Pinho, P., & Bastos, P. T. (2018). Inspiratory muscle training is effective to reduce postoperative pulmonary complications and length of hospital stay: a systematic review and meta-analysis. Disability and rehabilitation, 40(8), 864–882. https://doi.org/10.1080/09638288.2016.1277396\n\nNoronha, J., Samuel, S., Singh, V. P., & Prabhu, H. S. (2022). Effects of exercise-based prehabilitation in children undergoing elective surgeries: a systematic review. F1000Research, 10, 1262. https://doi.org/10.12688/f1000research.74493.2\n\nNo reporting guidelines / checklists for this protocol project reporting are explicitly stated as being followed (such as TIDieR, SPIRIT) - to enhance the quality and transparency of health research reporting, authors are strongly encouraged to explicitly state upfront within manuscript that they are following such guidelines within their protocol reporting, and that they in turn plan as part of the protocol to utilize an appropriate reporting guideline when preparing to share their results. It is suggest that a supplementary explicit statement is included, potentially within the last paragraph of the Introduction, that the subsequent protocol reporting is consistent with the SPIRIT 2013 checklist: ‘This article presents, following the SPIRIT 2013 framework criteria (https://www.spirit-statement.org/), the protocol for…’ (or similar to convey intent to reader; alternatively such a statement may be what leads off the Methodology / Protocol outlining section).\nIt is noted on page 8 that there is a Reporting guideline heading, and that a link is provided to another document, which is a completed but unlabelled SPIRIT checklist; however there are no identifying information to link that checklist to this protocol, and in fact the supplied page numbers (1-13) within that completed checklist do not align to this protocol reporting of 9 pages. Furthermore, within this completed checklist there are a number of criteria that are marked as ‘N/A’, without explanation – it is not clear for this reviewer why it would be considered that criteria 11b-d, 17a-b, 23, 27, 29, & 30 of the SPIRIT 2013 checklist are judged to be not applicable for addressing within this protocol reporting – this warrants commentary by authors.\nThe following specific comments are offered for author consideration:\nPage 3 of 10\nIntroduction, paragraph 1, line 8 – suggest amending to utilize previously defined abbreviation ‘…of the VSD.’\nIntroduction, paragraph 2, citations 8-14 discussed here all pertain to traditional cardiac surgeries associated with coronary artery disease or valvular dysfunction – the demographics and comorbidities of participants of such studies are vastly different to those being targeted in this current project. Whilst VSD repair is technically under the umbrella of cardiac surgery, it is somewhat misleading to reader to attribute such literature findings (of citations 8-14) to those having corrective surgery for congenital heart defects in their adolescence. Authors need to tailor this background context / synthesis to be of the relevant knowledge from the paediatric and/or adolescent cardiac surgery literature (even if it is limited). At the very least authors should acknowledge that there is limited external validity of existing adult literature to be translatable to the study cohort, and why.\nIntroduction, paragraph 3 – similarly this paragraph & its citation is only relevant to adults – citation 15 included only subjects >18 years of age & from the 8 included studies 7 were coronary artery bypass cohorts only, with the 8th a mixed cohort of mitral valve and coronary artery surgery case, and the gross average age across all these studies was 63 years and with a predominance of males. So the interventions and interventions presented are not necessarily aligned to the cohort of this project. It is acknowledged though by authors that there is limited literature but they don’t then extend to share any insights to what is the current incidence of post-operative complications in the adolescent VSD surgery cohort, what the current post-op physical therapy interventions consist of, what the use / role of IMT may be in the adolescent cohort more broadly?\nHowever more recent citations on same topic may have been more appropriate/relevant to have included? (albeit with same limitations – why did authors pin their sentiments against only #15)??\nCordeiro, A. L. L., Soares, L. O., Gomes-Neto, M., & Petto, J. (2023). Inspiratory muscle training in patients in the postoperative phase of cardiac surgery: a systematic review and meta-analysis. Annals of rehabilitation medicine, 47(3), 162–172. https://doi.org/10.5535/arm.23022\n\nXiang, Y., Zhao, Q., Luo, T., & Zeng, L. (2023). Inspiratory muscle training to reduce risk of pulmonary complications after coronary artery bypass grafting: a systematic review and meta-analysis. Frontiers in cardiovascular medicine, 10, 1223619. https://doi.org/10.3389/fcvm.2023.1223619\n\nDsouza, F. V., Amaravadi, S. K., Samuel, S. R., Raghavan, H., & Ravishankar, N. (2021). Effectiveness of inspiratory muscle training on respiratory muscle strength in patients undergoing cardiac surgeries: a systematic review with meta-analysis. Annals of Rehabilitation Medicine, 45(4), 264–273. https://doi.org/10.5535/arm.21027\n\nKendall, F., Oliveira, J., Peleteiro, B., Pinho, P., & Bastos, P. T. (2018). Inspiratory muscle training is effective to reduce postoperative pulmonary complications and length of hospital stay: a systematic review and meta-analysis. Disability and rehabilitation, 40(8), 864–882. https://doi.org/10.1080/09638288.2016.1277396\n\nThybo Karanfil, E. O., & Møller, A. M. (2018). Preoperative inspiratory muscle training prevents pulmonary complications after cardiac surgery - a systematic review. Danish medical journal, 65(3), A5450.\n\nIntroduction, paragraph 5, line 2 – suggest either correct typographical error (PowerBrethe) or more preferentially, amending to utilize previously defined abbreviation ‘…the PwB medic...’\nPage 4 of 10\nEthics and consent – authors have stated that study will be conducted with written informed consent from all participants; as participants are children/adolescents it is not clear here if the consent is from these minors and/or whether parental / carer / legal guardian consent is also provided. It is also not explicit as to whether this consent is obtained pre-operatively or post-operatively. Suggest supplementary information be included here to clarify. (It is only within criteria 4 of inclusion criteria [page 5] that it becomes evident that in fact it is parental consent, not participant consent, that is required.\nTrial design – is this really a RCT?? Both groups receive intervention involving IMT, just with different devices – there is no true control group or a standard comparator group (as IMT is not standard care in the chosen cohort). Commonly accepted convention is that the term “controlled” refers to the presence of a concurrent control or comparator group. Such studies have two or more groups – intervention and control. The control group receives no intervention, or another intervention that resembles the test intervention in some ways but lacks its activity (e.g., placebo or sham procedure, also referred to as “placebo-controlled” or “sham-controlled” trials) or another active treatment (e.g., the current standard of care). In this reporting, the standard care is not clearly outlined and within the protocol the variance between groups is insufficient, as it is the device to deliver the intervention (of IMT) that is the only difference and fundamentally the structure and prescription of the intervention in both groups is otherwise identical. If one does weight training of say biceps curls of 6 sets of 5 reps @ 40% of the 1RM, is not the training load the same whether using a bag of sand or a bag of sugar to provide the weight? Would one reasonable expect that performing the same task via the same prescription parameters produce the same impact on chosen outcome measures? The comparator difference between sand and sugar would be hard to substantiate. So then how do the authors hypothesize / propose that delivering IMT with the same training intensity, either with PowerBreathe or threshold devices, both acknowledged as pressure loaded devices, would perform differently and produce dissimilar outcomes? Authors have not provided any rationale as to how or why the two chosen devices would function any differently to deliver the desired IMT as prescribed.\nNo research question or hypotheses are offered by the authors within their protocol, although aims and objectives are shared. It would have been reasonable to have expected that a research protocol paper would include an overt hypothesis or research question.\nThe rationale to undertake this protocol is insufficiently prosecuted by the authors. Whilst authors acknowledge that research on physical therapy in those undergoing VSD closure surgery is limited, it is not established what current physical therapy practice is, whether current practice is effective, efficient, safe etc, or whether there are concerns that current practice may be associated with sub-optimal outcomes. Similarly, the justification for respiratory muscle training to be an adjunct or alternative strategy to usual/current/standard practice is not made. What is the problem (& its magnitude/significance) the anticipated findings from this project are hoping to address? Finally, the differentiation between two devices utilized to provide respiratory muscle training (PowerBreathe & Threshold devices) is insufficiently clear for the reader to appreciate why one may be preferrable over the other in the clinical cohort being studied. The authors fail to answer for the reader the fundamental ‘Why?’ and ‘So what?’ questions to substantiate the putting together of their research protocol…\nPage 4 of 10\nParticipants and recruitment, paragraph 1 – there is insufficient detail to allay concerns of potential selection / inclusion bias. What does ‘The inclusion and exclusion criteria for selection will be based on the cut-off values at baseline parameters when engaging participants’ actually mean? What baseline parameters, and what are the associated cut-off parameters? What is the rationale for a six-month recruitment phase? How does this relate/intercede with the sample size calculation provided on page 6?\nPage 5 of 10\nGroup information – as per comments above, the descriptors provided here for experimental and ‘control’ group interventions are identical in all parameters of the prescription framework (frequency, intensity, type, time, volume & progression), with the only difference being the piece of equipment used to provide the IMT; but both devices provide pressure loading, so what is really considered to be a stimulus for a differential response is not apparent.\nWhat physical therapy occurs before POD-3? Suggest some comments around this are included. What is the post-operative mobility program? When does ambulation commence? Is there a structured approach to post-operative mobility? Are there any restrictions to post-operative mobility, return to physical activity and/or sports? Would it be anticipated that the participants may return to schooling, or work, before data collection has been completed? There is no commentary within the protocol as to the standardization of ambulation, or even the recording of ambulation (frequency, distance, milestones, etc) or physical activity levels more broadly across the 7 weeks (4 weeks intervention & 3 weeks to final follow up) to be able to assess if this may be a confounder to the outcome measures. It is recommended that the authors may need to supplement the reporting of their protocol to address such potential confounders.\nIt is not clear what position the intervention will be performed in – will participants be standing or sitting for the IMT? If sitting, will it be supported sitting in a chair, or long sitting in bed/ on a plinth, or sitting unsupported over the edge of a bed/plinth? Will the IMT be delivered via a mouthpiece or via a mask (or will participants get to choose?). Will the IMT intervention (& conservative physical therapy management) be directly conducted by a therapist/researcher, or independently performed but under a form of supervision (i.e. participant is conducting the IMT within a hospital gym / ambulatory care setting, potentially in a group based scenario?\nWhen will participants be anticipated to be discharged from hospital after their surgery? It would presumably not be expected for them to stay admitted for the 4 weeks of the study, but regardless they are expected to return to the hospital twice daily to receive the intervention for the 4 weeks? Is this feasible, particularly as these are children/adolescents? Does this then create an inclusion bias such that anyone who resides not close to the hospital, and returns home after the surgery and thus unable to travel back to the hospital twice daily, will be excluded?\nWhat will be considered compliance with the intervention? If the intervention is prescribed as twice daily, every day for a 4-week period, then that is a total of 56 sessions – how many can be missed before a participant is withdrawn or considered ‘non-compliant’? Will researchers be recording attendance/ session completion data? How will any missed sessions or missed data be managed within the protocol is not described.\nPage 6 of 10\nOutcomes – who is planned to perform the assessment of the outcome measures? Will they be blinded to group allocation and independent to study objectives? Will this be a standardized, trained individual or a team of assessors? This should be clear for the reader, and to allow for repeatability and external validity considerations. What procedure will be followed to gather the MIP? Will subjects perform in sitting or standing etc? Again it is suggested that further details be included to assist clarity. Similarly, how will the 6MWT be conducted – indoors vs outdoors? Circular track vs point to point ? etc\n\n– suggest provide a supporting citation for procedure to be followed in this project (such as Holland, A. E., Spruit, M. A., Troosters, T., Puhan, M. A., Pepin, V., Saey, D., McCormack, M. C., Carlin, B. W., Sciurba, F. C., Pitta, F., Wanger, J., MacIntyre, N., Kaminsky, D. A., Culver, B. H., Revill, S. M., Hernandes, N. A., Andrianopoulos, V., Camillo, C. A., Mitchell, K. E., Lee, A. L., … Singh, S. J. (2014). An official European Respiratory Society/American Thoracic Society technical standard: field walking tests in chronic respiratory disease. The European respiratory journal, 44(6), 1428–1446. https://doi.org/10.1183/09031936.00150314)\nAnd same query on the conduct of the PFTs – suggest provision of a citation of the procedure/guideline to be followed in the conduct and interpretation of this outcome measure (e.g. Graham BL, Steenbruggen I, Miller MR, et al. Standardization of spirometry 2019 update. An official American Thoracic Society and European Respiratory Society technical statement. Am J Respir Crit Care Med 2019; 200: e70–e88. doi:10.1164/rccm.201908-1590ST Stanojevic, S., Kaminsky, D. A., Miller, M. R., Thompson, B., Aliverti, A., Barjaktarevic, I., Cooper, B. G., Culver, B., Derom, E., Hall, G. L., Hallstrand, T. S., Leuppi, J. D., MacIntyre, N., McCormack, M., Rosenfeld, M., & Swenson, E. R. (2022). ERS/ATS technical standard on interpretive strategies for routine lung function tests. The European respiratory journal, 60(1), 2101499. https://doi.org/10.1183/13993003.01499-2021)\nNo metrics of participant satisfaction, costs, device logistics (durability, cleanliness, ease of use, etc) are proposed to be captured. In a study which ultimately is simply comparing two devices delivering the same intervention (in the doses prescribed within the studied cohort), surely such parameters are required to be able to inform future consumers (and clinicians & researchers) as to which device may be preferable to pursue? Or are the authors anticipating a non-inferiority finding, and then being able to simply offer future patients a choice between devices to deliver any proposed IMT?\nPage 7 of 10\n‘Data collection, management and analysis’ is evident as a single line statement, but there are no paragraphs on data collection or management prior to details shared under an Analysis heading. As a protocol paper it would be expected that the specificity of the plans for data collection methods and data management be explicitly outlined for transparency and accountability (as per the SPIRIT checklist).\nAnalysis, line 1 -firstly ‘R studio’ is an editor (Integrated Development Environment), whilst ‘R’ (the software package) is what is used to do the analysis. It is suspected the author intentions are to use ‘R’. Suggest amending/supplementing to provide citation for the R software ' …using R software (Version 4.3.0 released April 2023) [R Core Team (2023). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.]' (NB authors please check the accurate version number you plan to use and reference accordingly, as already it is up to 4.3.2 as of 31st Oct 2023)\nLine 2 – correct typographical error ‘nanalysed’\nDiscussion\nIt is unclear to this reviewer what the intent of the current structure of the Discussion is; information presented here could easily sit instead within the context of the Introduction background/context aiding synthesis of existing knowledge and providing justification for this project and its protocol design. Whilst not imperative to have a Discussion within a protocol paper, if one is to be included then one may reasonable expect that the discussion aligns to aspects of the implementation of the proposed protocol – discussion around feasibility issues (anticipated or unanticipated), possible barriers to study conduct and proposed strategies to avoid and/or ameliorate, expected outcomes of the study - the protocol should indicate how the study will contribute to advancement of knowledge, how the results will be utilized, not only in publications but also how they will likely affect health care, health systems, or health policies.\nDissemination of results and publication policy should also feature (either within Discussion, or as unique sections)– whilst it is acknowledged that authors have included a Dissemination statement (on page 8), this has been targeted only at the protocol & findings being presented in conference proceedings. “The protocol should specify not only dissemination of results in the scientific media, but also to the community and/ or the participants, and consider dissemination to the policy makers where relevant. Publication policy should be clearly discussed- for example who will take the lead in publication and who will be acknowledged in publications, etc.” (https://www.who.int/groups/research-ethics-review-committee/recommended-format-for-a-research-protocol)\nPage 8 of 10\nWhat are the plans for data monitoring and auditing within this project? What is being used to define what constitutes an ‘adverse event’? Who will adverse events be reported to?\nThere are no details within the protocol reporting as to confidentiality, deidentification of data, plans for data storage and accessibility (post study) -the Data availability heading correctly states that there are no data presently, but the protocol needs to address how the incoming data will be managed…\nReferences\nSuggest reference list needs to be reviewed for consistency of style of capitalization of article titles.\n#18 – needs to be completed with correct full citation details: Menzes KKP, Nascimento LR, Avelino PR, Polese JC, Salmela LFT (2018) A review on respiratory muscle training devices . J Pulm Respir Med 8:451. doi: 10.4172/2161-105X.1000451\nIn a submission on this topic, other potentially relevant articles such as those listed below (non-exhaustive and non-prioritized listing) may have been pertinent and valuable inclusions within the Introduction and/or Discussion??(accepting they are not necessarily IMT focused, but more related to this study cohort and current practice)\nTurquetto, A. L. R., Dos Santos, M. R., Agostinho, D. R., Sayegh, A. L. C., de Souza, F. R., Amato, L. P., Barnabe, M. S. R., de Oliveira, P. A., Liberato, G., Binotto, M. A., Negrão, C. E., Canêo, L. F., Trindade, E., Jatene, F. B., & Jatene, M. B. (2021). Aerobic exercise and inspiratory muscle training increase functional capacity in patients with univentricular physiology after Fontan operation: A randomized controlled trial. International journal of cardiology, 330, 50–58. https://doi.org/10.1016/j.ijcard.2021.01.058\n\nWu, F. M., Opotowsky, A. R., Denhoff, E. R., Gongwer, R., Gurvitz, M. Z., Landzberg, M. J., Shafer, K. M., Valente, A. M., Uluer, A. Z., & Rhodes, J. (2018). A pilot study of inspiratory muscle training to improve exercise capacity in patients with fontan physiology. Seminars in thoracic and cardiovascular surgery, 30(4), 462–469. https://doi.org/10.1053/j.semtcvs.2018.07.014\n\nAbdulkarim, A., Shaji, S., Elfituri, M., Gunsaulus, M., Zafar, M. A., Zaidi, A. N., Pass, R. H., Feingold, B., Kurland, G., Kreutzer, J., Ghassemzadeh, R., Goldstein, B., West, S., & Alsaied, T. (2023). Pulmonary complications in patients with fontan circulation: JACC Review Topic of the Week. Journal of the American College of Cardiology, 81(25), 2434–2444. https://doi.org/10.1016/j.jacc.2023.04.036\n\nScheffers, L. E., Berg, L. E. M. V., Ismailova, G., Dulfer, K., Takkenberg, J. J. M., & Helbing, W. A. (2021). Physical exercise training in patients with a Fontan circulation: A systematic review. European journal of preventive cardiology, 28(11), 1269–1278. https://doi.org/10.1177/2047487320942869\n\nFerrer-Sargues, F. J., Peiró-Molina, E., Salvador-Coloma, P., Carrasco Moreno, J. I., Cano-Sánchez, A., Vázquez-Arce, M. I., Insa Albert, B., Sepulveda Sanchis, P., & Cebrià i Iranzo, M. À. (2020). Cardiopulmonary rehabilitation improves respiratory muscle function and functional capacity in children with congenital heart disease. A prospective cohort study. International Journal of Environmental Research and Public Health, 17(12), 4328–. https://doi.org/10.3390/ijerph17124328\n\nMcBride MG, Burstein DS, Edelson JB, Paridon SM. Cardiopulmonary rehabilitation in pediatric patients with congenital and acquired heart disease. J Cardiopulm Rehabil Prev. 2020;40(6):370–377.\n\nGauthier N, Curran T, O’Neill JA, Alexander ME, Rhodes J. Establishing a comprehensive pediatric cardiac fitness and rehabilitation program for congenital heart disease. Pediatr Cardiol. 2020;41(8):1569–1579..\n\nAkamagwuna U, Badaly D. Pediatric cardiac rehabilitation: a review. Curr Phys Med Rehabil Rep. 2019;7(2):67-80. doi:10.1007/s40141-019-00216-9.\n\nLongmuir PE, Brothers JA, de Ferranti SD, et al. Promotion of physical activity for children and adults with congenital heart disease: a scientific statement from the American Heart Association. Circulation. 2013;127(21):2147–2159.\n\nTikkanen AU, Oyaga AR, Riaño OA, Álvaro EM, Rhodes J. Paediatric cardiac rehabilitation in congenital heart disease: a systematic review. Cardiol Young. 2012;22(3):241–250\n\nDuppen, N., Etnel, J. R., Spaans, L., Takken, T., van den Berg-Emons, R. J., Boersma, E., Schokking, M., Dulfer, K., Utens, E. M., Helbing, W., & Hopman, M. T. (2015). Does exercise training improve cardiopulmonary fitness and daily physical activity in children and young adults with corrected tetralogy of Fallot or Fontan circulation? A randomized controlled trial. The American Heart Journal, 170(3), 606–614. https://doi.org/10.1016/j.ahj.2015.06.018\n\nClarke, S. L., Milburn, N. C., Menzies, J. C., & Drury, N. E. (2023). The provision and impact of rehabilitation provided by Physiotherapists in children and young people with congenital heart disease following cardiac surgery: A scoping review. Physiotherapy. https://doi.org/10.1016/j.physio.2023.09.001\n\nBeningfield, A., & Jones, A. (2018). Peri-operative chest physiotherapy for paediatric cardiac patients: a systematic review and meta-analysis. Physiotherapy, 104(3), 251–263. https://doi.org/10.1016/j.physio.2017.08.011\n\nGauthier, N., Curran, T., O’Neill, J. A., Alexander, M. E., & Rhodes, J. (2020). Establishing a comprehensive pediatric cardiac fitness and rehabilitation program for congenital heart disease. pediatric Cardiology, 41(8), 1569–1579. https://doi.org/10.1007/s00246-020-02413-z\n\nTran, D., Maiorana, A., Ayer, J., Lubans, D. R., Davis, G. M., Celermajer, D. S., d'Udekem, Y., & Cordina, R. (2020). Recommendations for exercise in adolescents and adults with congenital heart disease. Progress in cardiovascular diseases, 63(3), 350–366. https://doi.org/10.1016/j.pcad.2020.03.002\n\nFinally this reviewer is reminded of the following publication and musing of its relevance and message when considering this protocol:\nIllidi, C. R., Romer, L. M., Johnson, M. A., Williams, N. C., Rossiter, H. B., Casaburi, R., & Tiller, N. B. (2023). Distinguishing science from pseudoscience in commercial respiratory interventions: an evidence-based guide for health and exercise professionals. European journal of applied physiology, 123(8), 1599–1625. https://doi.org/10.1007/s00421-023-05166-8\nThe authors would do well to avoid such concerns potentially being within readers' perceptions by providing further details supporting their rationale and justification for their chosen study design and methodology protocol.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-939
https://f1000research.com/articles/12-935/v1	07 Aug 23	{ "type": "Genome Note", "title": "The complete mitochondrial genome sequence of Rikuzen flounder, Dexistes rikuzenius (Jordan & Starks, 1904)", "authors": [ "Jun Young Chae", "JinHo Kim", "Tae-Wook Kang", "Jinkoo Kim", "Hyung-Ho Lee", "Moo-Sang Kim", "Jun Young Chae", "JinHo Kim", "Tae-Wook Kang", "Jinkoo Kim", "Hyung-Ho Lee" ], "abstract": "In this study, the complete mitochondrial genome of the Rikuzen flounder (Dexistes rikuzenius) using next-generation sequencing and bioinformatics analysis is presented. The mitogenome was found to be a circular molecule 17,494 bp in length, containing 13 protein-coding genes (PCG), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and one control region (D-loop). Proportional nucleotide composition was A: 28.80% (5,039 bp), T: 26.51% (4,638 bp), C: 28.72% (5,025 bp) and G: 15.96% (2,792 bp), with a combined A+T content of 55.32%. The phylogenetic relationships of the taxon with 19 other flatfish species in the order Pleuronectiformes were then analyzed based on the 13 PCGs. Dexistes rikuzenius was most closely clustered with Cleisthenes herzensteini within this order, suggesting a close relationship.", "keywords": [ "Dexistes rikuzenius", "Pleuronectiformes", "Mitogenome" ], "content": "Introduction\n\nThe family Pleuronectidae belongs to the order Pleuronectiformes. There are 25 genera and 62 species in the family as listed in FishBase (Froese and Pauly, 2022), while Eschmeyer’s Catalog of Fishes (Fricke et al., 2022) reports 24 genera and 64 species. Further different classifications in the literature assume 23 genera and 61 species (Nelson et al., 2016), or 24 genera and 59 species (Vinnikov et al., 2018) based on genetic analysis. However, only 25 species in 18 genera have been reported to occur in Korea (MABIK, 2017).\n\nThe Rikuzen flounder (Dexistes rikuzenius), which is placed in this family, is a flatfish that lives on sandy or gravelly bottoms at depths of 42-200 meters (Yamada et al., 1995) and is distributed in the South Sea of South Korea, the East China Sea, and southern Hokkaido of Japan. The Rikuzen flounder is one of the rarest species in the family Pleuronectidae in South Korea. Its larvae are reported to be morphologically very similar to those of the stone flounder (Platichthys bicoloratus) (Lee et al., 2019), but little morphological and genetic information on this species is available. The mitogenomic data presented here are therefore expected to provide information that will aid in the efficient resource management of this species and further the molecular phylogenetic taxonomy of the family Pleuronectidae.\n\n\nMethods\n\nThe specimen used in this study was secured from Jeju South Korea (33°571’N, 126°256’E) and the storage facility of the Marine Fish Resource Bank of Korea in Pukyong National University were used to deposit under Voucher no. PKU 56422 (Jinkoo Kim, tjgk2002@gmail.com).\n\nThe genomic DNA (gDNA) was extracted from a fin of the specimen using the PureHelixTM Genomic DNA Prep Kit [Animal], Solution Type (NANOHELIX, Daejeon, South Korea). PCR was conducted using the fish universal primer set (Ward et al., 2005) to amplify the cox1 gene and the nucleotide sequence was analyzed by Macrogen (Daejeon, South Korea). A BLASTN (Johnson et al., 2008) search were utilized to confirm the species.\n\ngDNA (1 μg) was sheared using the S220 Ultra sonicator (Covaris, Woburn, USA). MGIEasy DNA library prep kit (MGI Tech, Shenzhen, China) was used for library preparation with according to the manufacturer’s instructions. Briefly, Fragmented gDNA was selected based on its size using AMPure XP magnetic beads and the fragmented gDNA was end-repaired and a-tailed at 37 °C for 30 min, and 65 °C for 15 min. Indexing adapter was ligated to the ends of the DNA fragments at 23 °C for 60 min. PCR was performed to enrich those DNA fragments that have adapter molecules after purifying the adapter-ligated DNA. Thermocycler conditions were as follows: 95°C for 3 min, 7 cycles of 98°C for 20 s, 60°C for 15 s, and 72°C for 30 s, with a final extension at 72°C for 10 min. The double stranded library is quantified using QauntiFluor ONE dsDNA System (Promega, Madison, USA). The library is circularized at 37 °C for 30 min, and then digested at 37 °C for 30 min, followed by cleanup of circularization product. The library is incubated at 30 °C for 25 min using DNB enzyme for making DNA nanoball (DNB). Finally, Library was quantified by QauntiFluor ssDNA System (Promega, Madison, USA). Sequencing of the prepared DNB was conducted on an MGISEQ-2000 sequencing platform (MGI Tech, Shenzhen, China) with 150 bp paired-end reads.\n\nThe raw sequencing data were deposited in the Sequence Read Archive (SRA) database (SRR21644379) (theMOAGEN, 2022b). Trimming was conducted using Cutadapt ver. 4.1 (Martin, 2011), and these trimmed data were assembled using the default option in the de novo assembler of the CLC Genomics Workbench (ver. 20.04; QIAGEN, Venlo, Netherlands). The circular form of the mitogenome obtained from the contig sequences was verified by mapping the filtered data onto the contig sequence using the “Map to Reference” tool in the Geneious software (ver. 2021.2.2; https://www.geneious.com). This final sequence was annotated using the MITOS Webserver (Bernt et al., 2013), and SnapGene software (ver. 5.3.2; GSL Biotech LLC; snapgene.com) was utilized for manually correcting the detailed annotation. Finally, the completed circular form of the mitogenome sequence was registered at the NCBI GenBank (OP066371). A maximum likelihood (ML) phylogenetic tree was constructed with MEGA 11 software (Tamura et al., 2021) using GenBank mitogenomes from all available 18 species of the family Pleuronectidae and one member of the family Bothidae, Bothus myriaster (Accession No. KJ433563), as an outgroup. All mitogenome sequences were collected from GenBank. The nucleotide sequences of PCGs were gathered from each mitogenome sequence and were aligned using the ClustalW multiple alignment tool in BioEdit under default options. The phylogenetic analysis was conducted using the GTR + G + I model with 1,000 bootstrap replicates.\n\n\nResults\n\nThe complete mitochondrial genome of the D. rikuzenius was a circular molecule, 17,494 bp in length (GenBank acc. no. OP066371) (Chae and Kim, 2022), and contained 13 PCGs, 22 tRNA genes, two rRNA genes, and one control region. Proportional nucleotide composition of the mitochondrial genome was A: 28.80% (5,039 bp), T: 26.51% (4,638 bp), C: 28.72% (5,025 bp) and G: 15.96% (2,792 bp), with a combined A+T content of 55.32%.\n\nAmong the 37 detected mitochondrial genes, nine of the PCGs and eight tRNA genes (tRNAAla, tRNAAsn, tRNACys, tRNAGln, tRNAGlu, tRNAPro, tRNASer and tRNATyr) were encoded on the L-strand, while all other genes were encoded on the H-strand. Most mitochondrial protein-coding genes had ‘ATG’ as their start codon, whereas only COX1 started with ‘GTG’. The seven PCGs (ATP6, ATP8, COX1, ND1, ND4L, ND5 and ND6) used ‘TAA’ as the complete stop codons, whereas COX3 used ‘TA’, and COX2, CYTB, ND2, ND3 and ND4 used ‘T’ as an incomplete stop codon. The latter two instances are presumably completed to ‘TAA’ by posttranscriptional polyadenylation (Ojala et al., 1981). The 12S and 16S rRNA genes were 949 bp and 1,714 bp long, respectively. The 22 tRNA genes ranged in length from 63 bp (tRNACys) to 74 bp (tRNALeu).\n\nThe results of the phylogenetic analysis show that D. rikuzenius is closely related to Cleisthenes herzensteini, and that these two are sister taxa to the two genera Limanda and Hippoglossoides (Figure 1). This is the only study to report a complete mitochondrial DNA sequence in the genus Dexistes. Mitochondrial DNA is a powerful tool for studying the evolution of genomes and for the identification of individual species. Our results will therefore be useful for understanding the phylogenetic relationships and taxonomic classification of the family Pleuronectidae, and are expected to contribute to the efficient resource management of the species.\n\nA maximum likelihood dendrogram was constructed using utilizing 13 PCGs from mitogenomes of 18 species in the family Pleuronectidae, and one species in the family Bothidae, Bothus myriaster (Accession No. KJ433563), as an outgroup. The GenBank accession numbers of mitogenome sequences are given in parentheses next to the species names. Node numbers correspond to Bayesian inference's posterior probabilities. The species of interest (Dexistes rikuzenius) is highlighted in bold and with an arrowhead.\n\n\nEthics and consent\n\nNo ethical approval is required for this study. We used a flatfish fin from a specimen that was previously collected by the MBRIS, outside of this study. This specimen was dead, and the sample was provided with permission by the MBRIS (permission no. 2022-130).", "appendix": "Data availability\n\nGenBank: Dexistes rikuzenius mitochondrion, complete genome. Accession number OP066371; https://www.ncbi.nlm.nih.gov/nuccore/OP066371.1 (Chae and Kim, 2022).\n\nBioProject: Dexistes rikuzenius. Accession number PRJNA882492; https://www.ncbi.nlm.nih.gov/bioproject/882492 (theMOAGEN, 2022a).\n\nSRA: Dexistes rikuzenius. Accession number SRR21644379; https://www.ncbi.nlm.nih.gov/sra/SRR21644379 (theMOAGEN, 2022b).\n\nBioSample: Animal sample from Dexistes rikuzenius. Accession number SAMN30941287; https://www.ncbi.nlm.nih.gov/biosample/SAMN30941287 (theMOAGEN 2022c).\n\n\nReferences\n\nBernt M, Donath A, Juhling F, et al.: MITOS: improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 2013; 69(2): 313–319. PubMed Abstract \| Publisher Full Text\n\nChae JY, Kim MS: Dexistes rikuzenius mitochondrion, complete genome. [Dataset]. GenBank. 2022. Reference Source\n\nFricke R, Eschmeyer WN, Van der Laan R: ESCHMEYER’S CATALOG OF FISHES: GENERA, SPECIES, REFERENCES. California Academy of Science; 2022. Reference Source\n\nFroese R, Pauly D: FishBase. World Wide Web Electronic Publication; 2022. Reference Source\n\nJohnson M, Zaretskaya I, Raytselis Y, et al.: NCBI BLAST: a better web interface. Nucleic Acids Res. 2008; 36(Web Server): W5–W9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee SJ, Kim JK, Ryu JH, et al.: Molecular identification and morphological description of larvae for ten species of the family Pleuronectidae (Pleuronectiformes, PISCES) from Korea. J. Korean Soc. Fish Ocean Technol. 2019; 55(4): 335–348. Publisher Full Text\n\nMABIK (Marine Biodiversity Institute of Korea): National List of Marine Species.2017; 397–399.\n\nMartin M: Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 2011; 17(1): 10–12.\n\nNelson JS, Grande TC, Wilson MV: Fishes of the World. John Wiley & Sons; 2016.\n\nOjala D, Montoya J, Attardi G: tRNA punctuation model of RNA processing in human mitochondria. Nature. 1981; 290(5806): 470–474. PubMed Abstract \| Publisher Full Text\n\nTamura K, Stecher G, Kumar S: MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021; 38(7): 3022–3027. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\ntheMOAGEN: Dexistes rikuzenius. [Dataset]. BioProject. 2022a. Reference Source\n\ntheMOAGEN: Dexistes rikuzenius. [Dataset]. SRA. 2022b. Reference Source\n\ntheMOAGEN: Dexistes rikuzenius. [Dataset]. BioSample. 2022c. Reference Source\n\nVinnikov KA, Thomson RC, Munroe TA: Revised classification of the righteye flounders (Teleostei: Pleuronectidae) based on multilocus phylogeny with complete taxon sampling. Mol. Phylogenet. Evol. 2018; 125: 147–162. PubMed Abstract \| Publisher Full Text\n\nWard RD, Zemlak TS, Innes BH, et al.: DNA barcoding Australia’s fish species. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 2005; 360(1462): 1847–1857. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamada U, Shirai S, Irie T, et al.: Names and illustrations of fishes from the East China Sea and the Yellow Sea. Overseas Fishery Cooperation Foundation; 1995." }	[ { "id": "255145", "date": "26 Mar 2024", "name": "Euna Jo", "expertise": [ "Reviewer Expertise genomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper reported the complete mitogenome sequence and performed phylogenetic analysis of flonuder fish, Dexistes rikuzenius. Overall, this is well written, but the following points require correction and amendment.\nAbstract: - PCG -> PCGs (protein-coding genes are plural form)\nIntroduction: - Unify either Korea or South Korea, throughout the manuscript. - To highlight the importance of this study, it would be better to state in the Introduction that there is only one species in the genus Dexistes, Dexistes rikuzenius, and that this paper is the first mitogenome report of the genus and species.\nMethods: - Please capitalize cox1 (to COX1) the same as the format you wrote in Results section. - Library preparation with according to~. -> Shouldn’t “with” be deleted here? - Fragmented gDNA was selected based on its size ~->Which size ranges were selected? - The fragmented gDNA was end-repaired ~ -> the \"size-selected\" gDNA was ~ - a-tailed -> A-tailed (caplitalize A) - is -> was. Unify the tense used in Methods. - Please add the accession numbers and references for mitogenomes of 18 species. - Why did you select the GTR+G+I model? Please describe it.\nResults: -Nnine of the PCGs ~ encoded on the L-strand -> Please add which genes they are. - Protein-coding genes -> PCGs (use abbreviation) - On the lenged of Figure 1, you wrote \"Node numbers correspond to Bayesian inference's posterior probabilities.\" Isn't it bootstrap values, not Bayesian inference's posterior probabilities? Please refer to Methods section you wrote and correct it.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] }, { "id": "276557", "date": "03 Jun 2024", "name": "Michael W Sandel", "expertise": [ "Reviewer Expertise Phylogenetic systematics", "ichthyology", "molecular ecology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis represents an adequate report of the mitogenome sequence of the Rikuzen flounder. The first paragraph of the introduction is factual, but not very interesting. Find \"QauntiFluor\" and replace with \"Quantifluor\" (x2). Wet lab and bioinformatic methods are appropriate and well documented.\n\nThe authors have adequately accomplished their task, but they have missed an opportunity to discuss two of the most peculiar aspects of Pleuronectiformes mtDNA: 1. The divergent mtDNA nucleotide composition bias resulting in non-monophyly of the group in previous studies.\n2. Certain flatfish taxa that exhibit control region duplication. If this species has only one control region, it seems that it must be unusually long (for a total mtDNA length of 17,494 bp). Note that most fish mitogenomes are about 500bp shorter. What accounts for this difference?\nThis paper will receive better readership if the authors place their results in a broader context within published literature.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-935
https://f1000research.com/articles/12-934/v1	07 Aug 23	{ "type": "Research Article", "title": "Anatomical variations of the sternum: sternal foramen and variant xiphoid morphology in dried adult human sternum in Ethiopia", "authors": [ "Alemayehu Shiferaw Lema" ], "abstract": "Background: The sternum exhibits unique anatomical variations with major clinical and forensic implications. This study is devoted to providing baseline epidemiological information about the sternal foramen and variant xiphoid morphology in Ethiopia. Two extremely interesting and unusual variations of the sternal foramen are also discussed. Methods: This observational study was carried out using dried adult human sternum obtained from skeletal remains samples brought for medicolegal examination over a period of 4 years. A total of 94 dried adult human sternums (66 males (70.2%) and 28 females (29.8%)) were obtained with an age range of 21 to 57 years and a mean age at death of 38.383 ± 11.3480 years. Dried human sternum specimens were morphologically examined, and morphometric parameters were recorded and photographed. Results: A sternal foramen was found in 18 specimens (19.1%); 17 were male and one was female. A single sternal foramen was observed in 83.3% (n=15/18) of the sternal bodies and 11.1% (n=2/18) of the xiphoid processes (both males). In addition, a double sternal foramen was observed in a single male specimen on the mesosternum and xiphoid process. The most common sternal foramen site was at the fifth costochondral junction level. The xiphoid process was present in 77 samples and ended as a single process in 83.1% (n=64/77) of samples. In 15.6% (n=12/77) of the samples, the xiphoid process was bifurcated and trifurcated in a single male (1.3%) specimen. Conclusions: The sternal foramen and variation in xiphoid morphology are common anatomical variations in Ethiopia. The findings of the current study highlight the necessity of strict precautionary measures during sternal procedures in this study population. In addition, such incidental findings during radiologic and autopsy procedures should be properly evaluated to avoid misdiagnosis and misinterpretation of such findings as traumatic or pathologic conditions.", "keywords": [ "Sternum", "Sternal foramen", "Xiphoid morphology", "Variations", "Sternal puncture", "Acupuncture", "Forensic", "Radiology", "Ethiopia" ], "content": "Introduction\n\nThe human sternum forms the anterior midline border of the thorax and is located adjacent to the pleura cavity, pericardial cavity and great vessels of the thoracic cavity.1 The human sternum comprises three components: the manubrium, the mesosternum (body), and the xiphoid process.1,2 Human skeletons have several variations that may require a distinction from pathologic alterations. The sternum is one of the parts of the human skeleton with the highest frequency of anatomical variation.2\n\nSternal foramen (SF) is a round or oval or even irregular bony defect in any part of the sternum that results from the incomplete union of the mesenchymal sternal bars during embryogenesis.2–4 SFs are usually asymptomatic and are frequently detected incidentally in radiological images, postmortem examinations and anatomy teaching specimens.2–6 Knowledge of congenital SF has various implications in clinical, acupuncture, and forensic practices.2–6\n\nAlthough SF is asymptomatic, its proximity to vital thoracic structures may pose a serious hazard during sternal procedures.2,3,5 In addition, SF can be misdiagnosed as lytic lesions or trauma in the interpretation of radiologic images and postmortem findings of skeletonized human remains.2,4–6 Therefore, awareness concerning sternal bone variations is important for radiologists and forensic experts to avoid misinterpretation.\n\nMoreover, sternal bone variation analysis is a simple and important potential source of data in the human skeleton identification process whenever there is recorded variation in the antemortem data of the victim.2–4 SFs have been detected in the manubrium, mesosternum and xiphoid process; however, they are more frequently observed in the lower portion of the sternum. The incidence of sternal foramina varies between different populations, ranging from 3.1 to 18.3%.1,2,4,5,7–18\n\nOn the other hand, the xiphoid process is the most variable portion of the sternal bone.5 Although educational textbooks describe the xiphoid process as pointed and elongated, it can be absent, broad, bifid, trifurcated, duplicated, or deflected and may contain a foramen. Such variations could be misinterpreted as an epigastric mass, fracture or traumatic fissure.1,5\n\nAlthough the distribution of these sternal variations shows discrepancy among different populations, data from Africans are scarcely reported. To my knowledge, no study has described sternal variations in the Ethiopian population. This study is devoted to providing baseline epidemiological information about the sternal foramen and variant xiphoid morphology in Ethiopia, comparing results with other populations and discussing the clinical and forensic significance.\n\n\nMethods\n\nThis observational study was carried out using dried adult human sternum obtained from skeletal remains samples brought for medicolegal examination from January 1, 2019, to December 31, 2022, in the Department of Forensic Medicine and Toxicology, St. Paul’s Hospital and Millennium Medical College in Addis Ababa, Ethiopia. All skeletal remains samples obtained during the study period of both sex and adult age groups that had human sternal bone were included in this study. Brocken sternal bone specimens and those without sex or age records were excluded from the study. Dried human sternum specimens were morphologically examined for the presence of SF, and their topology and morphometric parameters were recorded and photographed. The xiphoid process was classified as absent or present, and its terminal end was classified as single, bifurcated, or trifurcated. Samples were photographed, and SF measurements were obtained using a Vernier caliper with an accuracy of 0.02 mm. Data were analyzed using SPSS version 26.0. Continuous data were described in terms of the mean and compared using the Mann–Whitney U test. Categorical variables were described using frequencies and percentages and compared using the chi-square test. The level of significance was set at a p-value <0.05. The study was conducted as per the Declaration of Helsinki. Ethics approval was obtained from the St. Paul’s Hospital and Millennium Medical College institutional review board (ethical clearance reference no: PM 23/392). All information was treated anonymously and confidentially using unique identification codes rather than individual names and identifications. Waiver of informed consent was granted by the institutional review board, because of the anonymization of the data, the difficulty of the reaching next-of-kin during the examination, the potential value that the findings may add to clinical and forensic practices and the low risk involved in the use of the data.\n\n\nResults\n\nA total of 94 dried adult sternum specimens were available for the study. The 94 dried adult sterna samples examined constituted 66 males (70.2%) and 28 females (29.8%). SF was observed in 18 specimens, resulting in an incidence of 19.1% of the specimen. Among the 18 specimens with SF, 17 specimens (94.4%) were male, and a single specimen (5.6%) was female. There was a statistically significant difference between the presence of SF and sex (p=0.012). Ages at death ranged from 21 to 57 years, and the mean age at death was 38.383±11.3480 years. The sternal foramen was more frequent (61.1%) in individuals over 35 years of age, and there was a statistically significant difference between the presence and absence of the sternal foramen between younger individuals (<35 years) and older (≥35 years) individuals (p<0.001). However, its presence was not different in the younger (<35 years) and older (≥35 years) age groups at death in the pooled sex sample (p=0.250).\n\nA single SF was observed in the sternal body in 83.3% (n=15/18), while two (11.1%) single SF were detected in the xiphoid process (both males). In addition, a double sternal foramen was observed in a single male specimen on the sternal body and xiphoid process (Figure 1). However, there was no statistically significant difference between the topology of the SF (sternal body and xiphoid process) and sex (p=0.813). No foramen was observed on the manubrium or upper mesosternal segments proximal to the level of the fourth costochondral junction (CCJ). The most common site of SF was at the level of the fifth costochondral junction (CCJ) in 55.6% (n=10/18). This is followed by the fourth CCJ, sixth CCJ and xiphoid process, where SF is observed in 16.7% (n=3/18) of specimens at each level. However, there was no statistically significant difference between the topology of SF and both the vertical length (P=0.159) and transverse width (P=0.054) of SF. Four SFs (22.2% (n=4/18) with irregular bony margins and abnormal shapes were observed on the body of the sternum (Figures 2, 3 and 4). A single case of SF with a beveled margin was also documented (Figure 4). The mean transverse width of the SF was 7.08±3.325 mm (3-16 mm), and the vertical length was 7.88±4.285 mm (3-18 mm).\n\nThe xiphoid process was absent in 18.1% of specimens, including 13.8% males (n=13) and 4.3% females (n=4), and the majority (84%) of these individuals were below 30 years of age. There was no statistically significant difference between the presence and absence of the xiphoid process between younger individuals (<50 years) and older (>50 years) individuals (p=0.178) and sex (p=0.533). The xiphoid process ended as a single in 83.1% (n=64/77) of specimens, including 58.4% (n= 45) males and 24.7% (n=19) females. In 15.6% (n=12/77) of the samples, the xiphoid process was bifurcated and trifurcated in a single male (1.3%) specimen (Figure 5). However, no differences were observed between variations in xiphoid morphology with sex (p=0.670) or the presence of the sternal foramen (p=0.157).\n\n\nDiscussion\n\nThe sternum develops from mesenchymal condensation that forms paired sternal bars that were originally located on each side of the midline. Immediately after the attachment of the cranial ribs, these paired sternal bars begin to chondrify, migrate to the midline and fuse in a craniocaudal fashion. After chondrification, ossification centers begin to appear from the fifth month to early childhood in craniocaudal succession. These ossification centers in the body of the sternum develop into four transverse segments known as the mesosternum. The ossification centers for the mesosternal segments vary considerably. In general, the two proximal mesosternal segments have single centers, whereas the third and fourth segments have paired centers. Beginning in the third to the fourth year of life, the mesosternal segments fuse caudocranially and are typically completed in early adulthood.19,20 During childhood, single or paired ossification centers appear in the xiphoid process.4 Because of the presence of paired ossification centers in the lower portion of the sternal bone (mainly the lower mesosternal segments and the xiphoid process), the lower parts are the most variable portion of the sternal bone. Occasionally, the fusion of the paired ossification centers fails across their midline, resulting in a sternal foramen.19 There may be an incomplete fusion of the lower end of the sternum, resulting in a trifurcated or bifurcated xiphoid process.5\n\nThe incidence of SF varies in different studies from different populations and reported incidence data on SF from different populations ranging from 3.1 to 18.3%.1,2,4,5,7–18 Data from the literature on the incidence of SF are summarized in Table 1. The findings of the current study reveal that the incidence of SF in the Ethiopian population is 19.1%. This is inconsistent with many reported studies from American and European populations.8–10,14,15 Previous studies in Brazil, Greece, and East Africa documented a higher incidence. which is in agreement, although slightly lower than our finding.1,4,5,7,11 The variation in the reported incidence of SF could be due to geographic ethnic, racial, and genetic differences. This finding indicates that SF is a common congenital variation in Ethiopia and highlights the need for strict precautionary measures during sternal procedures in this study population. In addition, such incidental findings during radiologic and autopsy procedures should be properly evaluated to avoid misdiagnosis and misinterpretation of such findings as traumatic or pathological conditions.1,2,4,5\n\nConsistent with previous studies, significant differences were observed in which sternal foramina were more common in men than in women.3,6 In the current study, no foramen was observed in the manubrium or upper mesosternal segments proximal to the level of the fourth CCJ. The most common site of SF was at the level of the fifth CCJ, accounting for more than half of the specimens with SF. This is followed by the fourth CCJ, sixth CCJ and xiphoid process, where SF is observed in 3 specimens at each site. Double sternal foramen was observed in a single male specimen on the sternal body and xiphoid process. This is consistent with a study from Kenya in which SFs were predominantly observed in the lower sternal segments, but differed from studies from Turkey and Greece that revealed more SFs in the xiphoid process than in the lower mesosternal segments.4,5,10 This finding emphasizes the necessity of avoiding or adopting extreme caution during sternal puncture in the lower part of the sternum.\n\nSFs are usually asymptomatic and frequently detected incidentally on radiological images, surgical procedures, postmortem examinations and anatomy teaching specimens.1,2,4,5,10 SF is a common variation that has been ironically referred to as “a finding of no clinical significance”.2,21 Due to this, it is neglected in medical education, and only a few clinicians and acupuncturists are aware of SF.21,22 The proximity of SF to vital adjacent thoracic structures constitutes a fundamental parameter with which clinicians and acupuncturists should be familiar. The iliac crest is the preferred site for bone marrow aspiration; however, extraction from the sternum becomes inevitable in certain circumstances, such as dry taps from the iliac crest or a history of pelvic radiotherapy.6 On the other hand, the practice of acupuncture is considered inherently safe, but serious complications can sometimes occur.2\n\nIn the scientific literature, 14 cases of cardiac and pericardial injuries have been reported during sternal puncture for bone marrow extraction or acupuncture in the lower sternal segment, of which 8 were fatal. A lack of awareness of the sternal foramen was the root cause of all fatal cases.2 Therefore, during sternal interventions, avoidance of needle insertion in the lower thirds of the sternum and evaluation of previous radiographic scans or performing a preprocedural radiographic scan to rule out an SF are recommended.\n\nHalvorsen et al. are to be commended for emphasizing the clinical significance of SF and for stressing the need for awaring medical students and clinicians during anatomy lessons, as well as on demonstrations of sternal puncture techniques.21,22 In terms of acupuncture, the fourth and fifth CCJ levels correspond to commonly used acupuncture points and thus can pose great hazards when inserting acupuncture needles in persons with SF. The current study underpins the necessity for extra caution and avoidance of needle vertical insertion in the lower part of the sternum.2,6 Therefore, an acupuncturist must use a superficial oblique insertion technique to prevent injury to thoracic organs in the study population.\n\nRadiological evaluations can misdiagnose SF as lytic lesions or trauma. Therefore, it is vital for radiologists to be aware of this common variation and its radiographic characteristics to prevent misdiagnosis and subsequent mistreatment. When in doubt, radiologists should use computed tomography or magnetic resonance imaging, which best exposes the anatomical characteristics to enable discrimination, to differentiate SF from traumatic or osteolytic lesions of the sternum.2,3,6\n\nFurthermore, although SF is classically designated as a round or oval defect with a smooth margin, a study in Greece reported a single case of SF with an irregular margin and abnormal shape among five dried sternums with SF.3 In our study, four (22.2%) SFs with irregular margins and abnormal shapes were observed among eighteen dried sternums with SF. Such features can considerably complicate the radiological differentiation of SF and osteolytic lesions due to their remarkable similarity. Therefore, radiologists should be aware of SF even with irregular margins and abnormal shapes. The presence of such lesions must be analyzed with caution, and SF must be considered in the differential diagnosis.\n\nIn this study, an extremely interesting case of a sternal foramen with a beveled margin was observed. To my knowledge, this is the first description of a sternal foramen with a beveled margin. However, in this case, the observed beveling is in complete contrast to the classical feature of a sternal foramen. This characteristic, if present, can considerably complicate forensic interpretation. In medicolegal postmortem examination of skeletonized human remains, SF can be misinterpreted as a projectile entry wound or other penetrating injury or even osteolytic lesion from cancer or infection.2,3,6 If forensic experts and anthropologists are unaware of such anatomic variations, it may result in misclassification of the manner and cause of death.3 SF is a midline defect, usually on the lower part of the sternum, with a round, oval or irregular shape, covered with cortical bone, and sometimes with a beveled margin.\n\nAdditionally, if such variations are routinely documented in the medical record whenever incidentally detected during the radiologic assessment, they can contribute to preprocedural evaluation before invasive sternal procedures. Furthermore, documentation of sternal bone variation is a simple and important potential source of data in the identification process of skeletonized human remains. However, there have been reports of perplexing similarities among members of the same family, highlighting the need for caution in interpretation.4 Therefore, awareness of SF, together with routine documentation of such variations in antemortem medical and radiological records of individuals, can aid in accurate identification and investigation of the manner and cause of death.\n\nThe term xiphoid process is derived from the Greek word “xiphos”, which means a straight sword that corresponds to the pointed end of the sternum. However, the xiphoid process can be absent, broad, bifid, trifurcated, duplicated or deflected. Variant xiphoid morphologies, such as bifid or trifurcated xiphoid processes, are caused by incomplete fusion of the ossification centers.2,5 In the present study, bifid xiphoid processes occurred in 15.6% of cases, which is comparable to a study from Kenya.5 In addition, trifurcated xiphoid processes were observed in a single (1.3%) specimen. Such variations could be misinterpreted as an epigastric mass, fracture, or traumatic fissure.1,5 With increasing traffic-related injuries, clinicians should be aware of xiphoid variations to avoid misdiagnosis as a fracture or traumatic fissure. This necessitates special vigilance when assessing anterior chest wall injury, and it may therefore be considered a differential diagnosis. Furthermore, routine documentation of such variations, whenever detected incidentally, in antemortem medical records of individuals can aid in the identification of skeletonized human remains.\n\n\nConclusions\n\nThe sternal foramen and variation in xiphoid morphology are common anatomical variations in Ethiopia. The findings of the current study highlight the necessity of strict precautionary measures during sternal procedures in this study population. Clinicians, radiologists, and anthropologists need to be aware of these variations, and their presence should be taken into consideration in everyday practice. Moreover, the presence of irregular and beveled sternal foramina considerably complicates radiological and anthropological differential diagnosis. Therefore, such incidental findings during radiologic and autopsy procedures should be properly evaluated to avoid misdiagnosis and misinterpretation of such findings as traumatic or pathological conditions.", "appendix": "Data availability\n\nFigshare: Anatomical variations of the sternum: sternal foramen and variant xiphoid morphology in dried adult human sternum in Ethiopia, https://doi.org/10.6084/m9.figshare.23498958.v1. 23\n\nThis project contains the following underlying data:\n\n- Anatomical variations of the sternum: sternal foramen and variant xiphoid morphology in dried adult human sternum in Ethiopia.xlsx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nKirum GG, Munabi IG, Kukiriza J, et al.: Anatomical variations of the sternal angle and anomalies of adult human sterna from the Galloway osteological collection at Makerere University Anatomy Department. Folia Morphol. (Warsz). 2017; 76(4): 689–694. PubMed Abstract \| Publisher Full Text\n\nBabinski MA, de Lemos L , Babinski MSD, et al.: Frequency of sternal foramen evaluated by MDCT: a minor variation of great relevance. Surg. Radiol. Anat. 2015; 37(3): 287–291. PubMed Abstract \| Publisher Full Text\n\nGkantsinikoudis N, Chaniotakis C, Gkasdaris G, et al.: Morphological approach of the sternal foramen: an anatomic study and a short review of the literature. Folia Morphol. (Warsz). 2017; 76(3): 484–490. PubMed Abstract \| Publisher Full Text\n\nParaskevas G, Tzika M, Anastasopoulos N, et al.: Sternal foramina: incidence in Greek population, anatomy and clinical considerations. Surg. Radiol. Anat. 2015; 37(7): 845–851. PubMed Abstract \| Publisher Full Text\n\nEl-Busaid.: Sternal foramina and variant xiphoid morphology in a Kenyan population. Folia Morphol. (Warsz). 2012; 71(1): 19–22. PubMed Abstract\n\nChoi PJ, Iwanaga J, Tubbs RS: A comprehensive review of the sternal foramina and its clinical significance. Cureus. 2017; 9(12): e1929. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAshley GT: The human sternum: the influence of sex and age on its measurements. J. Forensic Med. 1956; 3: 27–43. Reference Source\n\nCooper PD, Stewart JH, McCormick WF: Development and morphology of the sternal foramen. Am. J. Forensic Med. Pathol. 1988; 9(4): 342–347. PubMed Abstract \| Publisher Full Text\n\nSchratter M, Bijak M, Nissel H, et al.: The foramen sternal: a minor anomaly–great relevance. ROFO Fortschr Geb Rontgenstr. Nuklearmed. 1997; 166(1): 69–71. PubMed Abstract \| Publisher Full Text\n\nYekeler E, Tunaci M, Tunaci A, et al.: Frequency of sternal variations and anomalies evaluated by MDCT. AJR Am. J. Roentgenol. 2006; 186(4): 956–960. PubMed Abstract \| Publisher Full Text\n\nBabinski M, Rafael F, Steil A, et al.: High prevalence of sternal foramen: quantitative, anatomical analysis and its clinical implications in acupuncture practice. Int. J. Morphol. 2012; 30(3): 1042–1049. Publisher Full Text\n\nIshii S, Shishido F, Miyajima M, et al.: Causes of photopenic defects in the lower sternum on bone scintigraphy and correlation with multidetector CT. Clin. Nucl. Med. 2011 May; 36(5): 355–358. PubMed Abstract \| Publisher Full Text\n\nSingh J, Pathak RK: Sex and age related non-metric variation of the human sternum in a Northwest Indian postmortem sample: a pilot study. Forensic Sci. Int. 2013 May 10; 228(1–3): 181.e1–181.e12. PubMed Abstract \| Publisher Full Text\n\nMacaluso PJ Jr, Lucena J: Morphological variations of the anterior thoracic skeleton and their forensic significance: radiographic findings in a Spanish autopsy sample. Forensic Sci. Int. 2014; 241: 220.e1–220.e7. PubMed Abstract \| Publisher Full Text\n\nBayaroğulları H, Yengil E, Davran R, et al.: Evaluation of the postnatal development of the sternum and sternal variations using multidetector CT. Diagn. Interv. Radiol. 2014; 20(1): 82–89. PubMed Abstract \| Publisher Full Text\n\nBoruah DK, Prakash A, Yadav RR, et al.: The safe zone for blinded sternal interventions based on CT evaluation of midline congenital sternal foramina. Skelet. Radiol. 2016 Dec; 45(12): 1619–1628. PubMed Abstract \| Publisher Full Text\n\nKuzucuoglu M, Albayrak I: Topographic evaluation of sternal foramen patients with thoracic computed tomography. Surg. Radiol. Anat. 2020; 42: 405–409. PubMed Abstract \| Publisher Full Text\n\nBolatli G, Dogan NU, Koplay M, et al.: Evaluation of sternal morphology according to age and sex in multidetector computerized tomography. Anatomy. 2020; 14(1): 29–38. Publisher Full Text\n\nFokin AA: Cleft sternum and sternal foramen. Chest Surg. Clin. N. Am. 2000; 10(2): 261–276. PubMed Abstract\n\nCunningham C, Scheuer L, Black S: Developmental juvenile osteology. Academic Press; 2016.\n\nWolochow MS: Fatal cardiac tamponade through congenital sternal foramen. Lancet. 1995; 346(8972): 442. PubMed Abstract \| Publisher Full Text\n\nHalvorsen TB, Anda SS, Naess AB, et al.: Fatal cardiac tamponade after acupuncture through congenital sternal foramen. Lancet. 1995; 345(8958): 1175. PubMed Abstract \| Publisher Full Text\n\nLema A: Anatomical variations of the sternum: sternal foramen and variant xiphoid morphology in dried adult human sternum in Ethiopia.xlsx. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "260376", "date": "03 Apr 2024", "name": "Andrea Sonaglioni", "expertise": [ "Reviewer Expertise Echocardiography" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 provided baseline epidemiological information about the sternal foramen (SF) and variant xiphoid morphology in Ethiopia, comparing results with other populations and discussing the clinical and forensic significance.\n\nThe Authors exhaustively described all the anatomical variants and clinical characteristics.of SF. The Authors reported an incidence of SF in the Ethiopian population of 19.1%; sternal foramina were more common in men than in women.\nGiven that SF is a common variation that has been ironically referred to as “a finding of no clinical significance\", it is usually neglected in medical education, and only a few clinicians and acupuncturists are aware of SF. However, it is important to differentiate SF from traumatic or osteolytic lesions of the sternum. Moreover, from a clinical point of view, when inserting acupuncture needles, given that the sternal foramen is more commonly localized in the lower part of the sternum, it is important to avoid a needle vertical insertion in the lower part of the sternum. An oblique insertion technique may prevent injury to thoracic organs\". I have only a suggestion for the Authors. In the Discussion section, after the sentence: \"An oblique insertion technique may prevent injury to thoracic organs\", the Authors could also add the potential usefulness of an echocardiographic monitoring during the needles' insertion procedure. In fact, the transthoracic parasternal long-axis view might be particularly useful for assessing the antero-posterior thoracic diameter, measured from the true apex of the sector (the point of entry of ultrasound) and the spine, the latter visualized behind the posterior wall of the descending aorta. The cardiac chambers are all visualized within this simple echocardiographic section and their integrity may be verified during the needles' insertion procedure. The Authors could cite following reference: Sonaglioni A, et. al., 2018 (Ref 1)\n\nIs the work 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": "230958", "date": "25 May 2024", "name": "Holger Wittig", "expertise": [ "Reviewer Expertise general forensic pathology", "forensic anthropology", "forensic radiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nScientific papers on morphological variations of the sternum are rare. Therefore, it is welcoming that this paper reports variations of the sternum in an Ethiopian population. The introduction mentions the high variability and, in the forensic context, its application for identification. However, corresponding references regarding identification are missing in the literature, such as: (Weiss G et al,2018) [Ref 1] or (Verna, E. et al, 2013) [Ref 2] or (Leite, V. M., et al,2020) [Ref 3] The statistical analysis of the data is perplexing. Presenting an average age with four decimal places (38.383 ± 11.3840 years) implies a level of precision that may be misleading and is highly uncommon. Age should not be specified beyond one decimal place, and a better choice would be to provide the median as a measure of central tendency. Foramina in the sternum result from incomplete fusion of sternal ossification centers during embryogenesis. Therefore, foramina in the sternum are congenital. Reporting significant age differences in the frequency of foramina can only be a random pseudo-result due to sampling errors. The quality of the photos is poor. The photos are unevenly illuminated (figures 2 and 3), the background is unclean (figures 1, 3, 5), and figure 4 appears blurry. Post-processing of the photos (black and white, background cleaning) or the use of scientific drawings is strongly recommended. Conventional X-ray images would also be an option. The statement asserting that variations in the sternum could contribute to determining the manner and cause of death lacks explanation. One can imagine that this might be possible in gunshot or stab injuries. However, anatomical variations seldom explain the cause of death. Especially in the Discussion section, there are many redundant, partially unsupported statements. The section should be significantly shortened. The epidemiological data on morphological variations is welcomed. However, in its current form, a substantial revision of the paper is needed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "276734", "date": "05 Jun 2024", "name": "Sven A Holcombe", "expertise": [ "Reviewer Expertise Computational anatomy and biomechanics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a thorough investigation into the presence of sternal foramen (SF) in adults from a localized Ethiopian population. The sampling methodology is sound and statistical analysis appropriate. The manuscript is clear and well written. The authors find that their studied population has an SF incidence of ~19%, marginally higher than the previously described highest incidence (~18%). While this is remarked upon (as an outlier), as a reviewer I am not inclined to suspect a regional association with the presence of SF unless the data across multiple studies starts to show more concrete patterns and at higher population counts. (e.g., a chance change of a single observation from this study would put this study back within the range of previous studies).\nNote also that Table 1 lists the most recent paper's population (Bolatli 2020) as coming from an Australian source whereas it seems to instead be from Turkey. Please review this table for accuracy. This is another reason for my reluctance to see a regional difference in SF presence: data just from one country (Turkey) but different populations/methodologies ranges from 4.5-16.8% across multiple studies. I would suspect variation in selection criteria or examination technique before other biological factors.\nOther than that, just a few typos as suggested changes.\nMethods: Brocken -> Broken\nResults: The xiphoid process ended as a single in 83.1%\nIn 15.6% (n=12/77) of the samples, the xiphoid process was bifurcated and trifurcated ...\nThe xiphoid process was bifurcated in 15.6% (n=12/77) of the samples, and trifurcated ...\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-934
https://f1000research.com/articles/12-933/v1	07 Aug 23	{ "type": "Systematic Review", "title": "Perspectives in fraud theories – A systematic review approach", "authors": [ "Christianna Chimonaki", "Stelios Papadakis", "Christos Lemonakis", "Stelios Papadakis" ], "abstract": "Background: Increasing progress is being made in the field of accounting fraud, and extensive theoretical research is needed to develop future research topics using trend analysis. Our research consists of a literature review that examines the most common fraud theories and attempts to interpret the characteristics of human behaviour that lead to fraud as well as current methods of detecting corporate fraud. Methods:\n\nWe searched the Scopus database for articles on a fraud theory. We analyse articles published between 2004 and 2022, using a keyword search for ‘Fraud Triangle’, ‘Fraud Diamond’, ‘Fraud Pentagon’, and ‘Fraud Hexagon’. Furthermore, we include all document types like articles, conference papers, reviews, book chapters, conference reviews, notes, and data papers. The investigation was limited to papers published in English from 2004 to 2022, not including the current year 2023, as documents are still being published. The last research was done at the end of January 2023. The results from the above criteria are to collect 302 papers. We used VOS program viewer in our bibliometric analysis. Results: According to our network analysis, the Fraud Diamond theory seems to be the most functional fraud theory. According to our findings of the published articles, the main human behavioural characteristics that can lead a manager to commit fraud are the components of the fraud diamond theory: capability - opportunity - pressure - rationalization. Thus, the fraud diamond theory analyses more than the fraud triangle, pentagon, and hexagon theories. So human behavioural characteristics have a positive effect and can lead to fraud in companies. Conclusions: Future research needs to analyse more Pentagon and Hexagon fraud theories, which are more recent and have not yet been analysed in detail. Also, future research needs to analyse more of the human behaviour characteristics related to the Pentagon and Hexagon fraud theories.", "keywords": [ "fraud theories", "corporate fraud", "network analysis" ], "content": "Introduction\n\nIn recent literature, accounting fraud is associated with sustainable and ethical management. Fraud scandals are widespread in the global economy. Fraudulent financial statements represent unethical management and harm the long-term profitability of a company and its shareholders. Sustainability and transparency are fundamental concerns for businesses. Transparency is an indispensable quality in the business world. According to agency theory, conflicts between managers and their stakeholders lead to fraud. As a result of inadequate corporate governance, managers still commit fraud today. By using fraud theories to interpret human behaviour, we combine ethical and sustainability concerns in our research. Existing accounting literature explains fraud theories and attempts to quantify and analyse human behaviour. Human behaviour is an essential factor in fraud theories and connects inseparably with fraudulent financial statements, as the preparators of financial statements are humans. Consequently, if they have the factors described in fraud theories, they can lead a company more easily to fraud. In addition, fraud theories attempt to explain why managers commit fraud. What is the motive for committing fraud? What is the impact of financial statement fraud on the economy?\n\nThe field of financial statement fraud continues to evolve, and extensive theoretical research is needed to develop future research topics using trend analysis. There are numerous articles in the literature that focus on the analysis of financial statement fraud and corporate fraud. For example, Hogan et al. (2008) examined the fraud triangle based on related articles on financial reporting fraud and showed the characteristics of a fraudulent company and the function of an auditor’s opinion in detecting and preventing fraud. Trompeter et al. (2013) extend the study of Hogan et al. (2008) earlier. They focus on administration, ethics, criminology, and psychology.\n\nIn addition, Amiram et al. (2018) discuss the findings and trends in the accounting fraud literature. They describe in more detail the various findings and methods found in the fraud literature. In addition, Uysal (2010) examined business ethics research articles using a bibliometric analysis. In addition, Uysal (2010) determined the citation frequency of business ethics-related articles and used a co-citation analysis to determine the communication patterns in this research area. Accounting literature researchers examine and synthesize numerous models, qualitative, quantitative, and textual analyzes to determine how accounting fraud can be detected. On the other hand, there are a small number of researchers who examine future issues and trends in accounting fraud research.\n\nIn a comprehensive literature review, the research contributes to the study of accounting fraud theories and indicates the trends in the literature. These are the research questions:\n\n1. Do a company’s ethical issues conflict with its sustainability issues? is the first research question.\n\n2. How can fraud be detected by studying human behaviour using fraud principles? is the second research question.\n\n3. What methods in the accounting literature are associated with theories of human behaviour and fraud? is the third research question.\n\nFraud theories\n\nIn the global economy, the phenomenon of accounting fraud and corporate fraud can be observed. Numerous fraud theories explain fraud as a factor of human behaviour. This paper examines fraud in light of the prevailing fraud theories.\n\nCressey examined the first theory of fraud (1953), where Cressy establishes a link between fraud and human behaviour. The ‘fraud triangle theory’ has been established as Cressey’s (1953) model in which three conditions that can lead to fraud are analysed; Opportunity, pressure, and rationalization. Fraud is essentially motivated by external forces. The motivation for managers to commit fraud is ‘pressure’. Cressey (1953), Albrecht et al. (2006), Lister (2007), and Manurung and Hadian (2013) all support the notion that non-financial and financial pressures motivate fraud by managers. In addition, according to SAS No. 99 (Ramos and West, 2003), external pressure, financial goals, personal financial need, and financial stability are motives for fraud by managers. According to Cressey (1953), Wilson (2018), Wolfe and Hermanson (2004), and Kelly and Hartley (2010), a fraudster’s opportunity lies in his or her capabilities. The fraudster can identify the structural weaknesses of the company and then break the trust. Inadequate corporate governance, a lack of internal control, and a lack of control by responsible personnel are conditions for opportunity development. According to SAS No. 99, there are three categories of accounting fraud, which are distinguished by organizational structure, sector and inadequate supervision. The final component of the fraud triangle is rationalization. Rationalization is an attitude based on a set of moral beliefs. The actions of fraudsters are justified by their beliefs. In summary, Cressey (1953) stated that the three components of pressure-chance-rationalization constitute the behavioural characteristic of a cheater.\n\nWolfe and Hermanson (2004) added a new component, ‘capability’, to the fraud triangle theory. Wolfe and Hermanson (2004) assumed that ‘capability’ refers to the personal skills and characteristics that lead administrators to commit fraud. The new fraud theory is known as the ‘Diamond Fraud’ theory. According to Wolfe and Hermanson (2004), the fraud diamond theory suggests that ‘opportunity’ opens the door to fraud. Motive and justification determine the individual who commits fraud. Therefore, the individual must have the ‘capability’ to recognize and see the ‘opportunity’ to commit fraud.\n\nFurthermore, Crowe (2011) extends the fraud triangle to include ‘arrogance’ and ‘competence’. The new fraud theory is referred to as the ‘Fraud Pentagon Theory’ and consists of five elements: ‘Opportunity - Pressure - Rationalization - Competence – Arrogance’. Crowe’s (2011) theory extends Wolfe and Hermanson (2004) by adding two new components ‘competence’ and ‘arrogance.’ More specifically a person’s ‘competence’ is his or her measure to perform a capability, in this cause, fraud. ‘Arrogance’, on the other hand, is a human behavioural trait that dominates an organization and overrides internal rules. This person is able to develop fraud strategies and has the goal of enriching themselves and winning.\n\nVoussinas developed the latest fraud theory (2019). This fraud theory is known as the ‘Fraud Hexagon Theory’ and consists of the following six components: stimulus, opportunity, rationalization, capability, collusion, and ego. According to Vousinas (2019), the explanation of these components is as follows: ‘Stimulus’ is the incentive for a leader to commit fraud; ‘Capability’ is the ability required to commit fraud; ‘Opportunity’ is the opportunity to commit fraud; ‘Ego’ is a characteristic of human behaviour; and ‘Collusion’ is the agreement of people to commit fraud and justify their actions, which is ‘rationalization’.\n\n\nMethods\n\nWe searched for documents with the fraud theories within as keywords in article titles, abstracts, and keywords. We perform a keyword search for ‘Fraud Triangle’, OR ‘Fraud Diamond’ OR ‘Fraud Pentagon’, OR’ Fraud Hexagon’ as each of the key theories previously discussed. Also, we search all open access types available to the Scopus database and all citation information. In addition, we select bibliographical information as affiliations, serial identifiers, publishers, editors, and some other information as tradenames and manufacturers, conference information, including references and accession numbers. Also, in this research, we include all authors who have examined fraud theories and all the subject areas, for example, ‘business management and accounting and ‘social sciences’.\n\nFurthermore, we include all document types like articles, conference papers, reviews, book chapters, conference reviews, notes and data papers. Also, we include documents in the final publication stage and articles in the press. We include countries such as the United States, Indonesia, Malaysia, Australia, United Kingdom, Canada, China, Ghana, France and Germany. The investigation was limited to papers published only in English from 2004 to 2022. We did not include in our research the current year 2023, as documents are still being published. The last research was done at the end of January. The results from the above criteria are to collect 302 papers.\n\nIn continuing for each document of 302 papers, we use the author’s keywords of each paper and the abstracts. Then we export the data in the CSV file and use the VOS viewer program for our bibliometric analysis.\n\nAfter importing the CVS file into the VOS viewer program, we choose the type of analysis and counting method. From this section, we analyse all the keywords of selected articles. The results of selected keywords appeared in Table 1. In this research, we created a network synthesis of keywords from 302 published articles. In creating the network, we only summarized the keywords that the researchers indicated occurred more than twice in the articles. Since authors sometimes use different words with similar meanings, we removed keywords with synonymous meanings. In more detail, keywords with synonymous meanings were excluded, such as ‘internal control’ and ‘internal controls’, ‘financial statement fraud’ and ‘fraudulent financial statements’, as shown in Table 1. Then a word cloud was created with the most frequent keywords from 2004 to 2022, as shown in Figure 1.\n\nThen we classify the keywords into clusters as shown in Table 5. A group of keywords is called a cluster and is included in the map. Clusters are not overlapping in the VOS viewer program. Clusters do not necessarily fully cover all keywords on a map. Consequently, there may be keywords that do not fit into any cluster. Thus, a keyword may fit into only one cluster. Clusters are identified by cluster numbers.\n\nFurthermore, we create a map based on text data. More specifically, we select all abstracts of the 302 papers related to fraud theories, and we use the binary country method to choose the threshold. We use the option to create a term co-occurrence map based on text data. Then we choose the field of abstracts and the most frequent words in analysing abstracts. The results are shown in Table 6. Also, we create clusters in the VOS viewer program as shown in Table 7.\n\nWe use multidimensional scaling to create the bibliometric map to increase the relatedness and similarity of elements (keywords) in a low-dimensional space. According to Van Eck et al. (2006) and Van Eck and Waltman (2007), the association strength for similarities (sij) is calculated by equation 1.\n\nFor every set of keywords (item) i and j, VOS involves as input a similarity sij (sij ≥ 0). The similarities sij as weights calculated on a ratio scale. The locales of keywords (items) in a map minimizing by equation (2).\n\nSubject to:\n\nThe purpose is the weighted sum to reduce the sum of the squares’ distance among every set of keywords (items). The weighted similarity among items is the squared distance between a pair of items. The average distance between two items equals one; to avoid solutions, all items have the exact location—the objective function given by equation 4, which is the ideal location of an item. The equation for the ideal location is shown in equation 4.\n\nThe ideal position of item i is defined as the weighted average of the positions of all other items. The ideal position of a keyword should occupy the most natural position that an item can have. Thus, the items appear to have the most desirable position that is closest to the ideal position described in Equation 2. The constraint in Equation 3 assumes that the positions of all keywords except the keyword (item i) are static and ignored. Equation 4 also describes the minimization of the objective function.\n\nIf the positions of all keywords i are static and the constraint is ignored, the minimization of the objective function has the effect of placing keyword i at its best position. Since the keywords have no static position and the solution is defined by the objective function and the constraint, we assume that the elements are not in the ideal position. So, according to the objective function, the keywords (elements) are placed no less than at the ideal position.\n\nSo, a map was created based on the counting technique and co-occurrence analysis with all keywords from 302 articles on fraud theories. The counting technique and co-occurrence analysis are used to determine the threshold value. The number of keywords to be selected is 48, and the lowest number of keywords is five out of the 988 keywords, 48 is the threshold. For each of the 48 keywords, the total strength of co-occurrence links with other keywords is estimated. The keywords with the largest total link strength are selected and listed in Table 4 below.\n\nMetrics and methods of human behaviour\n\nExternal pressures arise when managers want to respond to third-party demands. Managers may increase debt to external sources to remain competitive under these conditions. Another measure of external pressure in the fraud accounting literature is leverage and the ratio of total liabilities to total assets. Tiffani and Marfuah (2015), Manurung and Hardika (2015), and Skousen et al. (2009) suggest that the total debt/total assets ratio has a positive impact on financial statement falsification. Aprilia (2017) supports that a high debt to total assets ratio of a company can put pressure on the management team. When the company’s debt level is high, managers feel threatened by bankruptcy, which leads to pressure on management. External competitive pressure can cause managers to increase the company’s debt in order to stay in the market. Pressure can be referred to as another fraud factor.\n\nThe pressure/stimulus metrics for human behaviour created by Supri et al. (2018) and Sunardi and Amin (2018) provide evidence that financial goals can pressure management to achieve financial goals. In general, financial stability is a key issue for management. When financial stability is affected by economic conditions, managers feel pressured, which can lead to falsified financial statements. In the literature on accounting fraud, financial instability is measured by differences in total assets. If the differences in total assets are frequent in each year, it means that the company is in an unstable situation. Tiffani and Marfuah (2015), Manurung and Hardika (2015), and Skousen et al. (2009) also associate financial stability with falsification in financial reporting.\n\nPersonal financial needs are another human behavioural factor that can lead a company’s management to commit fraud. Tiffani and Marfuah (2015) pointed out that when the role of management is unclear, such as when the manager becomes part of the shareholders or participates in the board of directors’ commission, personal financial needs increase and may influence financial reporting. The ratio that can be used to measure own financial needs is the sum of shares held by insiders/total ordinary shares outstanding.\n\nReturn on equity (ROE) has been shown by Summers and Sweeney (1998), Brazel et al. (2006), Okoye et al. (2009), and Dechow et al. (2011) that it affects the percentage of fraud. A lower ROE means that the company has not attracted the attention of investors. In other words, the company management can edit the financial reports to attract more investors.\n\nSetiawati and Baningrum (2018) found that companies with negative return on assets (ROA) tend to have poor economic performance. In another study, Emalia et al. (2020) supported that negative ROA does not meet financial objectives and that a company’s management team can manage financial reporting to improve the company’s financial performance. The human behaviour capability studied by Beasley (1996) supports that external board members lead to more effective management oversight and serve as a fraud prevention measure. Albrecht et al. (2010) also confirmed Beasley’s (1996) statement.\n\nAccording to the literature, the last pressure factor is when a company seems to have liquidity problems. Persons (1995) proved that low current ratio leads to lower liquidity of a company’s assets and consequently a company is more vulnerable to short-term solvency.\n\nAccording to fraud theories, capability is a component that can lead to fraud. Wolfe and Hermanson (2004) and Manurung and Hardika (2015) show that board changes can be a temporary period that causes stress and can lead to fraud. In addition, effective monitoring is one way for organisations to reduce the likelihood of fraud. Beasly et al. (2010) support that an audit committee with independent members can reduce the likelihood of fraud. Tiffani and Marfuah (2015) also suggest that an effective monitoring mechanism can reduce the risk of fraud. The measure of effective monitoring is the percentage of independent committee members.\n\nSkousen et al. (2009) and Miller et al. (2012) investigated the possibility of human behaviour and demonstrated that claims have high inherent risk and can be manipulated. Their research was along the lines of Loebbecke et al. (1989), who reached the same conclusion. Thus, a high accounts receivable ratio may indicate manipulation in financial statements. Moreover, as described above, effective monitoring may measure the opportunity component of fraud theories.\n\nSykes and Matza (1957) suggested that a firm’s violation of internal regulations can be justified by rationalisation techniques. According to Skousen et al. (2009), rationalisation can be a measure when the auditor’s opinion affects the appearance of financial reporting. When the auditor’s opinion on the presentation of financial reporting includes the comment that it is not fair because it does not follow Generally Accepted Accounting Principles (GAAP), the management team tends to justify it (Lokanan and Sharma, 2018).\n\nAnother characteristic of human behaviour is integrity. Integrity can be measured by earnings management, sales history, and earnings growth. Revenue management occurs when management fails to meet financial goals. It is then possible to manipulate accounting principles (GAAP) to produce financial reports. Also, managers can exploit the flexibility of accounting rules and manipulate the level of earnings (Manurung and Hadian, 2013). In addition, integrity can be measured by sales performance. Assets support sales, so an increase in sales leads to an increase in assets (Brigham and Houston, 2006). If the sales growth ratio is positive, the company is viable. The sales growth ratio is also indicated as the market share or position of the company in the market. There are many ways for managers to manipulate sales, and sales growth ratio has increased in fraudulent financial reporting. Another measure of integrity is the earnings growth rate - usually measured by ROE (return on investment) or earnings per share. Generally, the profit growth rate measures the manager’s valuation. Managers can affect the cost of goods and sales sold, interest and operating expenses, and income tax. According to agency theory and the various conflicts, managers can manipulate all of the above ratios and present a company’s financial performance differently.\n\nThe metric used in the accounting literature for ego is how many images and information of the CEO are related to the company’s performance. According to Apriliana and Agustina (2017), the CEO applies the internal rules of a company. Thus, if a CEO is characterized by arrogance, this may act as a fraud factor.\n\nCollusion is described in the Hexagon Theory and supports the link between fraudulent financial reporting and collusion. Collusion occurs when more than two individuals engage in deceptive practices to achieve a goal for their benefit - the members’ agreement involves money or other benefits to facilitate their work. The law opposes collusion because it is only for personal gain. When collusion exists, the likelihood of fraud increases. The main characteristics of collusion are, first, payments to management or employees to gain money or goods, and second, the presence of an intermediary for the acquisition of services and goods.\n\nTable 3 summarizes all of the above measures used by researchers to detect fraud according to fraud theories. Many researchers have used regression models to examine the relationship between human behavioural characteristics and fraud theories.\n\n\nResults\n\nWe therefore captured 302 papers. Figure 1 shows the main keywords used in the search for fraud theories. Figure 1 shows the most frequent fraud theories authors have examined, and we help us determine the literature gap among fraud theories. Also, Figure 1 shows the relation or connection between fraud theories and the characteristics of human behaviour. In our bibliometric analysis, we use it as an additional result to answer the second research question.\n\nIn addition, fraud theories have attracted the attention of the research community over the years. As Publications on fraud theories from 2004 to 2022 are shown in Figure 2. Figure 2 shows all the publications contained in the sample of 302 articles as described in the methods section. We have not excluded any publication.\n\nTable 2 also shows the journals with the most publications from 2004 to 2022 on fraud theories. According to the interest in fraud theories, the journals have become more specific. According to the Scopus database, the journals that have published topics related to fraud theories and human behaviour are shown in Table 2 below. The criterion of the selection of this journal is the number of articles which have been published from the period 2004 to 2022.\n\nSkousen et al. (2009) examined the components of the fraud triangle based on SAS NO 99. Their research used a sample of fraud companies and matched them with non-fraud companies. Skousen et al. (2009) developed a broad range of variables as proxies of the components of the fraud triangle. They used the Univariate analysis to determine eight variables for the component of pressure and five variables for the component of opportunity. According to their results, change in assets, cash from operations -average capital expenditure/current assets, net cash flow from operating activities- cash dividends-capital expenditure are significant variables for external pressure. The variable of the percentage of ownership is significant for the component of personal financial need. For the component of opportunity, the nature of the industry and a dummy variable of CEO when simultaneously CEO hold, and managerial position are the most significant variables for the measurement of opportunity. Their results appear to improve over Persons (1995) to predict fraud substantially. The weakness of this research is the inability to explore significant variables for the components of rationalization.\n\nAlso, Manurung and Hardika (2015) studied empirical evidence for the components of the fraud triangle. According to their results, the most significant variables for detecting fraudulent financial statements are asset growth (for financial stability), ROA, the measurement of financial target and the leverage ratio as a measurement of external pressure. The study of Manurung and Hardika (2015) is consistent with Skousen et al. (2009).\n\nOn the other hand, Khamainy et al. (2022), examined the fraud diamond theory. Khamainy et al., the nature of industry and sales history significantly affect fraudulent financial statements. So, a higher increase in receivables and sales and the number of management-owned shares are suspicious ratios to investigating fraudulent financial statements. Effective monitoring negatively and significantly impacts fraudulent financial statements for the opportunity component. This result indicates that more members as independents can lead to more unbiased financial statements. Ratios of changes in total assets (a measure of financial stability), total debt/shareholder equity (external pressure), ROA (financial targets), earnings management (personal integrity) and changes of directors (capability) have no significant effect on the prediction of fraudulent financial statements. Khamainy et al. (2022), suggest further research to combine and compare more sectors as their research investigates only manufacturing firms.\n\nOkoye et al. (2009) examined the impact factor of the fraud triangle in the audit procedure. More specifically, Okoye et al. (2009) examined an audit plan as a framework for audit procedure. Their results are consistent with the fraud triangle factors and prove the importance of personal integrity. In addition, Brazel et al. (2006) examined the relationship between non-financial and financial data and concluded that fraud companies have a higher percentage of revenue growth.\n\nZainudin and Hashim (2016) investigated financial ratios to identify fraudulent financial statements. The results of this research show that ratios such as total debt/total equity, total debt/total assets, net profit/ revenue, receivables/revenue, inventory/total assets, working capital/total assets, revenue/total assets and the logarithm of current assets/total assets are significant indicators in the research of fraudulent financial statements. The weakness of this study was the small sample and the hand-collected data, which limited this research.\n\nIn another survey, Akbar (2017) examined the Pentagon theory for Indonesian companies. He concluded that the factors of pressure, which are: ROA (financial target), changes in total assets (financial stability), leverage ratio (external pressure), and institutional ownership, are the most significant, which can lead to fraudulent financial statements. The results of this research are in the same line as those (Apprilia, 2017) and (Tiffani & Marfuah, 2015). In addition, Quraini and Rimawati (2018), examined the fraud pentagon theory and concluded that ROA (financial stability), ineffective monitoring (opportunity), changes in director and auditor, institutional ownership and CEO’s picture in the annual report did not affect fraudulent financial statements. Also, their research suggests continuing with a sample of the private sector, as their research sample was public government firms. The following table summarizes all the above.\n\nWe use the counting method and co-occurrence analysis to answer the first two research questions, “Do ethical issues conflict with sustainability issues in a company?” In addition, the second research question is “How can fraud be detected by studying human behaviour using fraud theories?”\n\nTotal link strength indicates the number of articles in which the two keywords appear. The total link strength is a positive mathematical value. The larger this value, the stronger the link. According to our results, rationalization, opportunity, corruption, and pressure are the most important human characteristics that can lead to corporate fraud. Ethics also has a relatively high total link strength.\n\nThen we classify the keywords into clusters as shown in Table 5. A group of keywords is called a cluster and is included in the map. Clusters are not overlapping in the VOS viewer program. Clusters do not necessarily fully cover all keywords on a map. Consequently, there may be keywords that do not fit into any cluster. Thus, a keyword may fit into only one cluster. Clusters are identified by cluster numbers.\n\nNodes denote the keywords of each research article in a network diagram. A link in a network is one of the links between nodes in Figure 1. Keywords are nodes and have different attributes. Weight (link, total link strength, and occurrence) are key attributes. When keywords have been assigned to clusters, cluster numbers are an instance of an attribute. Numeric values represent these attributes. Weight attributes are non-negative values. Thus, the weight of a keyword should indicate the importance of the keyword. A keyword with a higher weight is interpreted as more significant than a keyword with a lower weight. There are also two weighting attributes in cluster analysis: The overall link strength and the links attribute. For a given keyword, the total link strength and links attributes indicate the number of links of a keyword with other keywords and the total strength of links of a keyword with other keywords.\n\nThus, according to our results, human behaviour and ethics are associated with fraud. The column of link attribute shows the number of keyword links with fraud theories. The total link strength attribute shows the total strength of keyword links of fraud theories.\n\nThe lowest number of occurrences of a keyword is 10 out of the 5982 entries; 182 meet the threshold. A relevance score is estimated for each of the 182 items. Based on this score, the most relevant keywords are selected and presented in Table 6 below. The number of keywords to be selected is 109.\n\nUsing the same procedure as described above, we classify the abstracts into clusters as shown in Table 7 - a set of keywords from the abstracts form clusters in the map. According to our results in Table 7, the keywords from the abstracts form four different clusters. The first cluster contains 13 keywords, “effect, ability, necessity, accounting fraud, board of directors, fraud diamond, financial target, financial stability, accounting fraud, effectiveness, external pressure, arrogance”, which are related to human behaviour and accounting fraud. It is also clear from the first cluster that financial goals and stability are key motivations for fraud. The second cluster establishes a link between the social impact of fraudulent financial statements and the prevention of fraud by auditors. The third cluster includes six keywords – ‘author, crime, corruption, cressey, financial fraud, fraud, behaviour’-and links human behaviour to corruption and crime. The last cluster contains only two terms - keywords: ‘employee fraud, asset misappropriation’ and connects asset misappropriation with employee fraud. So, from all the above clusters, we can conclude the relationship between human behaviour characteristic and fraudulent financial statements.\n\n\nDiscussion\n\nIn this study, we used network analysis to examine whether ethical issues are at odds with issues of corporate sustainability; second, whether fraud is detected by examining human behaviour by linking fraud theories; and third, what methods are used in the accounting literature and are linked to human behaviour and fraud theories. We used the Scopus database from 2004 to 2022; our sample includes 302 articles. As part of this research, we created a network synthesis of keywords from 302 published articles. In creating the network, we summarized only the keywords provided by the authors that occurred more than twice in the articles. Then, we created a map based on the counting method and coincidence analysis of all keywords from 302 articles related to fraud theories. We use the counting method and co-occurrence analysis. Then, we create a map based on text data with the same sample of 302 articles on fraud theories and use the binary country method to choose the threshold.\n\nHuge enterprises have more transactions than small and medium enterprises; consequently, accounting system control becomes more complex. Despite the convenience and usefulness of the accounting system, many transactions are easier to accounting errors and corporate fraud. Corporate fraud can cause a company to go bankrupt and lose the trust of investors and stakeholders. Companies therefore need to be critical and proactive to detect and prevent corporate fraud. Digital transformation has brought many changes to accounting, so fraud prevention has piqued the interest of managers and researchers.\n\nDetecting corporate fraud was difficult with previous technologies because they could not fully account for the various factors of human behaviour and the potential risk of developing digital forensic technologies. Big data analytics and artificial intelligence play a critical and pivotal role in corporate fraud detection because they can manage massive amounts of data. Therefore, artificial intelligence, big data analytics, blockchain, and machine learning are welcomed by practitioners and researchers as they can effectively detect corporate fraud. Therefore, researchers are investigating the new technologies to combat corporate fraud. Li et al. (2020) and Fassia (2019) use blockchain technology and big data analytics to predict corporate fraud. Li et al. (2020) also supports that big data analytics can uncover accounting errors and detect fraud by auditors and companies because artificial intelligence can detect fictitious transactions faster than auditors. In addition, another part of the researchers uses machine learning to develop a new model to detect corporate fraud (Bao et al., 2020; Brown et al., 2020).\n\nOur research attempts to fill the gap in the literature review because, according to Yu and Rha (2021), research investigating accounting fraud is increasing, but there is not enough research integrating practises and theories. This thesis contributes to the literature by examining human behaviour according to fraud theories and existing methods for detecting corporate fraud in the form of a traditional review. In this study, the most widely used fraud theories and the existing methods for detecting corporate fraud are explained.\n\n\nConclusions\n\nA company’s financial distress can be a fraud factor because management wants to improve the company’s economic performance (Aviantara 2021). Thus, payables, expense, and revenue accounts can be manipulated (Utami and Pusparini, 2019). Future research needs to analyse more Pentagon and Hexagon fraud theories, which are more recent and have not yet been analysed in detail. Also, future research needs to analyse more of the human behaviour characteristics related to the Pentagon and Hexagon fraud theories.", "appendix": "Data availability\n\nUK Data Service: All Fraud Theories: A Systematic Review Approach, 2004-2022. https://doi.org/10.5255/UKDA-SN-856474 (Chimonaki, Papadakis, and Lemonakis, 2023).\n\nThe project contains the following underlying data:\n\n• allfraudtheories.csv. (Final article details included in this study).\n\n• Explanationofourworkflow.odt. (Flowchart of selection process used in this systematic review).\n\nUK Data Service: PRISMA checklist and flow chart for ‘Perspectives in fraud theories – A systematic literature review approach’. https://doi.org/10.5255/UKDA-SN-856474 .\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAchmad T, Ghozali I, Pamungkas ID: Hexagon Fraud: Detection of Fraudulent Financial Reporting in State-Owned Enterprises Indonesia. Economies. 2022; 10(1): 1–16. Publisher Full Text\n\nAkbar T: The determination of fraudulent financial reporting causes by using pentagon theory on manufacturing companies in indonesia. Int. J. Bus. Econ. Law. 2017; 14(5): 106–113.\n\nAlbrecht C, et al.: The relationship between South Korean chaebols and fraud. Manag. Res. Rev. 2010; 33(3): 257–268. Publisher Full Text\n\nAlbrecht WS, Hill NC, Albrecht CC: The ethics development model applied to declining ethics in accounting. Aust. Account. Rev. 2006; 16 (38): 30–40. Publisher Full Text\n\nAltman E: I., 1968, Financial ratios, discriminant analysis and the prediction of corporate bankruptcy. J. Financ. 1968; 23(4): 589–609. Publisher Full Text\n\nAmiram D, et al.: Financial reporting fraud and other forms of misconduct: a multidisciplinary review of the literature. Rev. Acc. Stud. 2018; 23: 732–783. Publisher Full Text\n\nAnnisya M, Lindrianasari, Asmaranti Y: Pendeteksian Kecurang Laporan Keuangan Menggunakan Fraud Diamond. Jurnal Bisnis dan Ekonomi. 2016; 23: 1.\n\nAprilia R: Pengaruh Financial Stability, Personal Financial Need, Ineffective Monitoring, Change in Auditor Dan Change in Director Terhadap Financial Statement Fraud Dalam Perspektif Fraud Diamond Studi Empiris Pada Perusahaan Manufaktur Yang Terdaftar Di Bursa Efe. Jurnal Online Mahasiswa (JOM) Bidang Ilmu Ekonomi. 2017; 4(1): 1472–1486.\n\nApriliana S, Agustina L: The analysis of fraudulent financial reporting determinant through fraud pentagon approach. Jurnal Dinamika Akuntansi. 2017; 9(2): 154–165. Publisher Full Text\n\nAviantara R: Scoring the financial distress and the financial statement fraud of Garuda Indonesia with «DDCC» as the financial solutions. J. Model. Manag. 2021; 18(1): 1–16. Publisher Full Text\n\nBao Y, et al.: Detecting accounting fraud in publicly traded US firms using a machine learning approach. J. Account. Res. 2020; 58(1): 199–235. Publisher Full Text\n\nBeasley MS: An empirical analysis of the relation between the board of director composition and financial statement fraud. Account. Rev. 1996;443–465.\n\nBeasly M, Joseph V, Dana R, et al.: Diambil dari.2010. www.coso.org/documents/COSOFRAUDSTUDY2010_001.pdf\n\nBeneish MD: Detecting GAAP violation: Implications for assessing earnings management among firms with extreme financial performance. J. Account. Public Policy. 1997; 16(3): 271–309. Publisher Full Text\n\nBonner SE, Palmrose Z-V, Young SM: Fraud type and auditor litigation: An analysis of SEC accounting and auditing enforcement releases. Account. Rev. 1998;503–532.\n\nBrazel JF, Jones K, Zimbelman MF: What can nonfinancial measures tell us about the likelihood of fraud. Unpublished working paper. 2006.\n\nBrigham EF, Houston JF: Dasar-dasar manajemen keuangan.2006.\n\nBrown NC, Crowley RM, Brooke Elliott W: What are you saying? Using topic to detect financial misreporting. J. Account. Res. 2020; 58(1): 237–291. Publisher Full Text\n\nChimonaki C, Papadakis S, Lemonakis C: All Fraud Theories: A Systematic Review Approach, 2004-2022. Colchester, Essex: UK Data Service; 2023. [Data Collection]. Publisher Full Text\n\nChotimah C, Susilowibowo J: Pengaruh struktur modal, modal kerja dan pertumbuhan penjualan terhadap profitabilitas. Jurnal Ilmu Manajemen. 2014; 2(2): 422–433.\n\nCressey DR: Other people’s money; a study of the social psychology of embezzlement.1953.\n\nCrowe H: Why the fraud triangle is no longer enough. Horwath, Crowe LLP; 2011.\n\nDechow PM, Ge W, Larson C, et al.: Predicting material accounting misstatements.2011; 28: 17–82. Publisher Full Text\n\nDevy KLS, et al.: Pengaruh frequent number of ceos picture, pergantian direksi perusahaan dan external pressure dalam mendeteksi fraudulent financial reporting (studi empiris pada perusahaan farmasi yang listing di bei periode 2012-2016). JIMAT (Jurnal Ilmiah Mahasiswa Akuntansi) Undiksha. 2017; 8: 2.\n\nEmalia D, et al.: Dampak dari auditor quality, financial stability, dan financial target terhadap fraudulent financial reporting. Studi Ilmu Manajemen Dan Organisasi. 2020; 1(1): 1–11. Publisher Full Text\n\nHogan CE, et al.: Financial statement fraud: Insights from the academic literature. Audit. J. Pract. Theory. 2008; 27(2): 231–252. Publisher Full Text\n\nKelly P, Hartley CA: Casino gambling and workplace fraud: a cautionary tale for managers. Manag. Res. Rev. 2010; 33(3): 224–239. Publisher Full Text\n\nKhamainy AH, Ali M, Arif Setiawan M: Detecting financial statement fraud through new fraud diamond model: the case of Indonesia. J. Financ. Crime. 2022; 29(3): 925–941. Publisher Full Text\n\nLi C, Haohao S, Ming F: Research on the impact of artificial intelligence technology on accounting. J. Phys. Conf. Ser. 2020; Vol. 1486(3). IOP Publishing.\n\nLister LM: A practical approach to fraud risk: comprehensive risk assessments can enable auditors to focus antifraud efforts on areas where their organization is most vulnerable. Intern. Audit. 2007; 64(6): 61–66.\n\nLoebbecke JK, Eining MM, Willingham JJ: Auditors experience with material irregularities-frequency, nature, and detectability. Auditing-J. Pract. Theory. 1989; 9(1): 1–28.\n\nLokanan M, Sharma S: A fraud triangle analysis of the LIBOR fraud. J. Forensic Investig. Account. 2018; 10(2): 187–212.\n\nMahaputra INKA, Adnyana NK: Pengaruh rasio-rasio keuangan terhadap pertumbuhan laba pada perusahaan manufaktur yang terdaftar di BEI. Jurnal Akuntansi & Bisnis. 2012; 7(2): 243–254.\n\nManurung DTH, Hadian N: Detection fraud of financial statement with fraud triangle. Proceedings of 23rd International Business Research Conference. 2013; Vol. 36(8).\n\nManurung DA, Hardika L: Analysis of factors that influence financial statement fraud in the perspective fraud diamond: empirical study on banking companies listed on the Indonesia stock exchange year 2012 to 2014. International Conference on Accounting Studies (ICAS). 2015.\n\nMiller TC, Cipriano M, Ramsay RJ: Do auditors assess inherent risk as if there are no controls? Manag. Audit. J. 2012; 27: 448–461. Publisher Full Text\n\nPersons OS: Using financial statement data to identify factors associated with fraudulent financial reporting. J. Appl. Bus. Res. 1995; 11(3): 38–46. Publisher Full Text\n\nOkoye EI, Okafor TA, Ijeoma N: Impact of the fraud triangle on audit process: the Nigerian Accountant’s view. Univ. Adv. Res. J. 2009; 1: 130.\n\nQuraini F, Rimawati Y: Determinan fraudulent financial reporting using fraud pentagon analysis. J. Audit. Financ. Forensic Account. 2018; 6(2): 105–114. Publisher Full Text\n\nRamos MJ, West L: Fraud detection in a GAAS audit: SAS no. 99 implementation guide.2003.\n\nSetiawati E, Baningrum RM: Deteksi fraudulent financial reporting menggunakan analisis Fraud Pentagon: Studi kasus pada perusahaan manufaktur yang listed di BEI Tahun 2014-2016. Riset Akuntansi Dan Keuangan Indonesia. 2018; 3(2): 91–106. Publisher Full Text\n\nSihombing KS, Rahardjo SN: Analisis fraud diamond dalam mendeteksi financial statement fraud: studi empiris pada perusahaan manufaktur yang terdaftar di Bursa Efek Indonesia (BEI) Tahun 2010-2012. Diponegoro Journal of Accounting. 2014; 3(2): 657–668.\n\nSkousen CJ, Smith KR, Wright CJ: Detecting and predicting financial statement fraud: The effectiveness of the fraud triangle and SAS No. 99. Corporate governance and firm performance. Vol. 13. . Emerald Group Publishing Limited; 2009; pp. 53–81.\n\nSubramanyam KR: Analisis Laporan Keuangan. Edisi Kesebelas. Jakarta: Salemba Empat; 2017.\n\nSykes GM, Matza D: Techniques of neutralization: A theory of delinquency. Am. Sociol. Rev. 1957; 22(6): 664–670. Publisher Full Text\n\nSummers SL, Sweeney JT: Fraudulently misstated financial statements and insider trading: An empirical analysis. Account. Rev. 1998;131–146.\n\nSupri Z, Rura Y, Pontoh GT: Detection of fraudulent financial statements with fraud diamond. J. Res. Bus. Manag. 2018; 6(5): 39–45.\n\nSunardi S, Nuryatno Amin M: Fraud detection of financial statement by using fraud diamond perspective. Int. J. Dev. Sustain. 2018; 7(3): 878–891.\n\nTiffani L, Marfuah D: Deteksi financial statement fraud dengan analisis fraud triangle pada perusahaan manufaktur yang terdaftar di bursa efek Indonesia. Jurnal Akuntansi and Auditing Indonesia. 2015; 19(2): 112–125. Publisher Full Text\n\nTrompeter GM, et al.: A synthesis of fraud-related research. Audit. J. Pract. Theory. 2013; 32(Supplement 1): 287–321. Publisher Full Text\n\nYu S-J, Rha J-S: Research trends in accounting fraud using network analysis. Sustainability. 2021; 13(10): 5579. Publisher Full Text\n\nYusrianti H, Ghozali I, Yuyetta EN: Asset misappropriation tendency: Rationalization, financial pressure, and the role of opportunity (study in indonesian government sector). Humanit. Soc. Sci. Rev. 2020; 8(1): 373–382. Publisher Full Text\n\nUtami ER, Pusparini NO: The Analysis of Fraud Pentagon Theory and Financial Distress for Detecting Fraudulent Financial Reporting in Banking Sector in Indonesia (Empirical Study of Listed Banking Companies on Indonesia Stock Exchange in 2012-2017). 5th International Conference on Accounting and Finance (ICAF 2019). Atlantis Press; 2019.\n\nUysal ÖÖ: Business ethics research with an accounting focus: A bibliometric analysis from 1988 to 2007. J. Bus. Ethics.2010; 93: 137–160. Publisher Full Text\n\nvan Eck NJ , et al.: Visualizing the computational intelligence field [Application Notes]. IEEE Comput. Intell. Mag. 2006; 1(4): 6–10. Publisher Full Text\n\nVan Eck NJ, Waltman L: Bibliometric mapping of the computational intelligence field. International Journal of Uncertainty, Fuzziness and Knowledge-Based Systems. 2007; 15(05): 625–645. Publisher Full Text\n\nVousinas GL: Advancing theory of fraud: the SCORE model. J. Financ. Crime. 2019; 26(1): 372–381. Publisher Full Text\n\nWilson FM: Organizational behaviour and work: a critical introduction. Oxford University Press; 2018.\n\nWolfe DT, Hermanson DR: The fraud diamond: Considering the four elements of fraud.2004.\n\nZainudin EF, Hashim HA: Detecting fraudulent financial reporting using financial ratio. Journal of Financial Reporting and Accounting. 2016; 14: 266–278. Publisher Full Text" }	[ { "id": "197387", "date": "29 Aug 2023", "name": "Grigoris Giannarakis", "expertise": [ "Reviewer Expertise Management", "Accounting" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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's work is well-written and well-organized. The sections are well divided. They also provide an excellent literature review explaining the importance of perspectives on theories of fraud. Specifically, the authors pose three research questions whose answers they seek to explore. The first research question is whether the company's ethical issues conflict with its sustainability issues. The second research question is how fraud can be detected by examining human behavior using fraud principles. The third research question is: What methods in accounting literature are associated with theories of human behavior and fraud? Fraud in financial statement preparation is rising, and human behavior is associated with fraudulent activity. Investors, stakeholders, regulators, auditors, and other interested parties need to know how to interpret companies published financial statements.\nThe choice of samples for data collection demonstrates the authors' commitment to collecting robust and representative data. In addition, using the VOS Viewer program underscores their commitment to using modern tools to analyze literature effectively. Such methods are indicative of rigorous academic standards. Data collection sampling and the VOS Viewer program are also best practices in literature analysis.\nThe study's results align well with the research questions and demonstrate the careful approach of the authors. The results don't stand alone but pave the way for further studies, contributing to the scientific continuum.\nIn summary, this work is an essential contribution to the accounting and behavioral research field. It seamlessly combines theory with empirical research and provides stakeholders with valuable insights into the intricate web of human behavior and financial fraud. The research not only fills existing literature gaps but also points the way for future research in this area.\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": "225964", "date": "24 Jan 2024", "name": "Ni Wayan Rustiarini", "expertise": [ "Reviewer Expertise accounting", "fraud", "auditing" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. At the beginning of the paragraph, the author states that \"accounting fraud is associated with sustainable and ethical management\". However, the author does not clearly explain the relationship between these three factors theoretically and empirically. 2. The following sentence states, \"Existing accounting literature explains fraud theories and attempts to quantify and analyze humans\". This statement also does not clearly explain the statement, what is the literature, and who is the author? 3. The following statement states, \"Numerous articles in the literature focus on the analysis of financial statement fraud and corporate fraud\". What is unique or different about this research from previous ones? 4. The author addresses three research questions. I think the first question is unrelated to the second and third questions. This causes the author not to focus on the topic he wants to research. Authors are advised to focus on the second and third research questions. 5. In the introduction, the author did not explicitly convey the urgency or motivation of the research explicitly. Also, the manuscript does not include a clear objective and rationale. The author needs to describe research contributions. 6. The author must explicitly state the limitations of the topic to be researched, whether it is only financial statement fraud or includes other fraud (such as accounting fraud, procurement fraud, or academic fraud). Is the fraud discussed only in private companies, or does it include fraud in the public sector? 7. This manuscript needs to review more fraud theories, including explaining the process of evolution of these theories, from the fraud triangle to the fraud hexagon. 8. In the research method, the author uses a keyword search for 'Fraud Triangle', OR 'Fraud Diamond' OR 'Fraud Pentagon', OR 'Fraud Hexagon'. Do you only use these four phrases? Does the author also use the word \"fraud\" to track the articles used? There is a possibility that the article needed only uses the keyword \"fraud\". 9. What search method did the author use to answer the first research question? 10. This manuscript includes all document types, including articles, conference papers, reviews, book chapters, conference reviews, notes, and data papers. Can the author ensure all documents go through a good peer review process? Writers must use the PRISMA method to select the reference sources to answer research questions. 11. The author uses several countries, such as the United States, Indonesia, Malaysia, Australia, United Kingdom, Canada, China, Ghana, France and Germany. Why did the author only use ten countries, and how did he determine these ten countries? 12. The author needs to mention reference sources for the documents used. 13. In the results and discussion section, the author does not clearly outline the discussion related to the first research question. 14. The conclusion is concise. The author should summarize the overall research results, write down the implications, and present limitations and suggestions for further research.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? No\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] }, { "id": "212496", "date": "25 Sep 2024", "name": "Williams Kwasi Peprah", "expertise": [ "Reviewer Expertise Management", "Accounting and Finance." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article provides a comprehensive review of fraud theories, focusing on the evolution and applicability of models like the Fraud Triangle, Fraud Diamond, Fraud Pentagon, and Fraud Hexagon. By analyzing 302 articles published between 2004 and 2022, the authors assess how these theories explain the human behavioral characteristics that contribute to corporate fraud. The study employs bibliometric analysis using the VOS viewer to identify trends and gaps in the literature.\nStrengths: The article successfully compiles and evaluates the most relevant fraud theories, giving readers a broad understanding of the different models used to explain corporate fraud. The authors' use of keyword searches such as \"Fraud Triangle\" and \"Fraud Diamond\" ensures a focused and relevant literature review. The use of the VOS viewer for keyword mapping and cluster analysis adds value to the study. This visual approach to the literature provides a clear overview of trends and relationships between key terms in fraud research, making the findings more accessible to a broader audience. The conclusion effectively highlights the need for more research on the Fraud Pentagon and Hexagon theories, which are more recent but less explored. This identification of research gaps helps future scholars identify opportunities for further study. The article emphasizes the human behavioral factors behind fraud, adding a psychological dimension to the traditional accounting and governance-oriented discussion of fraud. This aspect enriches the understanding of fraud beyond mere financial metrics.\nAreas for Improvement: While the Fraud Diamond theory is identified as the most \"functional\" theory based on the analysis, the article does not sufficiently explain why this is the case compared to the Fraud Pentagon and Hexagon theories. A more balanced critique of these models could improve the objectivity of the findings. The study would benefit from a discussion on how these findings can be applied in practical settings, such as corporate governance or fraud detection. Although the article is thorough in its theoretical analysis, offering actionable insights for practitioners, auditors, or regulators would increase its utility. The exclusion of 2023 data may limit the timeliness of the article, given the rapidly evolving nature of fraud detection techniques, particularly with the rise of AI and blockchain. Including the latest research could provide a more complete picture of current trends in fraud theory.\nConclusion: The article presents a valuable and comprehensive review of fraud theories, with a particular emphasis on the behavioral aspects of fraud. It provides a solid foundation for future research, particularly regarding the underexplored Fraud Pentagon and Hexagon theories. However, a more balanced critique of existing models, clearer practical implications, and a more accessible presentation of the bibliometric analysis would enhance the article's impact.\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\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-933
https://f1000research.com/articles/12-931/v1	03 Aug 23	{ "type": "Study Protocol", "title": "Rapid, Economic, Acetic acid, Papanicolaou (REAP) staining technique analogous to the routine Papanicolaou staining (PAP) technique", "authors": [ "Prachi Alone", "Sahitya Vodithala", "Sunita Vagha", "Sahitya Vodithala", "Sunita Vagha" ], "abstract": "The current research compared classical Papanicolaou stain with REAP (Rapid, Economic, Acetic Acid Papanicolaou) stain to evaluate staining character, range, charge virtue, and sustaining of stain. The PAP Stain is the standard dye in support of shielding cell study for any cell study and is used in various research centers with several reductions. In certainty, PAP Stain has lowered squamous cell carcinoma incidence by 70%, particularly within wealthy provinces with effectively-designed broadcasting systems. But the stain has gone through several changes, moving from the uncreative formal mechanism -> gradual REAP PAP staining in which staining duration level is decreased, it improves the quality, it is cost-effective, easily available, and ethyl alcohol is temporarily superseded with 1% ethanoic acid in advanced nations with guarding programs with careful planning. It has been used to access FNAC (fine needle aspiration cytology). Because of its precise nuclear and nucleoli staining, PAP staining of FNAC and fluid smears is a trustworthy stain. FNAC is a straightforward, speedy, and reliable pre-operative assessment and diagnosis process that has sparked advancements in dyeing technology which utilizes minimal coloring count with a clear cell framework. Around 40 minutes are needed for the standard methodology for traditional PAP Stain. REAP Stain was developed to address this, improving quality while cutting staining time to about one minute. This study's objective was to evaluate the value of the Rapid-Pap stain utilizing quantifiable investigation. The effectiveness of cytoplasmic and nuclear staining, stain preservation, value, and overall course of action for the two techniques—conventional PAP stain and REAP stain—were collated. When compared to traditional Papanicolaou, REAP stain offers a good, quick, and affordable replacement for the screening of cells. The REAP technique also does a superb job for the maintenance of stains.", "keywords": [ "Conventional PAP Stain", "REAP Stain", "cervical (squamous cell) smears", "FNAC", "alcohol", "1% ethanoic acid", "Eosin Azure." ], "content": "Introduction\n\nGeorge Papanicolaou created the PAP dye, a vivid color examination of the cell tinge method, in the year 1942. Dr. Papanicolaou afterward updated his research in the years 1954 and 1960.1 As G.N. Papanicolaou initially studied the PAP stain, he went through numerous alterations, and was later modified by him in the year 1960.1 The Mallory aniline blue method, the Masson trichrome method, and the short methods can all be found in the history of the Papanicolaou staining method.2 In human medicine, Pap is the gold-standard staining technique for FNAC or exfoliate cells, which are used to diagnose neoplastic disorders.3\n\nSB Dighe (2005) published the REAP method.4 To compare the excellence of REAP blotching of spatter to conventional PAP staining using upcoming guidelines: Solar & protoplasmic blotching intensity, staining time, vanished success, lengthy duration of shade maintenance.5\n\nPapanicolaou stain has been modified to increase staining quality and/or to shorten staining time.6 PAP stains are accustomed to detecting and differentiating units from smears obtained in different physical fluids,7 gynecological smears,8 and FNAC9 smears from different organs. Various types of body fluids and needle biopsies are used to stain the specimens; the samples include gynecological Papanicolaou spatter, froth, grazing, cleaning, discharge, BAL, ascitic fluidic, pericardial fluidic, CSFs,10 malignant ascites, pleural effusion, joint effusion, semen discharge,11 FNAC,8 neoplasm affecting specimens, and matter containing loose cells. An application of spot-on tissue slides is connected with loose cell inspection. Papanicolaou spots are gold standard cells of the cervix covering up. It produces a metachronous, clear spotting response by distinct solar & cellular characteristics.\n\nConventional Papanicolaou Staining Procedure: Shift glass slides immediately out of the alcohol aether adhesive to 80% alcohol watery, then via 70% & 50% alcohol to D/W. For 4 minutes, ‘stain’ with ‘Harris hematoxylin’. Drain with D/W for a few seconds. Six times, bath through 0.25% Hydrochloric Acid in 50% eth-an-ol (20-60 sec). 6 minutes in flowing tap water Run through 50%, 70%, 80%, and 95% alcohol after rinsing with distilled water. 2 minutes staining in OG-6. Rinse in two 95% alcohol changes. For 2 minutes, stain in EA 36/EA 50. Rinse three times in 95% alcohol. Remove water from the abs. alc-oh-ol, then in similar pieces xylol and abs. alc; clear in dimethylbenzene and cover with DPx.12\n\nREAP P-A-P Staining Method: 1% ACID ACETIC ten soaks. Harris Haematoxylin 10 dips. [Previously heat to 60°C] Ten soaks in running H2O. ethanoic acid 1%, 10 dips for OG-6. 1%acetic acids ten soaks. Eosin azure-50 ten dips. 1.0% acetic - acid Meth-an-ol ten dips dissolvent ten dips perform mop up with every process. Install in DPx. In REAP, at every stage of alc-oh-ol in standard Papanicolaou was changed with 1%ethanoic acid.13\n\n• The spotting emulsion, which consists of Hematoxylin and spots the cellular nucleus. Harris hematoxylin was utilized by Papanicolaou within every 3 articulations for spots he proclaimed.14\n\n• The 2nd spotting emulsion (named OrangeG-6 dye) includes 95% ethanol and a trace of phosphor-tungstic acid. The OG-6 stands for Orange G, & the ‘6’ represents the conc. of phosphor-tungstic acid utilized; extra category encompassed OrangeG-5 and OrangeG-8).14\n\n• The 3rd spotting emulsion contains 3 stains Eosin-Y, Light-Green-SF-yellowish, Bis-marc brown-Y, within 95% eth-an-ol through the trace of phos-pho-tungstic acid & carbolic. Other formulations include EosinAzure-36, EosinAzure-50, and EosinAzure-65. The emulsions are labeled E. A, next to the count indicating the number of stains.15\n\nPhosphotungstic acid is applied to counterstains to modify the pH and aid maximize the color potency. This Eosin Azure contrast stain comprises Bis-marck-brown and phos-pho-tungstic acid, both of which settled down away from emulsion when combined, limiting usable lifespan concerning blending.16\n\nThis dye must produce very clear units, allowing often thick samples of merged units to be analyzed. Cellular nuclei should be frangible and blue-black in chroma, with well-defined histone designs. Cellular cytosol colors blue to green, while protein keratin gets – orange.17\n\n▪ Cell outline – Identifying a crisp, sharp cell outline with no darkness.\n\n▪ Nucleus outer view – to Identify transparently, sharply nucleus lining & chroma’s strength\n\n▪ Detailed nucleus – Detection of fragments, chroma-tin networks, & ‘nuclear inclusion\n\n▪ Distinction identifies the cell’s color distinction\n\n▪ Clear recognition – cytosol cells in the absence of particulate\n\n▪ Micronuclei – The recognition of well-defined micronuclei.\n\nThe benefits of REAP stain include: superior to normal Pap staining, good nucleus/cytosol stain improved color clarity, and low value.18 Acetic acid substitutes expensive eth-an-ol (25% of the complete price -of normal Papanicolaou stain), allowing for lasting duration color retention (>1 FY without dwindling), The technique is rapid, and the stain-ing of transitional cells such as RedBloodC/WhiteBloodC/Bacteria is safely stored, the nucleus/histones/karyon features = transparent/bracing, the cyno-philia/raised eosinophils within smears19 is equivalent to PAP stain, which breakdown RED blood Cells without alteration in tubular-cells shape.9 Due to its low value, it’s a viable option for regular PAP smear seen within a lump of cervix carcinoma identified in underdeveloped nations. The downside though REAP stain comprises discoloration observable within the smudge coat, especially observed cellular clumping, because of its low color accumulation.20 Some scientists believe that when slides are stored for more than 6 months, REAP stain provides lower color standards regarding improper storage.21 Also, stains – are best employed in resource-constrained contexts in which there is a price issue, rather than in exploration/triennial settings.15\n\nAshok Kumar Deshpande et al.4 conducted a study comparing Papanicolaou stain (PAP) with Rapid Economic Acetic acid Papanicolaou Stain [REAP] at the cellular level of the cervix.12 This research comprised 200 samples from persons of various ages. The study found that PAP Stain is substantially more time-consuming and costly than REAP Stain- lump scanning of cervical carcinoma. When juxtapose with PAP stain, these smears’ scenarios stained with REAP = highly transparent and free of detritus.\n\nAbhilasha Asthana et al. (2014) conducted research comparing the regular Papanicolaou staining procedure to the fast, economical, acetic acid, Papanicolaou (REAP) approach. This study covers a total of 100 samples from two separate sites, each with 50 sample sets. When compared to standard PAP stain, the REAP approach yields good colorful slides thus less expensive and takes less set-up considering lump scanning of mouth carcinoma.18\n\nRanu Roy Biswas et al. (2008) investigated Rapid Economic, Acetic Acid, Papanicolaou Stain {REAP} — “Is an acceptable substitute for traditional PAP Stain. There are 220 PAP smears from 110 participants in the research. According to the findings of the study, REAP stain, when compared to standard Papanicolaou stain, provides an appropriate, good, and speedy option for cytological screening at a low cost. Overall, absolute alcohol consumption is low. The stain preservation in the REAP process is also good. In our nation with a high prevalence of the illness, it may be regarded as a good alternative to traditional PAP stain for cell screening programs.13\n\nKalyani Raju et al. conducted research comparing convectional PAP stain with rapid PAP stain. The study covers 50 samples in all. According to the study, - the superiority of REAP ‘stain’ is as follows: prevailing over regular “Pap stain”, marvelous nuclear/protoplasm coloring more advance color-magnitude/clarity, worthwhile just like ethan-oic-acid replaces costly ethanol {25% -complete cost’s general quality Pap stain}, prolonged color safeguard [> one annual aside from vanishing], clever concept, varnishing of karyon/histones/nucleole characteristics – transparent comparable to Pap stain. Because of its low cost, it is a viable substituent for regular PAP smears under cervix carcinoma lump scanning in developed nations.22\n\n\nRationale\n\nFNAC is a technique whereby cells are obtained from a lesion using a thin bore needle and smears are made for cytological diagnosis.\n\nThe benefits of REAP stain over standard Pap stain include superior nuclear and cytoplasmic staining, improved color intensity and transparency, and lower cost. Acetic acid replaces expensive ethanol, which accounts for 25% of the cost of standard Pap stain, and provides long-term color preservation (more than a year without fading), a quick procedure, less time required, staining of non-epithelial cells like RBC/WBC/bacteria that are well preserved, nuclear/chromatin/nucleolus details that are clear/crisp, comparable cynophilia/eosinophilia of the cells.\n\n\n\n1. To determine the REAP staining span and PAP staining span.\n\n2. To screen Prolonged color maintenance Intensity of nuclear & cytoplasmic staining.\n\n3. To evaluate cost-effectiveness.\n\n4. To assess the superiority of staining of smears by REAP technique compared to the routine PAP technique\n\nPatients participating in the study will be asked to give prior written informed consent. Clinical history will be taken, an appropriate physical examination will be done on new patients and clinical details of the previously diagnosed cases will be obtained considering the inclusion and exclusion criteria. Fluids will be extracted from clinically questionable cases and forwarded to the Department of Pathology, Jawaharlal Nehru Medical College (J.N.M.C.), Sawangi Meghe, for histopathological evaluation. The specimens accepted in the Dept. of Pathology, J.N.M.C., will be dipped in alcohol–ether for fixation, fixed smear slides will be taken for diagnosis, and confirmed for further examination. Gross examination and diagnosis of the specimens will be done followed by the taking of appropriate fluid specimens. The smear is made from fluids and undergoes the REAP and PAP Stain.\n\nThe study is conducted among the common population of the Vidarbha district in AVBRH hospital. The study is about the Rapid, Economic, Acetic acid, Papanicolaou (REAP) staining technique analogous to the routine Papanicolaou staining (PAP) technique. The study is helpful in the awareness of cervical cancer among people. The conceptual research on cervical cancer will be done. The recruitment of the people is independently done and the awareness will be observed.\n\n\nMethods\n\nThe following study is a cross-sectional investigative study that will be conducted in the Cytopathological division of the Dept. of Pathology, Jawaharlal Nehru Medical College (JNMC), Sawangi (Meghe), Wardha, in collaboration with the Acharya Vinoba Bhave Rural Hospital (AVBRH), Sawangi (Meghe), Wardha, from 2021 to 2023. The institutional Ethical Committee approval has been obtained from Datta Meghe Institute of Medical Sciences (DMIMS (DU)/IEC/2022/1205). This investigation will take place after obtaining the patients’ written informed consent.\n\n\n\n1. Thirty (30) fluid specimens from patients accepted in the Department of General Pathology, Jawaharlal Nehru Medical College (JNMC), Sawangi (Meghe) were used in the study.\n\n2. Alcohol – ether fixed, smear on slides obtained from the specimens.\n\n3. Alcohol – ether fixative.\n\n4. FNAC instruments.\n\n5. PAP & REAP Staining assembly.\n\n6. Glass slides (Blue Star®). Dimensions: 7.5 × 2.5 centimeters.\n\n7. Binocular research microscope.\n\nIt is a observational cross-sectional study.\n\nThe study will be conducted in Aacharya Vinoba Bhave Rular Hospital Sawangi (Meghe), Wardha.\n\nSubjects of both sexes, aged 18 to 50 years getting diagnostic fine needle aspiration cytology (FNACs) done at the Acharya Vinoba Bhave Rural Hospital (AVBRH), Sawangi (Meghe), Wardha, will be included.\n\nCases under therapy, vulnerable personnel, patients in emergency conditions, ethnic minority groups, homeless people, nomads, refugees, and minors will be excluded from the study.\n\nAll necessary measures to control bias at all levels will be taken. As it is an observational study, all the samples which are routinely received in the section of cytopathology are taken into consideration for comparison between REAP staining technique analogous to the routine PAP technique. Institutional supply of dyes and chemical constituents present in dyes from different companies make minor modifications, that remain consistent, an essential part of staining protocol. Diagnostic search influences the findings of cells staining. The influence of content of result captions in response to diagnosis of samples. The purpose of study is to access the comparison between REAP staining technique analogous to the routine PAP technique for estimation of diagnostic accuracy for test samples.\n\nCochran formula for sample size estimation:\n\nN= Total patients of fluid cytology during the last one and half years = 3000\n\nThe data analysis will be done using a relevant Chi-squared test for understanding the comparative difference between groups and among groups will be compared. Whereas, the results obtained during the research will be analyzed using appropriate statistical methods (SPSS) by a qualified statistician.\n\nThe results of the study will be published in an indexed journal.\n\nThis study will evaluate the significance of REAP staining over the conventional PAP staining method. The study will demonstrate a considerable decrease in turnaround time for REAP as compared to conventional pap stain. This staining technique aids in an easy understanding of the pathophysiology of smears. The technique will also help in the assessment of the quality of the smear and will aid in providing easy and rapid diagnosis to patients.\n\nThe simplicity of the procedure (uniform 10 dips) also reduced the risk of human errors during staining will help researchers reduce the period of the staining procedure. The modifications that will apply to conventional pap stains have been described will suitable alternatives for REAP stains.\n\nThe study is yet to start.\n\n\nResults\n\nThe present study explores whether the qualitative and quantitative analysis of stained smears by REAP Stain is much better than conventional PAP Stain for nucleus and cytoplasm staining.\n\n\nDiscussion\n\nCervical cancer develops progressively over a certain amount of time.23 Most accidents take place in less developed countries where there are no reliable testing campaigns available. Some of the risk factors include acquired immunodeficiency syndrome, smoking, and human papillomavirus exposure. Treatment-related extended morbidity is common, however, most women with early-stage tumors can be cured. According to the outcomes of randomized clinical research, chemotherapy,24 and radiation therapy is considered a standard of care for cancer-developed patients and especially for women with locally advanced cancer, whereas its applicability to women in less developed nations is largely not tested.25\n\nIn 2006, Dighe SB et al., conducted research on the development of PAP stain and compared traditional PAP stain to REAP stain. The study evaluated the nuclear and cytoplasmic characteristics of both stains. The study concludes that REAP stain’s cytoplasm differentiation and transparency were ideal, and the nuclear features and chromatin pattern were visible.15\n\nIn 2008, Ranu Roy Biswas et al., the question of whether Rapid Economic, Acetic Acid, Papanicolaou Stain (REAP), a suitable substitute for traditional PAP Stain, has been examined. 110 participants provided 220 PAP smears for the study. According to the study’s findings, REAP stain offers an appropriate, excellent, quick, and affordable alternative to traditional Papanicolaou for cytological screening.26 Overall, very little alcohol is consumed. The REAP technique also does a superb job of stain preservation. In our nation, where the disease is quite prevalent, it might be thought of as a good substitute for the traditional PAP stain for cells screening program.13\n\nIn 2014, Abhilasha Asthana et al., compared the conventional Papanicolaou staining method with the quick, inexpensive acetic acid, Papanicolaou (REAP) method in a study. This study uses a total of 100 samples from two different sites, with 50 sets of samples per site. The study concluded that REAP method, as opposed to traditional PAP Stain, generates superior stained smears that are cost-effective and require less time for mass screening of oral cancer.18\n\nIn 2014, Shabnam Izhar et al. studied the effectiveness of a quick, affordable, acetic acid Papanicolaou stain as an alternative to traditional Papanicolaou stain in cervical smears. There are 737 smears in the study. According to the study’s findings, traditional PAP stain does a better job of color retention than REAP PAP stain, reduces turnaround time, and provides unmistakable cell shape due to superior staining quality.15\n\nIn 2015, Ashok Kumar Deshpande et al., Papanicolaou stain (PAP) and Rapid Economic Acetic acid Papanicolaou Stain (REAP) were compared in a study on cervical cytology. In total, 200 samples from persons of various ages were used in this investigation. The study found that PAP Stain required more time and money than REAP Stain for routine cervical cancer screening. When compared to PAP stain, the background of smears stained by REAP was incredibly clean and debris-free.27\n\nIn 2016, Bhagat P. et al. undertook research on the effectiveness of a modified, quick, affordable, acetic acid-based Papanicolaou stain. 102 liquid base cytology samples are included in Ref. 28. The investigation concludes that standard PAP staining produced good cytoplasmic staining. The study endorsed the adoption of the REAP approach as a practical substitute.29\n\nIn 2016, Kalyani Raju et al. studied the differences between the conventional PAP stain and the rapid PAP stain. The study uses 50 samples in total.30 The study came to the conclusion that REAP stain has several advantages over standard Pap stain, including superior nuclear/cytoplasmic staining with better color intensity/transparency, cost-effectiveness due to the substitution of acetic acid for expensive ethanol (which accounts for 25% of the cost of standard Pap stain), long-term color preservation (more than a year without fading), a quick procedure, and staining of nuclear/chromatin/nucleolus details that are clear and comparable It is a good substitute for the traditional pap smear in developing nations’ widespread cervical cancer screening programs due to its low cost.31\n\nIn 2018, Suresh Amin et al., conducted research on PAP Stain for mass screening of micronuclei in buccal smears. Identification and counting of micronuclei are made possible by the stained smear backdrop.32\n\nIn 2020, Garima Goel et al. completed research on Papanicolaou Stain, a cheap, quick, and suitable alternative to traditional PAP Stain for cervical smear staining. 100 smears from 50 female patients33 are included in the study. In comparison to traditional PAP stain, the study finds that REAP is a quick, affordable, and good approach for both cytoplasmic and nuclear staining with minimal alcohol use.34\n\nIn 2022, Karthika Panneerselvam et al. performed research on Papanicolaou, Acetic acid, and Rapid Staining techniques versus traditional staining techniques in the healthy oral mucosa. Eight patients and 160 slides were used in the investigation. The study’s findings show that even though the REAP staining technique had shorter staining times and costs, conventional staining methods were still preferred over them.29\n\n\n\n1. Inter-observer and Intra-observer flexibility.\n\n2. Practical errors during the process can affect the interpretation of staining.\n\n3. Loss of follow-up patients.\n\nApproved by the Institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research Sawangi (M) Wardha (ref. no. DMIMS (DU)/IEC/2022/1205).", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nKoss LG, Melamed MR: Koss’ diagnostic cytology and its histopathologic bases. Lippincott Williams & Wilkins; 2006.\n\nMarshall PN: Papanicolaou staining--a review. Microsc. Acta. 1983 May 1; 87(3): 233–243. PubMed Abstract\n\nMehta N, Modi L, Patel T, et al.: Study of cytomorphology of solid pseudopapillary tumor of the pancreas and its differential diagnosis. J. Cytol. 2010 Oct; 27(4): 118–122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsthana A, Singh AK: Comparison of the routine Papanicolaou staining technique with the rapid, economic, acetic acid, Papanicolaou (REAP) technique. Int. J. Med. Dent. Sci. 2014 Jul 1; 3: 484–489. Publisher Full Text\n\nSato M, Taniguchi E, Kagiya T, et al.: A modified rapid Papanicolaou stain for imprint smears. Acta Cytologica. 2004; 48(3): 461–462.\n\nBibbo M: Comprehensive Cytopathology. 2nd ed.Philadelphia: WB Saunders; 1991; pp. 881–906.\n\nPreethi S, Sivapathasundharam B: Will the modified Papanicolaou stain be the new stain for keratin? J. Histotechnol. 2015 Mar 26; 38(1): 9–13. Publisher Full Text\n\nHoda RS: Non-gynecologic cytology on liquid-based preparations: a morphologic review of facts and artifacts. Diagn. Cytopathol. 2007 Oct; 35(10): 621–634. Publisher Full Text\n\nChoudhary P, Sudhamani S, Pandit A, et al.: Comparison of modified ultrafast Papanicolaou stain with the standard rapid Papanicolaou stain in cytology of various organs. J. Cytol. 2012 Oct; 29(4): 241. Publisher Full Text\n\nKumar V, Abbas AK, Aster JC: Robbin’s basic pathology e-book. Elsevier Health Sciences. 2017 Mar 8.\n\nPawlina W, Ross MH: Histology: a text and atlas: with correlated cell and molecular biology. Lippincott Williams & Wilkins; 2018 Dec 7.\n\nDighe SB, Ajit D, Pathuthara S, et al.: Papanicolaou stain: Is it economical to switch to rapid, economical, acetic acid, Papanicolaou stain? Acta Cytologica. 2006 Nov 1; 50(6): 643–646. Publisher Full Text\n\nRoy Biswas R, Paral CC, Dey R, et al.: Rapid economic, acetic acid, Papanicolaou stain (REAP)-Is it a suitable alternative to standard PAP stain? Al Ameen J. Med. Sci. 2008; 1(2): 99–103.\n\nPawlina W, Ross MH: Histology: a text and atlas: with correlated cell and molecular biology. Lippincott Williams & Wilkins; 2018 Dec 7.\n\nIzhar S, Kaur R, Masih K: Efficacy of rapid, economical, acetic acid, Papanicolaou stain in cervical smears as an alternative to conventional Papanicolaou stain. J. Cytol. 2014 Jul; 31(3): 154–157. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBelgaumi UI, Shetty P: Leishman Giemsa cocktail as a new, potentially useful cytological technique comparable to Papanicolaou staining for oral cancer diagnosis. J. Cytol. 2013 Jan 1; 30(1): 18–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMehta N, Modi L, Patel T, et al.: Study of cytomorphology of solid pseudopapillary tumor of the pancreas and its differential diagnosis. J. Cytol. 2010 Oct; 27(4): 118–122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsthana A, Singh AK: Comparison of the routine Papanicolaou staining technique with the rapid, economic, acetic acid, Papanicolaou (REAP) technique. Int. J. Med. Dent. Sci. 2014 Jul 1; 3: 484–489. Publisher Full Text\n\nSato M, Taniguchi E, Kagiya T, et al.: A modified rapid Papanicolaou stain for imprint smears. Acta Cytologica. 2004; 48(3): 461–462.\n\nPrasaad PR: Short-duration Papanicolaou stain (SPS)—an alternative to conventional Papanicolaou stain in routine cytopathology? Comp. Clin. Pathol. 2017 Nov; 26(6): 1285–1288. Publisher Full Text\n\nAmin S, Patel N, Chattoo B: Oral cancers–Micronuclei as a biomarker of genotoxicity a population-based study to establish usable dynamic cut-off limits in tobacco users. Int. J. Mol. Immun. Oncol. 2019 Feb 16; 4(1): 9–12. Publisher Full Text\n\nThakur M, Guttikonda VR: Modified ultrafast Papanicolaou staining technique: A comparative study. J. Cytol. 2017; 34: 149–153. Publisher Full Text\n\nSalcedo MP, Phoolcharoen N, Schmeler KM: Intraepithelial neoplasia of the lower genital tract (cervix, vagina, vulva): etiology, screening, diagnosis, management. Comprehensive Gynecology. Elsevier; 2021 Jan 1; p. 637.\n\nYang GC, Hoda SA: Combined use of the “scratch and smear” sampling technique and Ultrafast Papanicolaou stain for intraoperative cytology. Acta Cytologica. 1997 Sep 1; 41(5): 1513–1518. PubMed Abstract \| Publisher Full Text\n\nWaggoner SE: Cervical cancer. Lancet. 2003 Jun 28; 361(9376): 2217–2225. Publisher Full Text\n\nEinstein AJ, Yang GC, Silberfarb JB, et al.: Effect of Ultrafast Papanicolaou Staining on. Anal. Quant. Cytol. Histol. 1997 Aug; 19(4): 361–367. PubMed Abstract\n\nDeshpande AK, Bayya P, Veeragandham S: Comparative study of Papanicolaou stain [PAP] with rapid economic acetic acid Papanicolaou stain (REAP) in cervical cytology. J. Evol. Med. Dent. Sci. 2015 May 21; 4(41): 7089–7095. Publisher Full Text\n\nBhagat P, Susheilia S, Singh K, et al.: Efficacy of modified rapid economic acetic acid-based Papanicolaou stain. Cytopathology. 2016 Dec; 27(6): 452–455. Publisher Full Text\n\nPanneerselvam K, Karthik RK, Ramadoss R, et al.: Rapid economical acetic acid Papanicolaou staining procedure versus conventional staining procedure in the normal oral mucosa. J. Oral Maxillofac. Pathol. 2022 Apr; 26(2): 285. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaju K: Evolution of pap stain. Biomed. Res. Ther. 2016 Feb; 3(2): 1–1. Publisher Full Text\n\nGill GW: Enviro-Pap: an environmentally friendly, economical, and effective Pap stain. Lab. Med. 2006 Feb 1; 37(2): 105–108. Publisher Full Text\n\nAmin S, Patel N, Chattoo B: Oral cancers–Micronuclei as a biomarker of genotoxicity a population-based study to establish usable dynamic cut-off limits in tobacco users. Int. J. Mol. Immun. Oncol. 2019 Feb 16; 4(1): 9–12. Publisher Full Text\n\nBibbo M, Hoda RS: Comprehensive Cytopathology. Int. J. Gynecol. Pathol. 1998 Jan 1; 17(1): 93. Publisher Full Text\n\nGoel G, Halder A, Joshi D, et al.: Rapid, economic, acetic acid Papanicolaou stain (REAP): an economical, rapid, and appropriate substitute to conventional pap stain for staining cervical smears. J. Cytol. 2020 Oct; 37(4): 170–173. PubMed Abstract \| Publisher Full Text" }	[ { "id": "204995", "date": "26 Sep 2023", "name": "Vijayashree Raghavan", "expertise": [ "Reviewer Expertise Dermatopathology", "Oncopathology", "Histomorphology", "Cytology", "Hemato-immunology and molecular biology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study objectives, design, protocol and material/samples are not clearly mentioned. There are many contradicting statements. The language used does not clearly state if the study has been done or yet to be carried out.\n\nAt one point it mentions a cross-sectional study, but the inclusion/exclusion criteria do not correlate and the study also compares the new REAP technique with Routine PAP technique - not comprehensible.\n\nThe study design/method must be appropriate (if it is a comparative study/case-control/experimental).\n\nSamples inclusion has to be very clear if all types of cytology specimen are to be included or restricted only to FNAC/Exfoliative smears.\n\nLanguage is poor with too much grammatical inappropriateness.\n\nThis study cannot be approved in the present format/protocol. It needs a major revision if the study must be done.\n\nModifications of PAP staining has been done long ago and many laboratories are routinely using REAP/Ultrafast PAP techniques. So, no new information is expected from this study.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] }, { "id": "205008", "date": "29 Sep 2023", "name": "Mitsuaki Okodo", "expertise": [ "Reviewer Expertise Oncopathology", "Cervical cytology", "Virology", "Molecular Biology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments: I do not agree with this study protocol. If this study is to be conducted, I believe it needs to be substantially revised.\nSpecific comments:\nThe abstract should explain why this protocol is interesting. However, most of the abstract is dominated by previous studies that are not interesting. Please specify the samples and methods used to evaluate the value of Rapid-Pap Stain.\n\nThe study protocol is not clearly described. Please describe it in detail to allow replication by others.\n\nREAP Stain is not a staining method known to everyone. Please provide the staining procedure. Since this is a research protocol and you are publishing it, there must be a difference between the existing REAP stain and the authors' staining method. Please clarify the difference.\n\nIt is not clear what evaluation criteria will be used to compare REAP stain and Pap stain samples. Please provide a detailed scoring methodology.\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? No", "responses": [] } ]	1	https://f1000research.com/articles/12-931
https://f1000research.com/articles/10-996/v1	01 Oct 21	{ "type": "Research Article", "title": "Hypothesis on the pathophysiology of syringomyelia based on analysis of phase-contrast magnetic resonance imaging of Chiari-I malformation patients", "authors": [ "Han Soo Chang" ], "abstract": "Background: Despite a number of hypotheses, our understanding of the pathophysiology of syringomyelia is still limited. The current prevailing hypothesis assumes that the piston-like movement of the cerebellar tonsils drives the cerebrospinal fluid (CSF) into the syrinx through the spinal perivascular space. However, it still needs to be verified by further experimental data. A major unexplained problem is how CSF enters and remains in the syrinx that has a higher pressure than the subarachnoid space. Methods: I analyzed phase-contrast MRI scans of 18 patients with Chiari-I malformation with syringomyelia undergoing foramen magnum decompression and 21 healthy volunteers. I analyzed the velocity waveforms of the CSF and the brain in various locations. The obtained velocity waveforms were post-processed using a technique called synchronization in situ. I compared between the preoperative data and the control data (case-control study), as well as between the preoperative and postoperative data (cohort study). Results: The syrinx shrank in 17 (94%) patients with good clinical improvement. In Chiari-I patients, the velocity of the tonsil was significantly larger than controls, but was significantly smaller than that of the CSF in the subarachnoid space, suggesting passive rather than active movement. The abnormal tonsillar movement disappeared after surgery, but the velocity waveform of the spinal subarachnoid CSF did not change. These results, contradicting the above mentioned hypothesis, required an alternative explanation. I thus hypothesized that there is a CSF channel between the fourth ventricle and the syrinx. This channel assumes one-way valve function when mildly compressed by the cyclical movement of the cerebellar tonsil. The decompression of the tonsils switches off the one-way valve, collapsing the syrinx. Conclusions: My hypothesis reasonably explained my data that clearly contradicted the existing hypothesis, and successfully addressed the above-mentioned theoretical problem. It will serve as a working hypothesis for further study of syringomyelia pathophysiology.", "keywords": [ "syringomyelia", "Chiari malformation", "pathophysiology", "hypothesis", "magnetic resonance imaging", "phase-contrast" ], "content": "Introduction\n\nSyringomyelia is a disease in which a fluid-filled cavity, a syrinx, is formed inside the spinal cord causing neurological symptoms. The mysterious pathophysiology of syringomyelia has fascinated many researchers for decades. Various authors have proposed a number of hypotheses to explain the mechanism of syrinx formation.1-13 However, these hypotheses contradict one another, and none seems to fully explain the pathophysiology of syringomyelia.\n\nThe current prevailing hypothesis of Oldfield et al., proposes that piston-like movements of cerebellar tonsils produce pressure waves in the cerebrospinal fluid (CSF), which drive CSF into the syrinx through the perivascular space.14 Despite being attractive, it is still a hypothesis and needs to be verified by new experimental data.\n\nAbove all, a fundamental question seems to remain unexplained. It is evident that the pressure inside the syrinx is higher than that of the CSF outside, which is required by the laws of physics15 and has been shown by direct measurements.7,16,17 No hypothesis so far, however, has explained how CSF enters and remains in the syrinx, which has higher pressure than the outside subarachnoid space.\n\nIn this article, I present my data obtained by detailed analysis of phase-contrast magnetic resonance imaging (MRI) of patients with syringomyelia associated with Chiari-I malformation. Based on these data, I propose a new hypothesis on the pathophysiology of syringomyelia, which attempts to address the above-mentioned problem.\n\n\nMethods\n\nIn this study, I dealt solely with patients with Chiari-I malformations who had associated syringomyelia in the cervical cord. The following patients were excluded: those with syringomyelia not related to Chiari-I malformation, those with syringomyelia with basal arachnoiditis, and those with Chiari-I malformation without syringomyelia.\n\nThis study, which was approved by the Institutional Review Board of Tokai University Hospital. No. 18-609, was a retrospective study on prospectively acquired data. Since January 2011, I have routinely incorporated phase-contrast studies into the cervical spine MRI of patients with Chiari-I malformation. The MRI studies were performed before surgery and at each postoperative follow-up visit, namely, at six months postoperatively, one year postoperatively, and then at intervals of one year postoperatively. These MRI studies used the same acquisition techniques described below.\n\nFrom January 2011 to April 2019, a total of eighteen compatible patients underwent foramen magnum decompression at our institution. These patients were enrolled in my study consecutively during the study period. Because one patient’s preoperative data was missing, we analyzed 17 preoperative MRI and 18 postoperative MRI. At the same time, I recruited 21 healthy volunteers from our hospital staff so that the mean age would be comparable with that of the patients. They underwent the same MRI studies.\n\nAll patients underwent foramen magnum decompression performed by the first author using the same surgical techniques, which consisted of a small suboccipital craniotomy of 2 × 2 cm, C1 laminectomy, and a Y-shaped dural incision followed by fascia patching. I did not perform intradural exploration, which was not necessary in the studied population with no basal arachnoiditis. Then, I replaced the free suboccipital bone flap over the decompressed dura with titanium miniplates, adjusting its angle so that it would not compress the dura. Then, I sutured the fascia patch on the edge of the bone flap. This manoeuvre maintained decompression by preventing postoperative scar tissue invasion.\n\nAll magnetic resonance images were obtained using a 1.5 Tesla scanner (Achieva, Philips Medical System, Best, The Netherlands). At each MRI session, phase-contrast images were obtained in the midline sagittal section with the movement in the cranio-caudal direction encoded into intensity with a velocity encoding (VENC) of 10 cm/sec. Data acquisition was triggered by the QRS wave of the patient’s electrocardiogram with the cardiac cycle divided into 25 or 35 segments. The detailed imaging parameters were as follows: TR 16 msec, TE 7.2 msec, flip angle 15 degrees, field of view 256 × 256, matrix 352 × 256, and slice thickness 5 mm.\n\nThe phase-contrast images of each subject were displayed on a computer monitor using an image-processing application (ImageJ version 1.52a, National Institutes of Health, Bethesda, Maryland, United States (RRID:SCR_003070)). Circular shaped ranges of interest (ROIs) were set at the following locations (Figure 1): the cerebellar tonsil, the spinal cord segment between the fourth ventricle and the syrinx, the ventral subarachnoid space at the level of the base of the odontoid process, the dorsal subarachnoid space immediately below the tonsil, the rostral portion of the syrinx cavity, and the medulla at the level slightly above the foramen magnum. In the controls, the same ROI settings were used except for the ROI at the syrinx, which was set instead at the cord segment at the level of the C3 vertebral body. The average flow speed of the pixels inside each ROI was measured at each time point of the cardiac cycle. Thus, six waveforms corresponding to the six ROIs were obtained for each MRI session and were stored in a computer file.\n\nCircles indicate the regions of interest on a mid-sagittal section of a magnetic resonance image at the craniovertebral junction. vs: ventral subarachnoid space; ds: dorsal subarachnoid space.\n\nSynchronization in situ\n\nThe obtained data started at the QRS wave of the electrocardiogram of each patient. However, the latency from the QRS wave to the initial rise of brain and CSF movements significantly varied among patients. This created difficulty when I tried to analyze the data in more detail. I needed to post-process the data so that the initial rise of the brain and CSF movements would be better synchronized. For this purpose, I developed a post-processing technique that I called synchronization in situ.\n\nFirst, I defined the CSF trigger point as the time point where the ventral subarachnoid CSF started to move caudally i.e. where the CSF velocity most rapidly changed in the caudal direction. The CSF trigger point could be identified for each MRI session using the ventral subarachnoid CSF waveform contained in the data file. Then, I standardized/synchronized the six waveforms of each MRI session in the following manner. First, to standardize the different number of time bins among the MRI sessions, I increased the number of time bins per cardiac cycle to 50 using linear interpolation. I then shifted and synchronized the six waveforms using ring buffers so that the CSF trigger point would become the middle point of the waveform. The post-processed data is available in a data repository.18,19 The software used for the data analysis is available in a software repository.19,20\n\nI compared the following three groups: (1) Preoperative studies of Chiari-I patients, (2) Studies of normal volunteers, and (3) Postoperative studies of Chiari-I patients at the last clinical visit. For each group, the mean velocities at the six locations were calculated at each time point in the cardiac cycle. The obtained waveforms were plotted for each group.\n\nI statistically compared the peak caudal velocity at each ROI. Comparisons were made between the controls and the preoperative Chiari-I patients and between the preoperative and postoperative Chiari-I patients. For the ROI of the spinal cord, I also compared the peak rostral velocity in addition to the caudal velocity because, as I describe below, paradoxical rostral movement of the spinal cord was observed in Chiari-I patients.\n\nFor the statistical analyses, I used the unpaired t test for the comparison between the controls and the preoperative Chiari-I patients and the paired t test for the comparison between the pre- and postoperative Chiari-I patients. For the comparisons of tonsillar velocity and CSF velocities in the dorsal and ventral subarachnoid space, I used analysis of variance with Tukey’s post hoc multiple comparison test. P values smaller than 0.05 were considered statistically significant.\n\nThe following computer packages were used for the statistical analyses: R version 4.0.4 (R Project for Statistical Computing, RRID:SCR_001905) and RStudio version 1.2.1 (RStudio, RRID:SCR_000432).\n\n\nResults\n\nThe mean and standard deviation (SD) of the age of the patients was 40.2 years (SD 16.0). There were fourteen female and four male patients. The mean (SD) age of the volunteers was 33.7 years (SD 9.8). There were seven female and fourteen male volunteers.\n\nThere were no surgery-related complications. The mean period from surgery to the final postoperative MRI was 633 days (SD 555, range 70–1700). The syrinx shrank in seventeen out of the eighteen patients (94%) with improvement of preoperative symptoms (Figure 2). In one patient, upper extremity pain persisted after surgery despite shrinkage of the syrinx.\n\nThe left column shows the preoperative images, and the right column shows the postoperative images.\n\nFigure 3 shows the mean velocity waveforms of the five ROIs (the medulla, which made almost no movement, was excluded) in preoperative Chiari-I patients. As shown in the figure, the cerebellar tonsil (red line) made a rapid caudal movement in synchrony with the rapid caudal flow of the cerebrospinal fluid in the ventral and dorsal subarachnoid space (blue and yellow line). The timing of this tonsillar movement was in synchrony with that of CSF movement (Figure 3).\n\nThe abscissa shows one cardiac cycle divided into 100% percentile. The ordinate shows the flow speed (cm/sec). The waveforms are synchronized so that the maximal caudal acceleration of the ventral CSF is placed at the 50th percentile\n\nThe peak caudal velocity of the tonsils was significantly smaller than that of the CSF in the subarachnoid space. The mean (SD) of the peak caudal velocity was 0.76 (0.47), 2.3 (1.7), and 3.5 (2.0) for the tonsils, dorsal subarachnoid space, and ventral subarachnoid space, respectively. These differences were statistically significant (F = 13.9, p < 0.001), and Tukey’s post hoc test showed that the difference of each comparison pair was statistically significant with p values of 0.02, <0.001, and 0.05 for the tonsil vs. dorsal subarachnoid space, the tonsil vs. ventral subarachnoid space, and dorsal subarachnoid space vs. ventral subarachnoid space, respectively.\n\nThe syrinx fluid (green line in Figure 3) also made a rapid caudal movement in synchrony with the CSF and the tonsil. This fluid movement appeared early in the time course and was almost simultaneous with the caudal flow of the CSF (Figure 3). There was no noticeable delay or phase shift between the commencement of caudal syrinx fluid movement and that of the subarachnoid CSF movement (Figure 3). On the other hand, the reverse flow in the cranial direction began much earlier inside the syrinx than in the subarachnoid space (Figure 3).\n\nA puzzling finding shown in Figure 3 was the rostral movement of the upper cervical cord (purple line) when all the other parts were moving caudally in patients. This paradoxical movement of the cord was barely seen in the controls (Figure 4). The difference was statistically significant (Table 1).\n\nThe prominent movement of the cerebellar tonsils seen in preoperative Chiari-I patients (red line in Figure 3) was absent in controls (red line in Figure 4). Paradoxical rostral movement of the upper cervical cord (purple line in Figure 3) observed in patients was also barely seen in the controls (purple line in Figure 4). These differences were statistically significant (Table 1). The CSF velocities in the subarachnoid space were also significantly larger in preoperative Chiari-I patients than in controls (Table 1).\n\nThe same abscissa and ordinate as in Figure 3. (The legend syrinx denotes the spinal cord at the C5 level.)\n\nFigure 5 shows the postoperative mean velocity waveforms. The most notable postoperative change was disappearance of tonsillar movement. On the other hand, the velocity profiles of the other areas analyzed did not significantly change from the preoperative profiles (Figures 3, 5, Table 1). Figure 6 shows the velocity waveforms of the tonsil and the dorsal CSF extracted from Figures 3 and 5 together with their 95% confidence intervals. It can be clearly seen that the tonsillar velocity was significantly smaller than the CSF velocity, and despite the postoperative disappearance of tonsillar movement, the CSF velocity did not change significantly (Figure 5, Table 1).\n\nThe same abscissa and ordinate as in Figures 3 and 4.\n\n\nDiscussion\n\nIn preoperative Chiari-I patients, the velocities of the cerebellar tonsil and spinal subarachnoid CSF were significantly elevated compared to those of controls (Figures 3, 4, Table 1). This finding agrees well with other studies in the literature.10,21-25 I could, however, clearly show that the velocity of the tonsil was much smaller (approximately one-third) than that of the subarachnoid CSF (Figure 6, Table 1).\n\nThe abnormal tonsillar movement disappeared after surgery (Figures 5, 6). This also agrees with previous studies.7,26,27 However, the postoperative subarachnoid CSF velocities remained elevated in my data (Figure 5, Table 1). Regarding this point, the literature showed conflicting results; the postoperative CSF velocity either decreased, increased, or remained unchanged.7,26,27 This variability may have been caused by some of the differences in surgical techniques such as the size of the craniectomy. The important point is that the syrinx shrank in 94% of my patients in whom CSF velocity postoperatively remained unchanged.\n\nAccording to the hypothesis of Oldfield et al.,7,14 the piston-like movement of the tonsil generates pressure waves in the spinal subarachnoid CSF, which drives the CSF into the syrinx through the perivascular space of the cord. However, these results cannot be interpreted in accordance with this hypothesis. First, my data clearly showed that the velocity of the tonsil was much smaller than that of the CSF (Figure 6). It is difficult to imagine that a slower moving object can become the source of a faster moving object. Second, the velocity of the subarachnoid CSF did not decrease after surgery despite the postoperative disappearance of tonsillar movement (Figure 6). If the increased CSF pressure is the cause of a syrinx, it is difficult to explain the shrinkage of syrinxes in my patients while the CSF velocities were unchanged postoperatively.\n\nTherefore, these data can perhaps be interpreted more naturally as follows. The herniated tonsils in Chiari-I patients reduced the cross-sectional area of the subarachnoid space at the craniovertebral junction. Consequently, the velocity of the CSF at the craniovertebral junction was elevated because of the so-called Venturi effect.15 The piston-like movement of the cerebellar tonsil is rather the result, not the cause, of this increased CSF velocity at the craniovertebral junction.\n\nIn that case, how does the abnormal tonsillar movement in Chiari-I patients relate to syrinx formation? Because it was the only parameter that changed when the syrinxes shrank, the abnormal tonsillar movement may well be closely associated with the pathophysiology of syringomyelia. It did not seem, however, that the two phenomena (tonsillar movements and syrinx formation) were mediated by CSF pressure waves in the subarachnoid space as postulated by Oldfield et al.\n\nTo better interpret these data, I made the following three premises, some of which may be rather controversial.\n\n\n\n1. The origin of the syrinx fluid is the CSF, and there is a channel connecting the syrinx cavity and the subarachnoid space.\n\n2. The central canal cannot be ruled out as a candidate for this channel.\n\n3. There must be some kind of one-way-valve mechanism to sustain the expanded state of the syrinx.\n\nA significant amount of evidence supports the first assumption. The composition of the syrinx fluid is the same as that of the CSF.28 Intrathecally administered contrast or tracer materials readily enter the syrinx cavity.29,30,31 Recently, Heiss et al., quantitatively analyzed the accumulation of intrathecally administered contrast material into the non tumor-related syrinx.30\n\nThe second assumption is controversial and needs to be discussed in detail in the next section.\n\nGardner and Angel1 and Williams32 originally assumed that CSF enters the syrinx through a patent central canal. However, this idea recently lost favor for several reasons.\n\n\n\n1. MRI scans do not show the communication between the fourth ventricle and the syrinx in most cases.\n\n2. Autopsy studies of syrinx patients presumably showed little relationship between the syrinx and the central canal.\n\n3. Autopsy studies of patients showed that the central canal progressively becomes obliterated as age increases.\n\nThe first point may be an invalid argument. The diameter of the central canal is in the order of 100 μm.33 The current resolution of MRI scans cannot clearly show a channel of this size.34 Therefore, the fact that MRI scans do not portray the communication between the fourth ventricle and the syrinx may prove nothing about the presence or absence of such a channel.\n\nAs to the second point, Milhorat et al.,35 published the largest autopsy series of 105 syrinx patients. The authors classified the cases into 47 communicating and 23 noncommunicating syrinxes based on the MRI findings. The other 35 cases were syrinxes of various etiologies. In these 23 noncommunicating syrinxes, 70% rostrally continued to a stenotic central canal and 30% continued to a patent central canal. According to the author’s description, the stenotic central canals did not seem to be obliterated. Simply put, 100% of the noncommunicating syrinxes rostrally continued to a patent central canal. Therefore, these data support, rather than exclude, the possible role of the central canal in the pathogenesis of syringomyelia.\n\nRegarding the third point, the central canal was conventionally deemed to be occluded in human adults.34 However, recent studies have shown that this occlusion is an age-related gradual process.33,36 Newman et al.,36 found in their study of 60 autopsy cases that the central canal was patent up to the fourth decade of life, while Yasui et al.,33 found that occlusion of the central canal occurred in somewhat earlier ages. Milhorat et al. reported in their study of 232 autopsy cases that occlusion of the central canal was seen only in four individuals.37 Storer et al.,34 considered that ”the study of the morphology of the central canal is difficult using only histological sections” because of its three-dimensional characteristic and proposed a computerized 3-D method of evaluation. Considering that Chiari-I malformation occurs in the pediatric population and in relatively early adulthood,3,38 the possibility that a patent central canal plays an important role in the pathogenesis of syringomyelia cannot be ruled out.\n\nThe third assumption was that there must be a one-way valve mechanism to explain the generation and maintenance of syringomyelia. This idea has been expressed by previous authors.6,39 However, the concept of the one-way valve mechanism has not been duly incorporated in the previous theories of syringomyelia. This concept, however, may turn out to be essential if I consider the following points. First, basic physical laws indicate that the syrinx in its distended state has higher pressure inside than outside.15 It can be intuitively understood that the inside pressure must resist the elastic force of the syrinx wall in addition to the outside pressure. A few experiments with direct measurement of the syrinx pressure have proven that interior pressure was higher than the outside pressure.17,39\n\nThe mean value is shown together with the 95% confidence intervals.\n\nTherefore, any theory of syringomyelia assuming a CSF channel between the syrinx and the subarachnoid space must explain the mechanism by which the syrinx maintains its expanded state against the pressure gradient. Without this mechanism, CSF will flow out from the higher-pressure syrinx cavity to the lower-pressure subarachnoid space until the syrinx is collapsed and the pressure gradient is equilibrated. Assuming the existence of a one-way valve is a reasonable idea to solve this problem.\n\nMy hypothesis on the pathophysiology of syringomyelia can be summarized as follows:\n\n\n\n1. There is a communicating channel between the fourth ventricle and the syrinx, most likely a patent central canal.\n\n2. This channel assumes a one-way-valve function when mildly compressed by herniated tonsils.\n\n3. Repetitive pressure waves pump CSF through this valve, thereby creating a syrinx distally.\n\n4. The foramen magnum decompression switches off this one-way valve by removing the local compression, leading to the collapse of the syrinx.\n\nThe idea of the central canal working as a one-way valve was proposed by du Boulay et al., in 1974.6 This long-forgotten idea needs to be reviewed in the context of the premises described above. The herniated tonsils in Chiari patients move like a piston in accordance with the cyclic CSF movements. I may analogize this piston-like movement to that of a ball in a ball valve. In other words, the CSF undergoes more resistance in the caudal direction than in the rostral direction. The data showed that the velocity of the tonsils and the CSF near the craniovertebral junction has higher peak velocity in the caudal direction than in the rostral direction (Figure 3). Because higher velocity means higher resistance according to the Venturi effect, the data suggest that there is unidirectional resistance to the CSF flow at the craniovertebral junction; namely, the resistance to the caudal CSF flow is higher than that to the rostral CSF flow. This phenomenon has been demonstrated by Williams et al., and he postulated that this unidirectional CSF resistance gives rise to a sucking mechanism that generates syrinxes.2,32 My hypothesis somewhat resembles Williams’ idea, but more detailed explanation is needed. I showed in our previous article12 that, when resistance to CSF flow in the subarachnoid space is increased at some point, the transmural pressure in the central canal is increased in the downstream segment. Therefore, with reciprocating flows across a point with unidirectionally increased resistance, increased intramural pressure will be repetitively generated in the central canal situated downstream to the increased resistance. This will in effect bring forth a one-way valve mechanism in the central canal. I have supporting evidence for this idea based on our simulation model11,12 and will report it in our future correspondence. My hypothesis can also clearly explain why the limited decompression at the foramen magnum is effective in shrinking the syrinx. By localized decompression at the craniovertebral junction, the compression of the cord is relieved, and the piston-like movement of the tonsils ceases together with the unidirection CSF resistance. This will turn off the one-way valve function of the central canal. The syrinx fluid will then flow out according to the pressure gradient, eventually collapsing the syrinx. My hypothesis solves the problem concerning the interpretation of my phase-contrast data. The only difference in the postoperative phase-contrast data was the disappearance of the tonsillar movement; there was no significant difference in the movement of the CSF in the subarachnoid space (Figure 6). Therefore, an explanation was needed about how the cessation of tonsillar movement caused the syrinx to shrink without significant changes in subarachnoid CSF movement. My hypothesis explains it in a straightforward way. The disappearance of the piston-like movement of the tonsils cancels the ball-valve mechanism functioning in the central canal. The subarachnoid CSF movement has no role in this causal relationship.\n\nOn the other hand, other hypotheses that regard the perivascular space as the CSF channel may face two major problems. First, these hypotheses will have difficulty identifying a one-way valve. There are no structures along the perivascular space that would function as a one-way valve. Nevertheless, if I cannot assume a one-way valve mechanism, the CSF in the syrinx will flow out according to the pressure gradient between the syrinx and the subarachnoid space. Second, even if there is a one-way valve in the perivascular space in the spinal cord, how does this valve cease to function after simple decompression at the craniovertebral junction? This may be a difficult question to answer. Thus, in my opinion, the perivascular space theories contain a serious theoretical problem.\n\nI reported here the paradoxical rostral movement of the upper cervical cord in Chiari-I patients. This movement can be observed in the movie of color-coded phase-contrast data of one representative patient.19,40 This phenomenon has not been previously reported in the literature. I only found similar rostral movement in a figure in the article by Hofman et al.,23 who studied Chiari-I patients using phase-contrast MRI. The authors, however, did not discuss this phenomenon or perform statistical analysis.\n\nThe phenomenon is paradoxical because all the other parts (ventral and dorsal CSF, cerebellar tonsils, and syrinx fluid) are moving caudally, while the cord segment between the fourth ventricle and the syrinx is moving rostrally. For this paradoxical cord segment movement to occur, there must be some force exerted on it. This force must be exerted by some adjacent tissue. The medulla makes almost no movement during this period, and the outside CSF is moving rapidly downward. The only possible source of this force may be the syrinx fluid on the caudal side of this cord segment. If I assume some CSF movement from the fourth ventricle to the syrinx through the central cord or some other channel, this phenomenon may become somewhat more comprehensible. Further studies will be needed to carefully examine this phenomenon.\n\nThe number of patients was relatively small. Although I obtained statistically significant results, the data might well be interpreted with a certain amount of caution. Although my hypothesis avoided the difficulty of existing hypotheses and explained my obtained data reasonably well, it still lacks sufficient direct evidence to claim its veracity. My hypothesis thus remains a hypothesis until further supporting evidence is obtained in the future. It will, however, serve as a working hypothesis for future studies of syringomyelia. The exact mechanism how the one-way valve mechanism appears in the central canal when the outside CSF movement is blocked in one direction is not described in this report. I am currently studying this point using computer simulation. The results of this study will be reported in a future article. Regarding the paradoxical rostral movement of the spinal cord, its velocity was relatively small. This interesting phenomenon needs further careful study. If a similar type of block of the CSF movement as caused by the cerebellar tonsil occurs in other locations in the spinal canal, syrinxes may be generated with the same mechanism. It is thus possible that my hypothesis be extended to explain the pathophysiology of syringomyelia associated with arachnopathy.41\n\n\nConclusions\n\nThe analysis of phase-contrast data of Chiari-I patients showed data that contradicted the existing hypothesis on the pathophysiology of syringomyelia. My hypothesis that the central canal assumes a one-way valve function when compressed by the cyclical movement of the cerebellar tonsils could reasonably explain the data and further explain how the CSF enters and remains in the syrinx that has higher pressure than the outside subarachnoid space.\n\n\nData availability\n\nDryad: Underlying data ‘Phase-contrast MRI data of 18 Chiari-I malformation patients and 21 controls’. https://doi.org/10.5061/dryad.37pvmcvm0.18\n\n- Data files: encoded_data.json\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: Extended data ‘Phase-contrast MRI data of 18 Chiari-I malformation patients and 21 controls. https://doi.org/10.5281/zenodo.5338940.40\n\nThis project contains the following extended data:\n\n- Video file: 57.mp4\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\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.5200009.20\n\n- File: analyze.py\n\nLicense: MIT\n\nSupplementary information: https://doi.org/10.5281/zenodo.5229173.19\n\n- File: README.txt\n\nLicense: Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nConsent\n\nWritten informed consent for publication of the patients details and their images was obtained from the patients.", "appendix": "References\n\nGardner WJ, Angel J: The mechanism of syringomyelia and its surgical correction. 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PubMed Abstract \| Publisher Full Text\n\nHeiss JD, Jarvis K, Smith RK, et al.: Origin of Syrinx Fluid in Syringomyelia: A Physiological Study. Neurosurgery. 2019; 84(2): 457–468. 1524-4040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi KC, Chui MC: Conventional and CT metrizamide myelography in Arnold-Chiari I malformation and syringomyelia. AJNR. Am J Neuroradiol. February 1987; 8(1): 11–17. 0195-6108. PubMed Abstract \| Free Full Text\n\nWilliams B: The distending force in the production of communicating syringomyelia. Lancet (London, England). July 1970; 2(7662): 41–42. 0140-6736. PubMed Abstract \| Publisher Full Text\n\nYasui K, Hashizume Y, Yoshida M, et al.: Age-related morphologic changes of the central canal of the human spinal cord. Acta Neuropathologica. March 1999; 97(3): 253–259. 0001-6322. PubMed Abstract \| Publisher Full Text\n\nStorer KP, Toh J, Stoodley MA, et al.: The central canal of the human spinal cord: a computerised 3-D study. J Anatomy. May 1998; 192(Pt 4): 565–572. 0021-8782. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilhorat TH, Capocelli AL, Anzil AP, et al.: Pathological basis of spinal cord cavitation in syringomyelia: analysis of 105 autopsy cases. J Neurosurg. May 1995; 82(5): 802–812. 0022-3085. PubMed Abstract \| Publisher Full Text\n\nNewman PK, Terenty TR, Foster JB: Some observations on the pathogenesis of syringomyelia. J Neurol Neurosurg Psychiatry. November 1981; 44(11): 964–969. 0022-3050. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilhorat TH, Kotzen RM, Anzil AP: Stenosis of central canal of spinal cord in man: incidence and pathological findings in 232 autopsy cases. J Neurosurg. April 1994; 80(4): 716–722. 0022-3085. PubMed Abstract \| Publisher Full Text\n\nSakushima K, Tsuboi S, Yabe I, et al.: Nationwide survey on the epidemiology of syringomyelia in Japan. J Neurol Sci. 2012; 313(1-2): 147–152. PubMed Abstract \| Publisher Full Text Reference Source\n\nHall P, Turner M, Aichinger S, et al.: Experimental syringomyelia: the relationship between intraventricular and intrasyrinx pressures. J Neurosurg. June 1980; 52(6): 812–817. 0022-3085. PubMed Abstract \| Publisher Full Text\n\nChang HS: Phase-contrast MRI data of 18 Chiari-I malformation patients and 21 controls. Zenodo. 2021. Publisher Full Text\n\nKlekamp J, Batzdorf U, Samii M, et al.: Treatment of syringomyelia associated with arachnoid scarring caused by arachnoiditis or trauma. J Neurosurg. February 1997; 86(2): 233–240. 0022-3085. PubMed Abstract \| Publisher Full Text" }	[ { "id": "161190", "date": "06 Feb 2023", "name": "Petra M. Klinge", "expertise": [ "Reviewer Expertise Chiari", "syringomyelia", "tethered cord", "hydrocephalus" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEditorial Note from F1000Research – 27th February 2023: A previous version of this peer review report raised concerns about the methodology and data. Following further review of the data, the reviewer chose to withdraw these concerns. The approval status has been changed from Not Approved to Approved with Reservations\n\nThe author proposes a new concept of syringomyelia formation in CM based on a tailored MRI simulation. The weight is on the MRI methodology that has been published and presented previously and is available in the Data Availability section of the article. Given that, the data suggest an additional intriguing hypothesis of syrinx formation when comparing Chiari to controls and pre and post-surgery. The authors propose a one-way valve mechanism because that is the only way to explain syrinx formation and reduction based on their finding of reduced tonsil velocity and unchanged increased CSF velocity in the spinal SAS post-surgery. Naturally, there is no anatomical or other hard evidence available to show that the one-way valve mechanism is anatomically relevant and existing, but given the MRI observations and calculations in this patient series, even though speculative of nature and not the proof or implementation of a new concept, it is a reasonable addition to the pathophysiology of syringomyelia, which is still under scrutiny. I would only ask the authors to be mindful, when they say \" that their hypothesis contradicts and or is a disproof of pre-existing theories\" because findings were interpreted based on different methods, tests and imaging algorithms. They should make a more balanced statement in this regard.\n\nThe author must avoid a narrative style “ I have…” , “ I conducted…” , “ I did and decided...”. The entire paper is more a narrative style, particularly when detailing the methods.\n\nThe MRI method may be reviewed by an MRI physicist or neuroradiologist. Also, the statistical methods need to be reviewed with a statistician as the paired and unpaired T-test with Turkey post-hoc seem too crude. ANOVA testing or multivariate testing seems more appropriate.\n\nIs the work 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": [ { "c_id": "10024", "date": "07 Aug 2023", "name": "Hans Soo Chang", "role": "Author Response", "response": "Thank you very much for reviewing our manuscript. We have improved the English expressions throughout the entire manuscript as suggested by the reviewer. We have also enhanced the explanation of the synchronization method described in our manuscript. We believe that it has become much more understandable. Our procedure merely involves synchronizing the waveforms obtained from each individual. This is simply a post-processing of data acquired from the MRI and has no connection with MRI acquisition techniques. We believe there is no need for a physicist to review our manuscript. Regarding the statistics, it's important to note that we carried out two distinct comparisons: (1) preoperative Chiari patients versus controls, and (2) pre- and postoperative studies of Chiari patients. In the first comparison, the data consist of two separate series, while in the second comparison, the data represent repeated measures from the same individuals. We believe it's entirely reasonable to conduct t-tests individually for these two comparisons. However, if we were, as the reviewer suggested, to perform a statistical analysis combining these three groups: pre- and postoperative Chiari patients and controls, we would face a problem as the data incorporate both individual and repeated measures. To overcome this issue, we would need to resort to a more advanced statistical analysis such as mixed-effects models. Nevertheless, we believe this would be excessive for the current situation. Therefore, we maintain that our approach of employing two separate t-tests is justified. We believe that our manuscript has improved significantly thanks to the comments from the reviewer. We would be grateful if she could take the time to review our manuscript again." } ] }, { "id": "163449", "date": "27 Feb 2023", "name": "Yuichiro Ohnishi", "expertise": [ "Reviewer Expertise Neurosurgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript investigated the differences of velocity waveforms of the CSF in various craniocervical locations between the preoperative and control cases, as well as between the preoperative and postoperative cases. Author described that the pressure inside the syrinx is higher than that of the CSF outside. There is the current prevailing hypothesis assumes that the piston-like movement of the cerebellar tonsils drives the CSF into the syrinx through the spinal perivascular space. Author also mentioned that a major unexplained problem is how CSF enters and remains in the syrinx that has a higher pressure than the subarachnoid space. Currently, it still remains unknown how CSF enters and remains in the syrinx, which has higher pressure than the outside subarachnoid space. Author propose a new hypothesis on the pathophysiology of syringomyelia.\nThis study is the single institution and small sample size. Introduction is compact. I think author’s surgery is solid. Discussion is too speculatively written. Overall, I am impressed with the author's hypothesis. Results showed that the postoperative tonsillar velocity was significantly improved. While, there was no significant changes of subarachnoid CSF, spinal cord, and medulla in postoperative velocity. The abnormal tonsillar movement disappeared after surgery. However, results were inadequate to answer author's question. The pressure, CSF velocity, tonsillar movement, anatomical connection to syrinx, and other factors are implicated in Chiari-I malformation.\nMy main concerns are below. 1) What is the reason that the velocity of postoperative CSF was not changed significantly. 2) The CSF velocity has the possibility to decrease the pressure of syrinx? 3) If so, you can explain this mechanism?\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": "10025", "date": "07 Aug 2023", "name": "Hans Soo Chang", "role": "Author Response", "response": "Thank you very much for reviewing our manuscript. We have revised our manuscript, improving English expressions throughout. We have also rewritten the discussion to ensure it has a better logical structure. As we stated in the discussion, our data contradicts existing hypotheses. Moreover, the hypotheses that propose CSF enters the syrinx via the perivascular space face a significant theoretical challenge: they need to explain how CSF enters and remains in the syrinx against the pressure gradient (with syrinx pressure being higher than the surrounding subarachnoid space). We believe that a complete overhaul of the theoretical framework is needed. We must construct our theory from the ground up. In this context, we have offered our hypothesis as an attempt to overcome this theoretical difficulty. We clearly stated in the discussion that our hypothesis is speculative and is not directly supported by our data. However, given the current theoretical impasse, we believe that presenting our hypothesis is justified, as it could serve as a working hypothesis, stimulating future studies. The underlying mechanism of our theory is presented in our revised manuscript more clearly. For further clarification, we have conducted a computer simulation study of CSF dynamics in the spine to support our theory. We have drafted a manuscript and submitted it to a journal. It provides a more detailed explanation of our hypothesis. Unfortunately, it is still in the review phase, so we cannot present a citation at this time. We believe that our manuscript has improved significantly thanks to the reviewer's comments. We would be grateful if the reviewer could take the time to review our revised manuscript." } ] } ]	1	https://f1000research.com/articles/10-996
https://f1000research.com/articles/12-927/v1	02 Aug 23	{ "type": "Study Protocol", "title": "Comparative evaluation of retention of record bases fabricated using conventional impression and modified digital scan for complete denture: An in vivo study", "authors": [ "Mahima Agrawal", "Anjali Borle", "Anjali Borle" ], "abstract": "Background: In prosthetic dentistry, making an impression to replicate the oral health and tooth morphology is regarded as a crucial step. It is essential to ensure that the intraoral state is replicated as exactly as possible throughout this process, because any mistakes or inconsistencies could significantly impact the quality of the entire denture. In secondary impression, it’s crucial to capture the resilient oral mucosa (oral vestibule and soft palate) in order to acquire a satisfactory retention. As an alternative to conventional impression, digital scanning has been widely used in complete denture procedure. Although the digital scanning of edentulous ridges is feasible, scanning resilient oral tissues in complete denture still cannot be supported digitally. This study focuses on modifying the digital scan and comparison of retention of record bases fabricated from these modified digital scan to the record bases fabricated using conventional impression. Objectives: To compare the retention of record base prepared from conventional impression to the retention of record base from modified digital scan. Methodology: A total of 18 patients with edentulous maxillary arches will be recruited. A modified intraoral scan of the maxilla will be made on nine patients and on the remaining nine patients, a conventionally border moulded impression with a custom tray will be made. A record base with heat polymerized acrylic resin will be made on the definitive cast. Using the modified scanned image, the CAD-CAM bases will be milled. A dynamometer will be placed onto the record base; this will simulate the dislodging forces, thus clinically testing record base retention. Expected outcome: The record bases from modified digital scan will show equivalent or higher retention compared to record bases from conventional impression. Conclusions: By the end of the study, we will know whether resiliency of tissues can be recorded by modified digital scans.", "keywords": [ "Tissue resiliency", "digital scanning", "conventional impression", "retention" ], "content": "Introduction\n\nIn prosthetic dentistry, making an impression to replicate the oral health and tooth morphology is regarded as a crucial step. One of the most essential and integral parts of dentistry is making an accurate dental impression. It is essential to ensure that the intraoral state is replicated as exactly as possible throughout this process because any mistakes or inconsistencies could have a significant impact on the quality of the entire denture.1\n\nThe generated casts are used for diagnostic purposes, treatment planning and prosthesis. In order for the impression to serve its purpose, it has to accurately represent oral tissues of the patient.2\n\nIn secondary impression, it’s crucial to capture the resilient oral mucosa (oral vestibule and soft palate) in order to acquire a satisfactory retention. Soft-tissue management, inappropriate impression tray selection, separation of the impression material from the impression tray, distortion of conventional impressions prior to pouring, and storage of conventional impressions for prospective cast remaking are issues with conventional impressions. Furthermore, patients have reported that conventional impressions make for an uncomfortable treatment experience, particularly for young patients and those with a strong gag reflex. These problems associated with conventional impression can be solved by impressions with digital scanners. Digital systems have certain drawbacks compared to conventional impression systems, such as the difficulty to accurately imprint mobile tissues and the tendency of the scan to modify the impression by diminishing trueness when imprinting the jaws.3\n\nIn the last years, digital scanning has been widely applied in complete denture fabrication as an alternative to conventional impression taking. The direct scanning of edentulous jaws has recently been developed, although they do not account for functional mucosal reflections. However, in several of these approaches dependability and reproducibility are under doubt. The two greatest drawbacks are the inability to take functional digital impressions and the lack of precision.4\n\nHowever, fully edentulous jaws are challenging to scan with an intraoral scanner due to the presence of nonkeratinized moveable mucosa and smooth surfaces covered with saliva. Moreover, only a monostatic image of the edentulous arches can be obtained by intraoral scanning; the resilient oral mucosa is not included.5\n\nThe conventional impression for complete denture fabrication involves loading the custom tray with either an elastomer or zinc-oxide eugenol impression paste after moulding the periphery’s plastic border. The molding of a peripheral border in function is still not possible with a digital scan. Thus, digital scanning can be modified to correctly register resilient oral tissues and the soft tissue areas in the posterior maxilla which aids in retention of maxillary complete denture.6\n\n\n\n• To evaluate the retention of record base prepared by conventional impression method.\n\n• To evaluate the retention of record base prepared from modified digital scan.\n\n• To compare the retention of record base prepared from conventional impression to the retention of record base from modified digital scan.\n\n• To evaluate the effectiveness, clinical outcomes of modified digital impression compared to conventional impression.\n\nIt will be an in vivo experimental cross-over study.\n\n\nMethods\n\nThis in vivo study will be conducted in the Department of Prosthodontics and Crown & Bridge, Sharad Pawar Dental College and Hospital, Sawangi, Wardha.\n\nInclusion criteria\n\n• Patients willing to participate in the study.\n\n• Patients having signed informed consent form.\n\n• Patients with well-formed edentulous maxillary arch.\n\nExclusion criteria\n\n• Patients with excessive maxillary ridge resorption.\n\n• Patients with bony undercut of more than 2 mm in the maxillary ridge will be excluded.\n\n• Patients with poor neuromuscular control.\n\n• Patients with malignancies.\n\n• Patients with flabby ridge, papillary hyperplasia.\n\n• Patients with epulis fissuratum.\n\nThe participants will be divided into two groups, each group containing nine participants.\n\nGroup I - Conventional impression\n\nGroup II - Modified digital scan\n\nA thorough intraoral examination will be carried out. In group I patients, conventionally border molded impression with a custom tray will be made and a definitive cast will be obtained. A record base with heat polymerizing acrylic resin will be made on the definitive cast. In group II patients, the maxilla will be scanned using an intraoral scanner. The scanned image will be modified by incorporating coding calculations according to the resiliency of the tissues. Using the modified scanned image, the CAD-CAM bases will be milled. A platform will be designed with a 45-degree inclination. A dislodging force will be applied via a dynamometer connected to a hook. This will simulate the dislodging force thus, clinically testing record base retention. The retention offered by the conventional record base and CAD-CAM milled record bases from the modified intraoral scan will be compared.\n\nPrimary outcome\n\nModified digital impression will result in more time efficient technique than conventional impression. By the end of the study, we will know whether resiliency of tissues can be recorded by modified digital scans.\n\nSecondary outcome\n\nThis study integrates the modified digital scans in the workflow of complete denture fabrication.\n\nFormula using mean difference\n\nPrimary variable (retention of the base)\n\nAs per reference articles.\n\nTotal samples required = 9 per group.\n\nTotal number of groups = 2 groups.\n\nAn unpaired t-test will be used for statistical analysis.\n\nThe modified scan technique may overcome the clinical difficulties during recording of functional depth of sulci and access to the posterior areas taking into consideration the resiliency of tissues during digital scanning procedure. Thus, the modified scan technique may help the clinician to overcome a major drawback of intra oral scanner for complete denture impressions achieving retention of the denture bases equivalent to conventional procedures.\n\nThe study has not started yet at time of writing.\n\n\nDiscussion\n\nIt is currently unknown whether intraoral scans for complete dentures are of sufficient quality as compared to traditional border-molded impression procedures.6 This study will focus on the comparison of the record bases fabricated by conventional impression and modified intraoral scan and whether they show any relevant clinical differences.6 According to the present study, a mucostatic digital scan does not record the resiliency of tissues.6 The clinician places the finishing line of the intraoral scan at the boundary between the attached gingiva and the movable resilient tissues on the outer edge of the edentulous arch.6 This may result in a underextended or overextended boundary.6 Thus, there is a need to post-process the scan of edentulous arches when making the record.7 Compared to conventional impression technique digital impression technique is more patient friendly.7 Modifications of the digital scan may overcome the shortcomings of the intraoral scan and improve the retention.8–10\n\nEthical approval received from Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha. ICE reference number: DMIHER(DU)/ICE/2023/850.\n\nThe procedure will be explained to all the patients willing to participate in the research. A written informed consent will be taken from the patients.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nI thank my Institution and my guide for helping me in conducting this study.\n\n\nReferences\n\nSason GK, Mistry G, Tabassum R, et al.: A comparative evaluation of intraoral and extraoral digital impressions: An in vivo study. J. Indian Prosthodont. Soc. 2018 Apr; 18(2): 108–116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbduo J, Elseyoufi M: Accuracy of Intraoral Scanners: A Systematic Review of Influencing Factors. Eur. J. Prosthodont. Restor. Dent. 2018 Aug 30; 26(3): 101–121. PubMed Abstract \| Publisher Full Text\n\nAswani K, Wankhade S, Khalikar A, et al.: Accuracy of an intraoral digital impression: A review. J. Indian Prosthodont. Soc. 2020 Jan; 20(1): 27–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalik J, Rodriguez J, Weisbloom M, et al.: Comparison of accuracy between a conventional and two digital intraoral impression techniques. Int. J. Prosthodont. 2018 Mar 1; 31(3): 107–113. Publisher Full Text\n\nMai HN, Lee DH: A digital technique to replicate edentulous arches with functional borders and accurate maxillomandibular relationship for digital complete denture. J. Prosthodont. 2020 Apr; 29(4): 356–359. PubMed Abstract \| Publisher Full Text\n\nChebib N, Imamura Y, El Osta N, et al.: Fit and retention of complete denture bases: Part II–conventional impressions versus digital scans: A clinical controlled crossover study. J. Prosthet. Dent. 2022 Aug 30; PubMed Abstract \| Publisher Full Text\n\nYuzbasioglu E, Kurt H, Turunc R, et al.: Comparison of digital and conventional impression techniques: evaluation of patients’ perception, treatment comfort, effectiveness and clinical outcomes. BMC Oral Health. 2014 Dec; 14(1): 1–7. Publisher Full Text\n\nChebib N, Kalberer N, Srinivasan M, et al.: Edentulous jaw impression techniques: An in vivo comparison of trueness. J. Prosthet. Dent. 2019 Apr 1; 121(4): 623–630. PubMed Abstract \| Publisher Full Text\n\nD’Arienzo LF, Casucci A, Ferrari M, et al.: Accuracy, time efficiency and operator preference in edentulous arch scanning: a preliminary report: a preliminary report. J. Osseointegration. 2021 Sep 29; 13(3): 164–170.\n\nAgrawal S, Kambala S, Borle A, et al.: Comparative evaluation of effect of microcurrent electrical stimulation on acupoints to control gag reflex in patients receiving prosthodontic treatment: An in vivo study. J. Indian Prosthodont. Soc. 2022; 22: 188–194. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "198641", "date": "04 Sep 2023", "name": "Gopi Chander", "expertise": [ "Reviewer Expertise Prosthodontics", "Dental materials" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nClarity and Organization: The content is generally well-organized, with distinct sections for the introduction, background, objectives, trial design, methods, outcomes, and other relevant information. This structure helps readers understand the purpose and context of the study.\n\nImportance of the Research Topic: The content effectively communicates the importance of accurate dental impressions in prosthetic dentistry. It emphasizes how errors in impressions can affect the quality of dentures, making it clear why this study is necessary.\n\nReference to Existing Research: The content includes references to existing research articles, which adds credibility and shows that the study is grounded in previous work. However, the references could be better integrated into the text to support specific claims and statements.\n\nObjectives: The objectives of the study are clearly stated, which is essential for guiding the research. They focus on evaluating the retention of record bases prepared through conventional impressions and modified digital scans, as well as comparing the two methods.\n\nEligibility Criteria: The inclusion and exclusion criteria for the study participants are well-defined, ensuring that the sample is appropriate for the research objectives. This enhances the study's reliability and relevance.\n\nIntervention Description: The content provides a detailed description of the interventions for the two groups, explaining how record bases will be prepared in each case. This clarity is essential for understanding the study methods.\n\nOutcomes and Sample Size Calculation: The primary and secondary outcomes are clearly specified, giving readers a clear understanding of what the study aims to measure. The sample size calculation is also provided, demonstrating a scientific approach to study design.\n\nStatistical Method: The use of an unpaired t-test for statistical analysis is appropriate for comparing the retention of record bases between the two groups. However, it might be beneficial to mention the significance level (alpha) and power (beta) explicitly.\n\nDiscussion: The discussion section highlights the limitations of intraoral scans for complete dentures and suggests that digital scans may overcome these limitations. However, it's important to note that this is a study protocol, and the results are not available yet. Therefore, this discussion should be based on existing research and hypotheses rather than on the study's findings.\n\nEthical Considerations: The content mentions that ethical approval has been obtained, ensuring that the study adheres to ethical standards.\nOverall, the content is informative and well-structured, but it's essential to remember that it represents a study protocol, and the results are pending. The discussion section should focus more on existing knowledge and hypotheses until the study is completed and results are available. Additionally, integrating references more effectively throughout the text would strengthen the content's credibility.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] }, { "id": "216446", "date": "23 Oct 2023", "name": "Mohamed Ahmed Alkhodary", "expertise": [ "Reviewer Expertise Prosthodontics", "dental implants" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe rationale for, and objectives of, the study are clearly described, and the study design is appropriate for the research question, however, sufficient details of the methods provided do not allow replication by others as the nature of the modified scan is not explained, the coding calculations according to the resiliency of the tissues are not displayed at all, and the hypothesis explaining their mode of action, or effect on the scanning process is not explained, further elaboration is needed on this specific detail as it represents the core of the work. The citation added just shows the previous efforts to register the peripheral tissues using intra-oral scanners in a physical way, your in-silico method needs further explanation.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "206850", "date": "23 Oct 2023", "name": "Tahir ullah Khan", "expertise": [ "Reviewer Expertise ORAL AND MAXILLOFACIAL SURGERY" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nINTRODUCTION\nIts well structured and written in scientific way. Gaps in the literature are identified and will be addressed by this project.\n\nMATERIAL AND METHODS\nOver all its well written but I will suggest to add the following points to make the study much better.\nINCLUSION CRITERIA: inclusion bias may be reduced by\nPatients alveolar ridge width should be same.\n\nSulcus depth should be same.\nPROCEDURE\nAll impressions/ procedures will be carried out by single consultant.\nMention trade name, company make of impression material, casting material and fabrication material.\nMention company name, make and specification of Scanners which will be used.\nIt will be better if three hokes are used for checking retention 1. Anterior. 2. Right posterior. 3. Left posterior.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-927
https://f1000research.com/articles/10-538/v1	06 Jul 21	{ "type": "Software Tool Article", "title": "multiomics: A user-friendly multi-omics data harmonisation R pipeline", "authors": [ "Tyrone Chen", "Al J Abadi", "Kim-Anh Lê Cao", "Sonika Tyagi", "Al J Abadi", "Kim-Anh Lê Cao" ], "abstract": "Data from multiple omics layers of a biological system is growing in quantity, heterogeneity and dimensionality. Simultaneous multi-omics data integration is a growing field of research as it has strong potential to unlock information on previously hidden biological relationships leading to early diagnosis, prognosis and expedited treatments. Many tools for multi-omics data integration are being developed. However, these tools are often restricted to highly specific experimental designs, and types of omics data. While some general methods do exist, they require specific data formats and experimental conditions. A major limitation in the field is a lack of a single or multi-omics pipeline which can accept data in an unrefined, information-rich form pre-integration and subsequently generate output for further investigation. There is an increasing demand for a generic multi-omics pipeline to facilitate general-purpose data exploration and analysis of heterogeneous data. Therefore, we present our R multiomics pipeline as an easy to use and flexible pipeline that takes unrefined multi-omics data as input, sample information and user-specified parameters to generate a list of output plots and data tables for quality control and downstream analysis. We have demonstrated application of the pipeline on two separate COVID-19 case studies. We enabled limited checkpointing where intermediate output is staged to allow continuation after errors or interruptions in the pipeline and generate a script for reproducing the analysis to improve reproducibility. A seamless integration with the mixOmics R package is achieved, as the R data object can be loaded and manipulated with mixOmics functions. Our pipeline can be installed as an R package or from the git repository, and is accompanied by detailed documentation with walkthroughs on two case studies. The pipeline is also available as Docker and Singularity containers.", "keywords": [ "machine learning", "multi-omics", "data integration", "data harmonisation", "multivariate analysis" ], "content": "Introduction\n\nA biological phenotype is an emergent property of a complex network of biological interactions. Since relying on a single layer of omics data to test a biological hypothesis results in an incomplete perspective of a biological system, interest in multi-omics data integration is steadily increasing as a means to decipher complex biological phenotypes.1\n\nWe illustrate these points with a hypothetical case of measuring protein and transcript levels in a same set of matched samples. Each of these omics data layers contain independent information. A correlation score is then obtained between expression levels of the two blocks of omics data, resulting in an interpretable association measure. While correlation scores are a primitive metric, especially in this context of protein and transcript,2 they represent an additional layer of data summarising valuable relationships. Identifying highly correlated features across independent blocks of omics data could potentially reinforce the validity of the result, while highlighting interesting features (strong positive or negative correlations) for further investigation [Figure 1]. Hence, exploiting such parallel measurements from a multi-omics perspective allows a more comprehensive and cohesive view of such complex and often dynamic systems, and this resolution would be expected to improve as more omics layers are added. Published multi-omics studies discovering novel biological insights which are not possible with single-omics data further supports our points.3-9 With the increasing volume of multi-omics data present in publicly accessible biological data repositories,10-12 multi-omics data integration is expected to be the core strategy of modern and future biological data analyses.\n\nThe rectangles represent different layers of omics data (e.g. proteome, transcriptome and lipidome) while the circles represent features within their respective omics data layer. Black single-line arrows show correlation between features within the omics data (e.g. a regulatory factor) while blue double-lines show correlation between features across different omics data layers. A powerful abstraction of the system under study can be obtained by reviewing multiple layers of omics data holistically.\n\nAs a result, methods have been developed to leverage the multitude of data modalities in characterising biological systems. While many tools are available, most of these methods are heavily customised to fit a specific experimental design, and are not generic enough to handle most use cases.1 Furthermore, many tools that claim to perform data integration actually perform high-level data aggregation, where datasets are processed individually and only summarised, high level information is analysed together.1 Of these algorithms, few perform data integration of multiple layers of omics data simultaneously, which we refer to specifically as “data harmonisation” to distinguish it from the more general term of “data integration”.1\n\nWhile some “data harmonisation” algorithms exist, it is important to note that at this time, no end-to-end pipeline or framework exists which allows the user to quickly and easily input unrefined data, run a pipeline and export output data which can be used for downstream analyses and further downstream analyses. Therefore, to facilitate this, we developed multiomics, a flexible, easy-to-install and easy-to-use pipeline.\n\nWe present a pipeline targeted at bioinformaticians called multiomics13 with some important features, implementing one of the state of the art tools in data harmonisation from the mixOmics R package.14 It is portable with multiple implementations, and can be installed as an R15 package or used by cloning the associated git repository.16 A series of diagnostic plots are generated automatically and compiled into a pdf file. There is seamless integration with mixOmics, where data generated by the pipeline is exported automatically as a R data object of mixOmics classes. As a form of checkpointing, the R data object is updated at every major stage of the pipeline, and can be loaded directly into the mixOmics suite of tools for further investigation or plot customisation. To increase reproducibility, command line arguments are also exported as a script file which can be rerun directly to reproduce the output. To improve usability, the option to provide command line arguments as a json file is also available.\n\nDetailed documentation is provided both within the source git repository and as vignettes in the R package. Multiple installation methods are shown in the git repository to maximise accessibility of our pipeline for users. Additionally, walkthroughs of two case studies are included. Complete and detailed examples of input data format are also provided, including a sample dataset which can be loaded directly from the R package. In this manuscript, we summarise these information and show a minimum working example to highlight some of the features of our pipeline.\n\n\nMethods\n\nQuick install\n\nYou can install this directly as a R package from gitlab:\n\n\n\nDocker17 and Singularity18,19 images are also available if the user prefers to use containers directly. Note that you typically need root access to run Docker, if this is not possible try Singularity.\n\n\n\nIf you don’t have root access, you can try Singularity. The Singularity image file is large and you may need to set $SINGULARITY_TMPDIR to a custom location with at least 1 GB of free space.\n\n\n\nManual install\n\nIf the above automated install steps do not work, detailed manual installation instructions are available in the source git repository at https://gitlab.com/tyagilab/sars-cov-2/-/tree/master for conda and R.\n\nYou may need to install mixOmics from source. Follow the installation instructions on https://github.com/aljabadi/mixOmics#installation:\n\n\n\nThe actual script used to run the pipeline is not directly callable but provided as a separate script. Running the following command will show you the path to the script. A copy of this is also available in the source git repository.\n\n\n\nExample input\n\nThree elements are the minimum required input for the pipeline [Figure 2]. First, at least two files corresponding to omics data blocks are required. Next, a file containing biological class information is required. Finally, a list of unique names labelling each data block is required. Examples of these input files and their internal data structure as they appear in the pipeline are shown.\n\n\n\nWe summarise pipeline installation steps and the flow of data through the pipeline. This figure was originally published on gitlab under a CC-BY-3.0 AU license and is reproduced here with permission.\n\nNote that column names and row names should be truncated to avoid bugs in the pipeline associated with name length. Furthermore, usage of non-alphanumeric characters in their names should be avoided as R quietly replaces these with. (periods).\n\nExamples of these data and class files for two case studies are included in the source git repository.\n\nThe pipeline is run with the command Rscript run_pipeline.R and passing a list of command line arguments either as strings of text or in a json file (recommended). Running the actual pipeline can take some time. The main bottleneck is parameter tuning which scales exponentially with the number of omics data blocks, but it is possible to disable this if the user wants to perform a test run or is already aware of the parameters. We note that R Data objects are periodically exported that allow for seamless integration with functions in the underlying mixOmics package when needed. A secondary bottleneck is data imputation, which scales with the number of components used and the dimensions of the input data. If needed, it is possible to impute and export this imputed data either with the pipeline or with the underlying mixOmics function, and then substitute that as input. The user can adjust the number of cpus if needed to speed up the process. Data imputation can be skipped if it is not required.\n\nCode for the pipeline can be examined in detail from the git repository or individual functions can be inspected directly after loading the R multiomics package.\n\nOutput files include a pdf file compiling all graphical output.20-24 Note that this can be quite large, especially if you have a large dataset. A graphml file is also exported for input into cytoscape.25 Due to the size and volume of plots, we provide a link to some example plots here. A manuscript using figures generated from this pipeline is also available for reference.26\n\nEach analysis generates a series of text files containing feature weights. In some ways, these are functionally analogous to differential expression analyses, where these coefficients summarise the features with the most phenotypically relevant information. At the same time, a table of feature correlations across multi-omics data is generated. Some examples of these are shown below:\n\n\n\nAn R data file containing all of the information above and a script containing command line arguments which can be used to reproduce the analysis are also exported to enable full reproducibility.\n\nExamples of these output files for two case studies are included in the source git repository.\n\n\nUse cases\n\nWe demonstrate a sample use case of our pipeline with reference to an earlier re-analysis of a published dataset.13,26 Our tool takes as input at least two data files present as tables of quantitative information, with samples as rows and features as columns. A list of names corresponding to the names of these data blocks are required. A file containing class information is also required as a list of newline separated values. Examples of these data and class files for two case studies are included in the source git repository. Other command line arguments are also possible pertaining to distance metrics of choice for prediction, number of features to select and others. A full description of these can be obtained by running Rscript run_pipeline.R -h, which will list every flag in detail. Because of the number of command line arguments, an option is provided to pass these parameters as a json file to the pipeline. Examples of these json files for two case studies are included in the source git repository.\n\nRegarding input data, some example data27 is provided as part of our R package.\n\n\n\nAlternatively, you may download this from our git repository directly. This is a subset of anonymised clinical data provided in a separate publication.27\n\nWe provide a fully processed dataset as a guide for the user. The steps below can be reproduced by downloading the R data object with the following command:\n\nInspecting the minimum required input (classes and data) reveals the following:\n\n\n\nFirst, data is filtered if associated options are specified by the user. Features with missing values across sample groups are discarded by default. The user can also choose to filter out features (columns) exceeding a certain threshold of missing values.\n\nImputing missing values is optional as PLS-derived methods can function without this step. However, we include this information in case the user would like to perform this step manually. Remaining missing values can be imputed by the user-specified --icomp flag. Imputation is effective when the quantity of missing values is <20% of the data. To investigate if the data has been significantly changed, the user can plot a correlation plot of the principal components before and after imputation. Since imputation can take a long time, especially for large datasets, the imputed data is saved by default and the user can load it in directly as input if desired.\n\nIf the study design is longitudinal (e.g. has repeated measurements on the same sample), then the --pch flag should be enabled by the user. The user should pass in a file with the same format as the classes file, but containing information regarding the repeated measurements.23,28 Providing this information allows the pipeline to adjust for this internally.\n\nMost of the parameters for the machine learning algorithms are specified by the user. These cover the three methods PLSDA (partial least squares discriminant analysis), sPLSDA (sparse PLSDA) and multi-block sPLSDA (also known as DIABLO). The underlying methods are implemented within the mixOmics software package and more information is available on their website http://mixomics.org/. For each method, a distance metric is specified, either “max.dist”, “centroids.dist” or “mahalanobis.dist”. Unlike PLSDA, sPLSDA and multi-block sPLSDA focus on selecting subset of the most relevant features and therefore require a user-specified list describing the quantity of features to be selected from the data. The number of components to derive for each method is also provided. For this section, several exploratory runs with a wide range can be carried out to find the optimal configuration of features, e.g. starting at 5,10,30,50,100, inspecting subsequent output and further narrowing the range. The user can specify a few additional special parameters to the multi-block sPLSDA (block.splsda) function. The linkage parameter is a continuous value from 0 to 1, and describes the type of analysis, with a value closer to 0 prioritising class discrimination and a value closer to 1 prioritising correlation between data sets. Meanwhile, setting the number of multi-block sPLSDA components to 0 causes the pipeline to perform parameter tuning internally. Note that this can take a long time, and scales exponentially per added block of omics data. The user can also specify the number of cpus to be used for parallel processing, which mainly affects parameter tuning. Using our example, these arguments are provided here:\n\n\n\nTo examine the performance of each method, “M-fold” or “leave-one-out” cross-validation is performed to generate error rate plots. To account for cases where sample classes are imbalanced, balanced error rates which simply averages the class-wise error rates are also calculated and shown [Figure 3].\n\nError rates are calculated by “leave-one-out” cross-validation implemented in mixOmics. These plots are generated for each analysis type (PLSDA/sPLSDA/DIABLO). An example showing error rates for DIABLO is shown here. This figure was originally published on gitlab under a CC-BY-3.0 AU license and is reproduced here with permission.\n\nResults are exported in a series of plots and compiled into a pdf [Figure 4]. They can also be accessed internally from our provided R data object.\n\n(a) Multi-block sPLSDA (DIABLO) plots for component 1 and 2 can be interpreted similar to a PCA except that the model aims to discriminate the sample groups. (b) Clustered image maps show the relationship between variables and omics data blocks. (c) Barplots of loading weights show the contributions of variables towards each biological condition for each block. (d) Circosplot depicts the high multivariate correlations between the selected features from each block. Line thickness indicates the strength of the correlation. This figure was originally published on gitlab under a CC-BY-3.0 AU license and is reproduced here with permission.\n\nPipeline output can be controlled by specifying a number of flags. By default, the pipeline deposits data in the current working directory. This behaviour can be easily modified. Setting outfile_dir specifies the master output directory. An R data object containing objects shown in the loaded RData file can be renamed with the rdata option, generating a file similar to the one used in this example. The plot flag defines the pdf file containing all graphical output as a multi-page pdf of all plots generated in the pipeline. A reproducible script is generated and named by the user with the args flag (this defaults to Rscript.sh).\n\n\n\nFinally, the pipeline has a limited check-pointing built-in. At each milestone in the pipeline, the relevant output is saved and written out as a RData file, similar to the one presented above. This allows the user to manually inspect the data and adjust it to their needs where needed. In the case of completed output, the user can further customise plots and data exports for publication or downstream analysis. Importantly, data objects are compatible with core mixOmics functions, and allows seamless integration with the mixOmics suite of tools if the user intends to extend or perform their own custom analysis workflows.\n\n\nData availability\n\nPrimary data was generated by third parties and is publicly available.27,29 For case study 1, translatome data is available from the source publication as Supplementary Table 1 and proteome data is available as Supplementary Table 2. For case study 2, the authors provided their data in a sql database.\n\nZenodo: Multi-omics data harmonisation for the discovery of COVID-19 drug targets. https://doi.org/10.5281/zenodo.4602867.13\n\nThis project contains the following data.\n\n• Documentation in markdown format describing pipeline usage on two case studies.\n\n• Input data files in plain text (see Source Data for more information).\n\n• Graphical output as pdf files and feature weights as text files.\n\n• Source code, including code to reproduce figures in this article and source code for the R package.\n\n• Docker file specifications for use with Docker and singularity images.\n\nGitlab: SARS-CoV-2.https://gitlab.com/tyagilab/sars-cov-2.13\n\n• Documentation in markdown format describing pipeline usage on two case studies.\n\n• Input data files in plain text (see Source Data for more information).\n\n• Graphical output as pdf files and feature weights as text files.\n\n• Source code, including code to reproduce figures in this article and source code for the R package.\n\n• Docker file specifications for use with Docker and singularity images.\n\nThe following underlying data is used in this article:\n\n• data_lipidome.tsv (Text file as raw input data (lipidomics) for case study 2.)\n\n• data_metabolome.tsv (Text file as raw input data (metabolomics) for case study 2.)\n\n• classes_diablo.tsv (Text file as raw input data (biological classes) for case study 2.)\n\n• RData.RData (R data object containing all input, intermediate and output data for case study 2.)\n\n• manuscript_figures (Example output plots that can be generated by the pipeline.)27,29\n\nCode and data is available under the MIT license. Documentation is available under the CC-BY-3.0 AU license.\n\nThe following extended data is available in the same repository:\n\n• data/case_study_1 (All raw input data for case study 1.)\n\n• data/case_study_2 (All raw input data for case study 2.)\n\n• results/case_study_1 (Example output data for case study 1.)\n\n• results/case_study_2 (Example output data for case study 2.)\n\nSimilar to underlying data, extended code and data is available under the MIT license. Documentation is available under the CC-BY-3.0 AU license.\n\n\nSoftware availability\n\n\n\n• Software available through R directly:\n\nThe actual script used to run the pipeline is not directly callable but provided as a separate script.\n\n• Source code available from: https://gitlab.com/tyagilab/sars-cov-2\n\n• Archived source code at time of publication: https://doi.org/10.5281/zenodo.4562009\n\n• License: MIT License. Documentation provided under a CC-BY-3.0 AU license\n\nThe specific version numbers of the packages used are shown below, along with the version of the R installation.\n\n\n\n\nAuthor contributions\n\nConceptualization, S. T, T. C; Data Curation, S. T, T. C; Formal Analysis, K-A. L-C, T. C; Funding Acquisition, K-A. L-C, S. T; Methodology, A. J. A, K-A. L-C; Project Administration, S. T; Resources, S. T; Supervision, K-A. L-C, S. T; Software, A. J. A, K-A. L-C, T. C; Validation, A. J. A, K-A. L-C, S. T, T. C; Visualization, A. J. A, K-A. L-C, Writing Original Draft Preparation, S. T, T. C; Writing Review & Editing, A. J. A, K-A. L-C, S. T, T. C.\n\n\nCompeting interests\n\nThere is no competing interest.\n\n\nGrant information\n\nS. T acknowledges the AISRF EMCR Fellowship by the Australian Academy of Science and Australian Women Research Success Grant at Monash University. T. C received funding from the Australian Government Research Training Program Scholarship and Monash Faculty of Science Deans Postgraduate Research Scholarship. K-A. L-C was supported in part by the National Health and Medical Research Council (NHMRC) Career Development fellowship (GNT1159458).", "appendix": "Acknowledgements\n\nThe authors thank the HPC team at Monash eResearch Centre for their continuous personnel support. This work was supported by the MASSIVE HPC facility. We acknowledge and pay respects to the Elders and Traditional Owners of the land on which our 4 Australian campuses stand.\n\n\nReferences\n\nChen T, Tyagi S: Integrative computational epigenomics to build data-driven gene regulation hypotheses. GigaScience. June 2020; 9(6): 1–13. 2047-217X. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaier T, Güell M, Serrano L: Correlation of mRNA and protein in complex biological samples. FEBS Lett. October 2009; 583(24): 3966–3973. 0014-5793. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMashima J, Kodama Y, Fujisawa T, et al.: DNA data bank of Japan. Nucleic Acids Res. October 2016; 45(D1): D25–D31. 0305-1048, 1362-4962. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAthar A, Füllgrabe A, George N, et al.: ArrayExpress update – from bulk to single-cell expression data. Nucleic Acids Res. October 2018; 47(D1): D711–D715. 0305-1048, 1362-4962. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen T, Philip M, Lê Cao K-A, et al.: A multi-modal data harmonisation approach for discovery of COVID-19 drug targets. Brief. Bioinform. May 2021. 1467-5463, 1477-4054. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRohart F, Gautier Be, Singh A, et al.: mixOmics: An r package for ‘omics feature selection and multiple data integration. PLoS Comput Biol. November 2017; 13(11): e1005752. 1553-7358. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmoot ME, Ono K, Ruscheinski J, et al.: Cytoscape 2.8: New features for data integration and network visualization. Method. Biochem. Anal. December 2010; 27(3): 431–432. 1367-4803, 1460-2059. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen T, Philip M, Lê Cao K-A, et al.: A multi-modal data harmonisation approach for discovery of COVID-19 drug targets. Brief. Bioinform. May 2021; 0(0): 0. 1467-5463, 1477-4054. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOvermyer KA, Shishkova E, Miller IJ, et al.: Large-scale multi-omic analysis of COVID-19 severity. Cell Systems. January 2021; 12(1): 23–40.e7. 2405-4712. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWesterhuis JA, van Velzen EJJ, Hoefsloot HCJ, et al.: Multivariate paired data analysis: Multilevel PLSDA versus OPLSDA. Metabolomics. October 2009; 6(1): 119–128. 1573-3882, 1573-3890. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "98916", "date": "29 Nov 2021", "name": "Javad Zahiri", "expertise": [ "Reviewer Expertise Bioinformatics", "computational genomics", "machine learning" ], "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 the present study, \"multiomics: A user-friendly multi-omics data harmonisation R pipeline\" the authors tried to develop a tool for multiple omics integration and analysis. The problem is of utmost importance. However, the tool needs more work to be suitable for publication. The major problem is that installation is not easy at all for non-expert users. I had a bunch of biological researchers to install the tool, but they couldn't.\n\nIn addition the below codes produce errors:\n\n> metab <- read.table( \"data_metabolomics.tsv\", sep=\"\\t\", header=TRUE, row.names=1)\n\n> data_names (the variable has not been defined)\n\n>download.file(url, \"RData.RData)\n\nAnother important point is comparing multiomics to other recent similar tools like MOVICS and CNet (among several tools) and showing the current tool's strength compared to others.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? No\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? No", "responses": [ { "c_id": "9927", "date": "17 Nov 2023", "name": "Tyrone Chen", "role": "Author Response", "response": "> In the present study, \"multiomics: A user-friendly multi-omics data harmonisation R pipeline\" the authors tried to develop a tool for multiple omics integration and analysis. The problem is of utmost importance. However, the tool needs more work to be suitable for publication. The major problem is that installation is not easy at all for non-expert users. I had a bunch of biological researchers to install the tool, but they couldn't. In addition the below codes produce errors: > metab <- read.table( \"data_metabolomics.tsv\", sep=\"\\t\", header=TRUE, row.names=1) > data_names (the variable has not been defined) >download.file(url, \"RData.RData) ---- We agree and acknowledge that we did not sufficiently test the installation process of the pipeline to be usable for non-expert users. Since the original submission, we introduced a new automated test involving a full run of the pipeline on a new case study (a recent example is publicly visible online here), made major changes to the internal logic of the pipeline, and improved the install experience for users. We also now use a new simplified case study, and provided the associated data internally within the package as well as through downloadable text files as a backup option. The latest version of the pipeline should now be installable and accessible following the instructions provided. Our publicly available github issue tracker remains open for any bug reports or feature requests. ---- > Another important point is comparing multiomics to other recent similar tools like MOVICS and CNet (among several tools) and showing the current tool's strength compared to others. ---- We agree that providing some rationale for using this tool over others available is useful for readers. We note that our previous review covered this topic in significant detail (Chen & Tyagi, 2020), and provides strong justification for its use, since it is one of the only methods that is generic in scope as well as input data, compared to most existing methods that make restrictive assumptions or require highly specific input data. In the latest manuscript text, we made the above point clear in paragraph 5, while the new paragraphs 2,3,5 together provide a summary of key points to justify its use over the current ecosystem of tools, and this information combined with the cited review places this tool in context of the field. Chen T., Tyagi S., Integrative computational epigenomics to build data-driven gene regulation hypotheses, GigaScience, Volume 9, Issue 6, June 2020, giaa064, https://doi.org/10.1093/gigascience/giaa064 ----" } ] }, { "id": "89102", "date": "08 Dec 2021", "name": "Arjun Krishnan", "expertise": [ "Reviewer Expertise Computational biology", "Bioinformatics", "Machine learning", "Software development" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, Chen and colleagues present an R pipeline for multi-omics data analysis that can potentially accept unrefined data and produce convenient outputs. The pipeline is available as an R package and as Docker/Singularity containers. It is built on top of and closely integrated with the popular mixOmics package. Though this work could be useful, in its current form, it is unfortunately unclear what the new contributions are.\nMajor comments\nThe authors talk about the “lack of a single or multi-omics pipeline which can accept data in an unrefined, information-rich form pre-integration and subsequently generate output for further investigation”. What does “unrefined and information-rich mean” mean? As this is the primary motivator for this new pipeline, it needs to be explained clearly, especially in terms of the content and structure of data that multiomics can accept but existing packages like mixOmics cannot?\n\nHow is this pipeline different from the one published by the same authors in Briefings in Bioinformatics: A multi-modal data harmonisation approach for discovery of COVID-19 drug targets (Chen et al. (20211)?\n\nThe proposed pipeline – multiomics – heavily relies on the mixOmics package for all its data preprocessing, multivariate analyses, and plotting. The authors note that the speed and memory bottlenecks (e.g. parameter tuning and data imputation) are still problems. So, is multiomics a convenient wrapper for mixOmics? What are the contributions of the multiomics pipeline in terms of features that are not already part of mixOmics or any other existing multi-omics packages?\n\nThe writing can be considerably tightened.\nThe first two paragraphs in Introduction can be condensed to a few sentences so that the practicalities of multi-omics data analysis can be brought up soon.\n\nFigure 1 is not contributing to the exposition and can be removed.\n\nWhat do the following statements on Page 4 at the beginning of passage 3 mean?\n“implementing one of the state of the art tools in data harmonisation from the mixOmics R package”.\n\n“It is portable with multiple implementations”\n\nFix: “run a pipeline and export output data which can be used for downstream analyses and further downstream analyses\".\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "9928", "date": "17 Nov 2023", "name": "Tyrone Chen", "role": "Author Response", "response": "> In this article, Chen and colleagues present an R pipeline for multi-omics data analysis that can potentially accept unrefined data and produce convenient outputs. The pipeline is available as an R package and as Docker/Singularity containers. It is built on top of and closely integrated with the popular mixOmics package. Though this work could be useful, in its current form, it is unfortunately unclear what the new contributions are. Major comments The authors talk about the “lack of a single or multi-omics pipeline which can accept data in an unrefined, information-rich form pre-integration and subsequently generate output for further investigation”. What does “unrefined and information-rich mean” mean? As this is the primary motivator for this new pipeline, it needs to be explained clearly, especially in terms of the content and structure of data that multiomics can accept but existing packages like mixOmics cannot? ---- Conventional biological pipelines involve multiple layers of data preprocessing and summarisation, where at each stage information is irreversibly lost. Therefore, in this context, unrefined and information rich refers to data in a primary form at a stage before heavy information loss occurs, such as matrices of molecular abundance data. Our pipeline takes these quantitative matrices as input, and can identify low-level correlations across individual molecules. We made this point clearer in introduction paragraph 5 of the latest version of the manuscript. We considered point (1) in relation to point (4), and made the above information clearer and more concise by reorganising the logic flow of the introduction. As a result, some paragraphs in the introduction are reordered or combined. ---- > 2. How is this pipeline different from the one published by the same authors in Briefings in Bioinformatics: A multi-modal data harmonisation approach for discovery of COVID-19 drug targets (Chen et al. (20211)? ---- Although the concept and function of the analysis workflow is identical, we saw the gap in the field of developing generic multiomics data integration solutions. Hence, we automated the workflow and the latest version contains major improvements to the internal logic and additional features since the original pipeline was first developed. A new simplified case study is available as well in the latest update and available on github. ---- > 3. The proposed pipeline – multiomics – heavily relies on the mixOmics package for all its data preprocessing, multivariate analyses, and plotting. The authors note that the speed and memory bottlenecks (e.g. parameter tuning and data imputation) are still problems. So, is multiomics a convenient wrapper for mixOmics? What are the contributions of the multiomics pipeline in terms of features that are not already part of mixOmics or any other existing multi-omics packages? ---- Data preprocessing steps such as the filtering of low-variance columns and formatting missing/zero values are our own custom additions and not part of the original mixOmics pipeline. These functions were written in collaboration with the original mixOmics authors and designed to create input compatible with mixOmics functions. (ref section: “Data preprocessing”) Regarding speed and memory bottlenecks, the latest version of the pipeline has been updated to align with the improved parallelisation in the latest version of mixOmics, and now also outputs a more comprehensive table of parameters for easy reuse to skip computationally expensive parameter tuning. To avoid the data imputation bottleneck, we save the imputation output for reuse, therefore imputation only needs to be run once instead of every pipeline iteration (ref section: “Running the pipeline”). We use mixOmics at the centre of our multiomics pipeline. Here, our key contribution is in the specific context of adding a layer of abstraction for non-expert users, resulting in an end-to-end pipeline for many independent mixOmics functions that can be run in a single command. This is non-trivial and conceptually similar to projects like nf-core (Ewels et al, 2020), where their main contribution to the field is achieving a high degree of automation to streamline complex bioinformatics pipelines. The main advantages are that it is both (a) accessible to new users who can generate a large quantity of relevant information in a single step, as well as being (b) convenient to expert users as an initial screen, as the internal data structures are identical and can be extracted for further analysis. Ewels, P.A., Peltzer, A., Fillinger, S. et al. The nf-core framework for community-curated bioinformatics pipelines. Nat Biotechnol 38, 276–278 (2020). https://doi.org/10.1038/s41587-020-0439-x ---- > 4. The writing can be considerably tightened. The first two paragraphs in Introduction can be condensed to a few sentences so that the practicalities of multi-omics data analysis can be brought up soon. ---- We considered point (1) in relation to point (4), and made the above information clearer and more concise by reorganising the logic flow of the introduction. As a result, some paragraphs in the introduction are reordered or combined. The information is also condensed as recommended. ---- Figure 1 is not contributing to the exposition and can be removed. ---- We believe that providing a figure targeting a general audience would be helpful, and note that this figure was used in multiple presentations to summarise the importance of multi-omics to a non-expert audience (including non-biologists), who found it helpful based on their feedback. In particular, the low-level correlations across individual molecules that we show are not a part of conventional multi-omics studies and is an important piece of information for most audiences, including biologists. We therefore decided to retain this figure. ---- What do the following statements on Page 4 at the beginning of passage 3 mean? “implementing one of the state of the art tools in data harmonisation from the mixOmics R package”. ---- This was redundant with a previous statement and is now removed. Original intention was to highlight the uniqueness of mixOmics as a generic data integration method. This point is now emphasised in the introduction section. ---- “It is portable with multiple implementations” ---- “Portable” refers to the usability of our package across different machines and operating systems. “Multiple implementations” referred to the original availability of our software as both a docker container as well as a R package. In the latest version of the manuscript, we removed “multiple implementations” since the latest version of our software does not use a docker container, as we considered this to be now redundant with the R package. As for “portability”, this still holds across different linux machines, but we did not test this for use on windows or mac, and we now clarified this point in the installation instructions for the github repository. We prioritised the linux version since the pipeline can be computationally expensive, and it would ideally be run on high performance compute clusters which run on linux. ---- Fix: “run a pipeline and export output data which can be used for downstream analyses and further downstream analyses\". ---- We thank the reviewers for catching this, now fixed." } ] } ]	1	https://f1000research.com/articles/10-538
https://f1000research.com/articles/12-925/v1	02 Aug 23	{ "type": "Systematic Review", "title": "Can yoga show better vascular outcomes than regular exercise among office workers: a scoping review", "authors": [ "Neha Susan Jibi Pattamukkil", "Poovitha P Shruthi", "Rajagopal Kadavigere", "Vaishali K", "Shivashankar K N", "Suresh Sugumar", "Sneha Ravichandran", "Koustubh Kamath", "Neha Susan Jibi Pattamukkil", "Poovitha P Shruthi", "Rajagopal Kadavigere", "Vaishali K", "Shivashankar K N", "Sneha Ravichandran" ], "abstract": "Background: Isolated systolic hypertension (ISH) in the elderly is mostly brought on by arterial stiffness with ageing, which results in a lack of vascular compliance. The main cardiovascular (CV) hazards for elderly persons are arterial ageing and high blood pressure (BP). The aim of this study was to investigate if endothelial function in young, middle-aged, and older, healthy persons would be improved by Bikram yoga, a heated form of hatha yoga. Methods: Randomised control trials on yoga, vascular function, exercise, and office employees were used in the study, which was done in four databases (Scopus, PubMed, Ovid, and Science Direct). Results: 3 of 103 articles were included. We identified improvement of vascular function, FMD and arterial stiffness using yoga intervention. While exercise does not significantly affect ventricular function, yoga significantly reduces heart rate (p = 0.031) and increases pulse pressure (PD). Yoga also results in a mean increase in left ventricular ejection time (LVET), which was observed in the yoga group. The improvement in myocardial function was more pronounced in the yoga group than in the exercise group, according to the between-group analysis, which revealed a significant difference in post-intervention rate-pressure product (RPP) between the yoga and exercise groups. With a P value of 0.004, the mean% Mean Arterial Pressure (MAP) was significantly lower, demonstrating that yoga was effective in lowering MAP. However, older adults, but not younger ones, experienced a significant increase in brachial artery FMD (Flow Mediated Dilation) following the intervention. Conclusions: Yoga interventions may improve vascular function, arterial stiffness, flow-mediated dilation, heart rate, RRP and MAP, with older adults experiencing a significant increase in brachial artery FMD, suggesting the effectiveness of yoga in improving myocardial function and lowering MAP, particularly in older adults.", "keywords": [ "Yoga", "Bikaram yoha", "hatha yoga Physical Activity", "Vascular Function", "FMD", "Exercise", "Sedentary lifestyle" ], "content": "Introduction\n\nEndothelial cells (ECs), vascular smooth muscle cells (VSMCs), the autonomic nervous system, adventitial tissues, including inflammatory cells, and vasa vasorum are only a few of the cell types that regulate vascular function.1 The gradual and quick development of novel biochemical markers and imaging techniques made a risk stratification for the start of cardiovascular disease or the onset of vascular events an extremely difficult process.2 Vascular homeostasis is disrupted by endothelial dysfunction, which raised the risk of cardiovascular disease and along with its related events.3 Due to its low impact and low joint stress characteristics, yoga may be a viable substitute for regular exercise among older persons.4\n\nWe previously reported that the most popular form of hatha yoga in the United States, non-heated hatha yoga, has not been shown to be beneficial in improving vascular function.5,6 Bikram (hot) yoga has shown a reduction in arterial stiffness in young people (ages 20 to 39 years), but not in middle-aged persons.7 The observed difference in myocardial function and vascular health between younger and older adults following yoga intervention may be attributed to factors such as the shorter duration8,9 of the intervention for the older adults, arterial ageing, and elevated blood pressure emerging as primary cardiovascular risk.8,9 As people age, structural and functional changes occur in the heart and blood arteries.10 As a mind-body lifestyle paradigm, isolated systolic hypertension (ISH) in the elderly is principally brought on by the loss of vascular compliance with ageing brought on by the stiffening of arteries.11–14 Several studies have demonstrated the positive effects of yoga programmes for both young as well as elderly persons’ cardiovascular health.15–17\n\n\nMethods\n\nThe Arksey and O’Malley framework16 is the foundation for this review. The following processes were taken: (1) defining the research issue, (2) locating studies that could address it, (3) choosing the study, (4) charting the data, and (5) reporting and summing the findings.\n\nBased on topic results and population, we divided this scoping study into two key research issues. Along with office professionals, young adults made up the target population.\n\nThe research topic and the desired result are shown in Table 1.\n\nSearch strategy\n\nStudies on vascular function, yoga, exercise, and office employees were the subjects of a systematic search that was undertaken in four databases (Scopus, PubMed, Ovid, and Science Direct) with the following Bolen operators in the search strategy [((((Yoga) OR (exercise) OR (Physical Activity))) AND (office worker) OR (Desk based workers)) NOT (children)]. The timeline was between 2000 and 2023. MeSH terms are listed in Table 2.\n\nThe investigation began in March 2023 and was finished on May 17, 2023. After the last round of searches was completed in front of a senior librarian, an inclusion analysis was carried out after May 20, 2023. Articles included focused on Yoga intervention effects, physical activity, vascular function, and improved body changes. The article’s language were English-based, and the articles covered the outcomes of exercise and yoga intervention while focusing on blood vessel diameter and velocity. The details are added in Figure 1.\n\nCharting of data\n\nThe Data chart done independently by authors, NS and KK. First we found the article based on the research question, the bias was rectified by SS using MS excel and Rayyan Software. The final data extracted was verified by SR. We tabulated the following information based on the publications we collected: authors, year, the nation in which the study was done, aims, participants, and activity break. Additionally, information about the control group, the washout time, and the major findings regarding hypertension, SB, and PA were collected.\n\n\nResults\n\nOnly three of the 103 publications that were discovered to address the research topic based on yoga, vascular function, and arterial width and velocity were located based on search results from four index databases, as shown in Figure 1. Two studies were conducted in USA, one study conducted in India. Three studies are specially focused on vascular function and arteries. The tabular data on SB, PA, hypertension, and its based treatments are shown in Table 3.\n\nGurunathrao S discovered that yoga significantly increases PD (p = 0.042) while decreasing HR (p = 0.031). While there is no difference between the exercise group and the yoga group in terms of ventricular function, the yoga group experienced a mean increase in Left Ventricular Ejection Time (LVET), and the yoga group experienced a significant decrease in RPP. There was no change in the walking exercise group but there was a significant decrease in mean % Mean Arterial Pressure (MAP) within the yoga group (p = 0.004).18\n\nThe study on the effect of Bikram yoga on endothelial function in young and middle-aged and older adults, examined how Bikram yoga influenced brachial artery flow-mediated dilation among two distinct age groups: young (age 18-39) and middle aged/older individuals (age 40-70).19 In the older group exhibited significantly higher baseline values for age and body mass, and they tended to have a higher baseline BMI compared to the young group. Surprisingly, the study observed no significant change in the brachial artery flow-mediated dilation after Bikram yoga in the younger group. However, in the older group, Bikram yoga demonstrated significant changes, indicating its effectiveness for this age category.19\n\nAccording to Hunter SD 1000.Editorial.Emerald.al there are no further significant group effects were found for post-intervention body weight, systolic and diastolic blood pressure, pulse, BMI, and mean blood pressure. In one study, after 12 weeks, brachial-ankle PWV in the hot (p = 0.52), thermoneutral (p = 0.75), and control groups remained stable (p = 0.31).9\n\n\nDiscussion\n\nReviewing the evidence, we found that the yoga interventions improved vascular function, flow-mediated dilation, and arterial stiffness.\n\nHeart rate (HR)\n\nRef. 18 the compared the results between a yoga group and an exercise group, specifically focusing on heart rate (HR) and PD. The following findings were observed:\n\n1. Yoga Group:\n\nWithin the yoga group, a significant reduction in HR was observed with a p-value of 0.031. This suggests that practicing yoga led to a measurable decrease in heart rate. Additionally, there was an increase in PD with a p-value of 0.042. This indicates that participants in the yoga group experienced an increase in discomfort during the intervention.\n\n2. Exercise Group:\n\nIn contrast to the yoga group, no meaningful change was observed within the exercise group for both heart rate and perceived discomfort. This implies that the exercise regimen employed in the study did not lead to significant alterations in these measures.\n\n3. Between Group Comparison:\n\nA significant mean change between the yoga and exercise groups was observed for both heart rate (p-value of 0.036) and perceived discomfort (p-value of 0.002). This indicates that the changes in heart rate and perceived discomfort differed significantly between the two groups. In other words, the effects of yoga on these measures were distinct from the effects of exercise.\n\nRegarding the interpretation of p-values, a p-value less than 0.05 is commonly used to determine statistical significance. In this study, the reported p-values (0.031, 0.042, 0.036, and 0.002) are all below 0.05. Therefore, the observed changes in heart rate and perceived discomfort are considered statistically significant, suggesting that these changes are unlikely to have occurred by chance.\n\nRate pressure product\n\nThe compared results between a yoga group and an exercise group, specifically focusing on rate-pressure product (RPP) as a measure of myocardial performance. The findings of the study indicate the following:\n\n1. Yoga Group:\n\nWithin the yoga group, a significant decrease in RPP was observed. The rate-pressure product reflects the workload on the heart, and a decrease in RPP suggests improved cardiac efficiency. This finding suggests that practicing yoga was associated with a beneficial impact on myocardial performance.\n\n2. Exercise Group:\n\nIn contrast to the yoga group, the exercise group did not exhibit significant changes in RPP. This implies that the exercise regimen employed in the study (walking exercise) did not result in notable improvements in myocardial performance, as measured by RPP.\n\n3. Between Group Comparison:\n\nThe group analysis revealed a significant difference in post-intervention RPP between the yoga and exercise groups. This suggests that the improvement in myocardial performance, as indicated by the decrease in RPP, was more pronounced in the yoga group compared to the exercise group. Therefore, the study implies that yoga may be a more effective treatment than walking exercise for enhancing cardiac performance.18\n\nArterial Stiffness\n\nA substantial decrease in mean% MAP was observed in the yoga group, with a P value of 0.004 indicating that yoga was effective in lowering MAP. In contrast, there was no discernible difference in the mean% MAP between the yoga and walking exercise groups. Yoga shows a positive effect on reducing MAP; walking exercises did not produce any significant changes.18\n\nThe study looked at how Bikrama Yoga affected young, middle-aged, and older people’ flow-mediated dilation (FMD) of the brachial artery.19\n\n1. Effect of Time and Age Interaction:\n\nThe study found that both the entire cohort and different age groups showed significant effects of time on FMD, indicating that FMD changed over the course of the intervention. Additionally, there was a significant time by age interaction, suggesting that the relationship between time and FMD differed across age groups. These findings highlight the relevance of considering age as a factor when examining the effects of Bikram Yoga on FMD.\n\n2. Age and Baseline Characteristics:\n\nAt baseline, older age groups had higher body mass and BMI compared to younger individuals. While body weight and BMI tended to decrease in the older age groups during the intervention, there were no significant changes in BMI, body mass, or body fat percentage in either age group as a result of Bikram Yoga. These baseline characteristics and lack of significant changes in body composition provide important context for interpreting the study’s results.\n\n3. Brachial Artery FMD:\n\nInitially, there were no baseline differences in brachial artery FMD between the young and older groups. However, following the Bikram Yoga intervention, only the older adults experienced a significant increase in brachial artery FMD. This suggests that Bikram Yoga may have a positive impact on FMD specifically in middle-aged and older individuals, while younger individuals did not exhibit significant changes in this measure.\n\nThe study you mentioned investigated the effects of hot yoga, thermoneutral yoga, and a control group on various outcomes, including brachial-ankle pulse wave velocity (PWV), blood pressure, BMI, body weight, and pulse.9\n\n1. Main Effects of Group:\n\nThe study found no significant main effects of group on post-intervention body weight, systolic and diastolic blood pressure, BMI, pulse, and mean blood pressure. This suggests that there were no significant differences in these outcomes among the hot yoga, thermoneutral yoga, and control groups after the intervention period.\n\n2. Brachial-Ankle PWV:\n\nAdditionally, there was no significant change in brachial-ankle PWV observed in the hot yoga, thermoneutral yoga, or control groups after the 129-week intervention period. Pulse wave velocity is a measure of arterial stiffness, and the lack of significant change suggests that neither type of yoga had a significant impact on arterial stiffness compared to the control group.\n\n\nConclusion\n\nBased on the research studies conducted, it has been consistently demonstrated that yoga has a positive impact on improving vascular function. However, it is important to note that further practice of yoga is necessary to continue experiencing these improvements in vascular function. Looking ahead, it is plausible to anticipate that yoga could serve as an excellent exercise option for office workers, potentially offering a range of benefits for their vascular health.\n\nThe field of yoga research faces several challenges that affect the ability to draw definitive conclusions. Firstly, there is a notable variation in the types of yoga interventions studied, including Bikram Yoga and unspecified practices. This lack of standardization in the type, duration, frequency, and intensity of interventions hinders the comparison of results and limits our understanding of the effects of yoga on desired outcomes. Secondly, the inclusion of participants from diverse age groups, such as young, middle-aged, and older individuals, introduces heterogeneity that can influence outcomes and complicate result interpretation, especially when considering factors like body mass index (BMI) and body weight. Conducting studies specifically targeting different age groups and accounting for relevant confounding factors would provide more valuable insights. Additionally, each study utilizes different outcome measures, such as heart rate, perceived discomfort, rate-pressure product, flow-mediated dilation (FMD), and pulse wave velocity (PWV), which provide insights into various aspects of cardiovascular health. However, employing standardized and widely accepted measures would enhance comparability and reliability across studies. Lastly, the duration of interventions varies widely, ranging from 12 weeks to unspecified time periods, and longer-term follow-up assessments are needed to comprehensively understand the sustainability and long-term effects of yoga interventions on the desired outcomes.\n\nFuture outcomes in yoga research include the establishment of standardized protocols for different types of yoga interventions, enabling better comparison of study results and definitive conclusions on the effects of yoga. Targeted studies focusing on specific age groups and considering confounding factors would provide more accurate insights. Emphasizing standardized outcome measures would enhance comparability and reliability across studies, facilitating meta-analyses and robust conclusions. Additionally, longer-term follow-up assessments beyond the current intervention durations would shed light on the sustainability and long-term effects of yoga interventions, providing a deeper understanding of their lasting benefits and potential risks. These efforts aim to address existing challenges and improve the quality and reliability of yoga research.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nShimokawa H, Satoh K: Vascular function. Arteriosclerosis, Thrombosis, and Vascular Biology. Vol. 34. . Lippincott Williams and Wilkins; 2014; pp. 2359–2362.\n\nBiomarkers as drug development tools: discovery, validation, qualification and use.\n\nChan SY, Mancini GBJ, Kuramoto L, et al.: The prognostic importance of endothelial dysfunction and carotid atheroma burden in patients with coronary artery disease. J. Am. Coll. Cardiol. 2003 Sep 17; 42(6): 1037–1043. Publisher Full Text\n\nOliveros E, Patel H, Kyung S, et al.: Hypertension in older adults: Assessment, management, and challenges. Clinical Cardiology. Vol. 43. . John Wiley and Sons Inc.; 2020; pp. 99–107.\n\nHunter SD, Tarumi T, Dhindsa MS, et al.: Hatha yoga and vascular function: Results fromcross-sectional and interventional studies. J. Bodyw. Mov. Ther. 2013 Jul; 17(3): 322–327. PubMed Abstract \| Publisher Full Text\n\nDaugherty A, Fisher EA, Taubman MB, et al.: Forty-Year Anniversary of Arteriosclerosis, Thrombosis, and Vascular Biology. Arterioscler. Thromb. Vasc. Biol. 2021; 41(9): 2353–2356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHunter SD, Dhindsa MS, Cunningham E, et al.: The Effect of Bikram Yoga on Arterial Stiffness in Young and Older Adults. J. Altern. Complement. Med. 2013 Dec; 19(12): 930–934. Publisher Full Text\n\nVlachopoulos C, Aznaouridis K, Stefanadis C: Prediction of Cardiovascular Events and All-Cause Mortality With Arterial Stiffness. A Systematic Review and Meta-Analysis. J. Am. Coll. Cardiol. 2010 Mar 30; 55(13): 1318–1327. Publisher Full Text\n\nHunter SD, Laosiripisan J, Elmenshawy A: Effects of heated and thermoneutral yoga interventions on arterial stiffness in middle-aged adults. Complement. Ther. Med. 2018 Oct 1; 40: 113–115. PubMed Abstract \| Publisher Full Text\n\nFleg JL, Strait J: Age-associated changes in cardiovascular structure and function: a fertile milieu for future disease. Heart Fail. Rev. 2012 Sep 2; 17(4–5): 545–554. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee HY, Oh BH: Aging and arterial stiffness. Circ. J. 2010; 74: 2257–2262. Publisher Full Text\n\nLaurent S, Boutouyrie P, Asmar R, et al.: Aortic Stiffness Is an Independent Predictor of All-Cause and Cardiovascular Mortality in Hypertensive Patients.2001. Reference Source\n\nPatil SG, Patil SS, Aithala MR, et al.: Comparison of yoga and walking-exercise on cardiac time intervals as a measure of cardiac function in elderly with increased pulse pressure. Indian Heart J. 2017 Jul 1; 69(4): 485–490. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlexander GK, Innes KE, Selfe TK, et al.: “More than I expected”: Perceived benefits of yoga practice among older adults at risk for cardiovascular disease. Complement. Ther. Med. 2013 Feb; 21(1): 14–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCramer H, Lauche R, Haller H, et al.: Effects of yoga on cardiovascular disease risk factors: A systematic review and meta-analysis. Int. J. Cardiol. 2014 May 1; 173(2): 170–183. PubMed Abstract \| Publisher Full Text\n\nDas K, Dhanakshirur G, Patil S, et al.: Comparison of the effects of yoga and lifestyle modification on grade-I hypertension in elderly males: A preliminary study. Int. J. Clin. Exp. Physiol. 2014; 1(1): 68. Publisher Full Text\n\nBharshankar JR, Bharshankar RN, Deshpande VN, et al.: Effect of yoga on cardiovascular system in subjects above 40 years. Indian J. Physiol. Pharmacol. 2003; 47: 202–206. PubMed Abstract\n\nGurunathrao S, If TD, If TD, et al.: Comparison of yoga and walking-exercise on cardiac time intervals as a measure of cardiac function in elderly with increased pulse pressure. Indian Heart J. 2017; 69(4): 485–490. Publisher Full Text\n\nHunter SD, Dhindsa MS, Cunningham E, et al.: ScienceDirect The effect of Bikram yoga on endothelial function in young and middle-aged and older adults. J. Bodyw. Mov. Ther. 2016; 6–10." }	[ { "id": "197281", "date": "23 Aug 2023", "name": "James Shadiow", "expertise": [ "Reviewer Expertise Exercise Physiology", "Diabetes", "Cardiovascular Disease", "Cellular Biology", "Endothelial Function", "Translational 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\nThis article aims to address the causal effects of novel forms of exercise, Bikram yoga, on metrics of cardiovascular health. It addresses an understudied topic, especially given that adherence to traditional forms of exercise is historically ineffective, leading to epidemics of metabolic and cardiovascular disorders within the United States of America. Despite the relatively small n size of research studies meeting inclusion criteria, readdressing critical flaws in the current manuscript may provide a valuable piece of literature that draws attention to a novel therapeutic supported to preserve or improve metrics of cardiovascular function in subsets of at-risk patient populations.\nAre the rationale for and objectives of the Systematic Review clearly stated?\nThe study's objective is represented in Table 1, comprising three research questions and paired outcomes.\nA singular research question that mends the current three would be appropriate for systematic reviews and improve readability.\n\nThere is no rationale for the first two research questions, including samples from office and desk-based workers. This can be sufficed through disseminating information involving sedentary lifestyles in the United States and leveraging the study of these workers to present an appropriate model typical of American workers.\n\nAdditionally, these outcomes are not defined, the methodologies of how these outcomes are constructed are not listed, and there is no context to the interpretability of meaningfulness of these outcomes. The authors might define an outcome: \"Rate pressure product is calculated by resting heart rate multiplied by systolic blood pressure. This metric indicates cardiac oxygen consumption and allows researchers and clinicians to understand the workload placed on a patient's heart at any given time.\"\n\nAre sufficient details of the methods and analysis provided to allow replication by others?\nNo, it is unclear why some studies that utilized a yoga intervention and assessed vascular function metrics included in the current manuscript were excluded. Below are studies that seem to meet the inclusion criteria (PMID):\nPatil et al. (20171). Patil et al. (20152). Saptharishi et al. (20093).\n\nThere is also a critical flaw in the research questions in Table 1 referencing the investigation of office and desk workers, the included studies in Table 3 do not mention this population.\n\nIs the statistical analysis and its interpretation appropriate?\nStatistical analysis: Yes, this systematic review utilized reported statistical analyses and therefore does not need to provide any novel statistics.\n\nInterpretation: No, a systematic review should set a clear outcome and then review the relevant literature to deliver a clear and concise answer to a singular specific question. This study rather individually summarizes three studies that each utilize a vast amount of varying central and peripheral vascular measures and therefore lacks a concise takeaway from these studies meeting the inclusion criteria.\n\nAre the conclusions drawn adequately supported by the results presented in the review?\nNo, the conclusion is over-reported. There is reported evidence in the included studies that state null findings and lack of significant change in response to Bikram interventions.\n\nAdditionally, the outcomes outlined in Table 1 do not align with those mentioned in the Abstract or Results, specifically LVET and RPP. RPP is a metric of cardiac oxygen consumption, while LVET is a metric for cardiac function. Although metrics of any portion of the cardiovascular tree are often related, this paper should either delineate peripheral vascular metrics or central cardiovascular metrics or provide a deduction into the causal improvement of one of these aspects on the other.\n\nThe limitations are well-understood and well reported.\n\nThe future outcomes are also exceptionally written.\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? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "197282", "date": "06 Sep 2023", "name": "Nantinee Nualnim", "expertise": [ "Reviewer Expertise Cardiovascular exercise physiology", "Cardiovascular risk factors and exercise", "Cardiopulmonary physical therapy" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study aimed to investigate the effect of Bikram yoga on vascular functions using a scope review. It’s interesting as Bikram yoga seems to be a novel form of exercise. However, there are many comments for this article.\nMajor comments:\n1. The topic of this article is not aligned with the study design. The studies that the authors recruit for the review did not mention that the participants are office workers. In addition, there was only one study that compares Bikram yoga with stretching exercise. It’s exaggerated to make a question on the article’s topic that “Can yoga show better vascular outcomes than regular exercise among office workers?”.\n2. Introduction:\n2.1: There are no rationales to support the objectives of this study mentioned in Table 1. The authors need to provide the information to support the importance of this study. For example, the potential benefits of Bikram yoga over traditional exercise, especially, aerobic exercise that is recommended by many international organizations to be the form of exercise to improve vascular function and essential hypertension. Why did the authors focus on office workers?\n\n2.2: There is no general objective of the study available in the introduction part.\n\n3. Methods:\n3.1: It’s unclear about the inclusion and exclusion criteria of the studies that the authors recruited for analyses.\n\n3.2: The 3 studies that were included in the analyses did not go along with the 3 objectives that were mentioned in Table 1. The population did not mention that they are office workers. The study design did not specify that they are a randomized control trial as written in the abstract. Why were some studies in the references excluded from the analysis as it investigated the effect of yoga on vascular functions?\n\n3.3: The number of the studies in the flow chart is a miscalculation. From 103 studies, the authors excluded 27 studies. Why did it leave only 27 studies for further investigation?\n\n3.4: The statistical analyses were not available. If it’s the systematic review, it should summarize the clear results for each research question.\n\n4. Results and discussion:\n4.1: There are no data analyses in the results. The authors just reported the findings from each study they recruited. It is not appropriate for a systematic review. They need to recruit relevant numbers of research to answer each of their research questions. They also need to synthesise and analyze data with appropriate statistical analyses.\n\n4.2: The discussion and conclusion of the study is over-reported. In addition, the information they provided is not aligned among the abstract, introduction, methods and results.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? No\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] } ]	1	https://f1000research.com/articles/12-925
https://f1000research.com/articles/11-1452/v1	08 Dec 22	{ "type": "Research Article", "title": "Acacia mangium: A promising plant for isolating anti-hepatitis C virus agents", "authors": [ "Tutik Sri Wahyuni", "Nida S. Sukma", "Adita A. Permanasari", "Chie Aoki-Utsubo", "Aty Widyawaruyanti", "Achmad Fuad Hafid", "Nida S. Sukma", "Adita A. Permanasari", "Chie Aoki-Utsubo", "Aty Widyawaruyanti", "Achmad Fuad Hafid" ], "abstract": "Background: Medicinal plants are potential resources for isolating drug candidates. Various plants have been reported to possess pharmacological effects including anti-hepatitis C activities. The current study examined the anti-hepatitis C virus (HCV) activities of Acacia mangium extracts in solvents with various polarities and further evaluated the mechanism of action of the extracts using Western blotting and combination treatment models. Methods: The leaves of A. mangium were extracted in two phases, first in ethanol and then in solvents with different polarities (n-hexane, dichloromethane, and methanol). HCV-infected Huh7it-1 cells were treated with the extracts at concentrations of 0.01, 0.1, 1, 10, 50, and 100 µg/mL. Results: The results revealed the strong anti-HCV activities of the extracts. The 50% inhibition concentrations (IC50s) of the ethanol, n-hexane, dichloromethane and methanol extracts were of 4.6 ± 0.3, 2.9 ± 0.2, 0.2 ± 0.3, and 2.8 ± 0.2 μg/mL, respectively, and no cytotoxic effect was detected. These extracts displayed stronger effects than the positive control ribavirin. The mode of action of the ethanol extract was evaluated at 30 µg/mL, revealing that the inhibitory effect was stronger on the post-entry step than on the entry step. Western blotting revealed that the extracts decreased NS3 protein expression, indicating that virus replication was suppressed. Further evaluation illustrated that combined treatment with the ethanol extract enhanced the anti-viral activity of simeprevir. Conclusions: These results indicated that A. mangium leaves could represent sources of anti-HCV agents.", "keywords": [ "Acacia mangium", "hepatitis C virus", "infectious disease", "medicinal plant", "medicine", "health" ], "content": "Introduction\n\nHepatitis C virus (HCV) infection is an acute or chronic liver disease. HCV infection has a high prevalence globally, and approximately 71 million people are at risk of liver cirrhosis or hepatocellular carcinoma attributable to chronic infection (Lange et al., 2014). To date, no effective hepatitis C vaccine has been developed because HCV is a commonly mutated virus (Pawlotsky et al., 2015; Pawlotsky Jm et al., 2018).\n\nHepatitis C treatment has evolved with the availability of direct acting antivirals, which have achieved sustained virological response (SVR) rates exceeding 90% (Javed et al., 2011). However, some low-income countries cannot access those therapies because of their costs, and the combination of interferon-alpha and ribavirin (RBV), which produces an SVR rate of 50%, remains in use. Combination treatment has also been reported to have serious side effects and risks of resistance, making this strategy less effective (Swain et al., 2010). Efforts to develop new agents for HCV are necessary. Further issues to overcome include the development of drugs that can inhibit the virus with fewer side effects and affordable prices for all countries. Therefore, it is necessary to develop affordable, safe, and effective HCV therapies.\n\nTraditional herbal medicine has become a popular treatment, and plants are among the primary components of such medicines. Our previous studies reported medicinal plants possessing anti-HCV activities (Adianti et al., 2014; Wahyuni et al., 2013; Wahyuni et al., 2014). Many medicinal plants have also been reported to inhibit HCV by inhibiting various steps of the HCV life cycle (Hussein et al., 2000; Ravikumar et al., 2011; Wahyuni et al., 2016).\n\nPlants in the Fabaceae family are frequently used by traditional healers to treat liver diseases, including HCV infection. The chemical compounds present in the Fabaceae family include saponins, tannins, flavonoids, proteins, stylbenoid, xanthones, terpenes (triterpenes, diterpenes), phytoalexin, galactonate, lactogenic agents (polyketide), and anthraquinone. Most of those compounds are reported to possess anti-viral, hepatoprotective, and anti-cancer activities (Roy et al., 2016). One genus of the Fabaceae family that has been demonstrated to inhibit HIV is Acacia, and the active species include A. nilotica (50% inhibitory concentration [IC50] = 40.5 μg/mL) and A. confusa (IC50 = 5 μg/mL) (Hussein et al., 2000; Lee et al., 2011).\n\nA. mangium contains alkaloids, flavonoids, polyphenols, glycosides, saponins, steroids, tannins, and terpenoids, and their leaves contain phenolic groups including tannins and flavonoids. A. mangium was reported to contain 2,3-cis-3,4,7,8-tetrahydroxyflavanone and teracacidin (Barry et al., 2005). This current study evaluated the anti-HCV activity of various extracts of A. mangium. In addition, the cytotoxicity of the extracts was evaluated. The mode of action was additionally assayed to determine the part(s) of the HCV life cycle inhibited by the extracts. Moreover, its mechanism of action was examined by Western blotting and combination treatment with current anti-HCV drug.\n\n\nMethods\n\nA. mangium leaves were obtained from an area in Mojokerto Regency, Indonesia. The plant was verified by an expert botanist from Materia Medica Indonesia, East Java (see the Underlying data (Wahyuni, 2022)).\n\nThe materials used in the bioassays were as follows: Huh7it-1 cells (Apriyanto et al., 2016); adapted variant of HCV strain (JHF1a) (Yu et al., 2010); Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO Invitrogen) supplemented with 10% of fetal bovine serum (FBS, GIBCO Invitrogen), 150 μg/mL kanamycin (Sigma-Aldrich), and non-essential amino acids (NEAAs, GIBCO Invitrogen); Dulbecco’s phosphate-buffered saline (GIBCO Invitrogen); formaldehyde (HCHO, Sigma-Aldrich); trypsin-EDTA (Sigma-Aldrich); 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT, Sigma-Aldrich); bovine serum albumin (BSA, Biowest); Triton X-100 Sigma-Aldrich); 3,3′-diaminobenzidine (DAB, Thermo Fisher Scientific); anti-HCV human antibody and HRP-conjugated goat anti-human Ig antibody (Thermo Fisher Scientific); RIPA buffer; polyacrylamide gel and polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA); β-actin antibody (MBL, Nagoya, Japan); and a chemiluminescence detection system (Bio-Rad; GE Healthcare, UK).\n\nA. mangium leaves (2 kg) were dried, ground into powder, and further extracted with two kind of extraction procesess. First, 200 g of powder was extracted by maceration process with a total of 2 liters of 96% ethanol and another 200 g was successively extracted with 2 liters of n-hexane, dichloromethane, and methanol. Specifically, 10 mg of the extract powder were dissolved in 100 μL of dimethyl sulfoxide to obtain 100,000 μg/mL stock solution (Wahyuni et al., 2013).\n\nHuh7it-1 cells were cultured in DMEM supplemented with 10% FBS, 150 μg/mL kanamycin, and NEAAs in 5% CO2 at 37°C and maintained for bioassay purposes. Cells were incubated at 37 °C for 2 days. Cells which showed more than 80% confluence were used for further bioactivity assay. The detailed protocol for cell passage is available at https://dx.doi.org/10.17504/protocols.io.n92ldpbd7l5b/v1.\n\nVirus stock was obtained by propagating HCV in Huh7it-1 cells. Culture supernatants at day 3, 5 and 7 after virus infection were collected. Virus titers were calculated by titration assay (wahyuni, 2013). In brief the virus harvested was diluted on x5, x25, x125, x625, and x3125 then put onto Huh7it-1 cells and incubated for 4 hours, the remaining virus was removed and refed with new medium for further incubation for 2 days. The infected cells were stained with DAB staining reagent and further calculated. The number of viruses represented as titer virus. The high titer virus stock (higher than 1x105) was chosen for anti-HCV assay. The stock was stored at −80°C until use (Wahyuni et al., 2018).\n\nAn anti-HCV assay was conducted using HCV-infected Huh7it-1 cells. Various concentrations of the extracts were mixed with virus solution (multiplicity of infection of 0.1) and inoculated into the cells at a final concentration of 0.01, 0.1, 1, 10, 50, or 100 μg/mL. The cultures were incubated for 48 h with 2 h of virus inoculation and further incubation for 46 hours at 37°C. The viral levels of the supernatants were examined by titration assay. Culture cell supernatant was collected, diluted 10x with medium and inoculated to the Huh7it cells. This was incubated for 2 days and the infected cells were calculated after the immunostaining process. The inhibitory effect of the extracts were calculated compared to the untreated control. The 50% inhibitory activity was conducted by SPSS software version 25 (Wahyuni et al., 2019; Wahyuni et al., 2018). Ribavirin was used as the positive control.\n\nHuh7it-1 cells were incubated with serial dilutions of the supernatant for 48 h. The cells were fixed with formaldehyde, stabilized with triton and subjected to immunostaining with primary antibody (human serum) and secondary antibodies (HRP-conjugated goat anti-human). The detailed immunostaining protocol is available in the Underlying data (Permanasari and Wahyuni, 2022a). DAB staining was performed to visualize the infected cells. The percent inhibitory effect was calculated by comparing the reduction of infected cells to the control (Wahyuni et al., 2018).\n\nThe mode of action assay was performed to examine whether A. mangium extract affected the entry or post-entry step of the HCV life cycle. Three parallel experiments were performed. First, the extract was only added to the cells during viral inoculation. Second, the extract was only added to the cells after viral inoculation. Third, the cells were treated with the extract both during and after inoculation. After 48 h of incubation, all supernatants were collected to examine their viral levels by titration assay. Viral supernatant was diluted with medium and inoculated to the Huh7it cells. Infected cells were calculated to further determine the percentage inhibitory against HCV. Anti-HCV activity was expressed by 50% inhibition concentration (IC50) (Hafid et al., 2017).\n\nThe MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to measure cytotoxicity. Huh7it-1 cells were incubated with various concentrations of the extracts for 48 h. Then, 10% MTT was added to the cultures for 4 h. Absorbance was measured at 560 and 750 nm to calculate the percentage cell viability relative to the control. SPSS software version 25 probit analysis was used to calculate the 50% cytotoxic concentration (CC50) (Wahyuni et al., 2018). The protocol of the MTT assay is available at http://dx.doi.org/10.17504/protocols.io.6qpvr4x5pgmk/v1.\n\nHuh7it-1 cells were treated with mixtures of the extracts (10 or 50 μg/mL) and HCV. After incubation for 2 days at 37 °C, the cells were collected, lysed, and protein levels were determined using a BCA assay kit (Thermo Fisher Scientific). Equal amounts of proteins were subjected to SDS–polyacrylamide gel electrophoresis followed by transfer to a polyvinylidene difluoride membrane. Samples were run in transfer buffer at 0.3 A for 30 minutes followed by processing to SDS running buffer at 0.1 A for 25 minutes. The membrane was applied into a blocking buffer of skim milk and reacted to antibodies. The primary antibody was an HCV NS3 mouse monoclonal antibody (clone H23; Abcam, Cambridge, MA, USA), and the secondary antibody was HRP-conjugated goat anti-mouse immunoglobulin. β-actin (MBL, Nagoya, Japan) served as the internal control (Permanasari et al., 2021; Widyawaruyanti et al., 2021). Membranes were incubated at room temperature for 1 hour in each antibody. NS3 protein expression was detected using an enhanced chemiluminescence detection system (GE Healthcare, Buckinghamshire).\n\nCombination treatment was performed by adding equal volumes of A. mangium extract and simeprevir (Toronto Research Chemical, Canada). Simeprevir was added at 0.25×, 0.5×, 1×, 2×, and 4×IC50 for monotherapy and combination. All treatments were performed for 48 h incubation. The IC50 of simeprevir when used in combination with A. mangium extract and monotherapy were calculated and compared using the SPPS probit assay version 25 (Wahyuni et al., 2020).\n\n\nResults\n\nAll A. mangium leaf extracts strongly inhibited HCV in a dose dependent manner (Figure 1). Inhibition concentrations of 50% of all extracts were calculated by probit analysis. Dichloromethane extract displayed the strongest effects, with an IC50 of 0.2 ± 0.3 μg/mL, whereas the IC50s of the extracts ranged 2.8–4.6 μg/mL (Table 1). While the positive control of ribavirin revealed the IC50 values of 10.4 ± 0.2 μg/mL. All of the extracts were demonstrated to possess stronger activity compared to the positive control of ribavirin. The raw data are available in the Underlying data (Permanasari and Wahyuni, 2022b).\n\nHuh7it-1 cells were cultured and inoculated with mixtures of the virus and each extract at various concentrations. Virus inhibition was calculated relative to the untreated control. The data represent the mean of three independent experiments.\n\n* Data represent the mean ± SD of three independent experiments\n\nThe cytotoxicity assay of the extracts observed no toxic effect in the Huh7it. The percentage of cell viability demonstrated that all extracts possessed cell viability higher than 80% in the concentration of 400 μg/mL. However, n-hexane and dichloromethane extract showed a reduction in the percentage of cell viability at the dose of 400 μg/mL (Figure 2). The raw data are available in the Underlying data (Permanasari and Wahyuni, 2022c).\n\nCells were treated with various concentrations of each extract of Acacia mangium. MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reagent was added after 2 days of incubation, and cell viability was examined using a microplate reader at wavelengths of 450 and 630 nm. The percent cell viability was calculated, and the 50% cytotoxic concentrations were determined by SPSS probit analysis. The data represent the mean of three independent experiments.\n\nThe mode of action assay was performed using three series of experiments. The result illustrated that the inhibitory effect was higher on the post-entry step than on the entry step. The percentage virus inhibition of post entry steps revealed more than 20% differences than the entry step (Figure 3). This result suggested that the extract dominantly affected post-infection processes such as virus replication, virus assembly, and virus release. The raw data are available in the Underlying data (Permanasari and Wahyuni, 2022d).\n\n(a) Cells were cultured with the ethanol extract of A. mangium (30 μg/mL) in three parallel experiments. First, cultured cells were treated with the extract only during inoculation (entry step). Second, cells were treated with the extract only after inoculation (post-entry step). Third, cells were treated with the extract both during and after inoculation. (b) The entry step comprises the processes of viral binding to host receptors, viral entry into the cells and endocytosis, whereas the post-entry comprises translation, replication, assembly, and release. (c) Percent inhibition of the entry step, post-entry step, and both steps. The data represent the mean ± SEM of three independent experiments\n\nEvaluation of the combination effect between the extract and simeprevir showed there is an enhancement of the effects of the extract to simeprevir, which is known as an HCV NS3 protein inhibitor. The addition of A. mangium extract increased the anti-HCV effect of simeprevir, as the IC50 of simeprevir when used in combination with A. mangium extract was reduced by 2-fold compared to that of simeprevir alone (Table 2).\n\nTo examine the mechanism of action of the ethanol extract, western blotting analysis was performed. The result demonstrated a reduction of the NS3 protein level due to the extract intervention. Immunoblotting revealed that treatment with A. mangium extract at 10 or 50 μg/mL decreased the NS3 protein expression by 40% and 95%, respectively, versus the control (Figure 4) (Underlying data, (Permanasari and Wahyuni, 2022e).\n\nHuh7it-1 cells were treated with a mixture of the extract (final concentration, 10 or 50 μg/mL) and virus. The cells were lysed in RIFA buffer, and an equal amount of proteins were separated by SDS–polyacrylamide gel electrophoresis.\n\n\nDiscussion\n\nThe study found that A. mangium possesses potential anti hepatitis C virus activity through some mechanism. The anti-HCV activities of the A. mangium leaves extracted using solvents of different polarities, namely 96% ethanol, n-hexane, dichloromethane, and methanol was examined. The data illustrated the strong anti-HCV activities of all extracts. Moreover, no toxic effect was observed according to the CC50 and selectivity index.\n\nEthanol is commonly used as a solvent in traditional drug development. Therefore, the strong anti-HCV activities of the ethanol extract and the lack of cytotoxicity provide necessary information for developing extracts of A. mangium as an alternative or complementary anti-HCV agent. However, we used various solvents with different polarities to further isolate the active anti-HCV compounds. The result demonstrated that the dichloromethane extract of A. mangium possessed the strongest inhibitory activity.\n\nMode of action analysis was performed as the first screening assay to determine the mechanism of A. mangium extract. The results illustrated that the ethanol extract more strongly inhibited HCV in the post-entry stage than in the entry stage. Virus production starts with binding of the virus to the host cell receptor, followed by virus entry and endocytosis. These processes are included in the entry step. The entry of HCV into hepatocytes is mediated by the viral E1 and E2 glycoproteins, which are the surface proteins of viral particles. HCV infection occurs through complex interactions of viral lipoviral particles with cellular factors, including low-density lipoprotein receptors, glycosaminoglycans, scavenger receptor class B type I, tetraspanin (CD81), claudin-1, and occludin (Dubuisson et al., 2008; Moriishi and Matsuura, 2003). Viral particles enter host cells through clathrin-mediated endocytosis, after which they are sent to the endosome. Meanwhile, the post-entry steps include translation, replication, and assembly. NS3–NS5 form a replication complex that produces new viral genomic RNA. Genomic RNA and HCV core proteins accumulate to form a nucleocapsid, which is excreted through the lumen side of the endoplasmic reticulum. After this excretion, the nucleocapsid can interact with very low-density lipoprotein (VLDL), followed by translocation to the Golgi for maturation. Mature HCV–VLDL complexes are released exocytically via the VLDL secretory pathway. Finally, new cells can be infected by released free HCV particles or by cell-to-cell transmission (Fénéant et al., 2014; Lindenbach and Rice, 2013; Zeisel et al., 2015).\n\nTo further clarify the mechanism of the anti-HCV effects of A. mangium extract, Western blotting was performed to evaluate the effect of the extract on NS3 protein. A. mangium extract decreased NS3 protein levels versus the control. NS3 is a non-structural virus protein that plays an important role in replication. It is an attractive target for HCV treatment. Inhibition of NS3 could result in decreased virus production. Moreover, the anti-HCV activity of the extract was evaluated in combination with the NS3 protein inhibitor simeprevir. A. mangium extract was demonstrated to enhance the inhibitory activity of simeprevir against HCV. This suggested that the extract potentiated the effect of simeprevir on secondary targets of HCV.\n\nChemical compounds play an important role in anti-HCV activities. Further isolation of the active compounds from A. mangium against HCV is needed. However, it has been reported that Acacia species are rich in polyphenols, flavonoids, alkaloids, saponins, and terpenoids. It was reported compounds in the genus Acacia include epicatechin, quercetin, proacaciaside I, and proacaciaside II (Figure 5). Those compounds were previously demonstrated to exhibit bioactivities such as anti-bacterial, anti-fungal, and anti-parasitic effects, which could contribute to anti-HCV properties (Chew et al., 2011; Rangra et al., 2019).\n\n(a) Auriculoside (https://pubchem.ncbi.nlm.nih.gov/compound/Auriculoside), (b) epicatechin (https://pubchem.ncbi.nlm.nih.gov/compound/72276), (c) quercetin (https://pubchem.ncbi.nlm.nih.gov/compound/5280343), (d) proacaciaside I (https://pubchem.ncbi.nlm.nih.gov/compound/102446075), and (e) proacaciaside II (https://pubchem.ncbi.nlm.nih.gov/compound/102446076).\n\n\nConclusions\n\nA. mangium leaf extracts possess strong anti-HCV activities without toxic effects. The extracts strongly inhibited the post-entry step, decreased NS3 protein levels, and enhanced the anti-HCV activities of simeprevir. These results suggest that A. mangium could be used to develop complementary and alternative treatments for HCV.", "appendix": "Data availability\n\nFigshare: Plant Determination of Acacia mangium.jpg (It provided the taxonomy information of Acacia mangium) https://doi.org/10.6084/m9.figshare.20973511 (Wahyuni, 2022)\n\nFigshare: Immunostaing HCV protocol. Immunostaining HCV.docx (It provided the detail steps of immunostainning process.) https://doi.org/10.6084/m9.figshare.20977168 (Permanasari and Wahyuni, 2022a).\n\nFigshare: in vitro activity of A. mangium against HCV. raw data figure 1.docx (It demonstrated the figure of cells with infected cells, the tables which showed number of infected cells and percent inhibition of the three replication of experiments.). https://doi.org/10.6084/m9.figshare.20977714 (Permanasari and Wahyuni, 2022b).\n\nFigshare: Cytotoxic effect of A. mangium extracts. cytotoxicity.docx (It provided the percent viability of extract ethanol, n-hexane, dichloromethane, and methanol https://doi.org/10.6084/m9.figshare.20977939 (Permanasari and Wahyuni, 2022c)\n\nFigshare: Untitled Item. Mode of action.docx (It provided the number of infected cells in three kind of inoculation method,entry, post entry and both.). https://doi.org/10.6084/m9.figshare.20977933 (Permanasari and Wahyuni, 2022d).\n\nFigshare: NS3 Acacia mangium inhibition. NS3 beta actin 4 (1).tif (It provided the raw data of western blotting assay) https://doi.org/10.6084/m9.figshare.21352095 (Permanasari and Wahyuni, 2022e).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe would like to thank Professor Hak Hotta (Konan Women's University), Dr. Takaji Wakita (National Institute for Infectious Diseases, Japan), and Dr. Yohko Shimizu (Kobe University) for providing Huh7it-1 cells.\n\n\nReferences\n\nAdianti M, Aoki C, Komoto M, et al.: Anti-hepatitis C virus compounds obtained from Glycyrrhiza uralensis and other Glycyrrhiza species. Microbiol. Immunol. 2014; 58(3): 180–187. 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Viruses. 2015; 7(11): 5659–5685. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "162586", "date": "21 Feb 2023", "name": "Ziwen Wang", "expertise": [ "Reviewer Expertise Discovery of novel antivirals based on 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\nCurrent study evaluated the anti-HCV activity of various extracts of A. mangium. In addition, the cytotoxicity of the extracts was evaluated. The mode of action was additionally assayed to determine the part(s) of the HCV life cycle inhibited by the extracts. Moreover, its mechanism of action was examined by Western blotting and combination treatment with current anti-HCV drug. The work is important for the development of novel antivirals, and the results are interesting. I recommended it to be indexed after minor revision.\nFor Abstract: Parts Background and Methods need to be reorganized, and the content does not match the requirements. Part Background only needs to introduce the research background. Part Methods does not need to write the specific operation process.\nFor Introduction: The research progress on the biological activity of Acacia mangium extract should be increased.\nTable 1: Extracts from different solvents show different activities because the chemical components in them have changed. Can you detect which components have changed by liquid chromatography? In addition, have you ever tested the biological activity of different batches of extracts from the same solvent?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9385", "date": "14 Mar 2023", "name": "tutik sri wahyuni", "role": "Author Response", "response": "Dear Reviewer, Thank you for the review comments. Herewith I submitted the revised version of manuscript following the reviewer comments. We revised the abstract section in the part of introduction and method. Other revision was also mentioning the bioactivities information of Acacia mangium and other Acacia genus in the introduction section. Other comments were regarding the HPLC profile of each extracts that have differences polarities. Not much information regarding this data, however some information of A. mangium compounds and other Acacia genus have been explained in the text. Comment regarding the examination of difference batch of extract have been done and gave the relatively the same activity. Best regards, Tutik Sri wahyuni, PhD." } ] } ]	1	https://f1000research.com/articles/11-1452

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