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https://f1000research.com/articles/12-506/v1	16 May 23	{ "type": "Research Article", "title": "Measuring linguistics of the wokototen chart made inductively by deciphering kunten materials", "authors": [ "Tomoaki Tsutsumi", "Koji Tajima", "Teiji Kosukegawa", "Tomokazu Takada", "Koji Tajima", "Teiji Kosukegawa", "Tomokazu Takada" ], "abstract": "In this paper, we focus on wokototen markings, which are a system of kunten annotations used to facilitate the reading of classical Chinese documents by Japanese readers. Using digitized data, we performed basic measurements of wokototen by using a chart that summarizes the wokototen markings of actual kunten materials described by Hiroshi Tsukishima, and we quantitatively clarified their characteristics. Kunten materials are classical Chinese books with annotations, called kunten, on the Chinese text. The wokototen is a type of kunten. In ancient East Asian countries, kunten systems were developed as a way of directly annotating Chinese documents so that they could be read and understood by non-native readers. For this reason, kunten materials and kunten are treated as historical sources for linguistic and historical research. The shape and position of a wokototen marking determines what kind of reading it indicates. The results of our basic survey quantitatively show that almost all the wokototen charts in actual kunten materials contain particles represented by “te”, “ni”, and “wo”, the most common shapes of wokototen are dots and shapes that can be written with a single stroke, such as ｜, ─, and ＼, and that the most common places to find these markings are to the right of characters in the horizontal direction and below characters in the vertical direction.", "keywords": [ "Kunten", "Reading Classical Chinese in Japanese", "Text Mining", "Digital Humanities" ], "content": "Introduction\n\nKunten is a Japanese system of text markings used to clarify the syntax and meaning of Chinese texts for Japanese readers. Previous research1–3 investigated how these marks can be handled on a computer. In this paper, we focus on a kunten system called wokototen that is used to annotate classical Chinese texts (kanbun), and we perform basic measurements by digitizing wokototen charts (wokototen-zu) that inductively summarize the markings added to actual documents.\n\nThe texts we are studying consist of classical Chinese texts annotated with kunten markings. These were once widely used, around from the Heian period to the Edo period in Japan, to promulgate social, cultural, and academic ideas in East Asian countries, where kunten systems were developed as a way of directly annotating these documents so that they could be read and understood by non-native readers.\n\nWokototen is one such system that was developed in Japan. It consists of marks placed inside or around the Chinese characters to indicate features such as grammatical particles, auxiliary verbs, and the readings of kanji characters. There are various flavors of wokototen associated with different schools and different annotators, and wokototen charts are used as means of inductively compiling the annotation marks used by different schools of wokototen or in different kunten documents.\n\nDocuments annotated in this way have existed from the Heian period to the present day, and wokototen is mostly found in Chinese classics, Buddhist scriptures, and Japanese books of the Heian and Kamakura periods. Since the materials that are the subject of our research contain complex information, this information must be preserved intact when they are digitized for analysis purposes. We therefore digitized the data by using a dedicated structured description method. For this paper, particularly with regard to this digitized data, we performed basic measurements of wokototen by using a chart that summarizes the wokototen markings of actual kunten materials described by Hiroshi Tsukishima,4 and we quantitatively clarified their characteristics.\n\n\nTarget of research\n\nWokototen markings are often found in kunten documents from the Heian and Kamakura periods, where they are used to indicate features such as grammatical particles, auxiliary verbs, and conjugated endings by means of variously shaped symbols such as dots (・), lines ( │ ), and hooks (└ ). These symbols can be placed at the four corners, inside, or around the strokes of a Chinese character, and their readings differ depending on their position and shape. For example, a dot to the upper right of a kanji has a different meaning than a line in the same position, or a dot to the lower right of the character. To uniquely identify the meaning of a wokototen markings, we need to know both its position and its shape.\n\nThere are various types of wokototen associated with different eras and different schools. For example, in one school, a dot at the upper right corner of a Chinese character is read as wo, but in another school, it is read as koto.\n\nThe decipherment and classification of wokototen has been the subject of several previous studies by researchers including Yoshinori Yoshizawa,5–7 Norio Nakata,8 Heiji Otsubo,9 Hiroshi Tsukishima,4,10 Yoshinori Kobayashi,11,12 and Masaji Kasuga.13 In particular, Norio Nakata8 compiled a set of 26 wokototen charts as materials for deciphering kunten materials.\n\nEven today, researchers specializing in wokototen are working to decipher kunten materials and study their historical background. Their general research method involves visually deciphering the wokototen and kana annotations in kunten materials to create Japanese transcriptions from which the contents of the materials can be understood. As a by-product of this process, a wokototen chart is generated as a key to the markings used in the source material. A wokototen chart not only summarizes the wokototen markings in the actual kunten materials, but also includes information on how the researchers understood these materials.\n\nAs shown in Figure 1, a wokototen chart indicates the reading of each marking according to its shape and position in a square frame (called a tsubo) corresponding to the location of a Chinese character. A wokototen chart typically consists of multiple tsubo, with each tsubo containing multiple wokototen. A collection of these wokototen charts is called a tenzushū (点図集).\n\nWokototen charts can be classified into two types according to the process by which they were created. One is a comprehensive chart, which is a collection of the wokototen markings used by each school. The other is an inductive chart, which is a collection of the wokototen markings used in a particular body of kunten material. The measurements in this study were made using the latter type of chart. For this paper, we performed basic measurements on 199 types of point charts contained in actual kunten materials as summarized by Hiroshi Tsukishima.4 By way of comparison, we also present the results of measurements made using the former type of point chart as described by Tomoaki Tsutsumi.1\n\n\nMethods\n\nThe data used in this study was based on wokototen charts data created from kunten materials created by Hiroshi Tsukishima, which were registered as of June 2022 in the Wokototen charts Database provided by the National Institute for Japanese Language and Linguistics (NINJAL). Further information on the database can be found on the NINJAL website and in reference [3].\n\nAs mentioned above, wokototen markings are used to annotate Chinese characters. The meaning of each marking is determined by its position, shape, and reading. In the data of NINJAL, the 'reading' of the wokototen has been entered in Japanese text. The ‘shape’ of the wokototen has been entered by replacing it with the similar character in Unicode. The 124 characters substituted in this database is shown in Table 1. The position of a wokototen is represented using a 7×7 square grid of cells with its origin at the center of a tsubo and the upper left and lower right corners at the coordinates (−3, −3) and (3, 3). Since wokototen markings are sometimes slightly separate from the kanji character, the center 5×5 square corresponds to the area occupied by the character, and coordinates in this region correspond to markings that overlap with the character. The outermost cells are used for positions that are separate from the kanji character. This digitization method of the readings, positions and shapes of the wokototen are described in detail in reference [1].\n\nThis method of expressing the position of the Kunten in coordinates has also been used in Korean studies of gugyeol,14–18 where a coordinate system of 5×5 squares is defined around each character. These methods of the Korean studies and the current one has the same concept but are not data compatible.\n\nIn this study, we performed measurements on 199 types of point charts (contained in actual kunten materials as summarized by Hiroshi Tsukishima4), relating to the reading, position, and shape of wokototen. The programme written in C# was used for the measurements.19 Excel 365 and Visual Studio Code (version 1.67) were used to view and compare the data from the database with the reference [4] and to correct the data. VisualStudio Community (version 17) was used to create the programme.\n\nThis program reads comma-delimited data, one per line, on the wokototen to be measured. It is a simple program that outputs the count of “reading,” “position,” and “shape” of the wokototen. A line of read data should be produced in the following format.\n\nMaterial Title,Tsubo No,Reading,Sharp,Position of X, Position of Y\n\nEx. 華厳経探玄記一巻,1,レ,・,-3,-3\n\nWe created this form of data by comparing data taken from the Wokototenzu database with the previous research.4 In some cases, there were multiple readings given for a single symbol in the wokototen chart. Examples are shown in the 'カ/ナ' in the top left-hand corner (-2, -2) of the fifth tsubo in Figure 1. In this case, we have divided the symbol into two separate totals. The one of wokototen of the shape ‘L’ reading ‘カ’ and the other of wokototen of the shape 'L' reading 'ナ. In the data created by adding these processes, the total number of target wokototen was 6411.\n\n\nResults\n\nThe readings of wokototen, such as “wo” and “koto,” were examined to determine how many were found in the target wokototen chart. As a result, there were 303 types of readings. Next, the number of these 303 types of readings on the wokototen chart were measured, and the top 10 types are shown in Table 2. This does not include 885 points for which no reading was noted.\n\nWe examined the shapes of wokototen markings such as ・ and │ to determine how many of each there were in the target wokototen chart. As a result, we found 124 different shapes. We then counted the occurrences of each of these shapes in the wokototen chart, and the top 10 shapes are shown in Table 3. Since the wokototen charts used in this study were hand-drawn, there were several shapes that were partially similar but with slight differences. In such cases, we counted the shapes as being of a single type. For example, the shapes 人 and 入 were grouped together as 人 and counted as the same shape.\n\nNext, we examined the shapes used to indicate the three most frequent readings, which were “te”, “ni”, and “wo”. As shown in Table 4, we found that these readings were represented by seven shapes: ・, \\, /, \|, ─, ◡, and :. The ・ shape was the most common.\n\nThe results of measuring the locations of wokototen markings are shown in Table 5 and are depicted graphically in Figure 2. In Table 5, the area with the red background shows the position of the square cells representing the location of the kanji character.\n\n\nDiscussion\n\nAs Table 2 shows, there are many particles and auxiliary verbs such as “te”, “ni”, “wo”, and “ha”. The most common reading was “te”, which had more appearances than the number of wokototen charts (199) because it sometimes appeared more than once in the same chart, and because there were only eight charts in which this reading was not mentioned. These were charts with an extremely small number of wokototen points. One example is Scroll 1 of the Biography of Yang Xiong Zhuan (漢書楊雄傳; yellow markings, 5th level), which has only one wokototen marking, Scroll 3 of the Mohe Zhiguan (魔訶止觀; green markings), which has eight, and Scroll 1 of Huiguo Heshang Zhi Bei (惠果和上之碑文; black markings), which has two. In cases where the markings appear in different colors, such as red/vermillion (shuten), black (bokuten), and white (hakuten), the symbol “te” was marked in a different color. The same is true for “wo” and “ni”, which explains why “te”, “wo”, and “ni” in particular are found in almost all wokototen charts.\n\nA comparison with the results of a survey1 of 26 major wokototen charts compiled by Nakata8 and Tsukishima4 for each school shows that there are differences in the types of readings that occur frequently. The most common readings in the major wokototen charts are “su”, “naru”, “nari”, and “tari”, but these occur less frequently in wokototen charts based on kunten materials. Of these readings, the most common was “su” with 166 occurrences, followed by “nari” with 149. The other readings “tari” and “naru” were the 22nd and 24th most frequent, with 80 and 77 occurrences, respectively.\n\nTable 3 shows that the overwhelming majority of wokototen markings are dots (・). There were six wokototen chart that did not contain dots. These were charts containing almost no wokototen, as in the Biography of Yang Xiong Zhuan, and charts where the \\ shape was used for the first tsubo instead of a dot, as in Scroll 1 of Tōdaiji Fujumonkō (東大寺諷誦文稿) and Scroll 8 of Myōhō Rengekyō (妙法蓮華経; the Lotus Sutra).\n\nIncluding the dot marking (・), which appeared most frequently, the wokototen shapes ・, \|, ─, and \\ that appeared more than 500 times are shapes that can be written with a single stroke. More complex shapes appeared less often, and the same trend was observed in the measurement results of the 26 main wokototen charts.\n\nThe results in Table 4 also show that readings with the highest number of appearances are often denoted by a single dot. Although other shapes are sometimes used, it is safe to say that these readings are almost always denoted by a dot in all schools. This is thought to be because particles that are important in reading Chinese texts as Japanese are often expressed in the simplest way, which means using a single dot, because of the large number of times they are added. In some cases, “wo” is represented using the ◡ shape, and these were the wokototen charts belonging to the fourth group in the classification according to Tsukishima.4\n\nFrom Table 5, it can be seen that wokototen are often drawn at the four corners and the center of a character. The most common location was the lower right corner (2, 2), where 709 wokototen were found. In the four corners of characters, where wokototen markings are often placed, markings were more commonly found in the lower corners in the vertical direction, and on the right side in the horizontal direction.\n\nNext, we examined the wokototen markings placed around the outside of the characters. More markings were found on the right side than on the left side. For example, there were 129 markings in the upper right external position (3, −3), but only one in the upper left external position (−3, −3). The most common positions of wokototen markings around the outside of a character were the top, middle and bottom on the outer right side, with the largest number (169) appearing in the middle at coordinates (3, 0). These results show that wokototen, like furigana, tend to be written to the right side of each character.\n\nThe above trends suggested by our measurements differ from the results obtained from the 26 main wokototen charts, where there was no difference in the placement of markings between the left, right, upper and lower positions. In addition, measurements of the 26 main wokototen charts showed that few of these markings are placed outside the characters, while in the actual materials it can be seen that many wokototen markings are placed to the right of each character. These differences may represent a trend caused by the actual addition of markings to kunten materials.\n\n\nConclusion\n\nWe have conducted basic measurements of wokototen markings as described by Hiroshi Tsukishima,4 who compiled a chart summarizing the wokototen markings applied to actual kunten materials. As a result, we have quantitatively demonstrated that almost all wokototen charts in actual kunten materials contain particles represented by “te”, “ni”, and “wo”. Our results also show that the most common shape used for wokototen is ・, and that markings with shapes of greater complexity are used in fewer documents. We found that wokototen are most often placed at the bottom and right of characters, and that when they are placed outside the characters, they appear mostly to the right side of them, showing the same tendency as that of Japanese annotations such as furigana.\n\nIn the present analysis of the trends in the Wokotenzu charts, it has been possible to identify an overall trend. However, it cannot be determined whether all individual Kunten materials show a similar trend. Further research into the content of individual Kunten Material will be required in the future.\n\nIn the future, we plan to use the information revealed by these basic measurements to develop a system that will help researchers to guess and present the locations of wokototen markings when deciphering kunten materials, and to automatically generate written transcriptions to help readers understand these materials. Another future task will be to extract the characteristics of wokototen by using large amounts of data, which have been difficult to compare and study using conventional manual research methods.", "appendix": "Data availability\n\nThe data for this study is owned by the National Institute for Japanese Language and Linguistics. The data of wokototen charts were used this study and can be obtained and search here: https://cid.ninjal.ac.jp/wokototendb/.\n\nThe data is available in accordance with the policy of the National Institute for Japanese Language and Linguistics. The policy is here: https://www.ninjal.ac.jp/english/utility/policy/.\n\nAnalysis code available from:\n\nhttps://github.com/TTMTMAK/CountProg_for_Wokoto/releases.\n\nhttps://github.com/TTMTMAK/CountProg_for_Wokoto/tree/master.\n\nArchived analysis code at time of publication: https://doi.org/10.5281/zenodo.7801472. 19\n\nLicense: Apache License, Version 2.0\n\n\nReferences\n\nTsutsumi T, Tajima K, Kosukegawa T, et al.: Computerized Method of KUNTEN and Quantitative Analysis of KUNTEN Using Computer. J. Inf. Process. 2018; 59(2): 278–287.\n\nTajima K, Tsutsumi T, Takada T: Quantitative Analysis of Kunten Materials for Interpreted Text Generation. J. Inf. Process. 2020; 61(2): 162–170.\n\nTsutsumi T, Tajima K, Kosukegawa T, et al.: Automatic Recognition of Wokototen Table using Kunten Database. J. Inf. Process. 2022; 63(2): 283–292.\n\nTsukishima H: Heianjidai Kuntenbon Ronko (平安時代訓点本論考), Kyukoshoin.1986.\n\nYoshizawa Y: Kokugo Kokubun no Kenkyu (国語国文の研究). Iwanami Shoten, Publishers; 1927.\n\nYoshizawa Y: catalogue of Tenpon (点本書目), Iwanamikouza Nihonbungaku (岩波講座日本文学). Iwanami Shoten, Publishers; 1931.\n\nYoshizawa Y: Kokugo Setsurei (国語説鈴). Publishing Department of Ritsumeikan University; 1931.\n\nNakata N: The Translation Part of The Philological Study of Kotenbon (古点本の国語学的研究 (総論編・訳文編)). Kodansha Ltd; 1954.\n\nOhtshbo H: A study of the kunten language (訓点語の研究). Kazamashobo; 1961.\n\nTsukishima H: A Study on the Reading of Chinese Words in the Heian Period (平安時代の漢文訓読語につきての研究). Publishing Department of The University of Tokyo; 1963.\n\nKobayashi Y: A Historical Study of the Reading of Chinese Classics in the Heian and Kamakura Periods (平安鎌倉時代に於ける漢籍訓読の国語史的研究). Publishing Department of The University of Tokyo; 1967.\n\nKobayashi Y: A Japanese Linguistic Study of Kakuhitsu Documents (角筆文献の国語学的研究), yukoshoin.1987.\n\nKasuga M: Kokunten no kenkyu (古訓点の研究). Kazamashobo; 1956.\n\nJinho P: Shuhon Kegonkyou dai36ten kuketsu no kaidoku (周本華厳経巻第36点吐口訣の解読)，Nikkan kanji kanbun juyou ni kansuru kokusaigakujutsuronbunshu (日韓漢字・漢文受容に関する国際学術会議論文集).2003; pp.127–158.\n\nChang K: Some Caracteristics of Point-attached Kugyol on Yuquishidilun (瑜伽師地論)，Nikkan kanji kanbun juyou ni kansuru kokusaigakujutsuronbunshu (日韓漢字・漢文受容に関する国際学術会議論文集).2003; pp.159–177.\n\nZai LC: Kyoto Kokuritsu Hakubutsukanzou Kegonkyo maki dai 17 no Kunten (京都国立博物館蔵『華厳経』巻第十七の訓点)，Diacritical language and diacritical materials，Kunten go gakkai.2005; pp.251–283.\n\nPaku J: Moji Seikatsushi no Kanten kara Mita Kuketsu (文字生活史の観点から見た口訣). Bungaku. 2011; Vol.12(No.3): pp.169–181.\n\nMiyoung O: Kankoku no Kuketsu Shiro oyobi KuketsuKenkyu no Genjo ni tsuite (韓国の口訣資料および口訣研究の現況について) ，Nikkan Kanbunkundoku Kenkyu (日韓漢文訓読研究). Bensei Publishing Inc.; 2014; 289–332.\n\nTomoaki T: TTMTMAK/CountProg_for_Wokoto: WokotoCount1.0.0. [Software]. Zenodo. 2023" }	[ { "id": "186344", "date": "17 Jul 2023", "name": "Brian Steininger", "expertise": [ "Reviewer Expertise Reception of Chinese literature in early and medieval Japan" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article presents a format for digitizing data on vernacular reading glosses in the okototen format, applied to classical Chinese texts in premodern Japan. Using this methodology, the authors compare data from \"comprehensive charts\" (prescriptive instructions for producing glosses kept by a particular school) and \"inductive charts\" (diagrams of actual glosses found in primary sources made by contemporary researchers--in this case, the work of Tsukishima Hiroshi). This is an important distinction. The statistical results of the survey contain few major surprises, though there is a somewhat interesting discrepancy in regard to the use of the space to the right of the character. As indicated in the conclusion, the major importance of this research is as foundational work building towards essential new tools for digitization and semi-automated analysis of kunten materials.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "186365", "date": "10 Aug 2023", "name": "Pádraic Moran", "expertise": [ "Reviewer Expertise Comparative glossing (annotation) practices generally", "specialising in the European Latin tradition", "but with an interest in Japanese glossing. Digital Humanities and computational analysis of texts." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a valuable study of wokotoken annotations, part of the Japanese kunten system of annotating Chinese/Sinitic texts. Its value lies in its computational approach. By counting the large number of complex wokotoken systems collected in Tsukishima 1986, the authors demonstrate very clear distribution patterns, which present new insights into Japanese annotation practices. The information is clearly and carefully presented, and the implications for future research (presented the Conclusion section) are very significant. This article will be of interest to specialists in kunten. It should also interest researchers on glossing more generally, since the computational approach adopted here could be a model for specialists in other traditions to follow.\n1) I saw just one significant error:\np. 5: ‘Examples are shown in the 'カ/ナ' in the top left-hand corner (-2, -2) of the fifth tsubo in Figure 1.’ This appears to be the third tsubo (middle left), not the fifth. (Also ‘カ/ナ’in the text is confusing, since the figure has the reverse: ナ/カ. Recommend to change the text accordingly.)\n2) I have two recommendations:\np. 5, table 1: Several shapes are followed by a sign ↑or ↓. These seem to require explanation.\np. 7, figure 2: Although the figure is extremely helpful, it is a little hard to read. The Y-axis runs from +3 on top to -3 on the bottom. However, the corresponding table 5 has -3 on top and +3 on the bottom. This is more intuitive, since -3 represents the top of a character. Recommend to reverse the Y-axis in figure 2.\n3) The following minor errors should be corrected:\np. 3: ‘around from the Heian period…’—sense of ‘around from’ is not clear; recommend to delete ‘around’ ‘charts are used as means’ ‘wokototen is mostly found’ > ‘wokototen are mostly found’\np. 4: ‘replacing it with the similar character in Unicode’—‘the’ > ‘a’ ‘The 124 characters… is shown’—‘is’ > ‘are’\np. 5: ‘This digitization method… are described in’—‘are’ > ‘is’ ‘These methods of the Korean studies’—‘These’ > ‘The’ ‘… has the same concept’—‘has’ > ‘have’ ‘The programme was written in C#’—‘The’ > ‘A’ ‘Sharp’ > ‘Shape’ ‘Examples are shown’ > ‘An example is shown’ ‘into two separate totals. The one of wokototen of the shape ‘L’ reading ‘カ’ and the other of wokototen of the shape 'L' reading 'ナ.’ > ‘into two separate items, one for the wokototen shape ‘L’ reading ‘カ’ and the other for the wokototen shape 'L' reading 'ナ’.’\np. 7: ‘six wokototen chart’—‘chart’ > ‘charts’\np. 8: ‘who compiled a chart’ > ‘who compiled charts’\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "174044", "date": "10 Aug 2023", "name": "Kiyonari Nagasaki", "expertise": [ "Reviewer Expertise Digital Humanities and Buddhist studies" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI acknowledge the potential significance of the research presented in this paper, which offers an insightful foundation for discussing matters related to pre-modern international exchange and translation. The findings are compelling, contingent upon further corroboration through subsequent research.\nHowever, having perused the manuscript in detail, I wish to raise two points for your kind consideration and, if possible, rectification.\nFirstly, our attention was drawn to the sequence of \"te\", \"wo\", and \"ni\". As per the frequency order established in your study, they should be listed as \"te\", \"wo\", and \"ni\". However, we have observed instances where the sequence appears as \"te\", \"ni\", and \"wo\". Unless there is a specific reason for this inconsistency, we recommend standardizing the order for improved coherence.\nSecondly, I noticed that the analysis carried out in this study could arguably be conducted without the use of digital technology. In order to enhance the position of your paper within the landscape of existing research and amplify its overall value, it would be beneficial to offer a clearer context. Specifically, it would be helpful if you could clarify whether no such analysis existed prior to the advent of digital technology, or if there was any similar analysis previously. If the latter is not the case, kindly offer a rationale for the absence of such analyses in the past. This contextual detail will further enrich the relevance of your work within the wider field of study.\nI request your thoughtful consideration of these points.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "186342", "date": "21 Aug 2023", "name": "Kristina Hmeljak Sangawa", "expertise": [ "Reviewer Expertise History of Japanese writing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes a system for digitising vernacular reading glosses of the wokototen type in classical Chinese texts, presents the results of quantitative measurements of the readings, shapes and positions of such glosses in summaries (compiled by H. Tsukishima) of wokototen markings used in actual kunten materials, and compares these findings with data from 26 wokototen 'comprehensive' charts of different schools compiled by Nakata and Tsukishima.\nNot surprisingly, the results show that simple shapes (dots or lines that can be written with one stroke) are the most common markings, with dots ('ten' in 'wokototen' literally means 'dot') being by far the most common; that the most frequent readings are marked by dots, and that most markings appear at the centre or at the four corners overlapping the character they mark. It was also found that markings not overlapping characters are most commonly found on the right of the character, as is still the case for phonetic guides (furigana) in contemporary Japanese.\nSome differences were found between the summaries of wokototen glosses in kunten materials and the 26 wokototen charts, with regard to the most frequent readings and the most frequent position of markings.\nThe quantitative findings are not surprising, but do empirically confirm common observations. As the authors note in their conclusion, the most important contribution of this research is the coding and processing method used to digitise wokototen markings, described in detail and complete with code that can be used in the future to extend the digitisation and semi-automatic analysis to other material.\nOne further important aspect of this article is that it is written in English. The majority of published research on Japanese vernacular readings of classical Chinese texts is written in Japanese, and is therefore not accessible to scholars from other areas of research such as the history of writing, language contact etc., who may not be versed in Japanese. This is also reflected in the list of references of this article, which only includes sources in Japanese (the first three in the list – previous papers by the same authors – are quoted with their English titles, but are actually papers published in Japanese with only an English title and abstract). Given the usefulness of the proposed method for digitisation and processing of text with complex markings and glosses, the fact that it is written in English makes it accessible to a wider audience and thus more likely to be used and built upon.\nThe terminology used follows the tradition of Japanese research on this topic and mostly consists of Japanese terms in romanised form. This is somewhat surprising, considering that two of the authors are also co-authors of a thoroughly explained proposal for English terminology to be used in research on vernacular readings of classical Chinese texts (Whitman et al. 2010, http://conf.ling.cornell.edu/whitman/WhitmanAlberizziTsukimotoKosukegawa2010Toward.pdf), where they propose using self-explanatory English translations instead of romanised renditions of Japanese terms, such as \"vernacular reading\" instead of kundoku or \"gloss\" instead of kunten. All terms in this article are, however, clearly explained and thus accessible also to readers who may not be familiar with the Japanese terminology.\nI have a few minor formal suggestions for a possible revised version of the article.\nJapanese long vowels could be romanised more consistently: in the list of references, the long vowel ō is sometimes spelled 'ou' following kana orthography (e.g. 14. Kegonkyou, 15. juyou, 16. Hakubutsukanzou), once 'oh' (Ohtsubo, but misspelled Ohtshbo), and sometimes not marked as long (Kazamashobo, Kegonkyo, Shiryo - misspelled as Shiro). The long vowel ū is not marked as such (kenkyu, ronbunshu etc.).\nThe reference list would be more reader-friendly in a more consistent and complete format, e.g. following the Generic style rules for linguistics [www.eva.mpg.de/linguistics/past-research-resources/resources/generic-style-rules/]. Specifically, author names would be more easily searchable and less ambiguous if given in both romanized form and Japanese script, and titles more accessible if quoted consistently with both the romanised form and the original Japanese script, followed by an English translation. In the present version of the article, works with English alternative titles (1, 2, 3) are cited only in English, some (6, 8, 10, 11, 12, 15, which originally have no English title) are cited in Japanese script and with an English translation of the title probably provided by the authors, without a romanised transcription of the Japanese title, while other titles (4, 5, 7, 13, 14, 16, 17, 18) are given in romanised form and Japanese script, but without an English translation of the title. For example, the following citation:\nOhtshbo H: A study of the kunten language (訓点語の研究). Kazamashobo; 1961\nwould be more informative and accurate in this form:\nŌtsubo H (大坪併治): Kuntengo no kenkyū (訓点語の研究) [A study of the kunten language]. Kazamashobō; 1961\nA few typos should be corrected:\np. 3 (Target of research) the meaning of a wokototen markings -> marking\np. 8 (Conclusion) Kunten -> kunten Kunten Material -> kunten material (Data availability) can be obtained and search -> searched\np. 9 (References) 6. catalogue -> Catalogue 9. Ohtshbo -> Ōtsubo /or/ Ootsubo 12. yukoshoin -> Kyūkoshoin 18. Shiro -> Shiryou\nNotwithstanding these suggestions for very minor corrections, the article is an important contribution to the study of Japanese vernacular readings of classic Chinese texts, and a welcome introduction of the proposed digitisation method to an international audience.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/12-506
https://f1000research.com/articles/12-505/v1	16 May 23	{ "type": "Research Article", "title": "A phenomenological analysis of the emotional experiences of graduate preservice student teachers in Indonesia", "authors": [ "Sunardi Sunardi", "Famala Eka Sanhadi Rahayu", "Desy Rusmawaty", "Istanti Hermagustiana", "Dyah Sunggingwati", "Sunardi Sunardi", "Desy Rusmawaty", "Istanti Hermagustiana", "Dyah Sunggingwati" ], "abstract": "Background: Addressing preservice teachers' emotions can help them develop the emotional connections necessary for successful teaching practicums and professional learning experiences. To understand preservice teachers’ emotions towards their surroundings, the researchers used an emotional geography framework to map out the source of their negative and positive emotions and how those feelings affect their beliefs, teaching style, and paradigms. Emotional geographies were divided into two categories (i.e., emotional distance and emotional closeness), which were further mapped into five major themes (i.e., sociocultural, moral, professional, physical, and political). Methods: This study examined emotional geographies of 15 graduate preservice student teachers of Teacher Training and Education Faculty of Mulawarman University during their teaching practicums. Moreover, this study used a qualitative design involving semi-structured interviews with 15 graduate students who completed their teaching practicums. Results: The participants experienced all emotional geographies (sociocultural, moral, professional, physical, and political) that reshaped their beliefs and teaching styles, especially related to teaching undergraduate students. These emotions are generally experienced when the participants deal with the students and cooperating teachers. Conclusions: It was found that teachers needed to be strict when instilling discipline in their students, develop friendships with their students to maintain classroom control, and require comprehensive preparation to create teacher's confidence and answer their students' questions. In effect, the theoretical framework of emotional geography when teaching undergraduates during practicums gave student teachers valuable experience to cope with all common teaching challenges and professional growth.", "keywords": [ "emotional geography", "student teachers", "teaching practicum", "teacher education" ], "content": "Introduction\n\nTeacher education can mold the personalities of a nation’s future generations. Therefore, high-quality teacher education aims to develop qualified, competent teachers in order to shape a country’s education future, which requires relevant teacher education courses that build and strengthen teacher competencies. One such program is a practicum, which is a required teacher education component from which preservice teachers can acquire professional growth and apply theoretical learning to practical situations (Hascher & Hagenauer, 2016). Preservice teachers refer to teaching candidates enrolled in post-secondary education-related degree programs who are receiving their initial training before practicing and have not yet taught in a classroom (Milner et al., 2003). In Indonesia, preservice teacher practicums are defined under the Ministry of Research, Technology, and Higher Education Regulation No. 55 of 2017 on Teacher Education Standards, paragraph 9, which states that all teacher training institutions must provide practicum programs that involve microteaching and school-based practicums.\n\nProfessional experience and/or teaching practicums are bridges between theory and practice and provide authentic preservice experiences for developing pedagogical skills and a familiarity with the teaching education landscape (Kaldi & Xafakos, 2017), which is critical because good teaching performances are related to strong pedagogical foundations (Hargreaves & Shirley, 2012). Therefore, teaching practicums provide student teachers with experience in teaching in terms of teacher education and preparation programs. Studies into preservice or student teachers can be valuable because such teachers have dual roles as both students and teachers, and their success in teacher education programs may reveal their potential as future educators (Lipka & Brinthaupt, 1999). Several studies have been conducted on preservice teachers, which involved assessing their knowledge and competence (Alkharusi et al., 2011; Petrovici & Masari, 2014; Ragawanti, 2015; Valli et al., 2013; Walton & Rusznyak, 2013), attitudes toward teacher programs and the teaching profession (Bhargava and Pathy, 2014; Mule, 2006), the challenges (Karunagaran & Saimin, 2019; Shah et al., 2021), anxiety (Agustiana, 2014; Daud et al., 2019), and perceptions (Adodo, 2013; Kumazawa, 2013; Sun & van Es, 2015; Thomas & O’Bannon, 2013; Zhu, 2017). While much of this study emphasizes the need to improve teacher education quality and preservice teacher abilities, few studies were noted to examine preservice teachers’ emotions during their practicums.\n\nEmotional effects have frequently been ignored because of a lack of awareness of the effects of teacher emotions on teaching quality. However, understanding teachers’ feelings could provide a more comprehensive perspective on teacher education effectiveness. Therefore, this study investigated the feelings that preservice teachers had during their teaching practicums and the influence of these emotions had on their teaching paradigms, beliefs, and teaching styles. One of the tools to assess teachers’ emotional geography in the classroom is by examining their emotion through Hargreave's emotional geography framework. Emotional geographies describe the patterns of closeness and distance in human interactions that shape the emotions we experience about relationships to ourselves, each other, and the world around us (Hargreaves, 2001b). Hargreaves (2001b) claimed that understanding teachers’ emotions is crucial because it can influence the classroom environment and the conduct of the learning activities.\n\nHargreaves’ (2001b) emotional geography framework can also be used to map a teacher’s series of feelings. Hargreaves (2001b) defined five emotional geographies (i.e., sociocultural, moral, professional, physical, and political) as geographical and experiential patterns of closeness/distance in human connections that form, configure, and color feelings about ourselves, the world, and one another. Various studies revealed that teachers encountered emotional geographies in their interactions with parents (Chen & Wang 2011; Sanhadi Rahayu & Asanti 2021), colleagues (Hargreaves, 2001a), students (Hargreaves, 2000), and principals (Lassila et al., 2017) as well as during the teaching process itself (Hargreaves, 1998a; Hargreaves & Tucker, 1991). However, the investigation of emotional geography of preservice teacher during the practicum has remained unseen by many scholars. Therefore, the present researchers want to fill this gap through this study.\n\nAs aforementioned, a teaching practicum provides real-life experience in teaching students in the classroom. However, preservice teachers encounter various obstacles and experience a variety of emotions during their teaching practice. They have to deal with various settings involving students and the sociocultural contexts in the institution in which they are teaching (Riesky, 2013). This study revealed the difficulties preservice teachers experienced due to their emotional beliefs (Riesky, 2013). Dotger et al. (2011) simulated parent–teacher and preservice teacher conferences to examine preservice teachers’ emotions, with the findings suggesting that their emotional geography could be classified into three distinct frameworks: professional, moral, and political. These emotional geographies revealed that preservice teachers were most apprehensive when engaging with parents about their children or the school, especially when the parents exhibited various emotions (e.g., support, concern, frustration, and fury). Rejeki Kristina, and Drajati (2018) profiled a female English instructor who taught in a rural location in Indonesia and developed an emotional geography sociocultural framework that depicted the participants’ feelings and discomfort about their cultural and social background and the rural environment in which they had been placed. Liu (2016) documented the experiences of an immigrant teacher in a professional community and how they overcame their emotional barriers to community understanding. These studies suggest that diverse emotional, physical, moral, sociocultural, professional, and political geographies shaped the teachers’ positive and negative feelings. Although numerous studies have examined the emotional experiences of preservice teachers during their teaching practicums (Dotger et al., 2011; Karunagaran & Saimin, 2019; Riesky, 2013; Thomas & O’Bannon, 2013), little attention has been given to their emotional geography as they interact with the students, their cooperating teachers, and other faculty members during their initial teacher education. Therefore, this study aims to investigate graduate preservice student teachers’ emotional geographies during their teaching practicums through Hargrave's emotional geography framework. Addressing preservice teachers' emotions has the potential to strengthen emotional bonds between preservice student teachers, cooperating teachers, and students, allowing them to develop the emotional connections required for fruitful teaching practicums and professional learning experiences during their initial teacher education.\n\n\nLiterature review\n\nA teacher’s emotions are indicators that can be used to explore aspects beyond the teacher’s professional relationships (Hargreaves, 2001a). Teaching is not only a cognitive and behavioral practice where elaborating teaching depends only on what they know and what they should do in the class. Instead, teaching involves another factor, such as their feeling when teaching, which can be a help or a threat for their professionalism.\n\nA teacher’s experience of teaching can be affected by their relationships with the students (Hargreaves, 2000), parents (Hargreaves & Lasky, 2004), their colleagues (Hargreaves, 2001a), principals, and educational changes (Hargreaves, 1998a, 1998b), with the feelings from these relationships affecting teaching styles, judgments, and the conduct of the class learning activities. Therefore, understanding these positive and negative emotions related to teaching, could reveal the underlying decision-making and problem-solving contexts in the classroom.\n\nUnderstanding teacher emotions is important for teachers with extensive teaching experience and student teachers during their teaching practicum. Teaching practicums allow preservice teachers to experience a real class and apply the knowledge they have learned. Following teaching plans may not be as easy as one believes and may evoke positive and negative emotions depending on how the threats in the class are perceived. These stressful situations guide the problems that need to be overcome, and the feelings being experienced could lead to changes in preservice teachers’ perceptions, beliefs, and teaching styles (Yuan & Lee, 2014).\n\nGraduate preservice teachers (GPSTs) are graduate students enrolled in master’s degree programs who are required to complete their teaching practicums before graduation (Geng et al., 2016). Although the term ‘preservice teachers’ is widely used, the preservice teachers in this study were slightly different because they were studying for their master’s degrees rather than bachelor’s degrees. Graduate English education program students are required to complete a teaching practicum comprising three to five classes with undergraduate students in similar majors and to teach the classes similarly to the undergraduate student lecturers. These teaching practicums provide GPSTs with additional teaching experience by allowing them to teach undergraduate students whose characteristics differ distinctly from the secondary school students they taught as part of their bachelor’s degrees, thus increasing their knowledge about dealing with adult students and positioning them for possible future employment as lecturers (Sunardi, 2018).\n\nA classroom is an emotional place. The students spend endless hours attending class, finishing projects, taking tests, and building social relationships contributing to life goals—holding a degree in education today has been more personal, social, or financially significant. Emotions like curiosity, interest, hope, pride, rage, anxiety, shame, confusion, irritation, and tedium are common, prevalent, and intense in these contexts (Pekrun & Linnenbrink-Garcia, 2015; Zhang & Zhu, 2008).\n\nThe significance of emotions in education extends to teachers, principals, and administrators in equal measure. For instance, teachers are responsible not only for imparting knowledge but also for instilling a passion for the subject and enthusiasm for learning (Pekrun & Linnenbrink-Garcia, 2015). Student teachers will learn emotions early in the teaching process, in this case, during their teaching practicum. Teaching practicum can project the real teaching atmosphere and give the student teacher valuable experiences to expose themselves to emotions in teaching. Typically, these feelings emerge when teachers' objectives, standards, and beliefs interact with those of other classroom participants during routine school activities (Hargreaves, 1998a). Thus, teachers experience various emotions, from happiness and pleasure when a lesson proceeds as intended to frustration and anger when working with difficult administrators (Schutz, 2014; Sutton & Wheatley, 2003). If they successfully generate enthusiasm for the course material, the motivational effects should extend well beyond the course. However, failing to maintain good teaching by standards will lead them to feel anxiety or boredom. (Pekrun & Linnenbrink-Garcia, 2015; Sutton & Harper, 2009; Titsworth et al., 2010)\n\n\nMethods\n\nThis study was approved by Universitas Mulawarman and Ethical Clearance Committee (Protocol Number: 061/UN17.5/PP/2020). Written informed consent was obtained from all participants.\n\nThis study investigated the emotional geographies of Graduate Pre-Service Student Teachers (GPSTs) majoring in English Education at Mulawarman University, who had finished their teaching practicum in 2019. There were 21 students who were first recruited and contacted for taking part in this study online via Zoom, but only 15 students were able to proceed with the interview. Six other students could not participate in this study due to limited access to the internet in their hometown as most of the students had returned home because of COVID-19 restrictions (Ministry of Education and Culture of Indonesia, 2020). Participant profiles are shown in Table 1.\n\nAll participants were enrolled in the TESOL teacher training program and had various previous education and teaching experiences. Most participants had been teaching for several years, both in formal education (e.g., teaching at school) and informal education (e.g., teaching in an English course). In contrast, three participants were relatively new to teaching, especially teaching university students.\n\nThis research was conducted from March to April 2020. Because of COVID-19 restrictions (Ministry of Education and Culture of Indonesia, 2020), the thirty to sixty minute semi-structured interviews were conducted online via Zoom; with two interviews conducted for each participant. Then, the participants were contacted by the researcher (S) to ask for their willingness and availability to participate in the study. The researcher (S) took the participants’ contact information from the postgraduate program of English Education and he also provided the link for the Zoom meeting. There were 15 students who were able and available to attend an interview with the researcher (S). The first interview was exploring their emotional geographies and in the second interview, researchers verified the interview transcriptions with the participants to ensure their answers were recorded correctly. In both interviews, the participants voluntarily gave consent to the use of their experience as data for this research. The transcript was double-checked by the participant to ensure the result were accurate.\n\nA phenomenological approach was adopted for data analysis and included three steps: coding the emotions, identifying and/or interpreting the causes of the reported emotions, and identifying the themes based on the emotional geography framework of Hargreaves (1998a). In the first coding emotion step, the interview transcripts were searched for passages that included information on teachers’ emotions, to which an emotional geography coding scheme was applied. The emotions (e.g., annoyance, happiness, and joy) were plotted as either positive or negative and coded accordingly. The second step in the analysis identified and/or inferred the cause of the participants’ reported emotions as reported by participants, the descriptions for which were paraphrased and described using qualitative content analysis. Statements describing similar causes were merged and a summary of the inferred causes was made for each identified emotion. This step involved scrutinizing the text passages within each code, such as motivation. The third step complemented the analysis by identifying the overall themes and the specific emotional geographies.\n\n\nResults\n\nHargreaves’ emotional geography framework was developed because teachers encounter scenarios that can result in positive and negative sociocultural, moral, professional, physical, and political emotions about their relationships with students, parents, colleagues, and the school principal throughout the teaching–learning process (Chen and Wang, 2011; Hargreaves 2000, 2001a, 2001b; Hargreaves & Lasky 2004; Lassila et al., 2017; Liu 2016), which then lead to the development of distinct emotional geographies with other parties. Results of the GPSTs’ interview analyses revealed that the GPSTs had developed emotional distance and closeness from their interactions with the students and cooperating teacher, which had exposed them to all five emotional geographies, the occurrences of which were appended to the respective participant interviews.\n\nSociocultural distance typically develops because of differences in cultures, habits, social standing, and economic levels. In this case, all participants expressed negative feelings about the students’ lateness, that is, punctuality was found to be a source of unpleasant emotions (e.g., frustration, anger, and feeling distracted and upset) (Prasetyarini et al., 2021; Bataineh, 2014). The class was able to function normally with few late students; however, class activities were significantly disrupted when over half of the class was late. Most participants stated that they had planned specific tasks for the class, and if the class began late, it affected all activities in the teaching plan, making the participants irritable (Zaid Bataineh, 2014). Explaining the materials again to the late students, which upset the participants’ concentration, was also necessary, as one participant stated:\n\nActually, the most emotional part was in the first meeting of teaching practicum; when I started the class, there were around 10 students who attended the class; I asked the leader of the class where her friends were and she said that they were still in the canteen and in other places, and then I asked her to contact her friends because we had to start the class now. For me, yeah, I know it’s like Indonesian habit, yeah, perhaps. Yeah, they were late, and then I had to wait around 15 minutes, and not all of them came to class that day (Participant 1)\n\nThis comment reveals that the GPST had experienced unpleasant emotions when the students were late as this had disrupted the lesson plan. However, most GPST participants felt that they had no right to be tough on the students because they were only graduate students, not lecturers (Boyce, 1997). Therefore, the sociocultural distance associated with student tardiness resulted in the participants experiencing political distance.\n\nThe participants were inevitably confronted with moral distance when they interacted with the students. Moreover, the participants believed that the students occasionally had different moral aims in mind during the interactions. The GPST participants expected the students to listen and pay attention; however, they occasionally encountered students who did not do so, making them feel distracted and enraged, especially when they notice their students were more concerned with their phones, as stated by one participant:\n\nIt’s really distracting because we are explaining in front of them and they focus on their phones, and we feel like they aren’t paying attention. We also wonder whether they understood the lesson because we don’t know their feelings about understanding the lesson, and if they were listening or not, so it was really distracting for me (Participant 5)\n\nAnother participant added the following:\n\nWhile I was teaching, I saw that one student was busy on their cell phone. In my heart, I felt so furious, I felt so angry because I’d been preparing my material for 2 hours, but they played with their cell phone, hello! (Participant 14)\n\nSeeing the students ignore them in favor of their phones made them feel humiliated and disrespected (Baker et al., 2012; Smale et al., 2021; Thomas & O’Bannon, 2013; Tindell & Bohlander, 2012). They felt that they had tried to do their best when teaching the class but had received disrespectful behavior in return. Inappropriate student behavior has commonly been reported as a source of negative feelings among preservice teachers (Baker et al., 2012; Karunagaran & Saimin, 2019; Thomas & O’Bannon, 2013) as they felt what they were doing was useless because they did not get the responses they wanted.\n\nThe most striking conclusion from this study is the emotional distance the participants felt about their professionalism. The GPST participants’ perceptions of their professional worth varied. The first point of contention was when they expressed doubts about their competence to educate the undergraduate students because the students had made them believe that their knowledge was insufficient. In response to this issue, the participants felt anxious during their initial period in the class and when teaching the students. As one participant stated,\n\nI get anxious right after thinking, ‘Will they like me?’ For example, will they accept me as their exchange teacher for the time I am going to teach them, but I am still a master’s degree student, so will they appreciate me? Will they accept me? I get this kind of feelings. (Participant 10)\n\nNot only did the participants cast doubt on their competence but they also acknowledged that they had limited comprehension of the teaching content at times (Agustiana, 2014). Consequently, the participants felt unable to respond when the students posed critical questions about issues. Another instance was when they believed they had not adequately prepared the content and lacked mastery of the material they had provided to the students. According to one participant, ‘Because there was a topic that I didn’t really understand, I tried to make my face look like I understood it and then I felt like no one noticed’ (Participant 3).\n\nTeachers should be well prepared for class and must comprehend and master the topics they are going to teach. However, preservice students occasionally have difficulties comprehending the content of their teaching materials and often do not have the time to talk with their cooperating teachers. These issues can cause students to have doubts and reservations about the teacher’s competence to instruct in the class and can affect subsequent lessons, even if the teachers are fully prepared afterwards. Therefore, teachers must be professionally prepared to create a favorable educational environment.\n\nFurthermore, the teaching practicums with undergraduate students majoring in English Education required the participants to communicate in English on daily basis. Regrettably, not all participants spoke English fluently and were therefore confronted with a professional divide, as commented on by one participant, ‘Maybe, generally, I have not been really satisfied with my speaking environment in the past years, something like that’ (Participant 13).\n\nThe participant expressed dissatisfaction with their speaking ability, which they believed was insufficient (Daud et al., 2019). Owing to a lack of confidence in their ability to communicate in English, they felt incapable of teaching the students. From the students’ perspective, English majors need the lecturer to be proficient in English teaching techniques; therefore, they would not feel they were ideal models for effective English as a Foreign Language (EFL) teachers if the students felt that participants were not competent lecturers. The questioning of a teacher’s ability occurs regularly in Indonesia, primarily because many EFL teachers are non-native English speakers. Thus, students are hesitant to speak English if they are only taught through textbooks; therefore, Indonesian preservice teachers should prioritize the English abilities needed to assist them in their teaching, that is, English fluency is a must for EFL teachers (Daud et al., 2019).\n\nAnother difficulty associated with professional distance was when the teacher’s plans did not align with the students’ qualities. Teachers are often confident that certain methods will be successful in the classroom; however, success is contingent upon whether these methods are appropriate for students. Therefore, professional distance occurs when reality does not match the teacher’s expectations, as reported by one participant:\n\nThis sometimes happens in our teaching because I had already planned to apply this method; however, when I applied this method and it was not related to the situation in the classroom, I had to directly find another way to match my method and the classroom situation. For example, when I prepared to conduct a group activity, such as a presentation, I saw that only one group was actively joining the presentation, so I had to change and present an activity that could interest all students in the classroom (Participant 5)\n\nThe teacher had arranged a group discussion and had anticipated that all students would fully participate; however, the classroom atmosphere or the students’ qualities did not align with the preplanned activities, meaning that the teacher had to choose whether to alter their activities or continue with them regardless of student engagement. When confronted with an unexpected event in the classroom, the decision to change activities needs to be made by the teacher based on their professional judgment, which also means that teachers should have alternative plans when their planned activities do not proceed as expected.\n\nWhen establishing an emotional bond between teachers and students, there inevitably needs to be an intimacy, regularity, and continuity of engagement between both parties (Hargreaves, 2001b). Physical closeness and distance are also related to the amount of time or space produced between the parties that interact (Liu, 2016). The physical theme evolved because of the interactions between the participants and other stakeholders (i.e., the students, cooperating teachers, and administrative staff). Three participants expressed anxiety when confronted with physical distance in their encounters with the students or the cooperating teachers.\n\nThe first physical distance occurred when preservice teachers were teaching undergraduate students, with a majority reporting feeling apprehensive when standing in front of students their age (Boyce, 1997). Their anxiety was because of a sense of inadequacy, unfamiliarity, and distrust. Their reactions to the physical distance were influenced by their first efforts at teaching in front of a class and by the professional distance they felt in the class (Agustiana, 2014; Shah et al., 2021; Zhu, 2017). Their uncertainty about their abilities and characteristics exacerbated their unfamiliarity with the students, which was why they were scared and experienced a physical distance from the students. As one participant stated:\n\nI think the biggest challenge for me was when I tried to explain something to them, given that was my first time teaching undergraduate students. I used to teach kids but not undergraduate students, so it was a big challenge. And then came the anxiety, right after I thought about whether they will like me? (Participant 10)\n\nThe aforementioned participant was concerned that the students would reject them because they were only a master’s degree student. This sentiment was shared by the majority of GPST participants in this study who were fearful that the students would question their abilities as they were only novice undergraduate teachers. These difficulties resulted in a range of emotions, including anxiety and nervousness.\n\nAnother difficulty related to physical distance occurred during the initial meeting of two participants with their cooperating teachers. Cooperating teachers are the classroom teachers with whom the participants needed to collaborate. Preservice teachers substitute for the cooperating teachers in three to five classes; therefore, maintaining a positive relationship with the cooperating teachers is a requirement so that the preservice teachers can discuss their concerns and seek and discuss solutions. However, not all participants had met their cooperating teachers in person, which created anxiety. As one participant put it:\n\n‘My coordinator was Mrs. A, then I was first felt bit scared because I was not very familiar with my cooperating teacher’ (Participant 7)\n\nThe participant encountered physical distance when communicating with their cooperating teacher because they had never met them (Boyce, 1997). Similar problems were raised by other participants who had never previously communicated with their cooperating teacher. They felt awkward initiating communication with someone they did not know in person as they felt it was necessary to meet their coordinating teacher in person first before discussing the teaching practicum. However, building a personal attachment before the teaching practicum was slightly difficult because not all preservice teachers lived in the same city as the university and their cooperating teacher. Therefore, they tried to contact them using messenger or social media but still felt awkward. This physical distance, in some cases, led to political distance as they felt very nervous when teaching in front of their cooperating teacher.\n\nSurprising information was found related to physical geography concerning how the preservice teachers felt about their student interactions. The teachers frequently attempted to create distance between themselves and the students to retain their professionalism. However, most of the GPSTs in this study believed that developing a close relationship with the students enabled them to better accomplish their teaching objectives. As one participant stated, ‘I really like teaching and I like to blend with the students’ (Participant 6). Another participant added the following:\n\nI tried to be friends with some of them, although they may feel awkward to befriend me; however, some of them were just like I told you, now I’m now friends with one of the students (Participant 9)\n\nThe participants believed that having personal contact with the students could gain their attention and respect; therefore, they attempted to develop positive relationships with the students through laughing, projecting a warm attitude, and providing personal communication. Even after the teaching practicum was completed, one participant stated that they enjoyed becoming friends with the students and developing relationships. The participants did not view their closeness to students to be a violation of their professionalism; rather, they felt that this closeness demonstrated their professionalism. It appeared that the study participants had divergent views on student–teacher interactions.\n\nThe final theme was political geography, which revealed the divergent views about the exercise of power (Liu, 2016). Political distance was observed when the participants questioned their power or authority in the class or when the teaching practicum coordinators were observing them.\n\nYeah, it happens. I asked the students an easy question, but there was no answer. I didn’t know if they really had no idea or they just were ignoring me because I was only a practicum teacher and not their real teacher. I was so angry because I felt disrespected. I wanted to go further, but I realized I had no authority for that as I was only a temporary lecturer for them, so I cannot exaggerate. (Participant 15)\n\nThis political distance made the GPST participants uneasy, which was also evident in their interactions with their cooperating teachers. They were nervous when they noticed the cooperating teacher, who they knew had greater power and authority, observing them in the class (Boyce, 1997). In comparison, one participant experienced a sense of political intimacy when their cooperating teacher bestowed complete authority on them and allowed them to teach the class without being monitored.\n\nWhat I taught in only 15 minutes at the beginning of the class, I could explain clearly and briefly to the students; I could communicate and interact well with the students. But when the teacher entered the class, I suddenly forgot the material. (Participant 13)\n\nPolitical distance was not found to only occur during the interactions between the participants and their cooperating teacher. Certain situations also made them feel helpless and had a major impact; however, they felt they could not deal with the situation. As one participant put it:\n\nDistractions in the classroom, such as from the students themselves or from the situation in the classroom, when I was conducting my teaching for the first time, the class was very hot because there was no air conditioning, so most students were not focusing and some people outside were being really noisy. (Participant 5)\n\nTherefore, as aforementioned, many GPST participants felt powerless when the classroom environment was not conducive. Classroom settings and the environment can have a substantial impact on class activity (Marzano & Marzano, 2003). Hot and noisy classrooms can be disruptive to both students and teachers and impair classroom activities (Prasetyarini et al., 2021). Therefore, the participant was unable to intervene because the classroom was hot and excessive noise was noted outside the classroom. The situation prevents students from concentrating on the materials provided by the participant. As a result, she maintains a political distance from the problem.\n\n\nDiscussion\n\nAfter examining the negative and positive emotions experienced by the GPSTs when teaching undergraduate students in their teaching practicum, the results were examined more broadly in light of Hargreaves’ (2001b) emotional geography concepts to determine how these emotions related to their teaching paradigms, beliefs, and styles. The GPSTs were confronted with various unfavorable sociocultural, moral, professional, physical, and political emotional barriers throughout their teaching practicums.\n\nThe first factor identified was sociocultural distance. Sociocultural distance is generally characterized by a separation of cultures, customs, social standing, and economic status (Hargreaves, 2001b). The GPST participants encountered a sociocultural distance because of the students’ punctuality, which was similar to the situation encountered by Ragawanti (2015), who discovered that the preservice teachers’ challenges revolved around activity management, with the most common difficulty being activity timing. Unlike students in primary and secondary schools, who typically have a set classroom and exit only at recess, university students spend most of their time outside the classroom waiting for their class to begin, which means a higher possibility exists that the students will arrive late to class. Having students arrive late irritated and angered the GPSTs as the lateness was interpreted as being disrespectful toward their authority as the practicum teacher (Prasetyarini et al., 2021; Zaid Bataineh, 2014). Thus, they questioned their authority and ability to discipline students who were late. Late students also disrupted the participants’ lesson plans and their ability to complete their well-designed exercises within the allocated time (Zaid Bataineh, 2014). Because many students arrived late to class, they wasted time and the GPSTs were unable to complete their planned tasks.\n\nThese late student episodes made the GPSTs feel that teachers should be rigorous when abiding by class regulations and that students should understand the implications of their tardiness and its effect on class activities. Consequently, the GPST participants chose to enforce stringent time limits and advised students that they would be unable to follow the class if they arrived late (Prasetyarini et al., 2021). Therefore, the GPSTs discovered that tight classroom regulations were necessary to ensure student punctuality.\n\nThe GPSTs felt moral distance when their aims differed from those of the students. Teachers may encounter student ignorance, excessive noise, silence (Ragawanti, 2015), and even poor behavior (Karunagaran & Saimin, 2019). The GPSTs said that they experienced feelings of moral distance when they observe that students’ attention was more focused on their phones than on their lessons, which they felt was disrespectful behavior. They admitted to being enraged, agitated, and preoccupied with the situation and believed that even though they had planned and prepared everything necessary to teach the students, the students lacked respect for them as teachers.\n\nThis moral distance encouraged the GPSTs to perceive the situation from both their and the students’ perspectives because they wanted to understand why the students disregarded them and preferred to play games on their phones than listen to the lesson (Baker et al., 2012; Smale et al., 2021; Thomas & O’Bannon, 2013; Tindell & Bohlander, 2012). Many of the GPSTs believed that it was either their teaching style that was boring the students or that their prepared activities were not suitably engaging. These beliefs compelled the preservice student teachers to admit that they needed to adapt their teaching approaches to make them more appropriate and appealing to the students in order to avoid boredom (Karunagaran & Saimin, 2019).\n\nProfessional geographies are formed when professional definitions and norms either divide the involved parties or allow them to collaborate on professional concerns. The GPSTs reported feeling professional distance in their relationships with their students and experienced certain emotions because they (1) had doubts about their abilities, (2) had not mastered the content, and (3) could not communicate fluently in English. The first difficulty arose when they mistrusted their abilities to teach the students. Most GPSTs in this study expressed similar concerns about the student’s perceptions of their abilities and were fearful that if they could not teach well, they would be ignored (Daud et al., 2019) and that their knowledge was insufficient to facilitate learning, all of which gave rise to anxiety, worry, and nervousness when teaching.\n\nThe second difficulty was because many of the GPSTs struggled to master the subject matter they were assigned to teach, and those who were confronted with this issue acknowledged that they lacked a thorough understanding of both the subject and the materials. While most attempted to comprehend the material, many were unable to do so but pretended to know it to avoid answering student inquiries (Agustiana, 2014). This was a legitimate concern regarding their professionalism. Teachers should be confident about their materials and activities before entering the class as this confidence can make it easier to control the class. Therefore, neglecting to master the content could foster a dislike toward the teacher. The GPSTs in this study acknowledged that they required additional time to become acquainted with the materials; however, most claimed that they had had only a limited amount of time to prepare and were therefore unable to do their best. They also assumed that learning the content would help them control their negative emotions in front of the students.\n\nFinally, the participants expressed dissatisfaction with their speaking abilities as a result of the professional distance (Daud et al., 2019). As EFL teachers, they were obligated to communicate in English; however, not all GPSTs in this study could communicate fluently in English, making them feel embarrassed and dissatisfied with their competence, primarily because most believed that English major students should have excellent teachers that can serve as EFL teacher role models. Most participants had shattered expectations because of their inability to communicate fluently with the students, enabling them to be aware of the necessity to practice their speaking in order to improve their teaching effectiveness as EFL teachers.\n\nPhysical geographies are concerned with time and place and connect or reduce relationships (Hargreaves, 2001b). The GPSTs encountered both physical distance and closeness in their interactions with the students and the cooperating teacher. They reported feeling apprehensive when teaching for the first time (Agustiana, 2014; Shah et al., 2021; Zhu, 2017), with their anxiety being exacerbated by their doubts about their ability to instruct the undergraduate students. A similar feeling occurred when they were required to communicate with their cooperating teachers for the first time. Several participants acknowledged that they had never met their cooperating teachers in person, which made contacting them initially awkward; however, this physical distance occurred only during the earliest stages of the relationship as they became calmer and more capable of reducing the physical gap when they had become better acquainted with the students and the cooperating teachers.\n\nThe participants perceived physical closeness during their teacher–student interactions despite their physical distance from the students. Most GPST participants believed that becoming better connected to the students as friends could assist them in maintaining class control. However, these beliefs contradict an earlier study that found that teachers preferred to maintain a professional distance between themselves and their students to remain professional (Liu, 2016). However, the GPSTs in this study defined professionalism differently than typical teachers, believing that being personally appealing and easy to get along with would be of benefit in their future careers. They felt that they needed to treat the undergraduate students differently because these students were adults who, unlike children and adolescents, could not be managed only by punishments or rewards. Given these disparate features, the GPST participants concluded that they needed to become friends with the students to enjoy the class and ensure success with the class activities.\n\nThe final theme was political geography, which is related to divergent views of power (Liu, 2016). The GPSTs reported experiencing unpleasant emotions when confronted with certain concerns (e.g., their authority in class, being observed by the cooperating teachers, and their inability to change unfavorable circumstances). The first political distance issue was inextricably linked to moral distance when the participants experienced negative feelings from the students’ behavior and powerlessness to intervene because they believed they lacked authority. Another concern was when the cooperating teachers were observing the participants (Agustiana, 2014; Boyce, 1997), with most GPSTs admitting that being monitored made them feel restricted because they were fearful of making errors (Daud et al., 2019). Several participants stated that they had been trembling when the cooperating teachers were observing them teach. However, most also felt that the observations made by the cooperating teachers’ could assist them. The final point was about the sense of impotence the GPSTs felt when confronted with unfavorable circumstances, the participants in this study complaint about the external factors like uncomfortable classroom which lead them to feel powerless since they have no authority or power to solve the problems.\n\nRegarding its limitations, the researchers identified a number of areas for improvement, including the sample method and the interview procedure. The participants in this study were taken randomly from one class of master's degree students who had finished their teaching practicum. Therefore, the researchers ware unable to characterize greater variance in the emotions displayed by the participants, as they were homogeneous. Consequently, the current study recommends that future research include a prior study through a survey and supplement it with an interview in which the researchers may determine which features are of interest. In addition, since the present research was conducted in the context of the COVID-19 pandemic, where the researchers were unable to meet the participants in person and conducted the interview via Zoom, the researchers encountered several difficulties, such as poor internet connectivity, which led to misunderstandings between the interviewer and the interviewee regarding the topic discussed; consequently, the answers of several participants cannot be utilized for further analysis. On the basis of this constraint, the researchers suggest that future studies conduct face-to-face interviews with the participants in order to clarify any misconceptions immediately. Furthermore, since this study relied on pre-service teachers' experienced during the teaching practicums which emphasized on their feelings only, it will be a better enrichment when the future research can correlate their experience with the way they cope with it, so called their self-regulation.\n\n\nConclusion\n\nThis study demonstrated the validity of using emotional geographies to describe the beliefs, paradigms, and styles of preservice student teachers. Hargreaves’s (2001a, 2001b, 2004) emotional geographies were applied to broaden the scope of the recorded examples. While the emotional geography concept has been previously applied to education, it has rarely been applied in preservice research. The current study demonstrated that the emotional geography concept could assist researchers to map preservice student teachers’ views during their practicums.\n\nThe teaching practicums with undergraduate students engendered some negative emotions, resulting in emotional distance. However, participants claimed that they could use these unpleasant emotions to improve their teaching styles and beliefs, concluding that teachers needed to maintain class discipline. Most also believed that teachers needed to present materials appealingly to avoid student boredom, that teachers should make friends with the students to maintain control in the classroom, and that thorough preparation was necessary to instill confidence in the students and be able to respond to their questions. Overall, however, both the positive and negative emotions experienced during their teaching practicum with the undergraduate students provided the GPST participants with significant experiences that will benefit them in their future careers.", "appendix": "Data availability\n\nFigshare: Interview Transcript of Graduate Student Teachers, https://doi.org/10.6084/m9.figshare.22276717.v2 (Sunardi et al., 2023a).\n\nThis project contains the following underlying data:\n\n• Interview with Graduate Student (Participant 1).docx\n\n• Interview with Graduate Student (Participant 2).docx\n\n• Interview with Graduate Student (Participant 3).docx\n\n• Interview with Graduate Student (Participant 4).docx\n\n• Interview with Graduate Student (Participant 5).docx\n\n• Interview with Graduate Student (Participant 6).docx\n\n• Interview with Graduate Student (Participant 7).docx\n\n• Interview with Graduate Student (Participant 8).docx\n\n• Interview with Graduate Student (Participant 9).docx\n\n• Interview with Graduate Student (Participant 10).docx\n\n• Interview with Graduate Student (Participant 11).docx\n\n• Interview with Graduate Student (Participant 12).docx\n\n• Interview with Graduate Student (Participant 13).docx\n\n• Interview with Graduate Student (Participant 14).docx\n\n• Interview with Graduate Student (Participant 15).docx\n\nFigshare: Analyzed Interview Transcript, https://doi.org/10.6084/m9.figshare.22191286.v2 (Sunardi et al., 2023b).\n\nThis project contains the following extended data:\n\n• Emotional Geographies fix.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 would like to acknowledge the Faculty of Teacher Training and Educational Studies, Universitas Mulawarman, Indonesia.\n\n\nReferences\n\nAdodo SO: Effect of Mind-Mapping as a Self-Regulated Learning Strategy on Students’ Achievement in Basic Science and Technology. 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Publisher Full Text" }	[ { "id": "299520", "date": "17 Jul 2024", "name": "Maria Olímpia Paiva", "expertise": [ "Reviewer 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, Here are some suggestions to make the article clearer, more objective, and of excellence:\nAbstract The objectives of the study should be more explicitly stated in the summary, as they are currently diluted between the \"Context\" and \"Method\" sections, and there should be a specific heading – \"Objectives\".\nIntroduction\n- Rewrite the underlined text to avoid repeating \"Hargreaves, 2001b\" too often: “Emotional geographies describe the patterns of closeness and distance in human interactions that shape the emotions we experience about relationships to ourselves, each other, and the world around us (Hargreaves, 2001b). Hargreaves (2001b) claimed that understanding teachers’ emotions is crucial because it can influence the classroom environment and the conduct of the learning activities.Hargreaves’ (2001b) emotional geography framework can also be used to map a teacher’s series of feelings. Hargreaves (2001b) defined five emotional geographies (i.e., sociocultural, moral, professional, physical, and political) as geographical and experiential patterns of closeness/distance in human connections that form, configure, and color feelings about ourselves, the world, and one another.”\n- As the article is based on Hargreaves' (2001) theoretical model of emotional geographies, it is essential that this model is further developed, along with its dimensions (socio-cultural, moral, professional, physical, and political). The author considers that teaching and learning are undoubtedly emotional practices, and consequently, the school space becomes an emotional geography. Hargreaves argues that such constructivist approaches to learning place greater emphasis on problem-solving and the application of knowledge, broadening the focus on creativity. In this context, emotional geographies describe the patterns of closeness and distance in human interactions, shaping the emotions we experience in our relationships with ourselves, others, and the world around us. Thus, emotion and cognition, feeling and thinking, intertwine in all social practices in complex ways.\n“However, the investigation of emotional geography of preservice teacher during the practicum has remained unseen by many scholars. Therefore, the present researchers want to fill this gap through this study” - First and third person references should be avoided. The information mentioned above should be addressed in the study objectives.\n“They have to deal with various settings involving students and the sociocultural contexts in the institution in which they are teaching (Riesky, 2013). This study revealed the difficulties preservice teachers experienced due to their emotional beliefs (Riesky, 2013).” Rewrite the underlined text to avoid repeating \"Riesky, 2013”.\n“A teacher’s experience of teaching can be affected by their relationships with the students (Hargreaves, 2000), parents (Hargreaves & Lasky, 2004), their colleagues (Hargreaves, 2001a), principals, and educational changes (Hargreaves, 1998a, 1998b), with the feelings from these relationships affecting teaching styles, judgments, and the conduct of the class learning activities.” Other authors' studies can be referenced in addition to those associated with Hargreaves.\nBefore presenting the empirical part of the study, it is suggested to briefly outline the study objectives.\nMethods The methodology should be guided by the headings \"Participants,\" \"Procedures,\" \"Instruments,\" and \"Data Analysis,\" under which all relevant information pertaining to each heading should be included.\nEthics This part concerning \"Ethics\" should be included under \"Procedures.\"\nParticipants Characterize the sample more comprehensively by including gender, years of service, age, and whether they work in public or private education, …\nResearch procedure The information in Table 1 is not easy to understand.\nDats analysis technique In the \"Data Analysis\" section, the \"Instrument\" used consists of the three mentioned stages (emotion coding, identification and/or interpretation of causes of reported emotions, and identification of themes based on emotional geography structure), based on Hargreaves (1998a). However, it is unclear how these three stages were adapted to the Indonesian context and what procedures were taken to validate these stages. The entire validation process and reliability of these three stages must be clarified to validate the study results effectively.\nLimitation and Implication Due to the known challenges in qualitative studies, it is suggested to propose additional considerations for future research to address the identified limitations, providing a more comprehensive and profound understanding of the emotional experiences of novice teachers and contributing to continuous improvement in teacher education. Examples to consider include: inclusion of various educational contexts; socio-cultural diversity; mixed data collection methodologies; longitudinal studies; enhanced communication tools; automatic recording and transcription; emotion analysis using AI tools; study of self-regulation strategies; assessment of pandemic impacts; hybrid teaching models; emotional support programs; and training in emotional self-regulation.\nConclusion Considering the importance of the topic, in addition to the conclusions presented, other relevant aspects for the training of future teachers can be considered. By incorporating these aspects into the training of future teachers, it is possible to prepare them in a more holistic and effective manner to face the challenges of contemporary education, promoting quality and student-centered teaching. Some suggested examples include: Development of Socioemotional Skills; Practices of Self-reflection; Integration of Educational Technologies; Continuous Professional Development; Diversity and Inclusion; Innovative Pedagogical Strategies; Collaboration and Teamwork.\n\nIs the work 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?\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": "12035", "date": "18 Jul 2024", "name": "Famala Eka Sanhadi Rahayu", "role": "Author Response", "response": "Thank you very much for your suggestions. I will definitely make an improvement based on what you suggested." } ] } ]	1	https://f1000research.com/articles/12-505
https://f1000research.com/articles/11-1413/v1	30 Nov 22	{ "type": "Genome Note", "title": "Draft genome sequences of three emerging beta-lactamase-producing Escherichia coli in the camel production system in Northern Kenya", "authors": [ "Rachael Gachogo", "Irene Karegi", "Brian Ogoti", "Victor Musyoki", "Dino Martins", "Frank Onyambu", "Joseph Kamau", "Irene Karegi", "Brian Ogoti", "Victor Musyoki", "Dino Martins", "Joseph Kamau" ], "abstract": "We report the draft genome sequences and annotation of three beta-lactamase-producing Escherichia coli (E.coli) strains isolated from fecal samples of healthy camels in Laikipia county, Kenya. This data adds to the online genome resources to support the ongoing antimicrobial resistance surveillance in the livestock-wildlife interface.", "keywords": [ "E.coli", "beta-lactamase", "AMR", "genome", "whole genome sequencing", "camel" ], "content": "Introduction\n\nAntimicrobial resistance (AMR), especially on the readily available beta-lactam antibiotics continues to threaten effective healthcare management and global economic success in livestock farming (Fashae et al., 2021; Kiiru et al., 2012). Recently, concerted efforts have been employed to explore the AMR situation following a One Health Approach. However, there still exists a gap in whole genome sequencing data on Escherichia coli (E.coli) harboring AMR genes in camels (Camelus dromedaries), a key component of livestock farming in arid and semi-arid regions of Northern Kenya.\n\n\nMethods\n\nHere, we report three draft genome sequences of beta-lactamase-producing E.coli isolated from camel fecal samples in Laikipia county, Northern Kenya. Fecal samples were collected from healthy camels reared under both ranching systems and pastoralism, transported in Cary Blair media (HiMedia Lab, Mumbai, India) and stored at 4oC awaiting processing.\n\nThe samples were cultured on MacConkey agar plate (HiMedia Lab, Mumbai, India) and incubated at 37°C for 18 hours. A single colony per MacConkey agar plate that displayed E. coli traits morphologically was identified through Gram staining and the IMViC (indol, methyl red, Voges Proskauer, and citrate) biochemical method and subjected to antimicrobial susceptibility testing (AST) against the beta-lactam antibiotic spectrum. Briefly, antimicrobial susceptibility testing (AST) was performed on Mueller Hinton agar (HiMedia Lab, Mumbai, India) using Kirby Bauer disk diffusion method. Antibiotics tested included, Ampicillin-10 μg (AMP), Chloramphenicol-30 μg (CHL), Tetracycline-30 μg (TCY), Gentamycin-10 μg (GEN), Streptomycin-10 μg (STR1), Trimethoprim-sulfamethoxazole-25 μg (SXT), Norfloxacin-10 μg (NOR), Ciprofloxacin-5 μg (CIP), Cefaclor-30 μg (CEC), Ceftriaxone-30 μg (CRO), Cefotaxime-30 μg (CTX), Cefuroxime-30 μg (CMX), Cefepime-30 μg (FEP), Amoxicillin–Clavulanate-20/10 μg (AMC), and Ceftazidime-30 μg (CA).\n\nProduction of beta-lactamase genes was assessed by conducting PCR to confirm genes encoding for CTX-M, TEM, CMY, SHV and OXA for the resistant isolates (Livermore et al., 2007). Briefly, a 20ul PCR reaction was prepared by adding 10ul of the Platinum II Hot-Start PCR Master Mix (Invitrogen, Carlsbad, CA, U.S), 0.4 ul of 10nmol of forward and reverse primers, 4ul of Platinum GC enhancer, 0.2ul nuclease free water and 5ul DNA. Cycling was performed on Veriti™ Dx 96-well Thermal Cycler (Thermofisher, Waltham, Massachusetts, U.S) using primers and PCR conditions as in Table 1. The respective gene fragment sizes were confirmed in 1% agarose gel (Invitrogen, Carlsbad, CA, U.S)\n\nGenomic DNA of beta-lactamase producing E.coli was extracted using Isolate II Genomic DNA Kit (Bioline) and sequencing performed using Oxford Nanopore Technologies (ONT). Briefly, manufacturer’s sample barcodes (Native Barcoding Expansion 1-12) (EXP-NBD104) and sequencing adapters (Ligation Sequencing kit) (SQK-LSK109)) were added following kit instructions. Sequencing was performed on MinION device using R9.4.1 flow cell to generate 186, 38153, and 31152 raw reads for the E.coli strains IPR_LC17, IPR_LC19 and IPR_LC20, respectively. Basecalling and demultiplexing of raw reads were performed using Guppy v3.6.1 (https://nanoporetech.com). Adaptors were trimmed using PORECHOP v0.2.4 (Wick et al., 2017) and poor quality reads, less than 500 bp and with an average quality score of below 10 were removed using NANOFLIT v2.8.0 (De Coster et al., 2018).\n\nE.coli PR_LC17 genome was assembled using Canu v2.2 (Koren et al., 2017) while E.coli (IPR_LC19) and E.coli (IPR_LC20) were assembled using Flye v2.9.1 (Kolmogorov et al., 2019). The taxonomy of the organism was confirmed using public databases for molecular typing and microbial genome diversity (PubMLST) (Jolley et al., 2018). Quality of the assembled genomes was assessed using QUAST v5.1.0 (Gurevich et al., 2013) and annotated using NCBI’s PGAP (Tatusova et al., 2016). The assembly metrics and annotation summaries are as shown in Table 2.\n\nThe project was approved by Institute of Primate Research (IPR) review committee (ISERC/10/2020).", "appendix": "Data availability\n\nIPR_LC20\n\nGenbank: Escherichia coli strain IPR_LC20, whole genome shotgun sequencing project, Accession number JAOZEZ000000000: https://www.ncbi.nlm.nih.gov/nuccore/JAOZEZ000000000.1/\n\nSequence Read Archive (SRA): Oxford Nanopore Reads of Escherichia coli in camel production systems in Northern Kenya, Accession number SRR21998714: https://www.ncbi.nlm.nih.gov/sra/SRR21998714\n\nIPR_LC19\n\nGenbank: Escherichia coli strain IPR_LC19, whole genome shotgun sequencing project, Accession number JAOZFA000000000: https://www.ncbi.nlm.nih.gov/nuccore/JAOZFA000000000.1/\n\nSequence Read Archive (SRA): Oxford Nanopore Reads of Escherichia coli in camel production systems in Northern Kenya, Accession number SRR21998715: https://www.ncbi.nlm.nih.gov/sra/SRR21998715\n\nIPR_LC17\n\nGenbank: Escherichia coli strain IPR_LC17, whole genome shotgun sequencing project, Accession number JAOZFB000000000: https://www.ncbi.nlm.nih.gov/nuccore/JAOZFB000000000.1/\n\nSequence Read Archive (SRA): Oxford Nanopore Reads of Escherichia coli in camel production systems in Northern Kenya, Accession number SRR21998716: https://www.ncbi.nlm.nih.gov/sra/SRR21998716\n\n\nReferences\n\nDe Coster W, D’Hert S, Schultz DT, et al.: NanoPack: Visualizing and processing long-read sequencing data. Bioinformatics. 2018; 34(15): 2666–2669. PubMed Abstract \| Publisher Full Text\n\nFashae K, Engelmann I, Monecke S, et al.: Molecular characterisation of extended-spectrum ß-lactamase producing Escherichia coli in wild birds and cattle, Ibadan, Nigeria. BMC Vet. Res. 2021; 17(1): 12–33. PubMed Abstract \| Publisher Full Text\n\nGurevich A, Saveliev V, Vyahhi N, et al.: QUAST: Quality assessment tool for genome assemblies. Bioinformatics. 2013; 29(8): 1072–1075. PubMed Abstract \| Publisher Full Text\n\nJolley KA, Bray JE, Maiden MCJ: Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications [version 1; referees: 2 approved]. Wellcome Open Research. 2018; 3: 1–20. PubMed Abstract \| Publisher Full Text\n\nKiiru J, Kariuki S, Goddeeris BM, et al.: Analysis of -lactamase phenotypes and carriage of selected -lactamase genes among Escherichia coli strains obtained from Kenyan patients during an 18-year period. BMC Microbiol. 2012; 12(1): 1. PubMed Abstract \| Publisher Full Text\n\nKolmogorov M, Yuan J, Lin Y, et al.: Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 2019; 37(5): 540–546. PubMed Abstract \| Publisher Full Text\n\nKoren S, Walenz BP, Berlin K, et al.: Canu: Scalable and accurate long-read assembly via adaptive κ-mer weighting and repeat separation. Genome Res. 2017; 27(5): 722–736. PubMed Abstract \| Publisher Full Text\n\nLivermore DM, Canton R, Gniadkowski M, et al.: CTX-M: Changing the face of ESBLs in Europe. J. Antimicrob. Chemother. 2007; 59(2): 165–174. PubMed Abstract \| Publisher Full Text\n\nTatusova T, Dicuccio M, Badretdin A, et al.: NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016; 44(14): 6614–6624. PubMed Abstract \| Publisher Full Text\n\nWick RR, Judd LM, Gorrie CL, et al.: Completing bacterial genome assemblies with multiplex MinION sequencing. Microbial Genomics. 2017; 3(10): e000132. Publisher Full Text" }	[ { "id": "157281", "date": "04 Jan 2023", "name": "Meenakshi S. Iyer", "expertise": [ "Reviewer Expertise Genomics", "Transcriptomics", "Anti-microbial resistance", "Protein sequence-structure-funcion relationships", "Database and algorithm development", "Big-data analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript the authors present a genome sequences of three E. coli strains isolated from camels in Northern Kenya. The importance of studying drug-resistant bacteria from livestock is obvious and these sequences are of course a very useful resource for the community. However I have a request for minor revisions. I found that the paper needs to address a few more points:\nThe authors need to provide more background on the need for sequencing E. coli samples from camel populations. There are some recent studies on beta-lactamase producing isolates from camle and other populations from Kenya. These studies (and more) can be referenced: Karegi I et al., 2022 DOI:https://doi.org/10.1016/j.ijid.2021.12.018; Anyanwu MU et al., 2012 https://doi.org/10.1155/2021/6630379; and Nueesch-Inderbinen M et al., 2020 https://doi.org/10.1016/j.sciaf.2020.e00274.\n\nThe results can be recorded in a separate section. Table 2 on genome annotation statistics can be moved to this section.\n\nThe results for the antibiotic susceptibility testing for the three isolates can be provided as a separate table.\n\nThe results of the PCR reactions for the 3 isolates describing which plasmids were detected from the samples need to be provided. These results can be corroborated with existing literature on the beta-lactamase encoding plasmid prevalence in Africa.\n\nIn Table 2, the statistics for partial genes detected in each strain can be listed. Overall, the authors can comment on the draft nature of the genome in the results section (% completeness, number of contigs etc.).\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": "162944", "date": "02 Mar 2023", "name": "Janet Midega", "expertise": [ "Reviewer Expertise Genomic epidemiology", "Microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide details of a study in which beta lactamase producing Ecoli from fecal samples are sequenced from Camels in Northern Kenya. Whilst this is an important study given the close interactions between human and camels in this part of Kenya, hence the likelihood of Animal to human transmission of beta lactamase producing E coli, It might help if the authors can write down a justification for why the study was conducted.\nWhy was there a focus on beta-lactamase producing E coli only? For example is it the most important isolate in this (human and camel) population?\n\nIn total how many camels are represented in the study or were these random fecal samples?\n\nIs it possible to be more precise about the location, for example provide GPS co-ordinates of the sampling locations - A county is a very wide area.\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/11-1413
https://f1000research.com/articles/12-502/v1	16 May 23	{ "type": "Research Article", "title": "Determinants of technical efficiency of smallholder farmers’ bread wheat production and implications of seed recycling: A stochastic frontier approach", "authors": [ "Yirgalem Eshete", "Bamlaku Alamirew", "Bamlaku Alamirew" ], "abstract": "Background: Due to decreasing productivity trends, enhancing farm level technical efficiency (TE) in bread wheat production (BWP) is central to Ethiopia's food security strategy. This requires increased BW productivity through the use of improvements in seeds and TE. However, there is some empirical research that examines the institutional, socio-demographic, economic, and agronomic determinants of the TE of BWP simultaneously. Therefore, the purpose of this study was to analyze determinants of TE and the implication of seed recycling on TE. Methods: The data was gathered from 450 randomly selected sample respondents via structured interviews, focus groups, and interviews with key informants. Data collection took place from December 15, 2021 to January 15, 2022. BW growing smallholder farmers were the major respondent of the study. Quantitative type of data including the number of inputs used, amount of wheat produced, and socio – demographic, economic, agronomic, and institutional variables were gathered and analyzed. The TE and factors affecting it were examined using a stochastic frontier production analysis with a trans-log functional specification. Results: The coefficients for labour (0.086), capital (0.22), fertilizer (0.042), weedicide (0.014), and machinery (0.005) were significantly positive at p<0.001 implying that an optimum increase to these inputs would increase BWP. The mean TE was found to be 79.99% indicating that farmers in the study operate 20.01% less than their potential. Nine of the hypothesized variables (69.2%) had significantly affected TE. Moreover, seed recycling reduced TE by 2.34%. Conclusion: Findings suggest that few farmers are able to grow BW either on the frontier or very close to it, which denotes high TE and poor access to improved seeds leading to seed recycling. Therefore, the farmer-to-farmer (F2F) extension method, which enables farmers to learn from their neighbors and create wider access to improved seeds through seed loans and vouchers are strongly suggested.", "keywords": [ "Agriculture", "Food Security", "Recycled seed", "Translog", "Technical Efficiency", "Inputs", "F2F (Farmer-to-farmer)", "Frontier" ], "content": "Introduction\n\nAgriculture has continued to be a crucial economic sector in developing countries for promoting growth, eliminating poverty, and providing food security. In Ethiopia, agriculture accounts for 41% of the country's GDP, 70% of the nation's industrial raw materials, 85% of the nation's food supply, and 80% of all jobs.1 However, this sector is characterized as low-tech, rain-fed, subsistence-based, and very conventional. Crop production plays a key part in both agriculture and the country's overall economic performance. It is the crop production sector's poor performance that has kept the country under vicious poverty cycle for generations.1–4 Therefore, increasing agricultural yield is not only essential but also a requirement for ending the vicious poverty cycle that has gripped the majority of people in least developed countries (LDCs) for millennia.5–8\n\nWheat has been widely grown in Ethiopia because of its many advantages and the country's good climatic circumstances. In Ethiopia, the total cereal production during the 2020/2021 production season was reported to be 302,054,260.6 quintals and wheat contributes 19.14% (57,801,306.00 quintals) of the total cereal production.9 In East Gojam Zone, the total cereal production is reported to be 17,095,145.90 quintals and contributes 27.98% (4,743,813.9 quintal) of the total cereal production in the zone.10 The percentage contribution of wheat to the total cereal production in the East Gojam zone (27.98%) is relatively high as compared to the regional (18.89%) and national (19.14%) averages.10 The national average wheat productivity was reported to be 30.46 qt ha-1 against 28.31 qt ha-1 for Amhara region. The average wheat productivity for the East Gojam zone was reported to be 32.12 qt ha-1 which is relatively higher than both the national and regional average.9 This suggests the significance of wheat production for the farmers livelihood in the research area. Ethiopia continues to be a net importer of wheat. It has been using imports to meet about 25% of its domestic demand. Wheat is the only grain that is nearly always imported into Ethiopia.11 This proves the importance of wheat as a researchable agricultural commodity in Ethiopia.\n\nThe Government of Ethiopia (GoE) in collaboration with its partners has been doing its level best to bridge the potential and observed productivity gaps through wider diffusion and scaling-up of improved seeds.12–16 Dissemination of agricultural technologies will not substantially improve productivity if farmers are not technically efficient. Technical efficiency (TE) in crop production refers to farmer’s ability to maximize outputs given a set of inputs and technology. Similarly, the degree of technical inefficiency reflects an individual farmer’s failure to attain the highest possible output level given the set of inputs and technology used.17,18 Improving productivity and TE of wheat production is at the center of researchers and policymakers in Ethiopia. This is due to the urgent need to improve the food security and livelihood situations of smallholder farmers. There are sufficient empirical evidences about production improvement as a result of new genetic and breeding innovations.19,20\n\nHowever, available literature with regard to TE tend to be quite generic.21–26 As a result, farmers in Ethiopia don’t know the exact amount of inputs (land, labour, oxen power, fertilizer, seed, capital, etc) that go into the production of a particular commodity or crop in which case a generic TE analysis may be sound. Yet, such generic analysis has the potential to hide crop specific and relevant information for policy uptake. This means that if care is taken in generating empirical evidences, doing crop – specific efficiency analysis may bring policy relevant information. This study therefore aimed at generating policy relevant information for bread wheat production (BWP) through TE analysis by estimating farmers level of technical inefficiency and its determinants. Moreover, about 90% of the farmers in Ethiopia rely on multiple times recycled seeds for bread wheat production. However, bread wheat seed recycling beyond five production seasons was considered as unproductive and unwise practice as continuous recycling of the same seed causes deterioration in seed quality. Yet, farmers in the developing world and Ethiopia are continuously using the same seed over several production seasons.5,27,28 The nexus between TE and bread wheat seed recycling has never been empirically investigated. As a result, farmers are continously making use of recycled seeds without knowing the implications of seed recycling on their TE. This study therefore has dealt with the nexus between seed recycling practices and TE of smallholder bread wheat producers. It has also aimed at investigating the determinants of farmers technical inefficiency.\n\n\nMethods\n\nThe proposal for this study was first reviewed and approved by the academic comission (AC) of the Center for Rural Development Studies during the 2020 academic year which is before the approval of the standard operation procedue (SoP) of the Addis Ababa University. The authors received retrospective institutional review board (IRB) approval for this study where the ethical review board of the Center for Rural Development Studies of the AAU reviewed the contents of the proposal, the associated research tools, and informed consents of the respondents retrospectively. As a result, the University found the proposal to be qualified for ethical clearnace and issued an IRB certificate dated on February 18, 2023 with an IRB number 002/02/2023 indicating that the proposal fulfills the standard requirements described in the SoP of the Addis Ababa University, College of Development Studies.\n\nThe trained enumerators of the study have properly read the consent form for the participants. The consent form clearly contains the objectives of the study and their participation is on voluntary basis that they are free to disengage if they are not comfortable. Participants were told that there is no direct benefit that will be provided for them as a compensation for the data that they are going to provide for the study. They were also told that the data collected will be published to inform policy makers and the research community and will not be used for any other purpose. Besides, they were informed that the analysis of the study did not identify participant individuals and will be anonymized by assigning a unique identification number. Yet, a significant number of participants have refused to put their signature on the consent form. This is due to the fact that the long held tradition and culture of the participant farmers’ don’t allow them to put their signature on studies like this. Besides, a substantial portion of the participants are incapable of reading and writing to sign on the written consent. Traditionally, farmers of the study area are skeptical in providing written consent. After assessing this situation and the minimal risk that the study may cause to human subjects, the ethical review board has confirmed the documentation of written consent can be waived. Thus, the data collection took place only when respondents of the study gave their consent at the beginning of each interview. As a result, this study has relied on verbal consent of the participants.\n\nThe common difficulty in collecting data from poor farmers’ in the rural setting is that farmers may feel that they will not be targeted for aid, credit schemes, welfare packages, and other development interventions based on the data they unfold to outside researchers. Fearing such vulnerability to exclusions, they often under report their annual production, plot size, number of animals, inputs used, and assets owned. On the contrary, farmers may also exaggerate certain variable values such as their age, family size, and number dependent family members hoping that they will be entitled for aid and other welfare benefit packages. Yet, the authors make sure that such participants expectation did not harm the quality of the data by employing different methods. First, most of the variables that could be under reported or exaggerated are registered by the local government. Thus, we have triangulated the reported values with what is locally registered. Second, we have recruited enumerators (development agents) working with the local community who have long years acquintance of the villages and the community members that participants don’t often hide information. The other challenge when collecting data using FGDs is that participants farmers’ may not feel comfortable to share their experience with development agents who often support them by providing inputs and agricultural advisory services. With the intention of avoiding this potential problem, the facilitation and transcription of the FGDs were made by the authors themselves than the local development agents.\n\nThis study was conducted among smallholder bread wheat growers in selected districts of the East Gojam zone of Ethiopia. There are two major crop growing seasons in the study areas. The major crop growing season is called ‘Meher’ in Amharic extends from May to September. The second and the shorter crop growing season is called ‘Belge’ extends from February to April. Smallholder farmers of the study areas culturally and historically grow bread wheat during the main season for several reasons. Lack of access to irrigation for all season production and the availability of sufficient rainfall during the main rainy season are the major reasons to heavily rely on ‘Meher’ wheat production. Agronomic recommendations such as crop rotation also can be cited as important reason to grow bread wheat during the main season only. Thus, we have considered smallholder farmers who grow bread wheat during the main production season. The list of bread wheat growers during the 2021/2022 major production season was obtained from the farmers’ training centers’ (FTCs) of the selected villages. These farmers grow bread wheat using different varieties that were introduced at different points in time. However, this study has considered a bread wheat variety largely grown by the dominant section of the farmers. Respondents were further stratified into fresh and recycled bread wheat seed users based on their experience in seed recycling. Besides, the data was gathered during the weekends and holidays during which farmers were not busy to provide information required for the study. Production inputs and agronomic practices applied by the respondents are important variables of this study. Thus, we intentionally undertook the data collection effective from December 15, 2021 to January 15, 2022 during which wheat will be harvested.\n\nThis study takes place in the East Gojam Zone of Ethiopia. East Gojam zone has a total of 18 Woredas and its headquarters, Debre Markos, is located 300 and 260 km far from Addis Ababa (the capital city of the country) and Bahir Dar (the capital city of the region), respectively. It is located at 10° 19' 60.00\" North, and 38° 00' 0.00\" East. The total population size of the East Gojam zone is estimated to be 2,358,051, out of which 1,154,740 and 1,203,311 were reported to be male and female respectively. Mixed farming system is the main occupation of farmers in the East Gojam zone. The area is dominated by mixed agricultural systems.29 Out of the 18 Woredas located in the East Gojam Zone, this study has especially considered Baso Liban and Debre Elias districts presented in Figure 1.\n\nSampling technique\n\nThis study has followed a multi – stage systematic random sampling technique in the selection of sample districts and respondents. The study districts and kebeles [1] were selected purposively. The potential for wheat production, absence of sufficient prior studies, availability of sufficient representative sample respondents who make use of similar bread wheat variety, and the long year’s acquaintance of the study places by the researcher were the reasons to consider these Woredas. Debre Elias and Baso Liban districts have 15 and 22 kebeles, respectively. The researcher has decided to consider eight kebeles (around 22% of the total kebeles) from both of the districts with strong conviction that these kebeles are sufficient and representative due to their homogeneity in agro-ecology, agronomic practices, and potential for BWP. As indicated in Figure 1, Yelemelmo, Lemechem, Dendegeb, Kork, and Dogemo kebeles were considered from Baso Liban district while Guay, Yekegat, and Debre Elias zuria kebeles were considered from Debre Elias district. The number of kebeles from each Woreda was determined using the probability proportional to size (PPS) formula specified below (Equation 1):\n\nWhere ni is the number of kebeles from each Woreda, n is total sample size of the study kebeles which is the sum of the sample size of the kebeles from the two Woredas (eight in this case), Ni is the total number of kebeles in each Woreda and N is the total number of kebeles in both Woredas (37 in this case). The specific sample kebeles were selected in collaboration with the development agents (DAs) based on their BWP potential and availability of farmers who make use of similar improved bread wheat production (IBWP) variety. Yamane’s sample size determination formula is often used in studies where the study population is finite and known. Henceforth, the total sample size of this study was also determined by using Yamane’s sample size determination formula,30 which is specified as below (Equation 2).\n\nn=1+N1+N(℮2)n=1+N1+N(℮2) Where n is the desired sample size; N is total number of population and, e = the level of precision or the quality of being careful and accurate which is equal to 0.05. Generally, the sample size for this study was 399 along with a 15% contingency which adds up to 459 total households. The analysis of the study has considered only 450 respondents as three of the respondents refused to provide information while six of the questionnaires collected were incomplete for analysis.\n\nSampling procedures and participants selection\n\nThe chief intention of this study was to investigate the major determinants of smallholder farmers TE in bread wheat production. Thus, we have first obtained a list of smallholder farmer bread wheat growers in the selected sites from the FTCs. From the list of bread wheat growing, it was found that 83.5% of the smallholder farmers grow bread wheat using a bread wheat variety called ‘Ogolecho’. Since agronomic practices, input use, and productivity is likely to vary by the nature of bread wheat variety, we have included farmers that make use of the same variety. Thus, farmers who make use of varieties other than ‘Ogolecho’ were excluded from the study. Besides, as the study deals with the implications of seed recycling on smallholder farmers TE, the respondents were further disaggregated into fresh (unrecycled) and recycled bread wheat seed users. Farmers who produce bread wheat using seeds reused more than five times were labeled as recycled seed users while those who produce bread wheat using fresh/unrecycled seed or recycled below or five times were considered as fresh/unrecycled seed users. It was found that about 247 (54.9%) of the respondents were recycled seed users while 203 (45.1%) were found to be fresh seed users. Figure 2 depicts the sampling procedures. These sample respondents were used for the face – to – face household interview using a structured interview schedule. Thus, bread wheat growing households were the unit of analysis under the current research.\n\nWe believe that key informant pluralism or diversity from different background or sector will allow us to capture varying perspectives about the determinants of smallholder farmers TE and problems of seed recycling. As a result, we have included key informants from different walk of life and responsibilities in relation to bread wheat production. Doing so has helped us to avoid a one – sided or biased information that may result by interviewing people from a particular division. We have first prepared a long list of potential key informants from stakeholders or actors involved in the value chain of bread wheat production. Then, we have narrowed down the list into the most relevant and knowledgeable informants. Hence, the sample respondents for the key informant interviews (KIIs) were purposively selected based on their experience and knowledge to the subject under consideration. Development workers, agronomists, seed experts, researchers, district bureau heads, seed suppliers, agro-dealers, and seed multiplication and marketing cooperative members who do have ample information due to their position and expertise were selected as key informants. These informants were considered as they are the most important actors across the value chain of bread wheat production. They are involved either in the inputs supply, business development support provision, organize the transport and marketing of agricultural inputs and products. The authors believe that inclusion of these individuals as key informants will assist to capture information that may be obscured during the quantitative data collection. These key informants were knowledgeable people who can talk on behalf of the farming community about the subject matter. These key informants were physically visited at their offices for the interview. These informants were busy experts and office position holders. Thus, we received appointments before the visit for the interview was made. The profile of the KII participants is provided in the supplementary table 1.31\n\nEfforts were made to compose the FGD discussants from the homogenous group that have some natural characterisitcs in common such as gender, residential locations, and seed recycling practices so that to ensure participants feel comfortable speaking in the group. We have followed two approaches in composing the focus discussion groups. First, we made sure that there is sufficient homogenity between groups so that to have comparable data among the groups. This approach has encouraged sense of uniformity and conformity among FGD particpants and assisted participants to reach on some consensus on issues raised. Second, we make sure that there is sufficient diversity within the groups. This has helped us to bring together participants that assume different roles, varying experiences and dissimilar backgrounds that may end – up in unexpected views and results. The selection the FGDs participants has involved organizing group of community members based on important criteria relevant to the objectives of the study and their consent to takepart in the discussions. The first criteria in grouping discussants was bread wheat seed recycling practice. In this regard, we had two group of FGD discussants that include fresh and recycled bread wheat seed users. The second group of the FGD discussants were composed of female and male headed households. The third group was composed of smallholder farmers who are member of improved bread wheat seed multiplication and marketing cooperatives.\n\nData types and sources\n\nThis study has employed both quantitative and qualitative data types. Quantitative type of data including the number of inputs used in bread wheat production along with different agronomic practices such as amount of fertilizer, man - days, seeds, plot size, amount of capital invested, amount of pesticide applied, weeding frequency, seed recycling frequency, and spacing between rows were gathered. The study has also considered different socio – economic and institutional quantitative variables such as gender, age, grade level, family size, dependency ratio, tropical livestock unit (TLU), land size, frequency of extension contact, amount of credit, and membership in seed multiplication and marketing cooperatives, respectively. The qualitative type of data included smallholder farmers knowledge and perception towards seed recycling, reasons for seed recycling, perception about the impact of seed recycling, barriers affecting seed replacement, and major challenges affecting technical efficiency and bread wheat productivity. These study variables were selected after extensive review of relevant literature, pre – data collection discussions made with experts and elders in the study districts, and long years acquaintance and experience of the researchers about the issue under consideration. These data were gathered both from primary and secondary data sources. Smallholder farmers, district development workers, agronomists, and subject matter specialists were the primary data source of this study. In addition, secondary data sources such as published research findings, journals, articles, statistical reports, official websites, government reports, dissertations, and thesis were consulted to triangulate and support the information obtained primary data sources. A systematic desk review was conducted to gather relevant information related to the objective of the study. The authors have heavily relied on important secondary data sources related to the technical efficiency of improved bread wheat production and its determinants including PubMed, Semantic Scholar, and Google Scholar platforms. Two typical search phrases, separated by the operator “OR,” were used: “technical efficiency” AND “bread wheat production in Ethiopia” or “technical efficiency” AND “determinants in Ethiopia.” We have applied this with the expectation that it will increase the likelihood that all studies conducted in Ethiopia would be found. For the systematic review, the authors have applied various articles inclusion and exclusion criteria including the following.\n\na) Articles concentrating primarily on technical efficiency or technical efficiency of bread wheat production were considered while articles related to other wheat or crop varieties were excluded from the review.\n\nb) Articles evaluating the seed recycling practices of smallholder farmers and/or determinants of the TE of smallholder farmers were included.\n\nc) The authors have focused on publications based on qualitative and quantitative empirical investigations.\n\nd) The authors also focused on articles where the country of data collection is in Ethiopia and/or in the Sub – Saharan Africa.\n\ne) The systematic desk review have emphasized articles published in scholarly journals and non-published documents such as government policies and strategies, agronomic training manuals, annual reports, and location specific agronomic recommendations.\n\nThe evidences obtained from these secondary sources were then checked against the findings of the current study. The authors have critically triangulate the data and findings between the current study and the secondary empirical evidences. We have further assessed the reasons and explanations for convergence, divergence, and complementarity of the data and findings between this study and the secondary data sources. Explanations were sought whenever we found strong degree of deviation between this study and the secondary sources. In general, the areas of divergence and convergence were further discussed.\n\nData collection methods\n\nFor a thorough understanding of the research problem, the study has used data collection methods employed in a mixed research design, which blends qualitative and quantitative research data. A mixed research design was used in order to compare the various viewpoints obtained from quantitative and qualitative data, as well as to demonstrate potential data convergence and divergence by combining the two database. We have applied convergent parallel mixed methods to gather quantitative and qualitative data, analyse each kind individually, and then compare the findings to see whether they support or refute one another. The main premise behind our preference for this approach is that both qualitative and quantitative data offer distinct forms of information. Authors apply this approach for the purpose of triangualtion and validation of findings from each method. As a result, the data for this study were collected using three important quantitative and qualitative data collection methods described below.\n\nTools translation and piloting\n\nThe tools used for both the quantitative and qualitative data were created in the English language based on extensive relevant literature reviews and discussions held with subject matter specialists. These tools were then translated into Amharic language using a group translation approach.31 The tools were given to two different certified and hired translators who independently translate the tools (meaning they don’t review each other’s translation). After the two translators complete the translation, a third translator has reviewed and reconciled the translation differences and produced a final translation. All the tools translated by all of the translators were submitted to the authors of this research and checked for their originality, meaning, and consistency. All of the tools used in this study incorporates informed consents that specifies the objectives of the study and seeks the consent of all of the respondents. The data collection was carried out under a complete private setting and keeps the principles of data confidentiality, privacy, and informed consent. Eight hired and trained enumerators were used for data collection. The training was provided by the corresponding author. The training encompasses line – by – line discussion of each questions, research ethics, and interviewing methods. Before starting the main survey, each enumerator was allowed to collect data from four randomly selected farmers that were expected to have similar characteristics with the sample respondents of the study. A debriefing session was then held after this field level pilot test and some modifications were made on the tools based on findings from the debriefing session. Finally, the hired enumerators were deployed to collect data under strict supervision of the corresponding author.\n\nQuantitative data collection method\n\nA structured questionnaire based interview was largely used as quantitative data collection method. The questionnaire used for the structured interview included questions with close ended response options only. The questions included in the structured interview were crafted in such a way that it enable us to capture the objectives of the study. Enumerators of the study were given the list of respondents identified using the procedures indicated above. Then they systematically select target respondents from the list at a regular interval to conduct a face – to – face interview using the structured questionnaire. The enumerators visit the selected household at his/her residential place for the interview. Enumerators of the study were trained to make several contact attempts for respondents who can’t be found at his/her residence for the interview. Yet, the maximum contact attempt was only three that all of the sample respondents were contacted for the interview during the first visit of the interviewers. The questions as well as their order was already scheduled that enumerators ask the questions accordingly and without change in the wordings of the questionnaire. The interview was conducting using the structured questionnaire detailed above that was translated into the local language (Amharic). The structure questionnaire both in English and Amharic along with the consent statement is available as extended data for this study.31\n\nQualitative data collection methods\n\nThis study has employed key informants’ interview (KIIs) and focus group discussions (FGDs) to gather qualitative data required to capture the objectives of the study. KIIs were carried out to get an in-depth understanding of the different issues pertinent to seed recycling, technical efficiency, and challenges related to bread wheat production. The KIIs were conducted with respondents such as development agents, subject matter specialists, researchers, and improved bread wheat seed suppliers who are believed to have ample information about the issues under consideration of this study due to their position, expertise, and leadership. A total of 14 key informants composed of four women and 10 men were approached for a face – to – face interview using the KII checklist. The interview was conducted at the office of each key informant. Appointments were taken through phone calls before visiting the key informant at his/her office so that they could have sufficient time to provide detail information. The data collectors approached and introduced themselves for the key informants and read the consent statement. After reading the consent statement, enumerators put a tick mark next to the option ‘yes’ to indicate that the respondent has verbally provided his/her consent to continue the interview. No key informant has declined to take part in the interview and his/her responses are recorded. The author himself was a note taker while a trained enumerator pose the questions from the KII checklist and facilitate the interviews. The data collection was conducted using a local language. The information obtained from the key informants was later transcribed by the authors. For the sake of confidentiality and privacy, pseudo names were used to identify the name of the KIIs participants. The maximum amount of time to finish the KIIs was 31 minutes.\n\nFGDs were conducted to examine the existence of consensus among the different group of discussants on certain common agricultural practices such as seed recycling, planting methods, spacing, and other agronomic practices. Besides, these qualitative data collection methods were employed with the intention of triangulating the data obtained from the quantitative survey. Issues raised for the FGDs were reasons or pushing and pulling forces for seed recycling, farmers perception on the consequences of seed recycling, knowledge and awareness of factors hampering or promoting their technical efficiency in bread wheat production.\n\nAs indicated in supplementary table 2, a total of six FGDs (three in each district) were conducted.31 All FGDs were properly recorded, transcribed, and translated into English by the corresponding author of this study. The highest group size for the FGDs was nine and a total of 49 discussants have participated in all of the FGDs. The FGDs were conducted at the FTCs assembly halls of each village. The date and the time for the FGDs was fixed to be on Sundays at 10:00 PM in consultation with the participants. Discussants prefer Sunday as it is observed as sabbath by the community that they don’t engage in other activities. The assembly halls where the FGDs took place were large enough with sufficient ventilation and lighting. It has allowed us to keep more than two meters distance between discussants so as to avoid risks of the COVID-19 spread. We also distributed face masks to participants to contain the spread of the virus. The group sitting was arranged in a circular setting so that all participants have a balanced eye contact with each other and can listen to what each other speaks. FGD was set up where each discussant of the FGDs were encouraged to feel free to speak on the issues raised by the facilitator. As a standard ethical procedure, we never keep personal identifying information such as names either in the notes or in the report. Rather we have used pseudo names so that the study doesn’t lose its human sense. Female facilitators and note takers were assigned in the female – headed group of FGD discussants so that participants will openly discuss questions posed to them. The composition of the FGD participants along with their characteristics is summarized and presented in the supplementary table 2.31 The data collection took place starting from September 15, 2022 to October 19, 2022. The longest discusstion time was 72 minutes while the shortest was 61 minutes. This could imply that there is a relatively uniform level of discussion across the FGDs.\n\nThe data collected for this study were analyzed using both descriptive and econometrics models. One stage Stochastic frontier analysis (SFA) with the translog functional form was used in identifying the factors significantly contributing to BWP and in estimating the contribution of the hypothesized parameters on the technical inefficiency. The analysis was made using the STATA Software, Version 17 (https://www.stata.com/). R software (https://posit.co/download/rstudio-desktop/ which is an open source and freely accessible software, is also capable of the same analysis used in this study. The sub-headings below are a summary of the data analysis methods employed in this study.\n\nDescriptive and inferential statistics\n\nThis study has frequently employed a number of descriptive statistics. It has used both measures of central tendency (mean, median, and mode) and measures of central dispersion (range, variance and standard deviation). Inferential statistics such as t-test and χ2 tests were frequently employed to check if there exists a difference in the different continuous and categorical variables considered in the study between farmers who recycled bread wheat seed and those who don’t recycle (use fresh) seed, respectively.\n\nTechnical inefficiency model of the SFA\n\nThe SFA, a parametric technique, and the data envelopment analysis (DEA), a non-parametric approach, are important methods for estimating the level of efficiency/inefficiency in production economics. The DEA is preferred because it accommodates multiple inputs and outputs in TE analysis. Nonetheless, DEA disregards the potential effects of random shock, such as measurement error and other sorts of data noise. The SFA, on the other hand, does not accommodate multiple inputs and outputs and is more susceptible to misspecification problems. The SFA, however, boosted its application in the examination of TE of agricultural production since it included stochastic components in the model.32–34 Thus, this study has opted for the SFA than the DEA.\n\nAgricultural production in developing countries is contingent upon factors that are beyond the control of the decision making units (DMU). External factors such as crop pests, disease infestation, drought, flood, frost/hail, and other exogenous factors which are beyond the control of the smallholder farmers do have significant effect on smallholder farmers’ crop production. Therefore, studies are expected to capture the effects of such uncontrolled variables. Moreover, studies are expected to account for technical errors that may arise due to measurement and observation during data collection. BWP in the study areas is reported to be hampered by the climatic variabilities that are least controlled by smallholder farmers’.18,34–41 In addition, sample respondents of this study were non-commercial and subsistence smallholder bread wheat growers’ who largely depend on family labour to operate their farm, and don’t have a tradition of keeping accurate records. Above all, the data for this study were collected using local units of measurement that their conversion into standard units of measurement may be erroneous. Thus, the data for certain variables like production, plot size, and amount of inputs used are likely to be susceptible to measurement and conversion errors.\n\nGiven the situations above, this study therefore adopts the SFA model as it enable the researcher to accommodate the effects of these uncontrolled variables and technical errors. The SFA was first proposed by Aigner, Lovell42 and Meeusen and Broeck.,43 and later modified by Jondrow, C. A. K. Lovell.33 The SFA model has also been used to model the technical efficiency effects of other observable explanatory variables.44 This approach splits the error-term (deviations) into two parts to capture purely random factors and are beyond the control of the DMUs. The first component is the technical inefficiency of the DMUs. The second component is the random shocks that among others include climatic variabilities, errors in measurement during data collection, and omitted variables. Therefore, the general stochastic frontier econometric model for this study is specified as below (Equation 3):\n\nWhere ln - refers to the natural logarithm, i - refers to the ith farmer in the sample (i = 1, 2, … .N), γi – refers to bread wheat yield (output) of the ith farmer, Xij – refers to the farm inputs used by the ith farmer, β0 – refers to the constant term, βi - represents the regression coefficient, while ϵi denotes the residual random error term which is composed of the following two elements (Equation 4).\n\nVi is a symmetric component that captures/accommodates the random error associated with random factors which are beyond the control of the DMUs such as weather variabilities like drought, frost/hail, flood, omitted variables, measurement errors, and other exogenous shocks. Ui is the one-sided component that capture deviations associated with factors under the control of the DMU (i.e., inefficiency). The model is such that the possible production Υi, is bounded above by the stochastic quantity, fxiβ* expVi; hence the term stochastic frontier. The random errors (Vi), where i = 1, 2 … N were assumed to be independently and identically distributed as N (0, σv2) random variable independent of the Ui′swhich were assumed to be non-negative truncations of the N (0, σu2) distribution (i.e. half normal distribution) or have exponential distribution. As far as TE is concerned, the variance parameters, σv2 & σu2 are of critical importance in this model. They are expressed as follows (Equations 5 and 6):\n\nWhere σ2 = overall wheat output deviations, ϒ is the ratio of farmers’ wheat output deviations due to technical inefficiency to the overall deviations. It ranges from 0 to 1, when ϒ=0 indicates that all output deviations are due to factors outside the farmers control (σu2= 0, thus σ2 =σv2), when ϒ = 1 indicates that all deviations are due to technical inefficiency (σv2 = 0, thus σ2= σv2). The TE of an individual farmer considered in this study can be defined as the ratio of observed or realized (actual wheat output) to the stochastic frontier output (potential output). The stochastic frontier output is the maximum output possible given the technology available and inputs used, it is given by (Equations 7 and 8):\n\nWhereTEi is technical efficiency, the inefficiency term Uiis always between 0 and 1, When Uiis equal to zero, then production is on the frontier,qi∗= expxiβ+Viand TEi=1, therefore a farmer is technically efficient. When Ui is greater than zero (Ui>0) the farmer is technically inefficient (TEi<1) since the production will be below the frontier. Factors affecting TEiwas previously estimated by using a two-stage process. In the first stage, the level of efficiency/inefficiency was measured using a normal production function. In the second stage, a probit/logit model was used to investigate the factors that determine levels of technical efficiency. However, since 2000, STATA, Limped, and Frontier computing packages which apply the Maximum Likelihood Estimate (MLE) are available which has enabled researchers to jointly estimate the Stochastic Frontier and the inefficiency model.17,40,45 Therefore, this study has employed a single stage approach where TE and its determinants are estimated together.\n\nEmpirical model specification of the production function\n\nThe SFM can take either the Cobb-Douglas (CD) production function or the Translog (TL) production function. The CD production function imposes restrictions on the farm’s technology by assuming constant production elasticities and setting the elasticity of input substitution to unity.32,46 In addition, it assumes a fixed return to scale and a linear relationship between the output and inputs used in production. The TL production function, however, assumes existence of nonlinear relationship between the output and inputs, and production elasticities are not constant. Researchers prefer the TL functional form due to its flexibility and does not impose assumptions about constant elasticity of production nor elasticities of substitutions between inputs.36,46,47 Thus, this study too opt for the stochastic frontier production function using the flexible TL specification and is specified as follows (Equation 9):\n\nWhere qi = wheat output per hectare of the ith respondent, ln=the natural logarithm, β0= Constant term, βi= regression coefficient of the ith variable, X1i= amount of labour (man-days ha-1) by the ith respondent, X2i= amount of capital for wheat production per hectare by the ith respondent, X3i= amount of total fertilizer applied (kg/ha) by the ith respondent, X4i= amount of land allotted for BWP (ha) by the ith respondent, X5i= amount of seed rate used (kg ha-1) by the ith respondent, X6i= amount weedicide applied (L ha-1) by the ith respondent, X7i= Machine rent (Ethiopian Birr) by the ith respondent, ε1 = is an error term and definedasVi−Ui\n\nVi = random effects (measurement errors, omitted explanatory variables) assumed to be independent of Ui, identically and normally distributed with zero mean and constant variance σv2. Uis=non-negative random error variables which are assumed to account for technical inefficiency among bread wheat producers of the study areas.\n\nUis - are the technical inefficiency effects which are assumed to be independent of Vi such that Ui is the non-negative truncation (at zero) of the normal distribution with mean Ui and Variance (δ2). The inefficiency model for this particular study is specified as follows (Equation 10):\n\nWhere Ui denotes the technical inefficiency of the ith farmer, while X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, and X12 denotes gender, age, grade level, family size, dependency ratio, tropical livestock unit (TLU), land size, hand weeding frequency, row spacing, frequency of extension contact, amount of credit, and membership in seed multiplication and marketing cooperatives, respectively.\n\nGeneralized likelihood ratio (LR) test\n\nThis study has employed the generalized LR test to select the appropriate functional form that best fits to the data, the appropriate distributional assumptions of the technical inefficiency term and the existence or non-existence of inefficiency on the model and others. One hypothesis has been tested with regard to the model specification using the generalized LR test, which is defined as follows (Equations 11 and 12):\n\nWhere, λ is the likelihood ratio (LR), L (H0) = the log likelihood value of the null-hypothesis; L (H1) = the log likelihood value of the alternative hypothesis; and ln is the natural logarithm. This has a chi-square distribution with degree of freedom equal to the difference between the numbers of estimated parameters under H1 and H0. Yet, where the test involves a γ, then the mixed chi-square distribution is used. The H0 is rejected when the estimated chi-square is greater than the critical value.\n\n\nResults\n\nThe findings presented here were based on the data collected from 14 KIIs, four FGDs, and 450 structured interviews. The profile of the KIIs and FGDs participants are summarized and presented in the underlying data materials of this study.31 Smallholder farmers’ wheat production and their TE is not solely contingent upon the quantity, quality, availability, and affordability of farm inputs. As a result, this study has hypothesized that there are multiple agronomic practices, plot level variables, demographic, socio-economic, and institutional factors affecting smallholder farmers wheat productivity and their TE. Thus, it is essential to present findings of the descriptive analysis prior to presenting results of the SFM as it will provide a vivid picture of the study area and sample farmers who grow bread wheat using recycled and fresh seeds. The descriptive statistics for all the variables considered in this study are presented in Table 1 below and it includes the mean values, the mean difference, the standard error, t-value, and p values. As detailed in Table 1 below, a significant difference was observed between fresh and recycled seed users in terms age, family size, dependency ration, TLU, land size, frequency of extension contact, seed recycling frequency, yield per hectare, weeding frequency, row spacing, seed rate, fertilizer rate, herbicide rate, and technical efficiency score at p=0.000. Yet, no significant difference was observed between these two groups in terms of grade level, amount of credit, and gender.\n\nThe GLR test has been conducted using the formula specified in equation 11 in order to select the model that best fits the data of this study. The test rejected the H0 (null hypothesis) that asserts the translog stochastic frontier production function can be reduced to a CD production function at 5% level of significance. This favors the application of the Translog functional form at the expense of the CD. Thus, earlier studies based on the CD model18,34,37,38,41,48 may be misleading and hence can’t be replicated here. The second hypothesis test was conducted to check the existence of inefficiency parameters. The null hypothesis was that technical inefficiency effects are not in the model (H0: γ = δ0= δ1= δ2= ….= δ13 = 0 (all wheat producer farmers considered in this study were efficient) was tested against the alternative hypothesis that inefficiency factors were in the model (H1: γ >0 and δi ≠ 0 for i= 0, 1, 2, …,13). The likelihood ratio test also rejected the null hypothesis H0 at 5% level of significance. Thus, it can be concluded that there exist technical inefficiencies in BWP which in turn implies that the traditional production functions that do not account for technical inefficiencies are inadequate for modelling BWP in the study areas. The estimated parameter for γ was higher than 1, implying that the variation in BWP was mainly attributed to variations inefficiency.31 The GLR statistics value confirmed that the σu2was strongly biased towardsσv2. The third hypothesis was carried out to check the H0 (null hypothesis) that the distribution of inefficiency can be reduced from truncated normal to half normal distribution μ=0. The result has turned out in rejection of the null hypothesis. This means that the distribution of the inefficiency term is truncated at 0 normal distribution which is significant at 5%.31\n\nThis study has employed the maximum likelihood estimates (MLE) of the TL functional form to investigate the major determinants of BWP difference among farmers. About seven variables were considered in the estimation of the frontier production function. The coefficients for the factors used in BWP were estimated under the full frontier production function (Maximum Likelihood Estimation). The coefficients presented in Table 2 below represents individual elasticities. These individual elasticities adds-up to 1.49 indicating that farmers in the study area are facing increasing returns to scale. This portrays the potential to further increase the extent of BWP. This also means that there exist a wide opportunity to increase bread wheat production with an increasing rate of return. The result of the MLE gave the value of the parameter estimations of the frontier model along with the value of log-likelihood function for the stochastic production function. Table 2 below is a summary of the MLE of the parameter of TL functional form of the stochastic frontier model.\n\n* p<.01.\n\n p<.05.\n\n* p<.1.\n\nIn this study, about seven hypothesized variables were considered in BWP function. Four of these variables (land, labour, capital, and fertilizer) had a significant effect in explaining the variation in BWP among smallholder farmers in the study area. The coefficient for labour measured in terms of man-days spent for BWP turned out to be significant and positive at p<0.01. This coefficient for labour implies that a 1% increase in man-days leads to a 0.086% increase in bread wheat productivity. This finding is backed by earlier studies that have revealed an increase in crop productivity is the result of intensive labour use for different farm operations and agronomic practices that augment production.37,38,41,49,50 Thus, the better wheat productivity in this study can also be attributed to the fact that farmers in the study area have allocated sufficient man-days for the various labour intensive agronomic practices such as maximum tilling frequency, row planting, weeding, and timely harvesting practices that enhance their productivity.\n\nSimilarly, the coefficient for capital invested measured in terms of Ethiopian birr was significantly positive at p<0.01. As expected, this coefficient indicates that an additional 1% rise in capital invested leads to a 0.22% increase in bread wheat productivity. This finding is backed by earlier studies. For instance, better annual income is reported to increase framer’s propensity to be productive as it enables them to have access to production supplementing inputs and technologies. The positive impacts of income on production enhancing agricultural innovation is frequently reported.7,8,16,18 The effect of capital investment was higher than any other variable considered in the production function of this study. The extremely significant effect of capital on BWP could have several explanations. First, it may serve farmers as an incentive to purchase production improving and efficiency augmenting technologies. Key informants of this study including seed suppliers and DAs have revealed that better-off farmers adopt technologies earlier than poor farmers. This could be because better-off farmers are well above the poverty line that they took the risk of trying a new innovation. Conversely, farmers who are already below the poverty line or closer to it could not have the appetite for production enhancing innovations for two obvious reasons. First, farmers who are already below the poverty line could not afford the purchase of production enhancing technologies as they don’t have sufficient capital for investment. Studies have also shown that poor farmers will not invest even on the cheapest farm innovations as they do have urgent consumption demand.12,14,51,52 Second, farmers closer to the poverty line do not have the incentive to try new innovations as risks of innovation failure will put them below the poverty line. This could be another explanation for the highly significant impact of capital investment in BWP. Besides, the agronomic practices in BWP are quite labour demanding. Higher income is likely to encourage farmers to make use of hired labour and machinery rent which in turn would improve production and efficiency of the farmers. This could be another explanation for a highly significant nexus between BWP and high capital investment.\n\nChemical fertilizer application was another important hypothesized determinant expected to have a considerable impact on bread wheat productivity in the study area. The coefficient for chemical fertilizer was positive and significant at p < .05 indicating that a 1% rise in chemical fertilizer application has contributed to a 0.042% increment in the amount of BWP per hectare. Several studies have revealed that the low wheat yield in Ethiopia is attributable to low fertilizer use, depletion of soil fertility due to continuous nutrient uptake of crops, and insufficient organic matter application. Besides, Ethiopia’s sole dependence on narrow imported fertilizer products (largely urea and di ammonium phosphate (DAP) is blamed to cause a considerable productivity loss. These two imported fertilizer products are rich sources of nitrogen (N) and phosphorus (P). But most Ethiopian soils lack other macro nutrients.53–55 The introduction of the blended NPSB fertilizer in addition to the existing types of fertilizer could explain the positive impact of fertilizer in wheat productivity of the study area. Despite the observed positive nexus between wheat productivity and fertilizer application, it is worth noting that farmers in the study area have applied fertilizer rates well below the recommendation. This could be due to the blanket fertilizer rate recommendation that did not take spatial differences into consideration. This might call for study area specific fertilizer rates related agronomic trials. Thus, future research should focus on this critical issue. Similarly,54 argue for a soil test based application of plant nutrient rather than the blanket recommendation of urea and DAP, and especially those containing sulfur, boron, and other nutrients is recommended in averting problems caused due to nutrient deficient soil.\n\nThe coefficient for land is interestingly significant and negative at p<0.01. This implies that an extra one-hectare land addition to the current wheat production will lead to a 0.03%productivity loss. This finding is diametrically opposite to the findings reported in multiple empirical investigations.13,17,38,39,56–58 Unlike the finding in this study, these studies found a significant positive association between land and crop productivity. The production theory of diminishing marginal utility states that an increase in input can increase the production of crop and other commodities up to certain optimum levels. Any additional input use after this optimum level will lead to a flat production. Further input addition after the plain/flat production curve will cause a decrease in production.59 This theory could be a plausible explanation for the current finding. Besides,60 has indicated that a major reason for the non-attainment of achievable yields is low fertility of the soils which is partly due to low use of fertilizers. But low fertilizer use was not observed in this study. The declining soil fertility and land fragmentation could explain the negative relationship between total wheat productivity and land size.\n\nThe coefficient for seed rate was not significant at the conventional probability levels indicating that seed rate did not contribute to total wheat productivity. As indicated in Table 1 above, farmers in the study place have used an average seed rate of 194.39 kg ha-1 which is higher than the recommendation by 44.39 kg ha-1. During the FGD, participants have raised that recycled seeds are used from reserves saved during the previous production, can be borrowed from relatives and friends, and are cheaper in the market. They have also confirmed that the productivity of recycled seed is believed to be inferior to the fresh seeds, unless compensated by applying extra seed rates. This farmers attitude of increasing productivity by making use of extra seed rates is against the recommendation and the established scientific theory of diminishing marginal productivity (DMP). The relatively cheaper market price for recycled seeds might have encouraged farmers to apply more amount of seed rate per hectare. Besides, higher seed rates application imply a risk aversion behavior of farmers to ensure good plant stand and survivalTavva, Aw-Hassan.16 This explanation can be adopted in this study too. Contrary to the current study, seed rate has shown the highest input effect on production and estimated elasticities. The positive contribution of seed rates on production and technical efficiency was attributed to the quality and quantity of the seeds.7,52,61 The insignificant contribution of seed rate in this study may therefore be explained by the inferior quality of the seeds and the seed recycling practices in the surveyed areas. Weeds remain a major challenge to increasing crop output as they compete with the crop plants for light, nutrients, and water. Such growth retarding weeds could not cause a significant harm if proper weeding frequency is followed.46 Thus, the application of weedicide was expected to increase total crops productivity. As expected, the coefficient for weedicide was positive and significant at p=0.007 indicating BWP is highly dictated by weed management.\n\nMachine rent was expected to have a significant positive impact on total factor productivity. As expected, a 1% change in machinery rent has led to a 0.005% change in bread wheat productivity. There are studies that support the current finding. Higher productivity, which requires greater power and mechanization, is one of the most important technologies that can increase production when resources are limited.62 This implies that mechanization leads to production improvement under sufficient supply of land, labour, and other resources. This may be a plausible explanation for the significant contribution of machinery on BWP under the current study. In addition, key informants of this study have repeatedly mentioned that the state sponsored harvester and threshing machineries has helped farmers for timely harvest and substantially reduce post-harvest losses. It is also worthy to note that out of the seven variables expected to improve bread wheat productivity, machinery rent has the least contribution. This could be due to the fact that these machineries (especially combine harvesters) were brought to the study areas from other regions on rental basis with expensive prices which is likely to increase the operation cost of BWP.\n\nIn Ethiopia, making use of freshly purchased improved seeds and improved seeds recycled for a maximum of five seasons are considered as improved seed use.27 Therefore, fresh seed use in this study refers to the use of freshly purchased seed and seeds recycled for a maximum of five seasons. This study has expected that multiple bread wheat seed recycling has a significant negative impact on the productivity and TE of smallholder farmers. Studies have shown that bread wheat seed recycling to a maximum of five production seasons can be considered as improved practice and don’t cause significant productivity loss. But, multiple seed recycling is reported to be a common practice in wheat growing areas of Ethiopia. For instance, about 84% of the farmers depend on recycled seeds while only 14% used new seeds. This practice produce seeds that have low germination percentage and is contaminated with weeds. The problem of recycled seed is serious in Ethiopia, where pathogens of the wheat rusts evolve quite rapidly, rendering farmers' varieties less resistant to disease. To avoid yield losses from disease, farmers should replace their wheat varieties frequently and regularly.8,27 Given these existing literatures, this study had the following hypotheses as far as the nexus between TE and bread wheat seed recycling is concerned:\n\nH0: diff = 0. This hypothesis assumes that there is no difference in mean TE between wheat producing farmers with fresh and recycled seed use.\n\nHa1: diff ! = 0. This is an alternative hypothesis with an assumption that the difference in mean TE between fresh and recycled bread wheat seed users is different from zero.\n\nHa2 = diff < 0. This is a second alternative hypothesis that expects the difference in the mean TE between recycled and fresh seed users is less than zero.\n\nHa3 = diff > 0. This is the third alternative hypothesis that expects the mean TE difference between recycled and fresh bread wheat seed users is greater than zero.\n\nAppendix Table 3 presented as an underlying data of this study summarizes the TE of fresh and recycled bread wheat seed users.31 This study has found that about 247 (54.89%) of the respondents make use recycled seeds for more than five production seasons while about 203 (45.11%) make use of either freshly purchased seed or recycled seeds below five times. The average TE of fresh and recycled seed users were 0.8123 (81.23%) and 0.7789 (78.89%), respectively. This can be interpreted as a typical fresh seed user and recycled seed user are operating 0.1877 (18.77%) and 0.211 (21.11%) less than his/her maximum TE and this difference is highly significant at p=.0000. Figure 3 presented in the supplementary material paints a clear picture about the relationship between seed recycling and TE scores.31 It offers a strong evidence that the variance in distribution of the TE scores between recycled and fresh seed users. A substantial proportion of fresh seed users had a mean TE score of greater or equal to 0.8165 while none of recycled seed users hit a TE score of this magnitude.\n\nThis study has also examined the marginal effect of seed recycling on the overall TE of BWP. As presented in the Table 3 below, a unit increase in seed recycling frequency is associated with a.001 decline in TE. Based on the ordinary least square (OLS) estimation of the Generalized Linear Model, the marginal effects of successive seed recycling up to four, eight, twelve, sixteen, and twenty times on the TE of BWP were predicted, respectively. Farmers who make use of fresh seeds (fresh or seeds recycled less than five times had a mean TE of 80%. Further seed recycling up to eight, 12, and 16 times will cause a 0.003, 0.007, and 0.01 loss in TE, respectively. Generally, it can be concluded that seed recycling and TE have a significant inverse relationship. This finding is backed by other empirical investigations.7,12,27,28,63,64\n\n* p<.01.\n\n p<.05.\n\nThus, from these findings, it is clear that the null hypothesis (H0: diff=0) that expects no TE difference and the fourth alternative hypothesis that expects a positive TE difference (Ha3: diff > 0) are rejected in favor of the 2nd (Ha1: diff ! = 0) and the 3rd Ha2: diff < 0) alternative hypotheses. This means that the assumption that seed recycling does not cause inefficiency in BWP does not hold water. In general, it can be deduced that seed recycling has caused considerable TE loss as compared to fresh seed. Recycled seeds are reported to be highly vulnerable to the most important wheat rusts, a group of diseases caused by fungal pathogens (Puccinia spp.), are stem rust (also called black rust), stripe rust (also called yellow rust) and leaf rust (also called brown rust). Stem and stripe rusts are the major biotic constraints to wheat production in Ethiopia, with frequently recurring epidemics. Studies revealed that recycled seeds are susceptible to weeds and pathogens.28,65 The biotic constraints and diseases may add financial and management burdens on farmers that might explain farmers technical inefficiency in this study.\n\n\nDiscussion\n\nThis study has considered about 12 different socio-demographic, economic, institutional, and agronomic determinants hypothesized to influence TE of BWP. The graphical abstract presented in Figure 5 and deposited as an underlying data provides a vivid pricture of the study variables.31 Among these variables, gender, grade level, plot size, and frequency of extension contact of the respondents did not have a notable contribution to BWP technical inefficiency. Figure 4 illustrates the nexus between TE scores and the different variables considered in the study. The coefficient for gender (being female) is negative but insignificant indicating technical inefficiency is not caused by gender differences. However, ample studies unfold that female headed households generally have less access to and control over different factors of production including land, credit, farm inputs, extension services, and irrigation water which contributes to their inefficiency in farm operations.46,66 A plausible explanation for the current insignificant impact of gender on technical inefficiency may be the traditional support system by which the community assist farm operation of widowed and divorced women. Moreover, participants of the female FGD have explained that helpless, single or unmarried, widowed, divorced, and women with disability are beneficiary of social welfare programs like the Productive Safety Net Program (PSNP) and other non-governmental organization (NGO) sponsored supports. These social welfare programs support women by providing farm inputs and emergency assistances in times of shocks and stresses that might have helped them to enhance their production and improve their TE. These same FGD participants have explained that there are women Village Economic and Social Associations (VESAs) that provide credit, saving, training, farm inputs, marketing services, labour, and social supports in times of misfortunes including crop production failures, livestock and human death. It was frequently mentioned that these VESAs have helped women to have access to and control over financial services which is not open to male headed households. The insignificant effect of gender on farmers technical inefficiency thus could be attributed to these support mechanisms. The finding in this study is backed by an earlier study of67 who have studied the TE of smallholder agriculture in developing countries.\n\nSource: Own Drawing (2022).\n\nThe age of the farmer was hypothesized to have a statistically significant positive effect on the TE of BWP. Age is considered as a proxy indicator of farmers’ experience in making use of improved practices and farm inputs. The coefficient for this variable is highly statistically significant with a negative sign at p<.01, which indicates that age of the farmer is inversely related with inefficiency. This implies that younger farmers tend to be more inefficient than older ones. From Table 4 presented below, it is clear that a one-year increase in farmers age reduces technical inefficiency by 0.13%. This could be due to the fact that longer years and experience in agriculture has helped older farmers to weigh the merits and demerits of efficiency enhancing practices and inputs which in turn motivate them for further adoption and improvement in production efficiency. In addition, longer years may bestow farmers the chance for better asset ownership and socio-economic status that embolden them to take risks in trying efficiency and production augmenting inputs and practices. Thus, the age of farmers can be considered as a vital socio-personal variable in improving efficiency of BWP. Similar finding was reported by other studies.13,37,41 However, few other studies are against this finding. For instance, it was found that older farmers are likely to be more conservative and hesitant to adopt new innovations and evade frequent experimentation with the new technologies that reduce their TEMesay, Tesafye.48 This study has also mentioned that farmers managerial ability decreases with age that reduce their productivity and TE.\n\n* p<.01.\n\n p<.05.\n\n* p<.1.\n\nEducation level of the respondent was expected to have a significant direct relationship with the farmers’ TE. Yet, the inefficiency coefficient found in this study is negative and insignificant. This means that farmers’ level of education measured in years of schooling attended don’t affect their level of TE. This is diametrically opposite to several established facts that have proven the highly significant positive impact of education on improving TE of farmers.16,41,48 These studies unfold that education improves the ability of the household to make informed and timely decision about production inputs. Educated farmers more often have better access to agricultural information and higher tendency to adopt improved inputs (like fertilizers and crop varieties) more optimally and efficiently. However, the discrepancy about the impact of education could have several explanations. The first explanation may be the difference in the level of education attained by farmers. The mean grade level attained by farmers of this study area is less than two years. The average years of schooling reported by the studies mentioned are higher than two years. Thus, few years of schooling in the study area could plausibly explain the insignificant contribution of education on farmers TE. Second, the difference in the scale of farm operation could be another explanation. For instance, the positive efficiency impact of education is observed in large and medium farms than small farms.40 However, this is against the established facts by the proponents of the small farm approach. For instance, studies have proved that land productivity of small farms was at least twice that of the largest ones. Small farms enable to make use of family labour intensively and also help in avoiding supervision constraints of managing a large hired labour force in big farms. Hence, small farms maximize return to land which is the scarce resource for them and for nation.68\n\nLarger family size has been associated with better productivity and TE. Studies unfold that households with larger family sizes have better advantage by being able to use labour resources to accomplish different labour demanding farm operations at the right time.20,69 Yet, higher household size has also been linked to less productivity and technical efficiency, when the actual family members are sick, unproductive, old, and disabled.18 These findings sheds light on the need to consider family size and dependency ratio separately. In this study dependency ratio refers to the number of dependent household members (which includes children less or equal to 14 years, elderly people greater or equal to 64 years, along with sick and people with disability) divided by the number of productive household members (which include working age people from 15 to 64 years). The same definition was used in several studies.70 Given these evidences, this study therefore has hypothesized that family size will have a highly significant contribution to TE while dependency ratio will end-up in curtailing farmers TE. The findings for both of these variables was congruent with prior expectations. From the coefficient presented in Table 4 above, it can be inferred that a 100% increase in family size reduces technical inefficiency by 50.2% at p<.05. On the contrary, a 100% increase in dependency ratio has increased farmers technical inefficiency by 1.9% at p<0.05. From these findings, it is also clear that the TE gains from an increase in family size can be cancelled by the efficiency loss as a result of higher dependency ratio. This finding is in agreement with the study made at Wolmera district of West Gojam in Ethiopia.18\n\nStudies have shown that bigger land size makes production operations difficult and costly to fulfill the production input requirements. Moreover, big farm owners with limited family and hired labour, draught power, and no access to labour saving technologies tend to poorly manage their plots.38,45 As a result, land size allocated for BWP was expected to have an inverse relationship with farmers TE. The coefficient for land size, however did not show any noticeable impact on farmers TE and was not in line with a priori expectation of the study. This means that the amount of land allocated for BWP did not affect farmers TE. Several findings are against what has been observed in this study area. For instance, a significant and positive impact of plot size on wheat producers’ inefficiency was reported in another study38. The same author has argued that the significantly high positive impact of plot size on technical inefficiency is due to the inherent nature of big plot sizes that hamper farmers ability to carry out important agronomic practices that must be done on time given the limited resources available at farmers disposal. On the contrary, farmers with larger area of cultivable land have the capacity to use compatible technologies that could increase their efficiency and enjoy the benefits of the economies of scale. The negative but insignificant coefficient for land size in this study could also be due to land fragmentation and declining soil fertility.13\n\nQuite often, livestock and crop production tend to be complementary. Animal wastes can be used as a soil conditioner while crop residues and remnants serve as livestock feed. Animals can be good source of cash and draught power which in turn enhance productivity and efficiency. Furthermore, animals can be used as a hedge and shock absorber to artificial and natural shocks and stresses.13 Thus, the number of animals owned expressed in TLU was hypothesized to have a highly significant positive impact on the TE of farmers. The parameter estimate was significant and negative indicating technical inefficiency decrease with increasing TLU at p<0.1. The parameter estimate has indicated that a 1% increase in TLU decrease farmers probability of being inefficient by 0.095%. This is in line with the prior expectation of the study.\n\nHand weeding frequency was expected to enhance production and TE of farmers in the study area. The coefficient for this variable is highly significant and negative implying a unit increase in weeding frequency reduces the probability of being technically inefficient by 0.05 at p<0.01. Farmers in the study area make use of both mechanical and chemical weed control methods that may have assisted them to enhance their production and TE. Earlier studies have also stated weed control as quintessential practice for efficient and profitable BWP. Weeds have direct and indirect impact on BWP. Directly weeds compete with the wheat crop for space, water, light, and nutrients while they harbor insects and disease-causing organisms indirectly.19,71\n\nThis study found that row planting and cluster farming is widely practiced in the study area. This is because the extension advisory service has been actively pushing farmers to move from the conventional broadcasting wheat sowing method on fragmented plots to row planting and cluster farming. However, earlier studies unfold that it is not only the sowing method that counts most in improving productivity and TE. The spacing between seeds and rows have also considerable impact on crop productivity and production efficiency.19,71 Given these findings, this study has assumed that the spacing between rows has a highly significant positive impact on the TE of BWP. This assumption holds water under the current study. A unit increase in the spacing between rows has reduced technical inefficiency by 0.005 at p=0.001. Given this finding, encouraging farmers to stick to keeping the optimum row spacing will lead to reduction in technical inefficiency and improve BWP.\n\nThe frequency of extension contact was hypothesized to have a significant positive impact in improving farmers TE in BWP. Unlike the prior expectation, the coefficient for extension contact turned out to be insignificant. However, this finding should be cautiously interpreted. The insignificant contribution of this variable could be due to its focus on other crop items than BWP. Key informant DAs have indicated that they have been intensively working on bread wheat advisory service for more than 30 years. Thus, they have shifted their attention to introduction of economically superior and industrial crops than conventional crops production. Extension may have significant impact in earlier stage of technology introduction when farmers lack sufficient information and skill. But at later stages, there will be information equilibria between the farmer and extension worker. Thus, this assessment has focused on later stage during which information, improved practices, and inputs for BWP has already been fully diffused and no new ways of BWP is generated. This late assessment may belittle the impact of the extension service at earlier time of improved bread wheat production technology introduction. This could explain the trivial contribution of the extension service. However, several findings reported the highly significant positive contribution of agricultural extension service in enhancing producers TE.18,72,73 The majority of farmers in Ethiopia are not exposed to multiple agricultural technology and information sources. It is only the state-led agricultural extension system that often provides them with agricultural advisory services and innovation.74\n\nEvidences from extensive literature review indicates that crop productivity and efficiency are enhanced when credit and other financial services are tailored to the needs of smallholder farmers.15,56,75,76 These empirical evidences further argue that farmers decision to invest and produce crops and other agricultural commodities is substantially influenced by access to a mix of financial instruments. Given these evidences, amount of credit was expected to produce a highly significant impact on the TE of BWP. Nevertheless, the coefficient for the amount of credit has turned out contrary to a priori expectation of this study. The amount of credit did not show any marked effect on the TE of BWP. This is due to the fact that the majority of the farmers in the study area don’t have access to formal financial institutions. This study has conducted FGDs with recycled and fresh seed users to understand the major challenges in BWP. Participants of the FGDs have frequently and unanimously explained that access to credit and financial services is very limited. These FGD participants further mentioned that it is only Amhara Credit and Saving Institution (ACSI) that provide credit service for the community. These participants have complained about the highest interest rate and the targeting criteria to access credit. It was mentioned that all farmers are not entitled to access credit. Land certificate, membership in the PSNP, assets ownership, and repayment ability are considered as major targeting criteria. It was also explained that farmers get access to credit from their own Rural Saving and Credit Cooperative (RuSACCO). The interest rate from the RuSACCO is mentioned to be fair as compared to other sources. But RuSACCO’s do not have the financial capacity to meet the financial requirements of all members. Thus, they give insufficient amount of credit to be repaid only within one year. Similarly, some credit arrangements reportedly discourage farmers from adoption of new agricultural technology, purchasing main farm inputs, or making other decisions that can increase their productivity and efficiency. This is due to expensive collateral requirements, unaffordable interest rates, lack of appropriate risk mitigation products, and supply driven nature of the financial services that don’t match smallholder farmers felt needs. The insufficient credit supply that has retarded agricultural production was reported in several studies.15,56,60,75,76 These facts on credit could explain the unmarked contribution of credit on the TE of BWP in this study area. Therefore, in order to increase the TE in BWP, farmers must be provided with sufficient amount of credit and easy access on favorable terms through formal institutions.\n\nThe study area is known for its wheat production suitability. Thus, the Ethiopian Seed Enterprise (ESE) and the Amhara Seed Enterprise (ASE) establish wheat seed multiplication and marketing cooperatives. There are evidences that producers’ cooperatives boost productivity and improve TE as they reduces transaction costs and play a significant role in input supply and the procurement of members produce in times of unfavorable market prices.77,78 Given these findings, this study has assumed a highly significant positive association between farmers membership in seed multiplication and marketing cooperatives and their technical efficiency. The coefficient is in line with the hypothesis. The coefficient indicates that the probability of being inefficient is reduced by 0.003 for members than non-members at p<0.05.\n\nFrom results of this study, the following recommendations are made\n\n• There are strong evidence that few farmers in the study areas are capable of producing bread wheat either on the frontier and/or extremely closer to the frontier that implies their high TE. Therefore, this study highly recommends a farmer – to – farmer (F2F) extension system that allow farmers to learn from their neighbor farmers on how to enhance their TE and improve their productivity. In this regard, Farmers’ Field School (FFS), farmers’ field days, excursions, and other group extension methods are strongly suggested. This could also be a financially viable informal technology diffusion and training modality for the existing extension system. Implementation of these group extension methods will also have a complementary effect on the face – to – face (individual) extension methods.\n\n• The number of man-days allocated and the amount of capital invested had significantly high positive contribution for wheat production. Therefore, improving labour use efficiency through training and increasing farmers’ access to financial sources and services is strongly recommended.\n\n• Respondents access to credit has helped farmers to substantially reduce their production inefficiency. This study therefore calls for an urgent policy design and implementation geared towards improving farmers financial access.\n\n• Dependency ratio has a considerably high contribution to farmers technical inefficiency. It is therefore quintessential to introduce labor saving technologies that would ease the work burden of families of with the highest dependency ratio induced by availability of family members with disability, old age, sick, and unproductive children.\n\n• Farmers continue bread wheat seed recycling that substantially curtail their productivity and efficiency. This is largely due to absence of supply, supply insufficiency, and expensiveness of early generation seeds (EGSs) and improved varieties. It is therefore highly recommended to encourage the establishment of seed multiplication and marketing cooperatives that could solve these problems. Besides, seed aids, seed loans, seed vouchers, and financial credits geared towards smallholder farmers seed security are strongly recommended as they can serve as an incentive for wider use of fresh and EGSs.\n\n• Social welfare programs and Village Economic and Social Associations (VESAs) have helped some sections of the bottom poor farmers to have access to and control over production augmenting and efficiency enhancing inputs and technologies. Yet, these social welfare programs and VESAs are few in number and benefit a minor portion of the society. Moreover, members of these VESAs support each other in times of agricultural labour requirement, crop production failure, livestock and human death. Yet, seed is not part of the VESAs support arrangement. Thus, it is highly recommended to scale – up and establish as many VESAs as possible and the multiple economic and social objectives in the existing VESAs should also be relaxed to include village level seed saving and seed loans provisions.\n\n• The findings in this study are based on a cross sectional data. Yet, farmers’ seed recycling practices tend to be dissimilar on longitudinal basis. This calls for the need to undertake similar empirical investigations using panel data in the future.\n\n\nConclusions\n\nThe elasticities from the MLE under the full frontier production function adds – up to 1.49. Thus, one can conclude that farmers in the study area are facing increasing returns to scale and there is a room to increase bread wheat production beyond its current level of production. In addition, the coefficients of the stochastic frontier production function for labour (0.086), capital (0.22), fertilizer (0.042), weedicide (0.014), and machinery (0.005) were positive and significant at p<0.001 implying that an optimum increase to these inputs would increase BWP. In other words, from these coefficients in the stochastic frontier production function it can be deduced that amount of labour employed (man – days), capital invested, fertilizer, weedicide, and machinery rent had significantly high contribution to the existing level of bread wheat production. However, the coefficient for land was negative and significant suggesting additional land allocation for BWP would likely reduce bread wheat productivity.\n\nThe mean TE was found to be 79.99%. The maximum likelihood estimates for the inefficiency parameter portrayed that farmers in the study area operates 20.1% less than their potential. It is therefore evident from this finding that there is an opportunity to increase the existing BWP without extra inputs allocation and through improving farmers TE or reducing their level of technical inefficiency. From the MLE, there is also strong evidence that few farmers in the study areas are capable of producing bread wheat either on the frontier and/or extremely closer to the frontier that implies their high TE. From the inefficiency analysis one can easily infer that longer years,79 higher number of animal ownership (TLU), credit amount, and membership in seed multiplication and marketing cooperatives have helped farmers to significantly reduce their technical inefficiency. Similarly, agronomic related parameters such as hand weeding frequency and row spacing had significantly enhanced farmers technical inefficiency. However, dependency ratio has significantly curtailed farmers level of technical efficiency. Also, from analysis of the nexus between seed recycling and TE, a typical recycled seed user was 2.34% less than fresh seed users in its TE. This testifies that seed recycling has significantly contributed to farmers’ inefficiency. Generally, it can be concluded that seed recycling and TE have a significant inverse relationship. Bread wheat seed recycling up to five times is considered as improved practices. However, a substantial TE difference was observed even among farmers who had recycled seed less than five times. Thus, one can conclude that the recycled seeds do have higher chance of contamination with impure and genetically inferior seeds that contribute to lower productivity and technical efficiency.", "appendix": "Data availability\n\nOSF: Determinants of technical efficiency of smallholder farmers bread wheat production and implications of seed recycling: The stochastic frontier approach. https://doi.org/10.17605/OSF.IO/8K6RJ. 31\n\nThe project contains the following underlying data:\n\n• Supplementary Material 5: Data for TE Analysis CSV.csv. (Anonymised responses from questionnaire).\n\n• Supplementary Material 6: Data for TE Analysis Stata.dta.\n\n• Supplementary Table 1. Profile of key information interview (KII) Participants.\n\n• Supplementary Table 2. Distribution of focus group discussion (FGD) Participants.\n\n• Appendix Table 1: Study assumptions and hypothesis test for model specification.\n\n• Appendix Table 2: Technical efficiency difference between fresh and recycled seed users.\n\n• Appendix Table 3: Distribution of technical efficiency score by seed recycling status\n\n• Variables key (Variables key for questionnaire headings in supplementary materials 5 and 6).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nOSF: Determinants of technical efficiency of smallholder farmers bread wheat production and implications of seed recycling: The stochastic frontier approach. https://doi.org/10.17605/OSF.IO/8K6RJ. 31\n\nThis project contains the following extended data:\n\n• Supplementary Material 3: English Questionnaire.pdf.\n\n• Supplementary Material 4: Amharic Questionnaire.pdf.\n\n• Figure 1: Map of the study areas.\n\n• Figure 2: Sampling procedures.\n\n• Figure 3: Technical efficiency of farmers with seed recycling.\n\n• Figure 4: Combined Graph of the 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\nAcknowledgments\n\nAuthors of the study are grateful for the men and women farmers engaged in the focus group discussions and the household survey. 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Publisher Full Text\n\nMeeusen W, Broeck JD: Efficiency Estimation from Cobb-Douglas Production Functions with Composed Error. Int. Econ. Rev. 1977; 18: 435–444. Publisher Full Text\n\nBattese GE, Coelli TJ: A Model for Technical Inefficiency Effects in a Stochastic Frontier Production Function for Panel Data. Empir. Econ. 1995; 20: 325–332. Publisher Full Text\n\nSultan S, Ahmed S: Technical efficiency and farm size productivity: micro level evidence from Jmmu and Kashmir. Int J Food Agric Econ. 2014; 2(4): 27–49.\n\nKibaara BW, Kavoi MM: Application of stochastic frontier approach model to assess technical efficiency in Kenyaâ€™s maize production. J. Agric. Sci. Technol. 2012; 14(1): 3–20.\n\nTeshome A, Abate E: Wheat technologies from where to where? The case of east Gojam zone of Amhara region, Ethiopia. Journal of Agricultural Economics and Development. 2013; 2(6): 226–236.\n\nMesay Y, et al.: Source of technical inefficiency of smallholder wheat farmers in selected waterlogged areas of Ethiopia: A translog production function approach. Afr. J. Agric. Res. 2013; 8(29): 3930–3940.\n\nAli A, Jan AU: Analysis of technical efficiency of sugarcane crop in Khyber Pakhtunkhwa: A stochastic frontier approach. Sarhad Journal of Agriculture. 2017; 33(1): 69–79. Publisher Full Text\n\nShamsudeen A, Kwame NP, Arkoh DS: Assessing the technical efficiency of maize production in northern Ghana: The data envelopment analysis approach. Cogent Food & Agriculture. 2018; 4(1): 1512390. Publisher Full Text\n\nDorosh PA, Mellor JW: Why agriculture remains a viable means of poverty reduction in Subâ€Saharan Africa: The case of Ethiopia. Dev. Policy Rev. 2013; 31(4): 419–441. Publisher Full Text\n\nMehmood Y, et al.: Do liquidity constraints influence the technical efficiency of wheat growers: evidence from Punjab, Pakistan. The Journal of Animal and Plant Sciences. 2020; 27(2): 667–679.\n\nBizuwork TD, Yibekal A: Optimizing blended (NPSB) and N fertilizer rates for the productivity of Durum wheat (Triticum turgidum L.var. durum) in Central Highlands of Ethiopia. Cogent Food & Agriculture. SOIL & CROP SCIENCES. 2020; 6: 1766733. Publisher Full Text\n\nEthioSIS (Ethiopian Soil Information System): Soil analysis report agriculture transformation agency [Unpublished]. Ministry of Agriculture and Agriculture Transformation Agemcy.2014.\n\nKidane G: Dryland agriculture production systems in Ethiopia. National Publishers; 2015.\n\nChandio AA, et al.: The nexus of agricultural credit, farm size and technical efficiency in Sindh, Pakistan: A stochastic production frontier approach. J. Saudi Soc. Agric. Sci. 2019; 18(3): 348–354. Publisher Full Text\n\nAbid H, et al.: Technical Efficiency of Wheat Production in Rain-fed Areas: A Case Study of Punjab, Pakistan. Pak. J. Agri. Sci. 2019; 49(3): 411–417.\n\nOmer GM: Bread wheat production in small scale irrigation users agro-pastoral households in Ethiopia: Case of Afar and Oromia regional state. International Journal of Agricultural Economics and Economics and Extension. 2015; 3(5): 144–150.\n\nColman D, Young T: Economics of agricultural production: Theoretical foundations. Principles of Agricultural Economics: Markets and Prices in Less Developed Countries (Wye Studies in Agricultural and Rural Development). Cambridge: Cambridge University Press; 1989; p. 5–29.\n\nAbdulai S, Nkegbe PK, Donkoh SA: Assessing the technical efficiency of maize production in northern Ghana: The data envelopment analysis approach. Cogent Food & Agriculture. 2018; 4(1): 1512390. Publisher Full Text\n\nTenaye A: Technical Efficiency of Smallholder Agriculture in Developing Countries: The Case of Ethiopia. Dent. Econ. 2020; 8(2): 34. Publisher Full Text\n\nMohammad AH, Mohammad AA, Abbas A: Impact of mechanization on technical efficiency: A case study of rice farmers in Iran. International Conference On Applied Economics (ICOAE) 2012.2012. Publisher Full Text Reference Source\n\nAtilaw A, et al.: Early generation seed production and supply in Ethiopia: Status, challenges and opportunities. Ethiopian Journal of Agricultural Sciences. 2016; 27(1): 99–119.\n\nBishaw Z, Struik PC, Van Gastel AJG: Farmers' seed sources and seed quality: 1. physical and physiological quality. J. Crop Improv. 2012; 26(5): 655–692. Publisher Full Text\n\nTolemariam A, et al.: Wheat Varietal Change and Adoption of Rust Resistant Wheat Varieties in Ethiopia from 2009/10 to 2013/14. Socioeconomics Program Working Paper 12. Mexico, CDMX: CIMMYT; 2018.\n\nGebre GG, et al.: Gender differences in the adoption of agricultural technology: The case of improved maize varieties in southern Ethiopia. Women's studies international forum. Elsevier; 2019.\n\nAnbes T: Technical efficiency of smallholder agriculture in developing countries: The case of Ethiopia. Dent. Econ. 2020; 8: 34. Publisher Full Text\n\nSchultz TW: Transforming Traditional Agriculture. New Haven and London: Yale University Press; 1964; xvi + 212. 45s.\n\nYohannis T, Tenaye A, Ganewo Z: Technical Efficiency of Agricultural Production in Ethiopia.2020.\n\nHarasty C, Ostermeier M: Population Ageing: Alternative measures of dependency and implications for the future of work. ILO Working Paper. ILO Working Paper.2020.\n\nWondimu T, Teshome A, Gezahegn BY: Economic Analysis of Tef Yield Response to different Sowing Methods: Experience from Illuababora Zone, Ethiopia. Journal of Economics and Sustainable Development. 2015; 6(1).\n\nAssefa W, Berhanu A, Demelash K: Participatory Evaluation and Selection of Bread Wheat (Triticum aestivum L.) Varieties: Implication for Sustainable Community Based Seed Production and Farmer Level Varietal Portfolio Managements at Southern Ethiopia. World Journal of Agricultural Research. 2014; 2(6): 315–320. Publisher Full Text\n\nTiruneh S, Yigesu AY, Bishaw Z: Measuring the effectiveness of extension innovations for out-scaling agricultural technologies. African Journal of Agricultural Science and Technology (AJAST). 2015; 3(7): 316–326.\n\nKassa B, Alemu D: Agricultural research and extension linkages: Challenges and intervention options. Ethiopian Journal of Agricultural Sciences. 2016; 27(1): 55–76.\n\nAbate GT, Francesconi GN, Getnet K: Impact of agricultural cooperatives on smallholders’ technical efficiency: empirical evidence from Ethiopia. Ann. Publ. Cooperative Econ. 2014; 85: 257–286. Publisher Full Text\n\nMartey E, Wiredu AN, Etwire PM: Impact of credit on technical efficiency of maize producing households in Northern Ghana, Selected Paper presented at the Centre for the Study of African Economies (CSAE) Conference.2015; p. 22–24.\n\nAltaye S, Mohammed H: Linking seed producer cooperatives with seed value chain actors: Implications for enhancing the autonomy and entrepreneurship of seed producer cooperatives in southern region of Ethiopia. International Journal of Cooperative Studies. 2013; 2(2): 61–65.\n\nGuth M, Katarzyna S-A: Economic resources versus the efficiency of different types of agricultural production in regions of the European union. Economic Research-Ekonomska Istraživanja. 2022; 33(1): 1036–1051. Publisher Full Text\n\nCentral Statistical Agency, Agricultural Sample Survey: Report on Area and Production of Major Crops (Private Peasant Holdings, Meher Season). Addis Ababa, Ethiopia: 2018; vol. I.\n\n\nFootnotes\n\n1 Kebele is the smallest administrative unit in Ethiopia." }	[ { "id": "174037", "date": "23 Jun 2023", "name": "Habtamu Taddele", "expertise": [ "Reviewer Expertise Climate Change and Rural Development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have raised important and noble research agenda for the scientific community. I have copied the title of this research in several search engines including Google Scholar, PubMed, and Google Semantic. I did not come across a single study that links bread wheat technical efficiency with seed recycling. The authors have captured with proper analytical methods the major determinants of smallholder bread wheat growers Technical Efficiency and clearly indicated the nexus with seed recycling. I do have strong conviction that the findings presented in this study will have paramount policy implications for people in the agricultural policy making circle and offers some pragmatic solutions to farmers and researchers engaged in improving wheat productivity and technical efficiency.\nCriteria 1: Is the work clearly and accurately presented and does it cite the current literature?\nI have checked the sources cited in the manuscript. I can assure you that the sources cited in the text are well acknowledged in the reference list. Moreover, the authors have summarized/paraphrased the sentences they borrowed from the different sources. The authors have applied reference management platforms/software and the reference style is well aligned to the scientific paper publisher’s standard. I believe that the manuscript has cited a sufficient number of articles relevant to the objectives of the article. I can confirm that the references cited are recent and not outdated.\nCriteria 2: Is the study design appropriate and does the work have academic merit?\nI can confirm that the study design is appropriate and has a strong academic merit. I can witness that the authors have used proper sampling design and provide sufficient details as to the context/setting under which the data collection was made. They have clearly indicated the data quality assurance mechanisms they have followed. I have also checked the underlying materials the authors have deposited in the Open Science Framework. I have seen the IRB approval along with the original and translated versions of the tools they have used in the data collection.\nCriteria 3: Are sufficient details of methods and analysis provided to allow replication by others?\nThe authors have used multiple sampling methods. They have employed different sampling methods including stratification, purposive, random, and probability methods. This blending of sampling methods is highly commendable because pluralism in the sampling method will assist them to get representative and unbiased samples. I have cross-checked the CSV data deposited by the authors for public access in the Open Science Framework (OSF). I found that the number of data deposited is congruent with the per-determined sample size.\n\nData Collection Methods: again they have employed quantitative (structured interview) and qualitative data collection (KIIs and FGD) methods. I believe this combination of the data collection methods will assist the authors to properly triangulate data. The data collection tools used in the study along with the rationale behind their use are well explained in sufficient details. The tools (both the original and the translated) are also deposited as an underlying data and are made openly accessible for public use.\n\nCriteria 4: Data analysis and interpretation:\nThe SFA applied by the authors was appropriate for the data type they have gathered. The type of analysis selected was first tested for its suitability with their data. The findings from the quantitative analysis were sufficiently backed and supported by findings from qualitative analysis. The interpretation of the analysis was very clear and excellent.\nCriteria 5: Are all the source data underlying the results available to ensure full reproducibility?\nI have checked the OSF link provided by the authors as far as the underlying data and extended materials is concerned. It is properly deposited and is made publicly available for reproduction. I found the way the main data was presented interesting due to the fact that it is properly and clearly coded and labeled that others can easily use it.\nCriteria 6: Are the conclusions drawn adequately supported by the results?\nI have proved that the conclusions are drawn based on the findings of the study. Commendable conclusions were reached based on the analysis of the data.\nFinal Assessment Decision\nApproved with minor editorial work as indicated below.\nSaying these, I strongly suggest the authors to consider the following minor editorial comments for further improvement of their manuscript:\nPlease be consistent in using terminologies like district vs woreda. Both are used frequently\n\nTitle: The title looks interesting. However, the following minor revision is suggested. “Determinants of technical efficiency of smallholder bread wheat production and implications of seed recycling: A stochastic Frontier Approach”\n\nAbstract: Background - Line 5: Please rephrase “However, there is some empirical research that examines ….” To “However, there are limited studies that examine…”\n\nMethods - Line 1:” The data was…” correct as “Data were…”\n\nMethods-line 5: Bread wheat growers were the major respondents… Did you include others? I think, for the questionnaire survey, only BW growers were the target group. If other respondents were involved, please justify.\n\nMethods-line 8. TE and factors affecting it, just “TE” is enough. Because, at this section, TE is the key term.\n\nResults-line 2. ‘Weedicide’ changes it with “Herbicide”. Because it is unusual and colloquial term in agronomy.\n\nKey word. Too many key words. I suggest to delete at least “Agriculture” and “Inputs”\n\nContext-line 3 (and throughout the document as well). Meher -use English (Autumn), to make it clear for international audience\n\nStudy area- --- The study takes place… need to be “the study was carried out….”\n\nI suggest to indicate your study area in range of coordinates (from -------- N, ---------E to -------N, ----------E). This is because the coordinate you put indicates only a point, not the study area coverage.\n\nSampling- “----by the researcher -----\" should be changed to “----by the researchers ---\". Use plural as there are two researchers\n\nLine 5: The researcher has decided…to ’The researchers decided”. Use past tense and plural.\n\nPage 6 line 1. Rewrite the equation n=1+N1…in scientific equation form (use Microsoft equation editor).\n\nSampling procedures and participant selection - Line 1: Change the word “chief” by “main”\n\nData types and sources - Consider the type and quantity of seed used rather than number of seeds/inputs Source for Tables in result section: Better to describe as “Researchers’ computation” rather than “own Computation”\n\nIn Table 2, you don’t need the last column as it is described below the table. In addition, merge the two columns for 95% CI. Also present all the tables in a more scientific way.\n\nFigure 3, the texts in the X-and Y-axes and the legend are with low contrast. May be, you have snapshotted it. Try to correct it. The same for other figures\n\nConclusion-line 1. Farmers are facing increasing returns to scale—this is a positive attribute. I suggest to use other word like “are experiencing” instead of “facing”\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "184180", "date": "10 Jul 2023", "name": "Vishal Dagar", "expertise": [ "Reviewer Expertise Technical efficiency and productivity 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\nDear authors,\nI'd like to congratulate you and your team for the good research work presented in your submitted paper for possible indexing in this prestigious journal. The topic is interesting, and I enjoyed it. However, before your work is recommended or accepted, a few comments must be included or addressed to improve the quality of your work. I have the following observations that may help improve your work: The authors must modify the following points in detail:\nIn the abstract, please include 2-3 special quantitative achievements from the findings of this study in the context of the environment by combining the research objectives and problems. Please limit your abstract to 250 words. Check spellings for many words that are misspelt or written in haste.\n\nThe introduction section needs a few more sentences to strengthen the article, and please include the research problem, objective, and novelty in the last paragraph of the Introduction section.\n\nInclude a few more sentences at the beginning of the introduction explaining your paper's contribution to the environment, climate change impact, and sustainability, as well as your attempts to deal with or present solutions to a specific problem/s and your unique contribution with this research paper.\n\nPlease also present the methodology section in a concise graphical format.\n\nThe literature review section is very weak; please revise it.\n\nPlease present your literature review in the form of a SmartArt chart.\n\nJust after the Methodology, please mention the societal benefits of your research in terms of evaluating its key determinant.\n\nIn 500-750 words, explain research problems, solutions, and the theoretical contribution of your study in the \"Results\" section.\n\nPlease include graphical presentations of your findings.\n\nDescribe why you placed this study in a separate section of \"Policy Suggestions\" just before the section of \"Conclusions.\"\nI found that the literature section is VERY weak, bring sustainability in your study, therefore it requires more studies to be reviewed. I suggest to you that you may include the following work:\nhttps://doi.org/10.1016/j.jclepro.2021.128585 https://doi.org/10.1016/j.jclepro.2021.128109 https://doi.org/10.1016/j.techfore.2023.122370 https://doi.org/10.1016/j.resourpol.2022.103169 https://doi.org/10.1016/j.heliyon.2021.e06952 https://doi.org/10.1016/j.energy.2022.123619 https://doi.org/10.1016/j.energy.2021.122365 https://doi.org/10.1016/j.eneco.2022.105884\nI think above all studies will make this study more relevant in bridging the gap with literature. Looking forward for your revised submission.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/12-502
https://f1000research.com/articles/10-1078/v1	25 Oct 21	{ "type": "Systematic Review", "title": "Academic entrepreneurial engagement for frugal innovation in higher education institutions: a systematic literature review", "authors": [ "Oluwayomi Toyin Ojo", "Magiswary Dorasamy", "Melissa W. Migin", "Jayamalathi Jayabalan", "Rajeswari R", "Soon Seng Tung", "Melissa W. Migin", "Jayamalathi Jayabalan", "Rajeswari R", "Soon Seng Tung" ], "abstract": "Higher education institutions (HEI) are faced with increasing challenges related to shrinking resources, high operation costs, the COVID-19 pandemic, decreasing student enrolment rates, and pressure to contribute to regional development and economic growth. To overcome such challenges, academics must move beyond their traditional functions of research and teaching and engage in entrepreneurial activities. Through engagement in entrepreneurial activities, academics can contribute to frugal innovation (FI) in private HEI (PHEI). The literature in this context emphasizes that academic entrepreneurial engagement (AEE) will lead to innovation, the identification of opportunities for new business ventures, financial rewards for institutions and academics, an impact on the economy, and the enhancement of social welfare. This study presents a systematic review of the literature and adopts the Transfield five-phase strategy to review the literature on AEE from the past two decades (2000–2020). A total of 1,067 papers on FI are obtained, only five of which focus on AEE. Moreover, papers related to AEE for FI are few. The study presents the research gaps, challenges, and potential factors for further research in this context. We conclude that FI for AEE in PHEI can be a game-changer for future sustainability. Moreover, we believe that the outcome of this review warrants further research.", "keywords": [ "Academic entrepreneurial engagement", "higher education institutions", "Frugal innovation", "Malaysia", "Private university", "Sustainability", "Game changer", "Systematic literature review" ], "content": "Introduction\n\nPrivate higher education institutions (PHEI) play an important role in a nation’s economic development and prosperity by supplying skilled workforce and generating basic knowledge for solving societal problems. Notwithstanding their strategic contributions, PHEI are challenged by dwindling resources and the need to contribute to the economy and social development.1 For instance, in Malaysia, PHEI are faced with rising operating costs, pressure to increase their global ranking, problems in balancing teaching and research task allocation, decreasing government funding and shrinking budget allocations for staff development and empowerment programmes.2\n\nMany PHEI in Malaysia incurred losses for years and are unable to access adequate capital.3 A recent report revealed that 55% of Malaysia’s PHEI incurred losses, and approximately 44% were financially insolvent. As a result, more than 5,800 academics are faced with an uncertain career outlook, and approximately 121,000 students are at risk of receiving poor-quality education.4 Besides, PHEI depend mainly on student fees to offset their operating costs. However, funding sources continue to decline. The annual budget of the National Higher Education Fund Corporation, which is a major provider of educational loans in Malaysia, was severely reduced owing to the high default and low repayment rates of existing loans.4 Thus, to survive, PHEI management must devise effective strategies to attain financial stability amidst dwindling resources.\n\nFurthermore, the COVID-19 pandemic created additional challenges and heightened the impact of existing problems encountered by PHEI. The Movement Control Order (MCO) and border closure policy to curtail the spread of COVID-19 resulted in a drop in student enrolment. Specifically, international student enrolment declined drastically owing to students’ inability to gain entry into the country.5 Prospective students also deferred or delayed their studies, whereas others were unable to pay tuition fees owing to loss of jobs and the economic recession. Inevitably, this situation will further put a strain on the cash flow of institutions5 and the economy. These challenges indicate the urgent need for PHEI management to restrategise to enhance their output and quality amidst dwindling resources.\n\nIn coping with the challenges associated with resource constraints and adversities, academics are expected to venture beyond their traditional responsibilities of conducting research, accomplishing administrative tasks and performing teaching activities and engage in entrepreneurial activities.6 By fostering entrepreneurial practices among academics, PHEI can stimulate innovative outcomes to improve economic growth, create jobs and research opportunities for graduates and support the educational ecosystem.7 PHEI establish initiatives to promote educational ecosystems that are conducive to entrepreneurial activity for academics.8–10 Hence, researchers paid attention to factors influencing university faculty members’ entrepreneurial intention, motivation and behaviour in intrapreneurial activities.11 Nevertheless, insights into the antecedents of academic entrepreneurial engagement (AEE) to enhance innovative performance are limited.\n\nEarly attempts to promote academic engagement in entrepreneurial activities can be traced back to the policy initiatives of the Bayh–Dole Act in the United States in 1980, which aimed to contribute to economic development and promote commercialisation and other forms of university technology transfer.12 Recent studies on AEE focused on different types of engagement, i.e. consultations, collaborations, contract research, training joint publications, advisory board membership, industry findings, conferences and so on.13 Moreover, researchers examined the determinants and outcomes of academic entrepreneurial intention.14 Academic entrepreneurial intention is underscored by capability factors such as entrepreneurial-related abilities, education, human capital and industrial experience.14\n\nAEE entails the commercialisation of scientific knowledge, including the transformation of knowledge into products and processes that may inevitably contribute to economic growth and innovation. AEE refers to academic engagement in activities beyond the traditional functions of research, administration and teaching, including formal and informal activities, such as collaborations with businesses and industries, new-firm creation, research output patenting, invention disclosures by academics to technology transfer offices, academic knowledge transfer, research output licensing, contract research, consulting, research collaborations, knowledge transfer mechanisms, student placements, training and continued professional development, leading to financial rewards for individual academics and institutions.15 Academic engagement in entrepreneurial activities supports knowledge transfer and technology commercialisation and can contribute significantly to entrepreneurial ecosystem development.\n\nPHEI are faced with increasing challenges related to shrinking budgets, adapting to changing contexts and contributing to economic development and competitiveness. Such challenges encourage universities to embrace significant transformation towards becoming an entrepreneurial entity.14 To overcome challenges, considerable pressure is placed on academics to become entrepreneurial. The literature ascertains the role of individual academics16 through their effective transfer of knowledge to form new ideas and inventions that can help universities achieve their entrepreneurial mission and promote sustainable development.\n\nCurrent studies on AEE focused on factors shaping academic entrepreneurial intention, exploring individual, organisational and institutional variables.17,18 However, social recognition, peer pressure and academic effective duty were identified as factors influencing academic entrepreneurial intention.13 Previous research explored various academic forms of engagement, such as spinoffs, firm creation, licences and joint ventures and so on.17,18 Other studies investigated factors motivating individual academics to start a business, such as their work routine, need for independence and desire to become wealthy.\n\nPrevious studies indicated that AEE is essential for creating an entrepreneurial university.19–21 Academic engagement in entrepreneurial activities can help enhance university global performance through frugal innovation (FI). FI may be a game changer for PHEI to leapfrog into the arena of effective academic entrepreneurial strategies based on limited resources. Hence, examining the potential of AEE to support PHEI is crucial.\n\nFI is also known as jugaad innovation, which demands rapid and rational adaptability to changing conditions.22,23 FI changed the nature of innovation as the ‘ability to accomplish more with less’, resulting in increased economic and societal value whilst reducing resource consumption.23 The main characteristics of FI are based on cost dimensions, such as reducing costs or purchase prices and the overhead on nonessential activities, increasing ‘value proposition’, decreasing unnecessary functions and features that are non-value adding and minimising the use of resources.24,25 Therefore, FI enables the significant reduction in resource consumption whilst achieving superior-quality standards and considering the goal of creating a frugal environment. The concept of FI requires further in-depth research using conceptual and empirical techniques.\n\nIn higher learning institutions (HLIs), some academicians and management personnel emphasise teaching and learning, student services and community participation, whereas others are concerned with achieving national goals by developing industry-oriented technical innovation, interacting with businesses and engaging in technology transfer. Clearly, HLIs contribute by engaging in R&D collaboration, skills development training, commercialisation through spinoffs and the co-creation of knowledge and ideas and entrepreneurial and technical expertise to overcome community challenges and attain national goals. Hence, emphasis is placed on exploring FI principles as a game changer for the sustainability of PHEI.\n\nThis study will provide academic researchers with insights into what has been done with regard to FI for AEE and PHEI to prioritise the factors that can influence AEE for FI in PHEI.\n\nGiven this background, the research questions of this study are as follows:\n\n1. Does a research gap exist in AEE studies to support FI pertaining to motivation, opportunity and the ability of academics in PHEI?\n\n2. What are the challenges encountered by academics and the key factors influencing the pursuit of entrepreneurial initiatives in PHEI?\n\n3. Can AEE for FI be a game changer for supporting constraints in PHEI?\n\nThe objectives of this proposal are as follows:\n\n1. To identify the research gaps in studies on FI for AEE pertaining to motivation, opportunity and the ability of academics in PHEI\n\n2. To investigate the challenges faced by academics in pursuing entrepreneurial initiatives in PHEI\n\n3. To explore the role of FI in AEE as a game changer for PHEI\n\nWe examine 1,067 papers on FI for AEE in PHEI and identify four major gaps in the literature. We believe that our findings warrant the attention of the research community.\n\n\nMethods\n\nThis paper was designed to present the identified research gaps and insights to examine FI as a game changer for AEE. Based on the literature, key factors influencing AEE to achieve FI in HLIs were explored. This literature review was based on the five stages of the systematic review as illustrated in Figure 1 proposed by Tranfield et al., 2003.26\n\nThis review aims to identify the research gaps, challenges and factors supporting FI for AEE in PHEI. In addition, this review aims to offer researchers a comprehensive review of previous works related to FI to support AEE initiatives, specifically in the form of a conceptual framework. Finally, the outcome of this review process will offer academic entrepreneurial communities a series of research ideas to advance the field.\n\nIn this stage, papers were examined for the selected keywords. The keywords included general key terms (‘AEE’) regarding specific keywords (‘FI for AEE in PHEI’). The focus of this review is to analyse research on AEE in PHEI. The factors and challenges mentioned in the literature were further classified into several categories.\n\nInclusion and exclusion criteria\n\nFigure 2 shows the inclusion and exclusion criteria for selecting the papers. The selected papers were those published in the past two decades, peer reviewed, related to AEE focusing on FI and of scholarly origin and either conceptual or empirical papers.\n\nKeywords\n\nWe focused on six major research areas, namely, (1) AEE, (2) FI, (3) institutions of higher learning (IHL), (4) motivation, (5) ability and (6) opportunity. For the first area, we included terms such as ‘Academic Entrepreneurial Engagement’ and ‘Frugal Innovation’. Each keyword set was combined with other keywords. Table 1 presents the keyword sets used for this research\n\nSearch strategy\n\nThe strategy used to collect the papers was based on five major online databases. The online databases were selected based on their wide range of social science research. Table 2 presents the results based on all the sources mentioned above and keyword sets. A total of two papers were listed when we used the keyword ‘Academic Entrepreneurial Engagement’. When we searched for ‘FI’, 1,067 papers were retrieved. When we used the keywords ‘AEE’ AND ‘Motivation’, two papers were listed. However, no papers were retrieved when we used ‘AEE’ AND ‘Opportunity’ and ‘FI’ AND ‘AEE Ability’. Finally, ‘AEE’ AND ‘Ability’ listed one paper. After careful selection based on the inclusion and exclusion criteria described above, we identified five papers relating to AEE and FI.\n\nFrom the sources mentioned above, we extracted the papers based on the extraction process in Figure 3.\n\nFigure 3 summarises the paper selection criteria for the review. From the databases and other sources, only AEE papers linked to motivation/opportunity/ability/FI/IHL were chosen for further review. The relevant papers based on the selection criteria are reported in the following subsections. From the 1,067 papers, we selected five for the final review.\n\nStages 4 and 5 of the Tranfield process are discussed in the following sections.\n\nInstitutional Review Board Statement: Research Ethical Committee (REC) of Multimedia University (EA1392021). The study was conducted according to the guidelines and approved by the REC of Multimedia University, Cyberjaya, Malaysia.\n\n\nResults\n\nBased on Tables 3 and 4, our findings revealed that no research was conducted in the area of FI for AEE related to motivation, opportunity and the ability of academics in PHEI. Most of the studies focused on factors shaping academics’ entrepreneurial intention, exploring individual, organisational and institutional variables.12,17,18 The studies on academic entrepreneurial intention were conceptualised from different theoretical perspectives, including theory of planned behaviour (TPB), social learning theory and motivational theories. Based on TPB, academic attitude, subjective norms and behaviour control were associated with entrepreneurial intention.27,28 Moreover, other factors such as perceived feasibility and desirability were determined to influence entrepreneurial intention, leading to actual behaviours.29,9 Therefore, further research is necessary to explore the role of motivation, ability and AEE opportunity for FI.\n\nChallenges\n\nSeveral challenges constrain academic engagement in entrepreneurial activities. Such challenges were categorised as individual factors, which constrained personal engagement; organisational factors, which were structural and systemic inhibitors of engagement; and process factors, which included institutional policies detrimental to academic entrepreneurial behaviour.\n\nBased on Table 5, existing studies highlighted the need for academics to assume job responsibilities beyond teaching and research as a major challenge to their engagement in entrepreneurial activities.13 Academics are expected to extend their research output to products and services suitable for the industry and society. Thus, their research outcomes should be commercialised by creating new firms without breaching current laws that govern their role within the university.30\n\nAcademics are challenged to develop the appropriate competencies to become entrepreneurial to help create a firm. Competence refers to the academic ability to identify resources to start a business.31 In addition, understanding the dynamics shaping the development of entrepreneurial competencies can improve capabilities to maximise resource use through FI, which means ‘accomplishing more with less’, thereby resulting in increased economic and societal value whilst reducing resource consumption.23\n\nFactors\n\nPrevious studies determined the relevant factors related to AEE. Regarding socioeconomic characteristics, gender, age, income and personality (proactiveness and optimism) were associated with academic entrepreneurial behaviour.32 Drawing on TPB, Wang et al.33 investigated the role of academic output and prior experience as individual factors and university reputation and supportive climate as organisational factors influencing AEE.34 identified cultural factors as contextual inhibitors of academic competencies for entrepreneurial engagement. These studies are further summarised in Table 6.\n\n\nDiscussion\n\nThe discussions are described in Table 7 to answer the research questions as shown below.\n\nLimitation of this study is on the number of keywords selected. Keywords selections are based on research focus. However, there is possibility of obtaining more articles if the keywords are expanded to field of study that are not specific in nature such as intention. This could possibly be publication bias.\n\nWe recommend future research to consider the following topics:\n\n• AEE factors involving the COVID-19 MOC and other related aspects\n\n• AEE for FI in PHEI focusing on adult learners\n\n• AEE using qualitative methods that may unveil in-depth knowledge within this context based on actual academic entrepreneur case studies\n\n\nConclusion\n\nThe purpose of this study is to notify academic entrepreneurial communities of the disparity in the implementation of FI to enhance AEE in studies published in the past 20 years. Our paper is based on an extensive review of the literature and the application of a methodology that includes five stages. This methodology is used to determine the scope and nature of FI to promote AEE. Despite the sizeable search result of 1,067 papers in the AEE domain, our search record is limited to five papers investigating FI for AEE. Based on our rigorous review of the five papers, we recommend further comprehensive research to determine whether FI is related to AEE and identify the challenges and factors supporting FI for AEE in PHEI and additional empirical work.\n\nPHEI that can achieve FI will be able to enhance their AEE and contribute to the economy and social development, thereby creating IR 4.0 educational ecosystems.\n\n\nData availability\n\nFigshare. Excel file- Dataset.xlsx. DOI: https://doi.org/10.6084/m9.figshare.14872554.v1.35\n\nFigshare. Guideline Checklist\n\nDOI: https://doi.org/10.6084/m9.figshare.16682626.v2.36\n\nThis project contains the following data:\n\n- This dataset is analysed for theories, type of papers, Academic entrepreneurial engagement factors and challenges, method and findings\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 Ministry of Higher Education, Malaysia, for awarding us the Fundamental Research Grant Fund (FRGS/1/2020/SS02/MMU/02/3) to conduct this research. The findings shared in this paper are part of this project.\n\n\nReferences\n\nLee MNN: Higher education in Malaysia: National strategies and innovative practices. Mass Higher Education Development in East Asia: Strategy, Quality, and Challenges. Springer International Publishing;2015; pp. 105–118. Publisher Full Text\n\nDahlan ARA, Ibrahim J, Razi M, et al.: Redesign business model options for university of the future and staying relevant in the fourth industrial revolution age.2018.\n\nNorliza MZ, Velantina A, Normazni A, et al.: Challenges A and evolution of higher education in malaysia. Int. J. Islam. Civilisational Stud. 2017; 4: 78–87. Publisher Full Text\n\nAziz A: Private colleges to suffer without loans. The Malaysian Reserve. 2019.\n\nMenhat M, Mohd Zaideen IM, Yusuf Y, et al.: The impact of Covid-19 pandemic: A review on maritime sectors in Malaysia. Ocean Coast. Manag. Aug. 2021; vol. 209: p. 105638. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerkmann M, et al.: Academic engagement and commercialisation: A review of the literature on university-industry relations. Res. Policy. Mar. 2013; 42(2): 423–442. Publisher Full Text\n\nMiller K, Alexander A, Cunningham JA, et al.: Entrepreneurial academics and academic entrepreneurs: A systematic literature review. Int. J. Technol. Manag. 2018; 77(1–3): 9–37. Publisher Full Text\n\nDavey T, Galan-muros V: Understanding entrepreneurial academics - how they perceive their environment differently.2020; 39(5): 599–617. Publisher Full Text\n\nGuerrero M, Rialp J, Urbano D: The impact of desirability and feasibility on entrepreneurial intentions: A structural equation model. Int. Entrep. Manag. J. 2008; 4: 35–50. Publisher Full Text\n\nVan Der Zwan P, Thurik R, Verheul I, et al.: Factors influencing the entrepreneurial engagement of opportunity and necessity entrepreneurs. Eurasian Bus. Rev. 2016; 6: 273–295. Publisher Full Text\n\nSiegel DS, Wright M: Academic Entrepreneurship: Time for a Rethink?. Br. J. Manag. Oct. 2015; 26(4): 582–595. Publisher Full Text\n\nLink AN, Siegel DS, Bozeman B: An empirical analysis of the propensity of academics to engage in informal university technology transfer. Universities and the Entrepreneurial Ecosystem. Edward Elgar Publishing Ltd.;2017; pp. 97–111. Publisher Full Text\n\nAbreu M, Grinevich V: The nature of academic entrepreneurship in the UK: Widening the focus on entrepreneurial activities. Res. Policy. 2013; 42(2): 408–422. Publisher Full Text\n\nNeves S, Brito C: Academic entrepreneurship intentions: a systematic literature review. J. Manag. Dev. Jun. 01, 2020; vol. 39(no. 5): pp. 645–704. Emerald Group Holdings Ltd. Publisher Full Text\n\nMirani MA, Yusof M: Entrepreneurial Engagements of Academics in Engineering Universities of Pakistan\|Elsevier Enhanced Reader. Procedia. 2015. (accessed May 28, 2021).Reference Source\n\nWright M, Phan P: The commercialization of science: From determinants to impact. Acad. Manag. Perspect. Feb. 01, 2018; vol. 32(no. 1): pp. 1–3. Academy of Management.Publisher Full Text\n\nBozeman B, Fay D, Slade CP: Research collaboration in universities and academic entrepreneurship: the-state-of-the-art. J. Technol. Transf. 2013; 38: 1–67. Publisher Full Text\n\nWright M: Academic entrepreneurship, technology transfer and society: where next?. J. Technol. Transf. 2014; 39: 322–334. Publisher Full Text\n\nEtzkowitza E, Websterb A, Gebhardtc C, et al.: The future of the university and the university of the future: evolution of ivory tower to entrepreneurial paradigm. Res. Policy. 2000; 29(2): 313–330. Publisher Full Text\n\nHannon PD: Why is the Entrepreneurial University Important?. J. Innov. Manag. Dec. 2013; 1(2): 10–17. Publisher Full Text\n\nZhao Z, Broström A, Cai J: Promoting academic engagement: university context and individual characteristics. J. Technol. Transf. 2020; 45(1): 304–337. Publisher Full Text\n\nRadjou N, Prabhu J, Ahuja S: Jugaad Innovation: Think Frugal, Be Flexible, Generate Breakthrough Growth. United State of America:Josse-Bass;2012.\n\nRadjou N, Prabhu J: Frugal Innovation: How to do more with less. London:Profile bo;2014.\n\nTiwari R, Kalogerakis K, Herstatt C: Frugal Innovations in the mirror of scholarly discourse: Tracing theoretical basis and antecedents.2016.\n\nWeyrauch T, Herstatt C: What is frugal innovation? Three defining criteria. J. Frugal Innov. 2016; 2(1): 1–17. Publisher Full Text\n\nTranfield D, Denyer D, Palminder S: Towards a Methodology for Developing Evidence-Informed Management Knowledge by Means of Systematic Review. Br. J. Manag. 2003; 14(3): 207–222. Publisher Full Text\n\nKautonen T, Van Gelderen M, Tornikoski ET: Predicting entrepreneurial behaviour: a test of the theory of planned behaviour. Appl. Econ. 2013; 45(6): 697–707. Publisher Full Text\n\nTrivedi R: Does university play significant role in shaping entrepreneurial intention? A cross-country comparative analysis. J. Small Bus. Enterp. Dev. 2016; 23(3): 790–811. Publisher Full Text\n\nFitzsimmons JR, Douglas EJ: Interaction between Feasibility and Desirability in the Formation of Entrepreneurial Intentions. J. Bus. Ventur. 2011; 26: 431–440. Publisher Full Text\n\nHalilem N, Amara N, Olmos-Peñuela J, et al.: ‘To Own, or not to Own?’ A multilevel analysis of intellectual property right policies’ on academic entrepreneurship. Res. Policy. Oct. 2017; 46(8): 1479–1489. Publisher Full Text\n\nDe Silva L: Academic Entrepreneurship in a Resource Constrained Environment - ProQuest.2012. (accessed Jun. 15, 2021).Reference Source\n\nSulaimon A-H, Adebiyi S, Amole B: Academic Enterpreneurship and Enterprenerial Development in Nigeria: Myth or Reality?. Vestn. Volgogr. Gos. Univ. Ser. 3. Ekon. Ekol. 2016; (4): 147–154. Publisher Full Text\n\nWang M, Cai J, Munir H: Promoting entrepreneurial intentions for academic scientists: combining the social cognition theory and theory of planned behaviour in broadly-defined academic entrepreneurship. Eur. J. Innov. Manag. 2020; 24(2): 613–635. Publisher Full Text\n\nGümüsay AA, Bohné TM: Individual and organizational inhibitors to the development of entrepreneurial competencies in universities. Res. Policy. Mar. 2018; 47(2): 363–378. Publisher Full Text\n\nOjo OT, Dorasamy M, Wigin MW, et al.: Excel file- Dataset.xlsx. figshare. Dataset. 2021. Publisher Full Text\n\nOjo OT, Dorasamy M, Wigin MW, et al.: PRISMA_2020_checklist- 1.docx. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "97934", "date": "23 Nov 2021", "name": "Murali Sambasivan", "expertise": [ "Reviewer Expertise Management Science", "Operations and Supply Chain Management", "General Management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article ‘Academic entrepreneurial engagement (AEE) for frugal innovation (FI) in higher education institutions: a systematic literature review’ highlights the need for private higher educational institutions (PHEI) to move beyond teaching and research to overcome the current challenges such as COVID-19 pandemic, shrinking resources and so on. Specifically, the article deals with the systematic literature review related to engagement in entrepreneurial activities by academics through frugal innovation (FI). The article concludes that FI for AEE in PHEI can be a game-changer for future sustainability.\n\nThe theme of the research ‘the idea of academic entrepreneurial engagement leading to innovation, identification of opportunities for new business ventures, financial rewards for institutions and academics, impact on the economy, and enhancement of social welfare ’ is interesting and requires further empirical research. This idea needs to be explored in both public and private higher learning institutions.\n\nI am highlighting below a few points that can be addressed to enhance the quality of the paper.\n\nMajor Points:\n\nThe introduction section must motivate the reader to read the complete article. Therefore, typically, the introduction section must address four questions: What do we know? What else do we need to know? Why do we need to know? What will be done in this research? The authors must revisit introduction based on these questions.\n\nWhy frugal innovation? Why not just, innovation?\n\nIntroduction must explicitly state the theoretical and practical contributions of this research.\n\nResearch questions and objectives are same. It is sufficient to have questions or objectives, unless they are different.\n\nWhy were motivation, opportunity, and ability used as keywords? Probably the keywords limited the number of papers selected.\n\nDiscussion section must be elaborated with more references and examples. I hardly see, any.\n\nMinor point:\n\nTo reedit the manuscript.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "7522", "date": "07 Feb 2022", "name": "Magiswary Dorasamy", "role": "Author Response", "response": "Review Comment: The introduction section must motivate the reader to read the complete article. Therefore, typically, the introduction section must address four questions: What do we know? What else do we need to know? Why do we need to know? What will be done in this research? The authors must revisit introduction based on these questions. The introduction section must motivate the reader to read the complete article. Therefore, typically, the introduction section must address four questions: What do we know? What else do we need to know? Why do we need to know? What will be done in this research? The authors must revisit introduction based on these questions. Response: Thank you for your comment. The introduction has been revised as suggested. Please see paragraph1, page 2-4 Reviewer Comment: Why frugal innovation? Why not just, innovation? Response: Thank you for your comment. The PHEI is facing resource constraints especially budget constraints. As such, frugal innovation will help PHEI to accomplish more with less. Please see paragraph 3, page 4 Reviewer Comment: Introduction must explicitly state the theoretical and practical contributions of this research. Response: Thank you for your comment. The theoretical and practical contributions have been addressed in the introduction section. Reviewer Comment: Research questions and objectives are same. It is sufficient to have questions or objectives, unless they are different Response: Thank you for your comment. We have retained the questions and removed the objectives. Reviewer Comment: Why were motivation, opportunity, and ability used as keywords? Probably the keywords limited the number of papers selected. Response: Our emphasis is to identify how AEE by way of motivation, opportunity and ability so that PHEI can achieve its goals (please see paragraph 2, page 4) Reviewer Comment: Discussion section must be elaborated with more references and examples. I hardly see, any. Response: Thank you for the comments. We did not cite the citations in this section as the discussion referred to the tables which contain the reference." } ] } ]	1	https://f1000research.com/articles/10-1078
https://f1000research.com/articles/11-868/v1	29 Jul 22	{ "type": "Research Article", "title": "Effects of vaccination against COVID-19 on the emotional health of older adults", "authors": [ "Christoper A. Alarcon-Ruiz", "Zoila Romero-Albino", "Percy Soto-Becerra", "Jeff Huarcaya-Victoria", "Fernando M. Runzer-Colmenares", "Elisa Romani-Huacani", "David Villarreal-Zegarra", "Jorge L. Maguiña", "Moises Apolaya-Segura", "Sofía Cuba-Fuentes", "Zoila Romero-Albino", "Percy Soto-Becerra", "Jeff Huarcaya-Victoria", "Fernando M. Runzer-Colmenares", "Elisa Romani-Huacani", "David Villarreal-Zegarra", "Jorge L. Maguiña", "Moises Apolaya-Segura", "Sofía Cuba-Fuentes" ], "abstract": "Background: The COVID-19 pandemic significantly impacted the mental and emotional health of the elderly, especially those from low to middle-income countries. However, COVID-19 vaccination may reduce this influence. Therefore, we aimed to estimate the effect of vaccination against COVID-19 on the emotional health of older adults. Methods: We selected a national, random, and stratified sample of non-hospitalized adults aged 60 to 79 years from Peru who intended to receive or had already received the COVID-19 vaccine during recruitment. During June and July 2021, the assessed outcomes were the fear, anxiety, and worry about COVID-19, general anxiety, and depression at baseline and after a month. We estimated the adjusted odds ratios (aOR) and 95% confidence intervals (95% CI) for each altered emotional health outcomes in those who had one and two doses, compared with those who were not vaccinated using multilevel logistic regression with mixed effects. Results: We recruited 861 older adults with 20.8% of loss to follow-up. At baseline, 43.9% had received only one dose of the vaccine, and 49.1% had two doses. In the analysis during follow-up, those who had two doses had less fear (aOR: 0.19; CI 95%: 0.07 to 0.51) and anxiety to COVID-19 (aOR: 0.45; CI 95%: 0.22 to 0.89), compared to unvaccinated. We observed no effects in those with only one dose. Conclusions: Two doses of COVID-19 vaccination in older adults improves their perception of COVID-19 infection consequences. This information could be integrated into the vaccination campaign as an additional beneficial effect.", "keywords": [ "Aged", "COVID-19", "Mental Health", "Anxiety", "Depression", "Peru" ], "content": "Abbreviations\n\nCAM: Centers for the eldery\n\nCAS: Coronavirus Anxiety Scale\n\nEsSalud: Social Health Insurance of Peru\n\nFCV-19S: Fear of COVID-19 Scale\n\nGAD-2: Generalized Anxiety Disorder\n\nPHQ-2: Patient Health Questionnaire\n\nPRE-COVID-19: Scale to measure worry for contagion of the COVID-19\n\n\nIntroduction\n\nThe COVID-19 pandemic has resulted a significant impact on the mental health of people worldwide.1 Approximately 15% of older adults had a mental health disorder before the pandemic (https://www.who.int/news-room/fact-sheets/detail/mental-health-of-older-adults), but older adults have reported greater declines in social communication, exercise, and finances during the pandemic years compared to young adults.2,3 In addition, the high contagiousness of COVID-19 and higher risk of death and complications in the elderly population,4,5 may have caused a worsening of sleep quality, well-being, depressive, and anxious symptoms, since the beginning of the pandemic.6 It is estimated that the damage has been especially profound in older adults in low- and middle-income countries compared to those in developed countries.7 Furthermore, the ministries of health from Latin American countries have not prioritized strategies or policies that deal with emotional and mental health problems during the pandemic, which could cause the impact on these aspects to be greater.8 For instance, Peru is one of the countries with the highest mortality rate from COVID-19 per million inhabitants in the world9 and it has had a significant economic and social impact.10 In this context, high levels of worry, anxiety, and fear of COVID-19 have been described in people living in Peru.11–13\n\nVaccination against COVID-19 has meant a change in the pandemic dynamics14 due to its proven effectiveness in reducing severe cases and deaths from COVID-19 in the general population and older adults.15,16 Mental health status during the pandemic could be related to COVID-19 vaccination in different ways (Figure 1). First, mood disorders such as stress, depression, and loneliness can decrease the immune system response caused by the COVID-19 vaccine.17 In addition, in older adults, having a mental disorder may be associated favorably18,19 or negatively20,21 with the willingness to be vaccinated against COVID-19. On the other hand, doubts about the COVID-19 vaccine correlate with high levels of anxiety, depression, and post-traumatic stress,22 which can cause acute episodes of anxiety immediately after receiving the COVID-19 vaccine.23 Finally, studies in the general population of the United States24–26 and in health professionals in Turkey27 suggest that receiving the COVID-19 vaccine may have a direct effect in reducing levels of anxiety and depression.\n\nOlder adults are a priority group to receive the COVID-19 vaccine28 which it may positively affect their emotional health (https://www.northwell.edu/coronavirus-covid-19/covid-19-vaccine-can-boost-mental-health-too). However, this population is underrepresented in studies evaluating this association. A better understanding of the relationship between vaccination against COVID-19 and emotional health would improve knowledge of the positive determinants of health in the population.29 The positive effect that vaccination against COVID-19 could generate in the older adult population would imply an improvement in their quality of life during the pandemic, and policies and promotion of vaccination could be oriented in this direction. So, it is necessary to know how receiving the COVID-19 vaccine could affect the perception of pandemic and risk of disease in older adults. The aim of the present study was to assess the effect of receiving the COVID-19 vaccine on emotional health in a representative group of older adults in Peru during the year 2021.\n\n\nMethods\n\nPeru is a country with a fragmented and heterogeneous health system. Among them, the Social Health Insurance of Peru (EsSalud) is one of the most important health systems in the country and is managed by the Ministry of Labor and Employment Promotion.30 EsSalud gives medical attention to formal workers, retirees, and their families. The executive function of EsSalud is divided through the 29 healthcare networks, representative of each region in the country. EsSalud provides health coverage to almost a third of Peruvians, including more than 1 890 000 older adults, of which 100 000 of them are users of the Centers for the Elderly (CAM, in Spanish) present in each healthcare network in the country.31 The CAMs provide health services that seek to improve the functional, mental, and social capacity of people aged 60 or older affiliated with EsSalud.32\n\nThe vaccination process against COVID-19 in Peru, organized by the Ministry of Health, was carried out by age groups, and began on April 16, 2021, with adults over 80 years old. Vaccination started on April 28, 2021, for adults over 70 years old, and on May 27, 2021, for adults over 60 years old. During that period, the BNT162b2 (BioNTech, Pfizer), ChAdOx1-S (Oxford, AstraZeneca), and BBIBP-CorV (Sinopharm) vaccines were available in Peru. However, the Peruvian Ministry of Health indicated that vaccination should be prioritized with BNT162b2 (BioNTech, Pfizer) for older adults with 60 years or older (https://cdn.www.gob.pe/uploads/document/file/1893194/Directiva%20%20Sanitaria%20N%C2%B0%20133-MINSA-2021-DGIESP%20.pdf). As a result, as of December 29, 2021, 80.8% of older adults who received at least one dose of the COVID-19 vaccine received BNT162b2 (BioNTech, Pfizer), while 10.5% and 8.7% had received the ChAdOx1-S vaccine (Oxford, AstraZeneca) and BBIBP-CorV (Sinopharm), respectively (https://www.minsa.gob.pe/reunis/data/vacunas-covid19.asp). However, the vaccination of older adults occurred during a political and health scandal in the country; Inoculation of the BBIBP-CorV (Sinopharm) vaccine candidate, outside the clinical trial, to 470 people in Peru, including health personnel and politicians.33,34 For several months, this caused a credibility crisis for COVID-19 vaccines, particularly BBIBP-CorV (Sinopharm).35\n\nA prospective cohort study was conducted that aimed to estimate the effect of the COVID-19 vaccine on the following emotional health outcomes of older adults affiliated to EsSalud CAMs: (a) Perception of fear, (b) anxiety, and (c) worry about COVID-19, (d) general depression, and (e) general anxiety.\n\nWe identified non-hospitalized adults aged 60 to 79 years affiliated to EsSalud and registered in the CAM available database at national level. The database represented 0.5% of all older adults affiliated to EsSalud. We included those older adults who had already been vaccinated or who had planned to be vaccinated against COVID-19 according to the Peruvian Ministry of Health vaccination schedule at a vaccination site in Peru. We excluded adults aged 80 years or older because more than one month had elapsed since the start of vaccination in this age group by the initiation of the recruitment. We also excluded those who had some impediment to adequate communication with the interviewer via the telephone call, were diagnosed with COVID-19 in the last three months, had symptoms related to COVID-19, or refused to participate during the interview. The recruitment period was from May 27 to June 30, 2021.\n\nBased on the recommendation of Cohen et al.,36,37 we consider an effect size of 0.20 standard deviations for the smallest effect36 estimated between any of the outcomes (perception score for fear, anxiety, and worry about COVID-19, general depression, and anxiety) and the exposure factor (unvaccinated vs. vaccinated with only one dose or vaccinated with two doses) in older adults. Assuming a significance level of 5%, statistical power of 80%, and equal variances between groups, we calculated a minimum sample of 788 older adults. Then, we corrected this value by a factor of 1.2, following the methodology proposed by Vititingghoff E. et al.,38 considering the primary analysis with adjustment for potential confounding variables. Thus, we obtained a minimum sample size of 946 older adults. Finally, we considered a rejection rate of 10% and a loss to follow-up rate of 10%; then, we planned to invite 1168 participants to the study. Further details are found in the extended data (Supplementary methods: Sample size calculation).39\n\nAfter excluding those who did not meet the selection criteria or did not have identification or contact data, we took 7 685 older adults from the registered CAMs into the database as a sample frame. From them, we chose a randomized and stratified sample for each of the 30 healthcare networks (n = 1 686). In addition, the sampling was carried out independently for two age subpopulations: 60 to 69 years (n = 846) and 70 to 79 (n = 840) years. For each subpopulation, we chose half of the calculated total sample size (allocation ratio 1:1). We decided to choose two age subpopulations because the recruitment period was during the start of vaccination of adults older than 60 to 69 years and one month after the initiation of vaccinations to adults older than 70 to 79 years (https://cdn.www.gob.pe/uploads/document/file/1893194/Directiva%20%20Sanitaria%20N%C2%B0%20133-MINSA-2021-DGIESP%20.pdf). The characteristics of the eligible population and selected sample are found in the extended data (Supplementary methods: Characteristics of the eligible population).39 To reduce non-response bias, we adjusted sample weights to account for non-response using weighting class adjustment.40,41 Further details are found in the extended data (Supplementary: Sampling weights calculations).39\n\nWe assessed five outcomes in emotional health: fear of COVID-19, anxiety about COVID-19, worry about COVID-19, general anxiety, and general depression, perceived by the respondents during the last two weeks before responding to the survey. Fear of COVID-19 was measured with the Spanish version of the Fear of COVID-19 Scale (FCV-19S) with seven items that are answered on a Likert scale from 1 (strongly disagree) to 5 (strongly agree).42 This scale measures the emotional and somatic fear response to COVID-19. It has adequate internal consistency (Cronbach’s α = 0.88) and convergent validity with other mental health covariates in two non-probabilistic samples of adults and older adults from Lima, Peru.42,43 The anxiety about COVID-19 was measured using the Spanish version of the Coronavirus Anxiety Scale (CAS), which measures persistent and excessive concern about COVID-19 that is accompanied by physical symptoms44 and has five items that are answered on a Likert scale from 0 (not at all) to 4 (almost every day).45 It is a unidimensional scale with adequate internal consistency (Cronbach’s α = 0.89 and 0.91), convergent validity with anxiety, and adjustment rates between females and males, and between older adults aged 60 to 65 years and 66 to 86 years. However, estimations in samples of adults and older adults from Lima, Peru, using Item Response Theory models, suggest that the instrument is more reliable in those with high anxiety levels to COVID-19.46,47 The outcome ‘worry about COVID-19’ was measured with the scale to measure worry about contagion of COVID-19 (PRE-COVID-19, in Spanish), which contains six items that are answered on a Likert scale of 1 (never or rarely) to 4 (almost all the time). This scale measures the degree of worry about possible COVID-19 infection in the respondent and how this concern affects their state of mind and their ability to carry out their daily activities. It was developed in a non-probabilistic sample of adults between 18 and 50 years of age from Lima and Callao, Peru, and it proved to be unidimensional, to have adequate content validity, internal consistency (ω coefficient = 0.90), and convergent validity with other mental health covariates.12 A higher total score on each scale means a higher respondent’s outcome level. Then, we dichotomized the total scores for each outcome and considered the upper quartile as a high outcome level.\n\nThe general anxiety outcome was measured with the Spanish version of the Generalized Anxiety Disorder (GAD-2), which contains two items that are answered on a Likert scale from 0 (never) to 3 (almost every day).48 A total score of two or more points can diagnose clinically relevant anxiety in older adults, with 67% sensitivity and 90% specificity.49 The general depression outcome was measured with the Spanish version of the Patient Health Questionnaire (PHQ-2), which contains two items that are answered on a Likert scale from 0 (no day) to 3 (almost every day).50 A total score of three or more points can diagnose clinically relevant depression in older adults without cognitive impairment with 79% sensitivity and 82% specificity.51\n\nWe asked about self-reported COVID-19 vaccination status (no dose, only first dose, and two doses of vaccine) and the time in days since they received each dose of the vaccine.\n\nIn addition, we asked about sociodemographic variables, previous mental health diagnosis and treatment, and personal and family history of COVID-19. In addition, we assessed the comorbidity with the Geriatric Comorbidity Index. This index measures the severity degree of 15 clinical conditions, classifying them from 0 to 4 each (0: no disease, 1: asymptomatic disease, 2: asymptomatic disease with treatment, 3: uncontrolled disease despite treatment, and 4: very serious or life-threatening disease). After presenting the severity degree classification and giving simple and standardized examples about each clinical condition, the interviewers asked the responders to identify their current situation for each clinical condition. According to these scores, the degree of comorbidity was grouped into classes: without comorbidity (all conditions absent), class I (one or more conditions with a severity degree of 1 or less), class II (one or more conditions with a severity degree of 2), class III (one condition with a severity degree of 3), and class IV (two or more conditions with a severity degree of 3, or at least one condition with a severity degree of 4).52\n\nWe conducted a pilot with trained interviewers for data collection for one day using a random sample from the sampling frame of approximately 120 participants. During the pilot, we evaluated the data collection capacity of the interviewers and the availability to participate of the selected older adults. Then, we identified and corrected deficiencies for formal data collection.\n\nAfter sampling, we assign an identification code to each selected older adult to facilitate recognition and monitoring within the program. Then, the interviewers contacted the selected participants through telephone calls, using the telephone numbers registered in the CAM database. Previously trained interviewers made the calls and collected data. In case of not answering two calls on two different days, the older adult was excluded from the study. During the call, the interviewers identified the older adult by asking them for their identity document number. Then, the older adult was invited to participate in the study by requesting their verbal informed consent. If the older adult agreed to participate in the survey, the interviewer asked about the selection criteria. Then, the interviewers collected the baseline data. The data collection interview took approximately 20 minutes.\n\nThe follow-up calls were made between July 1st and July 27th, 2021, considering the date of the baseline interview. During these calls, the interviewers followed the same procedure mentioned above. Again, we considered a lost record if the older adult did not answer the call twice on two days. Emotional health outcomes were asked directly using the questionnaires, but in a different order than before, with items in different places. At the end of the interview, they asked about vaccination against COVID-19.\n\nTrained interviewers registered the collected information through the Google Form platform (Questionnaire in Spanish and English in the extended data: Supplementary methods39), using the identification code of each participant. The principal investigator monitored this database every two days, looking for errors during data collection. In case of suspecting a wrong registration, we coordinated with the responsible interviewer to evaluate the need to re-register said entry.\n\nAll the information collected was automatically recorded in a Microsoft Excel 2021 sheet (Microsoft, WA, United States). Before the analysis, we joined the baseline database with the follow-up database considering the identification code of each older adult interviewed. We reviewed the database for inconsistencies in responses about vaccination. We considered the response in the baseline measurement as the valid one if it was inconsistent with the responses during the follow-up measurement. Then, we performed the descriptive analysis of the results in the total number of recruits and separately according to the vaccination status against COVID-19. We describe the relative and absolute frequencies of the qualitative variables and the mean ± standard deviation of the quantitative variables. The baseline prevalence of altered emotional health outcomes and their 95% confidence intervals (95% CI) were plotted for the total sample and separated according to COVID-19 vaccination status. We calculated the 95% CI with the logit adjust method for the design degrees of freedom.53\n\nWe assessed, in the baseline, the association between the vaccination situation and the outcomes in emotional health. We compared medians and interquartile ranges of emotional health outcome scores with the Kruskal Wallis test and the frequency of altered emotional health outcomes with the Chi-squared test. We estimated the odds ratios (aOR) and 95% CI for having adjusted altered emotional health outcomes by sex, age, time in days since receiving the last dose of vaccine (unvaccinated were assigned with zero), living with someone, comorbidity, history of emotional health, and history of COVID-19, using a logistic regression model.\n\nFinally, we assessed the association between vaccination status and altered emotional health outcomes, performing multilevel logistic regression models with mixed effects with a three-level structure. In addition to the first individual level of each measurement, we used random intercepts for the healthcare network and each individual as the other two levels. This analysis allows us to calculate the effects considering the stratum of the care network and the correlations of the two responses over time within the same individual, thus allowing a longitudinal and stratified assessment. In addition, we adjusted the regression model for the confounding variables: time in days since receiving their last dose of vaccine, sex, age, living with someone, comorbidity, history of mental health disease, history of COVID-19, and time in days since the basal measurement (The basal measurement records had a value of 0).\n\nIn all the previously mentioned analyses, we consider the study’s sample weights and the stratum using the svyset command. A value of p < 0.05 was considered statistically significant to reject the null hypothesis in all statistical tests. Statistical software STATA MP v17 (StataCorp, Texas, USA) was used for the analysis.\n\nEach participant gave verbal informed consent before being included in the study. The anonymity of the interviewees was always maintained, assigning each one an identification code. Therefore, the survey didn’t collect personally identifiable information. In cases where a participant had an acute event in her mental health, the interviewer immediately referred the participant to a psychiatrist free of charge who managed the event by telephone. The protocol is registered in the PRISA repository of the Peruvian National Institute of Health (ID code: EI00000001999), and it was approved by the Institutional Review Board of the Instituto Nacional del Corazón - EsSalud (Certificate of approval 25/2021-CEI).\n\n\nResults\n\nWe randomly selected and invited 1,686 older adults to the study. A total of 51.1% (n = 861) of them met the selection criteria for baseline measurement. Among the reasons for not participating in the study were not responding to the call (n = 695), having been diagnosed with COVID-19 in the last three months (n = 57), refusing to participate (n = 51), not wanting to be vaccinated (n = 11), have symptoms related to COVID-19 at the time of the interview (n = 8) and have been vaccinated in another country (n = 3). Then, 20.8% (n = 179) refused to participate or did not respond to the call for follow-up measurement (Figure 2). The frequency distribution of gender and mean age were similar between those who did not participate in the baseline measurement or during the follow-up measurement compared to the recruited patients (Table S1 in the underlying data: Supplementary results39).\n\nThe main characteristics of older adults aged 60 to 79 years affiliated with EsSalud’s CAMs are in Table 1. 54.5% of widowers and divorcees older adults had been in that status for less than a year. Since the pandemic’s start, 20.8% have been hospitalized among those ever diagnosed with COVID-19. On the other hand, among older adults who had at least one family member, whom they live, with a diagnosis of COVID-19, since the start of the pandemic, 17.2% of them had at least one family member who died due to COVID-19.\n\n† Some variables do not have 861 observations due to missing data.\n\n* Absolute mean and weighted mean.\n\n Proportion based on total people with a mental health disease history.\n\n* Proportion based on the total number of people living with someone.\n\n‡ p < 0.001.\n\n£ p < 0.05.\n\nRegarding the vaccination situation, in the baseline measurement, 43.9% of the respondents received only one dose with an average time of 15.6 days from the date they received the vaccine, and 49.1% received the two doses with an average time of 16.9 days from the date they received the vaccine. On the other hand, 5.4% of older adults reported having a previous diagnosis of a mental health disorder. However, we found the prevalence of general anxiety, assessed with GAD-2, to be 16.4% (95% CI: 14.1 to 19.1), and the prevalence of general depression, estimated by PHQ-2, was 8.0% (95% CI: 6.3 to 10.0). In addition, we found that those who had two doses of the vaccine had less fear of COVID-19, anxiety about COVID-19, worry about COVID-19, and general anxiety compared to those who were unvaccinated or those who had a vaccine. However, we didn’t observe a trend in the case of the outcome of general depression (Figure 3).\n\nDuring the baseline measurement, we observed that older adults who had two doses of the vaccine had less fear of COVID-19 (aOR: 0.27; 95% CI: 0.13 to 0.55) than those who were unvaccinated. Similarly, we observed a lower anxiety about COVID-19, worry about COVID-19, and general anxiety in those who had two doses of the vaccine, but without statistical significance. Meanwhile, those who had one or two doses of the vaccine were more likely to have general depression than those who were unvaccinated, although this was not statistically significant (Table 2).\n\nThe mean follow-up time for 661 older adults was 31.4 ± 0.14 days. Considering the one-month follow-up period, we observed that older adults with two doses of the COVID-19 vaccine had less fear of COVID-19 (aOR: 0.19; 95% CI: 0.07 to 0.51) and less anxiety about COVID-19 (aOR: 0.45; 95% CI: 0.22 to 0.89), compared to those who were unvaccinated. We observed similar results in the outcomes of worry about COVID-19 and general anxiety; however, there is high uncertainty about these estimates (Table 3).\n\n\nDiscussion\n\nWe hypothesize that vaccination has a causal effect in reducing fear, anxiety, and worry about COVID-19 and, also in general anxiety and depression. Our study, conducted in a cohort from a nationally representative sample of older adults affiliated to EsSalud, partially confirmed our hypothesis. We found evidence that receiving two doses of the COVID-19 vaccine, compared with unvaccinated, reduced the likelihood of high levels of fear and anxiety about COVID-19. However, we were unable to confirm these findings for any outcome in those who had received only one dose. To our knowledge, this is the first study that assesses the effect of vaccination on emotional health in a representative sample of older adults, using novel COVID-19 perception outcomes.\n\nHigh fear and anxiety about COVID-19 significantly decreased in those who had two doses of the COVID-19 vaccine than those who were unvaccinated. Interestingly, the scales that measured both constructs (FCV-19S and CAS, respectively) focus mainly on the emotional and physical reaction to thoughts related to COVID-19 and its possible contagion.54,55 However, the PRE-COVID-19 scale, which measured the worry about COVID-19, focused on daily dysfunction caused by thoughts about the possibility of getting COVID-19.12 This difference is relevant, as it would mean that vaccination could affect older adults by improving their mental well-being and reducing more intense psychosomatic symptoms of stress related to the pandemic (fear/anxiety of COVID-19)56; but without reducing daily thoughts and behaviors associated with the possibility of contagion (COVID-19 concern).\n\nOn the other hand, although we observed a slight decrease in general anxiety, the effect of vaccination on general depression and anxiety outcomes, measured with PHQ-2 and GAD-2, respectively, was not significant. However, previous studies conducted in adults from the United States,24 Turkey,57 and China,58 and health professionals from Turkey27 reported that those with at least one dose of the vaccine against COVID-19 have lower scores on the depression scales, measured with the PHQ-4, PHQ-9, and the Beck Depression Inventory, and on anxiety scales, measured with the GAD-7 and the Beck Anxiety Inventory. The mechanisms and causes of depression and anxiety in older adults are related to psychosocial factors of loneliness and loss, and neuroendocrine and vascular disorders.59,60 Previous mentioned studies included adults in general with low representation of older adults, so, according to our results, the effect of vaccination would not be sufficient to significantly reduce these outcomes in emotional health in older adults, since they would respond to other intrinsic and extrinsic factors that were not measured in the present study.\n\nOn the other hand, the evaluation of the effect of vaccination against COVID-19 on emotional health could be affected by the perception of the vaccine’s effectiveness. Older adults are particularly susceptible to fake news or misinformation,61 which could influence their perception of vaccination against COVID-19 and diminish its effect on their mental health. This effect should be evaluated in future studies. Similarly, the perception of effectiveness and vaccination intention could also be affected by the type of vaccine manufacturer, with the BNT162b2 vaccine (BioNTech, Pfizer) being the most preferred, and the ChAdOx1-S vaccine (Oxford, AstraZeneca) the least preferred in developed countries.62 Thus, even though most older adults were vaccinated with the BNT162b2 vaccine (BioNTech, Pfizer), the political scandal in Peru regarding the BBIBP-CorV (Sinopharm) vaccine33 may have partially affected the effect of vaccination on the emotional health of this population.\n\nWe observed the effect on emotional health from the second dose and not in those who had only one dose of the vaccine. This result is different from previous studies where all those who had at least one dose were included in the vaccinated group, regardless of whether they had both doses or not.24,27,57,58 Clinical effectiveness studies have shown the need for a second dose of the vaccine to have greater effectiveness in preventing mortality and severe disease from COVID-19 in older adults.15,63 This information was communicated promptly to the population, making most people aware of the need for a second dose, especially those willing to be vaccinated.64,65 This may explain that in the context where the communicational emphasis was placed on the need for the second dose of the COVID-19 vaccine, the effect on emotional health could mainly be observed in those who received two doses of the vaccine. Considering the high proportion of older adults who have had two doses of the vaccine,66 the effect of vaccination reported in other studies may have been carried by those who had both doses, compared to those who had only one dose of the vaccine against COVID-19.\n\nHowever, the presence of new variants of concern, such as B.1.1.529, could affect the population’s mental health67,68 due to their greater infectivity and immune escape from vaccination.69 Given this, the need for a third,70 or even a fourth,71 COVID-19 vaccine booster is currently under discussion. So, considering that the perception of the vaccine’s effectiveness correlates with the level of concern about the new variants,72 it is important to continue monitoring mental health in the most vulnerable populations such as the elderly, and its evolution during future vaccination policies against COVID-19.\n\nAs of January 2022, the two-dose vaccination rate in adults aged 60 years and older in different countries was around 80% (https://ourworldindata.org/grapher/covid-fully-vaccinated-by-age?country). Among the reasons for older adults to decide to be vaccinated is the fear of developing the disease and the perception of the vaccine’s effectiveness to prevent the disease.73,74 However, the lack of reliable information, the fear of possible adverse effects, and the limited access to receive the vaccines mean that many older adults do not get vaccinated or do not have the opportunity to get vaccinated.73 In this sense, the communication strategy to promote vaccination against COVID-19 could be complemented with the message of reducing fear and anxiety about being infected with COVID-19. Thus, integrating with other elements necessary to have an adequate vaccination rate, such as the empowerment of the first level of care and the availability and access to vaccines,75 it could improve citizen confidence in the vaccination process.\n\nCurrently, the COVID-19 pandemic has a significant impact on mental health, which will continue in the medium and long term.76 From the point of view of positive epidemiology, our results propose vaccination against COVID-19 as a positive determinant of mental health in older adults.29 So, vaccination against COVID-19 could contribute to the partial improvement of the emotional health of older adults. However, we must consider that the mental health of older adults depends on various intrinsic and extrinsic factors that not only respond to the COVID-19 pandemic.59,60\n\nThe interpretation of the results of this study must consider the following limitations. First, we were unable to obtain the planned sample size for the primary analysis, making the statistical power of our results insufficient to find statistically significant results. Thus, we do not rule out the effect of the vaccine on worry about COVID-19 and general anxiety, which should be evaluated in future studies. In addition, the low representativeness of the older adults affiliated with EsSalud registered in the CAM database, the high refusal to participate in the study, and the loss during follow-up could have caused selection bias. Thus, it is likely that older adults in CAMs have greater access to receiving the vaccine and to activities that improve their mental health, so the effect that we measured in the study may be overestimated. Additionally, the way to determine the vaccination status was by self-report, so the measurement of this variable could have been overestimated due to the social desirability bias. On the other hand, even though we did not conduct clinical interviews to evaluate the emotional health in the present study, we used different specific psychometric instruments for the perception of COVID-19 and general anxiety and depression. These tools have robust evidence of psychometric validity in our population of interest, making the constructs that we measured reliable.\n\n\nConclusions\n\nVaccination against COVID-19 with two doses in older adults reduces fear and anxiety about COVID-19, compared to those who were unvaccinated. However, we observed no effect in general anxiety and general depression, nor in those who only had one vaccine dose.\n\n\nData availability\n\nFigshare: Underlying data for ‘Effects of vaccination against COVID-19 on the emotional health of older adults’, https://doi.org/10.6084/m9.figshare.20134994.77\n\nThis project contains the following underlying data:\n\nData file 1: VacMentHe_DataBase.xlsx (The database has been anonymized and it has not distorted the scientific meaning.)\n\nFigshare: Extended data for ‘Effects of vaccination against COVID-19 on the emotional health of older adults’, https://doi.org/10.6084/m9.figshare.20135000.39\n\nThis project contains the following extended data:\n\nSupplementary material:\n\n• Supplementary methods:\n\n▪ Sample size calculation\n\n▪ Characteristics from the eligible population and selected sample\n\n▪ Sampling weights calculation\n\n▪ Questionnaire (in Spanish)\n\n▪ Questionnaire (in English)\n\n• Supplementary results:\n\n▪ Table S1. Comparison of selected sample, sample in baseline measurement and sample reached in follow-up at one month\n\n\nReporting guidelines\n\nFigshare: Strobe checklist for ‘Effects of vaccination against COVID-19 on the emotional health of older adults’, https://doi.org/10.6084/m9.figshare.20135051.78\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthor contributions\n\nChristoper A. Alarcon-Ruiz: Conceptualization, methodology, formal analysis, investigation, data curation, writing-original draft, writing-review & editing, visualization, supervision, project administration.\n\nZoila Romero-Albino: Methodology, investigation, resources, writing-review & editing, data curation, supervision, project administration.\n\nPercy Soto-Becerra: Methodology, formal analysis, investigation, resources, writing-original draft, writing-review & editing, data curation, supervision, project administration.\n\nJeff Huarcaya-Victoria: Methodology, investigation, resources, writing-review & editing, data curation, supervision, project administration.\n\nFernando M. Runzer-Colmenares: Methodology, investigation, writing-review & editing.\n\nElisa Romani-Huacani: Methodology, investigation, writing-review & editing.\n\nDavid Villarreal-Zegarra: Methodology, investigation, writing-review & editing.\n\nJorge L. Maguiña: Methodology, investigation, resources, writing-original draft, writing-review & editing, data curation, supervision, project administration, funding acquisition.\n\nMoises Apolaya-Segura: Methodology, investigation, resources, writing-review & editing, data curation, supervision, project administration, funding acquisition.\n\nSofía Cuba-Fuentes: Methodology, resources, writing-review & editing, supervision, project administration.\n\n\nConsent\n\nEach participant gave verbal informed consent before being included in the study because the recruitment and the data collection were by phone calls. During the informed consent, the interviewer acknowledged the research aims, the themes to discuss during the interview, the benefits and risks of participation in the study, and how the researchers will manage the collected data during the study. The Institutional Review Board approved this process and the verbal informed consent, which was documented during the phone call.", "appendix": "Acknowledgments\n\nTo the workers from the Gerencia de la Persona Adulta Mayor y Prestaciones Sociales, EsSalud for participating in the data collection process.\n\n\nReferences\n\nSantomauro DF, Herrera AMM, Shadid J, et al.: Global prevalence and burden of depressive and anxiety disorders in 204 countries and territories in 2020 due to the COVID-19 pandemic. Lancet. 2021 Nov 6; 398(10312): 1700–1712. PubMed Abstract \| Publisher Full Text\n\nGarcía-Fernández L, Romero-Ferreiro V, López-Roldán PD, et al.: Mental Health in Elderly Spanish People in Times of COVID-19 Outbreak. Am. J. Geriatr. Psychiatry. 2020 Oct 1; 28(10): 1040–1045. PubMed Abstract \| Publisher Full Text\n\nTurna J, Zhang J, Lamberti N, et al.: Anxiety, depression and stress during the COVID-19 pandemic: Results from a cross-sectional survey. J. Psychiatr. 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Publisher Full Text\n\nCaycho-Rodríguez T, Vilca LW, Carbajal-León C, et al.: Coronavirus Anxiety Scale: New psychometric evidence for the Spanish version based on CFA and IRT models in a Peruvian sample. Death Stud. 2021 Jan 10: 1–11. PubMed Abstract \| Publisher Full Text\n\nCaycho-Rodríguez T, Vilca LW, Peña-Calero BN, et al.: Measurement of coronaphobia in older adults: Validation of the Spanish version of the Coronavirus Anxiety Scale. Rev. Esp. Geriatr. Gerontol. 2021 Nov 12. S0211-139X(21)00177-3.\n\nGarcía-Campayo J, Zamorano E, Ruiz MA, et al.: The assessment of generalized anxiety disorder: psychometric validation of the Spanish version of the self-administered GAD-2 scale in daily medical practice. Health Qual. Life Outcomes. 2012 Sep 19; 10: 114. PubMed Abstract \| Publisher Full Text\n\nWild B, Eckl A, Herzog W, et al.: Assessing Generalized Anxiety Disorder in Elderly People Using the GAD-7 and GAD-2 Scales: Results of a Validation Study. Am. J. Geriatr. Psychiatry. 2014 Oct; 22(10): 1029–1038. PubMed Abstract \| Publisher Full Text\n\nCaneo C, Toro P, Ferreccio C, et al.: Validity and Performance of the Patient Health Questionnaire (PHQ-2) for Screening of Depression in a Rural Chilean Cohort. Community Ment. Health J. 2020 Oct 1; 56(7): 1284–1291. Publisher Full Text\n\nBoyle LL, Richardson TM, He H, et al.: How do the phq-2, the phq-9 perform in aging services clients with cognitive impairment? Int. J. Geriatr. Psychiatry. 2011 Sep; 26(9): 952–960. PubMed Abstract \| Publisher Full Text\n\nRozzini R, Frisoni GB, Ferrucci L, et al.: Geriatric Index of Comorbidity: validation and comparison with other measures of comorbidity. Age Ageing. 2002 Jul; 31(4): 277–285. PubMed Abstract \| Publisher Full Text\n\nDean N, Pagano M: Evaluating Confidence Interval Methods for Binomial Proportions in Clustered Surveys. J. Surv. Stat. Methodol. 2015 Dec; 3(4): 484–503. Publisher Full Text\n\nMertens G, Duijndam S, Smeets T, et al.: The latent and item structure of COVID-19 fear: A comparison of four COVID-19 fear questionnaires using SEM and network analyses. J. Anxiety Disord. 2021 Jun; 81: 102415.\n\nLee SA: Coronavirus Anxiety Scale: A brief mental health screener for COVID-19 related anxiety. Death Stud. 2020 Jul 2; 44(7): 393–401. PubMed Abstract \| Publisher Full Text\n\nChandu VC, Marella Y, Panga GS, et al.: Measuring the Impact of COVID-19 on Mental Health: A Scoping Review of the Existing Scales: Indian. J. Psychol. Med. 2020 Aug 18; 42(5): 421–427.\n\nBilge Y, Keles E, Baydili KN: The Impact of COVID-19 Vaccination on Mental Health. J. Loss Trauma. 2021 Aug 18; 27: 285–288. Publisher Full Text\n\nYuan Y, Deng Z, Chen M, et al.: Changes in Mental Health and Preventive Behaviors before and after COVID-19 Vaccination: A Propensity Score Matching (PSM) Study. Vaccines. 2021 Sep; 9(9): 1044. PubMed Abstract \| Publisher Full Text\n\nAlexopoulos GS: Mechanisms and treatment of late-life depression. Transl. Psychiatry. 2019 Dec; 9(1): 188. PubMed Abstract \| Publisher Full Text\n\nHellwig S, Domschke K: Anxiety in Late Life: An Update on Pathomechanisms. Gerontology. 2019; 65: 465–473. PubMed Abstract \| Publisher Full Text\n\nMoore RC, Hancock JT: Older Adults, Social Technologies, and the Coronavirus Pandemic: Challenges, Strengths, and Strategies for Support. Soc Media Soc. 2020 Jul 1; 6(3): 2056305120948162.\n\nMerkley E, Loewen PJ: The correlates and intensity of COVID-19 vaccine brand preferences in Canada [Preprint]. OSF Preprints. 2021 [cited 2022 Jan 6].Reference Source\n\nKissling E, Hooiveld M, Martín VS, et al.: Vaccine effectiveness against symptomatic SARS-CoV-2 infection in adults aged 65 years and older in primary care: I-MOVE-COVID-19 project, Europe, December 2020 to May 2021. Eurosurveillance. 2021 Jul 22; 26(29): 2100670. Publisher Full Text\n\nGoldfarb JL, Kreps S, Brownstein JS, et al.: Beyond the First Dose — Covid-19 Vaccine Follow-through and Continued Protective Measures. N. Engl. J. Med. 2021 Jul 8; 385(2): 101–103. PubMed Abstract \| Publisher Full Text\n\nStead M, Jessop C, Angus K, et al.: National survey of attitudes towards and intentions to vaccinate against COVID-19: implications for communications. BMJ Open. 2021 Oct 1; 11(10): e055085. PubMed Abstract \| Publisher Full Text\n\nKriss JL, Reynolds LE, Wang A, et al.: COVID-19 Vaccine Second-Dose Completion and Interval Between First and Second Doses Among Vaccinated Persons — United States, December 14, 2020−February 14, 2021. Morb. Mortal. Wkly. Rep. 2021 Mar 19; 70(11): 389–395. PubMed Abstract \| Publisher Full Text\n\nSu Z, McDonnell D, Ahmad J, et al.: Mind the “worry fatigue” amid Omicron scares. Brain Behav. Immun. 2022 Mar 1; 101: 60–61. PubMed Abstract \| Publisher Full Text\n\nJain A, Jolly TS: Omicron (B.1.1.529) COVID-19 Variant: A Mental Health Perspective on Lessons Learned and Future Challenges. Prim Care Companion CNS Disord. 2021 Dec 16; 23(6): 38797. Publisher Full Text\n\nRen SY, Wang WB, Gao RD, et al.: Omicron variant (B.1.1.529) of SARS-CoV-2: Mutation, infectivity, transmission, and vaccine resistance. World J. Clin. Cases. 2022 Jan 7; 10(1): 1–11. PubMed Abstract \| Publisher Full Text\n\nEroglu B, Nuwarda RF, Ramzan I, et al.: A Narrative Review of COVID-19 Vaccines. Vaccines. 2022 Jan; 10(1): 62. Publisher Full Text\n\nTylicki L, Dębska-Ślizień A, Muchlado M, et al.: Boosting Humoral Immunity from mRNA COVID-19 Vaccines in Kidney Transplant Recipients. Vaccines. 2022 Jan; 10(1): 56.\n\nTemsah MH, Aljamaan F, Alenezi S, et al.: SARS-CoV-2 Omicron variant: healthcare workers’ perceptions and beliefs of vaccine effectiveness and advocacy: a national survey during the first week of the World Health Organization variant alert [PrePrint]. medRxiv. 2021 Dec [cited 2022 Jan 6]; p. 2021.12.27.21268431. Publisher Full Text\n\nBhanu C, Gopal DP, Walters K, et al.: Vaccination uptake amongst older adults from minority ethnic backgrounds: A systematic review. PLoS Med. 2021 Nov 4; 18(11): e1003826. PubMed Abstract \| Publisher Full Text\n\nCaycho-Rodríguez T, Carbajal-León C, Vivanco-Vidal A, et al.: Intention to vaccinate against COVID-19 in Peruvian older adults. Rev. Esp. Geriatr. Gerontol. 2021; 56(4): 245–246. PubMed Abstract \| Publisher Full Text\n\nCastillo C, Villalobos Dintrans P, Maddaleno M: The successful COVID-19 vaccine rollout in Chile: Factors and challenges. Vaccine X. 2021 Sep 8; 9: 100114. PubMed Abstract \| Publisher Full Text\n\nEsterwood E, Saeed SA: Past Epidemics, Natural Disasters, COVID19, and Mental Health: Learning from History as we Deal with the Present and Prepare for the Future. Psychiatr. Q. 2020 Dec 1; 91(4): 1121–1133. PubMed Abstract \| Publisher Full Text\n\nDatabase: Figshare.2022 [cited 2022 Jul 4]. Publisher Full Text\n\nSTROBE CheckList: Figshare. figshare.2022 [cited 2022 Jul 4]. Publisher Full Text" }	[ { "id": "146036", "date": "30 Aug 2022", "name": "Oscar Rosas-Carrasco", "expertise": [ "Reviewer Expertise Internal Medicine", "Geriatric Medicine", "clinical research" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe ideal study to demonstrate the effectiveness of vaccination in mental health are intervention studies. However, this follow-up study may provide sufficient evidence to demonstrate the association between improved mental health status in the group that received two vaccines versus those that received no or one dose. Therefore, the knowledge provided by this study is relevant and should be reported.\nIn the introduction, the percentages of vaccination with three vaccines are described, in its majority it is commented that they were vaccinated with Pfizer, that the Ministry of Health gave preference to this vaccine for older adults, however, in the methods it is not specified if it was a exclusion criterion having been vaccinated with other types of vaccines. In this case, if they had been selected add this variable as it is confusing. On the other hand, the adverse effects of vaccines occur frequently, if they occur they could have an effect contrary to the reduction of fear, anxiety, etc. due to COVID-19. Could the effects or adverse reactions be quantified? If so, include them as another confounding variable in the methodology.\nIt is necessary to deepen in the section because no significant differences were found in terms of depression and anxiety. Perhaps a paragraph should be included on the fact that using general questionnaires for anxiety and depression decreases their sensitivity for the detection of very specific changes such as that of vaccination? Had questionnaires developed (for this work) been used for this specific purpose, a significant change could have been found.\nAnother line that can be included in the discussion is that the presentation of depression and anxiety and its change does not only depend on the vaccination against COVID-19, it depends on many factors that can exert a greater weight than the vaccination itself and that for this reason an impact could not be obtained, future studies with greater strength in the operational definition of the variables or with an intervention design could help to clarify these two aspects of mental health.\nThe part in the argument that three or four doses could further improve mental health status is well supported.\n\nIs the work 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": "9564", "date": "16 May 2023", "name": "Christoper A. Alarcon-Ruiz", "role": "Author Response", "response": "1. “The ideal study to demonstrate the effectiveness of vaccination in mental health are intervention studies. However, this follow-up study may provide sufficient evidence to demonstrate the association between improved mental health status in the group that received two vaccines versus those that received no or one dose. Therefore, the knowledge provided by this study is relevant and should be reported.” Response: Thanks. 2. “In the introduction, the percentages of vaccination with three vaccines are described, in its majority it is commented that they were vaccinated with Pfizer, that the Ministry of Health gave preference to this vaccine for older adults, however, in the methods it is not specified if it was a exclusion criterion having been vaccinated with other types of vaccines. In this case, if they had been selected add this variable as it is confusing. On the other hand, the adverse effects of vaccines occur frequently, if they occur they could have an effect contrary to the reduction of fear, anxiety, etc. due to COVID-19. Could the effects or adverse reactions be quantified? If so, include them as another confounding variable in the methodology.” Response: We included all participants independently of vaccine preference or the type of received vaccine (We are adding this information in the methods section). In addition, we had the variable vaccine preference in the database, and it was included in the multivariable analysis. However, we didnt collect information about the adverse effects of the vaccines, but we are acknowledging that as a limitation. 3. “It is necessary to deepen in the section because no significant differences were found in terms of depression and anxiety. Perhaps a paragraph should be included on the fact that using general questionnaires for anxiety and depression decreases their sensitivity for the detection of very specific changes such as that of vaccination? Had questionnaires developed (for this work) been used for this specific purpose, a significant change could have been found.” Response: We agree with the reviewer and add the following text to the limitations section: “Fifthly, we used short and general questionnaires to measure anxiety and depressive symptoms (i.e. PHQ-2 and GAD-2), which are not specific to vaccination settings. Therefore, it is possible that significant differences would have been found if instruments specifically designed for these settings had been used. Similarly, it is possible that the use of more comprehensive versions measuring depressive and anxiety symptoms, such as the PHQ-9 and GAD-7, which include emotional and somatic indicators, might have increased the variability of the measures and found significant results. However, we believe that using the PHQ-2 and GAD-2 captures the core symptoms of anxiety and depression.” 4. “Another line that can be included in the discussion is that the presentation of depression and anxiety and its change does not only depend on the vaccination against COVID-19, it depends on many factors that can exert a greater weight than the vaccination itself and that for this reason an impact could not be obtained, future studies with greater strength in the operational definition of the variables or with an intervention design could help to clarify these two aspects of mental health.” Response: We have included this information in the limitations section, as we believe it is an issue not covered in our study: “Sixth, it is possible that the change in outcomes such as anxiety or depressive symptoms in older adults was not solely dependent on COVID-19 vaccination. Social determinants of health or other factors may be more influential and not accounted for in our study (i.e., family support, economic status, or quality of life). Therefore, invite other researchers to design future studies with greater methodological rigour, taking into account the considerations mentioned in our limitations.”" } ] }, { "id": "159978", "date": "30 Jan 2023", "name": "Javier Mariani", "expertise": [ "Reviewer Expertise Cardiology - COVID." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report the results of an observational study assessing the association between vaccination status and mental health outcome variables at two-time points.\nOverall, the study is very interesting and the addressed theme is relevant. However, the manuscript has several issues that should be reconsidered.\nThe study aimed to evaluate the effects of vaccination on mental health outcomes, and this study design is not the most appropriate to accomplish that objective. The study evaluated the association between mental health measures and vaccination status (indeed participants were already vaccinated -the exposure- when outcomes were measured. This should be recognized and the paper reformulated consequently; that is, there are several causal relationship sentences, for example: “High fear and anxiety about COVID-19 significantly decreased in those who had two doses of the COVID-19 vaccine than those who were unvaccinated” (Discussion, second paragraph), that should be reconsidered.\n\nTable 1 does not have measures of association between groups and the variables in the table. Please, include p values or uncertainty measures that help to understand the distribution of baseline characteristics among study groups.\n\nThe analysis of the outcome as a dichotomic variable could affect the power to detect differences. As an example, the medians and IQR for general anxiety and depression are 0 for almost all groups (only the group one-dose has a general anxiety 75th percentile of 1); the dichotomization at 2 and 3 points, respectively, leaves most participants as 0. Also, the presentation medians and IQR in the results tables, do not represent the analyses, if this analysis strategy will be maintained, the tables should show the number and proportion of participants with the outcome. Other strategies, such as Poisson regression models (or some variant for zero-inflated data) could have some advantages.\n\nThere are no p values for association tests reported, please add this information to figure 3 and tables 2 and 3.\n\nThe unvaccinated group was small, which could affect the study's power to detect between-group differences.\n\nPlease, include comparisons between one-dose and two-doses groups in tables 2 and 3, and in figure 3.\nMinor comment\nCheck the spelling.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "9565", "date": "16 May 2023", "name": "Christoper A. Alarcon-Ruiz", "role": "Author Response", "response": "1. “The study aimed to evaluate the effects of vaccination on mental health outcomes, and this study design is not the most appropriate to accomplish that objective. The study evaluated the association between mental health measures and vaccination status (indeed participants were already vaccinated -the exposure- when outcomes were measured. This should be recognized and the paper reformulated consequently; that is, there are several causal relationship sentences, for example: “High fear and anxiety about COVID-19 significantly decreased in those who had two doses of the COVID-19 vaccine than those who were unvaccinated” (Discussion, second paragraph), that should be reconsidered.” Response: Thanks for the suggestion, we corrected the phrasing of our results, according to the study design. 2. \"Table 1 does not have measures of association between groups and the variables in the table. Please, include p values or uncertainty measures that help to understand the distribution of baseline characteristics among study groups.” Response: We added p values in Table 1 and described them in the results section. 3.“The analysis of the outcome as a dichotomic variable could affect the power to detect differences. As an example, the medians and IQR for general anxiety and depression are 0 for almost all groups (only the group one-dose has a general anxiety 75th percentile of 1); the dichotomization at 2 and 3 points, respectively, leaves most participants as 0. Also, the presentation medians and IQR in the results tables, do not represent the analyses, if this analysis strategy will be maintained, the tables should show the number and proportion of participants with the outcome. Other strategies, such as Poisson regression models (or some variant for zero-inflated data) could have some advantages.” Response: We agree with the suggestions. We excluded the analysis comparing medians and IQR of the outcomes. We are using a validated cut-off for all our outcomes, considering the interpretation of our results, considering them as clinical relevant outcomes for the participants. 4. “There are no p values for association tests reported, please add this information to figure 3 and tables 2 and 3.” Response: Thanks for the suggestion. However, we are reporting 95%CI of the estimates. These values are better to understand the statistical significance of our results. 5. “The unvaccinated group was small, which could affect the study's power to detect between-group differences.” Response: We acknowledge this situation as a limitation in the final paragraph of the Discussion section. 6. “Please, include comparisons between one-dose and two-doses groups in tables 2 and 3, and in figure 3.” Response: Thanks for the suggestions. We added these analysis in tabls 2 and 3, and included on the discussion of our results. The figure 3 already included this comparison." } ] } ]	1	https://f1000research.com/articles/11-868
https://f1000research.com/articles/11-225/v1	24 Feb 22	{ "type": "Systematic Review", "title": "Economic evaluations of maternal health interventions: a scoping review", "authors": [ "Katherine E. Eddy", "Alexander Eggleston", "Sher Ting Chim", "Rana Islamiah Zahroh", "Elizabeth Sebastian", "Chloe Bykersma", "Steve McDonald", "Caroline S. E. Homer", "Nick Scott", "Doris Chou", "Olufemi T. Oladapo", "Joshua P. Vogel", "Sher Ting Chim", "Rana Islamiah Zahroh", "Elizabeth Sebastian", "Chloe Bykersma", "Steve McDonald", "Caroline S. E. Homer", "Nick Scott", "Doris Chou", "Olufemi T. Oladapo", "Joshua P. Vogel" ], "abstract": "Background Evidence on the affordability and cost-effectiveness of interventions is critical to decision-making for clinical practice guidelines and development of national health policies. This study aimed to develop a repository of primary economic evaluations to support global maternal health guideline development and provide insights into the body of research conducted in this field. Methods A scoping review was conducted to identify and map available economic evaluations of maternal health interventions. We searched six databases (NHS Economic Evaluation Database, EconLit, PubMed, Embase, CINAHL and PsycInfo) on 20 November 2020 with no date, setting or language restrictions. Two authors assessed eligibility and extracted data independently. Included studies were categorised by subpopulation of women, level of care, intervention type, mechanism, and period, economic evaluation type and perspective, and whether the intervention is currently recommended by the World Health Organization. Frequency analysis was used to determine prevalence of parameters. Results In total 923 studies conducted in 72 countries were included. Most studies were conducted in high-income country settings (71.8%). Over half pertained to a general population of pregnant women, with the remainder focused on specific subgroups, such as women with preterm birth (6.2%) or those undergoing caesarean section (5.5%). The most common interventions of interest related to non-obstetric infections (23.9%), labour and childbirth care (17.0%), and obstetric complications (15.7%). Few studies addressed the major causes of maternal deaths globally. Over a third (36.5%) of studies were cost-utility analyses, 1.4% were cost-benefit analyses and the remainder were cost-effectiveness analyses. Conclusions This review provides a navigable, consolidated resource of economic evaluations in maternal health. We identified a clear evidence gap regarding economic evaluations of maternal health interventions in low- and middle-income countries. Future economic research should focus on interventions to address major drivers of maternal morbidity and mortality in these settings.", "keywords": [ "Economic evaluation", "cost-effectiveness", "cost-utility", "cost-benefit", "health economics", "maternal health", "maternal interventions" ], "content": "Introduction\n\nAn estimated 295,000 maternal deaths occur during pregnancy, childbirth, and the immediate postpartum period each year, as well as 2 million stillbirths and 2.5 million neonatal deaths1–3. Ensuring universal access to good-quality care for all women during pregnancy, childbirth and the postpartum period would prevent the vast majority of these deaths4–6. The World Health Organization (WHO) produces evidence-based global guidelines to help health services, clinicians and communities ensure that the best care can be provided to pregnant women, regardless of where they give birth. Since 2017, the WHO Department of Sexual and Reproductive Health and Research has embarked on a “living guidelines” approach to update recommendations in maternal and perinatal health7. Based on this approach, WHO’s portfolio of over 400 maternal and perinatal health recommendations is regularly assessed by an independent international panel of experts, to identify which recommendations are in most urgent need of updating, and if new recommendations are needed.\n\nDeveloping and updating WHO recommendations for global use involves explicit consideration of available evidence for a given intervention across several criteria, including: the balance of benefits and harms, how stakeholders value different health outcomes, acceptability, feasibility, equity and cost-effectiveness of the intervention8. Even when there is clear evidence that an intervention is beneficial, acceptable and feasible, policy makers must consider the resource implications of implementation at scale. Health budgets are finite and limited, meaning that adding (or expanding access to) an intervention has an opportunity cost that may result in detrimental reduction of another health intervention. In these instances, evidence on the affordability and cost-effectiveness of the intervention is critical to inform decision-making. The effectiveness evidence for a majority of WHO maternal and perinatal health recommendations are drawn from systematic reviews of randomised trials, however these reviews do not routinely evaluate outcomes related to resource needs or cost-effectiveness7.\n\nThere have been previous efforts to map economic evaluations across different maternal health interventions, though these have been narrowly focused on selected interventions. For example, a 2013 scoping review identified 36 studies on economic benefits of reproductive, maternal, newborn and child health interventions, but it was limited to cost-benefit studies from low- and middle-income countries (LMICs) only, excluded studies published before 2000, and did not consider all maternal and perinatal interventions recommended by WHO9. The 2016 Disease Control Priorities summarised cost-effectiveness evidence for selected, high-value maternal interventions, identifying 26 studies10. More recently, systematic reviews of economic evaluations have been conducted for single interventions as part of WHO recommendation updates11,12. Other reviews have focused on economic evaluations of certain categories of interventions in LMICs, such as health systems strengthening strategies, or programs to increase utilisation and provision of care13,14.\n\nA broad, contemporary synthesis of economic evaluations across a wide range of interventions would provide a critical resource for future updates of WHO maternal health recommendations. It could also provide a consolidated, navigable resource for policy makers, health managers, and clinicians to identify and consider evidence for decision-making in maternal health, including judgements around allocative efficiency and costing models for maternal health budgets15,16. Such a synthesis needs to be amenable to regular updating to reflect future changes in the underlying literature. Therefore, the aim of this project was to conduct a scoping review of primary economic evaluations of maternal health interventions to create such a database and to provide preliminary insights into the body of research conducted in this field.\n\n\nMethods\n\nA systematic scoping review was undertaken in this study. A scoping review is a type of research synthesis that aims to map literature on a particular topic or research area, providing an opportunity to identify types and sources of evidence to inform practice, policymaking and research17. This methodology was selected as we were seeking to examine the extent, range and nature of evidence on maternal health interventions and identify gaps in the literature, and not to formally summarise or pool data on cost-effectiveness of any single intervention18. This review was conducted in line with the Levac et al. scoping review framework19, which is an extended version of the Arksey and O’Malley framework20, and the PRISMA Extension for Scoping Reviews (PRISMA-ScR) reporting checklist (extended data E5)18. These frameworks help to ensure a consistent, thorough approach to the methodology of the review, and promote replicability. This protocol was registered and published on Open Science Framework (OSF) website21.\n\nFor this review, we considered only full economic evaluations – including cost-benefit analyses, cost-effectiveness analyses, and cost-utility analyses – to be eligible (Box 1). Studies with cost effectiveness data within, or alongside, randomised controlled trials of effectiveness were eligible. Systematic reviews of economic evaluations were not included. As this review focused on maternal health interventions, the population of interest was women who were pregnant or recently pregnant, in any stage of labour or childbirth, or in the postpartum period (up to 42 days). This review considered any intervention primarily aimed at improving maternal and perinatal health outcomes. This included any clinical, pharmacological, procedural, educational, or behavioural intervention implemented at any level (including individual, health care provider, community, facility, subnational or national levels). Pre-conception interventions, abortion-related interventions, interventions related to management of miscarriage or ectopic pregnancies, and interventions aimed only at newborns were not included.\n\n\n\nCost-benefit analysis (CBA)\n\nEconomic evaluations in which the cost of the intervention is related to a value of benefits that uses a common or equal unit of measure, typically monetary.\n\nCost-utility analysis (CUA)\n\nEconomic evaluations in which the cost of the intervention is related to a multidimensional measure of effectiveness which considers not only the outcomes but the valuation of benefits, i.e. a measure of utility such as QALYs or DALYs.\n\nCost-effectiveness analysis (CEA)\n\nEconomic evaluations in which the cost of the intervention is related to a single clinical or natural measure of effectiveness, e.g. deaths, cases.\n\nAdapted from: U.S. National Library of Medicine - Health Economics Information Resources: A Self-Study Course (Module 4)\n\nStudies were eligible regardless of what comparator was used and considered any perspective (including societal or health system perspectives). They were eligible if they reported any quantifiable health outcome alongside costs, though the key outcomes of interest were cost-benefit outcomes (where health effects are valued in monetary terms), cost per quality-adjusted life year (QALY) or disability-adjusted life year (DALY), and cost per condition averted or life saved. Eligible studies were those published in peer-reviewed journals conducted in any country. We excluded records published as letters, editorials, or conference abstracts. No language restrictions were applied; for studies published in languages other than English an initial translation was carried out using open-source software (Google Translate) for assessing eligibility. If the study was potentially eligible and this translation was inadequate for data collection, we sought assistance from multilingual colleagues.\n\nWe searched both specialist health economics databases (NHS Economic Evaluation Database and EconLit) and general medical and health databases (PubMed, Embase, CINAHL and PsycInfo) on 20 November 2020. For the period up to 2014, we limited searching to NHS EED, which provides access to over 17,000 economic evaluations of health and social care interventions. NHS EED collated results from weekly searches of MEDLINE, Embase, CINAHL, PsycInfo and PubMed until the end of December 2014. Economic evaluations added to NHS EED compare the costs and outcomes of two or more interventions using cost-benefit, cost-utility or cost-effectiveness analyses. NHS EED is available online but has not been updated since March 2015. Hence, for the period 2015 to 2020, we searched PubMed, EconLit, Embase, CINAHL and PsycInfo. The search strategies for these sources combine terms relevant to maternal health with terms related to economic evaluations (see extended data E1). Search terms for maternal health were derived from search strategies used by Cochrane Pregnancy and Childbirth to maintain and update their specialised register. Search terms for economic evaluations were derived from the search strategies used to populate NHS EED.\n\nIn consultation with an information specialist, we adopted a multi-phase approach to searching and screening records from PubMed. Phase 1 of the search was limited to records indexed with the most relevant MeSH term (Cost-Benefit Analysis). Phase 2 extended this to records indexed with other MeSH terms related to economics and costs. Phases 3a and 3b used free-text terms in the title/abstract limited to records not MeSH-indexed (i.e., the non-MEDLINE subset of PubMed). Phase 4 combined MeSH terms and free-text terms across all of PubMed. For pragmatic reasons, we adopted a sampling approach for the 16,135 unique records retrieved by phase 3b and phase 4 of the search, since we expected very few of these records to be relevant. We screened a 10% and 5% sample of phase 3b and phase 4, respectively. We similarly screened a 10% sample of 1025 NHS EED records obtained using non MeSH-indexed terms. Screening these sample records resulted in less than the pre-specified threshold of 3% being included in the review. Searches of Embase, CINAHL and PsycInfo were limited to records indexed with the appropriate subject indexing terms only. We also searched the WHO Global Health Library for any economic evaluations not identified from searches of the sources listed above.\n\nTitles and abstracts of all identified citations were deduplicated in EndNote and imported into Covidence software for screening. Two review authors independently assessed unique citations against the eligibility criteria. Potentially relevant articles were included for full text review and assessed for eligibility by two independent authors. At both stages, disagreements were resolved through discussion or consulting a third author. Where more than one paper reported on the same study (i.e. using the same sample and methods), the papers were collated to ensure the primary study was the unit of interest22.\n\nData extraction was conducted using a customised spreadsheet in Google Sheets. We extracted data on study characteristics, including: year, country, population of interest, period of intervention, context of care, intervention and comparator description, category of intervention, intervention mechanism, outcome measures, evaluation type and perspective, relation to WHO recommendation(s), cost year, currency, and data source. Country income levels were coded using World Bank data. We developed operational definitions for consistent coding of the extracted data (extended data E2). When coding the intervention mechanism of included studies, we used the Cochrane Effective Practice of Care (EPOC) classifications for health systems interventions. For each study, we searched the WHO website to identify whether the intervention or comparator considered by that study had a current WHO recommendation (for or against). If only part of the intervention was related to a recommendation (for example, when the study explored a package of interventions, of which one was a WHO-recommended intervention), that study was classified as partially linked to a WHO recommendation. All data were extracted by a single author, with a 15% sample independently reviewed by a second author. We conducted a series of consistency and validation checks for additional quality assurance. As this was a scoping review, no quality assessments of individual studies were performed. We reported findings on extracted variables using descriptive analysis with frequency tables and graphs on characteristics and coded categories of included studies as described above.\n\n\nResults\n\nWe identified 923 studies for inclusion in this review (Figure 1). The number of economic evaluations of maternal health interventions has increased over time, with over half of all included studies (489 studies, 53.0%) published from 2014 onwards, compared to those from 1984 to 2013 (434 studies, 47.0%) and just over a quarter of studies (239 studies, 25.9%) in the last three years (2018-2020) (Figure 2).\n\nThe economic evaluations were conducted in 72 countries (extended data E3: Table S123). Ten countries (United States of America [USA], United Kingdom [UK], Canada, Australia, Netherlands, China, South Africa, India, France, and Spain) accounted for nearly 70% (642 studies) of all studies (Table 1). The highest number of studies were from USA (313 studies), followed by the UK (119 studies), Canada (40 studies), and Australia (39 studies); 48 of the 72 countries had 5 or less studies. In total, 71.8% (663 studies) were conducted in high-income countries, with a further 21.3% (197 studies) in LMICs. The remaining 6.8% (63 studies) were conducted in multiple countries across different income levels (Table 2). LMICs with the highest number of studies were China (24 studies), South Africa (23 studies), and India (17 studies).\n\nStudies varied in the population of interest they focused on. We categorised studies based on the subpopulation of interest and identified 53 subgroups (extended data E3: Table S223). The most common were studies of women at risk of or experiencing preterm birth (57 studies), women undergoing caesarean section (51 studies) and women with HIV (48 studies) (Figure 3). Approximately half (465 studies, 50.4%) broadly considered any or all pregnant women or mothers, without specific restrictions or focus. More than half of studies related to interventions only in the antenatal period (543 studies, 58.8%), followed by the intrapartum period only (173 studies, 18.7%), and the postpartum period only (76 studies, 8.2%); the remainder were a combination of two or more periods (Figure 4). In terms of care setting, studies relating to outpatient services were most common (424 studies, 45.9%), followed by inpatient (224 studies, 24.3%), and a combination of both (147 studies, 15.9%) (Table 3). Only 115 studies (12.5%) related to interventions outside of healthcare settings, including community, home-based, or telemedicine interventions.\n\nWe identified 61 distinct categories of interventions, which we mapped to 10 broad topic areas (extended data E3: Table S323). The most common studies were those addressing prevention, recognition, and management of infection not specific or exclusive to pregnancy, such as HIV, Group B Streptococcus (GBS), and Hepatitis B (221 studies, 23.9%); labour and childbirth care (e.g. caesarean section) (157 studies, 17.0%); prevention, diagnosis, and management of obstetric complications (145 studies, 15.7%); screening and diagnosis of genetic disorders (109 studies, 11.8%); models of care (e.g. midwifery-led care) (103 studies, 11.2%), and routine antenatal and postpartum care (77 studies, 8.3%) (Table 4). In assessing interventions, we also identified 52 intervention mechanisms mapped to seven broad types (extended data E3: Table S423). The three most common were clinical interventions (379 studies, 41.1%), diagnostic tests (338 studies, 36.6%), and health system delivery arrangements (97 studies, 10.5%).\n\nOf the 923 studies in the review, 531 (57.5%) studies assessed an intervention or comparator related to a published WHO recommendation. For 258 studies (27.9%) the intervention was directly linked; for 217 studies (23.6%) the intervention was only partially linked; and for 56 studies (6.1%) the comparator was linked. A total of 392 studies (42.5%) assessed interventions and comparators for which there is no current WHO recommendation (Figure 5). Within the 258 studies where the intervention was directly linked to a current WHO recommendation, the most frequent interventions related to HIV management in pregnancy (54 studies); obstetric haemorrhage (23 studies); midwifery-led care (14 studies); syphilis in pregnancy (14 studies) and induction of labour (11 studies). Of those studies exploring interventions which were not the subject of a current WHO recommendation, categories including genetic screening (58 studies); premature labour/preterm birth (48 studies); vaccination in pregnancy (26 studies); caesarean section (23 studies); and Group B streptococcal disease (17 studies) were most common (extended data E3: Table S523).\n\nCost effectiveness analyses (CEA) using condition or intervention-specific measures of health effects accounted for more than half of all included studies (573 studies, 62.1%). 13 studies (1.4%) conducted a cost-benefit analysis, valuing health effects in monetary terms, and 337 studies (36.5%) conducted cost-utility analysis (CUA) valuing health effects using quality-of-life measures. Within the CUA studies, studies conducted in high-income countries primarily assessed quality-adjusted life years (QALYs) (206/209 studies) while those in LMICs primarily assessed disability-adjusted life years (DALYs) (61/90 studies), and the remaining CUA studies were conducted across multiple income levels. Included studies considered seven different self-reported cost-effectiveness perspectives, with some studies reporting more than one perspective (Table 5). Of the seven perspectives, health sector was the most reported (205 studies, 22.2%), followed by societal (176 studies, 19.1%), provider (98 studies, 10.6%), government (96 studies, 10.4%), third party funder (41 studies, 4.4%), payer unspecified (30 studies, 3.3%), and finally the patient (20 studies, 2.2%). Nearly one-third of studies did not specify the perspective used (302 studies, 32.7%).\n\n*Studies reporting more than one perspective are listed against each applicable perspective; as such, the percentages are not cumulative.\n\n\nDiscussion\n\nThis review identified and categorised 923 economic evaluations of maternal health interventions published over a 37-year period (the earliest study identified was from 1984). To our knowledge, this is the first such broad mapping of economic evaluations of interventions used during pregnancy, childbirth, and the postpartum period. The number of maternal economic evaluations have increased markedly in the last decade, with over half of included studies published since 2014. Included studies used diverse methods to explore a wide range of interventions, and the majority of studies presented evidence from high-income countries.\n\nComparison with other reviews of economic evaluations in maternal health similarly found that research in this area has increased. Previous reviews typically had a narrower focus, including studies focused on a single or specific set of interventions or evidence from specific settings9–14, and consequently identified a smaller number of eligible studies (typically less than 30). For example, a 2018 systematic review of health systems strengthening economic evaluations in maternal and perinatal health identified 24 eligible studies, 23 of which were published since 200013. A 2014 review identified 48 economic evaluation studies on utilisation and provision of maternal and newborn care in LMICs, of which 36 were published since 200014. These reviews, along with the upward trend of publications identified in our review, suggest increasing demand for economic evaluations in this topic area.\n\nEvidence from economic evaluations can be difficult to generalise across different settings, given differences in health system arrangements, payment models, and labour, equipment and medicine costs between jurisdictions24. Global estimates of maternal and neonatal mortality rates show that the vast majority of these deaths occur in LMICs1–3. In these contexts, health budgets are likely to be more limited, with difficult decisions to be made about which interventions to prioritise when resources are scarce. Affordability is also likely to be an issue for those countries where individuals and families are often required to cover the cost of healthcare (i.e. out-of-pocket costs). Despite these public health realities, this review found most economic evaluations were conducted in high-income settings; only 21% of included studies were set in LMICs, and seven high-income countries accounted for nearly two-thirds of available economic evidence. This is consistent with a 2013 scoping review of cost-benefit analysis studies pertaining to reproductive, maternal, newborn and child health in LMICs, which identified only 36 eligible studies9. Larger health budgets in high-income countries may be a driver for this, creating a stronger incentive to ensure value for money across higher overall health expenditure. The breadth of healthcare interventions available in high-income settings may also incentivise health economic research since there are more options to be considered by policymakers and insurers when allocating budgets. Nevertheless, this inequity in health economic research suggests efforts need to be better targeted to settings and health systems where the mortality and morbidity burden is greatest. Barriers to implementation of effective interventions in these settings are complex and diverse, but often include economic factors25. Greater investment in health economic evaluations for LMIC contexts – tailored specifically to the interventions used in these settings – would probably improve policy decision-making in these settings, yielding additional public health benefits.\n\nStudies considered a diverse range of interventions and patient sub-populations, such as women experiencing preterm birth, caesarean section, or HIV. However, a relatively small proportion of included studies related to the leading causes of maternal deaths globally26. Specifically, only 37 studies (4.0% of all studies) focused on obstetric haemorrhage, 33 studies (3.6%) on hypertensive disorders, 26 studies (2.8%) on infections that could lead to sepsis, and 7 studies (0.8%) on embolism – these four conditions comprise the leading direct causes of global maternal deaths. In this review, the most frequently studied interventions related to genetic screening and diagnostic tests (including for cystic fibrosis, trisomy disorders, and thalassaemia traits) (109 studies, 11.8%), HIV in pregnancy (including prevention of maternal-to-child transmission) (82 studies, 8.9%), preterm labour and birth (64 studies, 6.9%), and diabetes in pregnancy (38 studies, 4.1%). This may be related to the large proportion of studies conducted in high-resource settings, where maternal deaths are comparatively rare and economic research priorities may lie elsewhere27.\n\nWhen developing their recommendations, WHO prioritises interventions that are likely to have the greatest impact on reducing global maternal mortality and morbidity – as well as increasing the experience and wellbeing of women – and cost-effectiveness is a key consideration in developing these recommendations8. This review identified 258 studies that provide cost-effectiveness evidence on interventions directly linked to current WHO recommendations (extended data E3: Table S923). However, the majority of studies identified either did not relate (or relate only partially) to a WHO recommendation. This similarly suggests a dearth of economic evaluation research on those maternal health interventions of highest global priority.\n\nThis scoping review used a robust search in multiple databases, allowing us to identify a large number of studies across a broad range of interventions, settings and analytical designs. Adherence to the Levac et al. scoping review methodological framework19 and PRISMA-ScR checklist18 maintained consistency in our approach, while quality assurance and validation checks ensured data accuracy. Despite our best efforts, it is possible that some eligible studies were not captured. For example, while effectiveness trials may report on cost outcomes, this may not be clearly documented in the study abstract or main findings, making it difficult to detect. We also relied upon the NHS EED database to identify studies published before 2015. While our search from 2015 onwards focused on the same databases indexed by NHS EED, we are unable to fully assess the veracity of their eligibility assessment process and whether the two approaches meaningfully differed. An additional challenge in this review was in systematically classifying the population, intervention, comparators, and outcomes used across studies. For example, economic evaluations may involve the same target population, but report on different outcomes of interest, or consider different cost perspectives. With the data extracted in this review, we were not able to explore some economic analytical questions of public health importance (e.g. any differences in study findings across private vs public contexts), however, future expansion of this scoping review may allow us to do so.\n\nThis review was conducted to support WHO activities on living guidelines in maternal health7. In light of future updates to those guidelines, we intend to regularly update this review. In future updates, we anticipate incorporating quality assessments for individual studies that are generated from evidence syntheses of specific interventions, though there are acknowledged limitations in available tools for assessing quality of health economic literature28. The identification of studies in this review can be useful to maternal health guideline development or policy decision-making processes by providing a searchable, contemporary database of health economic evidence. This can be used to identify all available studies for specific interventions, subpopulations, and contexts. Further to this, the gaps in maternal economic evaluations that have been identified in this review can provide insights into where future research needs to be targeted.\n\n\nConclusion\n\nWe identified 923 economic evaluations of maternal health interventions, covering a wide range of subpopulations of women and health conditions. While the volume of economic evaluations has increased over time, there are significant disparities between available economic literature, and the causes and settings of maternal and newborn deaths. Future health economic research needs to focus on interventions to address the major drivers of maternal morbidity and mortality, and their implementation in limited-resource contexts. The review findings provide a comprehensive, and navigable resource for economic evidence to support maternal health guideline and policy development.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nZenodo: Economic evaluations of maternal health interventions: a scoping review (extended data repository). https://doi.org/10.5281/zenodo.603074523\n\nThis project contains the following underlying data:\n\nEconomic evidence in MH - Extended data E1 Search strategies.docx\n\nEconomic evidence in MH - Extended data E2 Operational definitions.docx\n\nEconomic evidence in MH - Extended data E3 Additional results tables.docx\n\nEconomic evidence in MH - Extended data E4 Extracted data for included studies.docx\n\nEconomic evidence in MH - Extended data E5 PRISMA-ScR checklist.docx\n\nLICENSE.txt\n\nData are available under the terms of the Creative Commons “Attribution 3.0 IGO” data license (CC BY 3.0 IGO).", "appendix": "Acknowledgements\n\nThis manuscript represents the views of the named authors only, and not the views of their institutions or organisations.\n\n\nReferences\n\nWorld Health Organization: Trends in maternal mortality 2000 to 2017: estimates by WHO, UNICEF, UNFPA, World Bank Group and the United Nations Population Division. 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PubMed Abstract \| Publisher Full Text\n\nHorton S, Levin C: Cost-Effectiveness of Interventions for Reproductive, Maternal, Neonatal, and Child Health. In: Black RE, Laxminarayan R, Temmerman M, et al. (eds) Reproductive, Maternal, Newborn, and Child Health: Disease Control Priorities. Third Edition. Washington (DC), 2016; 2. PubMed Abstract \| Publisher Full Text\n\nVogel JP, Wilson AN, Scott N, et al.: Cost-effectiveness of uterine tamponade devices for the treatment of postpartum hemorrhage: A systematic review. Int J Gynaecol Obstet. 2020; 151(3): 333–340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawrie TA, Rogozińska E, Sobiesuo P, et al.: A systematic review of the cost-effectiveness of uterotonic agents for the prevention of postpartum hemorrhage. Int J Gynaecol Obstet. 2019; 146(1): 56–64. 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Publisher Full Text\n\nEddy K, Vogel J: Cost-effectiveness evidence for maternal and perinatal health interventions: living scoping review. (protocol). Open Science Framework. 2020. Publisher Full Text\n\nGlenton C, Bohren MA, Downe S, et al.: EPOC Qualitative Evidence Syntheses: Protocol and review template. Effective Practice and Organisation of Care (EPOC), 2019. Reference Source\n\nEddy KE, Eggleston A, Chim ST, et al.: Economic evaluations of maternal health interventions: a scoping review (extended data repository). Zenodo. 2021. http://www.doi.org/10.5281/zenodo.6030745\n\nArnold M, Griffin S, Ochalek J, et al.: A one stop shop for cost-effectiveness evidence? Recommendations for improving Disease Control Priorities. Cost Eff Resour Alloc. 2019; 17: 7. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "163254", "date": "24 Mar 2023", "name": "Malte Sandner", "expertise": [ "Reviewer Expertise Labour", "Education", "Family", "Health Economics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article gives an overview about economic evaluations of maternal health interventions. The article includes 923 studies which meet the criteria applied by the researchers. These criteria are overall comprehensible and I guess the most relevant studies are included in the review.\n\nI think the authors should make clearer why the review is relevant. I think one reason for the relevance is to identify areas in which little research is conducted and where more research is necessary. I think the article is so far neglecting this aspect.\n\nFurthermore, the article should formulate certain areas where more research is needed. The authors go into this direction, but I think they can identify more particular gaps by interacting setting of care, subpopulations, country, country income level, and broad categories of maternal health interventions.\nFinally, I think the broad categories of maternal health interventions can be explained better. On which base are these broad categories chosen? How is the categorization conducted? I can imagine many interventions can also put in more than one broad categories of maternal health interventions.\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": "9661", "date": "26 May 2023", "name": "Katherine Eddy", "role": "Author Response", "response": "Reviewer comment 1: \"This article gives an overview about economic evaluations of maternal health interventions. The article includes 923 studies which meet the criteria applied by the researchers. These criteria are overall comprehensible and I guess the most relevant studies are included in the review.\" Response: Thank you for your comment. Reviewer comment 2: \"I think the authors should make clearer why the review is relevant. I think one reason for the relevance is to identify areas in which little research is conducted and where more research is necessary. I think the article is so far neglecting this aspect. Furthermore, the article should formulate certain areas where more research is needed. The authors go into this direction, but I think they can identify more particular gaps by interacting setting of care, subpopulations, country, country income level, and broad categories of maternal health interventions.\" Response: Thank you for your feedback, we agree that evidence mapping is a key reason for the review’s relevance and have included an amendment to the Introduction section highlighting this. We likewise agree that an important finding of the review is that there are gaps in the existing evidence base for economic evaluations of maternal health interventions. The discussion highlights the need for further research in low and middle-income countries as a matter of priority, and also suggests that there should be greater focus on interventions that address the key causes of maternal mortality and interventions that are the subject of a WHO recommendation. Further to this, we have added discussion on which geographic regions and population subgroups would benefit from additional focus. Reviewer comment 3: \"Finally, I think the broad categories of maternal health interventions can be explained better. On which base are these broad categories chosen? How is the categorization conducted? I can imagine many interventions can also put in more than one broad categories of maternal health interventions.\" Response: The intervention categories and broad topic areas for maternal health interventions in this paper were developed inductively from the included studies through discussion with study authors. This was an iterative process, during which operational definitions were developed to ensure consistent coding of the extracted data. Following coding, categories were reviewed against the operational definitions to ensure consistency. The methods section has been amended to include further detail regarding this process. The operational definitions of each intervention category are set out in Extended Data E2: Table S3. Categorisation for each study was determined during the data extraction process, as described in ‘Study selection, data extraction and analysis’. The first reviewer categorised each study, on the basis of the described intervention within the study. A 15% sample of data extraction (including this categorisation) was independently reviewed by a second author. A series of consistency and validation checks were conducted, which further confirmed that studies of the same intervention appeared in the some broad category. We acknowledge that some studies were relevant to more than one category of intervention, but that this would have resulted in double-counting in our analysis. Accordingly, we prioritised the most relevant category for each study for the purpose of analysis. An amendment to reflect this has been added to the limitations section." } ] } ]	1	https://f1000research.com/articles/11-225
https://f1000research.com/articles/12-116/v1	01 Feb 23	{ "type": "Review", "title": "Circadian clocks in health and disease: Dissecting the roles of the biological pacemaker in cancer", "authors": [ "Bridget M. Fortin", "Alisa L. Mahieu", "Rachel C. Fellows", "Nicholas R. Pannunzio", "Selma Masri", "Bridget M. Fortin", "Alisa L. Mahieu", "Rachel C. Fellows", "Nicholas R. Pannunzio" ], "abstract": "In modern society, there is a growing population affected by circadian clock disruption through night shift work, artificial light-at-night exposure, and erratic eating patterns. Concurrently, the rate of cancer incidence in individuals under the age of 50 is increasing at an alarming rate, and though the precise risk factors remain undefined, the potential links between circadian clock deregulation and young-onset cancers is compelling. To explore the complex biological functions of the clock, this review will first provide a framework for the mammalian circadian clock in regulating critical cellular processes including cell cycle control, DNA damage response, DNA repair, and immunity under conditions of physiological homeostasis. Additionally, this review will deconvolute the role of the circadian clock in cancer, citing divergent evidence suggesting tissue-specific roles of the biological pacemaker in cancer types such as breast, lung, colorectal, and hepatocellular carcinoma. Recent evidence has emerged regarding the role of the clock in the intestinal epithelium, as well as new insights into how genetic and environmental disruption of the clock is linked with colorectal cancer, and the molecular underpinnings of these findings will be discussed. To place these findings within a context and framework that can be applied towards human health, a focus on how the circadian clock can be leveraged for cancer prevention and chronomedicine-based therapies will be outlined.", "keywords": [ "circadian clock", "cancer", "night shift work", "early-onset cancer", "colorectal cancer", "Wnt signaling", "chronotherapy", "chronomedicine" ], "content": "Introduction\n\nBiological rhythms regulate daily, seasonal, and long-term oscillations that are essential for life on Earth. The circadian clock is an evolutionarily conserved pacemaker found in prokaryotes and eukaryotes that governs homeostatic circuits that are fundamentally required for host fitness and survival. The mammalian clock is functionally conserved to regulate sleep/wake cycles (Czeisler et al., 1980; Winfree, 1983), feeding/fasting rhythms (Damiola et al., 2000; Hara et al., 2001; Inoue et al., 1977; Stokkan et al., 2001; Vollmers et al., 2009), and a host of endocrine, metabolic, and immune functions (Green et al., 2008; Keller et al., 2009; Kitchen et al., 2020; Turek et al., 2005). The focus of this review is on mammalian clocks and their roles in health and disease, with a particular focus on clocks in healthy versus transformed cells. Recent evidence has cited multiple diverse and tissue-specific functions of the circadian clock in different cancer types such as lung, colorectal, hepatocellular, breast, and others (Chun, Fortin, Fellows et al., 2022; Dong et al., 2019; Janich et al., 2011; Lee et al., 2010; Papagiannakopoulos et al., 2016; Puram et al., 2016; Stokes et al., 2021). This review will provide a comprehensive overview of the divergent functions of the clock in cell cycle control, maintenance of genome integrity, and immunity in healthy tissues, in an attempt to deconvolute the elaborate cellular networks that the biological pacemaker impinges on. Additionally, the reported role of the circadian clock in different cancer types will be reviewed in the context of clinical and epidemiology data, pre-clinical in vivo mouse models, as well as mechanistic data from cell line-based studies.\n\n\nClocks in healthy tissues\n\nThe circadian clock is the internal biological pacemaker that controls cell autonomous 24-hour oscillations in gene expression programs that regulate organismal physiology (Figure 1). The central clock, which resides in the suprachiasmatic nucleus (SCN) of the hypothalamus, is responsive to photic cues and transmits autonomic and endocrine signals to synchronize tissue-specific peripheral clocks to the environmental light-dark cycle (Pando et al., 2002; Welsh et al., 2004, 2010; Whitmore et al., 2000; Yamazaki et al., 2000). Peripheral clocks are also entrained by external cues including temperature (Barrett & Takahashi, 1995; Brown et al., 2002; Gould et al., 2006; Huang et al., 1995; Ruoff et al., 2005) and food supply that serve to further fine-tune biological timekeeping (Damiola et al., 2000; Hara et al., 2001; Inoue et al., 1977; Stokkan et al., 2001; Vollmers et al., 2009). The circadian system is regulated by a tightly controlled transcriptional and translational feedback loop (TTFL) that encompasses a 24-hour day. The positive transcriptional activators of the circadian machinery, CLOCK and BMAL1, heterodimerize and bind to consensus E-box motifs located within promoters of core clock and clock-controlled genes (Ripperger & Schibler, 2006; Ueda et al., 2005). The core clock regulators of the negative arm of this TTFL, PERIOD (PER) and CRYPTOCHROME (CRY), are translated to repress the transcriptional activity of the CLOCK-BMAL1 complex (Duong et al., 2011; Michael et al., 2017; Nangle et al., 2014; Narasimamurthy et al., 2018). This entire transcriptional/translational feedback circuit drives the rhythmic periodicity of gene expression networks that govern endocrine function, metabolism, immune response, cell cycle control, and genome stability, many of which will be discussed below. Given the complex role of the clock in cellular metabolic control and nutritional challenge, and the host of recent publications covering this topic (Guan & Lazar, 2021; Reinke & Asher, 2019; Rijo-Ferreira & Takahashi, 2019; Verlande & Masri, 2019), this review will only cover clock-controlled metabolic alterations in the context of cancer.\n\nIn mammals, circadian rhythms are coordinated by the central circadian clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus (Inouye & Kawamura, 1979; Stephan & Zucker, 1972). The central clock receives photic cues and transmits endocrine and autonomic signals to synchronize tissue-specific peripheral clocks to the time of day (Pando et al., 2002; Welsh et al., 2004, 2010; Whitmore et al., 2000; Yamazaki et al., 2000). The circadian clock is regulated by a TTFL where CLOCK and BMAL1 drive transcriptional activation and PERIOD (PER) and CYPTOCHROME (CRY) feedback to repress this transcriptional activity. This TTFL regulates gene expression programs that modulate critical cellular processes needed to maintain homeostasis including cell division, maintenance of genome integrity, immunity, endocrine and metabolic functions. The circadian clock is implicated in regulating the growth and division of cells as the expression of cyclins is rhythmic (Graña & Reddy, 1995; Vermeulen et al., 2003). Circadian proteins also mediate the DNA damage response (Gery et al., 2006; Kang & Leem, 2014) and DNA repair including nucleotide excision repair (Gaddameedhi et al., 2011; Marteijn et al., 2014), base excision repair (Kozmin et al., 2005; Krokan & Bjørås, 2013), homologous recombination and non-homologous end-joining (Cotta-Ramusino et al., 2011; Shafi et al., 2021). In addition to regulation of cell division and DNA damage, the immune system is also tightly regulated by the circadian clock to promote efficient immunologic response to infection. Immune cells have functional circadian clocks (Keller et al., 2009; Silver et al., 2012) and the release of cytokines and chemokines is rhythmic (Gibbs et al., 2014; Pariollaud et al., 2018), as well as the release of immune cells into the bloodstream (Dimitrov et al., 2007; Méndez-Ferrer et al., 2008). This rhythmic secretion of chemokines facilitates time of day trafficking of immune cells into tissues (Gibbs et al., 2014; Méndez-Ferrer et al., 2008) which has been demonstrated to mediate the host response to infection (Kiessling et al., 2017) and disease (Gibbs et al., 2014; Kitchen et al., 2020). Lastly, metabolic processes, including glucose and lipid metabolism, cardiovascular health and endocrine hormone secretion are regulated by the circadian clock (Green et al., 2008; Verlande & Masri, 2019). Figure created using BioRender.\n\nCells regulate their growth and division using cell cycle checkpoints, which ensure timely progression of the cell cycle under normal physiological conditions, and also halt cell cycle progression in instances of DNA damage, erroneous mitosis, or environmental stressors (Collins et al., 1997; Kastan & Bartek, 2004). The duration and transition of each cell cycle phase is orchestrated by the activation of specific cyclin-dependent kinases (CDKs) by their respective cyclins (Graña & Reddy, 1995; Vermeulen et al., 2003). Whereas expression of CDKs remains relatively constant throughout the cell cycle, each cyclin peaks in expression in a staggered and coordinated manner to drive the cell cycle with appropriate timing (Graña & Reddy, 1995; Vermeulen et al., 2003). Given the rhythmic nature of both the cell cycle and the circadian clock, several studies have worked to investigate and define the connections between these two systems. It has been suggested that circadian rhythms and the cell cycle are tightly phase-coupled and oscillate with a 1:1 frequency in mouse fibroblasts (Feillet et al., 2014), and this synchronization has even been shown at the single cell level in mammalian NIH3T3 fibroblasts (Bieler et al., 2014). More recently, data suggests that the cell cycle and the circadian clock can synchronize each other bidirectionally in mammalian systems (Yan & Goldbeter, 2019). However, the extent of this coupling is still not well understood, as some studies report additional findings that the cell cycle and circadian clock can in fact operate independently, as demonstrated in Rat-1 fibroblasts as well as Lewis lung carcinoma cell lines (Yeom et al., 2010; Pendergast et al., 2010).\n\nGiven the intimate links between the circadian clock and the cell cycle, what are the molecular connections driving this interplay? Importantly, several studies have investigated connections between the circadian clock and the proto-oncogene c-Myc, a master regulator of cell cycle control that promotes cellular growth by driving cyclin expression and repressing CDK inhibitor activity (Burchett et al., 2021). In regard to transcriptional regulation of c-Myc, it has been reported that Bmal1-/- mice exhibited increased expression of c-Myc, whereas c-Myc expression was decreased in Cry1-/-;Cry2-/- mice (Liu et al., 2020), demonstrating a strong correlation between these networks. Additionally, c-MYC can impinge on circadian rhythms in U2OS cells, further highlighting the molecular crosstalk between the clock and the cell cycle that also integrates with cellular metabolic state (Altman et al., 2015; Shostak et al., 2016). In regard to regulation of MYC protein levels, it was demonstrated that CRY2 mediates MYC degradation and that Cry2-/- knockout induced increased proliferation and transformation rates (Huber et al., 2016).\n\nIn addition to circadian links to c-Myc, clock-dependent transcription has been observed for many other cell cycle modulators, including cyclin D, cyclin E, cyclin A, and cyclin B in human epithelium (Bjarnason et al., 1999; Fu et al., 2002). Likewise, G2/M regulator WEE1, which inhibits Cyclin B1-CDK1 activity, has oscillating protein expression and kinase activity and this oscillation was dampened in Cry1-/-;Cry2-/- mice (Gréchez-Cassiau et al., 2008; Matsuo et al., 2003). Another study found that CLOCK and BMAL1 knockdown leads to the suppression of WEE1 and thus an increased activation of apoptosis in human hepatocellular carcinoma cell lines, again confirming circadian influence on WEE1 regulation (Qu et al., 2023). Additional CDK inhibitors such as p21cip1/waf1 and p16-Ink4A also oscillate under circadian control and this rhythmicity was lost in Bmal1-/- mice or Per1Brdm1/Brdm1;Per2Brdm1/Brdm1 mutant mice, respectively (Gréchez-Cassiau et al., 2008; Kowalska et al., 2013). Taken together, these data highlight the important molecular links between the circadian clock and cell cycle control mechanisms.\n\nDue to a multitude of interactions between circadian proteins and cell cycle checkpoints and drivers, it is not surprising that circadian rhythm disruption can modify rates of cellular proliferation. For example, genetic clock disruption in mouse osteoblasts via Per1-/-;Per2m/m knockout or Cry1-/-;Cry2-/- knockout resulted in increased proliferation (Fu et al., 2005). Circadian clock disruption can potentially interfere with normal rates of cellular growth, introducing susceptibility to disease and cancer. Altogether, this data suggests that oscillations of clock proteins contribute to the proper expression of important cell cycle regulators that impact cellular proliferation.\n\nAnother important aspect of cell cycle regulation features the DNA damage response (DDR). Individual cells can receive tens of thousands of DNA lesions per day, which leads to replication errors, transcription blockage, and even permanent mutations if left unrepaired (Jackson & Bartek, 2009). Thus, the DDR has evolved to preserve genome integrity by recognizing various forms of DNA damage, stimulating DNA repair, inhibiting cell cycle progression until the repair is complete, and inducing apoptosis if the damage is irreparable (Giglia-Mari et al., 2011; Jackson & Bartek, 2009; Roos & Kaina, 2013).\n\nIn mammals, central DDR proteins ATR and ATM instigate the DDR by phosphorylating CHK1 and CHK2, respectively (Jackson & Bartek, 2009). Consequently, phosphorylated CHK1 and CHK2 activate transcription factor p53 to halt cell cycle progression (Ronco et al., 2017). It was reported that circadian proteins CRY1 and TIM modulate ATR and CHK1 activity in a time-of day dependent fashion, although it was found that Cry1-/-;Cry2-/- mouse embryonic fibroblasts (MEFs) still retained appropriate levels of checkpoint activity (Kang & Leem, 2014). Also, ATM and CHK2 form a complex with circadian protein PER1, and it was further demonstrated that PER1 knockdown reduced ATM-mediated CHK2 phosphorylation and dampened apoptotic response to DNA damage in human colon cancer cell line HCT116 (Gery et al., 2006).\n\nFurthermore, a bi-directional crosstalk between the circadian clock and p53 has been reported. The p53 tumor suppressor plays a key role in stimulating DNA damage checkpoints and a circadian oscillation of p53 transcription has been reported in human oral epithelium (Bjarnason et al., 1999). Concurrently, it has been demonstrated that p53 modulates circadian activity in mice by directly binding to the promoter of Per2 and repressing Per2 expression (Miki et al., 2013). Moreover, PER2 directly binds to the C-terminal end of human p53 and slows MDM2-mediated degradation of p53 (Gotoh et al., 2014), indicating multiple points of interplay between these two mechanisms. The consequences of clock regulation on p53 were evident in Per2m/m mutant mice that exhibited lower levels of p53, increased resistance to p53-mediated apoptosis, and higher sensitivity to γ radiation (Fu et al., 2002). p53 activates the CDK inhibitor p21cip1/waf1 to induce cell cycle arrest (Al Bitar & Gali-Muhtasib, 2019). As stated in the previous section, p21cip1/waf1 expression oscillates under circadian control, further demonstrating clock regulation of the DDR. Interestingly, it was reported that either Cry1-/- or Cry2-/- MEFs exhibit altered expression patterns of the p21cip1/waf1 transcript, Cdkn1a, in response to genotoxic stress via doxorubicin (Papp et al., 2015). This study further demonstrated that genotoxic stress can shift the CRY1/CRY2 ratio and consequently change circadian period length (Papp et al., 2015), again demonstrating the crosstalk between the DDR and the molecular machinery of the circadian clock.\n\nIn summary, these studies suggest that circadian proteins exert a wide influence on proper cellular response to the daily insults of DNA damage. Impaired DDR via clock disruption increases the likelihood of cell proliferation despite unresolved mutations, which is a major contributor to cancer progression. Furthermore, since DDR proteins are commonly targeted during chemotherapy to inhibit rapidly dividing cells, understanding the effects of oscillating circadian proteins and their impact on the DDR may result in enhanced efficacy of cancer therapeutics.\n\nThe circadian clock not only plays a role in regulating DNA damage checkpoints, but also affects the ability of cells to perform DNA repair. Cells are equipped with multiple repair pathways that act to maintain DNA sequence fidelity following damage from endogenous and exogenous sources. Interestingly, while each DNA repair pathway has several components, the activities of certain key components exhibit striking transcriptional regulation through the circadian clock (Sancar et al., 2010).\n\nNucleotide excision repair (NER) removes bulky chemical adducts that distort the DNA helix, most importantly UV-induced intrastrand crosslinks (Marteijn et al., 2014). NER relies on the xeroderma pigmentosum group A (XPA) protein to recognize the lesion and coordinate incision and removal by the XPF-ERCC1 endonuclease complex (Marteijn et al., 2014). It was recently described that XPA oscillates in a circadian fashion, thus repair activity after UV irradiation also follows circadian rhythmicity. However, this rhythmicity was lost in Cry1-/-;Cry2-/- mice, clearly demonstrating that NER activity is regulated by the clock (Gaddameedhi et al., 2011). As sunlight is the major source of UV radiation, this connection between DNA repair of UV-induced damage and the circadian clock is intriguing.\n\nBase excision repair (BER) uses a variety of different DNA glycosylases to remove damaged bases, leaving an abasic site that is subsequently processed by enzymes that carry out cleavage, gap-filling and ligation to restore DNA integrity (Krokan & Bjørås, 2013). The expression and activity of one such glycosylase, 8-Oxoguanine DNA glycosylase (OGG1), oscillates under clock control and OGG1 levels were disrupted in a human cohort performing shift work (Manzella et al., 2015). As OGG1 recognizes a specific type of oxidative damage (i.e., 7,8-dihydro-8-oxoguanine opposite cytosine or thymine), it remains to be seen why this enzyme has evolved to be clock-controlled. One interesting link is that OGG1 has been shown to be required to prevent mutations induced by UVA (Kozmin et al., 2005), suggesting an additional role of the clock in repairing DNA damage following sunlight exposure.\n\nDNA double strand breaks (DSBs) represent one of the most serious threats to genome integrity and multiple repair pathways have evolved for their repair, including homologous recombination (HR) and non-homologous end joining (NHEJ) (Chang et al., 2017; Sterrenberg et al., 2022). Due to the number of environmental and cellular sources of DSBs, understanding the role of the circadian clock in their repair is critical. Using HEK293T cells, CLOCK binding was found at several enhancer or transcriptional regulatory sites controlling DNA damage related genes including CDKN1A encoding p21, which mediates cell cycle arrest, as well as BRCA1 and RAD50, which play important roles in DSB repair (Alhopuro et al., 2010). Furthermore, CLOCK knockdown in human U2OS osteosarcoma cells resulted in abnormal cell cycle checkpoint response following irradiation and increased sensitivity to mitomycin C, indicative of a CLOCK-dependent response to repair DSBs (Cotta-Ramusino et al., 2011). CLOCK was also found to localize to laser-induced DSBs in U2OS cells, suggesting a potential direct role in the cellular signaling machinery required for DSB repair (Cotta-Ramusino et al., 2011). In addition to CLOCK, CRY1 is another circadian protein linked to DSB repair efficiency. CRY1 knockdown showed delayed DSB resolution in C4-2 and 22Rv1 cell cultures, and conversely, DSB resolution is enhanced upon treatment with the CRY1 stabilizer KL001 (Shafi et al., 2021). Transcriptomic analysis suggested that CRY1 regulated the expression of several major HR genes (including RAD51, BRCA1, and BRCA2) and other genes involved in NER, BER, mismatch repair (MMR), and NHEJ (Shafi et al., 2021). This study was specifically carried out in human prostate cancer cell lines as well as tissues from prostate cancer patients, and further studies are needed to define the role of CRY1 in regulation of DNA repair genes in other tissues, particularly those that are not hormone responsive.\n\nOverall, these studies show that the effect of the circadian clock on DNA repair is widespread across multiple repair pathways. Although the mechanistic links continue to be investigated, current data suggests that robust circadian rhythms contribute to optimal genome integrity. Further understanding of how the circadian clock potentiates faithful DNA repair through multiple pathways is paramount for developing strategies to both prevent cancer, and to establish better and less toxic treatments for patients undergoing chemo- and radiation- therapy that acts through damaging DNA.\n\nThe goal of the immune system is to be primed to respond to insult through a complex network of different organs, proteins and pathways. It may be advantageous for immune parameters to cycle with activity of an organism, potentially allowing for the host to respond more efficiently to infection. In support of this, the circadian clock has been shown to regulate key parameters of immunity including cytokine release, immune cell number and trafficking, as well as the inflammatory response.\n\nImmune cells, including splenic macrophages, dendritic cells (DCs), and B cells have been found to have cell-autonomous circadian clocks which directly control cellular immune function and timing (Keller et al., 2009; Silver et al., 2012). Cytokines and chemokines are small proteins that regulate the growth, activity and trafficking of immune cells and proper regulation of these proteins is essential for host immune defense. Importantly, the circadian clock has been linked to the production and release of cytokines and chemokines. Upon bacterial endotoxin stimulation, the secretion of TNFα and IL-6 by isolated ex vivo spleen derived macrophages was found to oscillate in a time-of-day dependent manner (Keller et al., 2009). Additional studies demonstrated an important role for Bmal1 in regulating cytokine response. Temporal gating of endotoxin-induced cytokine response in mice, a crucial feature of innate immunity, is dependent on the circadian clock as rhythmic gating of endotoxin response is lost in Bmal1-/- macrophages (Gibbs et al., 2012). This was found to be due to the suppression of Nr1d1, hereafter referred to as Rev-Erbα (Gibbs et al., 2012). An additional role of Bmal1 in regulating the immune response was identified with the genetic ablation of Bmal1 in bronchiolar cells that disrupted the rhythmic expression of the CXCL5 chemokine (Gibbs et al., 2014). These data suggest that the rhythmic release of cytokines is directly regulated by the circadian clock.\n\nIn addition to the rhythmic secretion of cytokines by immune cells, the circadian clock controls immune cell number and infiltration. For example, the number of hematopoietic stem cells (HSCs) and mature immune cells released from the bone marrow into the blood peaks at the beginning of the rest phase in mice (Méndez-Ferrer et al., 2008). In addition to the release of immune cells into the bloodstream, the circadian clock also modulates immune cell trafficking into tissues as evidenced by the rhythmic expression of CXCL5 and CXCL12 that regulate the trafficking and infiltration of neutrophils and HSCs, respectively (Gibbs et al., 2014; Méndez-Ferrer et al., 2008). Human studies provide additional evidence of circadian regulation of immune cell trafficking. Immune cells present in the blood of individuals on a normal sleep-wake cycle were compared to those on 24 hours of wakefulness (Dimitrov et al., 2007). It was found that the number of DCs and T cells in the blood is highly rhythmic and that sleep induced the expression of IL-12 which increased the number of monocytic DCs in the blood (Dimitrov et al., 2007). A more recent study found that individuals with blunted rest-activity rhythms exhibited increased inflammatory markers and elevated circulating white blood cells and neutrophils (Xu et al., 2022). These studies demonstrate a clock-controlled immune response through regulation of immune cell release into the bloodstream and trafficking into tissues.\n\nClock control of the immune system is critical for proper response to infection (Kiessling et al., 2017) and disease (Gibbs et al., 2014; Kitchen et al., 2020), and even vaccination (Cervantes-Silva et al., 2022). In support of this, mice infected with Salmonella enterica in the early rest period exhibited a high pathogen load and a stronger proinflammatory response (Bellet et al., 2013) and the magnitude of Leishmania parasitic infection in mice varied over 24 hours (Kiessling et al., 2017). These differences in infection and inflammation may be due to the time-dependent release of cytokines and immune cells. Indeed, the circadian expression of chemo attractants and the rhythmic infiltration of neutrophils and macrophages was lost in clock deficient macrophages (Kiessling et al., 2017; Sato et al., 2014). Additionally, pulmonary inflammation was found to be regulated by the rhythmic expression of the chemokine CXCL5 leading to time-of-day dependent neutrophil recruitment to the lung (Gibbs et al., 2014). Bmal1-/- bronchiolar cells lack this rhythmic CXCL5 expression leading to exaggerated inflammatory response and an impaired host response to Streptococcus pneumoniae infection (Gibbs et al., 2014). Bmal1 deletion suppresses Rev-erbα expression, and it was found that Rev-erbα-/- mice exhibit an exaggerated neutrophilic inflammatory response (Pariollaud et al., 2018). Furthermore, myeloid specific deletion of Bmal1 disrupts the diurnal trafficking of Ly6Hi inflammatory monocytes and promotes inflammation by inducing expression of monocyte attracting chemokines (Nguyen et al., 2013).\n\nOverall, these studies establish the circadian clock as a critical regulator of the immune response through the release of cytokines and the trafficking of immune cells. This leads to a time-of-day dependent proinflammatory response to challenge such as bacterial or pathogenic infection. Moreover, disruption of the circadian clock has the potential to alter the daily rhythm of the immune system and lead to various types of diseases, including cancer.\n\n\nConnecting the dots: The circadian clock and cancer\n\nIn the previous section, we described how the circadian clock regulates critical cellular processes including cell cycle control, the DNA damage response and repair, as well as immunity. These processes are included as ‘Hallmarks of Cancer’ that are dysregulated during transformation (Hanahan, 2022; Hanahan & Weinberg, 2000, 2011), which suggests that the circadian clock may be involved in tumorigenesis. In support of this, we describe how the circadian clock is associated with cancer by looking at epidemiological data, early-onset cancers, and the tissue-specific and cell-type dependent function of the clock in various model systems.\n\nAbout one quarter of the US population participates in night shift work (Alterman et al., 2013; Drake & Wright, 2017), which causes significant misalignment between the endogenous circadian clock and the sleep-wake cycle (James et al., 2017). Night shift work has been implicated as a risk factor for cancer and a systematic review of night shift work and cancer was recently reported (IARC, 2020). Several studies were identified that aimed to assess a correlation between night shift work and cancer risk, and these reports are summarized in Table 1. The most extensively studied association was between night shift work and breast cancer, and the majority of these reports found that night shift work increased the risk of developing breast cancer (Cordina-Duverger et al., 2018; Hansen & Lassen, 2012; Jones et al., 2019; Schernhammer et al., 2006). Several studies also noted an increased risk with duration of exposure and cumulative exposure to night shift work (Cordina-Duverger et al., 2018; Davis et al., 2001; Hansen & Lassen, 2012; Hansen & Stevens, 2012; Lie et al., 2011). In addition to breast cancer, shift work was also found to increase the risk of developing prostate, colon and rectum, lung, stomach, ovarian and pancreatic cancer. Although numerous studies cite a positive correlation between night shift work and cancer incidence, other studies report no effect (Jones et al., 2019; Koppes et al., 2014; Li et al., 2015; O’Leary et al., 2006; Pronk et al., 2010; Travis et al., 2016; Vistisen et al., 2017). There are multiple explanations for the contradictory data, including the lack of a standardized definition for night shift work, self-reporting collection process, and adjustment for confounding factors such as lifestyle. These limitations should be addressed and larger, more comprehensive studies are needed with multiple cancer types to define the epidemiological link between the circadian clock and cancer risk.\n\nThe previous section highlighted the potential increase in cancer incidence in populations that participate in night shift work, which is known to disrupt circadian rhythms. However, there is mounting concern for circadian disruption in the general population as the access to technological devices continues to increase. Gradisar et al. demonstrated that nine out of 10 individuals surveyed use a technological device in the hour before bed, with the use increasing in individuals under 30 years of age (Gradisar et al., 2013). Among the Japanese population, young adults between the ages of 15 to 20, were exposed to the highest intensity of artificial light-at-night (Chen et al., 2022). The exposure to dim light at night through the use of devices has been shown to disrupt circadian rhythmicity by suppressing melatonin and impairing sleep quality (Lee & Kim, 2019). This suggests that younger individuals may be exposed to more environmental factors that disrupt the circadian clock than older populations. Strikingly, the average annual increase in the incidence of all cancers in young adults aged 15 to 39 years old has continued to increase since 1975 (Miller et al., 2020). A review of 98 articles published between 1995–2020 found that the incidence of colorectal, breast, kidney, pancreas, and uterine cancer is increasing in younger age groups (di Martino et al., 2022). In addition to the increasing incidence, studies have also suggested that the underlying biology of cancer in young adults differs from the same cancer in children or older individuals (Tricoli et al., 2016, 2018). Altogether, this introduces the idea that environmental circadian clock disruption in younger populations may contribute to the increasing incidence of early-onset cancers, though further studies are needed to confirm this experimentally.\n\nIt is worth noting that the increasing trends of early-onset cancers are strongest for colorectal cancer (CRC). Between 1975 and 2010, there has been a steady decline in CRC incidence rates in adults over the age of 50. However, in patients aged 20 to 34, the incidence of CRC has continued to rise (Bailey et al., 2015). If this trend continues, it is expected that by 2030, the incidence of colon and rectal cancer in individuals aged 20 to 34 will increase by 90% and 124.2%, respectively (Bailey et al., 2015). It was also identified that the increasing incidence of CRC in younger populations was greatest among Hispanics and African Americans, suggesting an alarming cancer health disparity (Augustus & Ellis, 2018; Muller et al., 2021; Singh et al., 2014). Moreover, food intake is a powerful environmental cue synchronizing the circadian clock in peripheral tissues. Feeding mice only during the light phase, when mice are inactive, causes a phase shift in peripheral clocks of the liver, kidney, heart and pancreas (Damiola et al., 2000; Stokkan et al., 2001), demonstrating that the timing of food intake can disrupt the circadian clock. Nutritional challenge has also been shown to dynamically impact the circadian clock, including high fat diet and time-restricted eating (Acosta-Rodríguez et al., 2022; Chaix et al., 2019; Eckel-Mahan et al., 2013; Hatori et al., 2012). These studies establish the importance of diet and timing of food intake in maintaining robust circadian rhythms in peripheral tissues. It will be important to study the impact of alterations in these environmental and behavioral factors on the alarming increase in CRC rates in younger populations, as well as other cancer types.\n\nAlthough there is mounting evidence suggesting that the circadian clock is implicated in various types of cancer, the mechanism underlying the role of the clock in cancer is still being uncovered. Table 2 outlines significant findings that provide clues for how the circadian clock functions in various cancer types and model systems. Based on these studies, the circadian clock has been implicated in both the initiation and the progression of cancer through the regulation of oncogenic pathways, cell cycle control, DNA damage repair, stemness, immunity and metastasis (Figure 2). However, the effect of clock disruption on tumorigenesis may be tissue and model-specific. For example, knock out of the core clock gene Bmal1 in mouse models of solid tumors promotes tumor progression in CRC (Chun, Fortin, Fellows et al., 2022; Stokes et al., 2021), lung (Papagiannakopoulos et al., 2016), and other cancer types (Lee et al., 2010) but reduces the development of cutaneous squamous tumors (Janich et al., 2011). Furthermore, downregulation of Bmal1 in human glioblastoma stem cells halts their growth (Dong et al., 2019; Puram et al., 2016). As more studies are done in multiple cancer types and model systems, we may begin to better delineate the tissue-specific effects of clock disruption on cancer.\n\nIn normal tissue, the circadian clock maintains homeostasis through diverse functions including control of the cell cycle, genome integrity, immunity, and metabolism. Given the numerous roles of the circadian clock in maintaining physiology, it is not surprising that the clock has been implicated in cancer initiation and progression. Indeed, a large body of evidence has linked the circadian clock to processes that become dysregulated during tumorigenesis including the cell cycle, proliferation, genome stability, stemness, metastasis, inflammation, immunity, and oncogenic signaling pathways. Analyzing over 32 different cancer types, it was found that clock genes are associated with activation or inhibition of oncogenic signaling pathways including phosphatidylinositol 3-kinase (PI3K)/AKT and RAS/mitogen-activated protein kinase (MAPK) signaling pathways (Ye et al., 2018). Knockout of Bmal1 was shown to accelerate Apc LOH in a mouse model of CRC suggesting that the clock may be involved in maintaining genome integrity (Chun, Fortin, Fellows et al., 2022). With regards to the role of the clock in the cell cycle, mutation of Cry2 in MYC-transformed fibroblasts suppressed p53 and enhanced growth (Chan et al., 2021) whereas downregulation of BMAL1 and CLOCK in human glioblastoma stem cells induced cell cycle arrest and apoptosis (Chen et al., 2020; Dong et al., 2019), demonstrating a cancer and tissue-specific effect of the clock on tumorigenesis. The circadian clock has also been shown to regulate immunity and metastasis as clock gene dysregulation is correlated with increased inflammation (Gibbs et al., 2014) and T cell exhaustion (Wu et al., 2019). Chronic jet lag promotes an immunosuppressive microenvironment, enhances stemness, and increases cancer cell metastasis (Hadadi et al., 2020) and intravasation of circulating breast tumor cells was shown to have time-of-day frequency (Diamantopoulou et al., 2022) suggesting potential clock-control of metastatic seeding. A direct link between circadian immune function and anti-tumor immunity was demonstrated by clock-dependent trafficking of DCs to the tumor draining lymph node regulating circadian function of tumor-antigen specific CD8s and melanoma volume after engraftment (Wang et al., 2022). Lastly, the circadian clock has been implicated in metabolic pathways involved in driving cellular proliferation, especially related to the crosstalk between the clock and MYC signaling (Altman et al., 2015; Chun, Fortin, Fellows et al., 2022; Shostak et al., 2016; Stokes et al., 2021). Figure created using BioRender.\n\n\nHighlighting the circadian clock and CRC\n\nAs discussed in the previous section, there is a growing body of evidence suggesting a role for circadian clock disruption in various types of cancer. CRC is of particular interest as early-onset CRC is increasing at an alarming rate, faster than any other type of cancer (Augustus & Ellis, 2018; Bailey et al., 2015; Muller et al., 2021; Singh et al., 2014). Circadian disruption due to Bmal1 knockout was sufficient to drive CRC past the initiation stage (Chun, Fortin, Fellows et al., 2022; Stokes et al., 2021). The importance of multiple signaling pathways in CRC progression including Wnt, TGF-β, Notch, EGFR/MAPK and PI3K has been reviewed previously (Koveitypour et al., 2019). Here, we discuss how the circadian clock is disrupted in CRC and what molecular processes are governed by the clock that, when perturbed, result in CRC progression (Figure 3).\n\nCRC has been shown to be initiated by sequential mutations in known cancer-causing genes including APC, KRAS, p53, and SMAD4 (Drost et al., 2015; Li et al., 2014). Numerous studies have found that the circadian clock is involved in CRC initiation and progression. Importantly, circadian clock disruption promotes CRC pathogenesis in multiple mouse models (Chun, Fortin, Fellows et al., 2022; Stokes et al., 2021; Wood et al., 2008). Additionally, the core clock gene CLOCK was found to be mutated in 53% of CRC that display microsatellite instability and it was shown that CLOCK binds near DNA damage related genes p21, BRCA1 and RAD50 to mediate DNA repair, apoptosis, and cell cycle arrest (Alhopuro et al., 2010). Loss of Bmal1 in Apcex1-15/+ mice and intestinal organoids accelerated Apc LOH which drove transformation (Chun, Fortin, Fellows et al., 2022). These studies implicate the circadian clock in maintenance of genome stability and demonstrate a role for circadian clock disruption in promoting colorectal carcinogenesis. An increasing number of studies have also explored the relationship between CRC and circadian clock disruption through shift work, light-at-night, and diet. Night shift work in humans has been shown to increase the risk of developing CRC (Papantoniou et al., 2017; Schernhammer et al., 2003) and chronic jet lag, through exposure to light and night, increases CRC tumor burden in mice (Chun, Fortin, Fellows et al., 2022; Stokes et al., 2021). High-fat diet also disrupts molecular circadian rhythms (Eckel-Mahan et al., 2013; Hatori et al., 2012; Kohsaka et al., 2007). Given that HFD is known to enhance tumorigenicity of intestinal progenitors (Beyaz et al., 2016; Mana et al., 2021) and exacerbate CRC (Goncalves et al., 2019), the potential link with the intestinal clock warrants further investigation. Overall, compelling evidence implicates circadian clock disruption in CRC carcinogenesis, which suggests that night shift work, light-at-night, and diet could be potential drivers of CRC progression in humans, and especially in young-onset CRC. Figure created using BioRender.\n\nWnt signaling is an important pathway for many processes including development, proliferation, and apoptosis (Nusse & Clevers, 2017). This is especially relevant for the intestine, a highly regenerative organ where differentiated cells are replaced every four to five days by stem cells located in the crypt base (Van Der Flier & Clevers, 2009). The Wnt pathway is upregulated when Wnt ligands bind the frizzled receptor and trigger inactivation of the destruction complex, composed of Apc, GSK3-β and Axin (Neufeld et al., 2000; Orford et al., 1997; Rubinfeld et al., 1993; Su et al., 1993). As a result, β-catenin evades proteosome-dependent degradation, and shuttles into the nucleus to co-activate TCF-LEF mediated transcription of Wnt-dependent genes (Hoverter et al., 2014; Molenaar et al., 1996).\n\nThe intestine is a highly rhythmic organ and rhythmicity is important in functions such as peristalsis, permeability and secretion of digestive enzymes (Codoñer-Franch & Gombert, 2018). The molecular clock is involved in regulating intestinal circadian rhythms as core clock genes are expressed in many intestinal cell types including stem, progenitor, tuft, enteroendocrine and enterocytes (Habowski et al., 2020). Circadian rhythms of intestinal stem cells (ISCs) are thought to be driven, at least in part, by Wnt signaling secreted from differentiated cells. ISC clocks were responsive to both Wnt and Hippo signaling in the stem cell niche (Parasram et al., 2018). Furthermore, Paneth cells were found to rhythmically secrete Wnt and many Wnt pathway components have rhythmic expression (Matsu-Ura et al., 2016; Soták et al., 2013). This is likely to be essential for proper ISC function as Wnt signaling was found to couple the clock and the cell cycle in the intestine (Matsu-Ura et al., 2016) and the coupling of clock and cell cycle is conserved across multiple species (Hong et al., 2014a; Yang et al., 2010). In the intestine, the molecular clock was found to gate cell cycle progression and was important for ISC regeneration after DSS induced damage (Karpowicz et al., 2013; Matsu-Ura et al., 2016).\n\nMisregulation of molecular clock components have frequently been identified in human CRC. Multiple studies have reported decreased BMAL1, CRY1-2 and PER1-3, and increased CLOCK, CSNK1E and TIM in tumor tissue relative to matched healthy mucosa (Hong et al., 2014b; Krugluger et al., 2007; Mazzoccoli et al., 2011, 2016; Oshima et al., 2011; Zeng et al., 2014). This was also linked to disease progression as reduced BMAL1, PER1 or PER3 expression was associated with poor overall survival (Mazzoccoli et al., 2011; Zeng et al., 2014). Additionally, clock genes were found to be mutated in cancer and therefore might be involved in pathogenesis. A large fraction of CRC patients with microsatellite instability (MSI) had a mutation in CLOCK which could decrease CLOCK expression in MSI CRC cell lines (Alhopuro et al., 2010; Mazzoccoli et al., 2011).\n\nPre-clinical genetic mouse models have also demonstrated that clock disruption accelerates CRC pathogenesis. In the azoxymethane and dextran sodium sulfate (AOM/DSS) model of colitis associated CRC, the circadian rhythmicity of Per1, Per2, Reverb, Dbp and Bmal1 was reduced in tumors compared to healthy colon (Soták et al., 2013). In an ApcMin/+ model of CRC, tumors exhibited reduced overall expression of key clock components Rev-Erbα, Bmal1 and Per2, with complete loss of Per2 rhythm (Stokes et al., 2021; Yang et al., 2009). Furthermore, clock disruption was found to increase tumor burden when Per2 or Bmal1 were deleted in ApcMin/+ mice (Stokes et al., 2021; Wood et al., 2008). In a novel mouse model, where exons 1 to 15 in Apc were deleted in one allele (Apcex1-15/+), Bmal1 knockout increased tumor incidence, enlarged polyps and decreased survival (Chun, Fortin, Fellows et al., 2022). Additionally, environmental circadian disruption through use of a light shift paradigm or constant light increased both polyp formation and size of tumors in Apcex1-15/+ and Apcmin/+ mice (Chun, Fortin, Fellows et al., 2022; Stokes et al., 2021). Together these results suggest that circadian disruption can play a key role in driving pathogenesis of CRC.\n\nThe Wnt signaling pathway is highly mutated in human CRC with nearly all tumors containing inactivating mutations in APC or GSK3-β, or stabilizing mutations in CTNNB1 (β-catenin) (Kwong & Dove, 2009). Around 80% of sporadic human CRC contain a mutation in APC (Fearnhead et al., 2001). In many cases this is followed by mutation of the wild type allele, also known as loss of heterozygosity (LOH) (Fearnhead et al., 2001; Kwong & Dove, 2009). Due to its important role in the destruction complex, Apc mutation results in aberrant activation of the Wnt signaling pathway (Korinek, 1997; Morin, 1997; Moser et al., 1990). Apc mutations are sufficient to drive CRC initiation of adenoma growth (Cheung et al., 2010; Lamlum et al., 2000; Rowan et al., 2000; Zauber et al., 2016; Zhang & Shay, 2017). However, secondary driver mutations in key genes such as Kras, Tp53, and Smad4 are required for progression to adenocarcinoma (Drost et al., 2015; Fearon & Vogelstein, 1990; Vogelstein et al., 1988). The circadian clock has recently been identified as a secondary driver of CRC. Bmal1 loss was found to promote Apc LOH by increasing genome instability and resulting in Wnt signaling hyperactivation (Chun, Fortin, Fellows et al., 2022). Additionally, downregulation of PER2 in colon cancer cells increased β-catenin levels and cell proliferation (Wood et al., 2008). Increased β-catenin in CRC cell lines also enhanced PER2 degradation by upregulating β-Trcp, an E3 ubiquitin ligase component (Yang et al., 2009). As Apc is involved in regulation of cell-cell adhesion, microtubule stability, cell cycle and apoptosis (Fearnhead et al., 2001), clock disruption mediated Apc LOH could perturb multiple pathways important in CRC progression.\n\nAside from Wnt signaling, other pathways have also been implicated in clock mediated acceleration of CRC. The Hippo pathway regulates multiple key processes including proliferation, differentiation, tissue growth, and regeneration. A cascade of serine/threonine kinases act to sequester Yes associated protein (YAP1) and transcriptional activator with PDZ binding motif (TAZ) in the cytoplasm and prevent them from activating the pro-survival TEA DNA binding (TEAD) family of transcription factors. Dysregulation results in an increase in YAP/TAZ which is associated with many human cancers, mediating increased proliferation and metastasis (Calses et al., 2019). In an ApcMin/+ mutant mouse model, Yap1 was found to be required for the progression of early initiating cells by suppressing differentiation and promoting regeneration (Gregorieff et al., 2015). The Hippo pathway may be involved in clock mediated acceleration of CRC. Yap and Tead4 increased in ApcMin/+ Bmal1-/- mice and were associated with increased self-renewal (Stokes et al., 2021). Furthermore, Apcex1-15/+Bmal1-/- organoids had increased expression of YAP/TAZ pathway components compared to Apcex1-15/+ organoids (Chun, Fortin, Fellows et al., 2022). In summary, clock disruption accelerates the pathogenesis of CRC, and based on data from pre-clinical studies, the circadian clock likely impinges on several important signaling pathways that regulate intestinal biology.\n\n\nCircadian clock and prevention/treatment of cancer\n\nIn modern society, the necessity of night shift work and the presence of artificial light at night warrants a better understanding of the impact of circadian disruption on health and disease. Above, the literature defining circadian clock function in critical cellular processes and the role of circadian clock disruption in various cancer types was summarized. In this section, emerging ideas for how the circadian clock can be leveraged to both prevent and treat cancer are highlighted. For additional information on this topic, a more extensive review of chronotherapeutic approaches has recently been published (Sulli et al., 2018a).\n\nPromoting robust circadian rhythms through consistent sleep and feeding behavior is an important regulator of physiological health. However, night shift workers are faced with irregular activity-rest and feeding-fasting rhythms as well as artificial light at night exposure, all of which are known to disrupt the circadian clock. Key literature was reviewed above that aimed to define the correlation between night shift work and cancer prevalence. Although this body of literature requires more comprehensive studies to draw definitive conclusions, the importance of proper alignment of circadian rhythms has emerged as a key theme. Numerous studies cite a significant increase in cancer risk after long-term night shift work, typically 15–20 years (Åkerstedt et al., 2015; Hansen & Lassen, 2012; Wegrzyn et al., 2017). Risk was also seen to increase with duration of exposure (Davis et al., 2001; Hansen & Lassen, 2012; Hansen & Stevens, 2012; Lie et al., 2011). For example, in nurses who worked night shift for over five years, the risk of developing breast cancer increased from an odds ratio of 1.4 to 1.8 with increasing consecutive night shifts (Lie et al., 2011). In order to reduce the risk of cancer in night shift workers, the consecutive duration of night shifts may need to be limited as well as the cumulative exposure to night shift work.\n\nAlthough circadian misalignment is an unavoidable consequence of night shift work, recent research has recommended lifestyle interventions as a means of combating these effects. Night shift workers often disrupt their feeding-fasting patterns which has been shown to disrupt glucose metabolism (Spiegel et al., 1999). Night shift work has also been significantly associated with metabolic syndrome (Wang et al., 2014), which increases the risk for developing various types of cancer (Esposito et al., 2012). Therefore, increasing metabolic health through lifestyle intervention may combat the increased risk of cancer. One such intervention is time-restricted eating (TRE), which involves limiting the eating window to 6–12 hours per day (Manoogian et al., 2022). In a study with prediabetic men, limiting the feeding window to six hours for five weeks improved insulin sensitivity, blood pressure, oxidative stress, and appetite (Sutton et al., 2018). A recent study found that a 10-hour feeding window in 24-hour shift workers is a feasible intervention to reduce weight, improve cardiometabolic health, sleep quality, and mood (Manoogian et al., 2022).\n\nIt has been well established that night shift is associated with circadian misalignment, however, the general population is increasingly at risk of circadian misalignment through inconsistent eating patterns and artificial light-at-night exposure. Therefore, it may be beneficial to update cancer screening measures (Patel et al., 2022; Wolf et al., 2018). As the percentage of early-onset cancer increases, the screening age should decrease accordingly for early detection and prevention measures. Promoting robust circadian rhythms through consistent sleep, feeding, regulation of night shift work, and lifestyle interventions such as TRE may help improve parameters that impinge on human health and could offset the impacts of circadian misalignment on specific cancer types.\n\nCancer chronotherapy refers to the timing of an anticancer drug to increase efficacy and decrease toxicity. This approach is based on the rationale that the drug will be better tolerated at certain times of day based on the mechanism of action of the drug. A recent comprehensive review of chronomodulated chemotherapy has recently been reported (Printezi et al., 2022). In this systematic review, 11 of 18 studies found that chronomodulated chemotherapy significantly decreased toxicity while maintaining efficacy. More specifically, chronomodulated chemotherapy reduced side effects including nausea, vomiting, mucositis and leukopenia for nasopharyngeal carcinoma (Gou et al., 2018; Zhang et al., 2018), breast (Coudert et al., 2008), colorectal (Lévi et al., 1994, 1997) and endometrial cancer (Gallion et al., 2003). Reducing side effects is a critical aspect of patient care as it improves quality of life and often allows for higher or more frequent doses. Although chronomodulated chemotherapy does appear to reduce side effects, it remains elusive whether chronotherapy improves drug efficacy or prognosis. For example, only three studies report higher response rate and longer survival in chronotherapy treated groups (Gou et al., 2018; Lévi et al., 1994, 1997). However, two of the three of these studies report higher dose intensity in the chronotherapy treated group because of the reduced side effects. Therefore, it is unclear whether improved response is due to increased dose or a direct result of chronotherapy. A meta-analysis on factors impacting drug timing effects found that study size and whether or not the study was publicly registered as a clinical trial affected the reported efficacy of chronomedicine (Ruben et al., 2021), suggesting that further studies are needed. In summary, optimal timing of anticancer drugs appears to reduce toxicity but more mechanistic studies are needed to determine the clinical relevance of anticancer chronotherapeutic approaches on drug efficacy.\n\n\nConcluding remarks and future directions\n\nThe circadian clock is an evolutionarily conserved internal timekeeping system that maintains homeostasis within the body. In this review, we discussed the connection between the circadian clock and critical biological processes including cell cycle control, DNA damage response, DNA repair, and immunity. Disruption of these processes are known hallmarks of cancer (Hanahan, 2022; Hanahan & Weinberg, 2000, 2011), and we highlight the links between circadian clock disruption and cancer through clinical, epidemiological, and pre-clinical molecular studies. Though progress has been made to deconvolute the role of the circadian clock in cancer, this review highlights the divergent evidence linking circadian clock disruption with tumorigenesis. For clinical and epidemiological studies, these differing conclusions may be due to self-reporting, confounding factors, and non-standardized definitions of night shift work. For molecular and mechanistic studies, clock-controlled rhythmic expression is known to be tissue-specific, suggesting that the impact of circadian clock disruption would also be tissue specific. This makes drawing a simplified conclusion regarding the role of clock disruption on tumorigenesis difficult. Future studies are needed to systemically explore these tissue-specific differences and determine the role of clock disruption in each organ independently. These more comprehensive studies will yield a foundational understanding by which the circadian clock can be leveraged for cancer prevention and chronomedicine-based approaches.\n\nFinally, with the alarming rise in the rate of early-onset cancers and the necessity of night shift work in modern society, it is imperative to address the concern of circadian clock disruption in a growing population of individuals afflicted by circadian misalignment. In this review, we highlighted how individuals can promote healthy circadian rhythms by limiting the exposure to night shift work, lifestyle interventions such as TRE, and updated cancer screening. This list is not comprehensive and additional molecular studies are needed to guide our understanding of intervention approaches that can offset circadian clock disruption. Overall, the circadian clock presents a unique and underexplored connection between health and disease which has the potential for therapeutic value in cancer treatment.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAcosta-Rodríguez V, Rijo-Ferreira F, Izumo M, et al.: Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice. Science (New York, N.Y.). 2022; 376(6598): 1192–1202. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nÅkerstedt T, Knutsson A, Narusyte J, et al.: Night work and breast cancer in women: A Swedish cohort study. BMJ Open. 2015; 5(4): e008127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl Bitar S, Gali-Muhtasib H: The role of the cyclin dependent kinase inhibitor p21cip1/waf1 in targeting cancer: Molecular mechanisms and novel therapeutics. Cancers. 2019; 11(10). 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "162020", "date": "16 Feb 2023", "name": "Phillip Karpowicz", "expertise": [ "Reviewer Expertise Circadian rhythms", "stem cells" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 review and discuss a growing body of literature that has explored connections between circadian rhythm disruption and cancer. This is now a long-standing and growing field, thus the review is helpful in updating the reader on the many new research papers that have been recently published. The authors highlight new advances in the field, particularly in the effect of rhythm disruption in colorectal cancer. The review is well-written and well-cited, the sections covering epidemiological studies as well as cellular and molecular mechanisms are covered thoroughly. The tables help summarize many studies, and particularly Table 2 is a good resource.\nI have only the following minor comments/suggestions:\nIntro - The authors describe many studies which have found links between circadian clock genes and cell cycle regulators. In the intro (pg. 3), they might include a sentence mentioning that many of these studies were done in different tissues, and that the connections are tissue- or cell-specific. In addition, many studies have shown mechanisms occurring at the protein level rather than from the TTFL. For instance, the connections between Cry and cMyc, or Per and p53. The authors describe this area well and mention these issues but pg. 3 and Fig. 1 really only show the TTFL. The intro does not seem to reflect the complexity and themes covered in the review. Clarifying that clock genes can have cell-specific roles, and protein-based mechanisms aside from their role as transcriptional regulators would strengthen the intro.\n\nIn several parts of the manuscript, the authors mention the term “circadian misalignment” but don’t really define it. A couple of sentences defining what this means would help clarify (do the authors mean molecular clock misalignment, individual cell re-entrainment, or inter-tissue misalignment, or all three?).\n\nFigure 3 - With the finding that Bmal1 loss of function increases LOH, wouldn’t clock disruption be operating at the level of healthy epithelium/pre-cancer transition (where the Kras, APc, etc. mutation takes place)? The figure could indicate this contribution earlier than showing it at the stage past the pre-cancer step.\n\nPage 14 – the authors might put 'Clock disruption in CRC' section after 'The clock and aberrant signaling in CRC' section – to first explain the role of Apc in colorectal cancer before discussing the pre-clinical mouse models (Apc ex1-15 and Apc min). This would also fit better right after discussion of the Wnt pathway in the intestine.\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": "9620", "date": "17 Nov 2023", "name": "Selma Masri", "role": "Author Response", "response": "Response to Reviewer Comments Fortin et al., Circadian clocks in health and disease: Dissecting the role of the biological pacemaker in cancer F1000Research Reviewer #1 The authors review and discuss a growing body of literature that has explored connections between circadian rhythm disruption and cancer. This is now a long-standing and growing field, thus the review is helpful in updating the reader on the many new research papers that have been recently published. The authors highlight new advances in the field, particularly in the effect of rhythm disruption in colorectal cancer. The review is well-written and well-cited, the sections covering epidemiological studies as well as cellular and molecular mechanisms are covered thoroughly. The tables help summarize many studies, and particularly Table 2 is a good resource. We thank Reviewer #1 for their comments regarding the quality of our work and have addressed each of these comments in the text and highlighted the changes below. Intro - The authors describe many studies which have found links between circadian clock genes and cell cycle regulators. In the intro (pg. 3), they might include a sentence mentioning that many of these studies were done in different tissues, and that the connections are tissue- or cell-specific. In addition, many studies have shown mechanisms occurring at the protein level rather than from the TTFL. For instance, the connections between Cry and cMyc, or Per and p53. The authors describe this area well and mention these issues but pg. 3 and Fig. 1 really only show the TTFL. The intro does not seem to reflect the complexity and themes covered in the review. Clarifying that clock genes can have cell-specific roles, and protein-based mechanisms aside from their role as transcriptional regulators would strengthen the intro. This is an important point, and we are happy that the reviewer raised this issue. In the introduction (pg 3) and in Figure 1, we have highlighted the role of the circadian clock being cell- and tissue-specific as well as circadian regulation occurring at both the transcriptional and protein level. In several parts of the manuscript, the authors mention the term “circadian misalignment” but don’t really define it. A couple of sentences defining what this means would help clarify (do the authors mean molecular clock misalignment, individual cell re-entrainment, or inter-tissue misalignment, or all three?). We refer to “circadian misalignment” as misalignment between an individual’s internal circadian clock and the external cues such as light-dark and feeding-fasting cycles, and have now been defined in the text (pg 15). Figure 3 - With the finding that Bmal1 loss of function increases LOH, wouldn’t clock disruption be operating at the level of healthy epithelium/pre-cancer transition (where the Kras, APc, etc. mutation takes place)? The figure could indicate this contribution earlier than showing it at the stage past the pre-cancer step. This is a critical point. Our work and others have shown that circadian clock disruption alone does not induce tumor initiation (Stokes et al., Cell Mol Gastroenterol Hepatol; Chun, Fortin, Fellows et al., Sci Adv). However, other studies have shown that clock disruption alone does initiate tumorigenesis, through indirect mechanisms (Lee et al., PLoS One; Kettner et al., Cancer Cell). Importantly, Kettner et al. demonstrates that clock disruption initiates hepatocellular carcinoma through non-alcoholic fatty liver disease, which is known to predispose to tumorigenesis. Lee et al. aged clock disrupted mice before identifying cancer initiation, making it difficult to disentangle the effect of clock disruption on an aging phenotype versus the effect on cancer initiation. Therefore, we feel that clock disruption alone is difficult to link to cancer initiation in either of these studies. In the majority of studies, it is only when cancer has initiated that clock disruption progresses tumorigenesis. For example, mice with genetic or environmental clock disruption are exposed to gamma irradiation (Fu et al., Cell; Lee et al., PLoS One), inoculated with tumor cells (Filipski et al., Cancer Res) or crossed with a tumor-initiating mutation (Lee et al., PLoS One; Papagiannakopoulos et al., Cell Met; Pariollaud et al., Sci Adv; Wood et al., MCR) to observe the effect of clock disruption on tumor progression. This suggests that clock disruption may not be sufficient to initiate cancer but instead is sufficient to progress cancer. Therefore, with the lack of sufficient evidence suggesting that clock disruption functions at the level of cancer initiation, we have placed it after the pre-cancer step in the figure. We also discuss in the review the function of the circadian clock in maintaining stemness and regeneration in normal intestinal epithelium, and these elegant studies are discussed in detail. Page 14 – the authors might put 'Clock disruption in CRC' section after 'The clock and aberrant signaling in CRC' section – to first explain the role of Apc in colorectal cancer before discussing the pre-clinical mouse models (Apc ex1-15 and Apc min). This would also fit better right after discussion of the Wnt pathway in the intestine. We thank the reviewer for pointing this out. We agree with the reviewer on the order of these paragraphs and have made this change in the text." } ] }, { "id": "162021", "date": "02 Mar 2023", "name": "Kristin Eckel-Mahan", "expertise": [ "Reviewer Expertise Circadian rhythms" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 'Circadian clocks in health and disease: Dissecting the roles of the biological pacemaker in cancer', Fortin et al. provide a brief but well-balanced review of key molecular mechanisms discovered to date that are involved in the links between circadian clock disruption and cancer. The figures are nicely done and the tables are very helpful in summarizing the information. Overall, this is a nicely written article and I have only a few suggestions for improvement.\nConsidering the emphasis on early-onset cancers in the abstract, the authors may wish to draw a tighter connection between clock disruption and early-onset cancers in the manuscript. The authors give a nice molecular summary of CRC, the prevalence of which seems to be particularly pronounced in younger populations. The authors may wish to discuss more why CRC might show a tighter link to clock function than some of the other tumor types. In the context of CRC, the authors may want to add that intestinal tumors were also found in the Lee et al. (2010) study that they cite.\n\nI realize that the review does not focus on neuroblastoma; however, considering the discussion regarding BMAL1 and MYC, the authors may wish to cite Moreno-Smith et al. (2021)1, where MYC downregulates BMAL1 in MYCN-positive neuroblastomas and suppresses clock function.\n\nIn the paragraph discussing base excision repair, it would nice if the authors would provide additional details about the studies showing clock regulation of the enzyme OGG1 (is it tissue-specific, observed in vivo vs. in vitro, etc.?), especially considering the interesting links to shift work.\n\nWhen covering the Keller et al. (2009) paper discussing the role of the clock in splenic macrophages, the authors may wish to add that, like other cell types/tissues, much of the macrophage transcriptome is highly dynamic over the 24-hr. period.\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": "9621", "date": "17 Nov 2023", "name": "Selma Masri", "role": "Author Response", "response": "Response to Reviewer Comments Fortin et al., Circadian clocks in health and disease: Dissecting the role of the biological pacemaker in cancer F1000Research Reviewer #2 In 'Circadian clocks in health and disease: Dissecting the roles of the biological pacemaker in cancer', Fortin et al. provide a brief but well-balanced review of key molecular mechanisms discovered to date that are involved in the links between circadian clock disruption and cancer. The figures are nicely done and the tables are very helpful in summarizing the information. Overall, this is a nicely written article and I have only a few suggestions for improvement. We appreciate the insightful feedback from Reviewer #2 and we are pleased that this reviewer finds our study “helpful” and “nicely written”. Below we have made every effort to address these valuable comments. Considering the emphasis on early-onset cancers in the abstract, the authors may wish to draw a tighter connection between clock disruption and early-onset cancers in the manuscript. The authors give a nice molecular summary of CRC, the prevalence of which seems to be particularly pronounced in younger populations. The authors may wish to discuss more why CRC might show a tighter link to clock function than some of the other tumor types. In the context of CRC, the authors may want to add that intestinal tumors were also found in the Lee et al. (2010) study that they cite. The reviewer is correct that CRC incidence is on the rise in younger populations. CRC may show a tighter link to clock function due to the following reasons which we have expanded upon in the text (pg 9). Intestinal epithelial cells are highly regenerative and this regeneration is regulated by the circadian clock. Thus, the intestine is especially sensitive to disruption of circadian rhythms which can affect the coordination of cellular turnover (Stokes et al., CMGH; Yoshida et al., Chronobiol Int; Codoñer-Franch et al., WJG). The gut microbiota exhibits circadian rhythmicity and is negatively impacted by circadian disruption (Liang et al., PNAS; Heddes et al., Nat Commun). Alterations of the circadian clock, through timing of food intake and high fat diet, can disrupt the circadian clock in the intestine (Eckel-Mahan et al., Cell; Damiola et al., Genes Dev; Chaix et al., Cell Metab). I realize that the review does not focus on neuroblastoma; however, considering the discussion regarding BMAL1 and MYC, the authors may wish to cite Moreno-Smith et al. (2021)1, where MYC downregulates BMAL1 in MYCN-positive neuroblastomas and suppresses clock function. We thank the reviewer for bringing up this study and have included the study in Table 2 as well as the text (pg 5). In the paragraph discussing base excision repair, it would nice if the authors would provide additional details about the studies showing clock regulation of the enzyme OGG1 (is it tissue-specific, observed in vivo vs. in vitro, etc.?), especially considering the interesting links to shift work. This is an important point. The regulation of OGG1 has not been extensively studied. However, while one study found OGG1 to oscillate in both ex vivo and in vitro human lymphocytes (Manzella et al., Sci Rep), another found no such oscillation in human keratinocytes (Hettwer et al., Int J Cosmet Sci). This suggests a potential cell-type specific role of the circadian clock is regulating OGG1 expression. We have addressed these points in the text (pg 6). When covering the Keller et al. (2009) paper discussing the role of the clock in splenic macrophages, the authors may wish to add that, like other cell types/tissues, much of the macrophage transcriptome is highly dynamic over the 24-hr. period. We agree with the reviewer and have emphasized this point in the text (pg 7)." } ] } ]	1	https://f1000research.com/articles/12-116
https://f1000research.com/articles/12-500/v1	15 May 23	{ "type": "Research Article", "title": "Predictive factors of large liver cell carcinoma tumor response after first conventional transarterial chemoembolization in Indonesia according to the modified response evaluation in solid tumors criteria: a preliminary study", "authors": [ "Sahat Basana Romanti Ezer Matondang", "Liem Arinuryanto Lios", "Ibrahim Abubakar Hilmy", "Irsan Hasan", "Joedo Prihartono", "Liem Arinuryanto Lios", "Ibrahim Abubakar Hilmy", "Irsan Hasan", "Joedo Prihartono" ], "abstract": "Background: Various factors affect the response of large hepatocellular carcinoma (HCC) after transarterial chemoembolization (TACE), and the high demand for TACE procedures highlights the need for information and methods on the criteria for patient selection. Methods: Thirty-six HCC patients who were treated with conventional TACE between January 2014 and January 2020 and had pre- and postoperative imaging data were included in the analysis. The patients were categorized as tumor responders (complete or partial response) or nonresponders (stable or progressive disease) according to the modified response evaluation criteria in solid tumors (mRECIST). Eight predictive factors (Tumor diameter, Tumor amount, tumor vascularity, portal vein invasion, tumor location, presence of tumor capsule, Child–Pugh class, and alpha-fetoprotein (AFP) level) of tumor response were evaluated using multivariate analysis with logistic regression. Results: Only 42% of patients fell into the category of responders after conventional TACE. None of the Child–Pugh class B patients showed a tumor response. A multivariate analysis of the Child–Pugh class A patient group showed that a lesion diameter of 5–10 cm (OR: 8.78, 95% CI: 1.73–44.55; P = 0.009) are an independent predictor of tumor response after the first TACE procedure. Conclusions: A tumor diameter of 5–10 cm could be use as a predictor of tumor response after the first conventional TACE in Child–Pugh A patients, further researches are needed to confirm this finding.", "keywords": [ "Hepatocellular carcinoma", "HCC", "TACE", "Transarterial Chemoembolization", "Predictive factors", "mRECIST", "Modified response evaluation in solid tumors criteria", "Radiology" ], "content": "Introduction\n\nAccording to International Agency for Research on Cancer data, hepatocellular carcinoma (HCC) was the fifth most common cancer and the third most common cause of death from cancer globally in 2020.1 In Indonesia, there were 21,392 new cases of liver cancer in 2020, with a mortality rate of 7.7 deaths per 100,000 people.2\n\nHCC treatment options can be divided into curative and non-curative. Curative therapy is indicated for early stages and includes surgical resection, liver transplantation, and radiofrequency ablation.3 Non-curative (palliative) treatment aims to prolong survival by slowing tumor progression. Transarterial chemoembolization (TACE) is the most frequently used palliative procedure.1,3,4 The median survival rate of HCC patients receiving TACE ranges from 13.7 to 17 months.3,5 A systematic review of randomized clinical trials found that TACE provided better two-year survival than supportive therapy.6\n\nTumor response can be used as a measure of the efficacy of therapy and has been identified as an independent predictor of survival.6 The modified response evaluation criteria in solid tumors (mRECIST) system is used to determine the response to targeted tumor lesions, which is classified as complete response, partial response, progressive disease, and stable disease.7\n\nVarious studies have attempted to identify the predictive factors of tumor response after TACE. Hong et al. found that a tumor size of <4 cm, fewer than five tumors, hypervascular tumors, and the absence of portal vein invasion predicted a post-TACE tumor response.5 Mickey et al. reported that Child–Pugh class A patients responded better to tumors located in the right lobe and medial segment after TACE.8 Jeong et al. found that a tumor size of <5 cm and a single tumor were predictors of a complete response.9 Banangkoon et al. found that fewer than two sessions of selective TACE, a serum alpha-fetoprotein (AFP) level of <100 ng/mL, a cumulative tumor diameter of <30 mm, and unilobar involvement were predictors of a complete response.10 Wu et al. reported that the presence of a tumor capsule was a significant prognostic factor in HCCs with a tumor size of >5 cm.11\n\nAlthough several studies have assessed the determinants of tumor response, few such studies have been conducted in Indonesia. Loho et al. found that the average tumor diameter of HCC patients in Indonesia was 10.9 ± 4.9 cm and that more than 94% of patients had a tumor with diameter of >5 cm.12 The large tumor size of patients in Indonesia, along with long patient list for TACE procedures, and the large number of patients lost due to death made it difficult to achieve therapeutic targets. Thus, there is a need for research in determining factors to be considered in predicting tumor response to assist and hasten the prioritizing process for patients with better chance for response.\n\n\nMethods\n\nThis retrospective observational study used secondary data from HCC patients receiving conventional TACE in the radiology department of a tertiary hospital in Jakarta from January 2014 to January 2020. The inclusion criteria were HCC patients with target lesions measuring >5 cm who received conventional TACE (lipiodol and doxorubicin emulsion and embolization with a gelatin sponge) and had multiphase abdominal CT/MRI examinations within two months before the procedure and one to three months after the procedure. Patients with a previous history of systemic or locoregional therapy were excluded.\n\nIn this preliminary study, we evaluated eight factors, each divided into two categories: tumor diameter (5–10 cm or >10 cm), number of tumors (single or multiple), tumor vascularity (hypovascular or hypervascular), portal vein invasion (present or absent), tumor location (right/medial or left/bilateral), tumor capsule (present or absent), Child–Pugh class (A or B/C), and AFP level (<400 ng/mL or ≥400 ng/mL). Patients were divided into two groups according to tumor response status: responders (complete or partial response) and nonresponders (stable or progressive disease). Our aim was to determine whether these factors can be used as predictors of tumor response and assist in prioritizing patients for treatment. Due to the high rate of loss to follow-up, we evaluated tumor responses only after the first TACE.\n\nA total of 41 patients were identified in the hospital’s registry. Five patients were excluded due to previous HCC therapy (either surgical or interventional). Four of the remaining 36 patients' laboratory data were either incomplete or missing and thus their Child–Pugh class cannot be determined; however, one of those four patients had their Child–Pugh class in their diagnosis record, therefore they were still included in the Child–Pugh class analysis. The other three patients were also included in the tumor characteristics analysis because they had complete radiological data. (Figure 1) The patients’ tumor characteristics and responses were analyzed using a picture archiving and communication system (PACS). Demographic (sex and age), laboratory, clinical (etiology [hepatitis B, C, or neither] and cirrhosis status), and Child–Pugh class data were collected from the patients’ medical records. To eliminate bias, tumor analysis was performed independently by two radiologists blinded to the patients’ identities.\n\nTumor characteristics (diameter, number, location, vascularization, portal vein invasion, and presence of capsules) were assessed by the examining radiologist using PACS. The number of tumors and their locations were determined by the examining radiologist by examining either CT or multiphase MRI scans using PACS. The baseline tumor diameter was measured using the ruler function of PACS. In cases of more than one lesion, the five largest lesions’ diameters at most were added up. The post-TACE diameter measurements were based on viable lesions with the largest diameters. The presence of a tumor capsule was determined by the radiologist on CT or multiphase MRI as a thin, hyperattenuated or hyperintense layer in the delayed phase of the scan and as either a hyper- or hypoattenuated layer in the portal vein phase. Portal vein invasion, also measured in the portal vein phase, was defined as the obliteration of normal portal vein structure and/or intraluminal portal vein thrombosis.\n\nTumor response was categorized by comparing pre- and postoperative imaging using the mRECIST. Patients with complete or partial responses were categorized as responders (cases), while patients with stable or progressive disease were categorized as nonresponders (controls).\n\nAll data were analyzed using International Business Machines Corporation Statistical Package for the Social Sciences (IBM SPSS) Statistics version 20. Relationships between two categorical variables were assessed using the chi-squared test or Fisher’s exact test if the total sample size was too small. Relationships between categorical and numerical variables were assessed using the independent t-test if the distribution was normal or the Mann–Whitney U test if the distribution was not normal. Predictive factors with P-values of <0.20 in bivariate analysis were included in multivariate analysis using logistic regression with the Enter approach to construct a prognostic model with an alpha significance limit of 5%. Bilirubin and albumin levels were not analyzed as predictive factors but as components of the Child–Pugh classification system, which was assessed as a predictive factor. AFP was evaluated as an independent predictor.\n\n\nResults\n\nThe patients’ mean age was 59.7 ± 10.8 years. Most (72%) patients were male. Hepatitis B infection was found in almost two-thirds of the patients, whereas cirrhosis was found in only 10 patients. A total of 28 out of 33 patients (85%) were Child–Pugh class A, while the rest were class B. None were class C. The AFP levels of 64% of the patients were below 400 ng/mL. (Table 1) A tumor diameter of >10 cm was found in 22 patients, while a single tumor/lesion was present in 24 patients. Most (78%) patients had tumors in the right lobe or medial segment. Hypervascular tumors were found in 27 patients. Portal vein invasion was found in only nine patients. Tumor capsules were found in 15 patients (42%)(Table 2).\n\nThe bivariate analysis showed that a tumor diameter of 5–10 cm (OR: 8.5, 95% CI: 1.88–36.30; P = 0.004) could have an association with tumor response and therefore will be included in multivariate analysis. The presence of a tumor capsule (OR: 3.75, 95% CI: 0.9–14.81; P = 0.059) and Child–Pugh class A (OR: 9.58, 95% CI: 1.0–92.09; P = 0.131) were also included in the multivariate because their P value are <0.20. Conversely, the number and location of lesions, vascularization, portal vein invasion, and AFP levels did not significantly affect the response (Table 3).\n\n* Fisher’s exact test.\n\n† Chi square test.\n\nThe multivariate analysis considered predictive factors only for Child–Pugh class A patients because none of the Child–Pugh class B patients were tumor responders. A larger tumor diameter of 5–10 cm (OR: 8.78, 95% CI: 1.73–44.55; P = 0.009) was found to be an independent predictor of tumor response after the first TACE procedure (Table 4).\n\n\nDiscussion\n\nThis preliminary study aimed to find the factors that could be used as a predictor for tumor response, we evaluated eight factors that could be associated with tumor response and found a tumor diameter of 5-10 cm could be used as predictor.\n\nIn this study, we found that there were five Child–Pugh class B patients, none of whom showed a tumor response. While Miki et al. found that Child–Pugh class A and a tumor in the right lobe or medial segment were associated with a higher probability of complete tumor response. They also found that there were some patients with Child–Pugh class B that showed a response.8 Our study found that no patients from the Child–Pugh class B showed a tumor response. There are several factors that could explain this: first, only 15% out of 33 patients with Child–Pugh data were class B. Second, all of them had a tumor diameter >10 cm. The small sample size in this category and the large size of their tumor may explain why they showed no response.\n\nTumors with a diameter of 5–10 cm were associated with a higher probability of tumor response. This suggests that tumor diameter is an independent predictor of tumor response. This finding conflicts with previous studies reporting that a small tumor diameter (<5 cm) is a predictor of a complete response.5,9,11 But the fact that this study included no patients with a small tumor diameter (<5 cm) may explain the conflicting results.\n\nThe presence of tumors in the right lobe or medial segment was not shown to be an independent predictor in this study. Of the 29 patients with right/medial lobe tumors, 16 were nonresponders, and 14 had a tumor diameter of >10 cm, with an average of 15.9 cm. This finding differs from previously stated study by Miki et al. that showed an association between tumor in right lobe or medial segment and tumor response. The large diameter of the tumor in the right lobe or medial segment in this study might explain why tumors in the right lobe or medial segment did not predict a tumor response. Likewise, the number of tumors was not an independent predictor. Conversely, Jeong et al. and Hong et al. reported that a single tumor with a small diameter (<4 cm) predicted a complete response.5,13 In this study, all tumors were >5 cm in diameter. The difference in target sample’s tumor size might explain why the number of tumors was not a predictive factor.\n\nBanangkoon et al. found that an AFP level of <100 ng/mL was one of the factors that predicted a complete tumor response, the other being a tumor size of <30 mm.10 In this study, 21 patients had AFP levels of <400 ng/mL. Twelve of them were nonresponders, and eight had a tumor diameter of >10 cm, with an average of 15 cm. The combination of a large tumor diameter and an AFP level of <400 ng/mL might explain why these patients showed no tumor response.\n\nIn this study, the presence or absence of portal vein invasion and hypervascular tumors did not predict a tumor response. Conversely, Hong et al. reported that hypervascular tumors and the absence of portal vein invasion were predictors of tumor response.5 On the other hand, Wu et al. found that in tumors larger than 5 cm, vascular invasion did not predict a good tumor response; only the presence of a tumor capsule was associated with a favorable prognosis.11 In this study, 16 patients without portal vein invasion were nonresponders, 13 of whom had a tumor diameter of >10 cm, with an average of 17 cm. A total of 27 patients had hypervascular tumors. Fourteen of them were nonresponders, and 10 had a tumor diameter of >10 cm, with an average of 16.9 cm. The large tumor diameter may explain the lack of tumor response even in the absence of portal vein invasion and hypervascular tumors.\n\nIn this study, we found that the presence of a tumor capsule was not an independent predictor of tumor response. This conflicts with the findings of Wu et al.,11 who found that in tumors measuring >5 cm, only the presence of a tumor capsule was a favorable prognostic factor. The difference in finding could be due to the difference in sample size and that Wu et al. did not specifically examine for response after first TACE procedure, but after hepatectomy instead.\n\nThis research had several limitations. First, the number of subjects analyzed in this study was relatively small, therefore further studies are needed to confirm the validity of the findings of this study. Second, because it was a retrospective study, some laboratory data (albumin, bilirubin, prothrombin time, and AFP) could not be obtained, thus some patient’s Child–Pugh class could not be determined. Third, because no Child–Pugh class B patients showed a tumor response, the Child–Pugh class was excluded from the multivariate analysis.\n\nIn conclusion, in this study, a tumor diameter of 5–10 cm was an independent predictor of tumor response in Child–Pugh class A patients after the first conventional TACE procedure. Therefore, Child–Pugh class A patients with a tumor size of 5–10 cm could be prioritized for conventional TACE. We acknowledge that due to the small sample size analyzed in this study, further studies with larger sample sizes and multicenter designs (provided that they have uniform procedure protocols) should be conducted to confirm the results of this study.\n\nThis study was approved by the ethics committee of the hospital (No. KET-437/UN2.F1/ETIK/PPM.00.02/2020). Due to the study’s retrospective nature, the requirement for written informed consent was waived.", "appendix": "Data availability\n\nZenodo: Predictive factors of large liver cell carcinoma tumor response after first conventional transarterial chemoembolization in Indonesia according to the modified response evaluation in solid tumors criteria: a preliminary study (supplemental data), https://doi.org/10.5281/zenodo.7834809. 14\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 acknowledge the help of Eghar Anugrapaksi, M.D. in his role to interpret and confirm the statistical analysis of this study.\n\n\nReferences\n\nInternational Agency for Research on Cancer: All Cancers Source GLOBOCAN 2020. Int Agency Res Cancer; 2021. Reference Source\n\nInternational Agency for Research on Cancer: Indonesia Source GLOBOCAN 2020. Int Agency Res Cancer; 2021. Reference Source\n\nWhite JA, Gray SH, Li P, et al.: Current guidelines for chemoembolization for hepatocellular carcinoma: room for improvement? Hepatol Commun. 2017; 1(4): 338–346. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKobayashi T, Kawai H, Nakano O, et al.: Rapidly declining skeletal muscle mass predicts poor prognosis of hepatocellular carcinoma treated with transcatheter intra-arterial therapies. BMC Cancer. 2018; 18(1): 1–9. Publisher Full Text\n\nHu HT, Kim JH, Lee LS, et al.: Chemoembolization for hepatocellular carcinoma: multivariate analysis of predicting factors for tumor response and survival in a 362-patient cohort. J. Vasc. Interv. Radiol. 2011; 22(7): 917–923. Publisher Full Text\n\nLlovet JM, Bruix J: Systematic review of randomized trials for unresectable hepatocellular carcinoma: chemoembolization improves survival. Hepatology. 2003; 37(2): 429–442. PubMed Abstract \| Publisher Full Text\n\nLencioni R, Llovet JM: Modified RECIST (mRECIST) assessment for hepatocellular carcinoma. Semin. Liver Dis. 2010; 30(1): 052–060. Publisher Full Text\n\nMiki I, Murata S, Uchiyama F, et al.: Evaluation of the relationship between hepatocellular carcinoma location and transarterial chemoembolization efficacy. World J. Gastroenterol. 2017; 23(35): 6437–6447. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark HJ, Kim JH, Choi SY, et al.: Prediction of therapeutic response of hepatocellular carcinoma to transcatheter arterial chemoembolization based on pretherapeutic dynamic CT and textural findings. Am. J. Roentgenol. 2017; 209(4): W211–W220. PubMed Abstract \| Publisher Full Text\n\nBannangkoon K, Hongsakul K, Tubtawee T, et al.: Rate and predictive factors for sustained complete response after selective transarterial chemoembolization (TACE) in patients with hepatocellular carcinoma. Asian Pac. J. Cancer Prev. 2018; 19(12): 3545–3550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu TH, Yu MC, Chen TC, et al.: Encapsulation is a significant prognostic factor for better outcome in large hepatocellular carcinoma. J. Surg. Oncol. 2012; 105(1): 85–90. PubMed Abstract \| Publisher Full Text\n\nLoho IM, Hasan I, Rinaldi C, et al.: Hepatocellular carcinoma in a tertiary referral hospital in Indonesia: Lack of improvement of one-year survival rates between 1998-1999 and 2013-2014. Asian Pac. J. Cancer Prev. 2016; 17(4): 2165–2170. Publisher Full Text\n\nJeong SO, Kim EB, Jeong SW, et al.: Predictive factors for complete response and recurrence after transarterial chemoembolization in hepatocellular carcinoma. Gut Liver. 2017; 11(3): 409–416. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatondang SBRE: Predictive factors of large liver cell carcinoma tumor response after first conventional transarterial chemoembolization in Indonesia according to the modified response evaluation in solid tumors criteria: a preliminary study (supplemental data). [Data set]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "269141", "date": "13 Sep 2024", "name": "Shiro Miyayama", "expertise": [ "Reviewer Expertise TACE for hepatocellular carcinoma" ], "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. This study included many HCCs >10 cm. I think that it is very difficult to embolize HCCs >10 cm by a single cTACE session, because they are usually supplied by many arterial branches including extrahepatic arteries. I speculate that almost HCCs >10 cm in the present study might not be successfully embolized in a single TACE session. So, evaluation of technical success of cTACE (embolization of the entire tumor) is necessary before the discussion of the prognostic factor of tumor response. The authors should describe how they evaluated the technical success of cTACE in the present study.\n2. To treat large HCCs with cTACE, some special techniques, such as bland embolization followed by cTACE (Hidaka T, et al.2021 [Ref 1]), drug-eluting bead TACE followed by cTACE (Morishita A, et al.2021 [Ref 2]), and stepwise superselective cTACE (Miyayama S, et al.2010 [Ref 3], Miyayama S.et.al., 2019 [Ref 4]) have been reported. Please cite these papers and discuss the TACE techniques to embolize a large tumor safely and effectively.\n3. The authors compared the predictors of tumor response in the present study among several papers; however, some reported significant predictors were found in the study of cTACE for small HCCs. Therefore, the comparisons might not be meaningful because the tumor backgrounds in such reports might be extremely different from the present study. I speculate that the most significant predictor of tumor response in the present study might be “embolization of the entire tumor”.\n4. If many tumors were not successfully embolized in a single cTACE session in this cohort, the contents and discussion points of this manuscript should be changed to “difficulty in the whole tumor embolization of large HCCs in a single cTACE session.”\n5. The details of the cTACE procedure are unclear. Please add the details of cTACE techniques, for example, the use of a microcatheter and CT/cone-beam CT, a total dose of Lipiodol emulsions (water-in-oil or oil-in-water?) in a single session, the size of particle embolus, and additional cTACE through extrahepatic arteries. The endpoint of cTACE should also be clarified.\n6. cTACE for large HCCs might also cause severe complications. Please describe the complications in each patient.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-500
https://f1000research.com/articles/11-455/v1	25 Apr 22	{ "type": "Research Article", "title": "The signifying culture: An intercultural and qualitative analysis of Tiv and Yoruba folktales for moral instruction and character determination in children", "authors": [ "Olumide Olugbemi-Gabriel", "Mbasughun Ukpi" ], "abstract": "Background: In the study of African communities, folktales have remained a constant element. With their origin in the culture of oral storytelling, folktales have often been used by older age groups to guide and mould behavioural patterns in children. In ancient and traditional African societies, children were gathered at the end of the day by older members of the community for tales by moonlight sessions aimed at guiding their moral decisions. With globalisation and its consequent effects such as migration, dislocation and disindigenisation, the culture of communal folktale sessions is experiencing a quick death. This paper engages with the relevance of folktales as moral guides for children in African societies and as a renewed path to increased societal stability facilitated by morally set individuals. Methods: The folktales were randomly selected from a pool of Tiv and Yoruba folktales in Nigeria. Two animal-based folktales which are part of shared folk culture were picked from the Tiv society and one from the Yoruba society. The study follows a narrative and content analysis approach where the selected folktales are corroborated by four key informants, two males and two females within the ages of 50-65.\n\nResults: With particular focus on the benefits of promoting and re-introducing the folktale culture to encourage positive behavioural traits amongst individuals in the society, the study primarily highlights folktales as reflective of human life. In identifying this similarity, the character of children is largely influenced by the moral values inherent in these folktales.\n\nConclusions: There needs to be an increased use of media and audio-visual tools to expand the knowledge and accessibility of indigenous African folktales in order to preserve ethnic, national and social identity as well as to provide a moral compass for children.", "keywords": [ "Folktale", "culture", "morality", "Tiv", "Yoruba." ], "content": "Introduction\n\nAfrican culture has been portrayed in the media as a never-ending festival categorised by chant songs, dramatic dances and costumes, made from intricately designed African prints. Visitors to African countries are often caught searching for an authentic African experience, and such visits are sometimes seen as unfulfilling when these visitors find ordinary people leading ordinary lives. Although African culture has been commercialised and limited aspects highlighted, leaving other important parts fallow, post-colonial scholarship has used oral literature and folktales as a way of correcting colonial anthropology and reconstructing scholarship from an African point of view. For many centuries, folktales were important cultural means of expression, entertainment, education and bonding in African societies. A world of high modernity riding the waves of globalisation has eroded the place of most oral literary forms, and folktales in particular, with dire consequences for societies which hitherto relied on these forms for their intrinsic societal and ornamental values.\n\nHence, this paper’s ‘renewed’ interest in folktale as an embodiment of culture, identity and conveyor of moral instruction finds validation in Dundes and Bronner’s (2007, 55) affirmation of folktale as being inherently “autobiographical ethnography – that is, it is a people’s own description of themselves”. ‘Folk’ implies all humans as folk peoples, who tell the stories that reflect their societies, customs, traditions, cultural mores and worldview. Though some societies in Africa have held on much longer to the expression and values of folktale than others, the local and universal worth of this literary form in identity mapping and construction of culture remains constant.\n\nHowever, with the advent of globalisation, most cultural elements of language in erstwhile colonies have been adapted to suit the modern experiences of most postcolonial citizens and societies, thereby vitiating the place of folktales as a significant oral literary form. To this effect, folktales and oral storytelling are no longer popular in the day-to-day activities of most Africans. These practices have been largely replaced by television programmes and other forms of new media. This is not to argue that these programmes/media are completely unnecessary; but when their content re-enforces global culture to the detriment of indigenous content, the future of African moral values which were transmitted over many generations through folktales hangs in the threat of extinction.\n\nNigeria as a multicultural state offers a myriad of cultural practices representative of each culture and folktales serve as a recurring element in the midst of variation. The place of folktale as one of the expressive cultures connecting humanity has been established by Dundes and Bronner’s (2007, viii), who aver that: “folklore is prime evidence of culture, indeed of humanity … folklore is a people’s “symbolic autobiography,” folklore gives an “inside out” view of society. Folklore a mirror of culture, a lens for society, a key to behavior, a projection of mind.”\n\nEnongene Sone (2018, 147) also captures the essence of folktales and their role in African societies when he says, “Thus, the folktale was used, and is still being used in several parts of rural Africa, to comment on how the individual adheres to or deviates from the community’s behavioural norms. Seen in this light, the folktale, although a creation of the imagination, ultimately derives its material from the realities of society while simultaneously shaping people’s views and constituting and constructing society. As a result, the folktale can be said to be a good mirror of society”.\n\nIn appropriating the value of folktale as ‘prime evidence of culture’, this paper focuses on the folktales of two distinct Nigerian cultures to demonstrate the underlying similarities of cultural tools for morality. We do not claim that these two cultures give an overview of the entire Nigerian/African cultural scene. However, we attempt to converge the thoughts of the Tiv culture of Nigeria’s Middle Belt region and the Yoruba culture of Western Nigeria, thereby crossing geographical and socio-cultural boundaries to arrive at cultural symbiosis.\n\n\nMethods\n\nThe folktales were randomly selected from a pool of available written folktales in existing literature and oral folktales shared in home storytelling sessions in the Tiv and Yoruba societies of Nigeria. Two animal-based folktales which are part of shared folk culture were picked from the Tiv society and one from the Yoruba society. The selection of the folktales was based on popular animal characters found across African folklore including the hare and tortoise in order to establish a convergent point of analysis.\n\nThe study follows a narrative and content analysis approach where the selected folktales are corroborated by four key informants, two males and two females within the ages of 50-65. They were selected within this age range due to the role of elderly people in both societies as storytellers and an equal gender ratio was applied for a balanced input. The corroborative data consisting of the selected folktales was collected via audio recordings and then manually transcribed and translated literally. The folktales were then analysed against their didactic signification, moral relevance and engendering of social order.\n\nThe participants were aware that the data collected would be used for research and were clearly aware of their right to withdraw from the study or decline being reported in the study before, during and after the data collection.\n\nCulture debates have morphed and percolated over time, with earlier theorists such as Barthes (1972) and Foucault (1988), to later theorists such as Bhabha (1994) and Hall (1997), foregrounding from multiple perspectives, the place and influence of culture on peoples and their literary expressions. If Culture is said to be “the beliefs, rituals, and practices of a given social or ethnic group or nation” (Habib 2008, 172), then the general idea that culture deals with the basic life of a group of people, leads to further debates on what constitutes the pillars of culture; and what yardstick should be used in defining various groups culturally.\n\nIn highlighting the role of the physical environment, the ecological approach relates the ecosystem and its natural transformation over time as the defining factor for adaptation which produces culture. As such, the Yoruba culture reflects the way of life of the people; a way of life rooted in communal living by farming, hunting, fishing, and other socio-cultural and economic activities. The Tiv society on the other hand is also noted for farming due to adaptation needs in their migration history.\n\nJ. Steward’s extensive research into cultural change is documented in his book, Theory of Culture Changes (1955), wherein he gives insight into the relationship between environment and culture, emphasises the technological adaptations that people make as time progresses. Steward (1955) also touches on issues of resource exploitation and the concept of multilinear exploitation, which R.M. Netting, the author of Cultural Ecology (1986) critiques as being too broad for analysis.\n\nAlthough there are unique processes in various cultures, common linear and non-linear patterns can be identified. Overall, the analysis of texts or literary artefacts along the lines of culture “tends to stress what is specific or unique – in terms of time, place, and ideology – to a given cultural and literary movement” (Habib 2008, 173). Therefore, in this paper, the ideological positions and prevailing beliefs passed on through generations in folktales across two distinct Nigerian societies – Tiv and Yoruba – are used to signpost broader issues of class stratification, social cohesion, and the dynamics of power relations.\n\nHall’s (1997) work on cultural theory, specifically multiculturalism, encourages a multifaceted approach to examining culture. For him, imagination and representation lie at the core of understanding ourselves as part of a culture. According to Hall (1997, 576):\n\nCultural hegemony is never about pure victory or pure domination (that’s not what the term means); it is never a zero-sum cultural game; it is always about shifting the balance of power in the relations of culture; it is always about changing the dispositions and the configurations of cultural power, not getting out of it.\n\nThus, although a single approach to culture theory might be elusive, multiple approaches give room for comprehensive analysis.\n\nNigeria has experienced various phases of transition in the struggle to exist as an independent state, with its own belief systems and situational practice free from extreme external influences, which is often challenged and vitiated by internal conflicts arising from the existence of multiple cultures. At this point, the only way to achieve a tolerant cohesive cultural standpoint with regards to postcolonial Nigeria is to back-date the reality to pre-colonial Africa and find bearing from there. With the knowledge that this is physically impossible, the alternative would be to extract relevant traditions that foster progressive growth within a multicultural society. Using folktales to examine the moral consciousness of indigenous ethnic groups, the gap created by globalisation is exposed.\n\nFolktale or the act of storytelling is a way, in traditional African societies, of passing on tradition, moral codes of conduct, ethics, and maintaining social order. The storyteller’s position is fortified by his/her multiple mandates as a historian, entertainer, poet, musician and educator, duty-bound to transfer knowledge from one generation to another. In many African societies, the oral tradition forms serve important ethical and social purposes; hence, the assertion that the death of an African elder is the equivalent of an arsonist setting the library of a university ablaze.\n\nThe imperativeness of folktale or storytelling as an art form serving as an important instrument for entertainment, documentation and education in African societies has been documented by some scholars and writers (Achebe 1987; Insaidoo 2011; Obiechina 1993; Owomoyela 1997, 2004; Lawuyi 2012; Olugbemi-Gabriel 2015); while Dundes and Bronner (2007) have focused attention on folklore across world cultures. Adumbratively, Achebe in his epochal novel, Things Fall Apart, reveals the pivotal role which folktale plays in the entertainment and education of an African child. In chapter eleven of Achebe’s seminal novel, the didactic and entertainment values of folktale are privileged. According to the narrator, “Low voices, broken now and again by singing, reached Okonkwo from his wives’ huts as each woman and her children told folk stories” (67). It is night-time, Ekwefi, one of Okonkwo’s wives chooses to tell her only child, Ezinma a story based on the character of the notoriously popular animal trickster, Tortoise, whose shell is cracked due to acts of greed, treachery and subterfuge. “Once upon a time” the story begins, “all the birds were invited to a feast in the sky. They were very happy and began to prepare themselves … Tortoise saw all these preparations and soon discovered what it all meant.” The tales reveals how, out of uncommon greed, Tortoise betrays his benefactors (the birds as a group) and he is made to pay dearly for his action. Embedded in the act of storytelling for entertainment is the fact that, Ezinma is taught the virtues of contentment, honesty and the need to always place group interest above individualism.\n\nInstructively, folktales are usually told at nighttime, when the day’s work is over in order to emphasise the importance of the maxim: ‘work before play.’ Night-time storytelling also makes the storyteller the centre of attention because distractions are eliminated. With the timeless expression “Once upon a time”, which is a storyteller’s clever strategy of trapping time in timelessness or eternity, the attention of the listener is gained from the start of the story. In African folktales, time is quite fluid with no concrete time or place setting. Vagueness in time and place is intended to create opportunities for the listeners (mostly children) to associate the setting with their experiential world of fantasy and reality, wherein ghosts exist and humans are capable of good and bad acts. Ekefi’s folktale in Things Fall Apart is not only intended to entertain an innocent child after a hard day’s work; but it is equally an attempt by a mother, as the teacher, to pass on to her child a set of moral ethical codes necessary for good behaviour and social order. Thus, the African storyteller is not just an entertainer; s/he is also the archetypal educator, who exists to teach morals, and pass on societal values and ethos, which are crucial to the survival of the society. Furthermore, it is instructive to state that Achebe uses the novel to acknowledge the role of folktales in moral grounding within African societies. Foregrounding the relevance of folktale in today’s pedagogical teaching model, Dundes and Bronner (2007, 53) strongly advocate its use by teachers “as an instructional tool to develop tolerance” and build stellar character.\n\nIndeed, some scholars have affirmed the importance of folktales within the milieu of Yoruba and Tiv cultures. Lawuyi (2012, 1) holds that alo (folktale) does not only offer sociological imaginations and imaginings of the world of the Yoruba people/culture, the “clustering of imageries within any alo … can reveal insight into the social and political systematisation of not only the Yoruba but also the ‘Nigerian’ culture.” Udu and Udu (2013, 7) situate the centrality of folktale in the education of the Tiv child:\n\n“Folktales expose children to the richness of Tiv language, Tiv myths, songs, values and culture. They help children in both character and cognitive development by enabling them to learn good virtues such as tolerance, honesty, hard work, obedience to parents and the repercussions of being deviant.”\n\nGenerally, most African folktales portray characters which share similar perspectives, albeit sometimes, in different stories. Amongst the Tiv people of Nigeria, folktales are traditionally told in the communal hut called ‘Ate’ after dinner. The primary use of the folktales is to instil values that are beneficial to the growth of the children as they transition into new stages of life and also to entertain them. Whenever the children are gathered in the ‘Ate’, the dual significance of the folktale is not lost on them as partakers of a cultural milieu, which places high premium on instilling the right values and education in children to engender peaceful coexistence and social cohesion.\n\nThe use of animals in folktales is also a consistent feature of African oral storytelling. This is due to the relationship Africans have with animals as part of their ecosystem. Almost every part of life is interconnected with daily rituals involving animals, whether this is through the presentation of goats and cows during marriages or the use of dogs as hunting companions. This is why there are different animal characters across the regions of the continent.\n\nBy far, Ijapa, Tortoise is the most popular character in Yoruba folktale. Anansi the spider is the character closely associated with folktales from Ghana, Sierra Leone and Liberia, while the Hare is the most prominent animal trickster character in East, Central and Southern African folktales. These animal characters are usually tailored for particular regions to achieve an understanding of the inherent lessons embedded in the folktales. For the Tiv people, whose primary occupation is farming, animals are also prominent characters. But, unlike the Yoruba society which focuses on Tortoise as a character, the Tivs are particularly fond of using the Hare, who is a trickster in kwagh-alom tales (Hare tales) to present important lessons central to the wellbeing of the society.\n\nAlom stories have been documented by scholars such as Tsaaior (2015) who narrates a similar story of a he-goat who eats stolen yams and is unable to escape because of his distended stomach and is left behind by his friend, the hare. Another popular alom (Hare) story which is commonly found among a gathering of Tiv people focuses on the mystery of the missing pot of beans. At a time of famine in the kingdom, there is an increased need for work on the farm. Hare is married to a hardworking woman, who wakes up early and prepares food to take to the farm together with the equipment to be used. Hare on the other hand is lazy and does the bare minimum of work on the family farm, quite contrary to his position as the head of the family. As the folktale goes, on this particular day, Hare’s wife makes a special meal of beans before the family’s departure for the farm with a declaration that the meal would be eaten when they return from the farm. Hare, in his usual nature as a trickster, leaves the farm midway through the work, claiming that he has something urgent to attend to in the village. Of course, this is not true; his claim is a decoy for a more sinister intention. Hare makes his way home and eats half the pot of beans, which is supposed to be the family dinner. To cover his misdeed, Hare adds sand to what is left of the meal so that the pot would appear to still contain the quantity of beans prepared. Hare’s wife returns home after the day’s work and, unsuspectingly, brings out the pot of beans to serve the family dinner. To her consternation, she realises that someone has eaten half the food, replacing what is eaten with sand. She asks her husband if he is responsible for the acts of theft and subterfuge. Not known for honesty in the first place, Hare vehemently denies. Her children also denied culpability of the chicanery.\n\nIt is noteworthy that Hare’s wife’s enquiries are made through a song. However, the song is not an ordinary one as it serves the dual purpose of exposing and punishing the culprit:\n\nAlom a lu we\n\n(Hare if it is you)\n\nU yam alev u mor gbor gbin\n\n(That ate my beans, let your stomach grow well)\n\nWan ityondo a lu we\n\n(Hare’s child if it is you)\n\nU ya’m aleve mor daa laa\n\n(That ate my beans let your stomach swell)1\n\nAs she sings one after the other for the suspects, the innocent ones remain unchanged but when it is Hare’s turn, his stomach swells since he is the culprit.\n\nTheft is a serious moral ill and perhaps one of the first acts a child is warned against in almost all traditional African societies. The folktale uses the character of the Hare to bring to the fore the consequences of stealing. Through this method of associating terror with theft, children are frightened into being morally upright. This is an effective instructive and corrective measure. Through the course of connecting multiple moral lessons, the folktale also speaks against subterfuge, falsehood, indolence and selfishness. The conduct of the Hare signposts the elevation of self above others; which is an idea contrary to communalism, the guiding philosophy of many African societies. To these societies, when a person steals from his family as Hare has done, “the self is alienated from the common good, from the “other” which is framed in kinship. In return, the community makes move to ostracize or punish the thief” (Lawuyi 2012, 11). The punishment of a swollen stomach meted to Hare is therefore in order as it serves as a deterrent to anyone who may want to follow in his footsteps. Though metaphysically contrived, the manner of punishment reflects the belief that there are forces (benevolent and malevolent) ruling in the affairs of humans; a lesson elders want children to take to heart as part of their belonging in the society.\n\nThe tale also implies that if the Hare was a hardworking person, he would not need to scheme to take charge and/or steal from his own family. Hare’s signification of self (above others) and his indifference to his unenviable status (playing the second fiddle) negate the societal structure of expectations, which places the man as the head and defender of his family. Greed is also a concern in the folktale. If he had waited until everyone was home, he would have had a portion of the same meal. But because of his greed and self-centeredness, he contrives a subterfuge to steal from the common pot, without any consideration for the impact on others.\n\nFrom this, it is not difficult to see why folktales hold children of preliterate and traditional societies spellbound. If folktales in the past held such a strong influence on the behaviour of children the shift from folktales to TV shows as tools of moral education in modern societies brings forth an important question. Is there a problem lurking over our heads (as Africans) with the fading culture of storytelling and deployment of folktales for didactic purposes? Arguably, the erosion of traditional African means of moral instruction and teaching of values by westernisation and modernity have accentuated many vices such as greed, wanton materialism and theft in today’s societies. The abandonment by many Africans of Africa-oriented values such as communalism and moderation for the assimilation of western values such as individualism, materialism and capitalism, has created a generation of people who are a testament to the deculturalisation and disindigenisation of an entire race.\n\nThe Tiv people also have an aetiological folktale about Ikyarem, the green snake that saved them during their earlier wars. Tsaaior (2015, 11) documents this tale while highlighting generosity and good-naturedness as ideologies important to the Tiv people instructively with the name Ikyarem, which means friend. It is believed that during the early days of Tiv migration, the Tiv people came across the Ugenyi people of Cameroon. In the course of a clash between the two communities, the Tiv side was losing and they had to flee for respite. They found themselves at the Aya River and reminiscent of the biblical setting of the Israelites and the red sea, the Tiv are caught in the middle. With no means to cross the river and at the peak of exasperation, the green snake, Ikyarem, suddenly appears and stretches its full length from one end of the river to the other. The Tiv people are then able to use this makeshift bridge to cross over safely, thus providing them with a lifeline to continue their journey until they reached their present home, on the banks of River Benue. This folktale explains the reverence Tiv people have for the snake, which is believed to be harmless. It is considered taboo for any Tiv person to kill this snake.\n\nThere is a lot of doubt regarding the believability and credibility of the above story but it houses and promotes a special (sacred) part of Tiv history. This particular folktale creates awareness of the legendary Tiv resilience, which was ingrained in these people through their journey to finding a permanent home. According to Tsaaior (2015,10) tales such as these “also constitute a memory archive and documentary reservoir in Tiv society and foster an abiding sense of historical awareness”. He likens this to the biblical story of the parting of the red sea for the Israelites who were fleeing from Egyptians.\n\nOn the other hand, the folktale also shows a respect for animals and belief in reciprocity – one good turn deserves another; while it also promotes the idea that help can come from the most unexpected places. The noble theme that nobody should be discriminated against because of size, class, or because they appear weak, is given deserving prominence.\n\nIt should be noted that Tiv folktales have gone beyond the trite and common functions of entertainment and knowledge sharing to instil confidence and self-awareness in children. Beyond the existence of these tales, there is a lesson to be learnt in the way the stories are told. For larger groups, the tales are often told through dramatic performances like the kwagh-hir dance and, children in Tiv society are found in smaller circles retelling these stories through performances, which help in building confident personalities. The foundational years are important in moulding children’s personalities; and if the right systems are set in place from a young age, they have better psychological tools to deal with the various stages of growth into adulthood. Denga (1988, 15-16) argues that such tales aid in passing knowledge from one generation to another and mould the character of children who listen to these stories.\n\nIf culture can influence one’s way of life, then it is given that the foundational provisions of one’s culture could be understood as a loose framework for understanding other cultures and could create a bridge between other tribes and cultures Buttressing the intercultural realities of human existence, Evwierhoma (2002, ix) posits that “culture is often taken for granted because many see it as a common experience to all humans. But its dynamic nature gives it the flexibility to cross climes and races” (emphasis added). Interestingly, similarities have been observed between Tiv culture, particularly the language, and the cultures of other Bantu language speaking peoples. Clearly, there is similarities between ‘nyam’ the Tiv word for meat, and ‘nyama’ the word for meat in Swahili; alo, is Yoruba for folktale which, in Tiv, is alom. A Swahili or Yoruba speaker listening to a Tiv folktale that references meat can find a common ground in their understanding. Upon further interaction, they might even realise that more similarities are located within disparate cultural milieus. This paper therefore pays critical attention to the didactic use of folktales within Tiv and Yoruba cultural milieus to promote the idea of intercultural (as opposed to intracultural) evaluation of African folktales.\n\nThe use of animals to convey morals is not exclusive to Tiv people. Among the Yoruba, Ijapa (Tortoise) is a popular trickster in alo apagbe. For the purpose of clarity and elucidation, it is essential to distinguish the Yoruba dual words, alo apagbe from the generic and more popular mono word, alo in order to signpost the didactic significance of folktale among the people. When translated or transliterated, alo apagbe means ‘call and response story.’ In this sense, and within the milieu of Yoruba folktale, the storytellers and their listeners are active participants in the whole exercise, which usually involves singing and dancing in a way that underscores the recreational and educational values of folktales. Therefore, when a Yoruba adult tells folktale(s) to wide-eyed children, beyond entertaining them, the original intention is to instill valuable moral lessons on them through a ‘call and response’ strategy of engagement as the narrator prepares the children for life as responsible citizens in society. In signification, alo apagbe is intended to educate children, which is akin to the Western education model. The storyteller allows the children to be part of the storytelling, albeit in a controlled atmosphere where s/he retains authority over the entire activity. The purpose of folktale for education in Yoruba society is then realised through the critical feedback, which the storyteller gains from deploying the ‘question and answer’ model at the end of each story.\n\nIn Yoruba folktales, Ijapa (Tortoise) is well established as a cunning and indolent character. Being proverbially lazy, he lacks the capacity for work. In ‘Ijapa ati Igbin’ (Tortoise and Snail) folktale, the lazy Ijapa is newly married to Yannibo, the daughter of Igbin (Snail) and he is invariably saddled with the obligation of feeding another mouth. Daily and without any clear source of income or cultivated farmland, Ijapa returns home like every other farmer with robust tubers of yam, with which Yannibo prepares dinner for the young family. However, Igbin (Ijapa’s father-in-law) is experiencing a nagging and perplexing theft of yam, pepper, tomatoes and other vegetables from his farm. Naturally, unable to bear the implications of his farm being gradually decimated by a determined thief, Igbin sets a trap to nab the fellow. A day after the trap is set, Igbin arrives at his farm at dawn to find Ijapa, his beloved son-in-law, caught in the trap and in agony. Freshly dug tubers of yam together with vegetables are found close to where Ijapa writhes in shame and pain. Thus, the theft and perfidy of Ijapa are exposed in the most ignominious manner.\n\nDetermined to punish the thief by exposing him to opprobrium, Igbin drags Ijapa to a big baobab tree at the major intersection leading to different farmlands and ties him to the big tree, leaving no chance for escape. At daybreak, Ijapa is the butt of crude jokes, receives odious insults and excoriation from everyone who passed, and becomes a spectacle of shame. Ijapa’s crimes of indolence and theft are signposted as members of the community do not spare him from societal disapproval and opprobrium.\n\nHowever, at the close of the working day and much to the surprise and chagrin of the people on their return from the farm, they find Ijapa still tied at the same spot in great distress and exhausted from the weight of his shame and punishment. Instantaneously, the table turns against Igbin, who is communally dismissed as an Alaseju (an unpleasant Yoruba noun for anyone who has overdone something). ‘ju’ in the Yoruba word, ‘Alaseju’ connotes ‘too much’, and it usually carries negative implications in referential importance; in particular, whenever it is used, it stands as an excoriation of the receiver. Finding traction for this concept in Yoruba philosophy, Lawuyi (2012, 15-16) adds that “ju” calls attention to the limit of action, the end of individual freedom.” Therefore, ‘Alaseju’, in this context, refers to how Igbin undoubtedly crosses the societal boundaries of omoluabi (which is Yoruba for ‘a respectable and responsible personality’), iwontunwonsi (Yoruba for ‘moderation’) and molebi (Yoruba for ‘kinship’ or ‘family’); the three are central to the evaluation of one’s personhood in the society. Therefore, Igbin’s personal failings on the ethical landscape presuppose that Ijapa’s punishment is a matter which the people (and, by extension, the society) cannot leave to his sole discretionary power.\n\nWithin the context of Ijapa and Igbin folktale, two main issues are accentuated with regards to the centrality of omoluabi, iwontunwonsi and molebi on the moral landscape of Yoruba society. The issues of misdemeanour (theft in this case) and an appropriate but moderate punishment are given prominence among the Yoruba. Ijapa’s action (theft) is not tolerated by the community; hence it supports the disgrace and punishment mete out to him by Igbin, his father-in-law. In the society, any act of theft is evil and unacceptable because it encourages indolence and can easily create a recipe for social disharmony. On the vaunted danger of theft, societal codes dictate that when a thief is caught, appropriate punishment in relation to the act should be imposed, regardless of the kinship relationship between the parties. Igbin is therefore right to ignore the kinship relationship when punishing Ijapa for his crime.\n\nOn the other hand, the same society expects Igbin, as an omoluabi (responsible person) to be guided, and his actions tempered by the ethos of iwontunwonsi (moderation) in his search for justice against Ijapa, who doubles as his in-law. It should be noted and understood that it is Igbin’s violation of societal ethos and expectations on what constitutes appropriate punishment that ultimately turns the tables against him. The Yoruba sayings, “iwontunwonsi lo ye omoluabi” (a responsible person is guided by moderation) and “alo ni t’ijapa, abo ni t’ana re” (Ijapa gets the first blame, but the second blame is reserved for his in-law) exist as priceless reminders of societal expectations on how members must conduct themselves with dignity and moderation.\n\nThe moral lessons to be learnt here are not far-fetched. Clearly, the crime and punishment paradigm is well recognised and established in Yoruba society. But the culture, philosophy and ethics of the people also focus on the need for all persons to always endeavour to strike a balance between action and reaction. To achieve that balance, Iwa (character) is needed. Iwa is the linchpin of morality among the Yoruba and it is so central to behavioural expectations that it is further subsumed in the individual’s personality.\n\nAccording to notable scholar of Yoruba philosophy, Gbadegesin (1991, 79), Iwa is “perhaps the most important moral concept. A person is morally evaluated according to his/her Iwa - whether good or bad.” In regards to stealing, it can be said that “Ijapa ti s’owa nu” (Tortoise has lost character); therefore, he deserves the shame, insults, and punishment given to him by Igbin, and other members of the society.\n\nIn the same vein, Iwa and Omoluabi go together in Yoruba cosmology. A person is humanised through Omoluabi, but Iwa is central to the making of a person; that is, how they are viewed by other members of the society. Menkiti (1984, 176) opines that the becoming of a person in traditional African societies is a function of how well communal living and the discharge of obligations are emphasised by that person. One’s Iwa is ever present; it is an eefin (smoke), which cannot stay hidden. The centrality of Iwa to a person’s standing is codified in the Yoruba maxim, Iwa rere l’eso enia (character beautifies a person); in that sense, oozes and epitomises ugliness without Iwa. For exhibiting a lack of moderation in punishing Ijapa (his in-law), Igbin is stripped of the privileged status of Omoluabi, which Iwa grants a person in the society.\n\nIgbin’s indiscretion also underscores his failure to factor iwontunwonsin and molebi into the handling of Ijapa’s case. Iwontunwonsi is underlined by iforigji (forgiveness), which should have been activated ordinarily by the open fact that both parties are molebi (relatives) joined by marriage. In the eyes of the community, an omoluabi (Igbin earns that title because he is a hardworking farmer and responsible member of the community) should have been mindful of the fact that Ijapa does not steal to sell but in order to feed his family, which includes Yanibo, Igbin’s daughter. Seeing that Igbin’s inability to forgive and apply the necessary iwotunwonsin (moderation) could have easily led to the death of Ijapa, turning his wife (Igbin’s daughter) into a widow, the community swings into action by invoking its right to intervene and save the day. Thus, Igbin is compelled by communal disapproval to bring an end to Ijapa’s punishment on grounds that it violates the codes of iwontunwonsin, molebi and omoluabi, at the same time.\n\nThe story’s trajectory poignantly indicates that every member of the society in the Yoruba world is held to certain standards of responsibility in relation to action and reaction for the purpose of guaranteeing the dignity of person, peaceful coexistence and social order. Equally, the folktale illustrates that regardless of a person’s status in the society (whether as a violator of rule(s) or as one who enforces it), it is sacrosanct that the “principle specifying the limit of action, the correctness of procedure” (Lawuyi 2012, 12) is recognised and respected.\n\n\nConclusion\n\nThe examination of African folktales reveals that they play a great part in the culture of human development across ages and epochs; and it is now possible that the fading of these tales poses a formidable threat to human societies and Africa in particular. In any given society, activities are influenced by the collective ideology of its peoples and citizens. A flawed ideology and disposition to morality would affect the society accordingly. In understanding folktales as an essential part of a peoples’ cultural landscape, we are privileged to understand different communities and how they exist regardless of global influence. Thus, this study in investigating folktales as important cultural tools of informal education in Africa aligns with Dundes and Bronner’s (2007, 66) position that: “Folklore as a subject of study can be a most rewarding one. It does serve as a mirror of culture and it is a mirror well worth looking into.” Therefore, to sustain internal development in Africa in the age of globalisation, there is an urgent need to appropriate and promote relevant elements of her cultures, of which folktale is one, and putting up a determined fight to sustain these tangible and intangible cultures for character development and social harmony.\n\nDankarol and Agoom (2015) have given some suggestions on how to put a pause to the slow death of folktales and their didactic relevance within African societies. Both scholars believe that debates on core Tiv language issues, for instance, should be organised at the foundational stages; primary and secondary schools. Screenplays from Tiv folktales and competitions surrounding them should also be focused as a plausible way of encouraging development through folktales. Others include how the people/society can employ research on Tiv language with the purpose of sparking interest in it, in addition to the reintroduction of nighttime tales into our routines. These suggestions are valid in the sustenance of all endangered cultures in Africa and other places.\n\nIn extrapolating the suggestions, it is acknowledged that there is need to share folktales through the medium that appeals to the audience. Since audio-visual media is high on the list of acceptance and assimilation, it should be a default means of sustaining the culture of folktales. The reality of a technology-driven world brings to the fore the need to combine these advocated solutions with relevant means to gain and sustain the attention of the next generation with regards to folktales and the lessons they carry for all indigenous people. A basic step in this direction is the inclusion of more African language options on digital platforms enabling African folktales to retain stories in their original forms. User interaction interface in language and simulation games is also a progressive option for memory retention. With these measures, age-old positive aspects of culture espoused through folktales are protected and sustained, thereby having positive impacts on moral conducts and development in the concerned societies. The availability of folktales on digital platforms and in new media forms would serve to validate its ever changing yet constant nature. Moreover, it would also vitiate the position of those who hold the belief that folktales are relics of a bygone age and have dismissed its continued relevance in a world of high modernity. In its new forms, folktale would exist as a cultural totem of high identitarian value, especially in Africa where the essence of preserving intangible culture cannot be overemphasised.\n\n\nData availability\n\nDue to lack of consent from the participants, the transcripts from the storytelling sessions are not available.", "appendix": "References\n\nAchebe C: Anthills of the Savannah. Ibadan: Heinemann Educational Books; 1987.\n\nAchebe C: Things Fall Apart. Essex: Pearson Education Limited; 2008.\n\nBhabha HK: The Location of Culture. London: Routledge; 1994.\n\nBarthes R: Mythologies. New York: Hill and Wang; 1972.\n\nDankarol J, Agoom P: The Imperatives of Tiv Oral Poetry and its Influence on Oral Language Development Among Tiv Children. Global Journal of Art and Social Science Education. 2015; 3(4): 107–110.\n\nDenga D: Educational development in Tivland: Problems and prospects. Calabar: Rapid Educational Publishers;\n\nDowden H: Africa Altered States, Ordinary Miracles. Great Britain: Portobello Books; 2009.\n\nDundes A, Bronner SJ: The Meaning of Folklore: The Analytical Essays of Alan Dundes. Logan: Utah State University Press; 2007.\n\nEvwierhoma M: Issues in Gender, Drama and Culture in Nigeria. Lagos: Concept Publications; 2002.\n\nFoucault M: Technologies of the Self. Technologies of the Self: A Seminar with Michel Foucault. Martin LH, Gutman H, Hutton PH, editors. Amherst: University of Massachusetts Press; 1988; 16–49.\n\nGbadegesin S: African Philosophy: Traditional Yoruba Philosophy and Contemporary African Realities. New York: Peter Lang; 1991.\n\nHabib MAR: Modern Literary Criticism and Theory: A History. Malden, MA: Blackwell Publishing; 2008.\n\nHarper P: The Kwagh-hir of the People of Tiv: A Note on Dramatised History Telling and Constructions of Nature Among the Tiv of Southern Nigeria. Environment and History. 1997; 3(3): 371–376. Publisher Full Text\n\nHall S: Representation: Cultural Representations and Signifying Practices. London: Sage in association with the Open University; 1997.\n\nInsaidoo KA: Moral Lessons in African Folktales. Bloomington: Authorhouse; 2011.\n\nLawuyi OB: Ijapa and Igbin: A Discursive Meditation on Politics, Public Culture and Moral Languages in Nigeria. Ibadan: University Press PLC; 2012.\n\nMenkiti IA: Person and Community in African Traditional Thought. African Philosophy: An Introduction. 3rd Ed.Wright RA, editor. Lanham, Maryland: UP of America; 1984; 171–181.\n\nMurphy D: Francophone West African Cinema, 1955-69: False Starts and New Beginnings. Africa's Lost Classics: New Histories of African Cinema. Bisschoff L, Murphy DCambridge: Legenda; 2014; 50–62.\n\nNetting RM: Cultural Ecology. Prospect Heights, Ill: Waveland Press; 1986.\n\nObiechina E: Narrative Proverbs in the African Novel. Res. Afr. Lit. 1993; 24(4): 123–141.\n\nOkpewho I: Oral Literature in Africa: Backgrounds, Character, and Continuity. Bloomington: Indiana University Press; 1992.\n\nOlugbemi-Gabriel O: African Folktales and Social Order: A Study of Selected Yoruba Folktales. Engaging the Future in the Present: Issues in Culture and Philosophy. Ademowo AJ, Oladipo TD, editors. Ibadan: Hope Publications; 2015; 46–55.\n\nOwomoyela O: Yoruba Trickster Tales. Lincoln, Neb.: University of Nebraska Press; 1997.\n\nOwomoyela O: Àjàpá, Ajá the Dog, and the Yams. Myth and Knowing: An Introduction to World Mythology. Leonard S, McClure M, editors. Boston: MgGraw-Hill; 2004; 257–264.\n\nSone E: The Folktale and Social Values. Traditional Africa, Eastern African Literary and Cultural Studies. 2018; 4(2): 142–159. Publisher Full Text\n\nSteward JH: Theory of Culture Change: The Methodology of Multilinear Evolution. Urbana: University of Illinois Press; 1955.\n\nTsaaior JT: Telling the tale, telling the nation: tiv tales, modernity and the (re) construction of Nigerian nationhood. Southern African Journal for. Folk. Stud. 2015; 25: 2: 1–15.\n\nUdu T, Udu M: Integrating Tiv Socio-cultural Practices with Literacy Instruction for Peace and Social Order: What L2 Teachers Can and Should Do. Literacy and Reading in Nigeria. 2013; 14(1): 190–196.\n\n\nFootnotes\n\n1 This particular tale was sourced from a storytelling session at the Tyungu family home in Gboko on 27th December 2018. However other versions of the alom story have been recorded in Harper (1997) analyzing the relationship between hare stories and sacred masquerades." }	[ { "id": "137211", "date": "23 Jun 2022", "name": "Emmanuel Adeniyi", "expertise": [ "Reviewer Expertise African Literature", "Oral Literature", "Postcolonial Studies", "Yoruba Studies" ], "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 attempts an intercultural study of the Tiv and Yoruba folktales with a view to emphasizing the significance of the verbal art for moral rebirth in Africa. It is a good research idea, but the writers failed to address critical issues that ought to be the building blocks of their argument and interpretations. Looking at the methodology, I was expecting to see a qualitative analysis of data collected either through Focus Group Discussions (FGDs), interviews or other methods, but the writers only analyzed a few folktales and inserted their interpolations into the article without sound theoretical and empirical truths. To ask pointedly, what did writers do in their field trip? They possibly interviewed people, participated in folktales rendition, made recordings … Where are the data collected during their trip? Why are the interviews not transcribed? These are some of the questions that need to be addressed before considering this paper for indexing. While stating that they would engage in narrative and content analyses of their data, their analysis is more narrative-based. We are not told the model of CA they did. Is it relational or conceptual? Though it appears a conceptual analysis, their explanation of some lexical items in both Tiv and Yoruba is inadequate and may be difficult to accept as an example of CA. CA is done for specific purposes; what are the purposes of the methodology in this research? They are obviously missing. CA is more than giving themes to texts, it helps in delivering theoretically and conceptually structured arguments and interpretation. Honestly, that is missing here.\nAnother major issue with the paper is that the reader is left in the lurch regarding its purpose. While the writers set out to interrogate cross-cultural convergences using the media of folktales and totemic symbols, they fail to deliver on this. An intercultural study of this nature should also be comparativist. The writers hinted about this approach with their insertion of “convergence” study in their methodology. However, they failed to address this in the article. The article holds a lot of prospects. The topic is interesting, and its intercultural approach even makes it an exciting research idea worth pursuing. I don’t know of any study on Tiv/Yoruba cultural parallelism, yet this article has boldly delved into it. I think it is a good idea that should be pursued in African/a Studies.\n\nThe introduction doesn’t lay a solid background for the study. A promising work of this nature should be moored on critical interrogations of the issues pointed out in this section. It is too scanty, jerky and is deficiently blind to critical discussions that ought to strengthen the paper and indicate the enormity of research invested in it. Reading the article raises serious questions: What is the basis for converging or possibly comparing the two cultures? Why the choice of these cultures? Are there underlying cultural parallelisms, historical contiguities and any other analogous cultural elements uniting the two cultures? You can’t just claim your study investigates these cultures, paying attention to convergent patterns in them without identifying and justifying these parallel elements.\nHowever, I don’t recommend its indexing in its present form, due to some of the issues I pointed out in my review. It needs to be firmed up, taking into consideration a number of factors, including theoretical, methodological and analytical details. I recommend its complete overhaul and revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Partly", "responses": [] }, { "id": "135789", "date": "22 Jul 2022", "name": "Adeiza Isiaka", "expertise": [ "Reviewer Expertise Sociocultural and variationist linguistics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverview Thank you for the opportunity to read this fascinating paper. The topic is timely and the manuscript is well written. In general, the paper presents an innovative exploration of an ancient African fictive genre – that I am eager to see published.\nThrough a distilling of famous folktales from two ethnic milieus (Yoruba & Tiv), the paper expounds on the centrality of storytelling to cultural pedagogy in traditional African spaces. Part of its goal is interventionist – to theorize “folk-telling” as a defining African practice and as a conduit of knowledge, and a mirror of collective realities. The paper further establishes how through folktales, the old speaks to the new – the contemporary African social ecologies and their concomitant issues of class, power, and cohesion.\n\nComment to the authors\nFour informants were interviewed (2 males & 2 females). Barring word limit, relevant parts of the naturalistic data from these participants can be quoted to animate the analysis. Though the authors note that transcripts from the storytelling data could not published due to lack of consent. I suggest they state clearly if this was before or after the data was collected. Did the participants give a limited consent? Put differently, the statement under “Consent” and “Data availability” appear to contradict each other.\n\n“This study follows a narrative and content analysis approach…” I am wondering if the generous review of cultural debates in the paper can be de-emphasized, and instead highlight the core of narrative and content analysis. In other words, the discussion in the section on theory would need to circle back to the approach mentioned in the abstract.\n\nOn page 4 - in the paragraphs that begins with “Nigeria has experienced….”, an interesting point is made about the deployment of folktales towards restoring indigenous morality or precolonial African mores. Perhaps it would be possible to theorize this more strongly. My question to the authors is whether this can be advanced as a notion or conceptualized with a phrase that ties in with the broader interventionist discourse of folk-telling in Africa.\n\nThe last paragraph on page 5 signals a contrast between two animal characters in Yoruba and Tiv folktales – namely, the Tortoise and the Hare. It would be useful to learn more about these characters, and apart from farming, why the animals are distinctively prominent in stories from the two regions.\n\nThis is negligible: there are a few instances of semantic doubling in the paper (e.g., surprise and chagrin on pg.8).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-455
https://f1000research.com/articles/11-1409/v1	30 Nov 22	{ "type": "Research Article", "title": "Fatty acid composition on diet and carcasses, growth, body indices and profile serum of Asian redtail catfish (Hemibagrus nemurus) fed a diet containing different levels of EPA and DHA", "authors": [ "Netti Aryani", "Indra Suharman", "Saberina Hasibuan", "Nur Asiah", "Hafrijal Syandri", "Indra Suharman", "Saberina Hasibuan", "Nur Asiah", "Hafrijal Syandri" ], "abstract": "Background: The Asian redtail catfish Hemibagrus nemurus is a promising commercial aquaculture freshwater big-sized Bagridae catfish across Asian countries such as the Mekong, Malay Peninsula, and Indonesia. This study analysed the effect of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) supplementation in diets on changes in fatty acid compositions in feed and fish meat, lipid quality (atherogenic index and thrombogenic index), growth rate, body indicators, and serum metabolites of Hemibagrus nemurus juveniles.\nMethods: A total of 180 Asian redtail catfish (initial weight 54.80 ± 2.72 g) were fed four levels (0, 3,150, 6,300, and 9,450 mg of EPA+DHA/kg feed) sourced from fish oil. Diets were fed in triplicate in freshwater tarpaulin ponds, with 15 fish per tarpaulin pond. During the experiment, fish were fed 3% per day of the biomass weight. Results: Categorically, there were significant differences in the composition of fatty acids in the feed and fish meat. The atherogenic index was between 1.76 and 1.84, and the thrombogenic index was between 0.81 and 0.89 in all fish meat. Growth performance was significantly different between diets, while body indices did not make a significant difference between diets. The fish meat EPA and DHA showed positive linear relationships with diet EPA (p <0.001, r2 = 90%) and DHA diet (p<0.001, r2 = 85%). Serum metabolites among treatments D2 and D3 diet-fed feed for 60 days did not significantly differ. Glucose (GLU) levels had moderate relationships with triglycerides (TAG) (r2 = 65%), and GLU levels strongly correlated with low-density lipoprotein cholesterol (LDL-C) (r2 = 81%). Conclusions: Based on diets and whole-body carcass compositions, growth performance, and serum metabolites, Asian redtail catfish fed a diet containing 6,300 mg of EPA+DHA/kg feed are best for food safety.", "keywords": [ "Aquaculture", "Asian redtail catfish", "essential fatty acids", "growth rates", "serum metabolites" ], "content": "Introduction\n\nGlobal capture fisheries production from marine sources has stagnated in the last few decades.1,2 Therefore, food sources from freshwater fish farming are increasingly being recognized for their role in an environmentally sustainable and nutritionally sustainable food system.3,4 However, freshwater fish contain fewer omega (ω)-3 fatty acids than omega (ω)-6 polyunsaturated fatty acids (PUFAs), making them a healthy food.5,6 Therefore, fish feed freshwater must be provided with the addition of omega (ω)-3 FUPAs carried out sustainably. Several researchers have reported feed enrichment for freshwater fish, such as the addition of linolenic acid, fish oil, and soybean oil to Nile tilapia fish feed.6–8 Omega (ω)-3, as well as EPA and DHA for the feed of Atlantic salmon.9,10 Dietary lipid levels for juvenile Asian red-tailed catfish (Hemibagrus wyckioides) and silver barb (Puntius gonionotus) fingerling.11,12\n\nAquafeed is rich in numerous significant nutrients, such as amino acids, fatty acids, vitamins, and minerals. Of these, feed is rich in nutrients and can be used by fish to increase body weight and survival,13 disease resistance, and changes in the aquaculture environment.14,15 This factor could also increase feed efficiency, which is usually used as a success indicator of fish farming.16 However, better feed quality is related to the nutrients of the whole-body carcass and is beneficial for human health.1,4,5,8,14,17\n\nHemibagrus nemurus, commonly known as “Asian redtail catfish,” is a promising commercial aquaculture freshwater big-sized Bagridae catfish across Asian countries such as Mekong, Malay Peninsula, and Indonesia. Because of its dominant consumer preference, it is most abundant in ordo Bagridae.18 This species has a high growth rate, disease resistance, year-round reproduction,19 and wide adaptability to the cultivation environment.20,21 In recent decades, research on feed nutrition for the growth performance of Asian redtail catfish has garnered the interest of researchers, such as experiments on dietary protein levels to increase growth performance, feed efficiency, and survival rate of juveniles,22,23 and the utilization of salted trash fish meal in the diet as a substitute for fish meal.21 Substitution of fish meal with a mixture of by-catch and fish viscera meal mixtures.24 Additionally, the addition of turmeric (Curcuma longa) in artificial feed,25 and addition of Saccharomyces cerevisiae in commercial feed,26 as well as different lipid level on daily growth coefficient and feed conversion ratio of Asian redtail catfish, Hemibagrus wyckioides.11 Until now, there has been no information on the utilities of optimum EPA and DHA levels on the sensible diet for the growth rate and fish meat value of Asian redtail catfish. We hypothesized that supplemented levels of EPA + DHA via fish oil in the diet could improve the nutritional quality of dietary fatty acid and fish meat, feed efficiency, growth rate, body indices, and lipid profiles in the serum of Asian redtail catfish. Therefore, the first aim of this study was to investigate the effect of the EPA and DHA levels in diets on the fatty acid composition. The second aim was to analyse fish meat fatty acids, growth rate, feed efficiency, and nutritional quality of lipids.\n\n\nMethods\n\nThis study was conducted under the project entitled ‘Optimization of the use of EPA- and DHA-type fatty acids in the feed of Asian redtail catfish to strengthen food security after coronavirus disease (COVID-19)’. This research was funded by the Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia, which has been approved by the Institute for Research and Community Service, Universitas Riau (grant letter: 1633/UN19.5.1.3/PT.01.03/2022). The Ethics Community Research and Community Service Universitas Riau approved collecting and rearing of Hemibagrus nemurus juveniles with the ARRIVE guidelines, which have been stated in the letter of grant No. 1633/UN19.5.1.3/PT.01.03/2022, May 11, 2022. Additionally, according to Indonesian Legislation, the Asian redtail catfish were not categorized as a protected species. The research was carried out in the Hatchery Laboratory, Faculty Fisheries and Marine, Riau University for 60 days from May to June 2022. This study is reported in line with the ARRIVE guidelines.27 We have made efforts to alleviate the suffering of experimental animals in this study, except for some euthanized animal experiments that were carried out by piercing part of the fish brain. The total number of fish in the study was 180 fish consisting of 45 fish for each treatment. Each treatment consisted of 15 fish per replicate (replicate = 3). A total of three fish were anesthetized in each replicate for the analysis of whole-body flesh, and body indices. Before the fish was euthanized, it was soaked in fresh water at a temperature of 10°C for five minutes. The goal is for the fish to be calmer and pierce their brain easier. After that, the fish were put to sleep on a board with a cork bottom, and their brain was pierced with a large animal syringe (9G × 1 inch). Then the fish were dissected to measure their body indices. In addition, the flesh was collected to measure its fatty acid composition.\n\nAsian redtail catfish adapted for 30 days to standard, 2 mm pellet feed. The dietary composition (% dry weight) consisted of an 8.76% moisture level, 34.52% crude protein, 5.94% crude fat, 41.13% carbohydrates, 9.65% ash content and 3.85% crude fibre (Table 1). The total calories were 356.06 Kcal/100 g vitamin D (328.31 mcg/100 g). Minerals consisted of calcium (1,788.11 mg/100 g), phosphorus (1,081.764 mg/100 g), magnesium (298.42 mg/100 g), manganese (9.50 mg/100 g), sodium (549.30 mg/100 g), and zinc (14.10 mg/100 g).\n\nAfter adaptation, the fish were fed supplemented with fish oil (EPA + DHA) at a dosage of 0 g/kg feed (labelled D1). A total of 5 g/kg feed, consisting of 1,950 mg EPA and 1,200 mg DHA (labelled D2). The 10 g/kg feed consisted of 3,900 mg EPA and 2,400 mg DHA (labelled D3). The 15 g/kg feed consisted of 5,850 mg EPA and 3,600 mg DHA (labelled D4). EPA and DHA were mixed manually to the aquafeed to produce quality feed, which was then given to the animal experiment.\n\nFish samples were weighed using the AD-600i, with an accuracy of 0.01 g (ACIS model number AD-600i, China). The Indonesian Directorate of Metrology has approved the use of ACIS model AD-600i. Additionally, body length was measured with a measuring board with an accuracy of 1 mm. A total of 180 juvenile Hemibagrus nemurus were counted (initial body weight was 54.80 ± 2.72 g, and initial body length was 15.77 ± 2.5 cm). The age of the fish was 120-day post-hatching (120 DPH), whose sex has not been determined. Juveniles were obtained from the Hatchery Laboratory Faculty of Fisheries and Marine Science Universitas Riau. This species' health status was good, not a hybridization genetic modification. A total of 12 round tarpaulin ponds (diameter = 80 cm, height = 60 cm with a water volume of 400 litres) were placed in Hatchery Laboratory and equipped with continuous aeration. This experiment consisted of four treatments and three replicates, and each round tarpaulin pond was stocked with 15 juveniles randomly stocked. The Asian redtail catfish were fed pellets 2 mm in size supplemented with EPA and DHA, namely, D1, D2, D3, and D4 diets. Daily feeding was performed at 07:00, 13:00, 18:00, and 22:00 at a body weight rate of 3% per day for 60 experimental days. Every 20 days, five fish samples were taken from each tarpaulin pond. Before sampling, the fish fasted for 12 hours to vacate their visceral contents. Fish samples were anesthetized orally using clove oil, and body weight was measured to determine the amount of fed feed. Then, their weights were measured. After that, the fish were returned to their respective tarpaulin ponds according to treatment and replication.\n\nThe feed and whole-body meat of the fish of each treatment were examined utilizing a fatty acid composition by gas chromatography-mass spectrometry (GC-MS). The extraction of the total lipid was carried out according to the method described by Folch et al. (1957), as explained by Rajion28 utilizing a chloroform: methanol (2.1. v/v) solvent system. Transmethylation was carried out using 14% methanolic boron trifluoride. Transmethylation was carried out using 14% methanolic boron trifluoride. The derivatized fatty acid methyl esters (FAMEs) were separated on a Quadrex 007 series bonded phase fused silica capillary column (Quadrex Corporation, New Haven. CT, USA) (30 m × 0.25 mm ID, 0.20 mm film thickness, 007 Carwax/BTR) in a 5890 Hewlett-Packard Gas-Liquid Chromatograph (Hewlett-Packard Co., Avondale, PA). Individual fatty acids were recognized and measured by proportion with retention time and peak areas of FAMEs standards (Supelco 37 Component FAME mix and Nu-Check Prep Inc., GLC-569). The fatty acid composition of feed and whole-body meat of Asian redtail catfish were examined by Saraswanti Indo Genetech Laboratory, Bogor-Indonesia (SIG Laboratory, Accredited Testing Laboratory- LP -184-IDN).\n\nThe water quality values of the round tarpaulin ponds that were used to rear the Asian redtail catfish juveniles were recorded weekly. Water quality was measured at 10:00 AM at a distance inward of 10 cm from the water surface of each tarpaulin pond to detect the water temperature, dissolved oxygen, and pH. A thermometer (Celsius scale) was used to measure water temperature. Water oxygen (O2; mg/L) was measured by an oxygen metre (YSI Model 52, Yellow Instrument Co, Yellow Spring, OH, USA). The pH value of the water was calculated on a digital pH metre (Mini 0–14 pH IQ, Scientific Chemo Science Thailand). Additionally, we also measured the level of nitrate-nitrogen (NO3-N; mg/L), total alkalinity (mg/L), and hardness (mg/L) calculated according to standard American Public Health Association (APHA) procedures.29\n\nAll fish from each treatment and replicate (n=15 fish per replicate) were measured in length and weighed body weight separately for the final experiment. Initial body weight (IW), final body weight (FBW), weight gain (WG, %), and specific growth rate (SGR, %/day). The feed conversion ratio (FCR) and survival rate (SR) were analysed using the support formula as follows:\n\nFor the analysis of the body indices of Asian redtail catfish (Hemibagrus nemurus), three fish were sacrificed each in the tarpaulin pond, and their weight and length were measured. After that, it was immediately dissected to determine the Condition Factor (CF), Hepatosomatic Index (HSI), Viscerosomatic Index (VSI), and Liposomatic Index (LSI) as given below:\n\nThe nutritional quality of lipid AI and TI was calculated based on the equations30\n\nWhere:\n\nC12:0 = Lauric acid\n\nC14:0 = Meristic acid\n\nC16:0 = Palmitic acid\n\nC18:0 = Stearic acid\n\n∑MUFA = Sum concentrations of all unsaturated fatty acid\n\n∑n-6 = Sum concentrations of n-6 polyunsaturated fatty acid\n\n∑n-3 = Sum concentrations of n-3 polyunsaturated fatty acid\n\nIn the present study, to minimize stress on the experimental animals, the animals within the containers were lightly anesthetized with 1 ml/10 L clove oil for 2–3 min until the loss of coordination was visible. Afterward, the blood was collected by puncturing the caudal vertebrae tail vessels using a hypodermic needle of 1 ml (made in Indonesia). Briefly, blood samples from each specimen were placed in an Eppendorf tube, and then centrifuged at 3,000 rpm for 15 minutes (5804R, Eppendorf), and the supernatant serum rather than plasma was stored at -21°C. Then it was analysed at the Centre for Primate Animal Studies at IPB University, Bogor, Indonesia. Serum glucose (GLU) was analysed using the glucose oxidase method using commercial kit (Pathology Laboratory, the Centre for Primate Animal Studies, IPB University, Bogor, Indonesia), triglycerides (TAG) were measured using the GPO-PAP method, total cholesterol (TC) by the CHOP-PAP method, high-density lipoprotein (HDL) with direct method-select inhibition method, low-density lipoprotein (LDL) with direct method-surfactant removal were also measured using commercial investigation kit (Pathology Laboratory, the Centre for Primate Animal Studies, IPB University, Bogor, Indonesia), and the ratio of LDL-C and HDL-C were approximated as outlined previously.31\n\nTo determine the trial effect of supplemented EPA and DHA in diets, body meat, growth performance, body indices, and serum metabolite variables were measured using one-way ANOVA. The data from the experiment were analysed using SPSS (RRID:SCR_002865) 16.0 software package (SPSS, Chicago, IL). The data homogeneity was analysed with Levin's test and followed up with the post hoc Duncan’s multiple range test.32 Relationship between dietary EPA and DHA with whole-body meat; glucose levels, TAG levels, and LDL-C levels were analysed using Regression with curve estimation. For the figures presented, Microsoft Office Professional Plus 2019 was used.\n\n\nResults\n\nIn this study, the ∑MUFA in the four diets showed higher values than ∑SAFA and ∑PUFA. Regarding the ∑MUFA, the D1 diet had the highest value, followed by the D2, D3, and D4 diets with C-oleic acid (C18:1, n-9) abundance in all diets. The SAFA palmitic acid (C16:0) and stearic acid (C18:0) were abundant in all diets. However, in the PUFA, the higher composition was linoleic acid (C18:2 n-6) (Table 2).27\n\nThe Asian redtail catfish fed feed D1, D2, D3, and D4 showed higher levels of ∑MUFA in the body meat than in ∑SAFA and ∑PUFA. Regarding MUFA, oleic acid (C18:1) is the most abundant. Palmitic acid (C16:0) and stearic acid (C18) were significant concentrations of SAFAs. Conversely, linoleic acid (C18:2, n-6) abounds in PUFA. We noted that EPA and AA concentrations were lacking in all body meat, while DHA was higher (Table 3). EPA and DHA levels in feed have strong relationships with EPA and DHA levels in body meat (r2 = 0.897 for EPA; Figure 1 and r2 = 0.812 for DHA; Figure 2). AI and TI were significantly higher on the D3 diet than on the other treatments. Duncan's post hoc test showed that the AI and TI for fish fed D1 were not significantly different (p > 0.05) from those provided for D2 and D4 (Figures 3 and 4).\n\n1 Means ± SE (standard error) of three separate determinations, a b c d = significant in a row, nd= not detected\n\nEPA, eicosapentaenoic acid; FAME, fatty acid methyl ester.\n\nDHA, docosahexaenoic acid; FAME, fatty acid methyl ester.\n\nEPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. Note: A different superscript letter on each bar chart indicates a significant difference (P < 0.05). The same superscript letter on each bar chart does not show a significant difference (P > 0.05).\n\nEPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. Note: A different superscript letter on each bar chart indicates a significant difference (P < 0.05). The same superscript letter on each bar chart does not show a significant difference (P > 0.05).\n\nAll experimental diets given to Asian redtail catfish presented a significant (p < 0.05) effect on final body weight, body weight gain (%), and specific growth rate (%/day). The higher growth was contributed by the D3 diet, followed by a better feed conversion ratio (FCR). The mean values of CF, HSI, VIS, and LSI did not differ significantly (p > 0.05) among the D1, D2, D3, and D4 diets (Table 4). The survival rate of various diet experiments was insignificant (p > 0.05).\n\n1 Values are presented as mean ± SE, n = 3. Note: Numbers followed by different superscript of letters in the same row indicate a significant differences (P < 0.05). Numbers followed by superscript of the same letter in the same row showed no significant difference (P > 0.05). FL, final length; FW, final body weight; WG, weight gain; SGR, specific growth rate; FCR, feed conversion ratio; SR, survival rate; CF, condition factor; HSI, hepatosomatic index; VSI, viscerosomatic index; LSI, liposomatic index.\n\nThe serum GLU, TAG, TC, HDL-C, and LDL-C levels in fish fed a diet with no supplemental fish oil (control, D1) were significantly higher than those in fish-fed diets D2, D3, and D4. Glucose levels in experimental animals fed D2 and D3 and D3 and D4 showed no significant difference (p > 0.05), except for D2 and D3 diets. Additionally, LDL-C and HDL-C ratios indicated a significant effect (p <0.05) among the experimental diets (Table 5). The GLU level parameter also had moderate relationships with TAG (r2 = 0.652, Figure 5). Additionally, the GLU level parameter strongly correlated with LDL-C (r2 = 0.811, Figure 6).\n\n1 Values are presented as means ± SE, n = 3. Note: Numbers followed by different superscript of letters in the same row indicate a significant differences (P < 0.05). Numbers followed by superscript of the same letter in the same row showed no significant difference (P > 0.05). GLU, glucose; TAG, triglycerides; TC, total cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein cholesterol.\n\nGLU, glucose; TAG, triglycerides; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.\n\nGLU, glucose; LDL-C, low-density lipoprotein cholesterol; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.\n\nThe physicochemical water parameters in the tarpaulin ponds for rearing juvenile Asian redtail catfish were as follows: water temperatures ranged from 28 to 30°C, oxygen between 6.4 and 6.7 mg/L, and pH between 6.6 and 6.8. The water alkalinity increased from 60.5 to 67.5 mg/L HCO3, and the hardness varied between 67.5 and 69.5 mg/L HCO3.\n\n\nDiscussion\n\nFor food safety and to increase the efficiency of feed use in fish farming, it is necessary to use nutrient-rich feed, such as fatty acids, amino acids, minerals, and vitamins.7,33,34 According to previous research, commercial feeds do not contain complete nutrients such as EPA and DHA.4 Therefore, improving the nutrition of fish feed can be done by supplementing fish oil containing EFA and DHA. In this study, adding fish oil to feed directly correlated with the EPA and DHA composition for the D1, D2, D3, and D4 diets. The higher the level of addition of fish oil (EFA and DHA) to the feed, the higher the levels of EPA and DHA in the experimental diets.\n\nAdditionally, in all experimental diets, the MUFA level was higher than the SAFA and PUFA levels. By contrast, Nile tilapia feed supplemented with fish oil at 5, 10, and 15% showed higher SAFA levels than MUFA and PUFA levels.7 However, EPA and DHA supplementation in Atlantic salmon feed were 0.25, 0.50, 0.75, and 2%, and SAFA levels were lower than MUFA and PUFA levels. By contrast, EPA + DHA in feed was detected in 1.35, 3.33, 5.48, and 15.6% of the total fatty acid methyl ester (FAME).10 In this study, EPA and DHA addition to D1, D2, D3, and D4 diets were 0 g fish oil/kg feed, 5 g fish oil (consisted of 1,950 mg EPA and 1,200 mg DHA/kg feed), 10 g fish oil (consisted of 3,900 mg EPA and 2,400 mg DHA/kg feed), and 15 g fish oil (consisted of 5,850 mg EPA and 3,600 mg DHA/kg feed). The EFA + DHA values detected in the experimental feed D1, D2, D3, and D4 were 4.19, 4.53, 5.90, and 6.91%, respectively. Seemingly, EPA and DHA were detected in the D1 diet, even though it’s not added with fish oil, but levels were lower than D2, D3, and D4. However, it has been reported that EPA and DHA were not detected in commercial pellet feed.4 Conversely, EFA and DHA were found in commercial fish feed, which enriches coconut water and palm sap sugar, fermented with various fungi.\n\nThe ratios of DHA and EPA are determined by the levels of DHA and EPA in the feed. In this study, DHA/EPA ratios between 1.05 and 2.17 were lower in the D4 and D3 diets than in D1 and D2. In this context, a ratio of 1.05 in the D4 diet indicated that DHA and EPA were at their highest levels in the experimental diet. In this study, the DHA/EPA ratio differences within the experimental diet were due to the addition of EPA and DHA at different levels. Zhang et al.35 state that the diet's proportion of DHA/EPA must be precise to develop better feed formulations. For this case, the DHA/EPA ratio of the diet for maximum growth of Nile tilapia was 1.42,7 including 1.70 for giant gourami4 and 0.53 for Atlantic salmon.10 Supplementation with fish oil in aquafeed directly reflects the chemical composition of aquafeed and can influence DHA/EPA ratios.10,36 Arachidonic acid (ARA), EPA, and DHA are the main essential HUFAs in fatty acid compositions, and these elements may be preventively supplemented in the diet.37 In general, certain types of fatty acids are principal energy sources and crucial constructional portions for the diet.38\n\nIn this study, the fish feed supplemented with 10 g of fish oil (consisting of 3,900 mg EPA and 2,400 mg DHA/kg of feed) had the highest fat content in the whole-body carcass, 11.567%, with 39.074% SAFAs, 39.645% MUFAs, and 21.225% PUFAs. By contrast, the fat level in whole body carcasses fed the D1 diet with the lowest fat content was 9.20% with 39.231% SAFA, 42.514% MUFA, and 18.179% PUFA, respectively. This study found that MUFA levels were higher than SAFA levels in the all-body carcass of Asian redtail catfish fed fish D1, D2, D3, and D4. In Nile tilapia-fed pellets with fish oil added at 0, 5, 10, and 15% for 60 days, MUFA levels were also higher than SAFAs and PUFAs.7 However, Atlantic salmon in freshwater-fed feed were enriched at levels of 0.25, 0.5, 0.75, and 2.0% of EFA + DHA, and their whole-body carcasses also contained higher MUFA levels than SAFAs and PUFAs.10 By contrast, in wild specimens, sea bass (Dicentrarchus labrax) showed the highest level of PUFA compared with SAFAs and MUFA.39 In common carp (Cyprinus carpio), Rohu (Labeo rohita), and tilapia (Oreochromis niloticus) specimens captured in the Indus River, Pakistan, the levels of SAFA were higher than those of MUFA and PUFA.40 The total SAFA, MUFA, and PUFA in body meat depends on the supplemented fish oil, levels of DHA+EPA in the diet, and fish species. Additionally, it also depends on whether the body meat is from farmed fish or wild catch. Whether the SAFA, MUFA, and PUFA levels of Asian redtail catfish from cultured and wild captured sources are different is poorly understood.\n\nPalmitic acid (C16:0) was the major metabolite in the body meat, followed by stearic acid (C18:0). Palmitic and stearic acid levels were higher in fed fish D4. Oleic acid (C18:1) was identified as the essential MUFA in the fish carcass fed D1, D2, D3, and D4. The higher oleic acid (C18:1) in all carcasses could be due to its dominance in the pellet feed. Several authors reported that commercial fish feeds contain more oleic acid.4,7,39\n\nThe current study showed that Asian redtail catfish (Hemibagrus nemurus) fed feed supplemented with different levels of EPA and DHA obtained from fish oil contained higher EPA and DHA than control feed (D1). These results match those for other fish species.4,6,7 The EPA and DHA contents in the diet had strong relationships with the EPA and DHA contents in whole-body carcasses (r2 = 0.897 for EPA and r2 = 0.812 for DHA). This fact showed that adding EPA and DHA to the feed positively contributed to the EPA and DHA in the carcass of Asian redtail catfish. Regarding PUFAs, Asian redtail catfish freshwater can be considered a good source of n-3 series fatty acids, especially EPA and DHA, which have the highest content in fish fed a D3 diet. However, the levels were low, in line with the contents of EPA and DHA for Atlantic salmon freshwater fish,10 Nile tilapia,7 and common carp, Rohu, and tilapia.40 However, this level of EPA and DHA in freshwater finfish is lower than that in seawater finfish, such as sea bass and other fish species.5,39\n\nEPA and DHA are essential nutrients in a portion of healthy food, so finding an affordable source of PUFA from fish is crucial for consumer guidance because fish is one of the significant sources of EPA and DHA. The ratio of Σn-3: Σn-6 fatty acids was higher in fish fed D3 than in fish fed D1, D2, and D4, which showed that EPA and DHA supplements in the diet can improve carcass quality. Fish oil is one of the low-cost supplies of EPA and DHA. It is essential to aquafeed, especially in freshwater fish farming, such as Asian redtail catfish (Hemibagrus nemurus).\n\nIn the current study, the atherogenic index (AI) ranged from 1.76 to 1.84, and the thrombogenic index ranged from 0.81 to 0.89 in all whole-body carcasses fed feed D1, D2, D3, and D4. This appeared to be connected to a discrepancy in SAFA compositions among experimental diets. The AI levels in the whole-body carcasses of Asian redtail catfish fed feed D1 and D2 and D4 were insignificant, while fish fed feed D3 differed in D1, D2, and D4. The AI and thrombogenic index (TI) indices were significantly associated with the content of myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0), all of which are thrombogenic promotors.7 The Food Agricultural Organization and the Health World Organization recommend AI and TI values between 0.4 and 0.5. Even though the AI and TI scores of the Asian redtail catfish were higher than 0.5, we stated that consuming Asian redtail catfish flesh is a healthy food. In fish farming, AI and TI indices correlated with feed supplemented with fish oil, including EPA and DHA,7,10 feed quality used,4 fish species, and environmental factors.5,41\n\nFish feeds with different levels of EPA and DHA directly impact the growth rate of Asian redtail catfish juveniles. In this study, the final body weight, body weight gain (%), specific growth rate (%/days), and low feed conversion ratio were shown in fed fish D3. In this trial, enrichment pellet feed with EPA and DHA has a positive effect on the fatty acid composition of the feed. This factor causes the better growth rate of Asian redtail catfish. The SAA, MUFA, and PUFA contents were higher in feed supplemented with EPA and DHA. Higher fat levels in the D3 diet also increased the growth rate of Hemibagrus nemurus and have been observed in other species.4,10\n\nIn this study, feed supplemented with EPA and DHA at different levels sourced from fish oil did not affect the condition factor (CF) or body indices (HSI, VSI, LSI) of Asian redtail catfish during the 60-day experimental period. The results were the same as those for Micropterus salmoides and other species.42–45\n\nThe nutritional status and metabolism of reared fish indirectly reflect the blood biochemistry of fish. The protein, carbohydrate, and fat contents in food can change the levels of GLU, TAG, HDL, and LDL in blood serum and are closely linked to the activity of digestive enzymes. A consistent GLU level is one of the crucial indicators of human health.34 In the current study, the amount of GLU in the blood serum of fish was lower in fish fed the D2, D3, and D4 diets than in fish fed the D1 diet, with a constant level of GLU between 2.50 and 2.98 Mmol/L. However, TAG is the usual variety of fat that can be utilized behind time by the body for energy.46 The present study showed a difference in the serum TC, HDL-C, LDL-C levels, and LDL-C/HDL-C ratios, except between the D2 and D3 diets. These results indicated that EPA and DHA levels up to 9,400 mg/kg feed were conducive to energy storage for fish health and can be recommended for consumer food safety. This finding can minimize feed costs overall, whether it is fish oil or future sources of EPA + DHA, because the aquaculture sector needed 836 thousand tonnes of fish oil.47\n\n\nConclusions\n\nAsian redtail catfish (Hemibagrus nemurus) diet containing 6,300 mg of EPA + DHA via fish oil (diet D3) showed fatty acid compositions in the diets better than those other feed diets. The D3-fed feed to Hemibagrus nemurus also had a better effect on the fatty acid composition of body meat, nutritional quality of lipid AI and TI, growth rate, body indices, and serum metabolites. According to our research, the current inclusion of EPA and DHA via fish oil in fish feed is approximately 9,450 mg EPA + DHA/kg diet, which could be reduced as much as 3,150 mg EPA + DHA/kg diet. This finding could minimize the overall cost of aquafeed, whether it is fish oil or future sources of EPA + DHA.", "appendix": "Data availability\n\nFigshare: Fatty acid composition on diet and whole body, growth performance, body indices, and profile blood serum of Asian redtail catfish (Hemibagrus nemurus) fed a diet containing different levels of EPA and DHA. https://doi.org/10.6084/m9.figshare.21164425. 27\n\nThis project contains the following underlying data:\n\n- Table 1. Raw data list ingrediennts and proximate of feed.pdf\n\n- Table 2. Raw data fatty acid of experiment diet.pdf\n\n- Table 3. Raw data fatty acid of whole body of Asian redtail catfish over 60 daysdocx.pdf\n\n- Table 4. Raw data initial body weight of Asian redtail catfish.pdf\n\n- Table 5. Raw data initial body length of Asian redtail catfish.pdf\n\n- Table 6. Raw data final body weight of Asian redtail catfish each experimental diet.pdf\n\n- Table 7. Raw data final body length of Asian redtail catfish each experimental diet.pdf\n\n- Table 8. Raw Data of growth performance and body indices of Asian redtail catfish.pdf\n\n- Table 9. Raw data serum metabolites of Asian redtail catfishdocx.pdf\n\n- Authors ceklist Manuscriot No. 126487.pdf (completed ARRIVE checklist)\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 thank the Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia for supporting this study through the competitive grant's schema National Competitive Basic Research, 2022. The appreciation goes to the student and fish farmers who helped the authors to collect data in the field and laboratory.\n\n\nReferences\n\nTran N, Rodriguez, Chan CY, et al.: Indonesian aquaculture futures: An analysis of fish supply and demand in Indonesia to 2030 and role of aquaculture using the AsiaFish model. Mar. 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Aquaculture. 2008, 28; 1–4: 146–158." }	[ { "id": "157015", "date": "26 Jan 2023", "name": "Christopher Marlowe Caipang", "expertise": [ "Reviewer Expertise Aquaculture", "fish health management", "aquatic biotechnology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 evaluated the effects of various levels of EPA and DHA on selected growth and body composition indices of Asian redtail catfish. Results showed that the incorporation of 6,300 mg of EPA+DHA/kg in the diets gave the best effects on whole-body carcass compositions, growth performance, and serum metabolites of the fish.\nComments: This data obtained from this study is important in developing the aquaculture industry for this species of fish particularly in the establishment of commercial feeds. Comments and suggestions for the improvement of the manuscript are indicated below.\nIn the Conclusion (Abstract), why did the authors conclude that the level of 6,300 mg of EP+DHA in the feeds is the best for food safety? Please justify and support this claim with data from the work.\n\nPlease indicate the area or volume of the tarpaulin ponds so the stocking density could be determined.\n\nPlease justify the stocking of 15 fish per pond.\n\nThe use of commercial kits to measure blood parameters should be justified and authors should state whether these kits have been standardized for fish.\n\nIs there a need to write the formula of the different growth parameters in the text? Please explain.\n\nFrom Tables 2 - 5, the values of the SE were only placed in one column. Authors mentioned that they have triplicate in each treatment and 3 fish were sampled per replicate. Therefore, the number of fish per treatment should be 9. I suggest that each column (Control and D1-D4) should have its own SE. Kindly revise the tables accordingly.\nI hope that the authors will take these suggestions/comments when they revise the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9293", "date": "15 May 2023", "name": "Netti Aryani", "role": "Author Response", "response": "Although some studies recommended fat meat values between 0.5 and 1.0, no such guidelines exist for AI and TI. AI and TI values in the meat of Asian redtail catfish at 6,300 mg of EPA+DHA were 0.81 and 1.85. Foods with low AI and TI promote excellent human health, for example, by preventing human health coronary diseases. In the present study, the water volume of the tarpaulin ponds is 300 liters, with a stocking density of 15 fish per tarpaulin pond. Primate Animal Study Center, IPB University-Bogor, has standardized commercial kits to measure blood fish parameters. In the methods and calculations part, we revised the initial body weight formula, changed it to the final length (cm), and the final body weight (FBW) changed to the final weight (FW); we have presented in Table 4. Standard Errors (SE) In Table 2-5, we wrote down the results of data analysis from multiple comparisons of the Post Hoc Tests for each analyzed parameter." } ] }, { "id": "157014", "date": "31 Jan 2023", "name": "Rudy Agung Nugroho", "expertise": [ "Reviewer Expertise Animal Physiology", "Fish 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\nTitle\nGood title.\nAbstract\nPlease add the results: Condition Factor (CF), Hepatosomatic Index (HSI), Viscerosomatic Index (VSI), and Liposomatic Index (LSI).\nIntroduction\nBrief background and aims.\nMaterials and Methods\nTable 1, at proximate composition NEE should be NFE (Nitrogen Free Extract).\n\nTable 1: Please describe the content of the vitamin and mineral mix.\n\nThis species' health status was good. Please be specific on what categorize fish indicated as good in health status.\n\nDoes the feeding rate of 3% was optimum for this fish? Or any previous experiment mentioned that 3% is optimum for this fish?\n\nIt is stated in the methods that “Every 20 days, five fish samples were taken from each tarpaulin pond”. However, the results did not appear in the results section.\nResults and Discussion\nThe discussion of The link between digestive enzymes activity and the content of protein, carbohydrate, and fat contents which is related to the levels of GLU, TAG, HDL, and LDL in blood serum can be developed.\nConclusion\nSufficient.\nCurrent article is good and has a contribution in aquaculture field. However, the discussion need to be developed, based on the finding/results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9296", "date": "15 May 2023", "name": "Netti Aryani", "role": "Author Response", "response": "Abstract We state that whole-body indices such as Condition Factor (CF), Hepatosomatic Index (HSI), Viscerosomatic Index (VSI), and Liposomatic Index (LSI) were not significant between diets experimental, so the result we do not write in quantity in the Abstract. On the other hand, the number of words in the abstract is limited to 300 words. Methods We agreed formula NEE should be changed to NFE (Nitrogen Free Extract) as written on the note under Table 1. The vitamin mix contains vitamins A, 2,750 IU; Vit D, 550,000 IU; Vit E, 25,000 IU; Ca D-pantothenate, 25,000 mg, Mg, 25.000 mg; P, 50.000 mg. A feeding rate of 3% has been optimal for the growth of Hemibagrus nemurus in this study, although some researchers reported a range of 3 - 8%. Five fish samples were measured every 20 days, and their data were used for estimating the amount of feed given based on biomass weight (15 fish/pond) every 20 days. Hence, the results did not appear in the results section. Result and Discussion In this study, we did not analyze digestive enzyme activity, so the discussion related to the levels of GLU, TAG, HDL, and LDL in blood serum cannot get discussed yet; maybe in the future can be developed." } ] } ]	1	https://f1000research.com/articles/11-1409
https://f1000research.com/articles/12-498/v1	15 May 23	{ "type": "Study Protocol", "title": "Cross-cultural adaptation and validation of the Greek version of the Kerlan-Jobe orthopaedic clinic shoulder and elbow score in Greek overhead athletes", "authors": [ "Eleftherios Paraskevopoulos", "George Plakoutsis", "Maria Papandreou", "George Plakoutsis", "Maria Papandreou" ], "abstract": "Background: Overhead athletes frequently perform rapid and powerful throwing overhead strokes in positions at the extreme range of motion, increasing the risk of upper limb injury. The Kerlan-Jobe Orthopedic Clinic (KJOC) Shoulder and Elbow Score has shown to be a valid and reliable questionnaire that can be used for the assessment of the functional status of the upper limb of patients involved in highly demanding overhead sports. The KJOC has been translated into several other languages however, a Greek version of the KJOC is not available yet. Methods: The KJOC will be cross-culturally adapted into Greek following international guidelines. At least 100 overhead athletes with or without shoulder or elbow complaints will be recruited and asked to fill in the Greek version of the KJOC twice and the Disabilities of Arm, Shoulder and Hand Questionnaire (DASH) once. The internal consistency and the test-retest reliability will be examined using Cronbach’s alpha and the intraclass correlation coefficient (ICC), respectively. The standard error of measurement (SEM) and the minimum detectable change (MDC) will be calculated and possible ground or ceiling effects will be also examined. Convergent validity will be evaluated with the Greek DASH using Pearson’s correlation. Results: The results of this study will be presented in an article to be published later. Conclusions: This report describes the process of translation and cross cultural adaptation of the Greek version of the KJOC. We believe a study protocol will assist researchers in the field to improve the reporting of similar studies and as a result improve the quality of their studies.", "keywords": [ "Overhead athlete", "questionnaire", "patient-reported outcome", "elbow", "shoulder", "Kerlan-Jobe Orthopedic Clinic" ], "content": "Introduction\n\nIt is well known that overhead athletes frequently perform rapid and powerful throwing strokes above head-height during ball serving, in positions at the extreme range of motion, increasing the risk of shoulder or other upper limb injury (Paraskevopoulos et al., 2021a, 2021b, 2023). Nevertheless, the increased physical demands placed at the shoulder and elbow in overhead athletes may also affect physical performance during play (Clarsen et al., 2015). Despite the presence of symptomatology in this population, research has shown that they continue to train and compete irrespective of clinical symptom severity (Agresta et al., 2019; Clarsen & Bahr, 2014). Experts in the field support that prevention is key in the management of overhead athletes, including evaluation and monitoring of upper limb health and performance (Cools et al., 2015; Sukanen et al., 2022). Patient-reported outcome measures (PROM) are highly suggested in this clinical population for the assessment of functional performance (Paraskevopoulos et al., 2023).\n\nThe Kerlan-Jobe Orthopedic Clinic (KJOC) Shoulder and Elbow Score has shown to be a valid and reliable PROM that can be used specifically in overhead athletes (Alberta et al., 2010; Merolla et al., 2018). The KJOC was designed by Alberta et al. (2010) for the assessment of the functional status of the upper limb of patients involved in highly demanding overhead sports. The KJOC includes questions related to self-perceived ability to perform sport-specific movements such as, hitting a ball. The KJOC has been designed not only for symptomatic athletes but also for the assessment of the functional performance of healthy athletes as well (Sukanen et al., 2022). Lastly, the KJOC can also be used in order to examine the effectiveness of any intervention in overhead athletes with shoulder or elbow pathology (Sukanen et al., 2022).\n\nThe original version of the KJOC has been translated from English into several other languages, including Korean (Oh et al., 2017), Finish (Sukanen et al., 2022), Italian (Merolla et al., 2017), Turkish (Turgut & Tunay, 2018), Norwegian (Fredriksen & Myklebust, 2019) and German (Schulz et al., 2022). To date no comparable PROM exists in the Greek language, although overhead sports are highly popular in Greece. Thus, the aim of this study is to translate and cross culturally adapt the KJOC in Greek-speaking overhead athletes and examine the reliability and validity of the Greek version of the KJOC in this population.\n\n\nProtocol\n\nThe authors will collect data from multiple outpatient private clinics in Greece. To be eligible to participate in the study patients should meet the following criteria: 1) be adults (aged >18 years), 2) to compete and train at least two times weekly in an overhead sport, such as handball, volleyball, baseball, softball, basketball, water polo, tennis, or badminton and 3) to be able to natively communicate, read and write in Greek. Healthy and injured players with shoulder or elbow complaints will be included. Athletes will be excluded if they suffer from cognitive, communication, or psychological issues. Also, athletes suffering from neurological dysfunction or cardiovascular or pulmonary dysfunctions that result in functional limitations will be excluded. All participants will sign an informed consent form before participation. The study protocol has been approved by the ethics committee of the University of Peloponnese.\n\nA minimum sample of 100 participants will be considered adequate for the assessment of the internal consistency, floor and ceiling effects, construct validity, test-retest reliability, and measurement error based on the recommendation of the consensus-based standards for the selection of health measurement instruments (COSMIN) (Terwee et al., 2012).\n\nThe procedures for translation and cross-cultural adaptation of the KJOC will be based on previously reported studies (Beaton et al., 2000; Schulz et al., 2022). The first step includes the forward translation of the English version into Greek by two independent bilingual translators who are Greek in origin. One of the two forward translators is a Lecturer in physiotherapy with more than 30 years of experience in clinical physiotherapy and academic teaching. The other forward translator will have no physiotherapy or medical background and will be unaware of the existence of the KJOC. The two forward translators will compare their translations until a consensus is reached. A single Greek version of the scale will be formed from the two reports and the comments of the two translators. Again, another two bilingual independent translators will complete the two backward translations of the Greek version of the KJOC into English. The backward translators will compare the scale with the translated version in order to confirm whether the semantic, conceptual, and experimental equivalence are met.\n\nThe pre-final Greek version of the KJOC will be then pilot tested in 10 overhead athletes. From these 10 athletes, five will be symptomatic and five will be asymptomatic in the shoulder or elbow region. After completion of the 10 pilot pre-final versions of the KJOC an interview will be followed individually with all 10 athletes. Two physiotherapists with more than 10 years of experience will conduct the interviews. In each individual interview the participants will be asked to explain if the content of the pre final version of the scale is clear after reading the instructions, the items and responses. Furthermore, they will be asked to clarify whether parts of the scale are not clear and will provide suggestions for any possible modifications that may improve clarity.\n\nTest-retest reliability will be examined in the final Greek Version of the KJOC (Gr-KJOC). All participants (n=100) will be asked to complete the Gr-KJOC twice. The first time that they will complete the Gr-KJOC will be during their first contact with the authors and the second will be three to five days later. The interval of test-retest sessions in this study is specified in order to minimize recall and to be suitably short to guarantee clinical stability between testing sessions (Aljathlani et al., 2022). The clinical stability of the participants will be examined by asking each one whether they believed that their symptoms were the same in the retest session (Bennell et al., 2000). Only patients that answer that their symptoms are the same in the retest session will complete the Gr-KJOC for a second time. Completion of the Gr-KJOC twice should allow the investigators to examine test-retest reliability by comparing the results of the test and retest sessions. Internal consistency will be also assessed based on the degree to which separate items of the Gr-KJOC relate to each other (English & Keeley, 2014).\n\nThe construct validity will be examined after correlating the results of the Gr-KJOC with the Greek Version of the Disability of the Arm, Shoulder, and Hand (DASH) and the DASH Sports Module (DASH-SM) (Aljathlani et al., 2022). The Greek DASH has shown to be a reliable and valid instrument that can provide a standardized measure of patient-centred outcomes in Greek-speaking patients with upper limb disorders while the DASH-SM assesses symptoms and the functional status of the upper limb in sports settings (Themistocleous et al., 2006). The DASH contains 30 questions; 21 related to function, six related to symptom severity and three to social function. Each question is rated on a 5-point scale (1, no difficulty; 2, mild difficulty; 3, moderate difficulty; 4, severe difficulty; 5, unable). The questionnaire score is calculated by applying established formulas for the first 30 questions and the scores range from 0 (the best) to 100 (the worst) (Themistocleous et al., 2006). The DASH-SM contains four questions and the goal of the DASH-SM is to identify the specific difficulties that athletes might experience but which may not affect their activities of daily living and consequently may go “undetected” in the 30-item portion of the DASH (Aljathlani et al., 2022).\n\nThe mean duration for reading and completing the questionnaire by the athletes will be measured. Also, the duration needed for analysing the results of the questionnaire by one of the investigators and calculating the final score will be also measured. Questionnaire analysis will be conducted by one investigator in all cases.\n\nVerification of floor and ceiling effect will be made by percentage (>15%) of the participants who have obtained the minimum and maximum scores in the Gr-KJOC, respectively (Terwee et al., 2007).\n\nAll data will be analysed using IBM SPSS statistics (RRID:SCR_016479) 28.0. Descriptive statistics will be calculated and reported for all measures. The statistical level of significance will be set at p < 0.05. Normal distribution of the data will be examined via the Shapiro-Wilk test. Parametric tests will be selected for data with normal distribution and non-parametric tests will be selected for data without normal distribution.\n\nInternal consistency\n\nInternal consistency, as a degree of homogeneity of the single items of the Gr-KJOC, will be examined using Cronbach’s alpha. Internal consistency will be considered acceptable for the Gr-KJOC if the alpha value is going to be within the recommended range of 0.70 to 0.95 (Aljathlani et al., 2022; Prinsen et al., 2018).\n\nTest re-test reliability (measurement errors)\n\nThe Intraclass Correlation Coefficient (ICC) for absolute agreement will be used to examine test re-test reliability of each item and the total score of the Gr-KJOC. Measurement errors from the use of Gr-KJOC will be estimated from the Standard Error of Measurement (SEM) and the Minimum Detectable Change (MDC). For the SEM the following equation will be used: SEM = SD√(1-ICC). SD will be the pooled SD calculated by the following equation: SDpooled = √(SD12 + SD22)/2. Then, the MDC will be calculated using the following equation: MDC= SEM × 1.64 × √ 2 reflecting the smallest detectable within-person change in score (Aljathlani et al., 2022; Portney & Watkins, 2009; Valentín-Mazarracin et al., 2021).\n\nConstruct validity\n\nConstruct validity of the Gr-KJOC will be examined by correlating the results of the Gr-KJOC with the DASH and the DASH-SM. Spearman’s correlation will be used to examine construct validity. Correlation coefficients of 0.70-0.89, 0.40-0.69 and 0.10-0.39 will be considered as strong, moderate and weak, respectively (Schober et al., 2018).\n\nThe known group method analysis will also be used to assess a further aspect of construct validity and identify whether the Gr-KJOC can actually differentiate between symptomatic (playing with arm symptoms or unable to play due to arm symptoms) and asymptomatic athletes (playing without any arm symptoms), as previously performed (Schulz et al., 2022). In order to establish evidence of known-group validity, independent samples t-test and Mann–Whitney U test will be used. Also, Pearson’s effect size r will be calculated.\n\n\nConclusions\n\nThis study will translate and culturally adapt the original KJOC scale into Greek for the first time using a standardized approach. We aim to show that the Greek version of the KJOC will be reliable and valid to use for the assessment of the functional capacity of healthy and injured overhead athletes in the should or elbow. Our aim through this research process will be to develop a translated KJOC that will be used by researchers and health care professionals, as well as coaches in Greek-speaking athletic settings for assessing functional status, treatment outcome and return-to-sport ability in this population. Lastly, the KJOC can also be used to predict shoulder or elbow injuries since there is evidence to show that there is a relationship between lower KJOC scores and future in-season injuries for overhead athletes (Holtz & O’Connor, 2018).\n\nThe study protocol was approved by the Institutional Ethics Committee of the University of Peloponnese (Ref No. 7365).\n\nThe study has started. The study has not been completed and no data analysis has been performed.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAgresta CE, Krieg K, Freehill MT: Risk Factors for Baseball-Related Arm Injuries: A Systematic Review. Orthop J Sports Med. 2019; 7(2): 2325967119825557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlberta FG, ElAttrache NS, Bissell S, et al.: The development and validation of a functional assessment tool for the upper extremity in the overhead athlete. Am J Sports Med. 2010; 38(5): 903–911. Publisher Full Text\n\nAljathlani MF, Alshammari MO, Alsuwaygh MA, et al.: Cross-cultural adaptation and validation of the Arabic version of the upper extremity functional index. Disabil Rehabil. 2022; 44(19): 5656–5662. PubMed Abstract \| Publisher Full Text\n\nBeaton DE, Bombardier C, Guillemin F, et al.: Guidelines for the process of cross-cultural adaptation of self-report measures. Spine (Phila Pa 1976). 2000; 25(24): 3186–3191. Publisher Full Text\n\nBennell K, Bartam S, Crossley K, et al.: Outcome measures in patellofemoral pain syndrome: test retest reliability and inter-relationships. Phys Ther Sport. 2000; 1(2): 32–41. Publisher Full Text\n\nClarsen B, Bahr R: Matching the choice of injury/illness definition to study setting, purpose and design: one size does not fit all!. Br J Sports Med. 2014; 48(7): 510–512. PubMed Abstract \| Publisher Full Text\n\nClarsen B, Bahr R, Heymans MW, et al.: The prevalence and impact of overuse injuries in five Norwegian sports: Application of a new surveillance method. Scand J Med Sci Sports. 2015; 25(3): 323–330. Publisher Full Text\n\nCools AM, Johansson FR, Borms D, et al.: Prevention of shoulder injuries in overhead athletes: a science-based approach. Braz J Phys Ther. 2015; 19(5): 331–339. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEnglish T, Keeley JW: Internal consistency approach to test construction. The Encyclopedia of Clinical Psychology. 2014; pp. 1–3.\n\nFredriksen H, Myklebust G: Norwegian translation, cross-cultural adaptation and validation of the Kerlan-Jobe Orthopaedic Clinic shoulder and elbow questionnaire. BMJ Open Sport Exerc Med. 2019; 5(1): e000611. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoltz KA, O’Connor RJ: Upper Extremity Functional Status of Female Youth Softball Pitchers Using the Kerlan-Jobe Orthopaedic Clinic Questionnaire. Orthop J Sports Med. 2018; 6(1): 2325967117748599. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerolla G, Corona K, Zanoli G, et al.: Cross-cultural adaptation and validation of the Italian version of the Kerlan-Jobe Orthopaedic Clinic Shoulder and Elbow score. J Orthop Traumatol. 2017; 18(4): 415–421. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerolla G, Paladini P, Porcellini G: Assessment of return to play in professional overhead athletes subjected to arthroscopic repair of rotator cuff tears and associated labral injuries using the Italian version of the Kerlan-Jobe Orthopedic Clinic Shoulder and Elbow score. Musculoskelet Surg. 2018; 102(Suppl 1): 29–34. PubMed Abstract \| Publisher Full Text\n\nOh JH, Kim JY, Limpisvasti O, et al.: Cross-cultural adaptation, validity and reliability of the Korean version of the Kerlan-Jobe Orthopedic Clinic shoulder and elbow score. JSES Open Access. 2017; 1(1): 39–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParaskevopoulos E, Pamboris GM, Papandreou M: The Changing Landscape in Upper Limb Sports Rehabilitation and Injury Prevention. Sports. 2023; 11(4): 80. Publisher Full Text Reference Source\n\nParaskevopoulos E, Simeonidis T, Tsolakis C, et al.: The adjunctive benefits of mirror cross education on kinetic chain exercise approach in volleyball athletes with scapular dyskinesis. J Sports Med Phys Fit. 2021a; 62(1): 98–109. Publisher Full Text\n\nParaskevopoulos E, Simeonidis T, Tsolakis C, et al.: Mirror Cross-Exercise on a Kinetic Chain Approach Improves Throwing Performance in Professional Volleyball Athletes With Scapular Dyskinesis. J Sport Rehabil. 2021b; 31(2): 131–139. PubMed Abstract \| Publisher Full Text\n\nPortney LG, Watkins MP: Foundations of clinical research: applications to practice. Prentice Hall Upper Saddle River, NJ: Pearson; 2009; vol. 892. .\n\nPrinsen CAC, Mokkink LB, Bouter LM, et al.: COSMIN guideline for systematic reviews of patient-reported outcome measures. Qual Life Res. 2018; 27(5): 1147–1157. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchober P, Boer C, Schwarte LA: Correlation Coefficients: Appropriate Use and Interpretation. Anesth Analg. 2018; 126(5): 1763–1768. Publisher Full Text\n\nSchulz C, Eibl AD, Radovanović G, et al.: Cross-cultural adaptation and validation of the Kerlan-Jobe orthopedic clinic shoulder and elbow score for German-speaking overhead athletes. Physiother Theory Pract. 2022; 38(8): 1059–1070. Publisher Full Text\n\nSukanen M, Pajari J, Äyrämö S, et al.: Cross-cultural adaptation and validation of the Kerlan-Jobe Orthopaedic Clinic shoulder and elbow score in Finnish-speaking overhead athletes. BMC Sports Sci Med Rehabil. 2022; 14(1): 190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTerwee CB, Bot SD, de Boer MR , et al.: Quality criteria were proposed for measurement properties of health status questionnaires. J Clin Epidemiol. 2007; 60(1): 34–42. Publisher Full Text\n\nTerwee CB, Mokkink LB, Knol DL, et al.: Rating the methodological quality in systematic reviews of studies on measurement properties: a scoring system for the COSMIN checklist. Qual Life Res. 2012; 21(4): 651–657. Publisher Full Text\n\nThemistocleous GS, Goudelis G, Kyrou I, et al.: Translation into Greek, cross-cultural adaptation and validation of the Disabilities of the Arm, Shoulder, and Hand Questionnaire (DASH). J Hand Ther. 2006; 19(3): 350–357. PubMed Abstract \| Publisher Full Text\n\nTurgut E, Tunay VB: Cross-cultural adaptation of Kerlan-Jobe Orthopaedic Clinic shoulder and elbow score: Reliability and validity in Turkish-speaking overhead athletes. Acta Orthop Traumatol Turc. 2018; 52(3): 206–210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValentín-Mazarracin I, Nogaledo-Martín M, López-de-Uralde-Villanueva I, et al.: Reproducibility and Concurrent Validity of Manual Palpation with Rehabilitative Ultrasound Imaging for Assessing Deep Abdominal Muscle Activity: Analysis with Preferential Ratios. Diagnostics (Basel). 2021; 11(2). PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "263359", "date": "22 May 2024", "name": "Julia Fayão", "expertise": [ "Reviewer Expertise My doctoral research consists of an assessment of the shoulder complex in volleyball athletes" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 recommend extending the reliability testing time to up to seven days instead of five I suggest conducting responsiveness testing. It is important to mention whether there was authorization from the original author for the translation and validation. I recommend providing more information about the questionnaire that will be translated and validated into Greek. I suggest including \"study protocol\" in the title. As for my thoughts on the article, I would like to highlight its relevance in the context of cultural adaptation and validation of questionnaires for Greek high-performance athletes. This type of study is crucial to ensure the accuracy and reliability of measurements across different cultures and athletic contexts. Organizing my comments into 'Major' and 'Minor' points, I would like to address methodology, clinical relevance, and suggestions for improvement: Major: The methodology used for translation and validation is essential to ensure conceptual and linguistic equivalence of the questionnaire. The clinical relevance of this study lies in the need for specific assessment tools for Greek athletes due to the unique demands of their sports. The study has the potential to positively impact clinical practice and research in sports medicine by providing a validated and culturally adapted assessment tool. Minor: It would be helpful to provide more details on the specific methods used in translating and validating the questionnaire. A discussion of the study limitations and possible areas for future research would be valuable to contextualize the results. Thank you again for the opportunity to review 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? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "305665", "date": "05 Aug 2024", "name": "Maria Sukanen", "expertise": [ "Reviewer Expertise My area of research lies between sport and health sciences with a background in physiotherapy. I have expertise in conducting cross-cultural adaptation and validation research of patient-reported outcomes from a previous publication and current studies." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a study protocol about cross-cultural adaptation and validation process of the Kerlan-Jobe orthopaedic clinic shoulder and elbow score in Greek language. The protocol shows the efforts the authors have made with planning the research according to accepted standards. The protocol is mostly very clearly presented and below I have listed some minor comments and recommendations for the author’s consideration.\nIn the abstract (methods), I would replace the term ground with floor, as later on in the manuscript you refer to floor effect. In the abstract (conclusions), I recommend adding that this protocol also describes the assessment of psychometric properties of the Greek KJOC in addition to cross-cultural adaptation process. Many of the keywords are included in the study title, perhaps I would consider replacing e.g., shoulder, elbow and KJOC for example with psychometric properties and reliability. In the introduction you can add that KJOC has also been validated into Spanish language. Please also note that Finnish is written with two n’s. In the end of introduction, I would rephrase the study aim more precisely to match this study protocol. Under the subheading “Sample size”, it is perhaps not necessary to introduce the abbreviation COSMIN. Under the subheading “Translation and cross-cultural adaptation”, did only the translators work through the adaptation process of the Greek KJOC? Many times, multidisciplinary team input is of great value during cross-cultural adaptation. Under “Reliability” I would suggest extending the test-retest time interval closer to a week. In general, regarding the so far reported measurement properties of KJOC, the authors could briefly conclude what kind of validity/reliability results previous studies have obtained for the same variables you report that will be tested for the Greek KJOC. Under \"validity\", the authors provide general information regarding DASH as a comparison score, but this information is lacking for KJOC. I would recommend the authors to add a description of KJOC as well. Under “validity”, it is a great advantage that the authors have the possibility to use a translated and validated questionnaire as a comparison score to test validity. I would recommend the authors to follow the same order while describing measurement properties throughout the manuscript (abstract, data collection, analysis) e.g., starting with internal consistency, followed by validity, reliability, floor- and ceiling effect etc. This would make the storyline more logical for the reader. I would also add information of obtaining permission to move forward with the study from the original developer of the KJOC score.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-498
https://f1000research.com/articles/12-495/v1	15 May 23	{ "type": "Research Article", "title": "Hypertension and COVID-19 fractional derivative model with double dose vaccination", "authors": [ "Samuel Okyere", "Joseph Ackora-Prah", "Ebenezer Bonyah", "Bennedict Barnes", "Maxwell Akwasi Boateng", "Ishmael Takyi", "Samuel Akwasi Adarkwa", "Joseph Ackora-Prah", "Ebenezer Bonyah", "Bennedict Barnes", "Maxwell Akwasi Boateng", "Ishmael Takyi", "Samuel Akwasi Adarkwa" ], "abstract": "The prevalence of at least one underlying medical condition, which increases the likelihood of developing the severe COVID-19 disease, is found in 22 of the world's population. The primary underlying medical condition that contributes to COVID-19 problems in Ghana is hypertension. This work investigate COVID-19 in a population with hypertension taking into account double dose vaccination of susceptible individuals. The study modifies a previous model proposed in the literature to include double dose vaccination and Atangana-Baleanu-Caputo fractional derivatives is used to solve the model. We give few definitions of the ABC operator and determine the existence and uniqueness of the solution. Using COVID-19 data for the period February 21, 2021 to July, 24 2021, the model is tested. The dynamics of the disease in the community were shown to be influenced by fractional-order derivatives. Contrary to the previous model proposed in the literature, the vulnerable group saw a significant reduction in the number, which may be attributed to the double dose vaccination. We recommend a cost-effective optimal control analysis in future work.", "keywords": [ "Hypertension", "Fractional Derivative", "Basic Reproduction Number", "COVID-19", "Double Vaccination" ], "content": "Introduction\n\nIt is now known that COVID-19 significantly worsens the health of people with primary medical condition of hypertension.1 According to studies, those who have the difficulties are more likely to be given the COVID-19 and experience unfavourable effects.2–4 The populations most at risk for infections include the elderly and those with underlying illnesses such as hypertension, diabetes, coronary artery etc. At least one of the underlying medical disorders is present in 22% of the world’s population, which raises their risk of contracting the severe COVID-19 disease.5\n\nA significant medical disease that increases the chance of developing heart, brain, kidney, and other disorders is hypertension, often known as high blood pressure. One of the “silent killers” is hypertension. Most persons with hypertension are unaware of the problem because the illness may not have any symptoms or warning signs. Some of the symptoms include early-morning headaches, nosebleeds, irregular heartbeats, changes in vision, and ear buzzing.6\n\nSince COVID-19 first appeared in China, it has spread worldwide, causing approximately 614 million illnesses and over 6.5 million fatalities. Ghana revealed its first certified case on March 12, 2020. In Ghana, there have been 171,412 confirmed cases of COVID-19 with 1,462 fatalities reported to WHO between March 12, 2020, to March 29, 2023. A total of 22,384,226 vaccine doses have been given as of March 26, 2023. It also has most of the COVID-19’s specified fundamental problems, making it particularly vulnerable to the disease. According to estimates, there are 5.27 million hypertensive individuals in the country.7 In low- and middle-income countries, where the majority (two-thirds) of those with hypertension between the ages of 30 and 79 live, the condition affects 1.28 billion people worldwide, according to WHO.\n\nResearchers have used mathematical models to investigate and analyze the dynamics of infectious illness transmission. The future outcomes of diseases and potential interventions can be predicted using models, which provide a condensed depiction of reality. Without considering the patients’ underlying medical conditions, studies on COVID-19 that have employed mathematical models have concentrated on the kinetics of transmission in a population.8–22 Fractional derivatives have been suggested for use in mathematical models to study viral diseases.18,23–30 Fractional-order models are more accurate and reliable than integer-order models because they have more degrees of freedom. Predictions can now include past data thanks to the memory property.24,31\n\nTo explore the propagation of the COVID-19, some writers have also suggested fractional-order derivatives.18,29,30,32–36 The integer-order models of differential equations don’t seem to be as consistent with this illness as the fractional-order models do. This is so that the memory and heredity qualities that are inherent in different materials and processes can be described using fractional derivatives and integrals.16 Therefore, understanding and applying fractional-order differential equations is a growing necessity. Using fractional derivative, Ogunrinde et al.37 developed a dynamic model of COVID-19 and citizens’ responses from a portion of the population in Nigeria. Aba Oud et al.34 considered the effects of isolation, containment, and ecologic factors to develop a fractional epidemic model in the Caputo sense to analyze the dynamics of the COVID-19 outbreak in Pakistan. Ahmed et al.,32 suggested a fractional-order derivative to investigate the COVID-19 transmission kinetics in Wuhan, China. A susceptible-asymptomatic-symptomatic-recovered-deceased model was put up by Giangreco33 to study the COVID19 epidemic in Italy. They developed a mathematical model of the coronavirus illness 2019 (COVID-19) to examine the disease’s transmission and control mechanisms in the Nigerian population using the Atangana-Beleanu operator. A fractional-order epidemic model with the conventional Caputo operator and the Atangana-Baleanu-Caputo operator was suggested by authors in Ref. 29 for the propagation of the COVID-19 outbreak. To analyze the behavior of the COVID-19 in Ethiopia, Habenom et al.18 developed a mathematical model of Caputo fractional-order. To account for both government action and individual response, authors in Ref. 36 used a fractional derivatives model to create a mathematical model for COVID-19 dynamics. Yadav et al.6 investigated the fractional-order Covid-19 model using the powerful and successful analytical method known as q-homotopy analysis. A more thorough use of the fractional-order derivatives for simulating infectious diseases is provided in Refs. 10, 23–25, 30, 38, 39.\n\nMost countries are at war to halt the spread of COVID-19 and as such, have resulted to vaccinating susceptible individuals. According to the Ghana Health Service, about 41.7% of the total population have had at least one dose of vaccination and 32% of the population fully vaccinated against the COVID-19. As the globe seeks to find a cure for the disease, appropriate control measures remain one of the most efficient strategies to curb its spread.15 Authors in Ref. 40 formulated an optimal control model, to study the transmission of the COVID-19. Authors in Ref. 14 created a fractional-order derivative model formulated in the Atangana-Baleanu in Caputo sense to study the COVID-19 disease’s propagation. They incorporated the best controls in the model to decrease infections and expand the population that was susceptible. Atangana-Baleanu-Caputo derivative-based fractional optimum control model was proposed by authors in Ref. 15. They considered COVID-19 treatment, public awareness campaigns on social barriers separating infected people from susceptible, and social distance between these groups of people.\n\nIn this work, we build on earlier work in Ref. 41 by introducing a segment of the population that have received first and second dose vaccination and a proportion of the population having hypertension. This work was inspired by the COVID-19 works that are currently available in this literature.21,30,41,42 The classical model proposed in Ref. 41 does not adequately capture the dynamics of the virus’ non-local behavior. The model was built using the Atangana-Baleanu and Caputo derivatives, which have several desirable properties, such as a nonlocal and nonsingular kernel, which allows a better explanation of the crossover behavior in the model employing this operator. Other operators, including Caputo and Caputo-Fabrizio, which lack these characteristics, may or may not be able to accurately characterize the dynamics of the coronavirus.9 In this inquiry, we make use of the fractional-order derivatives from Ref. 24. It is innovative in this case to simulate utilizing the fractional derivative while considering the population with first and second dose vaccination and the vulnerable with hypertension. As far as we are aware, this has never been investigated.\n\nThe other sections are the method, numerical analysis, and conclusion. The proposed model in Ref. 41 is modified to include first and second dose vaccination. Atangana-Baleanu-Caputo fractional-order derivative was used to describe the model in the method section. we also list some few definitions and determine the uniqueness of the solution. The model is solved numerically in the numerical analysis section.\n\n\nMethods\n\nThe dynamics of COVID-19 transmission in a vulnerable population with first and second dose vaccination is examined in this part using fraction-order derivatives. Utilizing Atangana-Baleanu in the sense of Caputo stated in Refs. 24, 30 the fractional-order derivative is defined. The operator for fractional orders is defined as k, where 0 < k ≤ 1. Eight classes of individuals are distinguished in the model: Susceptible HS, individuals with hypertension Hh, Exposed HE.\n\nInfected with COVID-19 HI, COVID-19 patients with hypertension HC, Individuals with first dose vaccination HV1, Individuals with second dose vaccination HV2, and Recovered individuals HR. Figure 1 displays the model’s schematic.\n\nThe system is described below using the fractional-order derivatives.\n\nHS(0) = HS0, Hh(0) = Hh0, HE(0) = HE0, HI(0) = HI0, HC(0) = HC0, HV1(0) = HV1, HV2(0) = HV2, HR(0) = HR0.\n\nIn this part, the general fractional derivatives definitions concerning Atangana-Baleanu-Caputo is given.\n\nDefinition 1: Liouville and Caputo (LC) definition as in Refs. 24, 28\n\nDefinition 2: Let h∈T1x1x2,x2>x1∈01, then, the new Caputo fractional derivative as stated in Ref. 43 is:\n\nIf we let ζ=1−kk∈0∞ then =11−ζ∈01, the above equation assumes the form\n\nDefinition 3: Let h∈T1x1x2,x2>x1∈01, then, the new fractional derivative as defined by Ref. 43 is given as:\n\nLaplace transform of equation (6) is given by\n\nWhen k = 0, we do not recover the original function except when at the origin, the function vanishes. To avoid this issue authors in Ref. 43 proposed the following definition\n\nDefinition 4: Let h∈T1x1x2,x2>x1∈01, then, the new fractional derivative as defined by Ref. 43 is given as:\n\nWhen the function h(x) is constant, in equation (6), we get zero. The Laplace transform of equation (8) as given in Ref. 43 is:\n\nLet G(T) denote a banach space, where T = [0, b], and M=GT×GT×GT×GT×GT×GT×GT×GT with the norm HSHhHEHIHCHV1HV2HR=HS+Hh+HE+HI+HC+HV1+HV2+HR, where HS=Supπ∈THS,Hh=Supπ∈THh,HE=Supπ∈THE,HI=Supπ∈THI,HC=Supπ∈THC,HV1=Supπ∈THV1,HV2=Supπ∈THV2,HR=Supπ∈THR. Using the Atangana-Baleanu-Caputo integral operator on the system (1), we have\n\nApplying Liouville and Caputo (LC) definition of fractional-order derivatives gives,\n\nThe ABC derivatives only meets the Lipschitz requirement44,45 if\n\nHSt,Hht,HEt,HIt,HCt,HV1t,HV2t,HRt, have an upper bound.\n\nWe assumed that HSt and HS∗t are paired functions,\n\nConsidering\n\nEquation (13) simplifies to\n\nSimilarly,\n\nLipschitz condition is now valid. Now repeatedly applying system (10) gives,\n\nDifference of consecutive terms yields\n\nHSnt=∑i=0nΩHSnt,Hhnt=∑i=0nΩHhnt,HEnt=∑i=0nΩHEnt,HInt=∑i=0nΩHInt,HCnt=∑i=0nΩHCnt, HV1nt=∑i=0nΩHV1nt,HV2nt=∑i=0nΩHV2nt,HRnt=∑i=0nΩHRnt. Taking into consideration equations (15)–(16) and considering ΩHSn−1t=HSn−1t−HSn−2t,ΩHhn−1t=Hhn−1t−Hhn−2t, ΩHEn−1t=HEn−1t−HEn−2t,ΩHIn−1t=HIn−1t−HIn−2t,ΩHCn−1t=HCn−1t−HCn−2t, ΩHV1n−1t=HV1n−1t−HV1n−2t,ΩHV2n−1t=HV2n−1t−HV2n−2t,ΩHRn−1t=HRn−1t−HRn−2t,\n\nTheorem: The system (1) has a unique solution for t∈0b subject to the condition 1−kBkTi+kBkΓkbkni<1,i=1,2,3,.……,8 hold.15\n\nProof: Since HSt,Hht,HEt,HIt,HCt,HV1t,HV2t,HRt, are bounded functions and Equation (15)−(16) holds.\n\nΩHSt→0,ΩHht→0,ΩHEt→0,ΩHIt→0,ΩHCt→0,ΩHV1t→0,ΩHV2t→0 ΩHRt→0 as n→∞. Incorporating the triangular inequality and for any j, system (21) yields\n\nIn this section, we numerically investigate the behaviour of (1) using the Matlab program ODE45 Runge Kutta Method. The model is parameterized to investigate disease burden in a vulnerable population (specifically a population with hypertension) with double dose vaccination within the period February 24, 2021, to July 24, 2021 (from the beginning of the vaccination program in Ghana) (UNICEF). The 2021 Population and housing census revealed Ghana’s population is 30.8 million (Ghana Statistical Service), and 5.27 million people estimated to have hypertension.7 On this day, February 24, 2021, Ghana had 81,245 cumulative cases of COVID-19, with total deaths being 584 and 6,614 active cases. The recoveries stood at 74,047 (Worldometer). First and second doses had not started yet after receiving the vaccines. The first dose was rollout on March 2, 2021 (WHO) and second dose on May 7, 2021 (Ghana Health Service) which falls within the stipulated time. We assumed that the number of individuals exposed to the COVID-19 is twice the number of active cases. The number of hypertensive patients infected with COVID-19 is not known at the period, so we assume to be zero. Using the initial conditions N(0) = 30,800,000, Hh(0) = 5,270,000, HE(0) = 2×6614 = 13228, HI(0) = 6614, HC(0) = 0, HV1(0) = 0, HV2(0) = 0, HR(0) = 74047, HS(0) = N(0) − Hh(0) − HE(0) − HI(0) − HV1 − HV2 − HR(0) = 25436111, and the parameter values given in Table 1, the results of the simulation are displayed in Figures 2–9 for the period of 150 days within the stipulated period.\n\n\nDiscussion\n\nThe resulting solutions of the system (1) for 5 different values of k ∈ [0,1] at step-size 0.2 are shown in Figures 2 through 9. The figures denote the susceptible, susceptible with hypertension, exposed, infected, COVID-19 infected individuals with hypertension, recovered, first- and second dose vaccination compartments, respectively. With the introduction of vaccines into the model, it can be observed that the number of vulnerable individuals drastically reduces within the first 50 days for all the operator values (see Figures 2 and 6). The integer model (k = 1.0), shows the number of exposed, infected, and COVID-19 infected patients with hypertension reaching their highest peak, i.e., 150,000, 110,000, and 220,000 respectively, in the first 25 days (see Figures 3–5). On the other hand, a reduction in the fractional operator value revealed the number of exposed individuals reaching an early peak and lowest minimum even before the first ten days. The dynamics of the first and second dose vaccinations are depicted in Figures 8 and 9 respectively. A lower fractional operator value reveals a higher number of individuals receiving the first dose in the first five days. This number declines steadily thereafter. There is an increase in the number of recovered individuals and those receiving the second dose for the first fifty days (see Figures 7 and 9). This accounts for the reduction in the vulnerable population. The fall in the number of recoveries and individuals with the second dose may be due to the loss of immunity in those compartments.\n\n\nConclusion\n\nIn this study, the fractional-order derivative defined in the Atangana-Baleanu in the Caputo sense has been used to investigate a COVID-19 model that considers a population with hypertension as a primary condition and with double dose vaccination. We investigated, the solutions’ existence and distinctiveness. The numerical simulation showed different compartment dynamics for both integer and non-integer values of the fractional operator. The dynamics of the disease in the community were shown to be influenced by fractional-order derivatives. Contrary to the previous model proposed in Ref. 41, the vulnerable group (susceptible and susceptible with hypertension) saw a significant reduction in the number, which may be attributed to the double dose vaccination. We recommend a cost-effective analysis and optimal control model in future work.", "appendix": "Data availability Statement\n\nThe Ghana Health Service website, https://www.ghs.gov.gh/covid19, provided the data and information used to develop the mathematical model in this paper. This has been referenced in the text as. 50\n\nOSF: Output_data DOI:10.17605 / OSF.IO / K5PJ7 This project contains the following extended data:\n\n‐ MATLAB 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\nAcknowledgement\n\nThe reference 51 has an old version of this work which was submitted as pre-print.\n\n\nReferences\n\nCenters for Disease Control and Prevention: Underlying Medical Conditions Associated with Higher Risk for Severe COVID-19: Information for Healthcare Providers.2021. Reference Source\n\nGleason J, Ross W, Fossi A, et al.: The devastating impact of Covid-19 on individuals with intellectual disabilities in the United States. NEJM Catal. Innov. 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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. International Journal of Mathematics and Mathematical Sciences. 2022; 2022: 1–15. Article ID 7984818. Publisher Full Text\n\nPeter OJ, Panigoro HS, Abidemi A, et al.: Mathematical Model of COVID-19 Pandemic with Double Dose Vaccination. Acta Biotheor. 2023; 71(2): 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtangana A, Baleanu D: New fractional derivatives with non-local and non-singular kernel: Theory and application to heat transfer model. Therm. Sci. 2016; 20: 763–769. Publisher Full Text\n\nWorldometer: COVID-19, Coronavirus pandemic. Updated January 13, 2022. Reference Source\n\nColl C, Herrero A, Sanchez E, et al.: A dynamic model for a study of diabetes. Math. Comput. Model. 2009; 50(5–6): 713–716. Publisher Full Text\n\nIndexmundi: Ghana Birth rate, 2021. (accessed 11th December, 2021). Reference Source\n\nKouidere A, Labzai A, Ferjouchia H, et al.: A New Mathematical Modeling with Optimal Control Strategy for the Dynamics of Population of Diabetics and Its Complications with Effect of Behavioral Factors. J. Appl. Math. 2020; 2020: 1–12. Article ID 1943410. Publisher Full Text\n\nStokes EK, Zambrano LD, Anderson KN, et al.: Coronavirus disease 2019 case surveillance – United States, January 22–May 30, 2020. Morb. Mortal. Wkly. Rep. 69(2020): 759–765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang C, Wang J: COVID-19 and underlying health conditions: A modelling investigation’. Math. Biosci. Eng. 2021; 18(4): 3790–3812. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhana health service: COVID-19 cases. (Accessed: 20th November, 2021). Reference Source\n\nOkyere S, Prah JA, Bonyah E, et al.: A model of COVID-19 with underlying health condition using fraction order derivative. arXiv:2201.11659 [q-bio.PE]. Reference Source" }	[ { "id": "200955", "date": "05 Oct 2023", "name": "Khadija Tul Kubra", "expertise": [ "Reviewer Expertise Mathematical Modelling", "Heat and Mass transfer", "Numerical and analytical solutions of ODEs and PDEs. Fluid mechanics", "Numerical Simulations", "Geophysics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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's abstract reads well. The research, however, does not support the viewpoint emphasized in the abstract. Upon reading the abstract, one may find it interesting and persuasive. However, upon delving into the research itself, it becomes evident that the evidence presented does not align with the viewpoint highlighted in the abstract. The abstract may provide a concise and enticing overview of the topic, drawing readers in with its intriguing claims. Conversely, a closer examination of the research reveals that the data and analysis presented do not substantiate the assertions made in the abstract. It is important to critically evaluate the research itself rather than solely relying on the initial impression created by the abstract. This disconnect raises questions about the accuracy and validity of the claims made in the article.\n\nIn the abstract, the researcher made this claim \"The study modifies a previous model proposed in the literature to include double dose vaccination and Atangana-Baleanu-Caputo fractional derivatives is used to solve the model\", however, this research work shows no discernible modification. The lack of modification in the research work raises concerns about the credibility of the researcher's claim in the abstract. Without any visible changes to the model or inclusion of Atangana-Baleanu-Caputo fractional derivatives, it is unclear how the study supports or contributes to existing literature. This discrepancy calls into question the overall reliability and integrity of the article's findings.\n\nIn the abstract, the researcher made this claim \"By using COVID-19 data for the period February 21, 2021 to July, 24 2021, the model is tested\". The research, however, does not provide any such verified findings. This lack of tested results undermines the credibility of the study and raises doubts about the validity of the model. Without empirical evidence to support the claims made in the abstract, it becomes difficult to assess the practical implications and significance of the research.\n\nIn the KEYWORDS section, the researcher uses the term \"Basic Reproduction Number\". However, the proposed model does not include a calculation for the Basic Reproduction Number. Not even the researcher attempts any sort of qualitative analysis of the model's results. This work would be worthless without the inclusion of a qualitative analysis of the epidemic model. Without the inclusion of a calculation for the Basic Reproduction Number and the absence of any qualitative analysis of the model's results, the researcher's claim regarding the testing of the model using COVID-19 data becomes questionable. The lack of these crucial components significantly diminishes the value and reliability of the research findings.\n\nThe introduction is written very poorly. The introduction lacks clarity and fails to provide a concise overview of the research topic. It lacks a clear article's statement and fails to establish the significance of the study. Additionally, the introduction does not effectively capture the reader's attention or provide a compelling rationale for why the research is important. This weak start may undermine the reader's confidence in the overall quality of the article.\n\nNot even the researcher makes any attempt at a qualitative analysis for the model. Not equilibria, basic reproduction number and their stability, senstivity of parameters any of these. This lack of qualitative analysis is a significant limitation as it prevents a deeper understanding of the model's behavior and its implications. Without this analysis, it becomes difficult to assess the model's applicability and relevance to real-world scenarios. Additionally, the absence of sensitivity analysis limits the researchers' ability to explore the robustness of their findings and evaluate the potential impact of different parameter values on their results. There is no value to the work done if only the symbols are changed, so the qualitative analysis is essential.\n\nIn the Numerical Analysis section, the researcher claims that \"They numerically investigate the behaviour of (1) using the Matlab program ODE45 Runge Kutta Method\". In that case, why do they propose the ABC scheme for the model if numerical investigations are performed using the ode45 function of Matlab? In other words, this directly contradicts the researcher's abstract's main statement. It is important to note that while the researcher claims to numerically investigate the behavior of (1) using the ode45 function, it does not necessarily mean that this method is the most suitable or accurate for the given model. The proposed ABC scheme may offer alternative advantages such as improved accuracy, efficiency, or robustness compared to ode45. Therefore, it is crucial for the researcher to provide a clear justification for why they propose the ABC scheme despite conducting numerical investigations with ode45. No any clear connection between discussion, conclusion with the point of view offered in abstract. It is important for the researcher to establish a clear connection between their discussion, conclusion, and the point of view offered in the abstract. This ensures that the research findings are coherent and align with the overall objectives of the study. It is important for the researcher to address this discrepancy and provide a comprehensive explanation that ties all aspects of the study together.\nDue to the aforementioned gaps, I cannot recommend accepting your article for indexing. The researcher should revise their work to ensure a seamless flow of information and a strong connection between all sections. Additionally, they should provide a comprehensive explanation that effectively ties together all aspects of the study, addressing any discrepancies that may arise. Therefore, it is crucial for the researcher to revise and strengthen these aspects before resubmitting the article for indexing consideration.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": [] }, { "id": "214799", "date": "27 Oct 2023", "name": "Mohammadi B Jeelani Shaikh", "expertise": [ "Reviewer Expertise Mathematical modeling", "Fractional calculus", "Differential equation", "Numerical Analysis", "Approximation theory" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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) make a research article on“Hypertension and COVID-19 fractional derivative model with double dose vaccination”. The article yields interesting findings, and the material is appropriate for this journal. The list below includes a few observations. I can support its indexing, however I advise considerable revisions before it is indexed.\nThe entire document needs to have its grammatical and typographical errors fixed. Authors ought to edit it.\n\nCould you all provide a more specific research question or hypothesis that your study aims to address regarding the dynamics and treatment strategies with double dose vaccination. (specifically)\n\nThe writers need to remark on the results more effectively. The observed patterns should have a plausible physical explanation in addition to reporting what can be seen graphically in the figures. There needs to be more physical understanding of the discussion part.\n\nWhat is the aim of this review? The novelty is not mentioned in abstract.\n\nHow does the use of Atangana-Baleanu-Caputo fractional derivative enhance the model's ability to capture the complexity of covid model dynamics compared to conventional derivatives?\n\nThe text lacks any validation of the numerical results. Include a comparison of the results to earlier publications. Additionally, it needs to be emphasized in the abstract.\n\nAt the end of introduction section, research questions that highlight the research gap and novelty of the proposed problem.\n\nThe range of defined parameters needs to be added.\n\nWrite the conclusion more precise, It should highlight the novel finding in the current study. It should be point wise.\n\nIn practical terms, how might the insights from your model influence clinical decision-making or the development of new therapeutic approaches\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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-495
https://f1000research.com/articles/12-215/v1	27 Feb 23	{ "type": "Research Article", "title": "Efficacy of Moringa oleifera Lam. extracts and Pediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillus plantarum probiotic during starter period on growth performance of male broiler chicken", "authors": [ "Novita Wanda Karwanti", "Dynda Febriana Arumdani", "Andreas Berny Yulianto", "Tabita Dameria Marbun", "Anjum Sherasiya", "Mohammad Anam Al Arif", "Mirni Lamid", "Widya Paramita Lokapirnasari", "Novita Wanda Karwanti", "Dynda Febriana Arumdani", "Andreas Berny Yulianto", "Tabita Dameria Marbun", "Anjum Sherasiya", "Mohammad Anam Al Arif", "Mirni Lamid" ], "abstract": "Background: Probiotics and medicinal plants have been used to support human and livestock health. This research aimed to evaluate the potential of Moringa oleifera Lam. leaf extract on the growth of Pediococcus pentosaceus, Lactobacillus acidophilus and L. plantarum probiotic in vitro and to prove its potential as a feed additive, as an alternative to antibiotic growth promoters (AGP) to improve nutrient intake, body weight gain, feed conversion ratio and feed efficiency in broiler chicken starter phase. Methods: This study consisted of three sub studies: (1) Screening test for phytochemical compounds; (2) evaluation of several doses of Moringa extract (0%, 0.1%, 0.2%, 0.3% ). At the end of treatment, each colony was measured using the total plate count; (3) evaluation of probiotics and M. oleifera in vivo to prove growth performance on starter-phase broiler chicken. All results were analyzed by analysis of variance (ANOVA) then followed by the Duncan test. Results: (1) The phytochemical screening test M. oleifera extract contained flavonoids, saponins, tannins, triterpenoids and alkaloids; (2) M. oleifera extract at doses of 0.1%, 0.2%, 0.3% increased the growth of bacteria Pediococcus pentosaceus, Lactobacillus acidophilus and Lactiplantibacillus plantarum (p<0.05); (3) The use of probiotics, M. oleifera extract and their combination showed that body weight, body weight gain, feed conversion rate (FCR) and feed efficiency signifcantly differed (p <0.05) between the treatments, but there was no significant difference (p > 0.05) between the treatments on nutrient intake (dry matter, ash, crude protein, crude fiber and organic matter) on broiler chicken starter phase. Conclusions: The use of M.oleifera extract at doses of 0.1%, 0.2% and 0.3% increased the growth of P. pentosaceus, L. acidophilus and L. plantarum bacteria in vitro and the use of probiotics, M.oleifera extract and their combination by in vivo improved the growth performance on starter phase of broilers chicken.", "keywords": [ "Moringa oleifera extract", "public health", "probiotic", "growth performance" ], "content": "Introduction\n\nFeed comprises ingredients provided to poultries to meet necessary dietary requirements for livestock growth, development, and reproduction. Breeders administer antibiotic growth promoters (AGP) to improve production, trigger growth, and act as an antibacterial.1 However, the use of these growth promoters in animal production is gradually being restricted and banned in some countries. One antibiotic substitution which can be used in feed is probiotics.\n\nPhytobiotics are feed additives derived from pure plant materials. Phytobiotics are able to control micro-organisms in the digestive tract of poultry. Phytobiotics are able to increase metabolic activities in the body, so these phytobiotics can be used as feed additives in poultry. Phytobiotics have proven to have several functions, such as a growth-promoting effect, antimicrobial activity, anti-inflammation activity and improving performance.2–5 They allows to control micro-organism growth in the digestive tract of poultry by increasing the metabolic activities in the body, making them potential feed additives in poultry.\n\nPediococcus pentosaceus is a lactic acid bacteria (LAB). LABs are the bacterial group that ferments carbohydrates into lactic acid.6 Probiotics are microorganisms capable of improving growth and feed efficiency. Several probiotics improving production performance in broilers are P. pentosaceus, Bifidobacterium sp, Lactobacillus casei, and Lactobacillus acidophilus.7–12 P. pentosaceus is a Gram-positive bacteria, round-shaped, non-motile, not generating spores, and has negative catalase. As it grows, P. pentosaceus generates lactic acid and pediocin.13 Pediocin is a bacteriocin generated by P. pentosaceus, which, in a sufficient amount, can eliminate pathogenic bacteria such as Listeria monocytogens, Staphylococcus aureus, Escherichia coli, Vibrio alginolyticus, Pseudomonas stutzeri, and Aeromonas. P. pentosaceus bacterial growth requires nutrition with carbon, nitrogen, and mineral sources.\n\nMoringa oleifera belongs to the family Moringaecea. M. oleifera is very useful as a feed supplement for animals, as its leaves are highly nutritious. Moringa leaf carbohydrates reach 38.2 g for 100 g of Moringa leaf powder.14 Moringa also contains proteins, fats, fibers, calcium, magnesium, phosphorus, potassium, copper, iron, vitamin B1, vitamin B2, vitamin B3, vitamin C, vitamin E, and essential amino acids, e.g., arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and cysteine.15 Probiotics containing L. acidophilus, L. casei, L. lactis, and Bifidobacterium spp. and M. oleifera extract can increase the production performance of Peking ducks.16\n\nMoringa leaves are antioxidant, antibacterial, anti-inflamatory,17 and are rich in fats, proteins, vitamins, and minerals. Moringa leaves contain diversified phytochemicals such as flavonoids, saponins, tannins, phenols and alkaloids.17,18 The high-nutrient profile of Moringa suggests potential growth promoter and immunomodulatory effects.19 Moringa can be used as a source of micronutrients and as a dietary supplement in poultry.20\n\nNutrition consumed by poultry is used for maintenance and production. Feed efficiency in broilers is influenced by the level of feed consumption and body weight gain. High feed efficiency is produced due to low feed intake followed by a high rate of body weight gain. A high level of feed efficiency can reduce livestock production costs.\n\nThe study aimed to analyze the phytochemical properties of Moringa leaf extract and the ffectiveness of M. oleifera extract with different dosages and incubation times on in vitro bacterial growth of P. pentosaceus, L. acidophilus and L. plantarum probiotic, as well as their effect on growth performance on broiler chicken starter phase in vivo.\n\n\nMethods\n\nThis study was conducted in the Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya. The trial design was a complete randomized design consisting of four treatments: P0, P1, P2, and P3, with five replications.\n\nMoringa was extracted by mixing macerated moringa leaf with ethanol 70% and 96% as the solvent. Extraction with ethanol 70% can extract oligosaccharides.21 The phytochemical screening test was carried out in the form of the flavonoid test, tannin test, saponin test, triterpenoid test and alkaloid test.\n\nBacterial cell count measurement was conducted to measure the bacterial cell count of P. pentosaceus, L. acidophilus and L. plantarum before adding Moringa leaf extracts. A bacterial suspension comparison was performed using the McFarland 0.5 scale standard. After obtaining the same turbidity, a gradual dilution was conducted up to seven times to obtain a 101 bacterial concentration.\n\nA sterile test tube was filled with Aquadest 9 mL mixed with Moringa leaf extracts with the following treatments: P0: Without M.oleifera extracts; P1: 0.1% M.oleifera extracts; P2: 0.2% M. oleifera extracts; P3: 0.3% M. oleifera extracts. Furthermore, each test tube was added with 1 mL of each isolate and incubated for 24 h at 37°C, after each isolate bacteria was grown in the Agar MRS media using the pour plate method and incubated for 12 and 17 h. The growth of probiotic bacteria was calculated for the colonies.\n\nThe study material consisted of probiotics from W.P. Lokapirnasari’s and A.B.Yulianto’s collections and M. oleifera extract in drinking water. The commercial feed contained dry matter (86.00%), crude lipid (8.00%), ash (7.00%), crude protein (20.00 %), crude fiber (5.00%) and organic matter (93.00%). This study used a completely randomized design, using 100 day-old chicks divided into five treatments and ten replications; each replication contained two chicken. The treatments in this study were as follows: T0 = control, without feed additive; T1 = 1% P. pentosaceus; T2 = 1% L. acidophilus and L. plantarum; T3 = 0.5% P. pentosaceus + 0.5% L. acidophilus and L. plantarum; T4 = 0.5% P.pentosaceus + 0.5% L. acidophilus and L. plantarum + 1% M. oleifera extracts. The observed variables included dry matter intake, organic matter intake, ash intake, crude protein intake, ether extract intake, crude fiber intake, organic matter intake, feed conversion ratio and feed efficiency. All variables were calculated with the following7,22–24:\n\nThis research has obtained approval from the Ethics Review Team Brawijaya University through a letter from the Head of the Ethics Review Team number 057-KEP-UB-2020. The treatment in this study used probiotics and herbal extracts as feed additives which are safe to use and are thought to have positive and beneficial properties and to not cause pain in experimental animals. Variable taking does not slaughter experimental animals.\n\nAll data collected during this study were statistically analyzed under a completely randomized design. All data were tested for distribution normality and homogeneity statistics (analysis of variance). The data were analyzed using Statistical Design for Social Sciences (SPSS) v.22 (IBM Corp., NY, USA). Differences among means were detected using a one-way analysis of variance. The differences among means were determined using Duncan's test (p < 0.05).\n\n\nResults\n\nMoringa leaf extract was tested for its chemical composition using a phytochemical screening test. At this stage, five kinds of tests were carried out: flavonoid test, tannin test, saponin test, triterpenoids test, and alkaloid test. The results of the phytochemical screening tests are presented in Table 1.\n\nThe growth results of the probiotics P. pentosaceus, L. acidophilus and L. plantarum with several levels of M. oleifera extract showed significant differences between the treatments (p < 0.05) (Table 2, Figure 4).\n\nThe result of feed intake and nutrient intake of dry matter, ash, crude protein, crude fat, crude fiber and organic matter listed in Table 3. There was no significant difference (p > 0.05) between the treatments of feed intake, dry matter intake, ash intake, crude protein intake, crude fat intake, crude fiber intake, and organic matter intake in this study.\n\nThe result of body weight, daily body weight gain, feed conversion ratio, and feed efficiency are listed in Table 4. Body weight differed significantly (p < 0.05) between treatments. The control (T0) had the lowest body weight, which was different from all treatments. The average daily weight gain differed significantly (p < 0.05) between treatments. The control (T0), which differed from all treatments, had the lowest body weight gain. The FCR results revealed a significant difference (p < 0.05) between treatments. The feed efficiency results revealed a significant difference (p < 0.05) between treatments. T0 had the lowest feed efficiency value found.\n\n\nDiscussion\n\nThe phytochemical screening revealed flavonoids, tannins, saponins, terpenoids, and alkaloids which could serve as a natural source of antimicrobials. The results obtained from the flavonoid test were a change in color. There was a change in color to orange (Figure 1A). The orange to red color change is caused by the formation of flavilium salts. This indicates that the sample contained flavonoids.\n\nIn the tannin test, a positive result is indicated by a change in the color of the filtrate to green or blackish blue. In this test, the color of the filtrate changed to a dark green-black color (Figure 1B), so the sample was considered test-positive for tannin. The test for the presence of saponin was indicated by the appearance of foam. The foam indicated the presence of glycosides which have the ability to form froth in water as they hydrolyze into glucose and other compounds. In the saponin test carried out, foam formed for approximately 10 minutes; with the addition of one drop of hydrochloric acid, the foam did not disappear and the sample tested positive for saponin (Figure 1C).\n\nThe addition of concentrated sulfuric acid causes water to hydrolyze, which reacts with acetyl derivatives to form a red ring. A positive test for the presence of triterpenoids is indicated by a color change to red or purplish red. In the test conducted, the color changed to red (Figure 2), therefore the sample tested positive for triterpenoid.\n\nThe alkaloid test in this study used three reagents: Mayer’s reagent, Bouchardat’s reagent and Wagner’s reagent. The test with Mayer's reagent showed no white precipitate (Figure 3A), so the result is negative. Withthe Bouchardat reagent, a precipitate appeared and the color changed to orange (Figure 3B), therefore the result is positive. With Wagner's reagent, a brown precipitate appeared (Figure 3C). The precipitate was indicated as a potassium-alkaloid. Of the three reagents used, two of them were positive, so it can be concluded that Moringa leaves contains alkaloids.\n\nThe lowest growth of P. pentosaceus, L. acidophilus and L. plantarum was found for the treatment without M. oleifera extract (P0), which was different from all treatments. The fastest growth of P. pentosaceus was found with the addition of 0.2% M. oleifera extract (P2), followed by 0.3% M. oleifera extract (P3) and 0.1% M.oleifera extract (P1). The highest growth of L. acidophilus was shown with the addition of 0.3% M. oleifera extract (P3), followed by the addition of 0.2% M. oleifera extract (P2) and 0.1% M. oleifera extract (P1). The highest growth of L. plantarum was shown in the 0.3% M. oleifera extract (P3) treatment, which was not different from 0.2% M.oleifera extract (P2), while P2 was not different from 0.1% M. oleifera extract (P1) (Table 2).\n\nBacterial growth of P. pentosaceus L. acidophilus and L. plantarum was significantly improved after the addition of 0.1%, 0.2%, and 0.3% M. oleifera extract. This shows that the higher the M. oleifera extract concentration, the faster the bacterial growth of P. pentosaceus, L. acidophilus and L. plantarum. Microbial count difference was determined by the fermentation duration and nutrition availability. Primary nutrition required by P. pentosaceus, L. acidophilus and L. plantarum bacteria were carbon and nitrogen sources. Bacteria use carbon sources as the energy source and produced lactic acid while nitrogen is used to generate bacterial cell biomass.25\n\nM. oleifera extract contains the monosaccharides mannose, arabinose, xylose, and the oligosaccharides raffinose and stachyose.26 These oligosaccharides can be used as nutrition for bacterial growth.27 P. pentosaceus bacteria is an amylolytic bacterium that generates amylase and can hydrolyze carbohydrates into glucose. Glucose is a carbon source for bacteria as the energy fueling their growth and lactic acid formation. P. pentosaceus bacteria use oligosaccharides and monosaccharides in Moringa leaf as the energy source for fermentation.28\n\nP. pentosaceus is also classified into the proteolytic lactic acid bacterial group. Proteolytic bacteria are those capable of generating the protease enzyme to hydrolyze polypeptides in media into amino acids. Nitrogen is a constituting component of amino acids. Thus, bacteria can use amino acids as a nitrogen source for bacterial growth and DNA/RNA synthesis. Bacterial growth of P. pentosaceus on Agar MRS media ferments carbohydrates from Moringa leaf extracts into glucose and generate lactic acid. Metabolites of lactic acid are generated from glucose fermentation through glycolysis. P. pentosaceus metabolizes glucose into produce pyruvic acid through the Embden-Meyerhof pathway, which is then reduced into lactic acid by the lactase dehydrogenase enzyme and nicotinamide adenine dinucleotide phosphate (NADP).\n\nHigh bacterial growth followed the high lactic acid concentration on the fermentation media. The pH reduction and total acid level increase on the fermentation media is beneficial since it hinders pathogenic microbes. The growth of P. pentosaceus bacteria produces pediocin, a bacteriocin that inhibits the growth of pathogenic bacteria.8 P. pentosaceus incubated with M. oleifera extract could be used to improve the nutritional quality of rice bran.29\n\nM. oleifera extract’s beneficial effects as phytobiotics are considered to be due to their antioxidant properties. Phytochemical compounds such as flavonoids, saponins and tannins, exhibit antimicrobial activity.30 The phytochemical screening results in this study revealed flavonoids, tannins, saponins, triterpenoids, and alkaloids.\n\nThe feed intake in this study showed that there was no significant difference (p > 0.05) between the treatments with values 97.12-104.63 (g/chicken/day). The dry matter intake showed that there was no significant difference (p > 0.05) between the treatments with values 83.52-89.98 (g/chicken/day). The results of ash intake in this study showed that there was no significant difference (p > 0.05) between treatments with values 6.79-7.32 (g/chicken/day). The results of crude protein intake showed no significant difference (p > 0.05) between treatments with values 19.42-20.92 (g/chicken/day). The results of crude fat intake showed no significant difference (p > 0.05) between treatments with values 7.76-8.37 (g/chicken/day). The results of crude fiber intake showed no significant difference (p > 0.05) between treatments with values 4.85-5.23 (g/chicken/day). The organic matter intake results in this study showed no significant difference (p > 0.05) between the T0, T1, T2, T3 and T4 treatments with values 90.31 – 97.30 (g/chicken/day) (Table 3).\n\nBody weight was significantly different (p < 0.05) between the treatments. The lowest body weight was found in the control (T0), which was different from all treatments. For T1, T2, T3 and T4, there was no significant difference (p > 0.05), with values between 899.60-949.10 (g/chicken) in starter-phase broiler chickens.\n\nAverage daily weight gain was significantly different (p <0.05) between the treatments. The lowest body weight gain was found in the control (T0), which was different from all treatments. At T1, T2, T3 and T4, there was no significant difference (p > 0.05), with values between 62.52-70.60 (g/chicken/day).\n\nThe feed conversion ratio (FCR) results showed that there was a significant difference (p < 0.05) between the treatments. FCR values for T1, T2, T3 and T4 showed an improvement compared to the control (T0). The best FCR values were found for T2 (1.41) and T4 (1.48), which differed from all treatments. The FCR values at T1 and T3 showed no significant difference (p > 0.05), namely 1.60 and 1.65.\n\nThe feed efficiency results showed that there was a significant difference (p < 0.05) between the treatments. The lowest feed efficiency value was found for T0 (46.57%). The feed efficiency values for T1, T2, T3 and T4 showed an improvement compared with the control (T0). The best feed efficiency values were shown for T2 (71.30%) and T4 (70.26%), which were different from all treatments. The value of feed efficiency at T1 (62.92%) and T3 (60.45%) showed no significant difference (p > 0.05) (Table 4).\n\nThe results of this study are in accordance with other studies that showed the administration of Lactobacillus probiotic can improve gut health and contributes to increased growth performance.31 Probiotics play a role in modulating the balance of microbiota in the digestive tract and in developing intestinal health so that they affect feed consumption, digestibility, absorption of nutrients and improve feed conversion ratio and feed efficiency. The use of probiotics in poultry also plays a role in modulating the immune response, maintaining the health of the intestinal tract and influencing stress reduction.32–34\n\nOther studies have shown that the use of L. acidophilus poses an obstacle to the invasion of pathogenic bacteria and modulates the immune response in vitro and in vivo. This is because L. acidophilus produces bacteriocin. Additionally, probiotics that are classified as lactic acid will produce acid thereby lowering the pH in the intestine. In poultry, the use of L. acidophilus can balance the intestinal microflora and reduce the proliferation of pathogenic bacteria through antagonism and competitive exclusion.31,35 Protein availability can be increased by taking probiotics. Probiotics can improve the host’s health by increasing intestinal villi size and nutrient uptake.36\n\nThe use of probiotics can modify the intestinal ecosystem by decreasing pH through acid production by lactic acid probiotic bacteria and modulating enzyme activation in the digestive tract.37,38 Several factors can affect the colonization of probiotic microbes in the intestine depending on the availability of fermented substrates (prebiotics), pH in the intestine, frequency and dosage of probiotics, genetics, age, health, stress factors and the nutritional status of the host.39,40 This relates to the ability of probiotic micro-organisms to secrete enzymes such as protease, lipase, and amylase to help digest proteins, fats, and starch so that they can increase the availability of nutrients and help increase the digestion of feed nutrients.41\n\nM. oleifera extract contains phenolic compounds in insoluble and soluble bound forms. The bound phenolic compounds cannot be absorbed by the small intestine because they are bound by insoluble macromolecules such as cellulose, hemicellulose, structural proteins and pectin; these compounds enter the large intestine (colon), a fermentation by the intestinal microbiota occurs, which releases bound phenolic compounds. The released phenolic compounds can decrease pH, inhibit the growth of pathogenic bacteria and modulate the development of fermentative microflora so that it can improve host health.42,43 Moringa has no toxic effects and does not interfere with nutrient absorption.44\n\nIn this study, giving probiotics, M. oleifera extract and a combination of probiotics and M. oleifera extract during the starter phase of broiler chicken increased body weight and average daily weight gain, increased feed efficiency and improved feed conversion compared with control. This is because the addition of probiotics to poultry can modulate the balance of microbiota in the intestine, increasing feed digestibility and nutrient absorption. This is also influenced by the viability requirements of probiotics, where probiotics must be able to survive and not be damaged during processing to storage. The addition of probiotics increases nutrient bioavailability and improves poultry feed conversion. Probiotics must be able to survive various stress factors during processing, storage and digestion. Several different probiotic strains can exhibit wide variations in their abilities, such as efficacy as probiotics, stability of process and functional properties at the targeted site.45\n\nThe phytoconstituents present in M. oleifera are flavonoids, niazirinin, niazirin, proanthocyanidin, anthocyanins, kaempferol-3-O-(6”-malonyl-glucoside), β-sitosterol, β-sitosterone, 4-hydroxymellein, octacosanic acid.46 M. oleifera with ethanol extraction has a strong antioxidant activity.47 Hydro-alcoholic extract of Moringa leaves contain 21.1% polysaccharides (% of dry matter).44\n\nThe addition of a combination of probiotics and M. oleifera extract also resulted in better body weight gain, FCR and feed efficiency than the control. This is due to their proanthocyanidins content, which are condensed tannins (epicatechins and catechins) found in plants that provide protection against biotic and abiotic stresses, and act as antimicrobial, antioxidant, anti-inflammatory, vascular and cardiac activity.48–50\n\nThe use of M. oleifera extract improved FCR and increase feed efficiency due to the presence of secondary metabolites produced by the plant, which also play a role in giving flavor to feed, and are influenced by their ability to form complexes with macromolecules (polysaccharides and proteins) and metal ions.50,51 The use of probiotics and M. oleifera extract for feed efficiency agrees with Nikpiran et al. (2013)52 who found a better FCR with the addition of prebiotics to broiler rations compared to controls. This is thought to be related to the availability of substrates and the presence of a better balance of microbiota in the gut, resulting in better feed efficiency.53,54 The finding of increased average daily gain and improvement of FCR in this study by administering M. oleifera leaf extract to broiler chickens agrees with a previous study where the FCR value obtained was 1.41–1.65 and the average daily gain 62.52–70.60 g, while research by Akhouri (2013) using M. oleifera extract in starter phase broilers produced an FCR of 1.77 and average daily gain of 52.57 g.55 The results of our study are also in line with Sjofjan's (2021) that the level of probiotics increased body weight gain, body weight, feed intake of broilers and improved FCR.56\n\n\nConclusions\n\nThe phytochemical tests showed flavonoids, tannins, saponins, tripenoids, alkaloids. The use of M. oleifera extract at doses of 0.1%, 0.2%, and 0.3% increased the growth of P. pentosaceus, L. acidophilus and L. plantarum bacteria in vitro. The use of probiotics, M. oleifera extract and a combination of probiotics + M. oleifera extract used in vivo can improve the growth performance of starter-phase broilers chicken. Based on these results, the use of probiotics, M. oleifera extract or combination of both could be used as alternative antibiotic growth promoters in the poultry industry.", "appendix": "Data availability\n\nFigshare: Efficacy of Moringa oleifera Lam. extracts and Pediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillus plantarum probiotic during starter period on growth performances of male broiler chicken, https://doi.org/10.6084/m9.figshare.21802944.v3. 57\n\nThis project contains the following underlying data:\n\n- Widya PL excell.xlsx\n\n- Untitled2 revised in english.savRaw CFU.xlsx\n\n- Raw growth isolate-revised in english.sav\n\nFigshare: ARRIVE Essential 10 checklist for “Efficacy of Moringa oleifera Lam. extracts and Pediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillus plantarum probiotic during starter period on growth performance of male broiler chicken”, https://doi.org/10.6084/m9.figshare.21802944.v3. 57\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThis work was supported and funded by Ministry of Research and Technology of Higher Education (Kemenristekdikti), Chair of the Research and Innovation Institute, Rector Universitas Airlangga, Dean of Faculty of Veterinary Medicine Universitas Airlangga.\n\n\nReferences\n\nIsroli T: Yudiarti, Sugiharto: Biochemical and Blood Leukocytes Descriptions of 25 Days Age of Kampong Chicken Feed With Fungi Rhizopusoryzae. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrado-Rebolledo OF, Delgado-Machuca JDJ, Macedo-Barragan RJ, et al.: Evaluation of a selected lactic acid bacteria-based probiotic on Salmonella enterica serovar Enteritidis colonization and intestinal permeability in broiler chickens. Avian Pathol. 2017; 46: 90–94. Publisher Full Text\n\nLi Z, Wang W, Liu D, et al.: Effects of Lactobacillus acidophilus on the growth performance and intestinal health of broilers challenged with Clostridium perfringens. J. Anim. Sci. 2018; 9: 1–10.\n\nHamasalim HJ: Synbiotic as feed additives related to animal health and performance. Adv. Microbiol. 2016; 06: 288–302. Publisher Full Text\n\nLin WH, Yu B, Lin CK, et al.: Immune effect of heat-killed multistrain of Lactobacillus acidophilus against Salmonella typhimurium invasion to mice. J. Appl. Microbiol. 2007; 02: 22–31.\n\nFalaki M, Shargh MS, Dastar B, et al.: Effects of different levels of probiotic and prebiotic on performance and carcass characteristics of broiler chickens. J. Anim. Vet. Adv. 2011; 10: 378–384.\n\nKabir SL: The dynamics of probiotics in enhancing poultry meat production and quality. Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University. Int. J. Poult. Sci. 2009; 3: 361–364.\n\nAbd El-Hack ME, El-Saadony MT, Shafi ME, et al.: Probiotics in poultry feed: A comprehensive review. J. Anim. Physiol. Anim. Nutr. 2020; 104: 1835–1850. Publisher Full Text\n\nBomba A, Nemcova R, Gancarcikova S, et al.: Improvement of the probiotic effect of micro-organisms by their combination with maltodextrins, fructooligosaccharides and polyunsaturated fatty acids. Br. J. Nutr. 2002; 88: S95–S99. Publisher Full Text\n\nRehman A, Arif M, Sajjad N, et al.: Dietary effect of probiotics and prebiotics on broiler performance, carcass, and immunity. Poult. Sci. 2020; 99: 6946–6953. Publisher Full Text\n\nBedford M: Removal of antibiotic growth promoters from poultry diets: implications and strategises to minimise subsequent problems. Worlds Poult. Sci. J. 2000; 56: 347–365. Publisher Full Text\n\nShahidi F, Yeo J: Insoluble-bound phenolics in food. Molecules 2016; 21(1216): 1–22.\n\nde Rezende Mudenuti NV , de Camargo AC , Shahidi F, et al.: Soluble and insoluble-bound fractions of phenolics and alkaloids and their antioxidant activities in raw and traditional chocolate: A comparative study. J. Funct. Foods 2018; 50: 164–171. Publisher Full Text\n\nTeteh A, Lawson E, Tona K, et al.: Moringa oleifera leave: hydro-alcoholic extract and effects on growth performance of broilers. Int. J. Poult. Sci. 2013; 12(7): 401–405. Publisher Full Text\n\nYoha KS, Nida S, Dutta S, et al.: Targeted delivery of probiotics: perspectives on research and commercialization. Probiotics Antimicrob. Proteins. 2022; 14(1): 15–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKesharwani S, Prasad P, Roy A, et al.: An overview on phytochemistry and pharmacological explorations of Moringa oleifera. Pharm. Biosci. J. 2014; 26: 34–41.\n\nMoyo B, Oyedemi S, Masika PJ, et al.: Polyphenolic content and antioxidant properties of Moringa oleifera leaf extracts and enzymatic activity of liver from goats supplemented with Moringa oleifera leaves/sunflower seed cake. Meat Sci. 2012; 91(4): 441–447. Publisher Full Text\n\nShahidi F, Yeo J: Insoluble-bound phenolics in food. Molecules 2016; 21(1216): 1–22. Publisher Full Text\n\nTsao R: Chemistry and biochemistry of dietary polyphenols. Nutrients. 2010; 2: 1231–1246. Publisher Full Text\n\nShahat AA, Marzouk MS: Tannins and related compounds from medicinal plants of Africa. Medicinal Plant Research in Africa. Elsevier. 2013; 1: 479–555.\n\nXie DY, Dixon RA: Proanthocyanidin biosynthesis–still more questions than answers? Phytochemistry 2005; 66: 2127–2144. Publisher Full Text\n\nNikpiran H, Taghavi M, Khodadadi A, et al.: Influence of probiotic and prebiotic on broiler chickens’ performance and immune status. J. Nov. Appl. Sci 2013; 2: 256–259.\n\nBedford M: Removal of antibiotic growth promoters from poultry diets: implications and strategies to minimise subsequent problems. Worlds Poult. Sci. J. 2000; 56: 347–365. Publisher Full Text\n\nSalianeh N, Shirzad MR, Seifi S: Performance and antibody response of broiler chickens fed diets containing probiotic and prebiotic. J. Appl. Anim. Res. 2011; 39: 65–67. Publisher Full Text\n\nAkhouri S, Prasad A, Ganguly S: Moringa oleifera leaf extract imposes better feed utilization in broiler chicks. J. Biol. Chem. Res. 2013; 30(2): 447–450.\n\nSjofjan O, Adli DN, Harahap RP, et al.: The effects of lactic acid bacteria and yeasts as probiotics on the growth performance, relative organ weight, blood parameters, and immune responses of broiler: A meta-analysis. F1000Res 2021; 10.\n\nKarwanti NW, Arumdani F, Yulianto AB, et al.: Efficacy of Moringa oleifera Lam. extracts and Pediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillus plantarum probiotic during starter period on growth performances of male broiler chicken. Dataset. figshare 2023. Publisher Full Text" }	[ { "id": "167980", "date": "24 Apr 2023", "name": "Ugbo Emmanuel Nnabuike", "expertise": [ "Reviewer Expertise Medical Microbiology/Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article addresses the role of Moringa oleifera Lam. leaf extract on the growth of Pediococcus pentosaceus, Lactobacillus acidophilus and L. plantarum probiotic as an alternative to antibiotic growth promoters (AGP) in livestock. The report of this article also proved the importance of probiotics in combating the current antimicrobial resistance organisms ravaging the livestock industrials today.\nAbstract: The aim should capture the title; Summary of methods used need to be stated inside the abstract; Results should be written to flow with the methods used accordingly.\nIntroduction: Add Reference in last statement in paragraph three(3) and second to last paragraph; correct ‘ffectiveness’ to ‘effectiveness in last paragraph.\nMethod: Correct to; \"Phytochemical screening test was carried out to determine the presence of Flavonoid, tannin...\"\nResult: Good; but you need to put all the results together; some part of your result interpretation was added to the discussion.\nDiscussion: Result interpretation (Fig 1A, 2, 3) should be part of results. Cut the result interpretations and add to the Results part. Try to comparing your findings with the findings of previous researchers.\nConclusion: Ok\nI therefore recommend Approved with Reservations. The authors need to make the minor corrections pointed out on the reviewers report before indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9605", "date": "05 May 2023", "name": "Widya Paramita Lokapirnasari", "role": "Author Response", "response": "Revised from author Abstract: The aim should capture the title Revised: This research aimed to evaluate the efficacy of Moringa oleifera Lam. extracts and Pediococcus pentosaceus, Lactobacillus acidophilus, Lactobacillus plantarum probiotic during starter period on growth performances (nutrient intake, body weight gain, feed conversion ratio and feed efficiency) of male broiler chicken Summary of methods used need to be stated inside the abstract. Revised: Methods: This study consisted of three sub studies: (1) Screening test for phytochemical compounds. The flavonoid test was conducted by Bate Smith-Metcalf method and Wilstatter method. The Tannin test with Denis’ reagent. The saponin test was performed by applying the Forth method. Triterpenoid tests were performed by applying the Liebermann-Bouchard method and the alkaloids test was conducted by implementing the method of Mayer, Bouchardat and Wagner. (2) in vitro evaluation of several doses of Moringa extract (0%, 0.1%, 0.2%, 0.3% ),where each test tube was added with 1 mL of each isolate and incubated for 24 h at 37°C, after each isolate bacteria was grown in the Agar MRS media using the pour plate method and incubated for 12 h. The growth of probiotic bacteria was calculated for the colonies by using the total plate count; (3) evaluation of probiotics and M. oleifera in vivo to prove growth performance (nutrient intake, body weight gain, feed conversion ratio and feed efficiency) on male broiler chicken. All results were analyzed by analysis of variance (ANOVA) then followed by the Duncan test. Results should be written to flow with the methods used accordingly. Revised: Results: The results showed (1) the phytochemical screening test M. oleifera extract contained positive of flavonoids, tannins, saponins, triterpenoids and alkaloids; (2) in vitro evaluation of M. oleifera extract at doses of 0.1%, 0.2%, 0.3% increased the growth of bacteria P. pentosaceus, L. acidophilus and L. plantarum (p<0.05); (3) The use of probiotics in vivo evaluation, M. oleifera extract and their combination showed improve growth performance. The result of body weight, body weight gain, FCR and feed efficiency signifcantly differed (p <0.05) between the treatments, but there was no significant difference (p > 0.05) between the treatments on nutrient intake (dry matter, ash, crude protein, crude fiber and organic matter) on broiler chicken starter phase. Introduction: Add Reference in last statement in paragraph three(3) and second to last paragraph; Pediocin is a bacteriocin generated by P. pentosaceus, which, in a sufficient amount, can eliminate pathogenic bacteria such as Listeria monocytogens, Staphylococcus aureus, Escherichia coli, Vibrio alginolyticus, Pseudomonas stutzeri, and Aeromonas. P. pentosaceus bacterial growth requires nutrition with carbon, nitrogen, and mineral sources16,17,18 16. Pereira WA, Mendonça CM, Urquiza AV, Marteinsson VÞ, LeBlanc JG, Cotter PD, Villalobos EF, Romero J, Oliveira RP: Use of Probiotic Bacteria and Bacteriocins as an Alternative to Antibiotics in Aquaculture. Microorganisms. 2022;10(9):1705. 17. Khorshidian N, Khanniri E, Mohammadi M, Mortazavian AM, Yousefi M: Antibacterial activity of pediocin and pediocin-producing bacteria against Listeria monocytogenes in meat products. Frontiers in microbiology. 2021;12:709959. 18. Anh Thu NP, Hong Thuy DT, Nghia NH, Phuong Thao DT: Heterologous Expression of Pediocin PA-1 in Escherichia coli. bioRxiv. 2019;12:607630. They allows to control micro-organism growth in the digestive tract of poultry by increasing the metabolic activities in the body, making them potential feed additives in poultry6,7. 6.Yadav S, Jha R: Strategies to modulate the intestinal microbiota and their effects on nutrient utilization, performance, and health of poultry. Journal of animal science and biotechnology. 2019;10(1):1-1. 7.Krysiak K, Konkol D, Korczyński M: Overview of the use of probiotics in poultry production. Animals. 2021;11(6):1620. Nutrition consumed by poultry is used for maintenance and production. Feed efficiency in broilers is influenced by the level of feed consumption and body weight gain. High feed efficiency is produced due to low feed intake followed by a high rate of body weight gain. A high level of feed efficiency can reduce livestock production costs6,7,26,27. 6.Yadav S, Jha R: Strategies to modulate the intestinal microbiota and their effects on nutrient utilization, performance, and health of poultry. Journal of animal science and biotechnology. 2019;10(1):1-1. 7. Krysiak K, Konkol D, Korczyński M: Overview of the use of probiotics in poultry production. Animals. 2021;11(6):1620. 26. Jha R, Das R, Oak S, Mishra P. Probiotics (direct-fed microbials) in poultry nutrition and their effects on nutrient utilization, growth and laying performance, and gut health: A systematic review. Animals. 2020 Oct 13;10(10):1863. 27. Andriani AD, Lokapirnasari WP, Karimah B, Hidanah S, Al-Arif MA: Potency of probiotic on broiler growth performance and economics analysis. Indian Journal of Animal Sciences. 2020;90(8):1140-5. correct ‘ffectiveness’ to ‘effectiveness in last paragraph. Revised: The study aimed to analyze the phytochemical properties of Moringa leaf extract and the effectiveness of M. oleifera extract with different dosages and incubation times on in vitro bacterial growth of P. pentosaceus, L. acidophilus and L. plantarum probiotic, as well as their effect on growth performance on broiler chicken starter phase in vivo. Method: Correct to; \"Phytochemical screening test was carried out to determine the presence of Flavonoid, tannin...\" Revised: The phytochemical screening test was carried out to determine the presence of the flavonoid, tannin, saponin, triterpenoid and alkaloid. Result: Good; but you need to put all the results together; some part of your result interpretation was added to the discussion. Revised: The results of positive flavonoid test indicated by change in color, there was a change in color to orange. The interpretation of positive tannin, indicated by the color of the filtrate changed to a dark green-black color and the presence of saponin indicated by the foam. The interpretation of flavonoid, tannin , saponin were indicate in Figure 1A, B, C. The Figure 2 showed the result of positive triterpenoid test and the figure 3 showed the result of alkaloid Test [Mayer reagent (-), Bouchardat reagent (+) and Wagner reagent (+). Discussion: Result interpretation (Fig 1A, 2, 3) should be part of results. Cut the result interpretations and add to the Results part. Try to comparing your findings with the findings of previous researchers. Revised: These results are in accordance with the research of Kandeepan et al which showed that the results of phytochemical analysis of M.oleifera revealed the presence of alkaloids, flavonoids, saponins, tannins and terpenoids. M. oleifera leaf extracts contain a substantial amount of phenolic chemicals, which are principally responsible for antioxidant activities. Secondary metabolites with bioactive properties. M. oleifera leaves continue to be excellent sources of micronutrients and phytochemicals for the creation of nutraceuticals and functional foods32. Secondary metabolites extracted from M. oleifera leaves using chloroform, ethyl acetate, and ethanol contain bioactive substances such as such as steroids, saponins, tannins, flavonoids, terpernoids and phlobatannins33. 32. Kandeepan C, Sabitha M, Parvathi K, Senthilkumar N, Ramya S, Boopathi NM, Jayakumararaj R: Phytochemical screening, GCMS profile, and In-silico properties of bioactive compounds in methanolic leaf extracts of Moringa oleifera. Journal of Drug Delivery and Therapeutics. 2022;12(2):87-99. 33. Oladeji OS, Odelade KA, Oloke JK: Phytochemical screening and antimicrobial investigation of Moringa oleifera leaf extracts. African Journal of Science, Technology, Innovation and Development. 2020;12(1):79-84." } ] } ]	1	https://f1000research.com/articles/12-215
https://f1000research.com/articles/12-492/v1	15 May 23	{ "type": "Systematic Review", "title": "Role of nutritional supplements on oral health in adults – A systematic review", "authors": [ "Aishwarya Shodhan Shetty", "Ramya Shenoy", "Parul Dasson Bajaj", "Ashwini Rao", "Aparna KS", "Mithun Pai", "Avinash BR", "Praveen Jodalli", "Aishwarya Shodhan Shetty", "Parul Dasson Bajaj", "Ashwini Rao", "Aparna KS", "Mithun Pai", "Avinash BR", "Praveen Jodalli" ], "abstract": "Purpose: The word ‘diet’ usually encompasses the consumption of food that includes all the necessary nutrients required for the optimal functioning of the body. Nutritional supplements hold a supreme position due to the demanding lifestyles or medical conditions in this current era. Although medical literature has sufficient evidence exploring the effect of nutrients on general health, this systematic review aimed at specifically evaluating the role of nutritional supplements on oral health in adults. Methods: PRISMA guidelines were followed in conducting this systematic review where four electronic databases including Scopus, PubMed, EMBASE, and Web of Science were reviewed. Based on the inclusion criteria, four studies exploring the effect of oral nutritional supplements on oral health among the adult population were included in this systematic review. Results: This review generated evidence suggesting a positive effect of nutritional supplements on oral health. These findings were depicted in the form of a reduction in plaque scores, probing depths, gingival inflammation, and greater improvement in periodontal healing among individuals with higher intakes of nutritional supplements within recommended doses. Conclusions: The systematic review outlines the positive impact of consuming nutritional supplements in the recommended dosage on oral health. Additionally, this review stresses the necessity for interventional studies to further investigate the effects of nutritional supplements on oral health, particularly in regard to periodontal healing. PROSPERO registration: CRD42021287797 (27/11/2021).", "keywords": [ "Adults", "Health", "Nutrition", "Nutritional supplements", "Oral health" ], "content": "Introduction\n\nThe term “diet” generally refers to the entirety of the food that a person consumes, with the implication that it includes all the necessary nutrients essential for the body (Leech et al., 2015). Nutrition plays a vital role in meeting the fundamental requirements for maintaining good health, impacting both overall wellness and oral health significantly. Essential nutrients are chemical substances in food which cannot be synthesized in our body at all or in sufficient amounts but are vital for life, growth and tissue repair (Leaf & Weber, 1987; Mann & Truswell, 2017). It has been noted that taking essential nutrients before undergoing surgery and maintaining them at optimal levels can significantly enhance the effectiveness of the treatment. (Miresmaeili et al., 2015). This makes diet and nutrition an indispensable component in patient care (Sobotka & Forbes, 2019).\n\nVarious dietary nutrients play a significant role in the maintenance of oral health. Vitamin A plays a role in preserving the mucosal membranes, salivary glands, teeth, and protecting against cleft palate (Purvis et al., 1973). Despite the prevalence of vitamin A deficiency in developing countries (Haslam, 2019), it's important to recognize its positive effects on oral health. Vitamin E, with its anti-inflammatory action, helps to decrease the effect of oxygen-free radicals in the cells during the process of bone formation, thus having a positive impact on tooth formation (Esenlik et al., 2012; Sufarnap et al., 2020). Vitamin D plays a crucial part in absorption of calcium, magnesium and phosphorus from gut and insufficiency of vitamin D might lead to enamel and dentin hypoplasia and delayed eruption (Adegboye et al., 2010; Lowenstam & Weiner, 1985). Vitamin K has an association with fetal facial deformity (Howe & Webster, 1994). The role of ascorbic acid has been studied extensively in relation to collagen synthesis and therefore, vitamin C deficiency eventually leads to scurvy, which causes inflammation of the gingiva, commonly seen in populations who have limited intake of food like the elderly, and others include smokers and alcoholics (Onigbinde OO et al., 2021; Van Rooij et al., 2003). The marginal deficiency of ascorbic acid can also cause periodontitis (Alvares et al., 1981; Krasse, 1960). It has been observed that a daily intake of a 1 gram supplement of vitamin C was significantly associated with a decrease in sulcular epithelial permeability and an increase in collagen formation in humans (Leggott et al., 1986).\n\nDeficiency of folic acid is usually said to have its association with neural tube defects and a reduction in cleft lip/cleft palate has been observed when pregnant women take folic acid supplement (Burgess et al., 2018; Van rooij et al., 2004;). Folic acid deficiency also impairs the sulcular barrier function (Burt, 1982; Dhamo et al., 2021), and few studies have suggested that supplementation with folate might help in reducing the gingival fluid flow as well as the bleeding index. Vitamin B12 is known to contribute towards mucosal wound healing and facilitating bone health, both of which are necessary for recovery in cases of periodontitis. This fact is backed by studies which have demonstrated an inverse association between vitamin B12 and tooth count (Hugar et al., 2017; Zong et al., 2016). Calcium and vitamin D have a significant association with bone mineralization and osteoporosis where mineralization defects are seen in severe deficiency of vitamin D, while calcium causes negative calcium balance and bone loss. This loss of bone is apparent not only throughout the body but also in the alveolar bone (Adegboye et al., 2010; Jimenez M et al., 2014; Lowenstam & Weiner, 1985). Zinc is known to decrease the sulcular epithelial permeability by inhibiting the leukocyte activity, thereby decreasing gingival fluids and hence the gingival inflammation (Garcia et al., 2011; Rapisarda & Long, 1981; Vratsanos & Mandel, 1985). Diet is also known to have a considerable contribution towards prevention of particular oral tumors (Morse, 2005). There is evidence which suggests that individuals who consume adequate amounts of vegetables, whole grain foods and fruits, have a 40–80% lowered risk of developing oral or pharyngeal cancers in comparison to people who have a lower consumption of these food types (Franceschi et al., 1999; Mclaughlin et al., 1988).\n\nThere is a lack of literature on the consumption of nutrients and balanced diet on oral health. There are relatively fewer studies which explore the possible effects of oral nutritional supplements on oral health. Therefore, this systematic review aimed to evaluate the role of nutritional supplements on oral health in the adult population.\n\n\nMethods\n\nThis systematic review was conducted as per the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) (Shenoy, 2023) statement and the literature search was started in April 2021 and concluded in October 2021. The protocol of the present systematic review was registered with PROSPERO (CRD42021287797) (date of registration-27/11/2021).\n\nA complete literature search was performed across four electronic databases i.e. Scopus, PubMed, EMBASE and Web of Science. Peer-reviewed literature, either published in English language or with availability of translated text in English, were included for the present systematic review. The papers published in or after 2010 and which met the pre-determined inclusion and exclusion criteria were included in this systematic review.\n\nInclusion Criteria: Randomized controlled trials and cross-sectional studies with outcomes based on the effect of nutritional supplements on plaque score, gingival health and periodontal health among adult population were included in the present systematic review. Papers with full text access and either published in English language or with availability of translated text in English were included in this review.\n\nExclusion criteria: Studies on children and animal studies were excluded along with studies which utilized an intravenous mode of administration for nutritional supplements. Studies which explored the effect of nutritional supplements on general health as well as unpublished papers or study protocols were excluded from this review.\n\nThe search strategy followed across the four databases is presented in Table 1. Initially, the PubMed database yielded 235 search results, five articles were later excluded when a BOOLEAN term “NOT COVID” was added.\n\nThe databases were searched by two authors (AR, RS) using the search strategy, which was followed by removal of duplicates, if any. Following which title and abstract screening was performed by two authors (AR, RS) independently, where the authors responded with a ‘Yes’, ‘No’ or ‘Maybe’ for each entry based on the inclusion and exclusion criteria. Any disagreements were resolved with discussion by mutual consensus. The articles included following title and abstract screening, then underwent full-text review by two authors (AR, RS) independently. Disagreements were resolved through discussion with a third author (PD). The articles which qualified at this stage were finalized for quality appraisal and data extraction for this systematic review.\n\nFollowing full-text review, the studies included in this systematic review underwent quality assessment by two authors (AR, RS) independently. Among the four studies included for the present systematic review, three were cross-sectional studies while one was a randomized controlled trial. Therefore, the appropriate version of the Newcastle-Ottawa Quality Assessment Scale (Wells GA et al., 2021) was utilized for quality assessment of these studies respectively. This risk of bias tool covers three domains i.e., selection criteria, comparability criteria and outcome/exposure criteria, where every study is scored for each domain by giving stars. The selection criteria allotted with maximum of four stars, the comparability criteria with maximum of two stars and the outcome criteria with maximum of four stars.\n\nThe data from the full-text articles was extracted independently by two authors (AR, RS) using a pre-decided format which collected information pertaining to the authors, year of publication, study design, sample size, population studied, intervention or exposure provided, type of nutritional supplement intake, outcome measures, main findings, comparison groups if any, and limitations of the study. The data extracted by the two authors was jointly reviewed and was combined as one to fill out any missing information.\n\n\nResults\n\nThe PRISMA flow diagram representing the results of the review process is depicted in Figure 1.\n\nAmong the four studies chosen for this systematic review, one was a randomized controlled trial (RCT) while the other three studies were cross-sectional studies. The studies which were selected following full text review underwent quality assessment utilizing the appropriate Newcastle Ottawa Scale and the number of stars allotted to the selected study are depicted in Table 2. The summary of data extraction from these four studies is provided in Table 3.\n\n1 Maximum of 4 stars.\n\n2 Maximum of 2 Stars.\n\n3 Maximum of 4 Stars.\n\n* One star.\n\n Two stars.\n\n* Three stars.\n\n**** Four stars.\n\nThe nutritional supplements which were studied in the papers included in this systematic review included Orthomol vital f/m; ortho-mol pharmazeutische Vertriebs GmbH (Willershausen et al., 2011), calcium, vitamin D, fruits, vegetables, β-carotene, vitamin C, α-tocopherol, Eicosapentaenoic acid (EPA) Docosahexaeboic acid (DHA), α-linolenic acid (ALA), serum 25-hydroxy vitamin D concentrations as well as micronutrients like vitamins A, E, B1, B2, B6, B12, folate, iron, zinc, phosphorus, and caffeine. (Adegboye et al., 2013; Dodington et al., 2015; Luo et al., 2018). Only the RCT included in this systematic review included an additional nutritional supplement provided as an intervention, while the cross-sectional studies were based on dietary recall methods such as interviews and questionnaires which also considered additional nutritional supplements if taken by participants along with the dietary nutrients being studied.\n\nThe mean age of the participants in the three cross sectional studies was above 50 years, with older adults (65 years and above) being the prime focus in the study by Adegboye et al. (2013), while the mean age of study participants was 27.1 ± 3.0 years in the RCT (Willershausen et al., 2011) selected for this systematic review. Among the studies included in this systematic review, the effects of nutritional supplements on oral health were studied via outcome measures such as dental assessment including number of teeth, salivary flow and analysis, plaque score, probing depth, bleeding on probing, microbiological analysis, reduction in periodontal disease and severity.\n\n\nDiscussion\n\nThis systematic review intended to explore the effects of nutritional supplements on oral health in the adult population. The major findings of this systematic review revolved around the positive impact of nutritional supplements on oral structures, plaque scores and general inflammatory processes. A better understanding of the relationship between an individual's diet and oral health can be gained by recognizing the contribution of micronutrients in generating an adequate immune response (Moynihan, 2005).\n\nSeveral animal and human studies have very well demonstrated the modulatory role of specific micronutrients on the host's inflammatory response by decreasing inflammatory biomarkers and ultimately bone loss (Chapple, 2009). A similar trend was observed in one of the studies included in this systematic review by Willershausen et al. (2011) where the authors noted that the C-reactive protein (CRP) values, a marker of inflammatory processes, showed a lesser increase among study participants taking the nutritional supplements and a slightly positive effect on gingival inflammation as compared to the control group, where both groups consisted of dental students under high examination stress. In another study included in this systematic review, it was seen that the severity of periodontal disease increased with lesser intake of vitamins A, B1, C, E, iron, folate and phosphorus (Luo et al., 2018). Although the mechanism of action between nutrition and periodontal disease is not completely understood, Chapple et al. observed an inverse relationship between total antioxidant properties and periodontal disease, thereby shedding some light on the complex relationship between nutrition and inflammation leading to periodontal disease (Chapple et al., 2007). These findings are supported by evidence which recognizes the antioxidant properties of vitamins A, B1, C and E (Higashi-Okai et al., 2006; Muniz et al., 2015; Palace et al., 1999).\n\nThe study by Dodington et al. focused on observing the effect of dietary nutrients on periodontal healing among smokers and non-smokers with chronic generalized periodontitis following scaling and root planning. The authors observed a significant reduction in probing depth with higher intake of fruits and vegetables, α-tocopherol, vitamin C, β-carotene, EPA and DHA intakes among non-smokers while no such association was seen in smokers. This association can again be partly due to greater antioxidant intake in the form of fruits and vegetables in the diet. The findings of this study also suggested that whole-food sources, rich in anti-oxidants, are far more beneficial in comparison to supplements with purified compounds in promoting optimal periodontal healing. Beneficial outcomes of EPA and DHA supplements were also observed in participants in this study, which could be explained by the effects of a downstream metabolite of DHA, Resolvin D1, that has exhibited downregulation of inflammatory mediators in an in vitro study on periodontal ligament (Mustafa et al., 2013).\n\nAnother included study by Adegboye et al. observed protective effects of increased intakes of dietary calcium and dairy products (within recommendations) on the plaque scores among older adults aged 65 years and above with higher vitamin D intakes (more than 6.8 μg/d). This association was presented after adjusting for other confounding factors and participants with lower vitamin D intakes (less than 6.8μg/d) did not demonstrate this association between dietary calcium and plaque scores. This is in line with earlier evidence which is suggestive of an inverse association between higher intakes of calcium, dairy-products and vitamin D with dental caries and periodontitis (Dixon et al., 2009; Merritt et al., 2006). Since the effect of calcium intake was stratified according to vitamin D in the study by Adegboye et al., it seems that higher intake of vitamin D can improve the positive effects of increased calcium intake, likely by improving the absorption of calcium. This could be explained by the findings of Laky et al. who reported that deficiency in vitamin D levels was observed in larger proportions among individuals with severe periodontal disease in comparison with healthy adults (Laky et al., 2017). In another study included in this systematic review, the second highest level of vitamin D intake (3.2–6.0 μg), was found to be associated with reduced severity of periodontal disease in comparison to the highest level intake (≧ 6.0 μg), which indicates towards an optimal range of 3.2–6.0 μg when considering vitamin D intake (Luo et al., 2018). This explains the need for further investigation into the optimum levels of vitamin D for oral health promotion and periodontal disease prevention.\n\nThe results of the systematic review suggest that developing appropriate dietary interventions focused on oral health, especially periodontal health, might be beneficial alongside routine therapy in prevention of periodontal conditions. These results should be considered with caution due to the limitations of individual studies such as small sample size, high drop-out rates, lack of details regarding supplemental calcium intakes and self-reported dietary assessment method.\n\nThe present systematic review retrieved articles from the four databases. The majority of studies included had a cross-sectional design, highlighting the need for more interventional studies in this area. It is necessary to conduct further research to assess the effects of nutritional intervention on individuals with periodontitis, with the goal of developing effective nutritional therapies to manage periodontal inflammation.\n\n\nConclusion\n\nBased on the included literature, this systematic review highlights the beneficial effects of nutritional supplements on oral health when consumed in a recommended dose. These benefits of nutritional supplements were observed in the form of a reduction in plaque scores, gingival inflammation and probing depth and a greater degree of periodontal healing. This systematic review also wishes to highlight the need for interventional studies in this area to explore the effects of nutritional supplements on oral health, especially relating to periodontal healing. The review also stresses the necessity for interventional studies to further investigate the effects of nutritional supplements on oral health, particularly in regards to periodontal healing.", "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 ‘Role of nutritional supplements on oral health in adults – A systematic review’. https://doi.org/10.6084/m9.figshare.22709857 (Shenoy, 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\nAdegboye AR, Fiehn NE, Twetman S, et al.: Low calcium intake is related to increased risk of tooth loss in men. J. Nutr. 2010; 140(10): 1864–1868. PubMed Abstract \| Publisher Full Text\n\nAdegboye AR, Christensen LB, Holm-Pedersen P, et al.: Intakes of calcium, vitamin D, and dairy servings and dental plaque in older Danish adults. Nutr. J. 2013; 12(1): 1–5. Publisher Full Text\n\nAlvares O, Altman LC, Springmeyer S, et al.: The effect of subclinical ascorbate deficiency on periodontal health in nonhuman primates. J. Periodontal Res. 1981; 16(6): 628–636. PubMed Abstract \| Publisher Full Text\n\nBurgess LC, Phillips SM, Wainwright TW: What is the role of nutritional supplements in support of total hip replacement and total knee replacement surgeries? A systematic review. Nutrients. 2018; 10(7): 820. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurt BA: Diet and dental health; a study of relationships: United States, 1971-74.1982.\n\nChapple IL, Milward MR, Dietrich T: The prevalence of inflammatory periodontitis is negatively associated with serum antioxidant concentrations. J. Nutr. 2007; 137(3): 657–664. PubMed Abstract \| Publisher Full Text\n\nChapple IL: Potential mechanisms underpinning the nutritional modulation of periodontal inflammation. J. Am. Dent. Assoc. 2009; 140(2): 178–184. PubMed Abstract \| Publisher Full Text\n\nDhamo B, Jaddoe VW, Steegers EA, et al.: The association of maternal folic acid supplementation and prenatal folate and vitamin B12 concentrations with child dental development. Community Dent. Oral Epidemiol. 2021; 49(5): 445–453. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDixon D, Hildebolt CF, Miley DD, et al.: Calcium and vitamin D use among adults in periodontal disease maintenance programmes. Br. Dent. J. 2009; 206(12): 627–631. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDodington DW, Fritz PC, Sullivan PJ, et al.: Higher intakes of fruits and vegetables, β-carotene, vitamin C, α-tocopherol, EPA, and DHA are positively associated with periodontal healing after nonsurgical periodontal therapy in nonsmokers but not in smokers. J. Nutr. 2015; 145(11): 2512–2519. PubMed Abstract \| Publisher Full Text\n\nEsenlik E, Nazıroğlu M, Açıkalın C, et al.: Vitamin E supplementation modulates gingival crevicular fluid lipid peroxidation and antioxidant levels in patients with orthodontic tooth movement. Cell Biochem. Funct. 2012; 30(5): 376–381. PubMed Abstract \| Publisher Full Text\n\nFranceschi S, Favero A, Conti E, et al.: Food groups, oils and butter, and cancer of the oral cavity and pharynx. Br. J. Cancer. 1999; 80(3): 614–620. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcia MN, Hildebolt CF, Miley DD, et al.: One-year effects of vitamin D and calcium supplementation on chronic periodontitis. J. Periodontol. 2011; 82(1): 25–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaslam R: Vitamin and mineral supplements: Exploring how diet and supplements contribute to vision health. AJP. Aust. J. Pharm. 2019; 100(1183): 54–61.\n\nHigashi-Okai K, Nagino H, Yamada K, et al.: Antioxidant and prooxidant activities of B group vitamins in lipid peroxidation. J. UOEH. 2006; 28(4): 359–368. PubMed Abstract \| Publisher Full Text\n\nHowe AM, Webster WS: Vitamin K—its essential role in craniofacial development: A review of the literature regarding vitamin K and craniofacial development. Aust. Dent. J. 1994; 39(2): 88–92. PubMed Abstract \| Publisher Full Text\n\nHugar SM, Dhariwal NS, Majeed A, et al.: Assessment of vitamin B12 and its correlation with dental caries and gingival diseases in 10-to 14-year-old children: a cross-sectional study. International journal of clinical pediatric dentistry. 2017; 10(2): 142–146. Publisher Full Text\n\nJimenez M, Giovannucci E, Kaye EK, et al.: Predicted vitamin D status and incidence of tooth loss and periodontitis. Public Health Nutr. 2014; 17(4): 844–852. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrasse B: Effect of consistency of diet on bacteria in gingival pockets in dogs. Odont Revy. 1960; 11: 152–165.\n\nLaky M, Bertl K, Haririan H, et al.: Serum levels of 25-hydroxyvitamin D are associated with periodontal disease. Clin. Oral Investig. 2017; 21(5): 1553–1558. PubMed Abstract \| Publisher Full Text\n\nLeaf A, Weber PC: A new era for science in nutrition. Am. J. Clin. Nutr. 1987; 45(5): 1048–1053. Publisher Full Text\n\nLeech RM, Worsley A, Timperio A, et al.: Understanding meal patterns: definitions, methodology and impact on nutrient intake and diet quality. Nutr. Res. Rev. 2015; 28(1): 1–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeggott PJ, Robertson PB, Rothman DL, et al.: The effect of controlled ascorbic acid depletion and supplementation on periodontal health. J. Periodontol. 1986; 57(8): 480–485. PubMed Abstract \| Publisher Full Text\n\nLowenstam HA, Weiner S: Transformation of amorphous calcium phosphate to crystalline dahllite in the radular teeth of chitons. Science. 1985; 227(4682): 51–53. PubMed Abstract \| Publisher Full Text\n\nLuo PP, Xu HS, Chen YW, et al.: Periodontal disease severity is associated with micronutrient intake. Aust. Dent. J. 2018; 63(2): 193–201. PubMed Abstract \| Publisher Full Text\n\nMann J, Truswell AS: Essentials of human nutrition. Oxford University Press; 2017.\n\nMcLaughlin JK, Gridley G, Block G, et al.: Dietary factors in oral and pharyngeal cancer. JNCI: Journal of the National Cancer Institute. 1988; 80(15): 1237–1243. Publisher Full Text\n\nMerritt J, Qi F, Shi W: Milk helps build strong teeth and promotes oral health. J. Calif. Dent. Assoc. 2006; 34(5): 361–366. PubMed Abstract \| Publisher Full Text\n\nMiresmaeili A, Mollaei N, Azar R, et al.: Effect of dietary vitamin C on orthodontic tooth movement in rats. Journal of Dentistry (Tehran, Iran). 2015; 12(6): 409–413. PubMed Abstract\n\nMorse DE: Oral and pharyngeal cancer. Nutrition and oral medicine. 2005;205–221. Publisher Full Text\n\nMoynihan PJ: The role of diet and nutrition in the etiology and prevention of oral diseases. Bull. World Health Organ. 2005; 83: 694–699. PubMed Abstract\n\nMuniz FWMG, Nogueira SB, Mendes FLV, et al.: The impact of antioxidant agents complimentary to periodontal therapy on oxidative stress and periodontal outcomes: A systematic review. Arch. Oral Biol. 2015; 60(9): 1203–1214. PubMed Abstract \| Publisher Full Text\n\nMustafa M, Zarrough A, Bolstad AI, et al.: Resolvin D1 protects periodontal ligament. Am. J. Phys. Cell Phys. 2013; 305(6): C673–C679. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOnigbinde OO, Olajugba GO, Oyapero A: Dietary pattern, oral hygiene and self-reported gingival bleeding in pregnant women in Lagos State. Advances in Human Biology. 2021; 11(1): 73. Publisher Full Text\n\nPalace VP, Khaper N, Qin Q, et al.: Antioxidant potentials of vitamin A and carotenoids and their relevance to heart disease. Free Radic. Biol. Med. 1999; 26(5-6): 746–761. PubMed Abstract \| Publisher Full Text\n\nPurvis RJ, MacKay GS, Cockburn F, et al.: Enamel hypoplasia of the teeth associated with neonatal tetany: a manifestation of maternal vitamin-D deficiency. Lancet. 1973; 302(7833): 811–814. PubMed Abstract \| Publisher Full Text\n\nRapisarda E, Longo A: Effects of zinc and vitamin B 6 in experimental caries in rats. Minerva Stomatol. 1981; 30(4): 317–320. PubMed Abstract\n\nShenoy R: Role of nutritional supplements on oral health in adults – A systematic review. figshare.2023. Publisher Full Text\n\nSobotka L, Forbes A: Basics in clinical nutrition. Galen; 2019; (Vol. 1, No. 5th).\n\nSufarnap E, Siregar D, Lindawati Y: Effect of vitamin E supplementation on orthodontic tooth movement in Wistar rats: a prelimary study. F1000Res. 2020; 9: 9. Publisher Full Text\n\nvan Rooij IA , Ocké MC, Straatman H, et al.: Periconceptional folate intake by supplement and food reduces the risk of nonsyndromic cleft lip with or without cleft palate. Prev. Med. 2004; 39(4): 689–694. PubMed Abstract \| Publisher Full Text\n\nVan Rooij IA, Vermeij-Keers CK, Kluijtmans LAJ, et al.: RP Does the interaction between maternal folate intake and the methylenetetrahydrofolate reductase polymorphisms affect the risk of cleft lip with or without cleft palate. Am. J. Epidemiol. 2003; 157: 583–591. Publisher Full Text\n\nVratsanos SM, Mandel ID: Polyamines of dental plaque in caries-resistant vs. caries-susceptible adults. J. Dent. Res. 1985; 64(3): 422–424. PubMed Abstract \| Publisher Full Text\n\nWells GA, Shea B, O'Connell D, et al.: The Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomised studies in meta-analyses.\n\nZong G, Holtfreter B, Scott AE, et al.: Serum vitamin B12 is inversely associated with periodontal progression and risk of tooth loss: a prospective cohort study. J. Clin. Periodontol. 2016; 43(1): 2–9. PubMed Abstract \| Publisher Full Text" }	[ { "id": "176717", "date": "15 Jun 2023", "name": "Vijaya Hegde", "expertise": [ "Reviewer Expertise Oral Health", "Tobacco Induced Diseases", "Research and bioethics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments:\nThis is an important and well written paper about the usefulness of the Nutritional supplements on Oral health. The Introduction and methodology is well written.\nSpecific Comments:\nTitle:\n\nThis could be more specific. The mode of delivery of Nutritional supplement could be mentioned and also its impact on which aspect of Oral health.\n\nIntroduction:\nDefinition of ‘Nutritional Supplement’ could be added instead of Diet. Briefly mention the current uncertainties of the specific nutritional supplement on Oral health.\n\nMethods:\nKindly address the reasons for excluding the studies. Kindly mention if any software was used to extract the data. Were any steps taken to resolve inconsistencies? How was unclear data/information managed?\n\nResults:\nSummary statistics of the studies needs to be mentioned.\nDiscussion:\nKindly discuss the impact of the study results on policy or further research. The Final take away message, needs to be clearly spelled out in the discussion which is missing. Where there any limitations in the review process which need to be mentioned?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "9771", "date": "20 Jul 2023", "name": "Ramya Shenoy", "role": "Author Response", "response": "Dear Madam, The authors thank you for your effort and time given to improvise the article. Nutritional supplements commonly known as dietary supplements is used to make the title catchy. We have included recent studies available in the four major databases to elaborate on the effect of nutritional supplements on oral health. Articles were included based on inclusion and exclusion criteria. The data extracted by the two authors were jointly reviewed and combined as one to fill out any missing information. Excel 2016 is used to remove the duplicates. Detailed information on the included studies is shown in Table 3. There are no direct policy implications from this review. However, this review stresses the necessity for interventional studies to further investigate the effects of nutritional supplements on oral health, particularly in regard to periodontal healing." } ] }, { "id": "176715", "date": "20 Jun 2023", "name": "Suman Challa", "expertise": [ "Reviewer Expertise Oral health in Elders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe inclusion and exclusion criteria are well-defined. The search strategy is explained, but it would be helpful to know if any restrictions were placed on the language of the publications. They mention quality assessment was performed using the Newcastle-Ottawa Quality Assessment Scale, a well-recognized tool. However, the criteria for the selection of this tool should be explained. The data extraction process is justified, but it would be helpful to have more information on how discrepancies between authors were resolved. The article could benefit from a more detailed presentation of the results, including statistical analyses and effect sizes. The conclusion summarizes the findings but could provide more insights into the implications of the results for clinical practice or policy.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-492
https://f1000research.com/articles/12-491/v1	12 May 23	{ "type": "Case Report", "title": "Case Report: Challenging diagnostic of an accessory spleen mimicking a retroperitoneal tumor: A case report and review of the literature", "authors": [ "Alia Zouaghi", "Rim Miri", "Dhafer Hadded", "Meryam Mesbahi", "Haithem Zaafouri", "Mona Cherif", "Wassim Riahi", "Yazid Benzarti", "Sobhi Mleyhi", "Faker Ghedira", "Aslam Souli", "Chiraz Chamekhi", "Fatma Khanchel", "Aniss Ben Maamer", "Alia Zouaghi", "Rim Miri", "Dhafer Hadded", "Haithem Zaafouri", "Mona Cherif", "Wassim Riahi", "Yazid Benzarti", "Sobhi Mleyhi", "Faker Ghedira", "Aslam Souli", "Chiraz Chamekhi", "Fatma Khanchel", "Aniss Ben Maamer" ], "abstract": "Background: The accessory spleen is a congenital defect with an additional splenic tissue to the native spleen due to an incomplete fusion of splenic masses during the embryologic period. The preoperative diagnosis is usually mistaken for a retroperitoneal neoplasm. The aim of the present manuscript is to highlight the diagnostic difficulties of a retroperitoneal spleen. Case presentation: A 41-year-old woman was admitted to the surgical department of Habib Thameur Hospital, Tunis, for an abdominal mass discovered by auto palpation associated with a left lower quadrant pain. The computed tomographic scan showed a heterogeneous and partially necrotic retroperitoneal mass located along the left primitive iliac vessels. Arterial supply to the mass arose from the aorta and the left iliac vessel. The spleen was normal. MRI examination showed a left retroperitoneal heterogeneous and necrotic mass, surrounding the iliac vessels extended from the aortic to the left iliac bifurcation, hyper vascularized at the arterial time with a peripheral capsule intensely enhanced, and there were two other lateroaortic vascularized masses. The patient had a laparotomy resection of the retroperitoneal mass. The result of the specimen examination was an accessory spleen. The postoperative outcome was uneventful. Conclusion: An accessory spleen on the retroperitoneal position is extremely uncommon. Preoperative diagnosis is still challenging. The main misdiagnosis was neoplastic disease.", "keywords": [ "case report", "retroperitoneal neoplasm", "retroperitoneal mass", "accessory spleen", "retroperitoneal spleen" ], "content": "Abbreviations\n\nAS: accessory spleen\n\nCT: computed tomography\n\nMRI: magnetic resonance imaging\n\n\nBackground\n\nRetroperitoneal masses usually have a various spectrum of etiologies.1 Primary retroperitoneal ones are uncommon and divided into neoplastic and no neoplastic masses which are infrequent.2 They represent 15 to 20% of all primary retroperitoneal masses.2\n\nAccessory spleens (AS) are defined as a congenital defect with an additional splenic tissue to the native spleen due to an incomplete fusion of splenic masses during the embryologic period.3 They are located elsewhere than a normal anatomical situation.4 However, an ectopic spleen located other than in the left upper quadrant is an unusual situation.4 The retroperitoneal AS is a rare entity. It is usually misdiagnosed, and mistaken for a retroperitoneal neoplasm.5 Imaging findings are useful to characterize primary retroperitoneal tumors and to help the diagnosis of accessory spleen. Therefore, preoperative diagnosis is essential to avoid unnecessary surgery.5\n\nOur work highlights some of the diagnostic difficulties, describing a retroperitoneal AS removed on the suspicion of a retroperitoneal neoplasm considering that a preoperative pathological diagnosis could not be obtained.\n\n\nCase presentation\n\nWe report the case of a 41-year-old woman, with no past medical or family history, who presented in August 2020 with an abdominal mass discovered by auto palpation associated with a left lower quadrant pain. No other abnormalities were found in clinical examination of the abdomen. The results of routine biochemical and hematologic tests were normal. Computed tomography (CT) scan of the abdomen obtained after intravenous administration of iodine-containing contrast material showed a 73 × 57 × 37 mm heterogeneous and partially necrotic retroperitoneal mass located along the left primitive iliac vessels (Figure 1). The arterial supply of the mass came directly from the aorta. Multiple lymph nodes were identified around the mass and along mesenteric, aortic, and left primitive iliac vessels. The spleen was normal. We suspected a mesenchymal tissue tumor so that we decided to biopsy the mass. Ultrasound-guided fine-needle aspiration was done concluding hemorrhagic aspirate. T2-weighted magnetic resonance imaging (MRI) examination showed a left retroperitoneal heterogeneous and necrotic mass, surrounding the iliac vessels extended from the aortic to the left iliac bifurcation, iso-signal T1 and T2, hyper vascularized at the arterial time with a peripheral capsule intensely enhanced. The mass was vascularized by a supply artery native to the left primary iliac artery (Figures 2a, 2b & 2c) and there were two lateroaortic vascularized masses of 35 × 16 and 29 × 25 mm (Figures 3a, 3b). Abdominal organs were unremarkable.\n\nSince a retroperitoneal neoplasm of unknown origin had been recognized, imaging findings had suspected a vascular tumor: hemangiopericytoma or paraganglioma. The embolization of the arterial supply was judged risky since the coils could move into the iliac vessel and cause ischemia. After consent, the patient underwent laparotomy resection of the retroperitoneal mass in October 2020. A midline incision was made. After exploration of the abdomen, we found a hard solid 10 cm mass left to the aorta, repressing medially the left colon (Figure 4). There were no other abnormalities. We initially made a dissection and control on Suspensor Lake of left ureter and gonadic vessels in the retroperitoneum (Figure 5a). The left primitive iliac, external iliac and hypogastric arteries were also dissected and controlled so that we could ligate the supplying vessels on the left primitive iliac artery (Figure 5b). The dissection and control of the abdominal aorta permitted the ligation of an arterial supply with the mass and a para-aortic lymphadenectomy (Figure 5c). Finally, a total resection of the retroperitoneal mass was made (Figures 6, 7 & 8).\n\nGross examination of the resected specimen revealed a firm, encapsulated brown beige mass. Microscopic examination showed normal splenic parenchyma, no cell atypia, and no malignant cells (Figure 9). Immunohistochemical study showed that CD20 emphasizes white pulp, CD30 marked intensely red pulp, and CD23 marked the dendritic follicular cells. There was no malignancy on the resected lymph nodes. The post-operative outcome was uneventful, and the patient was monitored regularly.\n\nThe lymphoid tissue is distributed in white-blue star and red pulp (hematoxylin and eosin).\n\n\nDiscussion\n\nPrimary retroperitoneal tumors are uncommon with a large range of malignant or benign histological forms.6 There is a high rate of malignancy in these tumors so that the definitive diagnostic confirmation can only be made by the examination of the surgical specimens.6\n\nThe ectopic spleen must be differentiated from the accessory spleen, which is a common congenital abnormality defined by the presence of normal splenic tissue located in an abnormal situation, in addition to a normally placed spleen.3–5,7 AS have normal splenic histology and result from the failure of splenic cell fusion during the embryologic period.8 Its incidence varies from 10 % to 30%.5,9 Besides, an automatic implant of normal functional splenic tissue located in any compartment of the body outside the usual position can be classically seen. This entity is called splenosis and is mostly caused by abdominal trauma or surgery.9\n\nAs reported in the literature, AS are measured generally around 1 cm in diameter.5,7 Masses larger than 4 cm are rare.3 They could reach 3–4 cm in cases of splenectomy response to compensatory hypertrophy or an associated hematological pathology.6,8 However, in our case, the bulky mass exceeded this interval despite no history of splenectomy or any hematological anomaly. The AS is typically small, vascularized by branches of the splenic artery,10 and is usually located near the splenic helium or the pancreatic tail.4,5,9 However, one of the highlights of our case is the rarity of its location and vascular supply. The AS may mimic various tumors referring to its location such as a pancreatic tumor, adnexal tumor, retroperitoneal tumor.7 In the retroperitoneal situation, most of them are located on the left side except three cases reported by Maharaj et al, Kim et al, and Zhou et al, in which they were situated on the right retroperitoneal area.5,6,8\n\nThese spleens are usually asymptomatic, but they can be complicated by torsion, rupture, hemorrhage, or cyst formation.3 Representing a benign etiology, the diagnosis should basically be made preoperatively.8 However, knowing that malignant retroperitoneal tumors are more common than non-malignant ones, the differentiation between these tumors and the identification of an accessory spleen can only be made after surgery.8\n\nRetroperitoneal masses are diagnosed and characterized by referring to the enhancement pattern using radiological findings: Ultrasonography, CT scan, and MRI.1,7 But the diagnosis of the retroperitoneal accessory spleen is difficult and challenging on imaging.8,9 Radiological imaging can mislead the diagnosis of the accessory spleen to retroperitoneal tumors.4 CT scan findings demonstrate in typical cases, a mass with good margins similar to the splenic parenchyma in contrast.3 MRI can also be used to evaluate tissue characteristics and accessory splenic pedicles.3 In our case, imaging findings of the retroperitoneal mass led to misdiagnosis of the accessory spleen. They showed that the principal supply artery of the lesion was native of the left common iliac artery instead of the splenic artery according to the literature.11 On the other hand, the size of the mass in our case was much larger than reported in the literature, and its enhancement scanning pattern was different from the main spleen. Besides, percutaneous preoperative biopsy of the lesion was not sufficient to differentiate accessory spleen from other tumors, and it is not necessary when a soft tissue tumor is suspected.8\n\nNowadays, only nuclear medicine imaging can confirm the diagnosis using Technetium-99m heat-damaged red blood cell scintigraphy or Tc-99m heat-damaged autologous red blood cell SPECT-CT, which are the key means to differentiate accessory splenic tissue.3,11–13 This modality determines approximately the site of the accessory spleen, but it is only performed when there is a high suspicious index.13,14 So if there is any diagnostic doubt or difficulty based on the routine imaging modalities, especially in patients with the previous splenectomy, preoperative nuclear medicine scintigraphy could be investigated to exclude the presence of an AS or splenosis.14\n\nIn summary, when a difficulty is detected in diagnosing a retroperitoneal mass, only surgery can make a definitive diagnosis following the pathological examination of the specimen.5,14 In our work, according to the results of contrast-enhanced CT and MRI, we suspected hemangiopericytoma or paraganglioma. The challenge posed is that retroperitoneum can include a large variety of tumors. In our case, MRI findings suggested a vascular tumor since the mass was intimately surrounding vascular structures so that hemangiopericytoma was the etiology considered preoperatively in our patient. It is a rare vascular tumor native from the pericytes of Zimmerman and an unusual retroperitoneal mass that requires surgical exploration and excision to establish the final diagnosis of the accessory spleen after pathologic examination.15\n\nThe accessory spleen is usually misdiagnosed even using biological and imaging features and its management is challenging moreover controversial.8,10 When symptomatic or following a complication, such as torsion of its pedicle or its rupture or suspected malignant transformation, surgical resection is necessary.7 Surgical removal of an accessory spleen is also required when it is identified as splenectomy for the hematologic problem.6\n\nAccording to the literature, retroperitoneal accessory spleens are rare, and less than thirty cases were reported and published until 2013.3,7,14 A review of the literature was performed using the PubMed Database. We used “retroperitoneal”, “accessory spleen”, “retroperitoneal tumor” as keywords for the research. After excluding articles that did not report AS, and those who reported ectopic spleen or splenosis, and manuscripts not written in English, we found 38 cases of retroperitoneal accessory spleen between 1969 and 2020.\n\nThe analyzed articles included four cases of spleens larger than 7 cm, as in our case. According to the literature, this dimension is unusual, and these accessory spleens usually do not surpass 4 cm in diameter. The diagnosis was possible preoperatively in one case using technetium-99m-labeled heat-damaged red blood cell scintigraphy. This is the only case where this technique was performed. All the patients underwent a CT scan for diagnosis. MRI was used in two cases only. The differential diagnoses were various, and all cases were diagnosed accessory spleen postoperatively, after the pathological examination. As in our case, the literature highlights the challenge of reaching a diagnosis before surgery. So, when a retroperitoneal neoplasm is detected, surgeons should be conscious of a possible retroperitoneal accessory spleen. Table 1 shows the challenging course to reach the final diagnosis which leads to surgical resection in all cases.\n\n\nConclusion\n\nA wide spectrum of rare pathologies, including benign and malignant tumors, can be located in the retroperitoneum. The initial challenge in retroperitoneal diseases remains in the establishment of a diagnosis. Complete surgical resection is the only potential diagnostic modality. A retroperitoneal accessory spleen is extremely uncommon. The main misdiagnosis is a neoplastic disease so that the accessory spleen could be wrongly removed. Our case highlights the possibility of a retroperitoneal accessory spleen when investigating a retroperitoneal mass.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nRajiah P, Sinha R, Cuevas C, et al.: Imaging of Uncommon Retroperitoneal Masses. RadioGraphics. juill 2011; 31(4): 949–976. PubMed Abstract \| Publisher Full Text\n\nBartkowski DP, Ferrigni RG: Castleman’s Disease: An Unusual Retroperitoneal Mass. J Urol. janv 1988; 139(1): 118–120. Publisher Full Text\n\nPalumbo V, Mannino M, Teodoro M, et al.: An extremely rare case of an oversized accessory spleen: case report and review of the literature. BMC Surg. déc 2019; 19(1): 45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmer HZ, Chin C, Clarke K, et al.: Infarcted ectopic spleen presenting as retroperitoneal mass. J Minim Invasive Gynecol. sept 2007; 14(5): 660–662. PubMed Abstract \| Publisher Full Text\n\nZhou J-S, Hu H-P, Chen Y-Y, et al.: Rare presentation of a right retroperitoneal accessory spleen: A case report. Oncol. Lett. oct 2015; 10(4): 2400–2402. Publisher Full Text \| Free Full Text PubMed Abstract \|\n\nKim MK, Im CM, Oh SH, et al.: Unusual presentation of right-side accessory spleen mimicking a retroperitoneal tumor: Right-side accessory spleen. Int J Urol. août 2008; 15(8): 739–740. PubMed Abstract \| Publisher Full Text\n\nPorwal R: Retroperitoneal Accessory Spleen Presented As Metastatic Suprarenal Tumour- A Diagnostic Dilemma. J. Clin. Diagn. Res. 2015. Publisher Full Text\n\nMaharaj R: Right sided spleen laying retro-duodenal: A case report and review of the literature.2016; 6.\n\nRonot M, Aguiar D, Assalino M, et al.: Retroperitoneal ectopic splenic tissue: Imaging findings. Diagn Interv Imaging. sept 2016; 97(9): 935–937. PubMed Abstract \| Publisher Full Text\n\nLily WKM, Tony LK, Andrea AYW: Right sided retroperitoneal splenosis. Eur J Radiol Extra. oct 2010; 76(1): e19–e21. Publisher Full Text\n\nLi Y, Qiu X, Li W, et al.: Diagnostic challenge for giant left retroperitoneal accessory spleen: a case report. J Int Med Res. févr 2020; 48(2): 030006051987589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFelice M, Tourojman M, Rogers C: Right retroperitoneal splenosis presenting as an adrenal mass. Urol Case Rep. janv 2018; 16: 44–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBergeron E, Ratte S, Jeannotte S, et al.: The use of a handheld gamma probe for identifying two accessory spleens in difficult locations in the same patient. Ann Nucl Med. mai 2008; 22(4): 331–333. PubMed Abstract \| Publisher Full Text\n\nCh T, Kampantais S, Christopoulos P, et al.: Compensatory enlargement of an accessory spleen mimicking a retroperitoneal tumor: a case report. Hippokratia. 2013; 17: 185–186.\n\nChhaidar A, Zouari S, Bdioui A, et al.: Very rare localization of a retroperitoneal hemangiopericytoma revealed by lumbosciatalgia: A case report. Int. J. Surg. Case Rep. 2018; 53: 127–131. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSbrana F, Zhou D, Zamfirova I, et al.: Castleman’s disease: a rare presentation in a retroperitoneal accessory spleen, treated with a minimally invasive robotic approach. J Surg Case Rep. 1 oct 2017; 2017(10). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTjaden C, Werner J, Buechler MW, et al.: Reactive Hypertrophy of an Accessory Spleen Mimicking Tumour Recurrence of Metastatic Renal Cell Carcinoma. Asian J Surg. janv 2011; 34(1): 50–52. PubMed Abstract \| Publisher Full Text\n\nKapoor A, Jain A, Mahajan G, et al.: Elusive retroperitoneal accessory spleen. Indian J. Surg. 2004; 66: 298–299.\n\nRosenthal CL, Bishop MC: Accessory Spleen Presenting as a Retroperitoneal Tumour. Eur. Urol. 1981; 7(5): 314–316. Publisher Full Text\n\nRosenkranz W, Kamhi B, Horowitz M: Retroperitoneal accessory spleen simulating a suprarenal mass. Br J Radiol. déc 1969; 42(504): 939–942. PubMed Abstract \| Publisher Full Text" }	[ { "id": "221116", "date": "08 Nov 2023", "name": "Tomohiro Terasaka", "expertise": [ "Reviewer Expertise Endocrine disorders. Medical practitioner." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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,\nAt least there are more case reports other than their listed literatures regarding retroperitoneal accessory spleens1-5, and the large size (5-10cm) mass of ectopic spleen was not rare in the past literatures. With these matters, this article needs to be re-furbished more concerning past literatures and the sentence of this article, \"The analyzed articles included four cases of spleens larger than 7 cm, as in our case. According to the literature, this dimension is unusual, and these accessory spleens usually do not surpass 4 cm in diameter\".\n\nAs their report, I suppose it was necessary to exclude paraganglioma because if the mass is the case, during the excision of the mass in the operation the leak of cathecholamine possibly make the blood pressure violently fluctuated. With these figures, the necrosis in the mass and highly enhanced appearance are similar as paraganglioma.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-491
https://f1000research.com/articles/12-490/v1	12 May 23	{ "type": "Brief Report", "title": "Small studies in systematic reviews: To include or not to include?", "authors": [ "Abdallah El Alayli", "Preston Thomas", "Sara Jdiaa", "Razan Mansour", "Archana Gautam", "Millind A. Phadnis", "Ibrahim K. El Mikati", "Reem A. Mustafa", "Abdallah El Alayli", "Preston Thomas", "Sara Jdiaa", "Razan Mansour", "Archana Gautam", "Millind A. Phadnis", "Ibrahim K. El Mikati" ], "abstract": "Background: COVID-19 provided a real challenge for evidence synthesis due to the rapid growth of evidence. We aim to assess the impact of including all studies versus including larger studies only in systematic reviews when there is plethora of evidence. We use a case study of COVID-19 and chronic kidney disease (CKD). Methods: The review team conducted a systematic review of multiple databases. The review assessed the effect of CKD on mortality in patients with COVID-19. We performed a sensitivity analysis to assess the effect of study size on the robustness of the results based on cutoffs of 500, 1000 and 2000 patients. Results: We included 75 studies. Out of which there were 40 studies with a sample size of >2,000 patients, seven studies with 1,000-2,000 patients, 11 studies with 500-1,000 patients, and 17 studies with <500 patients. CKD increased the risk of mortality with a pooled hazard ratio (HR) 1.57 (95% confidence interval (CI) 1.42 - 1.73), odds ratio (OR) 1.86 (95%CI 1.64 - 2.11), and risk ratio (RR) 1.74 (95%CI 1.13 - 2.69). Across the three cutoffs, excluding the smaller studies resulted in no statistical significance difference in the results with an overlapping confidence interval. Conclusions: These findings suggested that, in prognosis reviews, it could be acceptable to limit meta-analyses to larger studies when there is abundance of evidence. Specific thresholds to determine which studies are considered large will depend on the context, clinical setting and number of studies and participants included in the review and meta-analysis.", "keywords": [ "Systematic Reviews", "Research Methodology", "Research Conduct", "Sample Size", "Decision-Making", "Plethora of Evidence", "COVID-19", "Choric Kidney Disease (CKD)" ], "content": "Introduction\n\nSince the onset of the COVID-19 pandemic, there has been an exponential increase in the number of publications. In a period of one year from October 2019 through October 2020, more than 48,000 peer reviewed articles were published on PubMed related to COVID-19.1 This was in addition to the thousands of pre-prints that were made available and used in decision making.2 This high and accelerated research output, although attempting to meet knowledge gaps, comes with a set of limitations and challenges. One of those challenges faced by the systematic review community is the added burden of screening, abstracting, and synthesizing a plethora of evidence. For this reason, some reviewers have been triaging included studies based on specific criteria including the study sample size.3 This abstract was previously presented in the 7th International Medicine & Health Sciences Congress (IMedHSC) at Paris, France in December, 2021. It is published as an online publication.4\n\nConceptually, systematic reviews are considered the best source of evidence as they aim to comprehensively identify and summarize all evidence for a specific clinical question.5 For that reason, ideally reviewers incorporate studies of all sample sizes, especially when evaluating rare diseases.6 Systematic reviews identify studies of different sample sizes and types. However, there is no empirical evidence that excluding smaller studies would affect or bias the pooled results of risk factors on prognosis in cases of plethora of evidence. This rationale can be justified by: larger studies 1) are usually of higher quality,7 2) provide more precise effect estimates,8 3) contribute a higher weight to calculation of the overall effect estimate when using meta-analyses methods.\n\nSensitivity analyses are often used to determine the robustness of systematic reviews results. They examine the effect of changing specific assumptions, methods, and other variables on the overall results.9 In this paper, we aim to assess the robustness of the results based on study sample sizes. We assess the difference between the overall effect estimates from studies of all sample sizes and those obtained after excluding smaller studies. We used a case study of the association between chronic kidney disease (CKD) and COVID-19 mortality.\n\n\nMethods\n\nThis study was done as a sensitivity analysis to a previously completed systematic review based on a case study of COVID-19 mortality in patients with CKD. We conducted this analysis to assess the effect of sample size in individual studies and to evaluate if it would affect the pooled effect estimate. The goal of the original review was to assess the outcomes of COVID-19 in patients with CKD.10 In the previous study, a team of reviewers (AE, PT, SJ, RM, AG) conducted a comprehensive systematic review in published databases (Embase, Epistemonikos, and Medline), and unpublished databases (Medrxiv, LitCOVID and SSRN) between January 1, 2020 to January 10, 2021. We also included patients from four registries; Hilbrands 2020,11 Holman 2020,12 Jager,2020,13 and Williamson 2020.14 We used a variety of keywords and subheadings on CKD and COVID-19 to identify all studies on the topic. Included studies were of any study type; cross-sectional, prospective, and retrospective cohort studies. The full search strategy can be found in supplementary data.15 As this review was based on existing literature, there was no involvement of patients or members of the public.\n\nReviewers included studies that report the risk of mortality among patients with CKD and COVID-19. Studies that did not report data on mortality, patients with CKD and COVID-19, or did not separate results for patients with CKD were excluded. The selection process was conducted using Endnote or standardized excl sheet. We grouped studies based on the type of effect estimate reported: hazard ratio (HR), risk ratio (RR), or odds ratio (OR). We pooled the data and created forest plots using a random effect model using RevMan 5.4.16 We assessed publication bias17 by performing a visual inspection to judge asymmetry of funnel plots. The full methods and results of the review were published separately.10 More studies were included in the original review as studies reporting outcomes other than mortality were also included. We extracted data using pre-tested excel sheet. Reviewers abstracted the following information from each study; first author name, date and country of publication, population characteristics (age, gender, prevalence of CKD), effect estimates with the associated 95% confidence interval (CI). Two reviewers separately completed screening and data abstraction and resolved disagreement by discussion or arbitration. To compare the effect of different sample sizes on the overall effect estimate we conducted a sensitivity analysis based on sample size per study. We utilized three different cut-offs to define smaller studies: 500, 1,000, and 2,000 patients. These cut-offs included 20%, 40%, and 50% of the studies.\n\n\nResults\n\n75 studies were eligible for inclusion out of 137 included in the original review with a total of 30,566,586 participants. 70 studies were cohort studies, four were cross-sectional studies and only one was a case control study. 65 studies were reported in published databases and 10 were reported in unpublished databases. Out of the 75 included studies, 40 had a sample size of >2,000 patients, seven studies with 1,000-2,000, 11 studies with 500-1,000 patients, and 17 studies had <500 patients. The grouping of those for the meta-analysis was based on the reported effect estimate, OR, HR, or RR (see Table 1). The flow chart of the original review is available in supplementary data.15\n\n* EE: effect estimate, HR: hazard ratio, OR: odds ratio, RR: relative risk.\n\nAcross the effects estimates RR, OR, and HR, there was an increased risk of mortality in patients with CKD and COVID-19. Based on all included studies of all sample sizes, the HR was 1.57 (95%CI.42-1.73), the OR was 1.86 (95% CI 1.64-2.11), and the RR was 2.58 (95% CI 1.08-6.16) (see Table 2).\n\n* EE: effect estimate, HR: hazard ratio, OR: odds ratio, RR: relative risk, CI: confidence interval.\n\nWhen excluding 17 studies that had < 500 patients, the HR was 1.54 (95% CI 1.39-1.71), the OR was 1.84 (95% CI 1.61-2.10), and the RR was 1.61 (95% CI 0.88 - 2.92). When excluding 28 studies that had < 1,000 patients, the HR was 1.48 (95% CI 1.33-1.65), the OR, was 1.77 (95% CI 1.54-2.02), and RR was 1.61 (95% CI 0.88 - 2.92). When excluding 35 studies that had <2,000 patients, the HR was 1.46 (95% CI 1.30-1.63), OR was 1.74 (95% CI 1.50-2.01), and RR was 1.61 (95% CI 0.88-2.92). All the effect estimates of the sensitivity analysis were very similar to the overall effect estimates (see Table 2). All forest plots can be found in supplementary data.15\n\nWhen assessing the funnel plots for studies reporting HR and OR, there was no suspicion of publication bias among the included studies (see Figures 1 and 2).\n\n\nDiscussion\n\nIn this paper, we assess the impact of limiting prognosis systematic reviews to large studies when there is plethora of evidence available using a case study of assessing chronic kidney disease (CKD) as risk factor for mortality among people infected with COVID-19. Pooled results were robust using different effect estimates (HR, OR, and RR) and different cutoff value to define smaller studies (500, 1,000, and 2,000 patients).\n\nThis study has multiple strengths. First, the review included a large number of studies (75) with a large number of patients (30,566,586 participants) and included studies using different effect estimates (HR, RR, and OR). This allowed the investigators to explore the effect of the sensitivity analysis on all these effect estimates. Second, there is no established cutoff or existing guidance to determine what is considered a smaller study. Hence, we assessed the effect of excluding smaller studies using multiple cutoffs. Some reviews exclude studies that included < 10 patients3 and some used other much larger cutoffs (e.g. < 10,000 patients).18 The decision about smaller versus larger studies should consider multiple factors. It would depend on the number of studies and the participants included in the systematic review and meta-analysis. Additionally, it has to consider the specific disease or condition being assessed by the systematic review. For example, rare diseases may not yield large sample size studies, and so the definition of ‘larger’ itself may vary. Therefore, such definition should be validated using sensitivity analysis by considering different cutoffs. Third, we assessed the relation between risk of bias and study size. We did not find any intrinsic difference between smaller and larger studies included in this paper. The risk of bias table for all studies can be found in the separate systematic review.10 In this systematic review the included studies’ risk of bias was not sufficient to explain the inconsistency in results. It would have been inappropriate to exclude smaller studies if they were the studies of higher quality (less risk of bias). The lack of clear association between study size and risk of bias allowed for non-biased results after exclusion of smaller studies. Finally, we also assessed the effect of publication bias on the results. One potential explanation of why smaller studies usually have higher effect estimates than the overall effect estimate, is a phenomenon referred to as ‘small study effect’.19 Small study effect usually occurs due to publication bias. As smaller intervention studies with higher effect size have a higher chance to get published and a larger treatment effect is needed to produce statistically significant results.20 In addition, studies with higher effect size are more cited and they have a higher chance to get captured in searches. The effect of this phenomenon of studies of prognosis is not clear. A systematic review conducted in osteoarthritis studies showed that the effect estimates from larger studies (>100 patients) is smaller than effect estimates derived from smaller studies and closer to the overall effect estimates.21 Another meta-analysis showed that the effect estimates from smaller studies had an exaggerated benefit when compared to larger studies.22 We did not have suspicion of publication bias when assessing the funnel plots visually. Studies with different effect estimates were distributed evenly for different study sizes.\n\nThe study has few limitations. We rely on assessing a single case example. The finding of this study me not be representative of the effect of excusing smaller studies in other reviews. However, We believe the large numbers we incorporated allow for making inference with enough certainty. Another limitation is that the case related to prognostic studies, which may not be generalizable to other types of systematic reviews. Additional empirical evidence and possibly simulation studies may help increasing the generalizability of these findings.\n\nThe conduct of smaller studies has been controversial as they lack statistical power to test a hypothesis and might expose patients to unnecessary harm and are more prone to methodological errors.22 Similar to what we are suggesting, some researchers suggested that smaller studies should be excluded from systematic reviews as they provide minimal power to the overall effect and they tend to potentiate publication bias.23 A paper by Stanley suggested using only the 10% most precise studies in the meta-analysis.24 Another study by Turner found little impact of underpowered studies on the overall effect in Cochrane reviews if at least two substantially powered studies were included.25 However, the inclusion of small studies in meta-analysis has been justified by the fact that these studies when combined will produce a significant effect besides their importance in evaluating rare diseases.6 This manuscript doesn’t aim to discourage the conduct of smaller prognostic studies. The conduct of this type studies is encouraged for any study size. In this manuscript we address the validity of excluding smaller studies in cases of plethora of evidence.\n\nIncluding only large studies has the risk of excluding potentially relevant evidence reported in small studies. For this reason, other solutions to accelerate the review process include the implementation of automated systems in the review process.26 A study showed that a semi-automated approach using a machine learning software reduced the consumption of time with low risk of missing articles.27 Many automated software’s are now available to assist reviewers in conducting systematic reviews, however, their use has not yet been widely adopted by reviewers.26 Additionally, excluding smaller studies may or may not have implications on decision making. This depends on factors such as the prevalence of the condition, baseline characteristics of patients, nature of the people important outcome. In this case study, the difference between the overall effect estimates when including all studies despite sample sizes and the effect estimates when excluding smaller sties was very small and statistically insignificant.\n\n\nConclusions\n\nOur results showed no major difference between the overall effect estimates from studies of all sample sizes and that obtained after excluding smaller studies. This is an exploratory work which shows anecdotal evidence that it might be acceptable to use evidence from larger studies for prognostic systematic reviews. This is especially relevant with the exponential increase in the number of publications (e.g., during the COVID-19 pandemic), as a matter of practical convenience. Although this may be true in cases of plethora of evidence (e.g., during the COVID-19 pandemic), caution must be made when using this evidence for policy and decision making. Additional work (e.g., simulation studies) is needed to further explore the effect of excluding smaller studies in multiple settings.", "appendix": "Data availability\n\nFigshare. Small studies in systematic reviews: To include or not to include? https://doi.org/10.6084/m9.figshare.21739295.v1. 15\n\nThe project contains the following underlying data:\n\n• Data supplement 1 Search Strategy Small Studies.pdf. (Search strategy for all databases in this study).\n\n• Data supplement 2 PRISMA flowchart Small studies.pdf. (Selection process flowchart for this study).\n\n• Data Supplement 3 Forest plots.docx. (Nine forest plots of comparison).\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\nMachovec KA, Ames WA: Evidence-Based Medicine in the Time of COVID: We Have a Problem. J. Cardiothorac. Vasc. Anesth. 2021; 35(5): 1274–1275. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFraser N, Brierley L, Dey G, et al.: The evolving role of preprints in the dissemination of COVID-19 research and their impact on the science communication landscape. PLoS Biol. 2021; 19(4): e3000959. Publisher Full Text\n\nHanson KE, Altayar O, Caliendo AM, et al.: The Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Antigen Testing. Clin. Infect. Dis. 2021; PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEl Alayli A, et al.: Small studies, to include or not to include in systematic reviews, when there is a plethora of evidence? Dec 2021. 7th International Medicine & Health Sciences Congress (IMedHSC), Paris, France. 2021. Publisher Full Text\n\nHaddaway NR, Pullin AS: The Policy Role of Systematic Reviews: Past, Present and Future. Springer Sci. Rev. 2014; 2(1): 179–183. Publisher Full Text\n\nHalpern SD, Karlawish JH, Berlin JA: The continuing unethical conduct of underpowered clinical trials. JAMA. 2002; 288(3): 358–362. PubMed Abstract \| Publisher Full Text\n\nEgger M, Smith GD: Misleading meta-analysis. BMJ. 1995; 310(6982): 752–754. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuyatt GH, Oxman AD, Kunz R, et al.: GRADE guidelines 6. Rating the quality of evidence–imprecision. J. Clin. Epidemiol. 2011; 64(12): 1283–1293. PubMed Abstract \| Publisher Full Text\n\nThabane L, Mbuagbaw L, Zhang S, et al.: A tutorial on sensitivity analyses in clinical trials: the what, why, when and how. BMC Med. Res. Methodol. 2013; 13(1): 92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJdiaa SS, Mansour R, El Alayli A, et al.: COVID–19 and chronic kidney disease: an updated overview of reviews. J. Nephrol. 2022; 35(1): 69–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHilbrands LB, Duivenvoorden R, Vart P, et al.: COVID-19-related mortality in kidney transplant and dialysis patients: results of the ERACODA collaboration. Nephrol. Dial. Transplant. 2020; 35(11): 1973–1983. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolman N, Knighton P, Kar P, et al.: Risk factors for COVID-19-related mortality in people with type 1 and type 2 diabetes in England: a population-based cohort study. Lancet Diabetes Endocrinol. 2020; 8(10): 823–833. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJager Kj KACNCCCS-AJEGLCFHMHAPKJL: Results from the ERA-EDTA Registry indicate a high mortality due to COVID-19 in dialysis patients and kidney transplant recipients across Europe. Kidney International;2020.\n\nWilliamson EJ, Walker AJ, Bhaskaran K, et al.: Factors associated with COVID-19-related death using OpenSAFELY. Nature. 2020; 584(7821): 430–436. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEl Alayli A:Untitled Item. Dataset. figshare. 2022. Publisher Full Text\n\nReview Manager (RevMan) [Computer program]: Version 5.3 ed. Copenhagen: The Nordic Cochrane Centre.The Cochrane Collaboration;2014.\n\nEgger M, Davey Smith G, Schneider M, et al.: Bias in meta-analysis detected by a simple, graphical test. BMJ. 1997; 315(7109): 629–634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin M, Lucas HC, Shmueli G: Research Commentary—Too Big to Fail: Large Samples and the p-Value Problem. Inf. Syst. Res. 2013; 24(4): 906–917. Publisher Full Text\n\nSchwarzer G, Carpenter JR, Rücker G:Small-Study Effects in Meta-Analysis.Schwarzer G, Carpenter JR, Rücker G, editors. Meta-Analysis with R. Cham:Springer International Publishing; 2015; p. 107–41. Publisher Full Text\n\nSutton AJ, Duval SJ, Tweedie RL, et al.: Empirical assessment of effect of publication bias on meta-analyses. BMJ. 2000; 320(7249): 1574–1577. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNüesch E, Trelle S, Reichenbach S, et al.: Small study effects in meta-analyses of osteoarthritis trials: meta-epidemiological study. BMJ. 2010; 341: c3515. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKjaergard LL, Villumsen J, Gluud C: Reported Methodologic Quality and Discrepancies between Large and Small Randomized Trials in Meta-Analyses. Ann. Intern. Med. 2001; 135(11): 982–989. PubMed Abstract \| Publisher Full Text\n\nAltman DG: Statistics and ethics in medical research: III How large a sample? Br. Med. J. 1980; 281(6251): 1336–1338. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStanley TD, Jarrell SB, Doucouliagos H: Could It Be Better to Discard 90% of the Data? A Statistical Paradox. Am. Stat. 2010; 64(1): 70–77. Publisher Full Text\n\nTurner RM, Bird SM, Higgins JP: The impact of study size on meta-analyses: examination of underpowered studies in Cochrane reviews. PLoS One. 2013; 8(3): e59202. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Altena AJ , Spijker R, Olabarriaga SD: Usage of automation tools in systematic reviews. Res. Synth. Methods. 2019; 10(1): 72–82. PubMed Abstract \| Publisher Full Text\n\nGates A, Gates M, Sebastianski M, et al.: The semi-automation of title and abstract screening: a retrospective exploration of ways to leverage Abstrackr's relevance predictions in systematic and rapid reviews. BMC Med. Res. Methodol. 2020; 20(1): 139. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "209173", "date": "10 Oct 2023", "name": "Cheng Xue", "expertise": [ "Reviewer Expertise Statistics. Nephrology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is a brief report of an example of sensitivity analysis that examines the impact of including or excluding small studies in a systematic review of COVID-19 and chronic kidney disease (CKD) mortality. It reports consistent and robust results across the sensitivity analysis, showing no major difference in the pooled effect estimates when excluding small studies. However, this is not a novel idea. Sensitivity analysis excluding small studies has been used in many systematic reviews. The results may be consistent or inconsistent depending on the quality of the included studies. So, this report relies on a single case example of CKD and COVID-19 mortality, which may not be generalizable to other types of systematic reviews or outcomes. It does not provide a clear rationale or justification for choosing the specific cutoffs for small studies, or how they relate to the prevalence and heterogeneity of the condition and outcome. It does not discuss the potential implications or limitations of excluding small studies for decision-making, especially for rare diseases or subgroups of interest. Therefore, including small studies may be better in most situations, for it could perform a sensitivity analysis that examines the impact of including or excluding small studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "279876", "date": "10 Sep 2024", "name": "Adrianna Lynn Westbrook", "expertise": [ "Reviewer Expertise I am a statistician and have contributed to studies on COVID. I do not have clinical knowledge of CKD." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 implications of including smaller N studies alongside larger N studies when designing a systematic review. Several cutoffs were explored to define small vs. large n studies and the results indicated including smaller N studies did not substantively alter the results of the systematic review. Though the authors point out that this is only one specific health issue, this type of study can help others who are compiling systematic reviews of other health topics.\nIs the work clearly and accurately presented and does it cite the current literature?\nIt’s not clear to me why one should exclude ‘low n’ studies by default – an n of 500 can be perfectly adequate if it is a rigorous and well-executed study. I worry the premise of the article reinforces the notion that bigger is always better even if the conclusions drawn do not support that assumption. I feel like more of the evidence for excluding small n studies is provided in the discussion when it would be better suited for the introduction.\nIf applicable, is the statistical analysis and its interpretation appropriate?\nWhy were these cutoff’s used? The 20%, 40%, 50% seem arbitrary – was there a rationale? Why not median and IQR to define cutoffs?\nDid any published studies come from the same registry with overlapping patients? If so, studies from the same source may have similar findings or similar biases – this seems to violate the assumption of independence. Authors should address this concern and discuss how it may impact the findings.\nAre sufficient details of methods and analysis provided to allow replication by others?\n\n// If applicable, is the statistical analysis and its interpretation appropriate?\nSample size is but one aspect of a study design that may be related to other key aspects of study design. For example, smaller N studies may be among CKD patients who have another specific comorbidity while larger studies may be more likely to be the ‘general population’ of CKD patients. Those smaller subpopulations may be more likely experience mortality – not because it’s a small N study but because of the inclusion criteria for that small N study. I would like the authors to talk more about the studies that were included – not just about N but other aspects of the participants, research methods, etc. I think this would enrich the analysis, especially with how we interpret the null findings (i.e., no differences in estimates when pooling across different cutoffs).\nMiscellaneous\nTypo on page 3 – “excl sheet”\nTable 1 headings could be made more clear. I believe “Studies with <1,000 patients” includes the “Studies with <500 patients” because (n=35) + (n=40) = (N=75), which is described as the total N for the sample. Still, can you add some notation to make it clearer, such as a footnote?\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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-490
https://f1000research.com/articles/12-489/v1	12 May 23	{ "type": "Research Article", "title": "Exploring Ghanaian medical students’ learning experiences during the COVID-19 lockdown: a case study of the University for Development Studies Medical School", "authors": [ "Anthony Amalba", "Bright Yammaha Amoore", "Sophia Ewuenye Adwoa Kpebu", "Bruce Ayabilla Abugri", "Victor Mogre", "Anthony Amalba", "Bright Yammaha Amoore", "Bruce Ayabilla Abugri", "Victor Mogre" ], "abstract": "Background: Coronavirus disease (COVID-19) continues to affect health professions education, especially in developing and middle-income countries, even though alternative educational measures have been sanctioned to continue educating students in their homes while observing physical distancing. Methods: A qualitative cross-sectional study design was adopted for the study. Participants among the four departments of a Ghanaian medical school were treated as clusters, and a voluntary response sampling approach was used to recruit students across the clusters to respond to self-administered online Google interview questions on students’ learning experiences during the COVID-19 pandemic. Results: The study found that lack of supervision, lack of access to school library resources, overload of syllabi and the interference of household chores were major factors that made online learning difficult and ineffective during the COVID-19 lockdown. Most participants (n=133, 67%) described online learning as completely inadequate, ineffective and an expensive mode of learning which may not develop the necessary competence and skills required for effective clinical practice. Conclusions: Notwithstanding this, almost all students believed that combining face-to-face and online learning will significantly improve medical education. The COVID-19 pandemic brought about the increased use of online learning in the health professions that was accompanied with significant challenges for students.", "keywords": [ "Covid-19", "health professions education", "learning experiences", "online learning", "lockdown" ], "content": "Introduction\n\nCoronavirus disease (COVID-19) is an infectious disease caused by a new coronavirus that has not been previously identified in humans. It was first identified in Wuhan, China on the 23rd of December 2019. According to UNICEF, since its discovery, its geographic distribution has continued to evolve. World Health Organization (WHO) reported that globally the outbreak affected 216 countries, with over 13 million cases and 500,000 deaths. Ghana recorded its first two cases from Norway and Turkey returnees on the 12th of March 2020, since then the numbers have continued to rise. On the 4th of May 2020, Ghana Health Service (GHS) reported 2,719 cases and 18 deaths occurring in 12 out of the 16 regions in the country within two months, amid three weeks of lockdown, intensive screening, and indefinite closure of schools for containment of the virus. Due to the infectious nature of the outbreak many countries attempted to observe the principle of social distancing through resorting to school closure which included health education institutions, to curtail the rising consequences of the outbreak. As shown in UNICEF Ghana report, this affected over 1.2 million learners, representing over 60% of the world’s student population.\n\nCOVID-19 continues to affect educational institutions in so many ways even though according to another UNICEF Ghana report alternative educational measures have been adopted through online learning, using various platforms such as zoom, WhatsApp, and Google Classroom in place of the traditional method of instruction (face-to-face delivery) to continue educating students in their homes while observing physical distancing. The approach comes with its challenges, especially in developing countries (low-and middle-income countries) as its ability to raise or maintain the expected quality of health professionals is a concern. For example, in the Philippines, the Commission on Higher Education (CHED) suspended the online form of instruction following resistance from students, including teachers who clamoured against the online mode of learning due to different factors including schools’ lack of preparation for the implementation of these due to inadequate access to internet and online educational resources (Toquero, 2020).\n\nFor a variety of reasons, many schools in the world including Ghana had also suspended all academic activities including clinical clerkships. One reason was to flatten the COVID-19 curve, another reason was to lessen the risk of exposure of student health professionals, although some students may be willing to put themselves at risk to provide support in combat (Ferrel & Ryan, 2020). According to literature, clinical skills training is a crucial moment in student developmental stage where clinical examination and reasoning skills, including therapeutic communication skills with patients, and multi-disciplinary teams are developed to enhance effective diagnosing and client adherence to treatment (Adebisi et al., 2020; Cecilio-Fernandes et al., 2020). All of these activities greatly diminished with an online learning environment, which gave no room for real-life clinical practice (Adebisi et al., 2020; Cecilio-Fernandes et al., 2020). Despite the growing trend of perceived challenges of online learning on the skills development of student health professionals, health educators across the globe continued to stress the need to protect students, arguing that the risks of exposure of students may be greater than the educational benefits of remaining in clinical settings or schools to learn about the new clinical entities, even though their services may be needed in times of healthcare worker shortage (Anderson et al., 2020). As many student health professionals were missing out on the valuable experiences of presentations, clinical rotations, and collaborative experiences of standards which helped previous generations to become doctors, it poses the question about how these students will evolve and integrate themselves into the medical community given the situation of a prolonged COVID-19 pandemic, or another future incidence of infectious disease (Ferrel & Ryan, 2020).\n\nDue to the above concerns, there are growing anxieties about the ability of online studies to produce the expected outcome of qualified health professionals, especially in developing and middle-income countries. Though several studies regarding COVID-19 have been conducted so far in relation to the medical field and other fields related to health sciences, very few studies have been undertaken to ascertain the impact of COVID-19 in the field of health professions education in Ghana (Sintema, 2020). There is thus the need to explore the learning experiences of student health professionals, especially during the adoption of online learning during the COVID-19 lockdown. This study leveraged on the impact of online learning on the professional development of student health professionals to draw recommendations for the overall improvement of the online learning system, with the aim of meeting the standards of health professions education in times of future crisis.\n\n\n\ni. How has COVID-19 impacted students’ learning during the COVID-19 lockdown?\n\nii. How do students perceive the role of online learning in building their competence for future practice?\n\n\nMethods\n\nThe study was conducted in the University for Development Studies School of Medicine (UDS-SoM), Tamale, Ghana from July-September 2020. It is affiliated to the Tamale Teaching Hospital where students receive clinical training. The UDS-SoM runs four programmes including an MBChB, Doctor of pharmacy, nurse of anaesthesia, and a master’s degree in public health. The school uses the problem-based learning (PBL) methodology of teaching for teaching and learning. This approach departs from the traditional teaching format that relies on instructor-formulated lectures with the student being the passive recipient rather than the active researcher of facts. Details of the curriculum is published elsewhere. Like other schools in Ghana and elsewhere, academic activity was brought to a close during the COVID-19 lockdown. There was a break in academic activities for a period as the university was contemplating how to continue with teaching and learning. Subsequently, online teaching tools were rolled out to complete the rest of the academic year.\n\nFollowing a qualitative study approach, participants of the study included all levels of medical students and first- and second-year pharmacy students, given that the pharmacy programme was new and had only two levels of students so far. A voluntary response sampling approach was used to invite participants to participate in the study by sending an electronic link to students’ WhatsApp group platforms. The electronic link contained an introduction to the study, consent procedures and a data collection tool. Those who agreed and consented to the study proceeded to respond to the data collection tool. Students were assured that their participation was voluntary and were at liberty to withdraw from responding to the data collection tool at any point in time. Anonymity and confidentiality were assured by the de-identification of the data collection tool. Data was collected using close-ended and open-ended questionnaires developed using Google forms. The open-ended questions provided students the opportunity to express their opinions and perceptions without restrictions. The open-ended questions were aligned to the research questions and were adapted from those of previous studies. Specifically, students’ perception and opinions on the impact of COVID-19 on their learning and the role of online learning in their professional development as future health care providers. Sex (male, female) which was self-reported by participants, students’ level of training (Level 100 to Level 600) and programme of study were assessed using close-ended questions. Pilot testing of the questions was done among a sample of 10 students to ensure easy comprehensibility. There were no changes made to the study after the pilot study. This study complies with the SRQR guidelines for reporting qualitative research.\n\nThe data collection and screening was performed by three of the research team members, BYA, VM and AA. Two team members (BYA and VM) coded the data and generated preliminary themes. The larger research team with expertise in health professions education (VM, AA and BYA), behavioural sciences (SEAK and VM) and qualitative research (SEAK, VM and AA) further deliberated on the suggested codes and themes before approving them as a research team. Differences identified were addressed and adjudicated where necessary.\n\nData was exported to Microsoft Excel. All texts were read and re-read by the research team. Coding was independently done by BYA and VM. The codes were then used to generate themes. Codes and themes generated were shared with the other members of the research team for expert advice. The findings were presented according to the themes that were generated.\n\nEthical approval was obtained from University for Development Studies Institutional Review Board (UDS/IRB/102/20) and participants who gave consent to be part of the study were assured of confidentiality and anonymity.\n\n\nResults\n\nA total of 202 students responded to the open-ended questionnaire (Amalba et al., 2023). Table 1 shows the general characteristics of the students. Participants had a mean age of 23.20 years, and the majority were male. There was no consideration of sex segregation in the design of the study and data analysis because this did not apply to this study, as both males and females had equal access to the online learning system in Ghana.\n\nMost of the students opined that their learning was made difficult and ineffective during the lockdown because they had no supervision from faculty members, given that they were learning from their homes. Students believed that, for effective learning to be achieved, there is the need for proper supervision and the lack of a supervised environment makes effective learning almost unattainable. This situation made some of them to feel lazy, less motivated and disinterested in learning.\n\n“I feel lazy to learn or even join for the classes because there's no supervision”.\n\n“It made me reluctant to study since I'm home”.\n\nSome students opined that, due to the restrictions to movement during the lockdown, their learning was made difficult because they did not have physical access to the University library. According to them the University library has relevant books and other learning resources that are not readily available online.\n\n“Learning was a little bit challenging especially in accessing the materials. Since it has denied me access to some learning materials due to restricted movement”.\n\nStudents also opined that the swift changes to their usual mode of learning to a situation of learning from their homes was sudden, leaving them confused, struggling and reluctant; this slowed learning at the initial stages. They were, however, quick to add that they managed to adapt to the changes as time went on.\n\n“It initially slowed my learning process but with time I adapted”.\n\n“Everything has moved at a slower pace”.\n\nRelated to the poor supervision, students indicated that learning was made difficult for them during the lockdown since their home environments were full of distractions and interferences, making them unconducive for effective learning. Students opined that their homes were not serene and were rather unfavourable and unsuitable for learning. Some said they were also distracted by house chores.\n\n“It has not been so great, the pressure at home does not permit effective learning.”\n\n“Very bad impact from COVID-19 on my studies. There is a whole lot of home distractions interfering negatively on my studies. Schools should reopen quickly for me to come to school. It has slowed me down…I'm at home and I have a lot of errands to run.”\n\n“Well, learning hasn't been easy. With all the chores at home, it has been one long bumpy ride. The home environment is not conducive for learning at this level, considering we (students’) aren't used to it.”\n\n“If I'm being honest, this pandemic has truly ruined my style of learning. This is because learning at home is not the same as in school. Schools provide a level of serene environment which is suitable for learning and not in the case of being home.”\n\n“COVID-19 has affected my studies negatively. This is because I find it very difficult to study when am at home. The home environment is not very conducive or favourable for learning”.\n\nFurthermore, students discussed that the lockdown led to a reduction in the amount of time needed to complete the 2019/2020 academic year. According to them, the reduction in the duration meant that contact hours were reduced resulting in overload of syllabi to learn within a short period of time. In addition, they believed some essential syllabi were not covered. All of these, according to the students impeded effective learning.\n\n“It has affected my studies negatively because we could not complete our curriculum for the 2019/20 academic year. It compressed the normal academic year schedule because of the break we had. It took a certain form of seriousness out. Also, I was forced to learn so much in a short period of time which didn't help”.\n\n“It has reduced the effectiveness of my learning. It has limited the number of effective contact hours and effective lectures”.\n\nTo make up for the lost instructional periods resulting from the lockdown, new teaching and learning technologies were adopted by both students and lecturers to complete the 2019/2020 academic calendar. Students reported that all their learning was moved online which led to serious challenges for their learning. Students’ opinions regarding the role of online learning on their career training was sought for. Among the 202 students, 198 responded to the question in which only 12% (n=23, 12%) believed online learning will be adequate to help provide them with the required competencies as health professionals. A good number of participants (n=21, 42%) were very sceptical about the ability of online learning to adequately prepare them given the practical-centred nature of their training. And a significant number of them (n=133, 67%) strongly described the online training as completely inadequate, ineffective and an expensive mode of learning even though a considerable number of them also believed an improved electronic learning system will contribute significantly, if not entirely, to the enhancement of the medical education system. Students further opined that the introduction of online learning tools resulted in limited opportunities to acquire practical and clinical skills that required face-to-face meetings with faculty. They recognized that online learning is deficient and cannot develop all the needed competence and skills required for effective clinical practice. Students asserted that, aside the theoretical syllabi, some skills can only be learned by hands on practice, which cannot be entirely acquired by reading and watching videos online. The following quotes further illuminates these views expressed by students.\n\n“Big No! Sincerely No! Certainly No! I don't feel like a medical student while sitting at home doing online studies. Home is certainly not a medical school. So, I can't be at home to learn medicine. At home where is the medical library? There is none. All the medical books of good quality and standard are in the medical school library. My lecturers are not with me at home. No other place can replace my medical school. I wish to be at school right now”.\n\n“Online learning cannot fully complete our training. Aside the theory aspect, medical training requires hands on practice which cannot be acquired online. And even if done online it’s not effective because there is the need for one to practice on something or someone and also see what’s being done well, however, materials for practical and skills are not available at home even if they are there, there is zero supervision”.\n\n“I also disagree because, online learning can never replace ward activities where students learn the skill of rapport building for effective communication geared towards diagnosis. Thus, the need for a blend of face-to-face lectures to make such a mark”.\n\n“It has negatively affected my learning. This is because, majority of the PBL is based on practical and skills training. All these things were eliminated during the online studies and the core mandate of the PBL, tutorials could not also be effective through this mode of learning, some of the courses are practical oriented and needs much time when emphasizing on it”.\n\n“However, having been unable to go to the wards have limited learning especially in a practical sense”.\n\n“It has really made it a bit tough because practical sessions are no longer held and I don't get to practice the skills I learn theoretically”.\n\n“It has been very bad due to the practical nature of my course. It has slowed down the learning process especially the skills and practical aspects due to zero contacts”.\n\nStudents indicated that the online learning mode that was rolled out by the University did not work so well for them because they were unable to undertake note revision of their lecture slides, given that they could always search Google for answers online, especially during the end of course assessments.\n\n“It has affected me negatively in that, it has slowed the level of learning and revision once answers can always be googled”.\n\nOthers described that the process has changed their way of learning.\n\n“It has changed the whole style of learning for me, I learn better when I go to the ward and observe what I have read being practiced but this was not the case and so it made it more difficult for me to appreciate and understand the topics I read.”\n\nStudents further discussed the absence of physical interactions with lecturers hindered their understanding of concepts since the technological issues provided limited opportunities to ask or answer questions during the teaching and learning process.\n\n“It has given me another aspect of learning but the Lecturer-student interaction is bad. This is as a result of the methods adopted in order for students to complete the academic year, and some lecturers send in only slides for us to study, it has slowed down learning because there are no lectures to attend for better understanding”.\n\nOpportunities for social and group interactions were also eliminated. Some students felt that the PBL methodology was not well incorporated in the online teaching and learning, in that, the group discussion that encourages teamwork was eliminated.\n\n“Badly, not able to meet for group discussion physically. More self-study, as well as group discussions…with minimal practical sessions with supervision”.\n\nStudents identified that online learning came with several cost issues since they will have to spend money to buy data to have internet connectivity. Apart from the cost issues students also opined that there were instances of poor internet connectivity. According to some students, learning became almost impossible with the adaptation of new technologies which was characterized by poor internet connectivity since some of them lived in remote areas of the country.\n\n“The current electronic learning system is costly and ineffective. This is proved by the challenges we experienced through the online learning process; poor network, expensive cost of data and the environment some of us find ourselves. Sometimes due to bad network, we were unable to hear what is being taught”.\n\n“COVID-19 has really slowed my learning pace and also cost me financially. it has been difficult with the online studies due to high cost of data. Oh! Hmm it's not been good at all. E-learning is expensive”.\n\n“Negatively, due to bad network and inability to join class. It has been difficult with the online studies due to poor network as it adversely affected my learning with detrimental consequences. It has been difficult, especially for students at the remote area, bad network has been a factor. As a result of the methods adopted in order for students to complete the academic year. Network challenges was a major factor and some lecturers send in only slides for us to study”.\n\nApart from the negative impacts of COVID-19 on learning, some students’ felt that there have been some positive experiences they have gained from the adaptation of the new technology in learning leading to the acquisition of new learning experiences.\n\n“It has broadened my scope on how to learn and communicate online through e-learning.”\n\n“It pushed me to explore different avenues to learn, the abrupt end to attending school/classes brought some apathy in studies but it gradually got better with the introduction of the online studies.”\n\n“For me, I think the impact is a positive one because it makes us do things in new ways, we thought was not possible in the past.”\n\n“It has made most of my learning virtual which requires 90% self-studies.”\n\n“It has made my learning very interesting. Adapting to different ways of learning through the online method.”\n\n“I would not say that it's been bad. It has been a nice experience with some form of challenges.”\n\n“I would say positive, if we consider that this has not been an ordinary time. It has changed the way I learn.”\n\nPositive especially with technological way of learning”\n\nSome students also believed that improved online learning can help to resolve some challenges and make important contributions to medical education. They further believed that the pandemic has impacted their learning positively by enabling them to self-direct their learning, appreciate better the problem-based methodology and focusing more on their books without any interferences as opposed to the usual attendance to lectures, and the hustles of postponement of lectures resulting in a lot of time wasting.\n\n“At first, I didn't think online learning could be useful for medical education, but now I am convinced if the network and internet access is stable it can positively contribute but not as compared to physical sessions.”\n\n“Online learning will ensure that when lecturers or students are indisposed or situations prevent them from coming to class, they can still hold their lectures online; I have realized that some lecturers are sometimes unable to honor their lectures, most of the time, such lessons are either rescheduled or totally cancelled. Since we [doctors] are dealing with human lives, I believe every information from lecturers are needed, so if they can't make it to class physically, they may be able to do it electronically. The online learning will help alleviate classroom acquisition problems in my school, and significantly cut down on the long hours spent in simple classroom lectures. Also, I learn best when I witness things, so through the video lectures submitted by my lecturers, I have been able to appreciate lessons more and I can revise what they say any time.”\n\n“It has just made me appreciate the student-centered skills nurtured during preclinical medical school as studies have become self-guided again”\n\n“It gave me more time to do self-study than usual times where you have to be going for lectures, tutorials and skills and sometimes lectures will be postponed or no venues resulting in a lot of time wasting. But studying at home, even when lectures are postponed or reschedule, I make proper use of the time than being on campus.”\n\n“I am mostly at home due to the COVID-19 protocols, I read more. Helped me focus more on my books.”\n\n“Reduced my onsite (bedside and tutorials) learning time increased time for my personal/private studies”\n\nSeveral students felt that they were able to learn without pressure and more conveniently on their own by exploring new technologies in learning.\n\n“I embraced virtual ways of learning, and learning was made comfortable and convenient.”\n\n“Disrupted our schedule, but introduced more convenient ways to explore technology for education.”\n\n“Honestly, it's better than in school since most of the stressors in school weren't at home. It helped us learn without pressure”.\n\n\nDiscussion\n\nIn this study we explored the impact of the COVID-19 pandemic/lockdown and its associated online learning on the professional development of student health professionals.\n\nA significant number of the participants (n=133, 67%) strongly described the online learning during the COVID-19 pandemic/lockdown as completely inadequate, ineffective and an expensive mode of learning. Students considered this mode of learning as a sudden, expensive, and challenging change, similar to previous findings reported from the Philippines, where both students and teachers strongly opposed the use of online learning during COVID-19 due to it being an abrupt and expensive initiative that they were not prepared for (Toquero, 2020). Students incurred extra cost to acquire the needed technological devices and internet services to be able to engage in online learning, which confirms the expensive out-of-pocket expenditure on internet data bundles that medical and pharmacy students in Nigeria also experienced (Adebisi et al., 2020). This partly contributed to the resistance met by online learning during the COVID-19 pandemic.\n\nAlso, due to the practical nature of the training of health professionals, most participants were very sceptical about the ability of online learning to adequately prepare them for future health care practice. For instance, they cited the loss of clinical skills training, laboratory training and the usual clinical rotations in hospitals due to social distancing as major setbacks to their skills development during the COVID-19 pandemic as also shown in a study by Cecilio-Fernandes et al., (2020). Our study further established the findings of previous studies that indicated that online learning poses major challenges to the training of future health professionals (Adebisi et al., 2020; Ferrel & Ryan, 2020).\n\nTime constraints and the overload of coursework were major effects of COVID-19 lockdown on the learning and professional development of participants in this study. Due to the closure of schools as part of the measure to reduce the spread of the COVID-19 virus, students stayed home for months without any academic work. This delayed the completion of the academic year and placed pressure on students and faculty to do more work in a limited time to catch up on the time lost. This was similar to the experiences of medical and pharmacy students in Nigeria whose studies were also halted, and major professional examinations suspended, due to their government’s measures to curb the spread of the virus (Adebisi et al., 2020). This could result in low performance in the professional examination post-pandemic and the general competency of these future health professionals and must be given the needed attention.\n\nOne challenge also revealed in this study is the lack of supervision and guided learning. The social restrictions during the pandemic took away the face-to-face interactions which included lectures, tutorials, skills training, laboratory training and hospital visitation sessions that students engaged in under the regular PBL module. The shift to online learning took away the guided learning and supervision students usually received from their lecturers in the regular PBL module. It also made it difficult for students to ask questions and get guidance on certain critical topics they found difficult to understand. This made some students feel lazy and less committed to their studies during the lockdown, as found in similar studies where medical students had difficulties in concentrating and learning effectively (Aftab et al., 2021; Cecilio-Fernandes et al., 2020). This poses a threat to the competencies and readiness of students in their future profession.\n\nQuite similarly, the closure of schools due to COVID-19 and the use of online learning during this period, resulted in less interaction between students and their lecturers as well as their peers. Participants indicated that they missed the regular skills training, laboratory training, hospital visitation and tutorial sessions that allowed them to get practical training and direct interaction with lecturers as well as their peers. As found in other studies, our study revealed that the use of the online lectures and demonstrative videos could not address the needed practical skills students gained from observing and engaging directly with their lecturers (Cecilio-Fernandes et al., 2020; Ferrel & Ryan, 2020). The collaborative learning at tutorial sessions was also missing and that took away the peer supported learning and social bonding of meeting in groups for tutorials. Thus, the social relations and mental well-being of students which is relevant for future practice was negatively affected (Adebisi et al., 2020).\n\nThe COVID-19 restrictions which led to the closure of schools also limited students’ accessibility to the physical library and other learning resources on campus. Health professionals require certain competencies and skills to be able to work diligently in their profession, therefore their training in school needs to equip them with the best of these competencies and skills. Access to library and other relevant resources complements the lectures and practical training to equip students with the needed knowledge and competencies for their professional practice. This confirms the findings in Nigeria which identified that it was difficult for students to find e-sources for some practical pharmacy and medical courses during the pandemic (Adebisi et al., 2020). The pandemic therefore challenged the comprehensive professional development of health professional students.\n\nAnother worrying situation students faced during the COVID-19 pandemic was the unfavourable learning environment at home. As schools were closed, students had to stay home for some months and resort to online lectures and self-study to cope with their academic work. According to our study, the home did not provide the best environment for effective learning for most students. There were several distractions like chores and other family engagements that did not give them the best study time. Most students also lived in communities that did not have good internet connectivity. Such students were unable to participate effectively in the online lectures the university resorted to. This resonates with in the findings of a similar study in Brazil (Cecilio-Fernandes et al., 2020). In addition to these challenges, frequent power outages were said to have affected the learning experiences of medical and pharmacy students in Nigeria during the pandemic (Adebisi et al., 2020). The social restrictions of COVID-19 therefore hindered the conducive learning environment needed for effective learning which further translates into effective professional development for practice.\n\nAmidst reports of the negative impact of COVID-19 on learning among students of health profession at the UDS, a few positive experiences were also identified. Generally, in this study, one positive impact of the online learning system adopted by the institution during the pandemic was the reduction in the person-to-person interactions, which minimized the spread of the virus among the university community as seen in other studies too (Anderson et al., 2020). This resonates with the findings of other studies that identified the large shift to online instruction as a means of limiting face to face interaction between teachers and the students, to prevent the spread of the virus and preserve lives for the needed future human resources (Toquero, 2020).\n\nAlso, about 12% of the study participants (n=23, 12%) believed that online learning during the COVID-19 pandemic was good. It helped both faculty and students to adapt innovative teaching and learning methods. Just as it was identified by Toquero (2020), this study also revealed that the online learning system adapted during the COVID-19 pandemic caused students to learn and make use of new learning modes to keep up with their learning. Thus, in a positive way, the situation challenged students and faculty to get out of their comfort zone of classroom interaction, to adapt other new technological methods of teaching and learning. Some participants stated that online learning will be adequate to provide them with the required competencies as health professionals if improved electronic learning systems are put in place by the university.\n\nSimilarly, the adaption of e-learning during the pandemic also made learning a more student-centred and self-motivated process for students. Our study found that amid the pandemic, students had more time on their hands to engage in self-directed learning. There were no physical classroom interactions which meant that students did not have to face the challenges of travelling to campus as well as missing and rescheduling lectures due to unavailability of lecture rooms or lecturers. This gave students enough time to do more self-studies. Students had more time to study on their own, at their own pace and in the comfort of their homes. This improved personal studies for some students as they were in better control of their studies and were able to explore new learning opportunities to learn new things as also shown in the findings of a similar study in the Philippines (Toquero, 2020).\n\n\nConclusion\n\nPractical clinical training is an essential part of health professional training. In the wake of technological development and innovative learning, it will be a good thing for higher educational institutions (including health professional training institutions) to identify and utilize innovative ways of teaching practical and clinical skills. This will help maintain the standards and quality of health professional training, even in the wake of other pandemics in future. Educational institutions need to improve technological and internet facilities in their institutions to improve online teaching and learning in this technological era. It is also important for faculty and students to have an effective system of communication in order to give and receive feedback and the needed support with online learning.", "appendix": "Data availability\n\nZenodo: Learning Experiences during COVID-19 lockdown, https://doi.org/10.5281/zenodo.7643568 (Amalba et al., 2023)\n\nThis project contains the following underlying data:\n\n• Learning Experiences during COVID-19 Lockdown.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\nAuthors wish to thank all students who spent their time filling the online items.\n\n\nReferences\n\nAdebisi YA, Agboola P, Okereke M: COVID-19 Pandemic: Medical and Pharmacy Education in Nigeria. Int. J. Med. Stud. 2020; 8(2): 162–164. Publisher Full Text\n\nAftab M, Abadi AM, Nahar S, et al.: COVID-19 Pandemic Affects the Medical Students’ Learning Process and Assaults Their Psychological Wellbeing. Int. J. Environ. Res. Public Health. 2021; 18(11): 5792. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmalba A, Amoore BY, Kpebu SEA, et al.: Exploring Ghanaian medical Students' Learning Experiences during the COVID-19 lockdown. [Dataset]. 2023. Publisher Full Text\n\nAnderson ML, Turbow S, Willgerodt MA, et al.: Education in a Crisis: The Opportunity of Our Lives. J. Hosp. Med. 2020; 15(5): 287–289. PubMed Abstract \| Publisher Full Text\n\nCecilio-fernandes D, Cândida M, Parisi R, et al.: The COVID-19 pandemic and the challenge of using technology for medical education in low and middle income countries.2020; 1–7.\n\nFerrel MN, Ryan JJ: The Impact of COVID-19 on Medical Education. Cureus. 2020; 12(3): e7492. Publisher Full Text\n\nSintema EJ: Effect of COVID-19 on the Performance of Grade 12 Students: Implications for STEM Education. Eurasia J. Math. Sci. Technol. Educ. 2020; 16(7): 1–6. Publisher Full Text\n\nToquero CM: Challenges and Opportunities for Higher Education amid the COVID-19 Pandemic: The Philippine Context. Pedagogical Res. 2020; 5(4). Reference Source" }	[ { "id": "187318", "date": "18 Aug 2023", "name": "Linda Tsevi", "expertise": [ "Reviewer Expertise Quality assurance in private higher education", "online 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\nComments to authors: The article examines salient information relating to experiences of students during COVID-19 lockdown in a higher education institution in Ghana. There are some insights and also some problems which are discussed as follows:\nThe clarity of the English language in the paper needs to be improved to enhance readability. There are a number of word repetitions in constructed sentences. It is strongly suggested that the authors use a professional language editor to improve the English and ensure the smooth readability of the article.\nIntroduction:\nThe ‘Introduction’ needs substantial work done on it. For instance, the introduction is written as if the pandemic is still in force. Since the WHO has indicated that COVID-19 is no longer a pandemic, and Ghana, as a country has lifted its COVID-19 restrictions, the introduction should reflect that update. The introduction will be easily readable in the past tense with updated information as most of the happenings indicated, are not being reckoned with in 2023.\n\nData in the ‘introduction’ should be restructured. For example, available statistics indicate that there were more than 500,000 COVID-19 related deaths globally. In the United States of America alone, the deaths were more than 500,000.\n\nPage 3. Line 24 - Authors should please change ‘According to literature’ to ‘Research indicates’.\n\nPage 3, paragraph 4 – Authors indicate ‘growing anxieties about ability of online studies…’ But the reader is not made aware of some of these ‘growing anxieties.’\n\nAuthors further indicate that ‘…very few studies have been conducted…’. This assertion is inaccurate as there are a number of related research done, even in the Ghanaian setting. A related paper is titled ‘COVID-19 and Medical Education in Ghana: Assessing the Impact’ by Asumanu E and Tsevi, L. There are additional related studies undertaken. The citations in the ‘introduction’ are scanty.\n\nMethods:\nParts of the methods are too verbose and require substantial rework.\n\nPage 4, Second paragraph, the authors indicate that it is a qualitative study, however the article has substantial quantitative information when one reads further. Authors should indicate if it is a mixed methods study.\n\nAuthors should explain all acronyms used in the paper and rewrite this section.\n\nThe Methods section does not have any reference/citation. This an academic paper and not an opinion piece so there is the need for citations to buttress statements made.\n\nResults:\nThe themes under the results section read like topics. The themes should give the reader a clear sense of content. The information in the results section needs to be adequately synthesized and succinct.\n\nThe way the quotations are interspersed in the paper make their reading very confusing. The reader cannot tell whether the quotation is from one participant or more participants. Authors need to do a lot of rewriting here.\n\nPage 5. No reference is made about Table 1 in the paper.\n\nThis section was not concluded properly. The information ends abruptly.\n\nDiscussion:\nThere is a vast disconnect in the high writing quality in this section when it is compared to the previous sections where the writing quality is poor. It reads as if this section was written by a different person.\n\nReferences:\nThe references are too scanty in-text. Authors require additional citations on studies done in related areas.\n\nGeneral Comments:\nThere is no proper review of literature in this paper so one wonders how the research questions were arrived at.\n\nAbstract needs to be rewritten to reflect the revised contents of the paper.\n\nThis paper requires substantial work before it can be considered for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-489
https://f1000research.com/articles/10-438/v1	03 Jun 21	{ "type": "Research Article", "title": "Factors affecting students’ entrepreneurial intentions and their differences based on gender, tribe, and parents’ occupation: a cross-sectional study", "authors": [ "Saidun Hutasuhut", "Thamrin Thamrin", "Reza Aditia", "Thamrin Thamrin", "Reza Aditia" ], "abstract": "Background: This study examines the impact of entrepreneurial knowledge and self-efficacy in increasing students' entrepreneurial intentions and their self-efficacy. Differences were analyzed in entrepreneurial intentions based on gender, tribe, and parents’ occupation. Methods: The data used in this research was collected from a survey, done by distributing questionnaires electronically to students of the Economics Education Study Program, Faculty of Economics, State University of Medan in the semesters 3, 5, and 7, (n = 307), with a response from 260 individuals. The analytical test used was the PLS-SEM and nonparametric Mann-Whitney and Kruskal Wallis tests. Results: Entrepreneurial knowledge and self-efficacy positively and significantly increased entrepreneurial intentions. Self-efficacy was not proven to act as a moderating variable, and there was no difference in entrepreneurial intentions between genders, tribes and parents’ occupations. Conclusions: There is a need to enhance the role of the family, so that women's entrepreneurial intentions will not fall lower than men’s. It should be noted, however, the role of the family was not discussed in this study. This research implies that it is necessary to embrace innovative entrepreneurship education, so as to increase the entrepreneurial intentions of women and reduce gender inequality in entrepreneurial activities. Further research needs to be conducted on the role of culture-based families in preparing their sons and daughters to become entrepreneurs.", "keywords": [ "Entrepreneurial intentions", "Gender", "Parents’ job", "Self-efficacy", "Tribe" ], "content": "Introduction\n\nThe prevailing patriarchal culture in Indonesia positions men as the bread winners of their families, while women are relegated to taking care of the household. This fact is very obvious from the Gender Empowerment Index in Indonesia in 2019, which is 75.24.1 Thus, one can say that the Indonesian patriarchal culture places obstacles on the pathways of women that hinder them from entering into the business world. Over the last three decades, entrepreneurship has emerged as an economic power in the world,2 and it has become an essential element for the economic growth and development of European countries.3 However, for developing countries, attention should be paid to factors that can influence an individual's intention to start a new business.4\n\nCurrently, the number of female students in Indonesia is 51.18%, which is higher than male students' at 48.82% (PDDikti.go.id). Therefore, this presents an excellent opportunity to increase the role of women in entrepreneurship through higher education. The readiness to enter the business world depends on the entrepreneurial intentions of those involved, and previous research has shows that, there are differences in entrepreneurial intentions between men and women,4,5 showing that. Entrepreneurial intentions of men are higher than womens.7–9 In particular, fear of failure has been highlited as a barrier for women who are non-entrepreneurs to release their entrepreneurial intentions.10\n\nIn recent years, there has been an increasing interest in the roles of cognitive approach and self-efficacy, since there is a direct relationship between educational background, knowledge through behavioral characteristics, and entrepreneurial intentions.11 Entrepreneurship education is essential because it significantly increases entrepreneurial knowledge,12 including forming, planning, running, or developing a business, which are skill that an entrepreneur needs. In addition, moderating the influence of gender on individual entrepreneurial orientation is required,13 and Sajjad & Dad14 have found a cultural connection to entrepreneurial intentions. Another factor that determines entrepreneurial intention is the level of self-efficacy. Self-efficacy is a belief that encourages individuals to do and achieve something.15 Self-efficacy can make someone have the desire to do something because they have the knowledge, skills, and ability to face various problems. Also, it is known that self-efficacy has a positive and significant effect on entrepreneurial intentions.16,17 Generally, parents expect their children to continue in the line of their professions, especially when they retire. Haq & Setiyani18 explained that entrepreneur parents influence their children's intention to become entrepreneurs, and a family of entrepreneurs has a significant impact on entrepreneurial intentions in college graduates. These families provide positive experiences that impact on their children's intention to become entrepreneurs.19\n\nThis study examined the predictor variables, which are related to students' interest in becoming entrepreneurs, in order to see if there are differences in entrepreneurial knowledge, self-efficacy, and entrepreneurial intentions between gender, parent's occupation, and tribe.\n\n\nMethods\n\nResearch design in this study is correlational design. In correlational research design, researchers use the correlation statistical test to describe and measure the degree of association (or relationship) between two or more variables or sets of scores and not attempt to control or manipulate the variables.20\n\nThis research included students at the Faculty of Economics, Medan State University, Indonesia. Data collection was carried out from September to November 2020 using an online survey platform.\n\nThe study population was all 307 students of the Economic Education Study Program, Faculty of Economics, State University of Medan. Only those willing to participate in the study (n = 260) were included. Notifications about this research were given during participants classes in which the students were invited to participate in the study.\n\nQuestionnaires were used to collect data on entrepreneurial knowledge, self-efficacy, and entrepreneurial intentions. The questionnaire measuring entrepreneurial knowledge and self-efficacy variables was adopted from Roxas,12 while the questionnaire measuring entrepreneurial intentions was adopted from the questionnaire developed by Linan & Chen.21 The three questionnaires used a scale of 1-5. A value of 1 means very weak, while a value of 5 means very strong in measuring the questions on each variable, respectively.\n\nThe occupation of parents was divided into entrepreneur and non-entrepreneur (entrepreneur = 1, non-entrepreneur = 0), while gender is divided into male and female (male = 1, female = 0).\n\nThe research data was processed by using Smart-PLS 3.0 and SPSS (version 25). Before statistical analysis was performed, normality assumption was tested using Kolmogorov–Smirnov test, and the result was that the data were not normally distributed. Therefore, the most suitable statistical analysis for the data was a non-parametric approach. To compare participants by tribe, Kruskal Wallis was used; for all other variables, Mann-Whitney was used. Meanwhile SEM-PLS was used to examine subgroups and interactions between variables.\n\nEthics approval was obtained from the Institute for Research and Community service of State University of Medan (approval number, IC220190/IC2RSE/LPPM/2020). Completion of the questionnaire also was taken as consent of the students to take part in the study.\n\n\nResults\n\nA total of 260 students returned the questionnaire;, male participants numbered 52 individuals, while female participants numbered 208 individuals. In total, 129 participants came from the Batak Toba tribe, 34 individuals came from Batak Karo, 15 were from Batak Simalungun, 4 were from Batak Dairi/Pakpak, 35 were Javanese, 7 were Melayu, 6 were Nias, 2 were Chinese, 14 were Batak Mandailing, 7 were Minang, and 3 were Aceh. A total of 4 individuals belonged to other tribes not described above.\n\nFor gender, there was no significant difference in the level of entrepreneurial knowledge, entrepreneurial intention, and self-efficacy (Table 1). When examining the students' tribe, there was also no significant difference between entrepreneurial knowledge, entrepreneurial intentions, and self-efficacy (Table 2). The number of students was predominantly Toba Batak tribe at 49.62% followed by Javanese 13.46%, Batak Karo 13.08%. We observed that the average score of students' entrepreneurial intentions from Chinese tribe was the highest at 4.085, and the lowest was Karo tribe at 3.451 (scale 1-5).\n\nWe observed that students' entrepreneurial intentions based on their parents' occupation have no significant difference. However, the average score of the entrepreneurial intentions, showed that for students whose parents have their own businesses scored 3.72, while students from white collar families scored 3.59. Familied who owned their own businesses consisted of entrepreneurs, farmers, and fishermen. Meanwhile, white collar families cooperated with other people or agencies such as state-owned enterprises employees, civil servants, soldier/police, and private employees.\n\nMeasurement models (outer)\n\nValidity\n\nThe validity testing for reflective indicators was observed from the convergent validity and average variance extracted (AVE). The correlation coefficient between the items score and the construct score (Table 4) showed that the loading factor PK1 - to PK 6 has a value that was above the recommended value of 0.7, so that all indicators were valid and fit the convergent validity. Another method to observe is the discriminant validity, which looked at the square root of AVE. The recommended value was above 0.5. The data showed that all the constructs contained in the research model have values of above 0.5 (see Table 4).\n\nReliability\n\nThe reliability test was done by looking at the composite reliability value of the indicators that measured the construct. The results of different tests and levels of entrepreneurial knowledge, entrepreneurial intentions, and self-efficacy based on gender, tribe and parents' occupation can be seen in Table 5 (composite reliability EI, SE, and EK are all 0.9). Reliability test can also be strengthened with Cronbach's Alpha, and for all constructs it is > 0.6.\n\nStructural model (Inner)\n\nThe inner model explains whether the research model that is built is a good model. In Table 5, the R-Square value that is obtained is 0.383. This explains that entrepreneurial knowledge and self-efficacy can explain entrepreneurial intentions by 38.3% and the remaining 61.7% is explained outside the research model.\n\nThis study examines whether entrepreneurial knowledge and self-efficacy play a role in increasing entrepreneurial intentions and it also examines whether self-efficacy act as moderating variables. The results obtained were: (1) Entrepreneurial Knowledge has a positive and significant effect on entrepreneurial intentions, and this can be seen from the P value, which is 0.000 (< 0.05); (2) The Path Coefficient value is positive, which is 0.268, and which indicates that the direction of the relationship between entrepreneurial knowledge and entrepreneurial intentions is positive; (3) Self-efficacy is proven to have a positive and significant effect on entrepreneurial intention, where p value is 0.000 (< 0.05).\n\n\nDiscussion\n\nThe desire to increase women's participation in Indonesia's political and economic sectors can be done through higher education. This desire gained momentum as women's participation in higher education continued to increase and women's achievements were on par with men. In fact, it has been observed that women's academic achievement is higher than men.22 In terms of self-efficacy, there is no difference between genders. Instead, women have a more adaptive approach when doing learning tasks.23,24 The problem lies in increasing women's intention to be active in the economic field and how to eliminate the fear of failure, which is a barrier to entering the business world. This study analyzed whether there are differences in the level of entrepreneurial knowledge, entrepreneurial intentions, and self-efficacy between genders. However, it was observed that there is no significant difference between the two. When viewed from the average score of entrepreneurial intentions, women have a slightly higher score, with a score of 3.69, as compared to men who have a score of 3.65. This is a valuable asset that should encourage women to become entrepreneurs, because the results of previous researches stated that the entrepreneurial intention of male students was higher than female students'8,9,25 as well as women having lesser intentions to starting a business.26 Therefore, this study has implications for how to design entrepreneurship education that can change the orientation of female students to become entrepreneurs.\n\nIn Indonesia, women are more interested in becoming teachers than entrepreneurs. In 2020, the number of female teachers was at a percentage of 69.84%, while that of male teachers was 30.16%. At the tertiary level, female students in education majors are also higher in number. This situation buttresses the fact that women's intentions to become entrepreneurs are still low. Davis & Shaver27 explained that women are less likely than men to increase entrepreneur numbers. One solution that can be proffered is for the government and all stake holders to provide entrepreneurship education to women so as to increase their entrepreneurial intentions. This is important because as Oosterbeek et al.28 and Westhead & Solesvik29 opined, entrepreneurship education is designed to be better able to increase the entrepreneurial intention of the male students. Gender stereotypes may make women to consider themselves as less competent than men in entrepreneurship.30 Not only that, barriers to women's entrepreneurship can be in the form of social, economic exclusion, and inequality.25\n\nIn this study, the respondents were divided into 12 tribes. The Toba Batak tribe consisted of the majority of the study population (49.62%), which was almost half of the respondents. This study found no significant differences in terms of the level of entrepreneurial knowledge, entrepreneurial intentions, and efficacy when viewed from the category of tribe. However, when viewed from the average score for the three aspects, it would be observed that the Chinese tribe had a higher scores than other tribes. However, the accuracy is low due to the minimal number of samples for the Chinese tribe. This information aligns with business practices so far, at least in Indonesia, which is more dominated by Chinese ethnic groups.31 When analyzed further, the number of students' in the ethnic groups of the Batak tribe was far greater, consisting of the Toba Batak, Karo, Simalungun, Dairi/Pakpak, and Mandailing Batak. The number of Batak respondents was 196 (75.38%). The Batak tribe adheres to traditional culture. In the patrilineal system, the men's position is more dominant than the women's position, and boys will become family heirs.32,33 Therefore, this inequality gives rise to the following question: does this position harm women's entrepreneurial intentions? Our study did not analyses this question; however, it will be interesting to further study the situation, especially the role of families in preparing their offspring for the future. Are there differences in educating children from various tribes? Is there any tribe that focuses more on educating its children to become entrepreneurs? These are questions that need to be answered.\n\nTable 1 shows no difference in entrepreneurial knowledge, entrepreneurial intentions, and self-efficacy between female and male students. It is hoped that women's entrepreneurial intentions will increase in order to encourage them to be better prepared to become entrepreneurs. This is because previous research show prove that men and women are both high achievers, but men are more likely to be accepted when applying for a job.24 Furthermore, women and men are treated differently in the workplace. Sometimes, women are treated less favorably than men.34 This situation should challenge women to focus more on entering into entrepreneurship rather than applying for jobs.\n\nEntrepreneurial intentions can also be seen from the perspective of the correlation between parents' work and the level of their children's entrepreneurial intentions. This is considered essential because it helps us to know how families educate children and prepare them for the future. Family is an effective and efficient place to build entrepreneurs, and the family can be a model for entrepreneurs.35 Parents who work as entrepreneurs are also known to be one of the factors that encourages children's intentions to become entrepreneurs.36,37 An entrepreneur family background encourages a higher entrepreneurial intensity score.38 Family support is also known to be positively related to the success of women entrepreneurs.39\n\nBased on Table 3, there is no difference in the entrepreneurial intentions between students who have entrepreneur parents or non-entrepreneur parents. This indicates that the work of parents has no impact on the level of students' entrepreneurial intentions. If we look at the average score of entrepreneurial intentions, students who have entrepreneurial parents only have a score that is 3.62% higher. This research's findings provide an early indication that parents do not direct their children to follow their work. On the other hand, it seems that parents often give freedom to their children to determine their own future work. The results are quite impressive because there is no difference in the entrepreneurial intentions, while other researches generally state that women's entrepreneurial intentions are lower.29,40 However, further investigation needs to be done to determine whether there is a role for informal education that takes place within the family system.\n\nFrom the results of the hypothesis testing, entrepreneurial knowledge has a positive and significant effect on entrepreneurial intentions. This explains that people in charge of running a business are better able to run the business. This condition encourages higher entrepreneurial intentions. The level of entrepreneurial learning achievement also contributes to students' entrepreneurial intentions,41 because entrepreneurship learning achievement is a description of the entrepreneurial knowledge that is owned.\n\nEntrepreneurial knowledge can be increased through entrepreneurship education. The European Union provides entrepreneurship education to equip knowledge and encourage entrepreneurial careers' attractiveness to the younger generation.42 Entrepreneurship education can develop entrepreneurial intentions.30,43–48 This research strengthens the theoretical foundation that increasing students' entrepreneurial intentions can be done by implementing entrepreneurship education. Entrepreneurship education should not be limited to business education, but it can start from primary education to higher education with a design that is adjusted to the level of education and the clusters of knowledge being held.\n\nFurthermore, self-efficacy is proven to have a positive and significant effect on entrepreneurial intentions. Thus, this study's results strengthen the research that stated that self-efficacy is positively and significantly related to entrepreneurial intentions.16,17 When students possess self-efficacy, it helps them to build a belief that enables them to do and achieve things. This belief is what drives the intention to become entrepreneurs. These findings explain that increasing entrepreneurial intentions can be done by increasing self-confidence. Self-efficacy can be built by increasing knowledge about business management through apprenticeships in the business world and successful entrepreneur models.\n\nMoreover, when the self-efficacy variable was used as a moderating variable for the entrepreneurial knowledge variable on entrepreneurial intentions, it was found that self-efficacy did not play a role in increasing entrepreneurial intentions. According to Bandura's15 definition of self-efficacy, self-efficacy refers to the belief in a person's ability to carry out the necessary actions to produce something. The cause of self-efficacy is not a moderating variable. It can be presumed that the level of self-efficacy is influenced by the level of knowledge about managing the owned business.\n\nSince our research did not address the perspectives of families, we do not know their role in preparing their children for their future professions, therefore the data obtained was limited to only students' perspectives. However, this issue is increasingly important because we need to know the differences in family roles based on ethnicity.\n\n\nConclusions\n\nThis study examined the impact of entrepreneurial knowledge and self-efficacy on entrepreneurial intentions and whether self-efficacy acts as a moderating variable. It also analyzed the level of knowledge, self-efficacy, and entrepreneurial intentions based on gender, ethnicity, and parent's occupation. This study's findings are: (1) Entrepreneurial knowledge and self-efficacy positively and significantly affect entrepreneurial intentions; (2) Self-efficacy is not proven as a moderating variable; (3) The level of entrepreneurial knowledge, self-efficacy, and entrepreneurial intentions did not differ significantly when it was observed between gender, ethnicity, and parents' occupation. Thus, efforts to increase women's role in entrepreneurship won't be too difficult to achieve because the entrepreneurial intentions of women are not lower than the men' as in previous studies. Nevertheless, we need to know the impact of informal education that occurs within the family system. Also, it will be interesting to know the differences that abound in families educating their children based on culture (patrilineal) and parents' work. This study's results cannot be generalized because it only involved one field of science, namely economic education. However, this study provides information on the importance of entrepreneurship education, so as to increase the entrepreneurial knowledge and self-efficacy of women. For this reason, it is necessary to create entrepreneurial education designs that can increase entrepreneurial intentions, especially for women.\n\n\nData availability\n\nFigshare: Students entrepreneurial intentions (sav).sav, https://doi.org/10.6084/m9.figshare.14578092.v1.49\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors thank for the willingness of respondents to filling out the questionnaire.\n\n\nReferences\n\nUNDP: Human Development Report 2019: beyond income, beyond averages, beyond today. 2019.\n\nRaposo M, Do Paço A: Entrepreneurship education: Relationship between education and entrepreneurial activity. Psicothema. 2011; 23(3): 453–457. PubMed Abstract\n\nFeder E-S, Niţu-Antonie R-D: Connecting gender identity, entrepreneurial training, role models and intentions. Int. J. Gend. Entrep. 2017. 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Publisher Full Text\n\nNowiński W, Haddoud MY, Lančarič D, et al.: The impact of entrepreneurship education, entrepreneurial self-efficacy and gender on entrepreneurial intentions of university students in the Visegrad countries. Stud. High. Educ. 2019; 44(2): 361–379. Publisher Full Text\n\nPaul Dana L: The education and training of entrepreneurs in Asia. Educ. + Train. 2001; 43(8–9): 405–416. Publisher Full Text\n\nSitompul R, Alesyanti, Ridwan M: Domestic violence as initiated by Batak culture in East Medan, Indonesia. J. Hum. Behav. Soc. Environ. 2020; 30(7): 835–842. Publisher Full Text\n\nVergouwen JC: The social organisation and customary law of the Toba-Batak of Northern Sumatra. Springer Science & Business Media. 2013.\n\nCleveland JN, Vescio TK, Barnes-Farrell JL: Gender discrimination in organizations, In: Discrimination at work Psychology Press; 2013, pp. 177–204.\n\nOoi KY, Ahmad S: A Study among University Students in Business Start-Ups in Malaysia: Motivations and Obstacles to Become Entrepreneurs. Int. J. Bus. Soc. Sci. 2012; 3(19): 181–192. Publisher Full Text\n\nGerry C, Marques CS, Nogueira F: Tracking student entrepreneurial potential: Personal attributes and the propensity for business start-ups after graduation in a Portuguese university. Probl. Perspect. Manag. 2008; 6(4): 46–54.\n\nNishantha B: Influence of Personality Traits and Socio-demographic Background of Undergraduate Students on Motivation for Entrepreneurial Career: The Case. of Sri Lanka. Endeavor to Dev. Educ. Entrep. Initial Exp. from Univ. Colombo. 2009; 49(2).\n\nPalmer C, Fasbender U, Kraus S, et al.: A chip off the old block? The role of dominance and parental entrepreneurship for entrepreneurial intention. Rev. Manag. Sci. 2019: 1–21. Publisher Full Text\n\nPowell GN, Eddleston KA: Linking family-to-business enrichment and support to entrepreneurial success: do female and male entrepreneurs experience different outcomes? J. Bus. Ventur. 2013; 28(2): 261–280. Publisher Full Text\n\nSantos FJ, Roomi MA, Liñán F: About gender differences and the social environment in the development of entrepreneurial intentions. J. Small Bus. Manag. 2016; 54(1): 49–66. Publisher Full Text\n\nHutasuhut S, Irwansyah AR, Aditia R: Impact of business models canvas learning on improving learning achievement and entrepreneurial intention. Cakrawala Pendidik. 2020; 39(1): 168–182. Publisher Full Text\n\nJakubczak J, Rakowska A: The role of education and cuture in the development of youth entrepreneurship in Europian Union. Manag. Knowl. Learn. Internaional Conf. 2013: 997–1004.\n\nIsmail M, et al.: Entrepreneurial Intention among Malaysian Undergraduates. Int. J. Bus. Manag. 2009; 4(10): 54–60. Publisher Full Text\n\nKusmintarti A, Thoyib A, Maskie G, et al.: Entrepreneurial characteristics as a mediation of entrepreneurial education influence on entrepreneurial intention. J. Entrep. Educ. 2016; 19(1): 24–37.\n\nPatricia P, Silangen C: The effect of entrepreneurship education on entrepreneurial intention in Indonesia. DeReMa (Development Res. Manag. J. Manaj.) 2016; 11(1): 67–86. Publisher Full Text\n\nPremand P, Brodmann S, Almeida R, et al.: Entrepreneurship education and entry into self-employment among university graduates. World Dev. 2016; 77: 311–327. Publisher Full Text\n\nTessema Gerba D: Impact of entrepreneurship education on entrepreneurial intentions of business and engineering students in Ethiopia. African J. Econ. Manag. Stud. 2012; 3(2): 258–277. Publisher Full Text\n\nZhang Y, Duysters G, Cloodt M: The role of entrepreneurship education as a predictor of university students' entrepreneurial intention. Int. Entrep. Manag. J. 2014; 10(3): 623–641. Publisher Full Text\n\nHutasuhut S, Aditia R: Students entrepreneurial intentions (sav). 2021. Publisher Full Text" }	[ { "id": "86581", "date": "28 Jun 2021", "name": "Emőke-Szidónia Feder", "expertise": [ "Reviewer Expertise Entrepreneurial intentions", "International entrepreneurship", "Strategic orientations", "International business" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research paper addresses the potential roles of entrepreneurial knowledge and self-efficacy in fostering entrepreneurial intentions. Furthermore, the study considered to analyse if there are significant differences between groups based on gender, tribe, and parents' occupation.\nBased on my opinion, the following observations should be considered and major improvements should be realised to the current form of the research paper:\nA more detailed background within the Abstract would be useful for readers.\n\nOriginality-related aspects should be emphasized within the paper.\n\nModerate editing of language and style is required.\n\nAuthors state the following: ”Gender Empowerment Index in Indonesia in 2019, which is 75.24.1”. The reference in the text is to the UNDP 2019 report, data in that report is for 2018 (report published in 2019). Also, there is a newer version/update on information for 2019 in the UNDP Report 2020 (http://hdr.undp.org/en/2020-report). Either year or reference should be corrected.\n\nThe literature review should be extended. There are several studies in the business/management field and psychology analysing the influencing role of knowledge, competencies, education, and of self-efficacy on enhancing entrepreneurial intentions. Controversial results should be highlighted, and even previous results of researchers in the field from Indonesia should be presented.\n\nAuthors state: ”This study examined the predictor variables, which are related to students' interest in becoming entrepreneurs...”. My observation would be that there are several potential predictors, while the authors considered just two of them (knowledge and self-efficacy). The phrase should be reconsidered by including some predictors or specific predictors.\n\nAuthors state: ”The questionnaire measuring entrepreneurial knowledge and self-efficacy variables was adopted from Roxas,12 while the questionnaire measuring entrepreneurial intentions was adopted from the questionnaire developed by Linan & Chen.21”. In my opinion, the authors referred to measurement scales, not questionnaires. Generally, researchers apply one questionnaire with several measurement scales. If there are multiple questionnaires in this case, several critical aspects should be discussed, like how they match between the three questionnaires they mention for the same respondent, how they stock personal individual data for matching, privacy aspects (GDPR), and so on.\n\nWithin the research model depicted in Figure 1, self-efficacy appears both as a predictor and moderator. In the case of a conducted research, self-efficacy can be a moderator for the causal relationship between knowledge and entrepreneurial intention; it cannot influence and moderate its own effect at the same time.\n\nRegarding Table 1 and 2, the authors should think about how relevant it is to compare a group of 52 and 208 respondents (regarding gender), respectively, and consider tribe, 129 and 4, 3, or 2 respondents.\n\nRegarding the results of hypothesis testing, there are 3 results mentioned, 1 and 2 regarding the same predictor. The authors should discuss either together or separately the sign and significance level of the causal relations in the case of the two predictors.\n\nRegarding the Discussion, if there are no differences between women and men regarding their entrepreneurial intentions, the authors should focus on why these entrepreneurial intentions do not turn into entrepreneurial activity or business creation in the case of women and how should this aspect be improved (e.g. opportunity creation, business context, dedicated programs, available budget/credits, etc.). Is really fear of failure the only blockage factor?\n\nThe Discussion also regards the role of parents and family in fostering entrepreneurial intentions - the case of existence or non-existence of entrepreneurial role models within the family. There is a slight controversy compared to the phrase stated in the Abstract, where the authors claim the following: ”…however, the role of the family was not discussed in this study”.\n\nThere are several limitations that should be mentioned and also some future research directions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9422", "date": "12 May 2023", "name": "Saidun Hutasuhut", "role": "Author Response", "response": "The authors would like to thank the reviewers for their precious time and valuable comments in the second round of review. We have carefully addressed all the comments." } ] }, { "id": "144001", "date": "18 Jul 2022", "name": "Boubker Omar", "expertise": [ "Reviewer Expertise Management Sciences", "Entrepreneurship" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study explores factors affecting students’ entrepreneurial intentions and their differences based on gender, tribe, and parents’ occupation. The topic of this study is very interesting. However, the following points are worthy of consideration to improve the manuscript’s comprehensibility.\nIt is unclear exactly the gap in knowledge that the authors intend to address. Accordingly, in the introduction section, the research gap and the motivation of the study need to be made clearer.\n\nThe literature review section can benefit from more studies that are extensive. Need to include more references. Some example papers that may be helpful are given below:\nMaslakçı A, Sürücü L, Şeşen H: Positive psychological capital and university students’ entrepreneurial intentions: does gender make a difference?. International Journal for Educational and Vocational Guidance1 Ramadani V, Rahman M, Salamzadeh A, Rahaman M, et al.: Entrepreneurship Education and Graduates' Entrepreneurial Intentions: Does Gender Matter? A Multi-Group Analysis using AMOS. Technological Forecasting and Social Change2 Boubker O, Arroud M, Ouajdouni A: Entrepreneurship education versus management students’ entrepreneurial intentions. A PLS-SEM approach. The International Journal of Management Education3\n\nThere is limited support from a specific theory within the hypotheses development. In my opinion, the inclusion of a theory describing the relation between entrepreneurial knowledge, self-efficacy, and entrepreneurial intention could provide a better guide to the study.\n\nThe research method should be clearly described in detail. In addition, the authors are invited to provide more clarification about the sampling technique used in this study.\n\nThe PLS-SEM approach was used to test the hypotheses, it is better to provide the reason for using this approach. In other words, why did you choose PLS-SEM instead of CB-SEM?\n\nIn the results section, the authors are invited to use the Fornell-Larcker criterion and the HTMT ratio of correlations in order to assess the discriminant validity of the measurement model.\n\nIt is necessary to introduce other essential criteria for checking the structural model, including; the effect size, the predictive relevance, and the Goodness-of-fit of the model.\n\nThe limitations and research directions are obviously not sufficient. The authors should include limitations that may be methodological or theoretical. In addition, what do you think a future study should take into account?\n\nSome minor English language editing is needed to correct the various grammatical errors present in the manuscript.\n\nAll the best.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9423", "date": "12 May 2023", "name": "Saidun Hutasuhut", "role": "Author Response", "response": "The authors would like to thank the reviewers for their precious time and valuable comments in the second round of review. We have carefully addressed all the comments." } ] } ]	1	https://f1000research.com/articles/10-438
https://f1000research.com/articles/12-485/v1	11 May 23	{ "type": "Policy Brief", "title": "The ethics of data self-reporting: important issues and best practices", "authors": [ "Niccolò Tempini" ], "abstract": "Background: This article examines the ethics of data self-reporting, in light of the extreme challenges thrown up by the COVID-19 pandemic. In many countries the public was asked to self-report personal social and health data often through the use of mobile apps, as various datasets were mobilised for the purpose of fighting COVID-19. Policy and implications: The article observes a number of dimensions that make the governance of self-reporting projects particularly thorny. The spectrum of self-reporting is extremely diverse. Projects can be scientific, commercial or other, can raise important privacy concerns, can be vulnerable to harm due to organised manipulation and poor governance. They change over time, with a tendency to function creep. The greatest scientific potential is through linkage between heterogeneous data sources; however, these practices are also the source of the highest risks for privacy and harms. Also, people take part for the most heterogeneous of purposes. Closely controlling their hopes, aims, and beliefs is usually beyond reach. This introduces various biases in the data. It can also introduce risks of self-harm because of the conclusions that people might draw. Lastly, participation in self-reporting is not equally distributed across society. When there are benefits associated in participation, it can exacerbate existing inequalities. Recommendations: The article illustrates three areas of emerging best practice in data governance: bottom-up models such as data trusts and data cooperatives; solidarity as a touchstone principle; and proactive research ethics processes and committees beyond public research institutions. These promising innovations deserve experimentation. Conclusions: Flexible and sustained ethical oversight is key. It is important to act proactively instead of reactively. Best practices must be adapted to the local setting and improved over time.", "keywords": [ "self-report data", "data studies", "data governance", "pandemic", "pandemic ethics", "data collection", "big data", "data self-reporting" ], "content": "Introduction\n\nAs the first wave of the COVID-19 pandemic washed through Europe in early 2020, governments desperate to get a grip and researchers and companies hoping to make direct impact into the issue all identified the use of self-report data (data that are collected by participants) as one of the key weapons digitalised societies have to make sense of a health phenomenon that is poorly understood, distributed, and urgently concerning.\n\nData had to be collected for at least a few different purposes, including symptom tracking and illness management; contact tracing (both at the national and private levels); and test result reporting. These purposes could be met in various combinations by many different offerings, and governments endorsed a spectrum of different technical methods for the collection, and legal requirements and implications tied to the use or non-use of these apps (Kitchin, 2020). Data also had to be collected through different levels of direct involvement on the part of the participant. Some of the data collection is automatically performed by the app once the participant has set it up; other data can only be produced if the user responds to specific questions.\n\nThe enthusiastic ‘technological solutionism’ (Morozov, 2014) with which some governments, including the UK, brandished a mobile application as a godsend for keeping the virus at bay can be levelled against their desperation to make their promises believable, the sacrifices digestible, and keep a stable social order at a time when the sky was falling. While uncritical ‘there’s an app for that’ arguments are easily dismissed, the more modest hypothesis, that self-report data can be a valuable resource in specific circumstances, is not. Data self-reporting for the study of health phenomena is a strategy that clinical researchers have been using for decades as well before the rise of the internet and personal computing, patients were asked to self-report symptoms and experience through paper-based questionnaires. The spectrum of patient experience is of course enormously varied so it goes without saying that the same approach will yield highly reliable results with self-reporting about one medical condition, while patients suffering from another condition might not be able to report good quality data.\n\nThe purposes for citizens to become a data contributor can be many; the general (im)mobilisation that a pandemic can bring to a population is an extraordinary force for project enrolment. Contact tracing apps risk reinforce social injustices, better protecting those who are already better off, while interfering with the livelihoods of more precarious, frontline workers (Ada Lovelace Institute and The Health Foundation, 2021). It is inadequate, as Lucivero and colleagues (Lucivero et al., 2021) remind us, that contact tracing apps are imposed on citizens on the grounds of a false dichotomy between individual privacy and public safety (Lucivero et al., 2021).\n\nOutside of pandemic times, data self-reporting has often been associated with individual and collective patient empowerment, and patient movements. In ‘normal’ time, it has long been clear that patients who are able to engage on large scale, distributed data collection exercises are better able to advance demands and push for a reorganisation of the research and policy agenda concerning their condition (Epstein, 1996; Rabeharisoa, Moreira, and Akrich, 2014). Data self-reporting becomes a way, then, to put things into motion ‘from the bottom up.’\n\nHowever, data self-reporting is also the basis on which tech giants have been building empires of surveillance now caught in the public eye for their potential to manipulate and interfere with the lives of individuals, communities and countries. A great deal of criticism has been levied towards project and rhetoric of web-based participation originating in the environs of the global tech industry, and disclosure of personal information, traits and behaviour through automated or manual self-reporting is seen by many as a driver of dis-empowerment and a new colonialism (Zuboff, 2019; H. Ekbia and Nardi, 2014). The pandemic has created opportunities for corporate technology platforms to further penetrate public health systems (Lucivero et al., 2020; Tempini et al., 2022), e.g., the co-development by Google and Apple of contact tracing methods to be used by the National Health Service (NHS) in its contact tracing app; leading to calls of ‘covidwashing’ (Kitchin, 2020); worries of data function creep where the platform monopolists are able to directly or indirectly make use of the data for private initiatives (Lanzing, 2021); and ultimately, the contribution to a general trend were collective dependence from the proprietary technology platforms of monopolists deprives the public from the ability to imagine and develop a future without it (Sharon, 2020).\n\nThis article relates to both the exceptional pandemic times and the dynamics of accelerated technology adoption and intensified data collection that they have generated; and the ‘state of play’ in normal times, when individual have been involved in voluntary and involuntary contribution of data by a range of projects from consumer grade e-commerce technologies to ‘cognitive surplus’ (Shirky, 2010) collective projects. In highly digitalised societies, the topic of the ethics and governance of self-report data is inevitably inexhaustible. But since the exacerbation of such ethical challenges caused by the pandemic and its responses, renewed calls have been made for evidence to be submitted to policy-makers. This brief offers a panoramic discussion of the key issues and articulates recommendations for self-reporting project decision-makers that are based on some of the latest innovations in data governance. The research that underpins this brief was funded by the AHRC under the grant ‘UK Ethics Accelerator: Coordinating and Mobilising Ethics Research Excellence to Inform Key Challenges in a Pandemic Crisis (AH/V013947/1)’. The early two-part report resulting from it informs this policy brief; it was made available on Zenodo (Tempini, 2022a, 2022b).\n\n\n1. Ethical issues\n\nThis section outlines the ethical issues that are outstanding to the pandemic experience and the decades preceding it during which web technology became mainstream. The second section takes stock of innovations that can help improve best practices with a view to tackle challenges outlines in the first section.\n\nData have a tendency to function creep: they often go on to be reused for many other purposes than the ones initially envisaged. The technology used to organise data collection tends to function creep too. This is true both in private and public sector projects. It is worrying because if data are not risk-managed in the best way, disclosures and leaks that lead to individual and group harm are more likely.\n\nIn the private sector, the emergence of surveillance capitalism (Zuboff, 2019), and the lively debate discussing it, has highlighted the deceitful and pernicious intrusions of privacy and the subsequent behavioural manipulations that US tech giants have built their empires from: an almost two decades long thread of surreptitious projects to take ever more and diverse data about users; flanked by an assertive and crafted rhetoric on the value of sharing, emancipation, community, and peer2peer entrepreneurship; that increased the ability of these giants to predict, modify, and generate human behaviour to the point that legitimate concerns have been raised as to the effects of big tech services on just about anything social including politics, markets, and mental health. The cost of surveillance capitalism, as Zuboff observes, is human futures. The model has been imitated by companies throughout the sector to the point that both practitioners and public, in different ways, are failing to imagine how tech can be built without relying on the sale of user profiles and behavioural modification. Innovative organisations focusing on privacy-first products are struggling to be seen and break through the current market chokehold. For this reason, the offer by Google and Apple to collaborate in developing an interoperable framework for contact tracing met mixed reception. In a time of great emergency, it was congenial for national COVID-19 response efforts to develop their apps on top of this tech giant-concerted framework. The UK tried to independently develop the same functionality for its NHS contact tracking app, only to backtrack. The tech giants were praised for munificence and sense of civic responsibility. The framework also employs gold standard privacy-preserving techniques approved by privacy experts. It was easy to see, however, how self-serving the move could be to white-wash the giants’ reputations with the very same broad framework they had been tarnished with – individual-level, comprehensive digital surveillance (Kitchin, 2020). The offer to help fits a history of manipulative attitudes to the public discourse.\n\nThe legitimisation of surveillance technology, methods, and frameworks as a response to the pandemic emergency has been such that even organisations who have been known to the public for nothing but scandals have had a chance at ‘covidwashing’. For instance, Israel’s contact-tracking app was developed by NSO Group, a secretive spyware organisation notorious for its services to authoritarian governments across the world (Kitchin, 2020). Similarly, Palantir and Experian, other actors of the digital economy of questionable ethics reputation, have also taken part. Observing how easily techniques from the most controversial commercial surveillance practices can be transferred all the way to state infrastructure for pandemic response should make us ask what we are dealing with. For companies such as Palantir, the opportunity was not only reputational, but also an opportunity to turn private technology into state infrastructure, with the commercial benefits that can ensue in the long run (Tempini et al., 2022). The means are the translation, adaptation, and re-deployment of technology, methods, and resources used in population surveillance. The aim is to become indispensable infrastructure of public health response and prepare state administration processes for further penetration through new drives of technologically, and organisationally, compatible systems provision. Case in point, after providing an analytics infrastructure crucial to the coordination of the covid response, Palantir now looks set to win an unprecedentedly valuable and wide-ranging contract for the provision of future NHS digital capabilities (Financial Times, 2022) extending from the infrastructure already provided during the pandemic. The strategy is right out of the infrastructure studies playbook.\n\nDespite the scandals revealed by a number of whistleblowers and leaks (e.g., Snowden on the PRISM program (Wikipedia, 2022a) reaction over recent years has been mixed and, so far, shy of introducing game-changing protections; such is the attraction that the public sector feels in respect to these new sources. The single-most impactful law recently issues on the topic, the European Union (EU) (UK) General Data Protection Regulation (GDPR), has had an uncertain track record as a sensitivising device, as broad patterns of use have not shifted. It provided great flexibility in implementation. In it, the opportunity for technology providers’ is implementation fragmentation. If every provider implements the regulation in a slightly different way (as in the myriad different ways to do ‘cookie’ language, explanation, and notification interface), user burden in expressing and articulating privacy preferences is highest. It is possible that it is rather a string of publicised scandals, such as the Facebook/Cambridge Analytica one, involving the threat of election manipulation and disinformation warfare, that has finally started to bite.\n\nInternet researchers, in the Internet and beyond, should also question and account for the ways in which they might be benefiting from such widely criticised ways to generate data, and the ways in which their own practice can relate to function creep issues: “Similarly, harvesting sensitive information from public–private environments such as social media may raise ethical issues, especially for research involving vulnerable populations who may have limited understanding of the implications of disclosing personal information on these platforms” (Clark et al., 2019).\n\nIn the public sector, large trends in networking and ubiquitous computing have brought about an explosion of digital watching as made possible by a highly granular fabric of recording devices; and a network of interoperable databases that are increasingly interconnected for fast, increasingly real-time, networked access. From CCTV cameras, which the UK has led the way in ubiquitous deployment and acceptance, to a panoply of digital listening devices, huge amounts of data can be made available to various agencies from the law enforcement to public administration. The value of data and the pressures to fuel the growth of a competitive digital economy have created incentives for governments to enable the regulated access to previously unavailable data even down to the individual level. The unveiling in Spring 2021 of plans by the UK Government for the regulated access and re-use of NHS England and Wales data (see General Practice Data for Planning and Research (GPDPR) reporting; (Machirori and Patel, 2021)) is the latest of a series of attempts to inject these data in the digital and research sector economies (Vezyridis and Timmons, 2017) and signals the determination on the part of policy makers to find the ‘right’ conditions that will make this digestible to the public. Neoliberal politics and New Public Management ideology of public administration have captured the imagination of policy makers in matters of technology innovation and management. They have made for social, political and financial pressures on state institutions to try to generate value out of any viable public asset. But the protracted occupation by private sector of public infrastructure provision has the effect of stifling imagination and discussion of alternative ways to develop public technology (Sharon, 2020; Tempini et al., 2022). For a long time, scholarship has further pointed out how technology infrastructure and standards have an inertia that makes radical revision as difficult as function creep is easy (Hanseth and Lyytinen, 2010). Once important social and organisational interdependencies have developed over information and communications technology (ICT) that saves as infrastructure, it is very difficult to unseat it. From the care.data debacle (REF) to the latest GPDPR move, NHS data have been one of the fields in which the pressure to data function creep is highest, raising the appeal for government to try to avoid the turbulence of inevitable questioning by trying to get plans pass scrutiny as quickly and silently as possible. As the Ada Lovelace Institute observes, this fits a pattern of a “decide, announce and defend” approach to the public focused on its persuasion instead of its involvement (Machirori and Patel, 2021; Ada Lovelace Institute, 2020). It is a self-defeating strategy, as each time, the plans have been halted by public debate that is only in part focused on the merits and demerits of the envisioned ways of using data and doing research with them; and is focused, in the rest, on public issues of governance, accountability, and trust. The GDPR’s roll out has now been indefinitely frozen (O’Donovan, 2021), as the size of deliberate opt-outs from a distrusting public was concerning. The pandemic has offered a few tell-tales on the issues and risks of data function creep. The UK government attempted to extend regulations of pandemic emergency that loosened constraints on the access to sensitive health data (Tempini et al., 2022; O’Donovan, 2021), but its failure to attend to public trust led to new failure (Wilson, 2021; Bharti et al., 2021).\n\nThe public deserves much better, more responsible approaches to data governance accountability and control of function creep. The public should be involved in the deliberation and articulation of values and aims embedded in projects and infrastructures using data about it (Bharti et al., 2021). Projects should be paramountly concerned with gaining public trust. At a minimum, Kitchin recommends that: “Citizens should know precisely what the app seeks to achieve and what will happen with their data. There should also be safeguards to stop control creep and the technology being repurposed for general or national security, predictive policing or other governance or commercial purposes” (Kitchin, 2020). And project managers should be concerned about the cumulative effects that straining public trust with exploitative or unaccountable data management can have in the long-term. In pandemic times, such risks are accelerated as function creep can ensue very quickly. An example is the COVID-19 Symptom Study, data self-reporting app ZOE (ZOE, 2022), launched by a private company and academics at the start of the first wave. Its funding was quickly secured, for the ensuing two years, by the government. Only few months later, when the research team started publishing promising results over the power of self-report symptom data to predict infections, authors observed the trend for the ZOE data to be “increasingly being linked to the public health response within the National Health Service (NHS) in the United Kingdom” (Drew et al., 2020). In the immediate, this could be seen as a good example of collective mobilisation and ingenuity in the face of exceptional challenge. But it poses concerns over the risks this shift could mean for the livelihood of individuals (See Box 1).\n\nThe ZOE COVID-19 Study is a self-report study of COVID-19 symptoms and patient experience centred around a smartphone app developed by a company, ZOE, and a team of nutrition and epidemiology researchers at King’s College, London (KCL). The app itself is an example of the speed at which existing technology, code and methods can be redeployed for new purposes. As the first wave hit the UK, the company refactored the app it had been developing for the study of nutrition into an app that could be used by covid patients to record their symptoms on a daily basis, for months, so as to be able to estimate the dynamics of the disease as it swept through society. ZOE and the KCL team led by Tim Spector are serial data self-report study leaders, having been known as The British Gut project, where they asked participants to provide funding, samples and data in exchange for personalised information and advice on gut health. The participation of contributors was already limited to the completion of a few well circumscribed tasks, with no direct input in project management and day to day operations; but it was still taken to be one of a crop of web-based projects that were breaking boundaries towards science democratisation (Del Savio, Prainsack, and Buyx, 2016).When ZOE COVID launched it was quickly downloaded by a great amount of people, thanks to the extraordinary momentum granted by the pandemic emergency (Varsavsky et al., 2021; Drew et al., 2020). It was also adopted in other countries (Kennedy et al., 2021). Contributors input demographic data upon registration, then are asked to submit a daily report of symptoms and covid test results. The data could then be used by researchers wanting to study covid patient experience; and estimate its demographic and geographic distribution as well as geographic and case count movements. Studies reported the self-reported data could be used to produce estimates that closely track those gained from best methods, leading researchers to conclude that data self-report can be a valuable complementary resource of data to be used by policy makers and public health officers in coordinating emergency response. They should be particularly useful, they add, to understand the situation in regions where best method estimates are not viable due to lack in testing capacity.The success of the ZOE COVID-19 app in providing a resource of good quality data was outstanding, but there remain open questions as to how feasible (and desirable) an initiative of such proportions would be outside pandemic time. The initiative commanded such extraordinary participation thanks to the extraordinary context in which many people found themselves; and benefited from the lockdowns keeping the population at home, and constant media coverage that kept the issue relevant and into focus for the many users that the app needs to keep daily reporting even when they feel well (to provide a baseline of negative symptomatology). It also benefited from sudden financial backing from the government, as the app was, in the researcher’s own words, “increasingly being linked to the public health response within the NHS” (Drew et al., 2020). As researchers observed (Varsavsky et al., 2021, p5), relatively high numbers of weekly active users are needed to confidently detect relatively small rises in case counts.However, there are various limitations and experts have been split over how much confidence can be put in these data to guide emergency response. Study researchers observe how the self-selected sample of respondents provides for a biased and non-representative sample of the population, with a trend towards more representation from the less vulnerable. It fits the experience of self-reporting initiatives in general. Events like the ‘pingdemic’ that unexpectedly requested so many citizens to self-isolate after technology had determined they had been exposed to a positive carrier could also suddenly pull the rug from under such initiatives. But other interventions can also interfere, such as vaccinations, or being clear of covid after infection, can provide a sense of security that makes some less interested. The same ZOE technology applied to Sweden, a country that had famously resisted the introduction of social distancing measures and lockdowns, had seen a lower uptake and again biased in favour of better-served areas (Kennedy et al., 2021) clustered around the universities running the study. It is potentially a warning sign for an approach that is recommended as valuable complement in the management of the emergency in the less well-resourced region of a country. People who have worse access to testing might be disadvantaged twice, if the surrogate measures also risk being worse.Worries of function creep and surveillance would not be easily assuaged by sudden partnerships with state institutions (Drew et al., 2020). Emergency response is a highly dynamic situation and even when the consequences of measurement are not taken directly to specific individuals, when public health officials are trying to coordinate a response which might include various measures affecting people’s livelihoods, we are already in a situation where self-report data have been repurposed to a new use and become what they were not initially meant to, e.g., a tool for population management. Experts warned about the potential emergence of incentives to lie; of worries of being reported to the police and in general of risk contributing data to the app; the potential consequences of these worries for the quality of data collection and of other related activities such as self-administered tests; and the consequences of the latter on the quality of policy making.\n\nThese are not only ethical concerns. They often become epistemological if people start to take countermeasures which in turn affecting the quality and reliability of the data (Bharti et al., 2021). As about any health researcher knows, opt-outs are not randomly distributed. Just as many other individual preferences and social facts, they introduce bias in experimental and observational research (Teira 2013). When sizeable, they quickly lead to issues statistical representation of the remaining sample. Thus, Bharti et al. (2021) stress how public involvement in the articulation of values and aims of a data project is not only politically beneficial but also leads to better outcomes. The GPDPR is a case in point, as the value of the proposition was tarnished by huge spikes in drop-out rates (O’Donovan, 2021). And as the controversies around the ‘pingdemic’ demonstrated, allowing an app to report information can give a third party the power to interfere with our everyday life at a latter point, on terms one might not be happy with. Perhaps ironically, many deleted the contact-tracing app once it started doing what it was supposed to do and ‘pinged’ them out of circulation. Contact-tracing apps are designed to discipline and reshape spatial movements and social connections (Kitchin, 2020), but they have an uneven impact. Essential workers are likely to face higher costs of ‘protecting others’ policies, if the government does not provide for any other forms of support. The complex dynamics of social systems (Wilson, 2021) mean that these workers will have very high incentives to flout unfair rules. Kitchin notes that data function creep can also be indirect. Location data that is automatically generated, for public health apps, by a smartphone operating system’s location; services can be shared by the operating system with other apps that also use location data; with the result of deepening the surveillance of a user within the ecosystem of data brokers and surveillance capitalism.\n\nResearch with Internet data has been raising ethical concerns related to consent and the distribution of harm and benefits for quite some time. Data science and artificial intelligence (AI) methods often rely on the use of large amounts of real-world data to train machines, and these data have often been taken where they are most easily available, e.g., much of the Internet. Internet users directly generate data as they post, record, and interact over platforms, but data are also continuously generated by the underpinning technological infrastructures as a matter of mere technical operation. The whole array of data generation and storage instances is impossible to keep track of (Clark et al., 2019). Intense debates have been generated over the ethical standards that new forms of digital research should adhere to (Petermann et al., 2022); including questions over the double standards that public- vs private-sponsored research follow. Questions of consent will be important for self-report data collection initiatives. If data could be reused multiple times a whole host of questions are raised as to what could be the appropriate mechanisms for granting consent for uses other than those originally envisaged at the point of data generation.\n\nCalls for ‘blanket’ consent that pre-emptively authorise a broad spectrum of re-use (and the milder version of ‘broad’ consent) have been heavily criticised for their vulnerability to misuse, capture and manipulation. Also heavily undermined have been individualistic arguments that envision each research participant deciding on the use of their own data as the most desirable ideal of data governance – most impractical but also unethical to dump all the responsibility onto isolated individuals. Organisational arrangements that require more complex governance procedures and accountability structures have been better received.\n\nSelf-report data, especially when health-related, are often very sensitive, giving rise to the potential for direct and indirect harm from misuse, manipulation, and profiling. It is thus important for contributors to be able to keep track and access contextually relevant information as to what is done with their data, and what is envisioned for the future. They should also be made aware that parties interested in accessing and using their data, because ethical standards and guidelines as to what are admissible re-uses of individual data change over time and across research domains and institutions, might not always think that contributors have a right to informed consent when:\n\n▪ the data are shared in a public setting seemingly without whatsoever expectation of privacy (for instance, tweets from public accounts)\n\n▪ there are no expectations that participants could be directly harmed from the research (for instance through anonymisation of the data at the point of collection)\n\n▪ when they are working with a private sector company that has served legally-compliant notifications – for instance, a fair processing notice that is GDPR compliant can pre-empt, at once, a whole host of uses and projects the company will undertake with the data.\n\nGiven the higher exposure to diverse and distributed expectations and engagements, high standards should be required of initiatives relying on self-report data. To be meaningful, question of consent should be renewed at major project milestones or updates. Participants should be actively invited to consider whether changing situations fit them and should not be expected to keep track and make sense of change by themselves. For instance, if the governance of a project changes due to change in ownership or management, self-report data should not be considered a conventional asset that can be sold on. Instead, they should be treated as objects that bind different people together in an economy of relational ethics (Prainsack, 2019a, 2019b; Birhane, 2021). Data generation and access should be minimised as much as possible. Many questions require functioning ethical oversight.\n\nSelf-report data collection initiatives can achieve impressive feats thanks to the economics of crowdsourcing, where a large number of contributors each invest only a small portion of their time to a great cumulative effect. However, it is not easy to run these initiatives over a long time, and how to do this will be particularly crucial for those initiatives whose data collection becomes more valuable the more it has taken place regularly and over a long period of time, e.g., data about health events and symptoms used for the understanding the spread of disease.\n\nThe emergency of the pandemic, with the emotional response and mobilisation it generated, along with the lockdowns and the way many people redirected their attention and free time to activities that can be completed at home, created one of the easiest scenarios for a self-report data collection project to succeed. Many people wanted to understand and learn about COVID-19 and needed reassurance and explanations on their daily experience. Many wanted to help. It was easier to accept intrusions of privacy, the boundaries and consequences of which were not fully clear. And still, even in the times of pandemic, several challenges arose to the motivation and distribution of participation. The UK ‘pingdemic’, and the consequent migration of many users deleting the contact tracing app from their phone, spoke of an unresolved tension on expectations and boundaries of the data reporting that contributors are voluntarily enabling; a tension that was only exacerbated by the experimental nature of the methods involved.\n\nA rich literature on participation in data self-reporting offers many points of concern to keep in mind for future self-reporting projects. The seminal ‘ladder of participation’ published by Sherry R. Arnstein (Arnstein, 1969) has since served to highlight how nuanced the concept of participation can be, open to manipulation and, as a result, how appearances can be misleading. An extensive literature has followed to question the concept of participation further. Self-reporting projects distributed over the web are often opaque as to their inner, complex workings, while very public about the moral economy that they want to draw on to mobilise support, with frequent calls to share for the common good, for altruism, emancipation, empowerment of oneself and their kin. On close scrutiny, participatory practices can turn out to be empty, tokenistic, or extractive ‘crowdsourcing’. Kelty and colleagues (Kelty et al., 2015) point out that participation on the web would be better understood as something that is not a linear spectrum along one axis, but rather as something multi-dimensional. In this way, the limited openness to participation of most web projects is easier to observe, with most projects restricting participation to one or few dimensions.\n\nIt is crucial that self-report data collection projects respect the effort and investment put in by contributors to make it possible by relating to them as invaluable partners and project stakeholders. Extractive models popular in the Silicon Valley, that promote the value of sharing and promise empowerment and emancipation, only to extract information from individuals while excluding them from benefits (H. R. Ekbia, 2016) and governance of a project, will come under fire and will be increasingly unpalatable, given the recent mood change in public sphere discussions on these matters. Participation in health research involves a particular kind of labour that involves turning one’s body into readable and available to observation and cognition (Brives, 2013; Milne, 2018; Cooper, 2012); and even in the case of unambiguously commercial projects where crowds are called on to contribute cognitive labour in exchange for financial retribution, as in Amazon’s Mechanical Turk, strong arguments have been raised that point to the exploitation that these models build on (H. Ekbia and Nardi, 2014; Irani and Silberman, 2013; Nardi, 2015).\n\nIt is beyond dispute that many projects have exploited contributors who are asked to volunteer time and information while they are kept out of any relevant sense of ownership of the results. In light of much literature, it is clear that it is difficult to develop a self-report data collection project without controversy. The asymmetries of projects with a very large, distributed base of contributors and a very small team of managers and developers will make for very sensitive politics of contribution and participation. Managers of data self-reporting projects should avoid exploiting a common double standard where the efforts of the contributor base are celebrated and recognised with the language of empowerment and ‘bottom-up’, or ‘patient-led’, research while at the same time, individual contributors are excluded from formal recognition when the research is published in peer-review outlets. Only a few projects have tried to recognise the individual contributors that a crowd is made of through authorship credits, and this not always be possible nor desirable. There are many other ways to formally recognise the contribution of the public in these projects other than scientific credit. Direct involvement in governance and management can be an alternative, as well as having formal and public ways to gather and respond to request, motions and value demands.\n\nIt is important, and even more so once out of pandemic time, for self-report data collection projects to consider very seriously how they can best and actively engage in transparent communication about all aspects of the project, including decision structures and finalities (See Box 2). The projects that do not cause controversy are likely to be those that engage in a sustained dialogue with the base of contributors; explain key changes in governance and direction of the project; demonstrate accountability to the contributor base at least as keenly as they strive towards other stakeholders such as sponsors, third party researchers, or regulators.\n\nHEAL-COVID is a study of post-hospitalisation COVID-19 symptoms and long-term outcomes. At the time of writing, it reports enrolment of over 1,000 patients who had been hospitalised with covid. Patients are asked to self-report data through a questionnaire that has been designed over only a few weeks by an interdisciplinary team of specialists, including specialists of patient participation. The urgency of the pandemic with its hospitalisation surges threatening NHS standards of care had a capacity for accelerating collaboration and overcoming challenges such as questionnaire copyright licensing, that in normal times can hold up the development of an experiment for much longer. Some licenses were obtained at a very quick turnaround. To find at record speed the patient representatives that could offer review and support in research design, the team was able to make use of Cambridge University hospital’s group. Patient involvement in questionnaire design focused the team’s efforts on designing a minimalist questionnaire of a minimum number of items – it was important not only to ask patients for feedback over questionnaire and workflow drafts, but also to have an open space where the patient could ask the questions they had come up with.Experts point out to contributor burden as both an ethical and epistemological challenge. Wasting time and energy of patients who are already challenged by a debilitating disease, many of whom have long-covid, should be avoided. But also, it is an epistemological challenge because over-burdening contributors diminishes the quality of responses, especially so when many patients are suffering from fatigue over a long time. This meant various trade-offs in deciding what symptoms patients should be asked about. What is important to patients from an experiential point of view might be different from other symptoms who could be warning signs of dangerous complications. Tests that are very valuable but very difficult to self-report well (e.g., a ‘six-minute walk test’) could be weighed against other less informative symptoms that are more intuitive and familiar to patients, such as mood changes. From a self-reporting perspective, reporting on symptoms that are important to patients and that patients can at the same time report on reliably was a key principle of questionnaire drafting.As they designed the study at the beginning of the pandemic, researchers had to make assumptions about the kind of patient that would be most likely to participate in the experiment. Experts note how the composition of the team, and the ecology of actors around them (funders, academics) was not representative of society’s demography. All things being equal, this can make the research more vulnerable to biases and assumptions baked in the research design and questionnaires. They also learned that the infection moved through society in unpredictable and ever-changing ways. While the first wave saw ethnic minorities and essential workers such as taxi drivers disproportionately hospitalised, by the time the third wave was sending patients into hospitals, they were disproportionately the unvaccinated. The consequences of these shifts for the validity of the questionnaire and how its items had been selected were unclear.It also makes more difficult to elaborate and take actions from the feedback about the study that is gathered from the participants; this will also come with hidden biases and assumptions, as researchers collect information only from the self-selected few who accepted to participate. Expert found it much more challenging to know about those who are not participating in the study – more likely to be vaccine and covid sceptics. It is unclear what are the consequences of collecting worse data about specific social groups. It is clear that much depends on how consequential the actions that can be taken from the data that are collected can be – a reason for caution when imagining the use of self-reported data in policy-making and crisis response.\n\nData can be used for purposes other than initially envisaged in many different ways. Contributors themselves, once engaged and active in the data collection, can repurpose or imitate the methods and inclusive rhetoric of the projects they joined, and extend aims of the data collection and networking taking place on a self-report data collection system to organise research on matters of their own interest.\n\nThere is a long history of this this kind of initiatives in health research, with perhaps the most historically significant one that captured by the pioneering work by Steven Epstein (Epstein, 1996), describing how in the midst of the US AIDS epidemic, the AIDS Coalition to Unleash Power (ACT-UP) (Wikipedia, 2022b) activists aiming to influence research on HIV and extend access to experimental therapies harnessed existing networks of civil rights activists to coordinate independent research, protest and sabotage.\n\nSince the rise of the web, techniques and methods for organising independent research that exploits available resources, data, and technology have grown more sophisticated. Examples have multiplied as we saw the emergence of self-report data collection beyond the domain of scientific research and into the domains of civic and political action, where data have been used by activists to mobilise evidence in support of their claims (Milan and Velden, 2016; Bruno, Didier, and Vitale, 2014); as well as artistic performances and other individual applications of data collection on the conduct of one’s life, in the culture of the Quantified Self movement (Sharon, 2017). In the field of health research, the self-organisation of data collection through web networks and technology has been called ‘patient-led research’ (Vayena and Tasioulas, 2013) in the wake of some celebrated examples (Paul Wicks et al., 2008; Paul Wicks et al., 2011) that excited commentators about the social web’s potential in scientific production.\n\nExciting as it might seem for ordinary individuals to take knowledge matters into their own hands, there are a number of thorny ethical issues associated with distributed contributors’ ability to organise data collection on the side (Vayena et al., 2015; Tempini and Teira, 2019; P. Wicks, Vaughan, and Heywood, 2014; Ledford, 2018). It is clear that people enrolled on these initiatives often interpret the data thus generated in order to make life decisions with potentially large consequences. For instance, patients might make changes in their treatment course, or take uncontrolled chemicals. And when contributors are many, different aims, expectations, levels of engagements and degrees of literacy on the subject matter are at play. Some will be much more vulnerable than others. They have many questions that they might seek to satisfy with what they find, whether the answer is provided through state-of-the-art methods and project level supervision or not.\n\nThese projects complete lack of ethical supervision, let alone of the sort that has long been required of publicly funded university research. No one will have a view as to who these people might be and how they should be supported. What these initiatives risk creating then is something short of well-developed solidarity, but rather, an ephemeral alliance and confluence of interests between individuals who might then be find themselves to face the consequences alone. The regulatory trend towards allowing patients more freedom to experiment with treatment courses means that the risk calculus is becoming more complex just as patients gain more freedom to do it themselves (Carrieri, Peccatori, and Boniolo, 2018; Navarro, Tempini, and Teira, 2021; Tempini and Teira, 2020).\n\nPandemic time is only likely to exacerbate the problem. With COVID-19 we have seen the emergence in social media and public sphere of a number of theories, since debunked, as to the causes of illness and more or less implausible treatments that are within easy reach of each citizen with a curious mind and misplaced scepticism. Many people are willing to shape actions and medical decisions based on information they find more or less casually. They are mostly on their own evaluating risks and kinds of harms that could arise. Spontaneous and ephemeral instances of self-report data collection are likely to compound this kind of issue, by buying credibility, and time, to implausible theories and methodologies. In several examples, patients eventually self-harm (Paul Wicks, Heywood, and Vaughan, 2012).\n\nSelf-harm is not the only relevant ethical issue. Another important issue is the potential for contributor-led initiatives to interfere with other, better designed clinical research, such as clinical trials, when participants decide to tamper with the experimental protocol by carrying out their actions on the side from it. The result could be the creation of noise, the jeopardisation or slowing down of the best research. These issues can create a rather uncomfortable situation for the managers and developers of the technology platforms these initiatives are relying on. An issue for them will also be how liabilities and best courses of action can be worryingly unclear.\n\nGiven that contributors can invest a significant amount of resources labouring to collect data (Milne, 2018; Cooper, 2012), develop expectations, and can face risk of undesirable consequences of participating in the data collection, it is really important that data are valued and used. As it has been made obvious by great amounts of literature, much of the value that has been ascribed to new kinds of data-intensive data collection, including the collection of self-report data, is due to the assumed possibility of reusing the same data multiple times for different purposes. Given that data can acquire new value when they are linked or juxtaposed with other data and questions, their value could be renewed as many times as people having access to them believe it is worth to do so.\n\nWhile the investment on the part of an individual contributor can be variable and small, the cumulative investment asked of society, or a relevant group within it, can be considerable; the more so the more contributors a project is able to attract. A self-report data collection project, therefore, will face demands of accountability as to the ways in which the data have been put to use; and whether the amount of effort it commanded was worth the good it generates.\n\nIf little is done with datasets that people might have a reasonable expectation more could have been done with, a tension can arise. Scholarship on data self-reporting has thus questioned (Prainsack, 2017; Tempini and Del Savio, 2019; Sharon, 2016) specific projects that harness the rhetoric of empowerment and participation but might not live up to expectations (see Box 3); and asked if those who directly contributing are then excluded from data governance decisions of the data, the incentive to maximise the use of the data might be weaker (Birhane, 2021; Prainsack, 2019b; Ernst Hafen, 2019; Tempini and Del Savio, 2019). Expert practitioners involved in health data self-reporting studies often stress the duty that a project has to make justice to the participant’s burden.\n\nPatientsLikeMe (PLM) is a social media network and platform of online communities centred on the patient experience. It gathers hundreds of thousands of patients suffering from thousands of different conditions. The patients gather to socialise, learn from one another and about their condition, and participate in health research. The platform, unlike most social media, is free of ads. The for-profit company sells research services and access to pseudonymised patient data. Its researchers have published many peer-reviewed publications, some very celebrated for the way they leveraged the web to produce scientific knowledge faster and cheaper than traditional methods. They made PLM one of the most hyped and promising social media in the health space. PLM promised a revolution of health research and care, a model of patient empowerment that would allow them to rewrite the research agenda of pharmaceutical sector, democratising health. This was hoped for especially in respect to ‘orphan diseases’ – conditions that, usually because of low patient numbers, fail to attract the level of investment and research necessary to develop treatments that make an impact. PLM was started as a community for ALS patients by a family affected by the condition who had been at the centre of some of the most dynamic activism and research around the disease – called ‘guerrilla scientists’ their early efforts had already attracted, before the founding of PLM, the attention of a Pulitzer-winning journalist (Weiner, 2004); and documentarists who took their story to Sundance (So Much, So Fast, 2006).The reality of everyday operations at PLM was more challenging than many commentators excited about what the social web could mean for knowledge production might have assumed. The collection of self-reported data from such a disparate base of contributors posed threw up many data quality issues related to the effort to bridge between the world of patient experience, knowledge, language, aims and expectations, and the world of standardised scientific observations, recording and communication, of data structures and taxonomies (Arnott-Smith and Wicks, 2008; Tempini, 2015; Paul Wicks et al., 2010; Frost and Massagli, 2008).The need to collect data that would be worth, at once, for patients and their personal sense of biographical trajectory; and for third parties interested in learning about them for socialisation, scientific or business purposes led to conflicting demands. At play were different definitions of what is a valuable direction for platform and what is necessary burden and attrition (Tempini, 2017). The platform was committed both to a bottom-up revolution of the health research industry; and to the development of a viable, self-sustaining business model in a highly competitive and dynamic health industry, where executive board members and venture capitalists have a way to focus one’s mind. Its monopolistic control over the self-report data, with little direct participation by patients in day-to-day operations and decisions, created an unresolvable tension (Tempini and Del Savio, 2019), and ambiguity towards some genuinely spontaneous patient-led initiatives that threatened to perturb the overall design (Tempini and Teira, 2019).\n\nThere are various reasons why a dataset could not be widely re-used. For instance, in the case of health data, their sensitive nature and the high risk of misinterpretation require not everybody should be given access. Also, the data that is actually collected might not be as good and reliable as initially expected by the researchers who design the exercise. Much depends on the everyday practical circumstances of a self-selected group of contributors, with different expectations, hopes and levels of literacy.\n\nHealth data can be particularly expensive to keep and govern. They require complex infrastructures to protect the data from unauthorised access. This means keeping up with continuously changing security standards and a quickly evolving risk and threat landscape; while at the same time, maintaining the knowledge that is required to ensure that the data remain valuable, of high quality, and that their peculiarities and qualities are well understood by those who directly re-use them (Demir and Murtagh, 2013; Tempini and Leonelli, 2018). In this respect, self-report data pose more challenges because they are often made available in a structural lack contextual of information and awareness of the specific situation in which they were generated by unknown, distributed volunteer contributors. Also, many data self-reporting projects, especially when funded through public research funds, can ran into issues of long-term maintenance and sustainability; it is important that plans are made early on as to how the data will be managed in the long run and how continuous funding and support for data governance functions should be secured.\n\nAt any rate, data self-reporting projects should attend to questions of contributor benefit; reflecting on how to feed back relevant and valuable information learned from the research done with the data, a task that becomes more difficult the wider and more diverse the contributor base is; actively rejecting forces pulling the relationship with contributors towards an extractive state, were contributors keep being asked to contribute data while a clear sense of benefit and utilisation is being lost. This attitude requires active vigilance on the part of project managers and a willingness to challenge abnormal power asymmetries, especially when the project is driven by private entities. Powerful tech giants have entrenched their economic might, to the detriment of the public good, on extractive relationships where ever more data is continuously collected in order to predict and generate behaviour.\n\n\n2. Actionable recommendations\n\nThis section takes stock of innovations that can help improve best practices with a view to tackle challenges outlined in Part I. The spectrum of self-reporting is extremely diverse and as such impossible to govern through a one size fits all approach; projects can be scientific and not, can raise important privacy concerns, and can be open to organised manipulation and poor governance of risks and harms; best practices and guidelines must be adapted to the local setting and remain open for improvement. The following are three areas of best practice that have been developed to help manage the ethical issues generated by data self-reporting projects. They are applicable to many different domains and institutional settings, and they should be seen as a complementary set of recommendations. They are all concerned with securing the representation of heterogeneous concerns, stakes and forms of knowledge in the day-to-day governance and decision-making of self-report data projects. This is a crucial step for a project to control for the key issues discussed in the previous section.\n\nManagers and developers of data self-reporting projects should consider what measures they can take to ensure data governance is accountable, inclusive, competent. Projects where data are extracted from a contributor base to then be shifted and shared according to the judgement of a self-selected few are more likely to enter controversy or make poor choices from an ethical point of view. These are the governance approaches privileged by private companies and tech monopolists who intend to control the way in which commercial value can be created by contributed data because they concentrate decision power in the hands of allegiant few.\n\nArguments in favour of individualised, distributed control of data that resonate with recent visions of decentralised web and organisational governance are problematic because of the burden and risks they put each individual deciding for themselves under. They imagine technological frameworks could make it possible for the data to be locked and unlocked by the individual they refer to, thus giving, in principle, total control over the data back to them. The real world might differ. Few have the expertise and time necessary to scrutinise proposal to use their data that might be put before them. Most are likely to take decisions that are against their best interests or principles because of poor or rushed judgement.\n\nIn recent years there has been a great deal of innovation in data governance frameworks that deliver a form of collective control on the projects, uses and aims the data are put to. These are the data trusts (Delacroix and Lawrence, 2019) and data cooperative (Ernst Hafen, 2019; E. Hafen, Kossmann, and Brand, 2014) approaches. They have been applied with particular promise in domains such as health data, where data are sensitive, requiring tight scrutiny, and highly valuable and desired by researchers and business alike.\n\nThese approaches reject the assumption that each individual is capable and best positioned to decide what projects are worth giving access to their data. They instead elaborate on forms of delegation whereby a number of research participants are appointed as delegates or trustees with data governance responsibilities and are charged with day-to-day decision making and governance. Depending on the setup, members can exercise their own decision over whether to participate in one project or another more or less often. These approaches can be used to take over and collectively govern and mobilise existing datasets that were already generated for other purposes, for instance, receiving healthcare; but can also be used to start and coordinate the collection of new data from the outset, in a ‘bottom-up’ fashion where the project is managed by participants. They are intended to afford better inclusivity, accountability and competent data management. Different individuals should be able to seek different levels of involvement according to their interest.\n\nNot all self-report collection projects need to be governed through a bottom-up approach such as data cooperatives or trusts and in many cases, data governance might be better managed through review boards composed of independent experts (see for instance, section 1.3) who might or might not be participating in the project themselves. The value of representative bottom-up data governance approaches is still in the potential for the project management to better articulate finalities, values and directions of a project in a way that better reflects the views and aims of its pool of participants, enacting the sort of relational ethics (Birhane, 2021) where experiential expertise is valued at the same time as professional expertise\n\nData governance includes the assessment of risks and benefits of ways of working with data to the best of knowledge and assumptions held by those involved in decision making. Assessing risks and benefits of different ways of using data necessarily involves making many assumptions about future developments and events. The exercise is uncertain, and it is possible even for the most suitable experts to make mistakes. This is true even when data are governed through a collective governance framework such as a data trust or cooperative. Harms and benefits will be very likely to distribute unevenly, and collective governance will not in itself make things right or fair if mistaken assessments lead to harm and benefits that are unfairly distributed in ways that could be mitigated.\n\nAs Barbara Prainsack argues (Prainsack, 2019a, 2019b), only when collective governance frameworks are combined with a commitment to the principle of social solidarity they can truly fulfil their revolutionary potential of alternative to the monopolistic extractive models pioneered by tech giants. To achieve this, data self-reporting projects need to accept the possibility of harm and prepare for its mitigation. With her colleagues (Prainsack and Buyx, 2017; McMahon, Buyx, and Prainsack, 2020) she calls for the creation of harm mitigation bodies that will operate harm mitigation measures. These, they suggest, could include issuance of apologies and stipulation of amendments to avoid future harm recurrence and financial support for the gravest of cases.\n\nThrough a combination of collective governance (see 1.1) and harm mitigation features, self-reporting projects can aspire at the implementation of a data donation economy that maximises data’s potential to be used for the common good.\n\nData donation has been used as a buzzword for broadly mimetic purposes by some exploitative projects, but it is not a fuzzy concept. There are a few conditions that must be met for a project to be based on data donation. Barbara Prainsack emphasises that for practices of data reporting and sharing to qualify as based on data donation, they need to exhibit relationality, indirect reciprocity and multiplicity. Relationality requires that the two parties (giver and receiver of data) mutually acknowledge the act of donation; this means that the data receiver (self-report data project manager) honours the work that the data donors (research participants) have done by “systematically considering the needs and interests of data donors and their significant others” (Prainsack 2019a:14); this can be reflected in the ways in which a project is organised to register, reflect and enact the views of its contributors. Relational ethics and bottom-up data governance, for instance, can go some way in this direction. But for the data donation economy to ensure that reciprocity is indirect and thus shared across the membership, harm mitigation measures are seen as a necessary instrument. It is through this combination of arrangements that data multiplicity (Prainsack’s way of pointing out the ability for data to be reused multiple times) can serve the common good. Data that are collected, for instance, through mainstream, status quo, commercial data governance practices (for instance, data collected through a user-generated data platform and used by a Silicon Valley giant for undisclosed commercial purposes) might still have multiple uses, i.e., multiplicity, but the economy of such a project would not be based on donation, because the data are not received as a gift and respected with the ethics gifts command.\n\nA crucially important function that officers of a data trust, data cooperative or data governance organisation form should need to provide is the oversight of ethical risks. At any rate, a data self-reporting project should implement an ethical risk oversight function that is adequate to its size and ambition. A project that aims to create a data resource that can be used time and again and is widely trusted by internal and external stakeholders is highly likely to need a formal oversight structure with clear governance procedures.\n\nThe time-tested standard model in this domain are the Research Ethics Committees that have been implemented for decades by universities in the UK, US (aka institutional review boards (IRB)), and the rest of the world. They normally require researchers who intend to embark on new research to submit a research ethics application. This is usually centred on a formal document (e.g., application form) that provides key information about the project aims, methods, resources; and evaluations of ethical risks together with a specification of measures planned to control for them. The application is reviewed by experts serving in the committee who should be able to judge whether the project is responsibly conceived, and the eventual ethical risks are acceptably approached, i.e., control measures are fit for purpose. If the application is rejected, the research cannot start.\n\nInstitutions can design, and have designed, research ethics oversight committees and processes differently to adapt to local conditions, but the above tend to be the main features, and this model has been adapted outside academia in the public and private sector as more organisations sought to control risks of their research and development (R&D) activities (including reputational risk, through the accountability that having a formal process affords). This means that there have been no off-the-shelf approaches to research ethics oversight, though there have been various guidelines.\n\nThe need for up-to-date guidelines and models has recently become apparent as some late developments in research methodology are challenging the research ethics status quo. A report by the Ada Lovelace Institute, the University of Exeter Institute for Data Science and Artificial Intelligence, and the Alan Turing Institute (Petermann et al. 2022) highlights the new challenges and the possible solutions that institutions should consider keeping their research ethics oversight function suitable (also, among others, explored by Ferretti et al., 2021; Jordan SR, 2019; Basl and Sandler, 2019; Clark et al., 2019). While for a long time RECs have been focusing on the ethical harms and risks that research could pose for the participants in a study (resulting from the origins of research ethics in medical research and the design of clinical experiments), new kinds of research employing cutting edge data science and artificial intelligence methods are bringing up emerging ethical issues. Here, the new innovations engendered by data self-reporting projects coordinated over the web are part parcel of these changes that need to be confronted.\n\nOf growing concern have been the long term consequences of research that might be developing methods and technology that can be easily re-deployed (recall ‘function creep’) across institutions, jurisdictions, countries, populations, and how they might have disparately unequal social impacts; along with a redefinition of issues of privacy, confidentiality, and consent at a time when researchers can source individual level data from the web or other easily available resources, without asking the individuals these data originated from.\n\nIssues are complex and their governance cannot be a box-ticking exercise. Methods that can be seen as gold standard in tacking some problems still require close and situated scrutiny. For instance, anonymisation can be a strong solution for granting privacy protection to individuals whose data widely available on the web is collected without their explicit informed consent. However, it is widely understood that the strength of anonymisation is always relative to the way in which the various other data sources a researcher can have access to, can lead to re-identification. Even when anonymisation is considered satisfactory for the purpose of privacy protection, in itself it offers nothing to protect individuals from the eventual unfair impacts of the outcomes of research once they are operationalised and deployed in new systems shaping individual movements, interactions, behaviours and access to services.\n\nIn recent years and in response to the emerging ethical challenges posed by the latest innovations in data science and AI, there has been growing demand for the active consideration of broader downstream impacts of such research. Research communities and academic societies have started to require researchers to submit research ethics statements on the broad societal impacts of their endeavours (Petermann et al., 2022). Leading commercial technology organisations have started to experiment with the implementation of internal ethics review processes for their commercial R&D projects, in an effort to improve their reputation and accountability when the public is questioning whether they can be trusted with running the socio-technical infrastructure of much public life. How much can commercial organisations be expected to follow together ethical and business principles, and successfully map, and manage, the broad landscape of social groups, needs and interests, is very much an open question. Criticisms of ‘stakeholder capitalism’ abound. But it can perhaps be argued that when self-report data power so many projects and systems across both the research and commercial worlds, and underpin affairs in the public sphere; and when so much of public life relies on complex infrastructures developed on methods and techniques of recent invention and never fully understood consequence; then, attempts at translating some of the ethical principles, expectations and methods developed to deal with the arising ethical issues of self-report data might be in order. The implementation of ethical oversight functions, which can take many forms, in new contexts is one such opportunity.\n\nQuestions around the re-definition of ethical standards and red lines are tightly linked to practical questions over the right training and composition of experts that REC should seek when evaluating projects with strong interdisciplinary components, whose risks are inherently more difficult to assess (Petermann et al., 2022); and complex projects involving multiple staggered undertakings where a technological solution builds from the previous. Questions that are open to debate and that will require careful consideration when implementing research ethics functions in organisations include also the scope, duration and frequency of interactions between research ethics functions and the researchers leading a project. These questions are not merely operational but really shape the quality of understanding and assessment that a research ethics function is able to deliver.\n\nBut at any rate, a form of research ethics oversight is needed in data self-reporting projects.\n\nHere again principles of bottom-up participation will be important. The world-leading public health databank SAIL (Ford et al., 2009), which has been managing dozes of highly sensitive, linked health datasets and making them available to hundreds of research projects, involves members of the public, together with domain experts, as reviewing members of research ethics applications.\n\nBottom-up participation will improve diversity of perspectives and sensitivity to assess bias and unequal impacts, and will be a required condition for a full implementation of collective governance approach involving data management through trust and cooperative; and design and operation of harm mitigation measures. Birhane (Birhane, 2021) emphasises, with the concept of relational ethics, how those communities and groups that are on the receiving end of a project’s impacts should be included in its governance, because they have a key epistemic privilege about its social outcomes – decisions should be taken with and through them, instead of without.\n\n\nConclusions\n\nImportant ethical questions on data self-reporting have arisen both before and during the pandemic. The pandemic saw various kinds of self-reporting apps taken up by extraordinary amounts of contributors and heightened our sensitivity to the issues that came forth then. But ‘normal times’ had already taught us much on issues of data governance, recognition and valuation that it is important to try to address as many of these issues at once to best prepare for the time after the pandemic. This manuscript discusses some of the key ones and recommends some of the most promising innovations to tackle them and to help with issues of public trust (Bharti et al., 2021). It argues that flexible and sustained ethical oversight is key. Best practices must be adapted to the local setting and improved over time. This is achieved by acting proactively instead of reactively, and by avoiding simplistic solutions. And it is achieved by increasing diversity (of both background knowledge and experience) in ethical oversight and project management.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nI would like to thank Paul Wicks, PPI for HEAL-COVID, who offered time to chat on the topic and input as an expert practitioner. The research that underpins this brief was funded by the AHRC under the grant ‘UK Ethics Accelerator: Coordinating and Mobilising Ethics Research Excellence to Inform Key Challenges in a Pandemic Crisis (AH/V013947/1)’. The early two-part report resulting from it informs this policy brief; it was made available on Zenodo (Tempini, 2022a, 2022b). I would like to thank the team at UK EA for their support and feedback on early versions of this manuscript.\n\n\nReferences\n\nAda Lovelace Institute: The Data Will See You Now: Datafication and the Boundaries of Health. London:Ada Lovelace Institute;2020. 978-1-8382567-0–8.\n\nAda Lovelace Institute and The Health Foundation. The Data Divide: Public Attitudes to Tackling Social and Health Inequalities in the COVID-19 Pandemic and Beyond. London:Ada Lovelace Institute;2021. 978-1-8382567-3–9.\n\nArnott-Smith C, Wicks P. PatientsLikeMe: Consumer Health Vocabulary as a Folksonomy. AMIA Ann. Symp. 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Profile Books;2019." }	[ { "id": "173168", "date": "18 May 2023", "name": "Edward S Dove", "expertise": [ "Reviewer Expertise health privacy law", "research ethics governance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 really enjoyed reading this Policy Brief on the ethics of data self-reporting. It is generally written well and and references a good number of relevant sources. It makes some helpful recommendations for engaging in data self-reporting projects better and with deeper respect for the rights and interests of those who self-report their data.\nThere are few areas I think can be strengthened, however.\nI'm unclear on how the terms (data) ethics and (data) governance are used in this paper. What are considered ethical issues and what are considered governance issues, and are the recommendations more broadly framed as governance solutions to ethical issues? If so, is that problematic?\n\nI think it would be very helpful to include a summary box at the end of the paper that summarises the identified (ethical) issues and the specific recommendations, and how they are linked, e.g. which recommendation (some? all?) can address each issue identified in section 1? This would help address the journal's requirement that published articles (policy briefs) provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader.\n\nThe introduction uses the term 'article' but perhaps 'policy brief' is more accurate? That said, I'm used to policy briefs being much, much shorter (around 2000 words max) -- 8000+ words can hardly be said to constitute a 'brief'! Be that as it may, it would be further helpful to elucidate the specific policy recommendations, as distinct from a general overview of 'emerging best practice'. And at times, I was confused because I'm not clear how an existing best practice can be made better (if it needs to be improved, can it really be called a 'best practice'?) and which aspects are specifically best practices as opposed to a general model or infrastructure.\n\nI was unclear what \"The strategy is right out of the infrastructure studies playbook\" sentence means.\n\nOn p. 7, you write, \"To be meaningful, question of consent should be renewed at major project milestones or updates.\" But is consent really feasible in data self-reporting projects, particularly those that involve entire swathes of the population? Might we be putting too much faith in consent to do the ethical work?\n\nOn p. 7, I don't understand the sentence: \"Location data that is automatically generated, for public health apps, by a smartphone operating system’s location; services can be shared by the operating system with other apps that also use location data; with the result of deepening the surveillance of a user within the ecosystem of data brokers and surveillance capitalism.\" It seems some words are missing. I would suggest re-writing.\n\nA smaller comment is that there are a fair number of minor typos (e.g. reference to GDPR rather than GPDPR at one point; a bracket that states 'REF' rather than the specific literature reference) and grammatical errors that another careful proofread should be able to address. Also, I would avoid seasonal references ('Spring 2021' on p. 5) as this can cause confusion across our hemispheres. Best to just specify the month or when it falls within the calendar year (e.g. 'early', 'mid', 'late').\nI hope these review comments are helpful. Again, I really enjoyed reading this.\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": [] }, { "id": "238847", "date": "27 Mar 2024", "name": "Barbara Prommegger", "expertise": [ "Reviewer Expertise Information systems" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper explores the ethics surrounding data self-reporting, including challenges posed by the COVID-19 pandemic. Positioned as a policy brief, it underscores the significance of ethical considerations in self-reported data collection, highlighting issues such as functional creep and consent and privacy concerns, and provides recommendations that can help limit the potential harm of misuse of self-reported data.\nPositive Aspects: I enjoyed reading the paper as it addresses a highly relevant and timely topic, given the increasing risk of misuse of data that might be publicly available and/or self-reported by individuals. The author provides many practical examples which makes the article highly interesting and entertaining to read.\nAreas for Improvement:\nConceptualization of Self-Reported Data: While there is a short description of self-reported data at the beginning of the article (page 1), there is still a need for clarification regarding the definition of self-reported data, as the examples provided in the paper differ significantly. For example, symptom tracking for a COVID-app (page 3) differs from tweeting on a public account (example page 7), and as a consequence also the ethical issues and recommendations based on the different forms of self-reported data might differ from each other. For example, while in the first case, individuals might not fully understand, how their data gets used but still participate in data collection, in the second example users might not even know that they actively “report” data that will be used later. By delineating different forms of self-reported data based on factors such as awareness and participation of individuals, readers can better understand the nuances involved. Additionally, establishing a clear definition and contextual framework for self-reported data would aid comprehension of the paper. Clarity of Context: The paper occasionally intertwines various contexts concerning the collected data, including research, government, and industry. Similar to the mix of different kinds of self-reported data, these different contexts may entail different ethical concerns and recommendations. For example, collecting self-reported data for research purposes is usually highly regulated (as the author also describes on page 13), while the usage of publicly available tweets in the industry might not be that controlled. It would be beneficial to distinguish between these contexts or abstract to a higher level to identify common ground and provide ethical concerns and recommendations accordingly to enhance clarity. Role of COVID-19 Context: While the relevance of the COVID-19 crisis is evident in the introduction, its connection to subsequent sections becomes somewhat obscured. Also, the provided examples in the boxes are interesting and relevant, but they are only loosely connected with the rest of the text. A more seamless integration of COVID-19-related aspects throughout the paper, including the examples provided, would enhance coherence and strengthen the paper's overall argument. Mapping Concerns to Recommendations: Currently, it is challenging for readers to discern how the concerns raised correlate with the actionable recommendations provided in the paper. Strengthening the link between these segments would facilitate a clearer understanding of how to address each concern explicitly, thereby enhancing the paper's practical utility. Recommendations: In the current version of the article, it is somewhat unclear at which target group the recommendations are aimed at, i.e. who should actually implement these recommendations. I therefore suggest clarifying the level of the target group to which the individual recommendations are addressed (global, national, institutional and individual level), which will make them more relevant and actionable for a broader audience.\nMinor Suggestions: To improve comprehension of the paper, I would suggest including tables in the results section providing overviews of 1) the ethical concerns, 2) the recommendations, and 3) how both map. This way, readers who simply want to skim the paper will still be able to gather the most important takeaways of the text.\nI enjoyed reading the paper and appreciate its focus on addressing important, timely matters surrounding self-reported data. I hope that the paper will continue to improve based on the review provided.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Partly\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Partly\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "254857", "date": "11 Apr 2024", "name": "Richard Milne", "expertise": [ "Reviewer Expertise Sociology and bioethics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 presents a sociologically informed analysis of ethical issues concerning data self-reporting, with a primary focus on health data. Additionally, it provides a review of emerging practices aimed at mitigating potential negative impacts of data self-reporting on individuals' lives, from which it derives some recommendations for a governance framework.\nPOSITIVE ASPECTS: The analysis is generally well-written, rich in pertinent and important observations. It is also ambitious, as it aims to explore how the COVID-19 pandemic may have exacerbated ethical issues related to health data self-reporting more broadly.\nKEY AREA OF IMPROVEMENT: The key area of improvement is refining the overall focus and structure of the analysis and improving the use of evidence/examples to support the analysis and recommendations.\nThe first part of the paper discusses ethical issues related to data self-reporting both within and outside the context of the COVID-19 pandemic. The second part considers examples of best practices and actionable recommendations. The analysis in the first part brings together various contexts, methods, and technologies of self-reporting, it could benefit from clearer signposting to elucidate how these elements are interconnected. Additionally, it was not always evident how the discussion in the second part on best practices and recommendations directly relates to the ethical issues addressed in the first part. Overall, the analysis tends to prioritise breadth over depth, which may need a better balance between the two.\nFor example: The section ‘Self-report and self-harm’ develops a clear analysis of how the COVID-19 pandemic has exacerbated ethical issues in relation to data self-reporting. The section ‘Consent and privacy’ doesn’t seem to reference the COVID-19 pandemic. The section ‘Contribution and participation’ doesn’t seem to address ethical issues and instead offers recommendations.\nThe section on 'Function creep' stands out in the paper due to its length compared to the other sections. The author uses the concept of 'surveillance capitalism' to contextualise the phenomenon of 'function creep'. While the analysis effectively highlights the negative implications of both terms through an extensive discussion of the societal, political, and economic landscape surrounding surveillance, digitalisation, and the use of public data, there's an opportunity to improve it by specifically addressing the ethical concerns arising from the expansion of monitoring infrastructures for data self-reporting, particularly within public health. This seems to be important because, in the section on 'Self-report and self-harm', the author appears to discuss the exchange of data in a bottom-up initiative in a positive light. Without a thorough examination of the unique characteristics of data self-reporting and the emerging ethical issues associated with it, the analysis seems to attribute ethical concerns to the reputation of organisations extending their infrastructure, rather than to the infrastructures themselves. A more technical exploration of data tracking operations and an in-depth analysis of each aspect would shift the ethical discussion from reputational concerns to the technical domain, thereby holding all involved parties equally accountable for their actions.\nA good reference for this is refer [1].\nBelow are some other points:\nThe discussion on the relation between ethics and epistemic knowledge is interesting. The analysis is in paragraphs: ‘Function creep’, ‘Box 1: Zoe’ and ‘Box 2: Heal Covid’. Could this be brought together as a standalone ethical issue? The author argues that ethical practices mean reliable knowledge, while raising an observation on the truthfulness of information disclosed when individuals participate in self-reporting. The author writes: “Experts warned about the potential emergence of incentives to lie”. This is an interesting point that could be explored further by engaging with this literature. What are the incentives to disclose deceitful information about oneself in practices such as Zoe App as compared to let’s say centrally managed survey like Heal Covid? What is the relevance for data self-reporting and for using it to inform action?\nDefinition of self-reporting. The author writes that 'the spectrum of self-reporting is extremely diverse' and that 'projects can be scientific, commercial, or others' (in abstract). I think this is left too general. The specific contexts, methods, devices, and uses of data self-reporting matter to formulate analysis of ethical issues and governance frameworks. For example, consider the differing ethical implications and governance requirements between contact-tracing apps tracking location and disease status (as in positive or negative COVID-19 test), and apps like Zoe App, which tracked COVID-19 symptoms. Does this difference in data self-reporting lead to different ethical considerations, implication for policy and recommendations? Incorporating in-depth analysis within example boxes would help anchor the discussion in current practices.\nExample Boxes. The use of boxes to emphasise important projects is great. However, the choice of ‘Patient like me’ as an example within the box raises some questions, as it doesn’t directly relate to the COVID-19 pandemic unlike the other examples. Also, the specific function of these boxes isn't entirely clear. While they provide additional information, the analysis within them often mirrors the main text too closely. A more effective use of the boxes could involve providing a technical description of the examples, focusing on their relevance to self-reporting. This would offer readers a deeper understanding of how these projects operate within the context of data self-reporting.\nRisk mitigation / Direct action? The analysis of the second part seems more focused compared to the first. However, I am uncertain about the author's rationale for choosing bottom-up, participatory, and direct action initiatives to developing governance frameworks aimed at centralised data-self reporting projects. It's important to clarify whether the primary objective is risk-mitigation or achieving social impact. Additionally, incorporating empirical examples demonstrating how self-reporting adversely affects individuals' livelihoods is essential. Without such examples, our ethical considerations may remain purely theoretical and lack practical application.\nThere is also scope to link the discussion of governance frameworks back to the concerns raised. How, for example, would governance structures such as data trusts address problems of function creep? This might also recognise that the indeterminacy of future data uses is a long-standing problem for medical research data – particularly genomic data – and that governance arrangements have evolved in response to this.\nLanguage. Generally well-written but needs proof reading. There are a few expressions that are perhaps unsuitable for the type of publication such as “get a grip” (p.3), “sky was falling” (p.3), “pull the rug” (p.6). The use of paragraphs is a bit erratic. Some paragraphs are quite lengthy and cover lots of ground, some are shorter and perhaps need further development in the next stage of writing.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Yes\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Yes\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-485
https://f1000research.com/articles/10-275/v1	06 Apr 21	{ "type": "Method Article", "title": "Organizing gene literature retrieval, profiling, and visualization training workshops for early career researchers", "authors": [ "Fatima Al Ali", "Alexandra K Marr", "Zohreh Tatari-Calderone", "Mohamed Alfaki", "Mohammed Toufiq", "Jessica Roelands", "Basirudeen Syed Ahamed Kabeer", "Davide Bedognetti", "Nico Marr", "Mathieu Garand", "Darawan Rinchai", "Damien Chaussabel", "Fatima Al Ali", "Alexandra K Marr", "Zohreh Tatari-Calderone", "Mohamed Alfaki", "Mohammed Toufiq", "Jessica Roelands", "Basirudeen Syed Ahamed Kabeer", "Davide Bedognetti", "Nico Marr", "Mathieu Garand", "Darawan Rinchai" ], "abstract": "Developing the skills needed to effectively search and extract information from biomedical literature is essential for early-career researchers. It is, for instance, on this basis that the novelty of experimental results, and therefore publishing opportunities, can be evaluated. Given the unprecedented volume of publications in the field of biomedical research, new systematic approaches need to be devised and adopted for the retrieval and curation of literature relevant to a specific theme. Here we describe a hands-on training curriculum aimed at retrieval, profiling, and visualization of literature associated with a given topic. This curriculum was implemented in a workshop in January 2021. We provide supporting material and step-by-step implementation guidelines with the ISG15 gene literature serving as an illustrative use case. Through participation in such a workshop, trainees can learn: 1) to build and troubleshoot PubMed queries in order to retrieve the literature associated with a gene of interest; 2) to identify key concepts relevant to given themes (such as cell types, diseases, and biological processes); 3) to measure the prevalence of these concepts in the gene literature; 4) to extract key information from relevant articles, and 5) to develop a background section or summary on the basis of this information. Finally, trainees can learn to consolidate the structured information captured through this process for presentation via an interactive web application.", "keywords": [ "Literature profiling", "Science education", "Concept extraction", "Data visualization" ], "content": "Introduction\n\nPeer-reviewed publications constitute the body of knowledge upon which biomedical research is based. This resource is essential for the generation of novel hypotheses and the design of studies, experiments, and trials and is thus key to the discovery process itself. However, the volume of literature on any given specific research topics has grown dramatically in recent years, making it increasingly challenging for a single person to manually survey the relevant literature in its entirety, and consequently to acquire the foundational knowledge needed for discovery research. Hence, developing a solid foundation of skills for literature retrieval and profiling is of critical importance for early-career biomedical scientists. These competencies will, for instance, be needed: 1) to acquire a sound knowledge base through developing the ability to compile and summarize large volumes of literature. 2) to develop data interpretation skills as well as become able to assess novelty and potential impact of a given finding; and 3) to develop scientific writing skills and become able to write background material on specific topics.\n\nPublicly available omics profiling data provides ideal material for training new generations of biomedical researchers.1–3 One of the “collective omics data” training modules that we have developed follows a reductionist analysis and interpretation workflow using publicly available transcriptome profiling data as the source material.4 The first activities that form part of this training module involve literature retrieval and profiling for a given candidate gene.\n\nIn this article, we describe a detailed stepwise approach that we have developed running literature profiling training workshops. The training program that we have devised takes advantage of publicly available omics data.4 The training module presented here focuses on retrieval of gene-centric literature. Supporting material such as sets of slides, templates, and a handout, is also provided along with an illustrative use case. Notably, although the training activity that is presented focuses on gene-centric literature retrieval and profiling, the skills and approaches can be adapted to any other application (e.g., focusing on disease-centric, or pathway/process-centric literature).\n\n\nMethods\n\nThe hands-on training exercise described here is suitable for implementation as part of a broader course on research methodology, or as a stand-alone workshop. The training is appropriate for undergraduate, graduate and post-doctoral trainees and no prior bioinformatics experience is required in order to participate. The time commitment depends on the level of experience of the attendees, the overall organization of the workflow and the volume of literature associated with the candidate gene(s) selected. For instance, for a gene with ±1,000 associated articles and five participants each focusing on a different theme, the training could be covered in one introductory session (40 min), and three two-hour hands-on sessions. The format and content of these sessions are described in more detail below. These would cover:\n\n• Introductory session\n\n• Step 1 – Retrieving the relevant literature\n\n• Step 2 – Extracting concepts\n\n• Step 3 – Generating literature profiles\n\n• Step 4 – Developing interactive data visualizations\n\n• Step 5 – Writing a narrative\n\nIn addition to covering workshop pre-requisites and time commitment, announcements for such workshops can also list the sets of skills trainees are expected to develop including:\n\n• Literature retrieval (development of advanced PubMed queries).\n\n• Literature profiling (information extraction, determination of keyword frequencies).\n\n• Data visualization (structuring and presenting information in an interactive format).\n\n• Assimilation of biomedical knowledge (synthesis and presentation).\n\n• Scientific publication (optional: manuscript preparation and peer-review).\n\nTrainees are required to undertake some independent preparation between each of the sessions. Alternative formats are possible depending on the needs of the participants and a range of biomedical themes (e.g., diseases, pathways, or cell types) could be selected as the focus of the literature workshop. The endpoint for such workshops may include interactive literature profiling representations generated as part of the hands-on training activities and/or a peer-reviewed publication that, for instance, could build on such a resource.\n\nThe introductory session is designed to provide participants with an understanding of basic concepts and present an outline of the training curriculum. In addition, this session should present the overall rationale and teaching objectives of the training program. The introduction should also define the endpoint of the workshop activity as the development of a web resource that permits the visualization and exploration of structured literature profiles for a gene of interest (described in detail below, taking ISG15 as an illustrative case). This should be followed by an overview of each of the steps that will be undertaken during the hands-on session. These are outlined above and described in more details below. It should also provide participants with an opportunity to ask questions before the start of the hands-on sessions. Ideally, the presentation should last no longer than 40 minutes and an illustrative case could serve to support the presenter’s narrative. A generic presentation is provided (Extended data File 1).34\n\nAs a first step, all the literature that is relevant to a given gene of interest must be identified. This forms the body of literature that will be subjected to literature profiling in subsequent steps. Most researchers will already be familiar with PubMed, the search engine hosted by the US National Center for Biotechnology Information (NCBI) (https://pubmed.ncbi.nlm.nih.gov/). While this tool is straightforward to use, developing queries that will permit the comprehensive retrieval of literature associated with a given theme can be more complicated.\n\nIt is important to design a PubMed query that will permit the retrieval of all the literature that is available for the gene of interest. Challenges when retrieving literature associated with a given gene include capturing all aliases and variations that may exist in addition to the official gene names and symbols. For instance, 14 aliases were used to develop a query for the gene officially known as “ISG15 Ubiquitin Like Modifier”. Failure to include all the aliases can lead to under-representation of the literature and erroneous judgments that may have major consequences (e.g., when gaging the novelty of a given finding and its suitability for publication). The use of synonyms among gene names and aliases can also result in high proportions of false positive results, particularly when aliases are common language terms (e.g., CAMEL genes [official symbol: CTAG2] or WARS genes [official symbol: WARS1]). If the literature retrieved in this step misses relevant records for the candidate gene (= false negatives), the resulting literature profiles will be incomplete. If the literature retrieved contains records that are irrelevant (= false positives), the resulting literature profiles will be inaccurate. For these reasons, it is important to optimize PubMed queries used for gene literature retrieval. Notably, the same principle applies when generating PubMed queries for any given topic. For instance, when querying PubMed to identify literature associated with inflammation, terms such as “inflammatory” or “inflamed” should also be captured.\n\nThe endpoint of this step is the development of an optimized PubMed query for a given gene. Any criterion can be used for the selection of a candidate gene to be assigned to a trainee or group of trainees. However, a significant body of literature should be associated with the gene in question. For the purposes of illustration in this article, we have selected ISG15.\n\nPractical activities for this step:\n\nThe official gene name, symbol and all aliases are retrieved from the GeneCard website (e.g., for ISG15: https://www.genecards.org/cgi-bin/carddisp.pl?gene=ISG15). Although gene names may also be retrieved from different sources, the advantage of using GeneCards is that information has already been compiled from reference databases such as NCBI Entrez Genes, Uniprot or genenames.org.\n\nPubMed queries are built using the official gene name, official symbol, and all aliases for the gene of interest as search terms, and by using the appropriate Boolean operators (AND, OR, NOT), field restriction tags, and suitable syntax. For instance, in a query using multiple search terms, the Boolean operator OR must be capitalized in between each keyword. The field restriction tag [tw] (or alternatively the more restrictive [tiab]) should be added after each term (field restriction tags must be in square brackets), in order to limit the search to titles and abstracts (e.g., ISG15 [tw] OR IFI-15K [tw] OR IFI15 [tw]), or [pt] to restrict/exclude the search of a particular publication type (e.g., NOT review [pt]). Quotation marks should be employed when compound words should appear as an exact phrase in the search (e.g., “ISG15 Ubiquitin Like Modifier” [tw]). And for reference, a wide range of training material can also be found on the NCBI website:\n\nhttps://pubmed.ncbi.nlm.nih.gov/help/\n\nhttps://learn.nlm.nih.gov/documentation/training-packets/T0042010P/\n\nThe PubMed query is run, and quality checks are performed among the publications that were returned. This is to identify search terms that may be too permissive and return false positive results. For instance, short three-character acronyms tend to be more problematic, as are terms that are otherwise used as part of a common language (examples provided above).\n\nIf necessary, the query is optimized by addressing problematic terms. Removing the ambiguous or problematic term altogether may be a solution, but this could also lead to false negative results (missing literature that is actually relevant to the gene of interest). As a compromise, the search term could be retained but optimized by adding a keyword that would restrict the search (e.g., provided below for HUCRP and UCRP). One may also find in some instances that the list of aliases known for a given gene is incomplete and needs to be amended (e.g., below).\n\nIllustrative case:\n\nThe optimized query (searching all words and numbers in the title, abstract, other abstract, MeSH terms, MeSH subheadings, publication types, substance names, personal name as subject, corporate author, secondary source, comment/correction notes, and other terms) for ISG15 is as follows:\n\nISG15 [tw] OR “ISG15 Ubiquitin Like Modifier” [tw] OR \"Interferon-stimulated gene 15\" [tw] OR \"IFN-induced 15-kDa protein\" [tw] OR IFI-15K [tw] OR “Ubiquitin Cross-Reactive Protein” [tw] OR \"Ubiquitin-Like Protein ISG15\" [tw] OR (HUCRP [tw] AND \"Cross-Reactive Protein\" [tw]) OR G1P2 [tw] OR IP17 [tw] OR (UCRP [tw] AND \"Cross-Reactive Protein\" [tw]) OR \"Interferon-Induced 17-KDa/15-KDa Protein\" [tw] OR \"Interferon-Stimulated Protein, 15 Kda\" [tw] OR \"Interferon-Induced 17 Kda Protein\" [tw] OR IFI15 [tw] OR IMD38 [tw] NOT review [pt].\n\nThis query returned 1,186 results (as of September 1st, 2020). Optimization included the addition of aliases for this gene that were not captured in the GeneCard database but identified by reviewing some of the results (e.g., notably, the search argument Interferon-stimulated gene 15\" [tw], which alone retrieves over 230 entries in PubMed). The acronyms HUCRP or UCRP also proved a source of false positive results (CPR standing for cross-reactive protein, rather than C-reactive Protein). This was rectified by adding the AND “cross reactive protein [tw]” argument to each of the ambiguous acronym.\n\nA large body of literature can be associated with a given gene. It may be useful then to employ a systematic “cataloguing” or indexing approach. For this step, workshop participants may first define themes under which concepts, and keywords associated with these concepts, will be categorized (e.g., themes could be ’Human diseases and pathogens’,’Tissues’,’Cell types’, or ’Cellular processes’). Participants would in turn scan titles of articles associated with ISG15, looking for concepts linked to the theme of interest. For example, under’Human diseases and pathogens’, a concept could be ’liver cancer’ and associated keywords could be’liver cancer’,’hepatic carcinoma’, ’hepatocellular carcinoma’,’HCC’, or’liver carcinoma’. The output for this activity would be lists of concepts and keywords associated with the gene literature organized under different themes. It is the prevalence of these concepts that will be measured in the subsequent step (generation of literature profiles).\n\nNotably, this process could be repeated for other themes (or different themes could be assigned to each of the participants). If the literature is extensive and time is limited, it would also be possible to divide the literature into subsets (e.g., by batches of 100 articles, with participants assigned different batches to work on for the same theme). If the literature is sparse, all available articles for the gene may be used rather than just focusing on those articles including search terms in titles (i.e., using [tw], instead of [ti] in Step 2a). However, it may be generally preferable for a workshop to be based on selected genes with a relatively abundant literature (e.g., >100 articles returned when restricting the search to titles).\n\nPractical activities for this step:\n\nA subset of the literature is retrieved, restricting the search to titles (using the optimized query from Step 1, and substituting the field restricting argument [ti] for [tw]).\n\nA given theme is assigned to, or selected by, participants (e.g.,’Human diseases and pathogens’,’Tissues’,’Cell types’,’Biomolecules’,’Pathways’,’Biological processes’).\n\nConcepts relevant to the theme in question are identified in the titles of articles retrieved by the query designed in a) (e.g., liver cancer, HIV)\n\nThe concepts and associated keywords (e.g.,’hepatocellular carcinoma’, ’liver carcinoma’,’hepatic cancer’, for the concept’liver cancer’ or’virus’,’viral’,’human immunodeficiency virus’ for the concept HIV) are recorded in a spreadsheet (example and templates are available in Extended data File 2).35\n\nIllustrative case:\n\nThe subset of the ISG15 literature in which the official gene name, symbol or aliases are present in titles is retrieved. The query below is adapted to retrieve only the literature for which gene name and aliases are present in titles (using [ti]).\n\nISG15 [ti] OR “ISG15 Ubiquitin Like Modifier” [ti] OR \"Interferon-stimulated gene 15\" [ti] OR \"IFN-induced 15-kDa protein\" [ti] OR IFI-15K [ti] OR “Ubiquitin Cross-Reactive Protein” [ti] OR \"Ubiquitin-Like Protein ISG15\" [ti] OR (HUCRP [ti] AND \"Cross-Reactive Protein\" [tw]) OR G1P2 [ti] OR IP17 [ti] OR (UCRP [ti] AND \"Cross-Reactive Protein\" [tw]) OR \"Interferon-Induced 17-KDa/15-KDa Protein\" [ti] OR \"Interferon-Stimulated Protein, 15 Kda\" [ti] OR \"Interferon-Induced 17 Kda Protein\" [ti] OR IFI15 [ti] OR IMD38 [ti] NOT review [pt]. This query returned 312 results (as of September 1st, 2020).\n\nTitles are then parsed from the ISG15 literature for concepts corresponding to the theme: “human diseases and pathogens”. The concepts and associated keywords retrieved for the “human diseases and pathogens” category are listed in Extended data File 235 (Excel File: Extraction of concepts Tab).\n\nDetermining the relative prevalence of concepts among the literature associated with a given gene can be useful. For instance, when assessing the novelty of a finding (e.g., change in transcript or protein abundance associated with pathogenesis). Moreover, such an exercise and the information being derived would also be useful in other instances when writing general background/summary about the gene for a report or a manuscript.\n\nWith the two previous steps completed, determining the prevalence of concepts in the literature associated with a given gene can be achieved quite simply. For this, the literature query developed in Step 1 is modified to narrow the search and retrieve literature associated with the concepts identified in Step 2. The endpoint for this activity is a table showing the frequency of articles for these concepts in the literature associated with a given gene (e.g., Extended data File 235 [Excel File: Literature Profiling Tab]).\n\nNotes: Participants may also be encouraged to explore different types of visual representations for this type of data. Treemaps or word clouds can for instance show relative prevalence, while other types of graphs, such as 2D bubble graphs, may also show article frequencies (e.g., Figure 1). Another exercise could involve visualization of changes in the abundance of the literature associated with the selected concepts over the years. For this purpose, queries can be amended to add a range of publication dates with the field restriction tag [dp] (e.g., adding AND 2000:2010 [dp] to the query).\n\nTreemap representation of the relative prevalence of concepts associated with the “human diseases and pathogens” theme among the ISG15 literature.\n\nPractical activities for this step:\n\nThe literature query from Step 1 is employed (using the field search restriction [tw]).\n\nThe Boolean argument AND is added, followed by search terms corresponding to keywords related to one of the concepts identified in Step 2 (e.g., “Liver cancer”, “Liver carcinoma”, “Hepatic carcinoma”, etc.).\n\nQuotation marks, field search restriction and the Boolean OR are added, so that the notation would read as follows: … AND (“Liver cancer” [tw] OR “Liver carcinoma” [tw] OR “Hepatic carcinoma” [tw], etc.)\n\nThe query thus constructed is run, and the number of articles retrieved recorded (for instance in a spreadsheet: Extended data File 2).35 Steps b through d are repeated for the rest of the concepts identified in Step 2.\n\nVisual representations of the literature profiles are generated.\n\nIllustrative case:\n\nThe prevalence of concepts among the ISG15 literature associated with the theme “Human diseases and pathogens” is given in Extended data File 235 (Excel File: Literature Profiling Tab) and represented visually in Figure 1 (also in Extended data File 235 [Excel File: Generating plots Tab]).\n\nValuable insights can be gained from visual representation of information. And this could be further facilitated when the information underlying these visual representations can be accessed interactively by the end user.\n\nTo produce such interactive visual representations in the context of training workshops, we recommend using the Prezi web application (https://prezi.com). This tool has been developed to create presentations in which it is possible to zoom in and out between levels of information. This gives users the opportunity to visualize the prevalence of concepts while at the same time allowing them to “drill down” into each individual concept in order to access relevant underlying information. The endpoint for this activity is the production of a “hierarchical circle packing chart” representing the relative abundance of concepts in the literature for a given gene and theme (Figure 2A, https://prezi.com/view/zCedrcYaAEUAON1VeEUi). Such a resource gives users access to underlying reference information for each of the concepts (Figure 2B) and can be made available publicly.\n\nAt the highest level, the representation permits visualization of the relative abundance of concepts associated with the “human diseases and pathogens” theme for the ISG15 gene. The color and size of the circles is a representation of the number of articles retrieved for a given concept (as indicated in the figure key). B. Zooming into each of the circles permits access to another level of information, such as links to PubMed results as well as screen captures of articles relevant to a specific topic (e.g., the relevance of ISG15 as a biomarker). An interactive version of this presentation can be accessed via: https://prezi.com/view/zCedrcYaAEUAON1VeEUi/\n\nFor the practical activity designed for this step, participants will need to register with Prezi and create an account to access and edit the interactive presentations. They can do so free of charge by selecting the basic account at: https://prezi.com/pricing/basic/. Ideally, this should be completed ahead of the training session. If instructors have access to a paid subscription, they can create an unlimited number of presentations and invite individual participants to collaborate with editing rights. Otherwise, the participants can create their own presentation and invite the instructors as collaborators. They would also need to copy and paste material from a master template in their own Prezi account. Thus, this alternative solution would be workable, but probably not ideal.\n\nPractical activities for this step:\n\nParticipants are given access to a Prezi presentation that contains a template and illustrative example (e.g. https://prezi.com/view/u65ZqHn9ZBJx1VKHqfZA/). As indicated above, one such presentation could be created and made available for each participant. It could serve as their own “Sandbox”, to familiarize themselves with the application and use as a starting point to develop an interactive resource. Multiple users can also work simultaneously on the same shared presentation. It would thus also be possible to create one presentation for a group of participants to work on cooperatively.\n\nStarting with the template and following the illustrative example, participants can build a circle packing chart for concepts identified in Step 3.\n\nThe size and color of the circles is determined by the frequency of articles in the gene associated literature related to a given concept (as shown in Figure 2A).\n\nThe circles can be arranged manually to create a visually attractive representation.\n\nUnderlying information is then added to each of the circles and accessed by zooming in to different levels (Figure 2B). This information could include, for instance:\n\n• The gene symbol and concept;\n\n• The query link;\n\n• Number of articles retrieved on a specified date;\n\n• Result link to those articles;\n\n• Screen capture and links pointing to articles relevant to a specific topic (see Step 5 below).\n\nIllustrative case:\n\nThe circle packing chart representation for ISG15, focusing on the “Human diseases and pathogens” topic is shown Figure 2. The Prezi can be accessed for interactive exploration via: https://prezi.com/view/zCedrcYaAEUAON1VeEUi/\n\nA screen recording demonstrating how to add underlying information to a Prezi circle packing chart can be accessed via: https://soapbox.wistia.com/videos/2tmC1VeQyr\n\nWriting material for a report or manuscript can be one of the motivations for profiling the literature associated with a given topic. This is a skill that is worth developing as scholarly output is the measure by which productivity is measured in academic research settings. For instance, it can be at first important to be able to identify the information that is worth capturing and communicating (e.g., types of molecules measure, species, or comparator groups) The last hands-on activity of the workshop consists in capturing such information and using it as a basis for writing up a narrative about the gene of interest.\n\nA specific topic would be selected as the focus of the activity to limit the amount of literature that would need to be covered. One such topic could, for example, be the relevance of a gene (in this case ISG15) as a biomarker for diagnostic applications within the overall theme of “Human diseases and pathogens” defined in Step 2. Participants could then focus on the most prevalent disease (e.g. in the case of ISG15 literature, three diseases have >50 articles and ten diseases have >20 articles). Literature queries corresponding to the diseases that have been selected would then be modified to retrieve only those most likely to contain information about ISG15 and its relevance as a biomarker.\n\nPractical activities for this step:\n\nConcepts most prevalent in the gene associated literature for the theme of interest are selected (e.g. concept = Hepatitis C infection, within the theme = “Human diseases and pathogens”).\n\nThe query developed to retrieve literature for a given concept is modified to restrict the search in order to retrieve articles relevant to a given topic (e.g. relevance of the gene as a biomarker). The Boolean “AND” is appended at the end of the query along with relevant keywords in parenthesis separated by the Boolean “OR” (e.g. “AND (biomarker OR biomarkers OR diagnostic OR diagnosis OR prognostic OR prognosis)”.\n\nArticles are reviewed and those deemed relevant to the topic are selected (e.g., those in which changes in abundance of the gene product in clinical specimens are reported).\n\nRelevant information is captured from the abstract and/or full text and recorded in a spreadsheet (e.g. analyte name, species, data generation methods, comparator groups, etc.). In some cases, the full text of articles may not be accessible (e.g., paywall), and the abstract has only incomplete information. This can lead to the absence of important information in the “capture” spreadsheet, rendering the findings unusable. Participants can then be reminded of the importance of using best practices when reporting findings (e.g., mentioning the comparator group, species or the specific factor being measured – being protein or RNA). It may also be an opportunity to discuss the merits of publication in open access journals.\n\nA function in the spreadsheet (column X) concatenates the information entered in the previous step to automatically generate standard sentences.\n\nThe standard sentences are collated and edited to produce a cohesive paragraph or section that is relevant to the topic. For the final step, attendees may choose to “polish” the automatically generated text themselves, with the possibility of having the instructor making final edits. It would also be possible to use the services of a professional copyeditor.\n\nIllustrative case:\n\nGene of interest: ISG15\n\nTheme: “Human diseases and pathogens”.\n\nTopic: Biomarker/diagnostic relevance.\n\nFocusing on ISG15 and “human diseases and pathogens” as a theme, with biomarker/diagnostic/prognostic as a topic, the main diseases identified in step 3 were selected (>20 articles; Figure 2A, red circles and orange circles). Starting with the query developed for retrieving ISG15 literature relevant to Hepatitis C virus, the search is restricted by adding the expression “AND (biomarker OR biomarkers OR diagnostic OR diagnosis OR prognostic OR prognosis)”. Three articles were returned. From each article, relevant information available in the abstract, and in the full text where possible/necessary, is recorded in a spreadsheet (Extended data File 3).36 The information in question relates specifically to changes in abundance of ISG15 measured in clinical specimens (since it is directly relevant to the topic that was selected). The spreadsheet automatically generates “standard sentences” by concatenating the information that is entered in the spreadsheet. The steps described above are repeated for other diseases (>20 articles were returned within the ISG15 literature). Automatically generated sentences are compiled and edited to produce the final version of a written section describing the potential relevance of ISG15 as a biomarker in clinical settings.\n\nText that is automatically generated by filling out the spreadsheet from Extended data File 336 is provided below (Part A). It is broken down by human disease or pathogen. PubMed IDs are provided in lieu of reference. Gaps are apparent in the sentence where input is missing from the spreadsheet (usually information is not available in the abstract and access to full text is not open). It is followed by a narrative that was prepared from this automatically generated text (Part B). In this case the narrative was prepared by a professional scientific copyeditor.\n\nStep 5e: Automatically generated text (Part A):\n\nHepatitis C Virus:\n\nThe abundance of ISG15 proteins measured by … … ……… … .. is increased in vivo in human liver in patients with HCV infection compared to uninfected controls [26833585].\n\nThe abundance of ISG15 Transcripts measured by … … …… … … is increased in vivo in human liver in patients with HCV infection compared to uninfected controls [26833585].\n\nThe abundance of ISG15 Transcripts measured by multiplex branched DNA assay is increased in vivo in human PBMCs and liver cells in patients with HCV infection who were unresponsive to IFN treatment compared to patients with HCV infection who were responsive to IFN treatment [23588721].\n\nThe abundance of ISG15 measured by … … ……… … … is increased in vivo in human Liver in patients with HCV infection associated with an unfavorable HCV genotype 1, a high hepatic HCV load and a low antiviral response to IFN compared to patients who did not present such characteristics [20639253].\n\nHIV:\n\nThe abundance of ISG15 IFN stimulated gene, ISG15-mRNA and ISG15-SNPs measured by TaqMan assays is increased in vivo in human PBMC in untreated HIV-1 patients HIV infection compared to healthy donors [26563749].\n\nThe abundance of ISG15 expression measured by … … ………… … … is decreased in vivo in human PBMC in patients with long-term antiretroviral therapy HIV infection compared to uninfected controls [26563749].\n\nThe abundance of ISG15 Transcripts measured by multiplex branched DNA assay is increased in vivo in human PBMCs and liver cells in patients with therapeutic responses in 20 HIV/HCV genotype-1 subjects undergoing HCV treatment compared to patients with HCV infection who were responsive to IFN treatment [23588721].\n\nThe abundance of ISG15 Protein measured by … … …………… … .. is increased in vivo in mouse models Brain in mouse models of acute and chronic neuronal injuries HIV infection compared to global ischemia and traumatic brain injury, and in transgenic mice overexpressing HIV gp120 protein [23090498].\n\nMelanoma:\n\nThe abundance of ISG15 Proteins measured by immunohistochemistry is increased in vivo in human melanoma cell clones and DC “in patients with” tumor compared to [12067988].\n\nBreast cancer:\n\nThe abundance of ISG15 Proteins measured by immunohistochemistry is increased in vivo in human immune cell markers “in patients with” invasive BC patients with long-term follow-up compared to … … ………… … .. [33073304].\n\nThe abundance of ISG15 Proteins measured by … … ……………… … . is increased in vivo in human mammary tumors “in patients with” mammary tumors compared to normal mammary tissue [30500379].\n\nThe abundance of ISG15 Immunoregulatory and antitumor measured by shRNAs is ISG15 inhibits breast tumor growth and increases NK cell infiltration in vivo in nude mice tumorigenesis in patients with Cancer compared to … … …………… … … [25749047].\n\nThe abundance of ISG15 molecule measured by … … …………… … … is highly expressed in vivo in human numerous malignancies “in patients with” breast cancer immunotherapy compared to [22057675].\n\nThe abundance of ISG15 Proteins measured by semiquantitative real-time PCR, cDNA dot-blot hybridization and immunohistochemistry is overexpressed in vivo in human invasive breast carcinomas and normal breast tissues “in patients with” breast carcinoma cells compared to normal breast tissue [18627608].\n\nThe abundance of ISG15 Proteins measured by short hairpin RNA-mediated knockdown is increased in vivo in human tumors “in patients with” breast cancer ZR-75-1 cells decreased CPT sensitivity compared to [18566215].\n\nThe abundance of ISG15 Proteins measured by short hairpin RNA-mediated knockdown is reduced in vivo in human tumor cells selected for resistance to CPT “in patients with” breast cancer compared to … … …………… … … [18566215].\n\nSystemic Lupus\n\nThe abundance of ISG15 mRNA level measured by quantitative polymerase chain reaction is high in vivo in human whole blood cells “in patients with” 28 patients newly diagnosed with SLE compared to with 10 patients with undifferentiated connective tissue disease, and 22 healthy volunteers [28204879].\n\nThe abundance of ISG15 measured by … … …………… … … is induced in vivo in human plasmablasts/Plasma cells “in patients with” active systemic lupus erythematosus compared to … … …………… … … [27357150].\n\nHBV\n\nThe abundance of ISG15 Proteins measured by … … …………… … … is high in vivo in human HBV-related HCC tissues “in patients with” hepatitis B virus (HBV) compared to non-tumor tissues [26770308].\n\nStep 5f: Edited narrative, based on the standard text (Part B):\n\n“Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin family, which includes ubiquitin and ubiquitin-like modifiers (Ubls). Ubiquitin and Ubls are involved in the regulation of a variety of cellular activities, including protein stability, intracellular trafficking, cell cycle control and immune modulation. ISG15 has been implicated as a biomarker with diagnostic relevance for a number of human disorders, including cancer (melanoma and breast cancer) and autoimmune diseases (SLE), as well as infection with pathogens such as HBC, HCV and HIV.\n\nThe expression of ISG15 has been implicated in a wide range of human cancers, although the roles of ISG15 in tumorigenesis and responses to anticancer treatments remain largely unknown. In patients with breast cancer, ISG15 is overexpressed at both the mRNA and protein levels in mammary tumor tissue compared to that in normal mammary tissue.5,6 Increased ISG15 protein expression has also been detected in human melanoma cell lines.7 These findings indicate the potential of ISG15 as a tumor biomarker. ISG15 protein expression is upregulated in immune cell markers from patients with invasive breast cancer8 as well as dendritic cells from melanoma patients.7 ISG15 is also highly expressed in breast cancer patients undergoing immunotherapy.9 Short-hairpin RNA-mediated silencing of ISG15 expression has been shown to inhibits breast tumor growth and increase NK cell infiltration a nude mouse model of tumorigenesis.10 Furthermore, shRNA-mediated knockdown of ISG15 increased the resistance of human tumor cells to CPT.11 These findings indicate that ISG15 is also a candidate biomarker of the responsiveness to immunotherapy among patients with cancer.\n\nISG15 plays a key role in the host antiviral response. As such, ISG15 is implicated as a diagnostic biomarker of viral infection. Studies in patients with HCC have shown high levels of ISG15 protein in human HBV-related HCC tissues compared to the levels detected in non-tumor tissues.12 Furthermore, upregulated expression of ISG15 was observed at both the mRNA and protein levels in liver tissue samples from patients with HCV infection compared to the levels detected in uninfected controls.13 Similarly, the abundance of ISG15 transcripts was found to be increased in human PBMCs and liver cells in patients with HCV infection who were unresponsive to IFN treatment compared to the levels in corresponding IFN-responsive patients.14 In addition, high levels of ISG15 in the liver of patients with HCV infection were associated with an unfavourable HCV genotype 1, a high hepatic HCV load and a low antiviral response to IFN compared to patients who did not present such characteristics.13 These findings indicate the potential of ISG15 as a biomarker of IFN treatment response in patients with HCV infection.\n\nIncreased ISG15 expression has also been reported in mouse models related to HIV infection. ISG15 is upregulated in transgenic mice overexpressing the HIV gp120 protein.15 Moreover, in mouse models of acute and chronic neuronal injuries with HIV infection, ISG15 protein in the brain was increased compared to the levels detected in mice with global ischemia and traumatic brain injuries.15 The importance of ISG15 as a biomarker of HIV infection and treatment response has also been revealed in human studies. Compared to healthy donors, TaqMan assays revealed high ISG15 expression (mRNA and protein) as well as an increased frequency of ISG15-SNPs in human PBMC from untreated patients with HIV infection.16 Moreover, ISG15 expression decreased in human PBMC in patients with HIV infection after long-term antiretroviral therapy.16\n\nThe key importance of ISG15 in the human immune system is also reflected in its role in autoimmune diseases, such as SLE. High levels of ISG15 transcripts were detected in human whole blood cell samples from 28 patients newly diagnosed with SLE compared to with 10 patients with undifferentiated connective tissue disease, and 22 healthy volunteers,17 indicating the potential of ISG15 as a specific biomarker of SLE. Moreover, the upregulation of ISG15 in human plasmablasts/plasma cells from patients with active SLE indicate that ISG15 can be used as a marker of disease activation in patients in remission.18\n\nThus, an increasing body of evidence supports a role of ISG15 as a biomarker with diagnostic relevance for a number of human disorders.”\n\n\nImplementation\n\nAs proof of principle, a workshop was implemented using the step-by-step guide and supporting information provided with this paper. It was led by FAA and AKM, who participated in the development of the training curriculum, but had no prior experience running such training activities. The workshop took place on January 26, 2021. In total, 29 graduate students from Hamad Bin Khalifa University took part. It was offered as one three-hour class as part of an “Introduction to Data Science” course. Due to Covid-19 restrictions the workshop was run remotely using WebEx. As no information was collected from workshop participants (i.e., no surveys, or questionnaires), the activity was not considered to constitute human research and therefore no ethical approval was required. The generic introductory presentation in Extended data File 134 was adapted to provide more specific context, and notably explain how, CCR1, the target gene for this workshop was selected (for illustrative purposes this introductory presentation is also made available here: Extended data File 4).37 FAA and AKM, the two co-instructors, also prepared a handout to guide participants through the different steps (Extended data File 5),38 specifically for the CCR1 use case. Several days prior to the workshop, participants had been asked to create a Prezi “Basic” account and a template was created and shared with each one of them ahead of time. Following the introductory presentation (10 minutes) participants carried out hands-on activities following each one of the tasks described in the handout, with each step corresponding to one working session. Given time constraints, material prepared ahead of time by FAA and AKM was provided to the participants at the end of each working session so that they could move on to the next. Ideally sessions should span multiple days in order for participants to complete the assignments. However, the more intensive schedule implemented here had the advantage of introducing literature profiling concepts and approaches to a large group of trainees in a time-effective manner. The opportunity to continue work later on, on their own time being also available to them.\n\n\nConclusions\n\nEffectively harnessing biomedical literature is one of the most fundamental skills required by biomedical researchers. Given the current rate at which articles are published, developing more systematic approaches to literature profiling based on defined principles may prove especially beneficial to early-career biomedical researchers.\n\nHere, we present a training workflow as well as supporting material that may be re-used/adapted for the organization of ‘Gene literature retrieval, profiling and visualization’ training workshops. Hands-on activities range from literature retrieval and optimization of PubMed queries, to the development of interactive resources and authoring original material focusing on a specific topic.\n\nA number of the steps described could easily be automated. For instance, the concatenation of official gene names and aliases for retrieving gene-specific literature using PubMed (Step 1). Indeed, such tools are under development by our group and will be made available in the future. These tools would not only to save time, but also minimize user error. Nevertheless, some of the steps, such as optimization of search queries or extraction of information from abstracts will always require some critical evaluation and decision making that cannot be automated. Refraining from the use of such automated tools in a training workshop may also be a deliberate choice on the part of the instructor if the emphasis is initially on the development of competency in completing the steps involved throughout the process.\n\nHere, we focused on gene-associated literature, simply because the training curriculum that is currently under development is based on the reuse of publicly available omics data. A similar approach could be employed to profile the literature associated with any given disease, pathway, molecular process, or drug, for instance.\n\nWe aim to further develop an illustrative case that would lead to the publication of a review of the ISG15 literature employing the literature profiling and visualization approaches described herein. We also plan in the future to develop and make available guides and supporting material for the implementation of hands-on training workshops focusing on other topics, which include: 1) retrieval, visualization and interpretation of gene-specific transcriptional profiles19–21 2) constitution and curation of themed public dataset collections22–30 3) global analysis of large-scale omics data.31–33 These hands-on training activities would build in part on the skills developed through the implementation of the literature profiling workshop that we have presented here.\n\n\nData availability\n\nNo newly generated data are associated with this article. Information was retrieved via the literature search engine PubMed (https://pubmed.ncbi.nlm.nih.gov/).\n\nThis project contains the following extended data:\n\nFigshare: Literature profiling workshop, introduction session slides (S File 1), https://doi.org/10.6084/m9.figshare.13669070.v2.34\n\nFigshare: Literature profiling workshop: steps 1-3 (S File 2), https://doi.org/10.6084/m9.figshare.14160329.v3.35\n\nFigshare: Literature Profiling Workshop: Step 5 (S File 3), https://doi.org/10.6084/m9.figshare.14161484.v1.36\n\nFigshare: Literature profiling workshop: HBKU handout (S File 4), https://doi.org/10.6084/m9.figshare.14166395.v1.37\n\nFigshare: Literature profiling workshop: HBKU intro presentation (S File 5), https://doi.org/10.6084/m9.figshare.14166500.v1.38\n\nData are available under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) license.", "appendix": "Acknowledgements\n\nWe wish to thank all the students from Hamad Bin Khalifa University (HBKU) for participating in the workshop that served as a proof-of-principle for the use of the implementation guide described in this paper. We also thank the Division of Genomics and Translational Biomedicine at the College of Health and Life Sciences at HBKU for hosting this workshop.\n\n\nReferences\n\nMargolis R, Derr L, Dunn M, et al.: The National Institutes of Health’s Big Data to Knowledge (BD2K) initiative: capitalizing on biomedical big data. J Am Med Inform Assoc. 2014 Dec; 21(6): 957–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Horn JD, Fierro L, Kamdar J, et al.: Democratizing data science through data science training. Pac Symp Biocomput. 2018; 23: 292–303. PubMed Abstract \| Free Full Text\n\nGarmire LX, Gliske S, Nguyen QC, et al.: The training of next generation data scientists in biomedicine. Pac Symp Biocomput. 2017; 22: 640–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaussabel D, Rinchai D: Using “collective omics data” for biomedical research training. Immunology. 2018; 155(1): 18–23. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMackeh R, Boughorbel S, Chaussabel D, et al.: -A curated transcriptomic dataset collection relevant to embryonic development associated with in vitro fertilization in healthy individuals and patients with polycystic ovary syndrome. F1000Res. 2017; 6: 181. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlazkova J, Boughorbel S, Presnell S, et al.: A curated transcriptome dataset collection to investigate the immunobiology of HIV infection. F1000Res. 2016; 5: 327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBougarn S, Boughorbel S, Chaussabel D, et al.: A curated transcriptome dataset collection to investigate inborn errors of immunity. F1000Res. 2019; 8: 188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahman M, Boughorbel S, Presnell S, et al.: A curated transcriptome dataset collection to investigate the functional programming of human hematopoietic cells in early life. F1000Res. 2016; 5: 414. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRinchai D, Presnell S, Vidal M, et al.: Blood Interferon Signatures Putatively Link Lack of Protection Conferred by the RTS,S Recombinant Malaria Vaccine to an Antigen-specific IgE Response. F1000Res. 2015; 4: 919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRawat A, Rinchai D, Toufiq M, et al.: A Neutrophil-Driven Inflammatory Signature Characterizes the Blood Transcriptome Fingerprint of Psoriasis. Front Immunol. 2020 [cited 2020 Dec 7]; 11. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nRinchai D, Altman MC, Konza O, et al.: Definition of erythroid cell-positive blood transcriptome phenotypes associated with severe respiratory syncytial virus infection. Clin Transl Med. 2020; 10(8): e244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaussabel D: Literature profiling workshop, introduction session slides (S File 1). figshare. Journal contribution. 2021. Publisher Full Text\n\nChaussabel D: Literature profiling workshop: steps 1-3 (S File 2). figshare. Journal contribution. 2021. Publisher Full Text\n\nChaussabel D: Literature Profiling Workshop: Step 5 (S File 3). figshare. Journal contribution. 2021. Publisher Full Text\n\nChaussabel D: Literature profiling workshop: HBKU handout (S File 4). figshare. Journal contribution. 2021. Publisher Full Text\n\nChaussabel D: Literature profiling workshop: HBKU intro presentation (S File 5). figshare. Journal contribution. 2021. Publisher Full Text" }	[ { "id": "86220", "date": "15 Jun 2021", "name": "Gary S. McDowell", "expertise": [ "Reviewer Expertise Early career researcher experiences", "training and professional development." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors discuss the need and provide a curriculum for early career researchers to develop the ability to survey and synthesize the background literature to a field in an appropriate manner. This is demonstrated with publicly available “omics” data as an example.\nThis article gives an excellent description of a training workflow that is suitable and adaptable for training in systematic literature profiling. The article provides clear illustrative use case examples that are very helpful in demonstrating the results of the training workflow, and provide excellent materials for others to work from in developing similar materials.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "96508", "date": "20 Oct 2021", "name": "Valerie Matarese", "expertise": [ "Reviewer Expertise I am a former biomedical researcher with 25 years' experience as a biomedical editor in different capacities and 15 years' experience as instructor of research writing at the PhD level. I am also a scholar of academic editing." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverview This method article describes a short workshop (3 hours) with five stated teaching objectives: bibliographic research in PubMed on gene-related topics (“retrieval”); extraction of relevant keywords (“concepts”) from article titles; “profiling” (counting the number of articles per concept); using the data and online tools to create interactive graphics (“visualization”); and “writing a narrative review” using a semi-automated method in a spreadsheet. I welcome a workshop description for teaching bibliographic research, and I am sure that workshop trainees will enjoy learning to make treemaps and circle packing charts. However, I have substantial reservations about the last objective and thus, as peer reviewer, I cannot endorse the method as a whole.\nRegarding F1000Research questions The rationale for the workshop is clearly given, but no valid justification is given for using a spreadsheet to write a narrative review (fifth objective) for the Introduction section of a research article or for a review article. The conclusions about the method and its performance are supported in part by the findings presented in the article, but the generated nonsense text is clear evidence that the proposed method for automated text generation should not be published.\nMajor comment The fifth objective, “Writing a narrative”, is associated with two skills that workshop trainees can expect to learn: “Assimilation of biomedical knowledge (synthesis and presentation)” and “Scientific publication (optional)”. However, as described in the manuscript, the workshop is unable to fulfill these promises at all. In this fifth part of the workshop, participants are instructed to read the “abstract and/or full text and record in a spreadsheet” a series of data. They then use the spreadsheet’s concatenate function to generate “standard sentences” and collate these into a “cohesive paragraph or section”. Finally, the text is “polished” by the students, an instructor or a “professional scientific copyeditor”. There are enormous problems with all of this.\nFirst and foremost, the method is proposing the automated generation of text for publication based primarily on abstracts (“full text where possible/necessary”) and without critical appraisal. Only in the conclusions of the manuscript is there any minimal mention of this problem: “Nevertheless, … extraction of information from abstracts will always require some critical evaluation and decision making that cannot be automated” (emphasis added). I cannot endorse a method for the computer-assisted writing of research articles and reviews, as it is unscholarly, wasteful to the reviewers and (if published) readers who will have to sift through substantial nonsense text, and damaging to knowledge advancement.\nThat this method can generate nonsense text is seen in the two pages of “Illustrative case”. Part A (page 11) shows the computer-generated “standard sentences” which are close to gibberish, for example:\nThe abundance of ISG15 Proteins measured by immunohistochemistry is increased in vivo in human immune cell markers “in patients with” invasive BC patients with long-term follow-up compared to … … ………… … .. [33073304].\nPart B (page 12) shows the “edited narrative” which “was prepared by a professional scientific copyeditor”. Given both the poor science and poor English grammar and style of this narrative, I doubt that a professional editor worked on it. Furthermore, the use of an editor to create meaningful text from the “standard sentences” in Part A would amount to ghost writing. Here is just one example from the supposedly edited text:\nCompared to healthy donors, TaqMan assays revealed high ISG15 expression (mRNA and protein) as well as an increased frequency of ISG15-SNPs in human PBMC from untreated patients with HIV infection.\nTaqMan assays do not inform on protein levels. The frequency of a SNP cannot change unless there is gene duplication (what changes is the allele frequency). There is no need to use “human” to describe cells from HIV patients. There is a misplaced modifier before “TaqMan” and an unwanted hyphen.\nI therefore feel that, for a revised version of this manuscript to be approved, the fifth objective should be deleted along with its two associated skills (impossible to achieve in a short workshop). In addition, a paragraph should be added with comments on the need for early career researchers to learn to read the scientific literature at different levels (from scanning to critical appraisal of the full text) and to appreciate that scientific findings must be replicated and validated before they can be considered “true”.\nMinor comments The title is a hard read, because there are six words between “Organizing” and what gets organized. I suggest: Organizing training workshops on gene literature …. for early career researchers”.\nThe word “profiling” is used to the point of being meaningless. It is used six times in the 20-line Introduction, in the context of literature, omics and transcriptome. It should be deleted after “omics” and “transcriptome”, as these words are best used as nouns, not adjectives describing the vague “profiling”. Regarding “literature profiling”, this non-standard expression should be defined early in the text, not in the Methods section (bottom page 3). Here, profiling means “information extraction” from titles and “determination of keyword frequencies”. This definition should be given in the Introduction as soon as “literature profiling” is mentioned.\nThroughout the text: The use of conditional mood (“this session should present”, “the presentation should last” and so on) is unnecessary, burdens the text and slows reading. The simple present tense can be used in most cases. A method is not a recommendation (“should”) but a description (“is”).\nThroughout the text: The use of italics in the “illustrative cases” makes reading difficult, especially because of the extensive use of quotation marks and parentheses. Italics should be reserved for emphasis on small pieces of text. Here, instead, a block quote (indented text) would be better. Text boxes can also help.\nPage 2: “some independent preparation”. Does this mean independent study, exercises, getting organized technically?\nPage 2: Ref. 34 is cited right after Ref. 4. It should be renumbered 5. I confused it with Refs. 3,4 (missing a comma). Similarly Ref. 35 is cited too early, on page 6.\nPage 3, line 3: “on any given specific research topics” should be changed to “on any given research topic”. Topic should be singular. Specific is unneeded and redundant with “given topic”.\nPage 3: “for a gene with ± 1000 associated articles and five participants”. This phrase is too concise to have meaning, as a gene cannot have participants.\nPage 4, Step 1. No mention is made of NCBI’s Advanced Search Builder; MeSH terms; or truncation by asterisk. The manuscript should explain why these advanced features are not taught.\nPage 4, bottom: “The field restriction tag [tw] (or alternatively the more restrictive [tiab]) should be added after each term… to limit the search to titles and abstracts”. Actually, it is [tiab] that limits the search to titles and abstracts. The broad [tw] searches almost everything (see https://pubmed.ncbi.nlm.nih.gov/help/#tw).\nPage 5: “examples provided above”. Where? Vague cross-references like this make reading difficult.\nPage 6: “For instance, when assessing the novelty …”. This incomplete sentence is lacking a subject-verb pair. Similar problem on page 13, bottom.\nPage 6, bottom: “The Boolean argument AND is added”. AND is a Boolean operator, not argument.\nPage 6, bottom: ‘… AND (“Liver cancer”[tw] OR “Liver carcinoma”[tw] OR “Hepatic carcinoma”[tw], etc.)’. A query cannot contain “, etc.”. It should be removed, and a final period added to the sentence.\nPage 9, Step 5. This part is excessively wordy and suggests an uneasiness with the concepts. We read “is the measure by which productivity is measured”, “it can be at first important to be able to identify information that is worth capturing” and “A specific topic would be selected as the focus … to limit the amount of literature that would need to be covered”.\nPage 13: Webex. Not WebEx.\nPunctuation\nComma after “Supporting material” in the third paragraph of Introduction. Comma after “This is a skill that is worth developing” (page 9).\n\nReplace ± with “about” (first paragraph of Methods).\n\nHyphens: no hyphen after -ly adverbs, e.g. “early career researcher”. Use hyphens in “gene-associated literature”, “false-positive result” and “false-negative result” (but not in “false positives” or “false negatives”).\n\nThere are many instances where the required space between words is missing, especially when a quotation mark is used, e.g. “For example, under’Human diseases and … associated keywords could be’liver cancer’,’hepatic carcinoma’, …” (page 5). The entire manuscript should be checked and spaces added where needed. A hint is that the curly quotation mark is in the wrong direction before the word when a space is missing.\n\nDouble quotation marks should be used in all cases except when nesting is needed. Nesting should be used on page 5 where the sentence:\nThis was rectified by adding the AND “cross-reactive protein [tw] argument to each of the ambiguous acronym.\n\nshould be changed to:\nThis was rectified by adding the ‘AND “cross-reactive protein [tw]”’ argument to each of the ambiguous acronyms.\nor more simply:\nThis was rectified by adding ‘AND “cross-reactive protein [tw]”’ to each of the ambiguous acronyms.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "9475", "date": "11 Jul 2023", "name": "Damien Chaussabel", "role": "Author Response", "response": "Overview This method article describes a short workshop (3 hours) with five stated teaching objectives: bibliographic research in PubMed on gene-related topics (“retrieval”); extraction of relevant keywords (“concepts”) from article titles; “profiling” (counting the number of articles per concept); using the data and online tools to create interactive graphics (“visualization”); and “writing a narrative review” using a semi-automated method in a spreadsheet. I welcome a workshop description for teaching bibliographic research, and I am sure that workshop trainees will enjoy learning to make treemaps and circle packing charts. However, I have substantial reservations about the last objective and thus, as peer reviewer, I cannot endorse the method as a whole. We appreciate the time spent reviewing this work and the constructive feedback given. The reason we are coming back to revising this article a little bit late is because we wanted first to complete the development of a more rounded training curriculum. As a result, a study guide and use case have recently been published in F1000Research (https://f1000research.com/articles/11-994, https://f1000research.com/articles/11-1294). Literature profiling remains nonetheless a major component of this training curriculum and could be run as stand-alone training/workshop. Also, we also wanted to circle back to this manuscript at this time and address all outstanding comments and add references to the more recent work. We have addressed the main comment raised in the review by eliminating the step in the workflow that involved automatically generating text from the information provided by workshop participants. We did not intend for this step to cause controversy and were simply trying to practically train early-career scientists in countries where English literacy may not be as high as in more privileged countries. The question then becomes, how can these brilliant scientists overcome the language barrier and fulfil their potential? The approach we attempted, as an experiment, was only meant to be a first step towards trainees becoming independent writers, eventually able to produce literary work without artificial crutches. However, we admit that the approach may have been flawed or misguided (or both!), and we acknowledge your point. We are happy to remove this step from the manuscript. Regarding F1000Research questions The rationale for the workshop is clearly given, but no valid justification is given for using a spreadsheet to write a narrative review (fifth objective) for the Introduction section of a research article or for a review article. The conclusions about the method and its performance are supported in part by the findings presented in the article, but the generated nonsense text is clear evidence that the proposed method for automated text generation should not be published. Thanks for raising these concerns. We have now removed the steps that pertains to automated text generation (5e and 5f). We feel that capturing relevant information and recording it in a spreadsheet is still a worthwhile exercise. Indeed, participants can learn in the process what information they should be looking for in the work of others and make sure to report in their own (e.g. abstracts often fail to mention in what species the work is performed or if abundance of protein or transcripts is measured). We now write instead as step 5e: “The information captured in the spreadsheet will serve as a synopsis that participants will be able to rely upon for developing a written narrative, which could be used in an introduction or review article.” The spreadsheet has also been amended accordingly, removing the functions used to generate text. And the text that was automatically generated has been removed from the manuscript. We retained the written narrative, simply prefacing the paragraph with the following statement: “Narrative, based on the information captured in the spreadsheet: “ Major comment The fifth objective, “Writing a narrative”, is associated with two skills that workshop trainees can expect to learn: “Assimilation of biomedical knowledge (synthesis and presentation)” and “Scientific publication (optional)”. However, as described in the manuscript, the workshop is unable to fulfill these promises at all. In this fifth part of the workshop, participants are instructed to read the “abstract and/or full text and record in a spreadsheet” a series of data. They then use the spreadsheet’s concatenate function to generate “standard sentences” and collate these into a “cohesive paragraph or section”. Finally, the text is “polished” by the students, an instructor or a “professional scientific copyeditor”. There are enormous problems with all of this. First and foremost, the method is proposing the automated generation of text for publication based primarily on abstracts (“full text where possible/necessary”) and without critical appraisal. Only in the conclusions of the manuscript is there any minimal mention of this problem: “Nevertheless, … extraction of information from abstracts will always require some critical evaluation and decision making that cannot be automated” (emphasis added). I cannot endorse a method for the computer-assisted writing of research articles and reviews, as it is unscholarly, wasteful to the reviewers and (if published) readers who will have to sift through substantial nonsense text, and damaging to knowledge advancement. That this method can generate nonsense text is seen in the two pages of “Illustrative case”. Part A (page 11) shows the computer-generated “standard sentences” which are close to gibberish, for example: The abundance of ISG15 Proteins measured by immunohistochemistry is increased in vivo in human immune cell markers “in patients with” invasive BC patients with long-term follow-up compared to … … ………… … .. [33073304]. Part B (page 12) shows the “edited narrative” which “was prepared by a professional scientific copyeditor”. Given both the poor science and poor English grammar and style of this narrative, I doubt that a professional editor worked on it. Furthermore, the use of an editor to create meaningful text from the “standard sentences” in Part A would amount to ghost writing. Here is just one example from the supposedly edited text: Compared to healthy donors, TaqMan assays revealed high ISG15 expression (mRNA and protein) as well as an increased frequency of ISG15-SNPs in human PBMC from untreated patients with HIV infection. TaqMan assays do not inform on protein levels. The frequency of a SNP cannot change unless there is gene duplication (what changes is the allele frequency). There is no need to use “human” to describe cells from HIV patients. There is a misplaced modifier before “TaqMan” and an unwanted hyphen. I therefore feel that, for a revised version of this manuscript to be approved, the fifth objective should be deleted along with its two associated skills (impossible to achieve in a short workshop). In addition, a paragraph should be added with comments on the need for early career researchers to learn to read the scientific literature at different levels (from scanning to critical appraisal of the full text) and to appreciate that scientific findings must be replicated and validated before they can be considered “true”. We amended Step 5 as described above, removing altogether any notion of automated text generation, while keeping the step that consists in capturing and recording key information in a spreadsheet. If necessary, even this could be removed, but again as stated above we feel it is a worthwhile exercise. We amended our learning objectives, removing the two points mentioned in your critique and writing instead: Extraction of biomedical information (capturing information in a structured format). We also added wording highlighting the need for participants to go beyond scanning titles and abstracts and read research papers: “Thus, early career researchers need to develop the ability to read the scientific literature at different levels: from “scanning” a large number of articles to reading the full text version for a more in depth understanding.” And for the record – since integrity is no small matter in science - we maintain that a professional and reputable science editor did work on this text, as we had stated earlier; even though as pointed out the service rendered was maybe not of the highest quality in this case (we have been otherwise been quite happy with them). Minor comments The title is a hard read, because there are six words between “Organizing” and what gets organized. I suggest: Organizing training workshops on gene literature …. for early career researchers”. Thank you for the helpful edits, which have been incorporated throughout the text and to the best of our abilities. We also took your advice and changed the title of the article to improve legibility. The word “profiling” is used to the point of being meaningless. It is used six times in the 20-line Introduction, in the context of literature, omics and transcriptome. It should be deleted after “omics” and “transcriptome”, as these words are best used as nouns, not adjectives describing the vague “profiling”. Regarding “literature profiling”, this non-standard expression should be defined early in the text, not in the Methods section (bottom page 3). Here, profiling means “information extraction” from titles and “determination of keyword frequencies”. This definition should be given in the Introduction as soon as “literature profiling” is mentioned. We have deleted profiling when associated with omics data and defined literature profiling early on in the introduction. Throughout the text: The use of conditional mood (“this session should present”, “the presentation should last” and so on) is unnecessary, burdens the text and slows reading. The simple present tense can be used in most cases. A method is not a recommendation (“should”) but a description (“is”). That makes sense and we have edited the text throughout. Throughout the text: The use of italics in the “illustrative cases” makes reading difficult, especially because of the extensive use of quotation marks and parentheses. Italics should be reserved for emphasis on small pieces of text. Here, instead, a block quote (indented text) would be better. Text boxes can also help. Text boxes sound ideal. We will reach out to the editor to see if this is feasible. Page 2: “some independent preparation”. Does this mean independent study, exercises, getting organized technically? We clarified this point by rewording this sentence, that now reads: “Trainees can be asked to perform tasks between each of the sessions.” Page 2: Ref. 34 is cited right after Ref. 4. It should be renumbered 5. I confused it with Refs. 3,4 (missing a comma). Similarly Ref. 35 is cited too early, on page 6. Indeed, we will communicate this to the editor so it can be addressed in the formatted document. Page 3, line 3: “on any given specific research topics” should be changed to “on any given research topic”. Topic should be singular. Specific is unneeded and redundant with “given topic”. This sentence has been edited accordingly. Page 3: “for a gene with ± 1000 associated articles and five participants”. This phrase is too concise to have meaning, as a gene cannot have participants. We have edited this sentence to read: “For instance, for a gene with about 1,000 associated articles, five participants could work together on this same gene, each focusing on a different theme.” Page 4, Step 1. No mention is made of NCBI’s Advanced Search Builder; MeSH terms; or truncation by asterisk. The manuscript should explain why these advanced features are not taught. Indeed, thanks for the suggestion. We have re-written this section and mention the availability of the advanced search builder and provided the corresponding URL. We also pointed to training material available on the NCBI website. In our experience truncation by asterisk can produce unwanted results and increase false positives, also did not want to recommend using it in this case. Page 4, bottom: “The field restriction tag [tw] (or alternatively the more restrictive [tiab]) should be added after each term… to limit the search to titles and abstracts”. Actually, it is [tiab] that limits the search to titles and abstracts. The broad [tw] searches almost everything (see https://pubmed.ncbi.nlm.nih.gov/help/#tw). Yes, this is correct, and this entire section has been rewritten for clarity. We found the [tw] field to be effective in preventing PubMed from trying best guesses when few entries are returned. [tiab] can be used when users wish to further restrict the search. But both should provide good results for our purpose. Page 5: “examples provided above”. Where? Vague cross-references like this make reading difficult. Thanks for pointing this out. We are now providing those examples instead. Page 6: “For instance, when assessing the novelty …”. This incomplete sentence is lacking a subject-verb pair. Similar problem on page 13, bottom. This has been corrected. Page 6, bottom: “The Boolean argument AND is added”. AND is a Boolean operator, not argument. Indeed, this has been corrected. Page 6, bottom: ‘… AND (“Liver cancer”[tw] OR “Liver carcinoma”[tw] OR “Hepatic carcinoma”[tw], etc.)’. A query cannot contain “, etc.”. It should be removed, and a final period added to the sentence. This has been corrected as well. Page 9, Step 5. This part is excessively wordy and suggests an uneasiness with the concepts. We read “is the measure by which productivity is measured”, “it can be at first important to be able to identify information that is worth capturing” and “A specific topic would be selected as the focus … to limit the amount of literature that would need to be covered”. We rewrote and condensed this paragraph. Page 13: Webex. Not WebEx. This has been corrected. Punctuation Thank you, that was very useful, and we have now made the necessary edits throughout. Comma after “Supporting material” in the third paragraph of Introduction. Comma after “This is a skill that is worth developing” (page 9). Replace ± with “about” (first paragraph of Methods). Hyphens: no hyphen after -ly adverbs, e.g. “early career researcher”. Use hyphens in “gene-associated literature”, “false-positive result” and “false-negative result” (but not in “false positives” or “false negatives”). There are many instances where the required space between words is missing, especially when a quotation mark is used, e.g. “For example, under’Human diseases and … associated keywords could be’liver cancer’,’hepatic carcinoma’, …” (page 5). The entire manuscript should be checked and spaces added where needed. A hint is that the curly quotation mark is in the wrong direction before the word when a space is missing. Double quotation marks should be used in all cases except when nesting is needed. Nesting should be used on page 5 where the sentence: 'This was rectified by adding the AND “cross-reactive protein [tw] argument to each of the ambiguous acronym.' should be changed to: 'This was rectified by adding the ‘AND “cross-reactive protein [tw]”’ argument to each of the ambiguous acronyms.' or more simply: 'This was rectified by adding ‘AND “cross-reactive protein [tw]”’ to each of the ambiguous acronyms.'" } ] } ]	1	https://f1000research.com/articles/10-275
https://f1000research.com/articles/10-481/v1	16 Jun 21	{ "type": "Research Article", "title": "The balancing act: A qualitative study on remote work and childcare during the COVID-19 lockdown in South Africa", "authors": [ "Salome Scholtz" ], "abstract": "Background: The coronavirus disease 2019 (COVID-19) pandemic has impacted many employees' world by forcing some to remote work whilst also caring for children. Methods: This study explored the remote working experiences of child caregivers in balancing work and childcare during the South African COVID-19 lockdown period (from 26 March 2020 to 31 December 2020). A qualitative descriptive design was employed to collect data through an online qualitative questionnaire with semi-structured questions. A final sample of 22 participants was included using purposive and snowball sampling. Results: Findings indicate that employees experienced difficulty balancing work and childcare, which prompted them to use personal and organisational resources to restore work-life balance. These resources included people, spaces and time. Conclusions: This study provides insight for employers and healthcare workers into how remote workers balanced work and childcare and can promote conversations or interventions on improving employee remote work experiences - especially in the unique South African context.", "keywords": [ "Caregiving", "COVID-19", "lockdown", "remote work", "work-life-balance", "work experiences", "working from home." ], "content": "Introduction\n\nThe novel coronavirus disease 2019 (COVID-19) is an infectious viral disease that has shaken the world of work through a COVID-19-all-remote work experiment (Liang, 2020; Tavares, 2017). This experiment resulted in specific sectors’ employees using their homes as workplaces, relaxation spaces and schools for the first time (Oakman et al., 2020). According to the Organisation for Economic Cooperation and Development (OECD) (OECD, 2020), work productivity has declined on individual and organisational levels due to the lack of childcare support, violations of family-work boundaries and social isolation. South Africa has the highest number of COVID-19 infections on the African continent (Mwai, 2020; South African Corona Virus portal, 2021). Additionally, the population is under a unique threat during the pandemic with its high HIV and tuberculosis infections, diabetes and shared substance abuse (Egbe et al., 2019; Egbe & Ngobese, 2020).\n\nAfter the first positive COVID-19 case was reported in South Africa on March 5, 2020 (Stiegler & Bouchard, 2020), the infection rate quickly increased and lead to drastic measures taken by the South African government, namely: a nationwide lockdown starting on March 26, 2020 (South African Government News Agency, 2020). According to The Human Sciences Research Council (HSRC) (2020), the middle class South African citizens adhered to the lockdown regulations well; staying home and working remotely, which according to Putro and Riyanto (2020), is a highly desired work arrangement for this professional group. However, as in-person schools and childcare centres were closed during some phases of the lockdown, childcare responsibilities increased, thereby blurring the boundaries between work and home (Shockley et al., 2020). This study sought to explore the experiences of these South African employees in balancing remote work and childcare during the 2020 South African lockdown to identify opportunities where managers and healthcare workers can lend support to these employees.\n\nLockdown is a situation where people are not allowed free movement in an area due to danger, such as that posed by the COVID-19 pandemic. The South African lockdown started on level five, the strictest lockdown level, indicating a high COVID-19 infection rate and low health system readiness (Businesstech, 2020). The lockdown was soon extended, and the country only entered lockdown level one on September 20, 2020 (Mwai, 2020; Stiegler & Bouchard, 2020). During the level five lockdown, all activities such as dog-walking, attending school, outdoor exercises, the use or purchase of alcohol and cigarettes (public consumption) were banned (Stiegler & Bouchard, 2020), making South Africa one of the African countries with the strictest lockdown regulations (Stiegler & Bouchard, 2020). The restrictions slowly eased with every level after that, with lockdown level three (May 31), for example, allowing alcohol retailing, religious activities and exercise (South African Coronavirus Portal, 2020a).\n\nDuring the lockdown, certain employees found themselves remote working for the first time by conducting work at home or a place other than that of the organisation’s usual site (Baruch, 2000; Cambridge Dictionary, 2021). This work arrangement can potentially promote various personal and professional benefits, for example, a better work-life balance for employees (Sarbu, 2020), improved company culture, communication and productivity (Tuyo, 2020). Baard and Thomas (2010) found that South Africans experienced improved organisation loyalty, productivity and work-life balance when working from home. Choudhury (2020) adds that remote workers enjoy the freedom to work from home and experience a higher quality of life, improving their overall happiness. Organisations also benefit by increasing employee engagement with happier and more productive employees and lower office hire expenses. However, Anderson and Kelliher (2020) warn that remote work benefits differ when enforced and full-time. Their research indicates that the benefit of autonomy and gratitude employees experience from willingly remote working can be lower when employees are deprived from the choice to do so. This lowered remote work benefit can especially be seen in mothers due to less control of when and how work is conducted in the midst of family responsibilities when employees are forced to work from home (Anderson & Kelliher, 2020).\n\nFirst-time remote workers in South Africa experienced work overload and performance pressure despite the positive aspects of working remotely (Matli, 2020). This work overload was especially true when employees’ home responsibilities increased, and work-life boundaries became vague (Sarbu, 2020). Due to the governmental lockdown, parents had to work from home while fulfilling their children’s teacher and parent roles (Casale & Posel, 2020). Thus, a tremendous burden was placed on families (Del Boca et al., 2020) as they balanced their responsibilities in unexpected ways (Limoncelli, 2020) and work hours were influenced by what happened within homes (Casale & Posel, 2020). In academia, for example, some researchers have experienced a decrease in publications during the lockdown, which could be due to juggling childcare and working from home (Hedding et al., 2020). Research shows that one parent’s need to stay home and care for the children has made it difficult, especially for women, to work during the COVID-19 pandemic (Queisser et al., 2020). According to Odek (2020), the school closures restricted South African women as many women had to stay home and care for children instead of working (Odeku, 2020).\n\nSouth African child caregivers fulfilled these roles of parent, teacher, and employee until August 3 2020, when schools gradually opened, starting with Grade 12 learners (Enca, 2020). The largest group of learners, grades R to four, six, nine, 10 and 11, started attending school on August 24, and the final grades five and eight returned on the 31st (Makou, 2020). However, as most schools could not fulfil the requirements for social distancing in 2021, half of the learners still spend some time at home during the week. Some schools accept learners on a ‘week on’ – ‘week off’ basis, and other schools allow learners to attend school every second day. This type of arrangement led to learners attending half the school days of the second part of the school year in 2020 (van der Berg, 2020). Working from home was also complicated by childcare as it interfered with conducting remote work, as some employees feared clients might infect their children if they met at home (Odeku, 2020). Adding to this was caring for those who have contracted the COVID-19 virus and expanding work hours (Khwela-Mdluli & Beharry-Ramraj, 2020). Therefore, Khwela-Mdluli and Beharry-Ramraj (2020) and Odeku (2020) urged South Africa to devise systems to support the health and work equality of South African remote working women. This system is especially imperative during school closures and women carrying the burden of care and remote work for their families (Odeku, 2020).\n\nInternational research also shows that most of the additional work during lockdown fell on women (Sevilla & Smith, 2020). A Spanish study found that women experienced higher work and family conflict than men (Lopez-Núčez et al., 2021). In contrast, Del Boca et al. (2020) indicated that childcare was distributed equally between Italian parents whilst home activities were still primarily the responsibility of women. Time spent with children during the lockdown was also less if the parents returned to workplaces after the lockdown (Del Boca et al., 2020). Research by Shockley et al. (2020) investigated the gender roles whilst remote working and caring for children. Their results showed that dual-earner couples assumed gendered roles during the lockdown. In contrast, men only occasionally looked after children (e.g. when wives were in meetings or one day a week), with most or all childcare duties falling on wives. They also found women had high relationship tension, low family cohesion and poor job performance. Men also experienced some impact on job performance, presumably due to marital strife due to unequal distribution of household duties (Shockley et al., 2020).\n\nAdditionally, couples tried alternating days of work and care with one partner working in the morning and the other in the evening; this, however, still impacted their job performance and may not help those who work remotely full-time (Shockley et al., 2020). In India, women experienced higher emotional exhaustion than men caused by personal life interference whilst working from home (Bhumika, 2020). Putro and Riyanto (2020), on the other hand, found that Asian sandwich generations, i.e. persons caring for both their parents and their children, experienced relief from work conflict as their parents could assist in childcare while they remote worked. South African households, however, are unique compared to international families due to parental absence (Hall, 2019). According to Hall (2019), South African children make up 34% (19.7 million) of the total South African population, and of these children, only 34% live with both their biological parents (Hall, 2019; Hall & Mokomane, 2018). Thus, children are often raised by relatives, without fathers or live separate from their siblings (Hall, 2019).\n\nThe COVID-19 pandemic has left many businesses in turmoil and managers losing sleep over their employees’ struggles (Zaki, 2020). At the onset of the COVID-19 pandemic, UNICEF encouraged employers to provide time and support to new remote working parents through, for example, reassessing workplace policies, flexible work time, good hygiene at workplaces and giving assistance to cope with stress (UNICEF, 2020). Despite the opening of schools and childcare centres, Watson (2020) warns human resource (HR) workers should not think that all children have returned to school. With the COVID-19 outbreak seen as continuous (Jones, 2020) and the unpreparedness and weak infrastructure of South African schools, many parents cannot or do not send their children to school full-time or at all (Amnesty International, 2021). This is further supported by South African homeschooling agencies such as Impaq that have experienced a rise in homeschool applications to 5000 more learners in August of 2020 (Businesstech, 2020). Many parents have adopted this learning mode as their new permanent schooling option after the national lockdown (Businesstech, 2020). To combat this change in work-life, some international HR firms have engaged in conversations with their employees and encouraged larger organisations to conduct surveys to gain insight into employee experiences in August 2020 (Watson, 2020). Croce (2020) encourages communication from organisation leaders, specifically regarding showing empathy to employees’ experiences as an effective way to minimise the anxiety and consequences of COVID-19 in the workplace. The benefits of these types of inquiries can be seen in industries such as the automotive industry, where more managers have grown comfortable with flexible work schedules and arrangements, especially for employees with children, following the pandemic (Muller & Burden, 2020).\n\nRemote work holds various benefits for employees, such as the opportunity to arrange their work schedule to reconcile their work-family conflict, especially for those employees who also fulfil caregiving roles (Sarbu, 2018). However, COVID-19 has forced many employees to work from home under the worst conditions (Bloom, 2020). Bloom (2020) states that no employee should be forced to work from home due to variation in employees’ desire to do so and their different home environments. Choudhury (2020) and Bloom (2020) therefore encourage knowledge workers to take the time and attention to overcome the potential hurdles to remote work during the COVID-19 lockdown in order to continue remote working and reap its benefits post COVID-19. This attention is imperative, as working at home may become the norm for many employees in the foreseeable future (Oakman, 2020), and is becoming a reality for South Africa as it experiences a flux in infections (South African Coronavirus Portal, 2020). Therefore, it may be prudent to investigate the experiences and possible strategies caregivers have employed during the first lockdown to determine the role an organisation and healthcare workers can play in improving remote work whilst caregiving. Thus, the research question will be: What is the remote working experiences of child caregivers in balancing work and childcare during the South African COVID-19 lockdown period?\n\nThis article adds knowledge to research with regards to how South African employees managed remote work and child caregiving during the COVID-19 pandemic. Findings may allow managers and organisations insight into the homework lives of caregivers and possibly promote conversations and interventions on how they can assist these employees in remote working. Helping remote workers will ease the burden on employees and this study is especially imperative as South African households can differ vastly from international homes (Hall, 2019), making South African employee experiences unique. This study will also contribute to knowledge about the qualitative descriptive research design, a valid research method in need of methodological transparency in literature (Doyle et al., 2020). Additionally, this study utilised an online questionnaire that is not often used for this type of design (Kim et al., 2017) and may provide support for online questionnaires as data collection methods in these times of social distancing.\n\nResearch purpose and research question\n\nThis study’s general aim is to explore the shared lived experiences of South African employees with regards to how they conducted or balanced their work whilst fulfilling caregiver roles during the COVID-19 lockdown. Based on the posed research question and aim, the following specific objectives were explored:\n\n• Who were the role players that promoted or hindered balancing remote work and childcare during the COVID-19 lockdown?\n\n• What organisational or personal resources have employees used in balancing their remote work and childcare during the COVID-19 lockdown?\n\n• What role could organisations play in assisting employees with balancing remote work and childcare?\n\n\nMethods\n\nA qualitative research method was followed in this study. Therefore, the researcher focused on how participants organise themselves in their contexts and make sense of these contexts through naturalistic research using words (Nieuwenhuis, 2016a).\n\nThis study was conducted through a qualitative descriptive research design. Qualitative descriptive designs aim to create straightforward descriptions of participants’ experiences (Sandelowski, 2010) and are not focused or committed to a theoretical analysis of data (Doyle et al., 2020) or increasing conceptual understanding (Chafe, 2017). According to Bradshaw et al. (2017), this design presents data in terminology closely related to the research question whilst recognising the participant experiences’ subjective nature. Thereby determining the who, what and where of a phenomenon (Sandelowski, 2000) and providing information that can easily be used for future interventions (Neergaard et al., 2009; Sullivan-Bolyai et al., 2005). The philosophical underpinning of the qualitative descriptive design is pragmatism (Neergaard et al., 2009). Pragmatism refers to making decisions on how research should be conducted based on the research aims or objectives (Ormston et al., 2014). As this study aims to merely describe caretakers’ experiences whilst remote working to find areas where organisations and healthcare workers could assist these employees, this philosophical stance is deemed adequate.\n\nAccording to O’Brien et al. (2014) researcher characteristics such as personal attributes, qualifications, participant relationship, presuppositions and research method, can influence the research and should be reflected on by the researcher. Thus, the following researcher characteristics should be noted by the reader when considering the results of this study. The researcher is a white female with a PhD in Psychology in her late twenties, who performed this study as part of her post-doctoral fellowship research for a South African University. A researcher-participant relationship was followed throughout this research where the researcher aimed for ethical research behaviour and reporting of participant experiences. This relationship was initiated when an invitation with a link to the online questionnaire was posted on two large social media sites. Participants who chose to take part in the study were anonymised by the online data collection system Typeform.com. Participants had the opportunity to contact the researcher if they had questions regarding the online questionnaire, however, no contact was initiated. Thus, no direct interaction took place between the researcher and the participants during the research process. From a personal perspective, the researcher shared three characteristics with the participants. Firstly, the researcher is a South African citizen who lived in South Africa during the 2020 lockdown period. Secondly, the researcher is also a white female, making her part of the largest gender group that took part in this study. Lastly, the researcher has a PhD and forms part of the education level group of participants named “PhD”.\n\nBased on the researcher’s community values of a traditional household structure and the read literature in preparation for this study, it was possible that there was the presupposition that women would be the primary caretakers during the COVID-19 lockdown. However, a co-coder whose community and household background differs significantly from the researcher by being modern and with an open family structure, was used to combat potential bias. Based on the lack of insight into the chosen topic, a qualitative method was chosen to perform this study. The researcher’s experience regarding this method consists of a Master’s degree in research psychology conducted using a qualitative research method and a PhD focused on the use of research methodology.\n\nThis research was completed online by participants between 16 March 2021 and 17 April 2021. Thus, participants were instructed to complete the questionnaire in an environment comfortable to them, where they had online access. Employees from all sectors were able to participate in the study, making the organisational setting all sectors with remote working employees during the COVID-19 lockdown. To allow for persons from all organisations to take part in the study and to adhere to social distancing measures in South Africa, an online questionnaire was deemed appropriate.\n\nThe sampling strategy consisted of the purposive and snowball sampling methods. Purposive sampling is generally used when conducting qualitative descriptive research (Palinkas et al., 2015) and refers to selecting participants who know the specific research phenomenon (Ritchie et al., 2009). Additionally, snowball sampling typically consists of the researcher sending the questionnaire to one or two persons and identifying others with the same characteristics for further research (Maree & Pietersen, 2016). For this study, participants who self-identified with the following characteristics completed the online questionnaire: South African employees that have remote worked full-time or part-time during the 2020 lockdown period whilst caring for one or more children. The researcher posted an invitation for potential participants on social media sites Facebook and LinkedIn. This invitation included an informed consent form that listed the required participant characteristics to take part in this study. Therefore, individuals who do not use these social media platforms or have access to the internet to complete the internet survey were excluded. The sample size was determined by data saturation which is indicated when further coding is not possible, the ability for new information has been achieved, and there is enough information to replicate the study (Fusch & Ness, 2015).\n\nEthical clearance\n\nThis study received ethical clearance from the Economic and Management Sciences Research Ethics Committee (EMS-REC), North-West University, Potchefstroom Campus, South Africa (NWU-00018-21-A4) on February 26 2021. Participants were provided with informed consent forms before taking voluntary part in the study to describe the purpose, extent and data access of their participation.\n\nThe researcher was responsible for gaining ethical and scientific clearance to conduct the study and invite participants to participate. Furthermore, the researcher was also available to participants and answered any questions regarding the research. Lastly, the researcher’s most important role was to describe the participants’ experiences.\n\nTextual data were collected on the online survey platform typeform.com and extracted in a Microsoft Excel Worksheet format (Professional version 2016) for data analysis. This data has been stored on an online open access repository (Figshare), where anonymous outputs can be shared with other researchers. The researcher posted an invitation to the research on the identified social media platforms indicating aspects such as voluntary participation, the contact information of the researcher and the participants’ right to withdraw their participation at any time. Participants who wished to complete the questionnaire clicked on the provided questionnaire link, which directed them to the informed consent form and questionnaire on typeform.com. Participants were only able to complete and submit the questionnaire after agreeing to the informed consent form by clicking the “Agree” button. Typeform.com sent the data to the researcher for content analysis. Data collection commenced on 16 March 2021 and ended 17 April 2021. Data analysis and co-coding was performed from 18 April 2021 to 1 May 2021. A report was compiled based on the findings from data analysis.\n\nOnline approaches are becoming popular for qualitative descriptive studies and offer researchers flexibility and ease of participation (Doyle et al., 2020). According to Eckerdal and Hagström (2017), qualitative questionnaires can be helpful in various research disciplines by providing rich material. The qualitative questionnaire used in this study consisted of semi-structured questions based on the research question and study objectives. Semi-structured questions define the line of inquiry (Nieuwenhuis, 2007b), and for this study, five questions were included:\n\n• Briefly describe your general experience of balancing work and childcare during the COVID-19 lockdown.\n\n• Describe the assistance you had (if any) in balancing working from home and childcare?\n\n• What organisational or personal resources have you used to balance working from home and child care during the COVID-19 lockdown?\n\n• Where did you work best whilst working from home? (Describe the environment and circumstances)\n\n• How could your organisation have assisted you in balancing remote work and childcare?\n\nThe final sample consisted of 21 participants (four male; 18 female), with the youngest participant being 25 and the eldest being 59 (mean = 39 years; mode = 30, 34, 36). The sample consisted of White/Caucasian participants caring for a mean of two children. The specific sample characteristics are listed in Table 1.\n\nData were extracted in a Microsoft Excel Worksheet (Professional version 2016) from the online data collection platform: Typeform.com. that anonymised participants. Questions were presented as headings, and participant answers were compiled under each relevant heading. Participants received participant numbers based on the Excel Worksheet line number their answers were logged in. Data were coded in this Microsoft Excel Worksheet and kept secure on the researcher’s password-protected laptop. Coding took place on the same worksheet as the participant answers for each question to provide an audit trail.\n\nAnalysis of qualitative descriptive studies is data-driven and does not consult pre-existing epistemological or philosophical perspectives (Lambert & Lambert, 2012). Content analysis is commonly used for qualitative descriptive designs as it limits the interpretation of results from the researcher (Hesse-Biber & Leavy, 2011). In accordance with the study aim, this content analysis was based on exploratory work and provided a simple report of common aspects mentioned by participants (Green & Thorogood, 2004). Specifically, an inductive, text-driven (Krippendorf, 2013) content analysis was applied wherein the researcher searched for variation and similarities in the data to finally produce descriptive categories or themes (Graneheim et al., 2017). The frequency with which some codes occurred were also considered and described as per classical content analysis (Leech & Onwuegbuzie, 2008). The straightforward description of the qualitative descriptive data is a crucial strength of this design as researchers can more easily agree on the findings when limited interpretation was applied (Kim et al., 2017). Four stages were followed for data analysis as indicated in Table 2 (Bengtsson, 2016):\n\nTrustworthiness allows readers to have confidence in what the researcher has written about a phenomenon (Stahl & King, 2020). Various criteria exist on how to promote the trustworthiness of a study; for this research, the following four critical measures of Lincoln and Guba (1985) were employed: credibility, dependability, transferability and confirmability. Credibility refers to the extent that participants’ experiences are represented through the researcher and can be recognised by other researchers and readers (Tobin & Begley, 2004). The researcher promoted credibility through prolonged engagement with the data, frequent debriefing with her supervisor and a thick description of the phenomenon (Nieuwenhuis, 2016). Dependability of research findings was ensured through creating an audit trail (Lincoln & Guba, 1985) of the decision-making process by keeping a journal. This allows other researchers to see the decisions the researcher made in analysing the data (Nieuwenhuis, 2016b). The transferability of qualitative research does not refer to generalising findings to a broader context but rather to what extent readers can connect findings to their own experience and to what extent the participants reflect the study context (Lincol & Guba, 1985). To achieve transferability, the researcher provided a detailed description of the South African lockdown context, participants who cared for children and remote worked during this time and a qualitative research approach. Additionally, participants were purposefully chosen to represent the phenomenon under study. Lastly, confirmability of the study shows the extent to which the study findings are shaped by the participants and not the researcher biases or motivations (Lincoln & Guba, 1985). The researcher provided a reflection on her own characteristics to highlight possible biases, included quotes from participants to support findings and an audit trail of the research process and data analysis (Nieuwenhuis, 2016b).\n\n\nResults\n\nFindings of participant experiences are presented through descriptions that exhibit themes and categories (Kim et al., 2017). Overall the qualitative findings derived from the content analysis showed that employees experienced remote working and providing childcare during the COVID-19 lockdown period as a strenuous task: it was “difficult” (P11), “… stressful” (P6), “very challenging” (P19) and “chaos” (P22) (Scholtz, 2021). Participants often followed their expression of its difficulty with information on balancing work and family life:\n\n[I] t was stressful. I could not fully give my attention to my work and my child simultaneously” (P6) and “A balancing act between school requirements and work requirements. Extra demands placed by both work and school on parents which results in work time shifts and working later in the evening and earlier in the mornings when surroundings are quiet” (P14).\n\nAnother participant described the experience as:\n\n[I] t is hard to balance work and childcare because the children need attention and household duties need to be completed, also the work isn’t being done by someone else. It is a race against the clock to have happy and provided for kids and work completed by the end of the day. Mostly work has to be done after the kids go to bed (P8).\n\nOnly three participants described their general experience as positive stating that is was “easy” (P8) or “good” (P1; P18).\n\nThe role players that promoted or hindered balancing remote work and childcare during the COVID-19 lockdown\n\nWhen balancing work and childcare at home, participants received assistance mostly from their partners. This helped employees to divide work and family responsibilities: “My wife and I took turns to manage different responsibilities” (P20), and devised schedules “Schedule between my spouse and myself” (P16). However, in some cases this assistance was limited: “My husband was there to assist, but only when he came home after work in the evening” (P9). Other family members also assisted in childcare to create opportunities for employees to complete their work such as “The older kids helped a lot with the smaller child. The live in grandparents was great assistance in balancing the house work and assisting the older children” (P15). The environment played a role for one participant as they “[live on] a farm, it was not a problem” (P8), whereas one employee received help from a community member, namely: “from [a] pastor” (P23) in balancing their work and family. Five participants did not receive any assistance.\n\nThe organisational or personal resources employees used in balancing their remote work and childcare during the COVID-19 lockdown\n\nEmployee resources consisted of people, technology, time and their houses. Specifically, employees utilised organisational resources such as technology through “[Microsoft] Teams” (P1), and “online organisation resources … ” (P3) in balancing remote work and childcare. Personal resources included “Homeschooling via internet” (P22), “ … home internet … ” (P20), “TV and DVDs … ” (P2) for entertaining children whilst working. People were also seen as both an organisational and personal resource to achieve balance between remote work and childcare, for example: “Family and employees had to help” (P4) and “Personal resources include my husband being able to take 1 child with to his work so there were days I only had 1 toddler to look after” (P9), or “… grandparents to assist with child care when possible …” (P14).\n\nTime slots as they became available for employees to complete their work were also employed as a valuable resource. For example, Participant 14 indicated that they worked “from 19:00 to 00:00 when all was quiet and I could focus on my work” and Participant 19 worked “[i] n the mornings, while toddlers slept and middle childhood child did home Montessori work, so that there was uninterrupted time to work quickly”.\n\nHome spaces available for work were used or created; specifically, participants listed private spaces such as “ … [a] room where I closed the door” (P2), or a home office: “ … turned our spare room into an office” where “… our son knew he mustn’t disturb us” (P20). The shared home spaces were used to perform childcare responsibilities while working like in the case of Participant 6 “In our living room, where I could keep an eye on my child playing outside/inside” or the “[i] n the lounge or dining room table when the childrens home work also needed attention” (P15).\n\nLastly, employees restructured aspects of their home or work life to fit with their unique remote work contexts. For example, Participant 19 applied a “… stricter routine for children, chores for children. Work done while children take naps and did homework” to change her home life to fit with her work requirements. Participant 10, on the other hand, changed her mode of work: “Area (especially outside if possible) where kids can be play around you” to fit with her home or family requirements.\n\nThe role organisations can play in assisting employees with balancing remote work and childcare\n\n“They need to give more autonomy …” (P20) was a key feature of improving remote work and childcare for the sampled employees. This need for autonomy was expressed through more than half of the participants that listed “Flexible hours” (P13), or “Workable timeframes and deadlines” (P11) as a way organisations can assist in balancing their work and family lives. One participant described how flexible work hours was granted by their organisation during the lockdown: “There was a deadline, but it didn’t matter at what time or how I got the work done, as long as it was finished by the deadline” (P19). In addition to flexible work hours, employees indicated that meetings as a mode of organisational communication were at times unnecessary “To not waste my time with unnecessary meetings and trainings that can be communicated through email.” (P14) and that “minimising meetings that can be emails” (P4). Lastly, those that were not allowed to work remotely full-time expressed a desire to do so: “Working from home” (P) or having “Childcare at work” (P2). However, participants did acknowledge that full-time remote work was not always possible: “Depending on the organisation. Consulting require face time with clients and interaction with team members. Working in isolation is not always feasible. Companies should remain fluid and flexible however employees should remain loyal and focused” (P16).\n\n\nConclusions/discussion\n\nThis study aimed to explore the experiences of employees whilst remote working and providing childcare during the 2020 COVID-19 lockdown in South Africa. The descriptive qualitative design showed that this South African sample’s experiences were similar to many other international samples in that employees struggled to juggle work and family life during lockdown (Qian & Fuller, 2020). Employees did not report disliking remote working; on the contrary, those who could only remote work part-time expressed their desire to do so full time. However, the lack of control over how they conducted their remote work made it challenging in the midst of childcare responsibilities. To combat this difficulty, the participants depended on certain role players as well as organisational and personal resources.\n\nUnsurprisingly, the most significant role players lived with participants (e.g. partners, live-in grandparents and older children), which may be due to social distancing restrictions. Partners were the most popular role players and provided assistance based on availability and on schedules devised by couples. Creating schedules to conduct work or care for children can promote remote work; however, the choice of who worked and who cared for children during the pandemic was reportedly still burdensome for couples (Qian & Fuller, 2020). Additionally, a study conducted in November 2020 found that despite men’s involvement in childcare during the pandemic, South African female employees still took on most childcare responsibilities (Casale & Shephers, 2020), which was also found in this study. Some female participants either received no assistance, worked whilst caring for children, had to wait for their partners to become available (i.e. return from work) or worked at night when children were asleep. Globally, gender inequality at work was as expected based on research indicating that women were often those who could not work due to childcare and that women are often part of the in-person labour market, which was hard hit by the lockdown (Kochhar & Barroso, 2020; Lemieux et al., 2020). This study contributes to research in those South African women who remained employed during the lockdown through remote working still took on the bulk of the childcare responsibilities.\n\nTo achieve a balance between work and childcare, employees used personal resources such as people and spaces. Specifically, there were employees that chose private versus shared spaces to conduct their remote work at home. Those who chose private spaces strengthened the barriers between work and family life by trying to keep these two aspects separate by, for example, informing children not to disturb them when they are in their home office. Alternatively, some of the employees actively diminished these barriers by combining childcare and work, such as working outside while children played or working while children completed their homework in shared spaces such as dining rooms. This finding concurs with other research; whereas some employees prefer a separation between work and non-work roles, others choose to blur these roles (Gonsalves, 2020). The type of workspace employees chose could, therefore, possibly be dependent on employee preference (Gonsalves, 2020). Additionally, participants in this study also add to literature that the type of work they had to complete influenced their chosen workspace as, for example, private spaces helped them to focus.\n\nFurthermore, based on the experiences and work hours, most employees often did not seem to control their work schedules at home. They usually only had time to work when childcare responsibilities and meetings forced them to work. Work-family conflict has been found to influence involuntary variable work schedules and vice versa and has been linked to lowered job satisfaction, perceived stress, psychological distress and greater turnover intention (Kaduk et al., 2019). However, if remote working employees follow a variable work schedule that they choose, i.e. “temporal flexibility” (Gonsalves, 2020), they experienced higher work satisfaction (Kaduk et al., 2019). Temporal flexibility has many benefits, such as decreasing work/family role blurring and improved employee health (Moen et al., 2016). Additionally, having control over their schedules can improve employee remote work experiences whilst also promoting the benefits of remote working for both the employee and the organisation (Kaduk et al., 2019). Therefore, organisations and healthcare workers are faced with an opportunity to improve South African employees’ remote working experiences and reap the benefits of this new mode of work through flexible work hours. Especially considering that parents are part of the group most willing to remote work (Bloom, 2014) and most of the current study sample of child caregivers highlighted the need for creating flexible work hours.\n\nEmployees also stated that management could focus on adapting their mode of communication through limiting meetings or changing meeting times to accommodate most employees where possible. Zoom meetings have been found to require more emotional effort and cognitive load than in-person meetings due to the absence of non-verbal cues and the need for increased eye contact and focus (Sander & Bauman, 2020). Additionally, employees with children have the added anxiety of a child interrupting or walking in during meetings (Sander & Bauman, 2020). However, online meetings are unavoidable in the new remote workplace and employers are encouraged to use alternative forms of communication such as detailed documents or phone calls (Sander & Bauman, 2020) to create online meeting breaks for employees. Other research found turning off video when not speaking in meetings or employing other technologies such as virtual reality headsets can assist in Zoom fatigue (Reinach, 2020). In this sample specifically, employees preferred communication to be through email rather than online meetings. New standards of working together and communicating are called for to adapt to the post-COVID-19 workplace (Wiatr, 2021).\n\nThe boundaries between work and home are in normal circumstances porous, with one often creeping over to the other (Dunn & McMinn, 2021). This research concludes that employees used resources such as people, time and spaces to repair or uphold the balance between work and family by re-establishing these boundaries or intertwining work and family life in the new and abnormal work circumstances brought on by COVID-19. For managers, it is clear that South African remote working employees who care for children require flexible work hours and variation in the modes of communication to promote balance and thus well-being in their new remote working lives. Healthcare workers on the other hand, are encouraged to use findings to conduct further research into how resources can be employed to promote work-family balance for new remote workers.\n\nHighlighting the possible areas of assistance for remote workers is imperative as childcare experience whilst remote working can have long-lasting repercussions for gender equity (Qian & Fuller, 2020). Fear for child safety and the lack of capacity in certain schools indicate that some parents will still be required to work remotely despite the opening of schools and businesses (Qian & Fuller, 2020). Thus, organisations are encouraged to use the collected data to improve the remote working experiences of child caregivers, especially female employees, to prohibit growth in the gender gap.\n\nThis study was limited by a small heterogeneous sample, thus findings should be seen within the context of the included participants. Future studies should attempt to conduct studies with larger samples of participants from different contexts. The online qualitative nature of this study should also be considered, as there was limited opportunity to probe for further information through this method; however, as this is at a time of remote working in the pandemic, an online questionnaire was deemed appropriate. Despite the lack of probing, the researcher experienced the descriptive design to be easily applied to online data collection, and it is recommended to other qualitative researchers with similar research aims. Future researchers are also encouraged to conduct similar studies or more in-depth research to confirm findings from this study or employ quantitative studies to reach larger groups of participants on this topic.\n\n\nData availability\n\nFigshare: Remote work and childcare dataset – Controlled.xlsx. https://doi.org/10.6084/m9.figshare.14545998 (Scholtz, 2021).\n\nThis project contains the following underlying data:\n\n- Remote work and chilcare dataset - Controlled.xlsx (raw data file)\n\nFigshare: Remote work and childcare dataset – Controlled.xlsx. https://doi.org/10.6084/m9.figshare.14545998 (Scholtz, 2021).\n\nThis project contains the following extended data:\n\n- Remote work and childcare Informed consent and questionnaire.pdf (participant consent and questionnaire forms)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)", "appendix": "References\n\nAnderson D, Kellihner C: Enforced remote working and the work-life interface during lockdown. 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Van Schaik Publishers; 2007; (pp. 81–93).\n\nNieuwenhuis J: Introducing qualitative research. In: Maree K (Ed.), First steps in research. Van Schaik Publishers; 2016a; (pp. 50–69).\n\nNieuwenhuis J: Analysing qualitative data. In: Maree K (Ed.), First steps in research. Van Schaik Publishers; 2016b; (pp. 104–130).\n\nOakman J, Kinsman N, Stuckey R, et al.: A rapid review of mental and physical health effects of working at home: how do we optimise health? BMC Public Health. 2020; 20: 1825. Publisher Full Text\n\nOdeku KO: The plight of women entrepeneurs during COVID-19 pandemic lockdown in South Africa. Gender Behav. 2020; 18(3): 16068–16074. Reference Source\n\nOrmston R, Spencer L, Barnard M, et al.: The foundations of qualitative research. In: Ritchie J, Lewis J, McNaughton NC, et al. (Eds.), Qualitative Research Practice. A Guide for Social Science Students and Researchers. Sage Publications; 2014; (2nd ed., pp. 1–25).\n\nPalinkas LA, Horwitz SM, Green CA, et al.: Purposeful sampling for qualitative data collection and analysis in mixed method implementation research. Adm Policy Ment Health. 2015; 42: 533–544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPutro SS, Riyanto S: How Asian Sandwich Generation Managing Stress in Telecommuting during Covid-19 Pandemic. Int J Scientific Res Engg Development. 2020; 3(3): 487–492. Reference Source\n\nQian Y, Fuller S: COVID-19 and the gender employment gap among parents of young children. Canadian public policy. 2020; 46(S2): 89–101. Publisher Full Text\n\nRitchie J, Lewis J, Elam G: Designing and selecting samples. In: Ritchie R, Lewis J (Eds.), Qualitative research practice: A guide for social science students and researchers. SAGE Publications; 2009; (pp. 77–85).\n\nSandelowski M: Whatever happened to qualitative description? Res Nurs Health. 2000; 23: 334–340. PubMed Abstract \| Publisher Full Text\n\nSandelowski M: What’s in a name? Qualitative description revisited. Res Nurs Health. 2010; 33: 77–84. PubMed Abstract \| Publisher Full Text\n\nSander L, Bauman O: 5 reasons why Zoom meetings are so exhausting. Conversation. 2020; 5. Reference Source\n\nSarbu M: The role of telecommuting for work-family conflict among German employees. Res Transportation Economics. 2018; 70: 37–51. Publisher Full Text\n\nScholtz S: Remote work and chilcare dataset - Controlled.xlsx. figshare. Dataset. 2021. Publisher Full Text\n\nSevilla A, Smith S: Baby steps: The Gender Division of childcare after COVID19. CEPR Discussion Paper No. 14804. 2020. Reference Source\n\nShockley KM, Clark MA, Dodd H, et al.: Work-family strategies during COVID-19: Examining gender dynamics among dual-earner couples with young children. J Appl Psychol. 2020; 106, 15–28. PubMed Abstract \| Publisher Full Text\n\nStahl NA, King JR: Expanding approaches for research: Understanding and using trustworthiness in qualitative research. J Developmental Education. 2020; 44(1): 26–29. Reference Source\n\nStiegler N, Bouchard J: South Africa: Challenges and successes of the COVID-19 lockdown South Africa: challenges and successes of containment linked to COVID-19. Ann Med Psychol (Paris) J. 2020; 178(7): 695–698. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSullivan-Bolyai S, Bova C, Harper D: Developing and refining interventions in persons with health disparities: the use of qualitative description. Nurs Outlook. 2005; 53(3): 127–133. PubMed Abstract \| Publisher Full Text\n\nTobin GA, Begley CM: Methodological rigour within a qualitative framework. J Adv Nurs. 2004; 48: 388–396. PubMed Abstract \| Publisher Full Text\n\nTuyo D: The unexpected benefits of remote work. USA: The Union Times; 2020.\n\nWiatr A: Flexible working arrangements- current conditions and research directions. Business Management 1. Reference Source\n\nWatson R: HR firm shares ideas to support working parents. Grand Rapids Business J. 2020, August 21: 1–3. Reference Source\n\nZaki J: Harvard business review: Leadership & managing people. Don’t just lead your people through trauma. Help them grow. 2020. Reference Source" }	[ { "id": "168534", "date": "20 Apr 2023", "name": "Auditya Purwandini Sutarto", "expertise": [ "Reviewer Expertise Industrial or organizational psychology", "ergonomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGENERAL COMMENTS This is an interesting study that explores how employees balance their unprecedented remote work and childcare tasks amidst the COVID-19 pandemic in the South African context. As an exploratory study, the authors used a qualitative study design to answer some related issues. This study was conducted two years ago; hence, it is important to add implications for more current contexts. Besides, I have some queries to improve the quality of manuscript\nTITLE\nGiven a limited sample and design of study, a term \"exploratory\" may be added in the title\nINTRODUCTION\nThe author can replace the earlier work of Tavares (2017) with other references (more recent) relate to remote work amidst the pandemic.\n\nA clear identification how this current study will address the gaps or contribute to the existing remote work of knowledge is required. It may be directed to South Africa or global context.\nMETHOD\nHow does the author deal with the representativeness of South African remote workers when all the participants are \"White/Caucasian\"?\n\nIt is necessary to include only relevant information to the readers which contribute to their understanding of your chosen methods. The paper could be streamlined, omitting, or reducing unnecessary explanations of basic procedures of the qualitative study unless they are unfamiliar to the readers.\nRESULTS\nThe author should provide context for selected quotes. For example, explaining who the participants and their background.\n\nSince the sample size is relatively small, it is possible to make a table consisting detail characteristics of the participants, rather than merged into a summary (ID Participant, Age, #of children & age, education level, etc). This will help the authors contextualize their selected quotes.\n\nThe presentation of the main themes under each research question is less unclear.\nDISCUSSION\n“The descriptive qualitative design showed that this South African sample’s experiences were similar to many other international samples in that employees struggled to juggle work and family life during lockdown (Qian & Fuller, 2020)” >> More citations are needed.\n\nDo you have any suggestions for the implications of this study beyond the pandemic, as currently COVID-19 would no longer be a public health emergency and most countries tend to re-shift into conventional (office) work arrangements.\n\nIt is necessary to put the conclusion in the end of text, as a separate section from the Discussion, summarizing key findings how the study’s findings answer your research questions.\n\nMore discussion regarding sub-theme and final themes. The author may refer to the following works related to family or child care while remote work during the pandemic to enrich the discussion:\n- Barriga Medina et al., (2021)1 - Bernhardt, Recksiedler & Linberg (2022)2 - Çoban (2022)3 - Schieman, Badawy, Milkie & Bierman (2021)4 - Wang, Liu, Qian & Parker (2020)5\nREFERENCES Many citations are missing in the reference list. Please re-check.\nMatli (2020), Khwela-Mdluli and Beharry-Ramraj (2020), Muller & Burden, 2020, (Sarbu, 2020), Choudhury (2020), (Oakman, 2020), O’Brien et al., (2014) , Nieuwenhuis, 2007b Lemieux et al., (2020), Krippendorf, (2013)\nPRESENTATION\nProofread 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? 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": "9652", "date": "26 May 2023", "name": "Salome Salome", "role": "Author Response", "response": "Thank you for reviewing my article; I believe your suggestions have improved its quality. I have included your suggestions as follows: TITLE The title indicates that this is a qualitative study which, per definition, is exploratory. INTRODUCTION Tavares (2017) reference has been removed. The article's contribution is indicated above the research aim. METHOD The sample was a convenience sample, open to all South Africans, and identified as a limitation of the study. DISCUSSION Supporting references were added Post-COVID-19 lockdown implications were added The conclusion is placed as required by the article's guidelines Thank you for the references; I have added these to the discussion." } ] } ]	1	https://f1000research.com/articles/10-481
https://f1000research.com/articles/12-482/v1	11 May 23	{ "type": "Brief Report", "title": "High copy number of multi-drug resistance genes in spent coffee grounds used in organic composting", "authors": [ "Jinpao Hou", "Yuen Ting Chiu", "Kit-ling Lam", "Ki-ying Kwong", "Johnny Hoi-lung Lau", "Lawal M. Marafa", "Stephen Kwok-wing Tsui", "Ian Wing-yin Mo", "Ping Lung Chan", "Jinpao Hou", "Yuen Ting Chiu", "Kit-ling Lam", "Ki-ying Kwong", "Johnny Hoi-lung Lau", "Lawal M. Marafa", "Stephen Kwok-wing Tsui" ], "abstract": "Background: The demand for organic composts made of animal manure and food waste, such as spent coffee grounds, is increasing as organic farming is gaining popularity. One of the potential public health hazards of using these organic composts is the dissemination of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Animal manure is generally believed to be the primary source of these bacteria and genes since antibiotics are commonly used in animal farming. However, there is little concrete evidence supporting this claim. Here, we examined this argument by characterising the resistome of the raw materials, namely, chicken manure (CF), spent coffee grounds (COFFEE), and chopped grass (GRASS) commonly used in Hong Kong for preparing organic composts. Methods: Three independent samples of the three raw materials were collected from an organic farm in Hong Kong. DNA of the samples were extracted and sequenced by metagenomic sequencing. The resistome of the samples was analysed using the resistome analysis pipeline ARGs-OAP v2.0. The abundance and the richness of the ARGs among the samples were compared using one-way ANOVA with Tukey post-hoc test. Results: COFFEE carried the highest copy number of ARGs per cell (2.771–3.539) and was significantly higher than that of CF (1.991–2.617) and GRASS (0.491–0.537). Multi-drug resistance genes predominated the resistome of COFFEE, for which the richness of the resistome was the lowest among the three materials (150–154 ARG subtypes). It was significantly lower than that of CF (203–229 ARG subtypes) but not that of GRASS (153–203 ARG subtypes). Conclusions: Contrary to the general belief that animal manure is the primary source of antibiotic resistance genes in organic composts, it was found that COFFEE carried the highest copy number of ARGs among the three materials, and multi-drug resistance genes predominated the resistome of COFFEE.", "keywords": [ "Antimicrobial resistance", "Antimicrobial resistance genes", "Coffee grounds", "Food safety", "Multi-drug resistance", "One-health", "Organic compost Resistome" ], "content": "Introduction\n\nOrganic vegetables are gaining popularity among customers due to the general belief that organic food is healthier and safer than vegetables grown with chemical fertilisers. The increased demand for organic vegetables prompted many small-scale farms to practice organic farming. In addition, many amateur and urban farmers also practised organic farming as a hobby.1\n\nTo fertilise farm soil with exogenous carbon and nitrogen at optimal carbon-to-nitrogen ratio2 and to reduce pathogen loads,3 organic composts are typically prepared by composting nitrogen- and carbon-rich organic materials.4 Typical raw materials for preparing organic composts include animal manures, protein-rich vegetation, and carbon-rich vegetation. For example, Meng et al., demonstrated that co-composting pig manure with spent mushroom substrate and rice husks increased the maturity and nutritional content of the compost product5 and Gurtler et al., reported that composting manure effectively reduced the quantities of pathogenic microorganisms in organic composts.6\n\nIn order to ensure that there is no carry-over of chemicals through organic composts and to standardise the quality of the produces, a stringent set of criteria on the origins and quality of these raw materials are in place in many organic farms of industrial scale or accredited organic farms under the National Organic Plan of the US7 or other accreditation schemes. However, instead of following these stringent criteria, many farmers from small-scale organic farms or amateur and urban farmers often prepare their organic composts with raw materials that are easily accessible, such as animal manure in the farms, food wastes, and green waste, in order to reduce the production cost. In addition, standardisation in quality is not a significant concern for these farmers since many of their produces were sold to a small and specific group of customers or for personal consumption.8\n\nAlthough the use of organic compost can prevent the chemical hazards associated with chemical fertilisers and pesticides, organic composts in organic farming may increase the risk of disseminating antibiotic-resistant bacteria and antibiotic resistance genes (ARGs).9 For example, Lam et al., found a larger number of antibiotic-resistant bacteria and higher minimal inhibitory concentration (MIC) in organic lettuce sold in Hong Kong compared with the lettuce grown with chemical fertiliser and hydroponic methods.10 It is thus evident that antimicrobial resistance (AMR) contamination in organic compost can pose a public health risk and warrant further investigation and surveillance.\n\nAnimal manure is generally believed to be the primary source of these antibiotic-resistant bacteria and ARGs.11 This belief originates from the fact that antibiotics are frequently used for promoting growth and curing veterinary diseases,12 and antibiotic-resistant bacteria are being selected and tainting animal manure. For example, Pan et al., analysed swine manure samples from 21 concentrated animal feeding operations in China and found that the detection frequencies of tetracyclines, sulfonamides and macrolides were 84.9–96.8%, 84.9–96.8% and 4.8%, respectively.13 The presence of bacteria carrying antibiotic-resistant genes (ARGs) has been widely observed, and manure has been identified as the primary source of ARGs in organic vegetables.14\n\nAlthough antibiotic-resistant bacteria and resistome in various types of animal manure have been well characterised, few studies explored ARGs in other raw materials of organic composts. The contribution of these raw materials to the AMR risk of organic composts remains to be explored. Here we conducted a preliminary study to measure the ARG abundance and richness (the number of ARG subtypes) in three raw materials commonly used for preparing organic composts in small-scale organic farms in Hong Kong, which are chicken manure (CF), spent coffee grounds (COFFEE), and plant litter (chopped grass, GRASS). Surprisingly, our result indicated that, among the three raw materials, COFFEE harboured the highest ARG abundance with genes conferring resistance to multidrug being the predominating ARG subtypes. Our data challenged the general belief that animal manure is the primary source of ARGs in organic composts and suggested that other raw materials used for preparing organic compost may have a major contribution to the AMR risk of organic composts.\n\n\nMethods\n\nThree independent samples were collected for each raw material. The three raw materials were collected from a private organic farm in the New Territories, Hong Kong (22.30 N 114.08 E). CFs originated from a chicken farm in the New Territories, Hong Kong. COFFEE were collected by the farmers from café and coffee shops near the farm. The government collected the GRASS from various public parks and transferred them to farms.\n\nTotal genomic DNA was extracted from 0.1 g of each sample using NucleoSpin Soil mini kit (MACHEREY-NAGEL, Germany; Cat No.: MNG-740780.50; Lot No.: 2006/002). The DNA concentration and quality were determined by spectrophotometric analysis (NanoDrop 2000c, Thermo Scientific, USA) (RRID:SCR_018042) and agarose gel electrophoresis, respectively. Library preparation and metagenomics DNA sequencing of all samples were performed by Novogene Bioinformatics Technology Co, Ltd. Briefly, DNA samples were randomly sheared into short fragments and end repaired. Poly-A tail and Illumina adapter were then added to the fragments (Illumina Inc., San Diego, CA, United States). The libraries were checked with Qubit and quantified by real-time PCR. Size distribution detection of the libraries was performed using bioanalyzer. Quantified libraries were then pooled and sequenced on Illumina HiSeq 2500 (RRID:SCR_016383).\n\nRaw reads were quality-filtered using Trimmomatic v0.39 (RRID:SCR_011848)15 with the following parameters: leading or trailing low quality (Phred score < 3) or N bases were trimmed off; bases were trimmed off if the average quality score dropped below 15 in any 4-base sliding window; sequences were removed if they were less than 36 bp in length. The quality-controlled reads were subject to ARG identification using the resistome analysis pipeline ARGs-OAP v2.016 by aligning to the SARG v2.0 database with an ARG type-subtype-hierarchical structure. ARG types were quantified into the unit of copies of ARGs per cell by normalising ARG abundance to the cell number,17 which was calculated from the estimation of universal essential single copy marker genes (ESCMGs).18 The resulting ARGs were classified into 24 types, which denoted the type of antibiotic against which the gene confers resistance, and 1,204 subtypes, which indicate their functional gene annotation.\n\nThe statistical significance of ARGs abundance and diversity between the three raw materials was determined by one-way ANOVA followed by Tukey post-hoc test using GraphPad Prism (RRID:SCR_002798) version 9.5.1 for macOS (GraphPad Software, San Diego, California USA). A difference with a p-value < 0.05 was considered significant.\n\n\nResults\n\nARGs in CF, COFFEE, and GRASS samples were quantified using resistome analysis pipeline ARGs-OAP v2.0. A total of 21 ARG types and 420 subtypes were identified. Notably, the result indicated that COFFEE had the highest abundance of ARGs (average abundance 3.20 copies/cell), followed by CF (average abundance 2.20 copies/cell) and grass (average abundance 0.519 copies/cell) (Figure 1a and Table 1). The total ARG abundances were significantly different between any of the two raw manure types (Tukey's HSD, adjusted p = 0.017, 0.001, and 9.76 x 10-5 for CF vs. COFFEE, CF vs. GRASS, and COFFEE vs. GRASS, respectively; Figure 1b). In addition, multi-drug ARGs were most abundant in COFFEE (average abundance 2.4 copies/cell) compared to the other two raw material types. Aminoglycoside, MLS, sulfonamide, tetracycline, and chloramphenicol were the predominant ARG types in CF, where they together accounted for 85.1% of the total ARG abundance. The ARG alpha diversity represented by the total number of ARG subtypes (i.e., richness) varied from 150 to 229 across all samples (Table 1), with CF having a higher diversity than the COFFEE group (Tukey's HSD, adjusted p = 0.008, Figure 1c).\n\n(a) The ARG type profiles among samples from raw materials. Stacked bar charts show the abundances of ARG types based on cell number normalisation. ARG types were colour-coded as indicated. The chart was generated using the ggplot2 package in R (v.4.0.3). Bar plots showed one-way ANOVA analysis of the ARG type abundance (b) and the ARG subtype richness (c). n =3, p < 0.05. ARG, antibiotic resistance gene; CF, chicken manure; COFFEE, coffee ground; GRASS, chopped grass; MLS, Macrolide-lincosamide-streptogramin.\n\nARG, antibiotic resistance gene; CF, chicken manure; COFFEE, coffee ground; GRASS, chopped grass.\n\n\nDiscussion\n\nThe demand for organic composts is increasing as organic farming is gaining popularity. Organic composts are often made of GRASS as the carbon sources and animal manure and other protein-rich materials, such as COFFEE, as the nitrogen sources. Since antibiotics have been frequently used in animal farming, residual antibiotics and ARGs are commonly found in animal manures. Thus, animal manure is generally believed to be the primary source of ARGs found in organic composts. However, there is little concrete evidence to support this belief. Therefore, this study examined this idea by characterising the resistome of the raw materials commonly used to produce organic composts in Hong Kong. To our surprise, instead of the animal manure, COFFEE carried the highest copy number of ARGs among the three raw materials used, and multi-drug resistance genes predominated its resistome. The abundance of multi-drug resistance genes in COFFEE was also the highest among the three raw materials. The high abundance of ARGs in COFFEE may be due to the infestation of fungi in COFFEE. These results challenged the general belief and highlighted the potential role of coffee grounds and possibly other plant-based food wastes in AMR dissemination. It is worth noting that a recent study also reported that corn stalk residue might add antibiotic-resistant bacteria to organic compost.19 The high number of ARGs in COFFEE also leads to the speculation about whether ARG contamination is common in coffee, coffee beans and other coffee products. Further investigations are needed to confirm this study's findings, gauge the prevalence of AMR contamination in coffee and coffee products, and evaluate the role of food waste and green waste in contributing to the dissemination of AMR along the food chain and in the environment.\n\nNo human, animals, nor plant are involved.", "appendix": "Data availability\n\nNCBI BioProject: Resistome analysis of raw materials of organic composts. Accession number PRJNA949012; https://identifiers.org/NCBI/bioproject:PRJNA949012. 20\n\n\nReferences\n\nReganold JP, Wachter JM: Organic agriculture in the twenty-first century. Nature plants. 2016; 2(2): 15221. PubMed Abstract \| Publisher Full Text\n\nVandecasteele B, Reubens B, Willekens K, et al.: Composting for increasing the fertiliser value of chicken manure - effects of feedstock on P availability.2016.\n\nSelvam A, Xu D, Zhao Z, et al.: Fate of tetracycline, sulfonamide and fluoroquinolone resistance genes and the changes in bacterial diversity during composting of swine manure. Bioresour. Technol. 2012; 126: 383–390. Epub 20120320. PubMed Abstract \| Publisher Full Text\n\nBernal MP, Alburquerque JA, Moral R: Composting of animal manures and chemical criteria for compost maturity assessment. A review. Bioresour Technol. 2009; 100(22): 5444–5453. Epub 2008/12/31. PubMed Abstract \| Publisher Full Text\n\nMeng X, Liu B, Xi C, et al.: Effect of pig manure on the chemical composition and microbial diversity during co-composting with spent mushroom substrate and rice husks. Bioresour. Technol. 2018; 251: 22–30. Epub 20170914. PubMed Abstract \| Publisher Full Text\n\nGurtler JB, Doyle MP, Erickson MC, et al.: Composting To Inactivate Foodborne Pathogens for Crop Soil Application: A Review. J. Food Prot. 2018; 81(11): 1821–1837. PubMed Abstract \| Publisher Full Text\n\nNOP Handbook: Guidance & Instructions for Accredited Certifying Agents & Certified Operations. In: Agricultural Marketing Service USDoA, editor.2021.\n\nMcDougall R, Kristiansen P, Rader R: Small-scale urban agriculture results in high yields but requires judicious management of inputs to achieve sustainability. Proc. Natl. Acad. Sci. 2019; 116(1): 129–134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKyakuwaire M, Olupot G, Amoding A, et al.: How Safe is Chicken Litter for Land Application as an Organic Fertilizer? A Review. Int. J. Environ. Res. Public Health. 2019; 16(19). Epub 20190920. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLam KL, Kong WP, Ling PY, et al.: Antibiotic-Resistant Bacteria in Hydroponic Lettuce in Retail: A Comparative Survey. Foods. 2020; 9(9). Epub 20200921. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLima T, Domingues S, Da Silva GJ: Manure as a Potential Hotspot for Antibiotic Resistance Dissemination by Horizontal Gene Transfer Events. Vet Sci. 2020; 7(3). Epub 20200813. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMshana SE, Sindato C, Matee MI, et al.: Antimicrobial Use and Resistance in Agriculture and Food Production Systems in Africa: A Systematic Review. Antibiotics (Basel). 2021; 10(8). Epub 20210813. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPan X, Qiang Z, Ben W, et al.: Residual veterinary antibiotics in swine manure from concentrated animal feeding operations in Shandong Province, China. Chemosphere. 2011; 84(5): 695–700. Epub 20110331. PubMed Abstract \| Publisher Full Text\n\nZhang YJ, Hu HW, Chen QL, et al.: Transfer of antibiotic resistance from manure-amended soils to vegetable microbiomes. Environ. Int. 2019; 130: 104912. Epub 20190617. PubMed Abstract \| Publisher Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. Epub 20140401. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYin X, Jiang XT, Chai B, et al.: ARGs-OAP v2.0 with an expanded SARG database and Hidden Markov Models for enhancement characterisation and quantification of antibiotic resistance genes in environmental metagenomes. Bioinformatics. 2018; 34(13): 2263–2270. PubMed Abstract \| Publisher Full Text\n\nYin X, Deng Y, Ma L, et al.: Exploration of the antibiotic resistome in a wastewater treatment plant by a nine-year longitudinal metagenomic study. Environ. Int. 2019; 133(Pt B): 105270. Epub 20191101. PubMed Abstract \| Publisher Full Text\n\nNayfach S, Pollard KS: Average genome size estimation improves comparative metagenomics and sheds light on the functional ecology of the human microbiome. Genome Biol. 2015; 16(1): 51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStaley ZR, Schmidt AM, Woodbury B, et al.: Corn stalk residue may add antibiotic-resistant bacteria to manure composting piles. J. Environ. Qual. 2020; 49(3): 745–753. Epub 20200318. PubMed Abstract \| Publisher Full Text\n\nHong Kong Metropolitan University: Resistome analysis of raw materials of organic composts. [Dataset]. NCBI BioProject. 2023. Reference Source" }	[ { "id": "213433", "date": "02 Nov 2023", "name": "Tam Tran", "expertise": [ "Reviewer Expertise I am a microbiologist interested in studying antimicrobial resistance in the environment." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors investigated resistomes of three different raw materials that are commonly used in organic composts using shot-gun lllumina sequencing technology. The authors found that spent coffee grounds carried the highest copy number of antibiotic resistance genes (ARGs). Although the results were a bit unexpected as animal manure is commonly found as a primary source of ARGs, my biggest concern is that the data provided here were very limited. Therefore, I recommend the authors should tone down their findings. It is important to highlight this limitation in their abstract and discussion.\n\" Here we conducted a preliminary study to measure the ARG abundance and richness (the number of ARG subtypes) in three raw materials commonly used for preparing organic composts in small-scale organic farms in Hong Kong, which are chicken manure (CF), spent coffee grounds (COFFEE), and plant litter (chopped grass, GRASS)\".The authors only had samples from a single farm, therefore this sentence needs to be rephrased.\n\n\"Three independent samples were collected for each raw material.\" Did they get sampled on the same day? How was the sampling done? If they were sample on the same day, I do not expect they are much different from each other. Therefore, the evident would be very weak.\n\n\"CFs originated from a chicken farm in the New Territories, Hong Kong.\" Does this chicken farm use any antibiotics?\n\n\"Novogene Bioinformatics Technology Co, Ltd\" where (city, country) is this company located?\n\n\"Thus, animal manure is generally believed to be the primary source of ARGs found in organic composts. However, there is little concrete evidence to support this belief. \" This statement is not very accurate. There have been a lot of studies that investigated ARGs in animal manure, and showed that ARGs were found abundantly present. This is due to the overuse of antibiotics in these livestock farms.\n\nIf COFFEE is actually the primary source of ARGs instead of manure, this would be very alarming because the product has been just directly consumed by human. And for this reason alone, it is more important to track down the contamination source, and where or how it happened.\n\nIs the work 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? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-482
https://f1000research.com/articles/12-303/v1	20 Mar 23	{ "type": "Research Article", "title": "Use of non-intrusive laser exfoliation to improve substance uptake into citrus leaves", "authors": [ "Luis Ponce Cabrera", "Ed Etxeberria", "Pedro Gonzalez", "Teresa Flores Reyes", "Ed Etxeberria", "Pedro Gonzalez", "Teresa Flores Reyes" ], "abstract": "Background: Despite the presence of stomata in leaves, foliar application of agrochemicals can be extremely inefficient due to the low permeability of leaf cuticular surfaces to polar compounds. Methods: This study introduced a laser-based “wax exfoliation” method to facilitate the penetration of substances into the leaf and, together with enhancing their uptake into the phloem and subsequent transport across tissue. This investigation demonstrated the effectiveness and non-invasive properties of laser exfoliation to improve the penetration of foliar-applied substances into citrus leaves. Results: This work presents the use of laser energy to exfoliate the cuticle of a leaf, with the highest energy density of 0.76 J/ cm2 resulting in 85-90% exfoliation across the entire laser-spot area. The infrared wavelength of the erbium laser is specifically chosen to target the wax cuticle without causing damage to the underlying epidermal cells. This selective ablation allows for increased penetration of therapeutic compounds into the leaf and transportation throughout the plant's vasculature. This is demonstrated using a fluorescent glucose analog applied to the laser treated leaves, showing increased penetration and transport throughout the leaf. Conclusions: Our findings demonstrate that the use of laser technology for the foliar application of agrochemicals provides significant advantages, including improved foliage uptake of therapeutic compounds. The method of cuticle exfoliation presented in this study is highly effective and non-intrusive, limiting its effects to the cuticle only. Future work should focus on the development of prototypes for in-field applications, including testing at longer distances as the Er:YAG laser does not require a lens for this application.", "keywords": [ "Citrus", "foliar sprays", "foliar uptake", "laser light" ], "content": "Introduction\n\nOne of the most prevalent methods used in modern-day agriculture to improve crop health, and hence yield, is the foliar application of agrochemicals.1 However, several barriers cause retardation and interfere with the efficient penetration and utilization of these substances. For example, the leaf surface is coated by a waxy cuticle that serves as a barrier for the prevention of water loss and pathogenic entry into the plant body. Due to its water impermeable nature, the cuticle also prevents the entry of externally applied soluble compounds such as most agrochemicals. The movement of substances into the leaf occurs primarily through the stomata,2 located mainly on the less-exposed abaxial side of citrus leaves.3 This property reduces the functional surface area leading to reduced agrochemical penetration through the foliar route. As consequence, considerable quantities of applied chemicals end up in the plant’s natural environment and can generate undesirable ecological impacts.\n\nAn example of research focused on foliar application of substances into plant leaves is the study of agrochemicals to treat citrus Huanglongbing (HLB),4,5 a bacterial disease of citrus that has spiraled the Florida citrus industry to the brink of disappearance and actively spreading across other regions of the world on a large scale.6 In the case of HLB infections, therapies considered against the causative bacterium Candidatus liberibacter (CLas)7 include treatment with antimicrobial agents as a promising alternative to enhance the crop’s lifespan.5 However, response to such treatments is diminished due to challenges associated with the application process and the ensuing slow penetration into the phloem, the conductive tissue where bacterial populations aggregate within infected citrus plants.8\n\nA novel alternative was recently identified to enhance penetration of agrochemicals into the leaf. This system involves the generation of leaf perforations of approximately 250 μm in diameter using a CO2 laser directly focused upon the leaf surface. The perforations not only puncture the cuticle but also perforate the epidermis and few layers of underlying palisade parenchyma.7 When applied on treated leaves, penetration of sample substances was increased over 2,000% over untreated leaves.9\n\nThe use of laser micro-perforations as a plant pharmacodynamics-enhancing technique has several drawbacks. First, apart from the cuticle, laser-induced pores also affect the underlying leaf epidermis and palisade parenchyma due to the inevitable removal of material from both areas.9,10 Second, this methodology requires highly focalized lasers to achieve maximum efficiency within field conditions, which is technologically complex due to the intrinsic irregularity of leaf surfaces, their random orientation, and depths within a tree.\n\nThe goal of this investigation was to devise a lesser invasive mechanism to enhance substance uptake into the leaves using specific laser beams. Our study describes a novel methodology for enhancing the penetration of agrochemicals into citrus leaves without the drastic requirement for physically perforating leaf tissues. This novel technique is based upon epidermal water content-dependent selective absorption of Erbium laser light. Partial separation of the waxy cuticle is successfully obtained across an area of several square centimeters through the application of a single laser beam. Since there is no damage afflicted to the leaf epidermal tissue, the cuticle rapidly regenerates within a brief period, thereby recovering its protective functions.\n\n\nMethods\n\n‘Valencia’ orange leaves from greenhouse-reared plants of approximately one meter were selected. ‘Valencia’ orange is one of the main species affected by Huanglongbing (HLB) disease, and its leaves are covered by a thick cuticle layer that can impede the absorption of foliarly applied treatments, making it an ideal model organism. Laser treatments were applied to attached leaves and these remained on the tree for post-laser application in order to provide the live conditions. Treated leaves were consequently detached and transported to the laboratory for further analysis.\n\nIt is well-established that leaves have a high-water content including the epidermis. Since water has a strong absorption band in the 3000 nm wavelength region, an in-house Er:YAG laser (fundamental wavelength = 2940 nm; 200 μs pulse duration) was used for foliage irradiation from a distance of approximately 30 cm. Each laser treatment consisted of a single shot performed without specific focusing of the laser on the leaf surface. In order to discern the varying effect of laser intensity on the leaf wax cuticle three laser energy levels were employed, with a spot area of 0.78 cm2. This spot area was determined by the diameter of the laser crystal used in the experiment (1cm), as no focusing element was employed. This allowed for a direct measurement of the effect of laser intensity on the leaf wax cuticle, without the added variable of a focusing element. No lens was used to focalize the laser and the beam divergence was 0.1 mrad, consequently disregarding the necessity for standardizing irradiation distance in such assays. In all cases, one single pulse sufficed to obtain the effect of wax cuticle exfoliation. The laser energies tested for the removal of wax across the investigated leaf foliage ranged from 0.3-0.76 J/cm2, depending upon the laser energy level.\n\nTo visualize the penetration of applied substances into the leaf, a fluorescent analog of glucose solution (NBDG: 2-NBDG = 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose. λ Ex/Em (nm) 465/540) was applied to the leaf surface. The NBDG solution was prepared and used at a concentration of 30 mM.9\n\nMicroscopic observations were performed using a Carl Zeiss™ Axio Scope A-1® microscopy platform equipped with a Canon™ EOS Rebel T3i® camera and a Carl Zeiss™ AxioCam ICc 1®. Low magnification images were obtained using a Zeiss™ Stemi SV11® fluorescent stereoscope (Carl Zeiss Microscopy™+ GmbH, Göttingen, Germany).\n\nFor High-magnification observations of the plant samples with fluorescence, a Carl Zeiss AxioScope A1 fluorescent microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany) equipped with a Zeiss Axio Cam ICc1, with filter Set 43 or Rhodamine filter from Zeiss (Ex: BP 545/25, Em: BP 525/50) for red and green fluorescence, was used.\n\nLow magnification images were observed under a Wild Heerbrugg stereoscope (Wild Heerbrugg Instruments, Ltd., Heerbrugg, Switzerland) using a Green –Only bandpass filter, Stereo Microscope Fluorescence Adapter SFA-LFS-GO (NIGHTSEA, Electron Microscopy Sciences, Hatfield, PA. 19440). Images were captured with a Canon PowerShot S3 IS (Martin Microscope Co., Easley, SC). The procedures and techniques described in this manuscript have been thoroughly described to allow for replication of the results. All relevant materials, steps, and parameters have been included to ensure full reproducibility of the experiment.\n\n\nResults\n\nThe effect of increasing laser energy levels applied to the leaf surface was studied by treating 20 leaves from the same tree with three different energy levels. The results of these pulses on three representative leaves are presented in Figure 1. The yellow areas in Figure 1 represent the modified leaf cuticle according to the level of energy applied. Exfoliation of the leaf cuticle increased proportionally with laser intensity level. For the highest energy density value at 0.76 J/cm2, the exfoliation effect was achieved across (85-90%) the entire laser-spot area (Figure 1c).\n\nFigure depicts three different levels of laser energy-intensity. a) 0.3 J/cm2, b) 0.5 J/cm2, c) 0.76 J/cm2.\n\nA close-up of a treated portion of a leaf using 0.76 J/cm2 energy density is shown in Figure 2. The waxy material, which appears relatively smooth in untreated leaves (Figure 2a), aggregated and segregated after treatment, forming clean breaches into the epidermis (Figure 2b). Under these conditions, only the cuticle was affected as the green underlying epidermal layer of cells remained undisturbed by the energy applied. The image in Figure 2c (black-and-white) was generated using the color tool from Power Point® (Microsoft™, USA) to facilitate the estimation of percent exposed areas. Regarding such estimations, the online tool ‘Coolphptools’ was employed,11 to calculate the percentage area per color. The black-color areas, which corresponded to wax cuticle exfoliated regions, was estimated to cover as 0.61, indicating a 61% success rate in cuticle exfoliation of the total laser-irradiated area. Table 1 shows the average percentage of exfoliated area for each energy level.\n\nIt can be appreciated that the exfoliated are is correlated to the energy density of the pulse.\n\nThe infrared wavelength inherent to the erbium laser, which is identical to the absorption peak of water, allows for considerable laser-energy absorption by such water content. This phenomenon provokes the rapid heating and exerting of pressure that ‘pushes’ the wax cuticle outwards. Since such a laser-energy/wavelength is not absorbed within the epidermal tissue or any other plant constituent, consequently, there is no irreversible physical damage imposed upon the leaf structural integrity, apart from cuticle exfoliation (temporary separation). The mechanism of selective ablation of plant parts/elements was previously utilized for the removal of cactus spines, where it is possible to pulverize and extract such spines through rapid heating of water content present within glochids.12\n\nFigure 3 presents a fluorescent view of a laser treated leaf using a green filter. In Figure 3a, the areas where the cuticular wax was “lifted” or exfoliated appear green in color. The fluorescent green color represents autofluorescence of undamaged epidermal cell walls. These open irregular-shaped areas range from several tens to hundreds of micrometers. The beige areas correspond to the exfoliated wax clumps. A cross section of a treated area in presented in Figure 3b. The figure distinctly shows the “lifting” of the epidermal wax on the upper side of the leaf. The bright spots represent autofluorescence of the vascular tissue.\n\nFigure 4 is a diagrammatic representation of a laser treated leaf highlighting the leaf epidermis prior to (Figure 4a) and following (Figure 4b) application of a laser pulse. In Figure 4, the grey areas represent the cuticle whereas the underlying cells include the epidermal cells and the palisade cells. Post-laser treatment, the cuticle appears ‘lifted’ (or partially detached) as a result of the pressure exerted by the laser-excited water (Figure 4b). Through such areas having detached and raised cuticles, therapeutic compounds can easily penetrate into the leaf epidermal layer and then follow its pathway to the plant transport system.\n\nTo demonstrate the effectiveness of the laser treatments in allowing externally applied hydrophobic substances to penetrate the leaf and travel throughout the plant vasculature, we applied a fluorescent NBDG to laser treated areas (Figure 5). The images are viewed under green/red control untreated leaf is presented in Figure 5a. Conversely, Figure 5b depicts a treated leaf two hours after laser treatment and NBDG application. The externally applied fluorescent NBDG is visible throughout the leaf, especially in the veins containing the plant vascular tissue, clearly indicating successful solution penetration and distribution across the majority of leaf surface area.\n\n(a) control untreated leaf, and (b) laser-treated leaf two hours after application of fluorescent NBDG. The circle indicated by the arrow is the laser spot application area. Figures were taken using an I-phone with a green-fluorescent filter.\n\n\nConclusion\n\nThe application of agrochemicals through the foliar route remains a “gold-standard” therapeutic administration route for enhancing crop productivity, treatment of diseases, and pathogen/parasite circumvention and prophylaxis. However, despite its wide application, penetration through leaves remains quite inefficient, causing dramatical environmental impact as > 90% of applied agrochemical doses by the foliar route are not absorbed by the plant and eventually lead to a detrimental impact on the immediate plant environment.1 The use of laser technology for foliar application of agrochemicals contributes with a plethora of advantages. Aside from the improving foliage uptake of most therapeutic compounds/agrochemicals, it results in the reduction in agrochemical losses/wastage and eventual detrimental impact on the treated plant’s immediate environment. The high efficacy provided by the cuticle exfoliation method presented in this communication eliminates perforation of live tissue by limiting its effect to the cuticle only, therefore, providing a less intrusive method for substance penetration. Future work requires the development of prototypes for in-field application conditions. This will include testing at longer distances based on the fact that the Er:YAG laser does not require a lens for this application.", "appendix": "Data availability\n\nOpen Science Framework: Use of non-intrusive laser exfoliation to improve substance uptake into citrus leaves. DOI: 10.17605/OSF.IO/6M43P. 13\n\nThis project contains the following underlying data:\n\n• Statistics exfoliated area (Percentage exfoliated area and pulse energy density for all samples in this study.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nFernandez V, Eichert T: Uptake of hydrophilic solutes through plant leaves: Current state of knowledge and perspectives of foliar fertilization. Crit. Rev. Plant Sci. 2009; 28: 36–68. Publisher Full Text\n\nBurkhardt J, Basi S, Pariyar S, et al.: Stomatal penetration by aqueous solutions—An update involving leaf surface particles. New Phytol. 2012; 196: 774–787. PubMed Abstract \| Publisher Full Text\n\nRee HS, Hirano E: The density of stomata in citrus leaves. J. Agric. Res. 1931; 43: 209–222.\n\nKwakye S, Kadyampakeni DM, Morgan K, et al.: Foliar micronutrient applications ot emhance growth and yield of Huanglongbing (HLB)-affected sweet orange. Soil Sci. Soc. Am. J. 2022. Publisher Full Text\n\nVincent CI, Faraj Hijaz F, Pierre M, et al.: Systemic Uptake of Oxytetracycline and Streptomycin in Huanglongbing-Affected Citrus Groves after Foliar Application and Trunk Injection. Antibiotics. 2022; 11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDala-Paula BM, Plotto A, Bai J, et al.: Effect of Huanglongbing or greening disease on orange quality, a Review. Front. Plant Sci. 2019; 9: 1976. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJagoueix S, Bove JM, Garnier M: The phloem-limited bacterium of greening disease of the proteobacteria is a member of the alpha subdivision of the Proteobacteria. Int. J. Syst. Bacteriol. 1994; 44: 379–386. PubMed Abstract \| Publisher Full Text\n\nBrodersen C, Narciso C, Reed M, et al.: Phloem Production in Huanglongbing affected Citrus Trees. HortScience. 2014; 49(1): 59–64. Publisher Full Text\n\nEd Etxeberria E, Gonzalez P, Fanton Borges A, et al.: The Use of Laser Light to Enhance the Uptake of Foliar-Applied Substances into Citrus (Citrus sinensis) Leaves. Appl. Plant Sci. 2016; 4(1): 1500106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEtxeberria E, Gonzalez P, Bhattacharya P, et al.: Determining the Size Exclusion for Nanoparticles in Citrus Leaves. Proc. Fla. State Hort. Soc. 2016; 51: 732–737. Publisher Full Text\n\nColorextract software. Reference Source\n\nPonce L, Arronte M, de Posada E , et al.: Laser has proven its de-thorning Capabilities in Food Products. Ind. Laser Solut. 2009; 6: 6–9.\n\nCabrera L, Etxeberria E, Gonzalez P, et al.: Use of non-intrusive laser exfoliation to improve substance uptake into citrus leaves.Reference Source" }	[ { "id": "167110", "date": "28 Mar 2023", "name": "Humberto Cabrera", "expertise": [ "Reviewer Expertise Laser", "optics", "photothermal spectroscopy", "digital holography microscopy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 laser-based “wax exfoliation” method to facilitate the penetration of substances into the leaf and, together with enhancing their uptake into the phloem and subsequent transport across tissue. Authors demonstrated the effectiveness of the method to improve the penetration of foliar-applied substances into citrus leaves. One important property is to be non-invasive and more effective when compared to traditional laser treatment methods developed by same authors before using CO2 laser. The feasibility of the proposed method was validated by the experimental results obtained in citrus leaves. However, would be very interesting to see the results in the plant some period after treatment. The simple design without focusing lens and low cost laser makes this system attractive for practical applications. Some important discussions such as the productivity and practical massive implementation are not given. Discussions on other limitations of the system can better inform the readers on the potentials and applicability of this technology. Overall, this is a concise and clearly written manuscript and can be indexed after some major corrections listed below:\nAs a matter of comparison, I suggest to add a leaf without laser treatment (the same leaf prior to treatment) in Figure 1.\n\nI would suggest statistical study about the plant grow and if it helps crop plants grow faster and helps to produce more crops, compared to non-treated plants.\n\nHow much affect the pulse duration on the efficiency of the exfoliation? Is it possible to do such study if the laser is available or there is no any sense? I suppose that with 200 μs the process will be mainly ablative, however some thermal effects can be also present. I suggest more detailed explanation in the text and description of the physical process.\n\nIn conclusion part authors stated that: “This will include testing at longer distances based on the fact that the Er:YAG laser does not require a lens for this application.” Considering the divergence of laser beam (mrad) I would like to know which will be such long distance (more than 30 cms) in order to keep the necessary energy densities and results achieved in this work.\n\nWill be interesting if there is a description about efficiency and productivity compared with other methods. The laser beam is small, and if extensive zone are treated, the question about productivity and efficiency should be considered (when long distances and scanning procedure are applied).\n\nAnother important point here is also an estimation of the cost of the device or the prototype for massive implementation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9557", "date": "14 Apr 2023", "name": "luis ponce cabrera", "role": "Author Response", "response": "General comments: The reviewer is correct that it would have been interesting to see the results in the plant for a period after treatment, but this is beyond the scope and objectives of this work. We are presenting a method that allows the uptake of substances to be created and have demonstrated that there is an improvement. However, the consequences of the treatments can be very different depending on the substance applied. Each substance will require a specific study as the doses and timing will be different. Regarding the productivity, it is also too early to tell as it will depend on how much substance is required and how many entry points will need to be opened in the plant. It may be sufficient to treat just one leaf in some cases, while in other cases it may be necessary to treat multiple leaves. Answers to the referee's questions: Regarding the referee's suggestion to include a leaf before treatment in Figure 1, as can be seen in Figure 5, an image of the leaf where the treatment area is visible before (a) and after (b) treatment has already been included. As mentioned in the general comments, a statistical study on how the plant grows and whether this helps farmers is beyond the scope of this article. This method can be used to improve the capture of many different substances, each of which will require a specific study. Regarding the pulse duration, the available experimental setup does not allow for modification of that parameter, but we agree with the referee that it is worthwhile to conduct a study in the near future to optimize it. However, the penetration of the fluorescent glucose demonstrates that for the specific pulse duration we have used, the process significantly increases the capture of the substance, which was the main objective of this work. We did not include a discussion of efficiency and productivity because the development of prototypes applicable under field conditions is future work. However, we have modified the final part of the conclusions to include an estimate that gives an idea of the feasibility of this method. The answer to this question is related to the previous one. As mentioned earlier, the productivity for each specific substance will depend on many factors, and it is an analysis that is beyond the scope of this work. The cost of the device will be related to parameters such as power, pulse energy, scanning system, and others, as well as the size of the market. A rough estimate could be a few thousand dollars. The financial viability will depend on how effective the method is in saving on agrochemicals or improving plant treatments. It is still too early to make an accurate economic evaluation." } ] } ]	1	https://f1000research.com/articles/12-303
https://f1000research.com/articles/12-480/v1	10 May 23	{ "type": "Review", "title": "Molecular technologies ending with ‘omics’: The driving force toward sustainable plant production and protection", "authors": [ "Tshegofatso Dikobe", "Kedibone Masenya", "Madira C Manganyi", "Tshegofatso Dikobe", "Kedibone Masenya" ], "abstract": "As the global population is surging, the agricultural industry is required to meet the food demand while simultaneously providing eco-friendly sustainable crops that can withstand numerous abiotic and biotic stresses. The current era requires high-throughput biotechnology approaches to alleviate the current plant production and protection crisis. Omics approaches are regarded as a collection of high throughput technologies ending with “omics” such as genomics, proteomics, transcriptomics, metabolomics, phenomics and epigenomics. Furthermore, omics provide the best tactic to increase high quality crop production yield. A body of evidence has shown that microbial diversity, abundance, composition, functional gene patterns, and metabolic pathways at the genome level could also assist in understanding the contributions of the microbial community towards plant growth and protection. In addition, the link between plant genomes and phenotypes under physiological and environmental settings is highlighted by the integration of functional genomics with other omics. However, application of single omics technologies results in one disciplinary solution while raising multiple questions without answers. To address these challenges, we need to find new age solutions. For instance, omics technologies focusing on plant production and protection. Multi-layered information gathered from systems biology provides a comprehensive understanding of molecular regulator networks for improving plant growth and protection, which is supported by large-scale omics datasets. The conclusion drawn from the in-depth information is the holistic integration of multi-disciplinary omics approaches to pave the way towards eco-friendly, sustainable, agricultural productivity.", "keywords": [ "agriculture", "biotechnology", "genomics", "metabolomics", "multi-omics", "transcriptomics", "proteomics" ], "content": "Introduction\n\nIn this era, global population growth outstrips agricultural production which might lead to food insecurity, world hunger, poverty, and depopulation. The Food and Agriculture Organization of the United Nations1 reported that in 2020, 811 million people globally experienced hunger which is an increase of up to 161 million people from 2019.1 It was projected that the global population will surge by 25% by 2050, thus agricultural production will need to be increased by 49% in order to meet the food demand.2 Food security incorporates nutritional quality as well quantity, which are essential requirements to meet an increasing global population and reduce hunger. However, food security is strongly inhibited by abiotic and biotic factors that negatively affect crop yield and production.3 In addition, several challenges contribute to the global food security crisis with climate change at the forefront.2,3 Climate change has worsened the situation with unstable rainfall patterns, extreme temperatures (high or low), salinity, pathogens and pest severity, distribution and resistance to pesticides.4\n\nAccording to the United Nations (UN), this is the “decade of action” whereby the 17 Sustainable Development Goals need to transform our world and be achieved by 2030 such as combating poverty.5 How do we develop food systems that are resilient and sustainable, in order to feed the billions of people globally? This is the burning question that the scientific community are working on tirelessly. To address these challenges, high-throughput biotechnology approaches has become the focal point of many strategies to improve plant production and protection. Moreover, omics provide the best tactic which will aid an increased high-quality yield in crop production.6 Omics is a multi-disciplinary science-based discipline that investigates the interaction of various organisms with their environment and such characterization is gathered from the genome profiles, transcriptomes, proteomes, metabolomes and a variety of other related “omes”. A combination of omics technologies have proven to be valuable in agriculture, by exploring plant systems response pathways to various abiotic and biotic factors.7 In addition, multiple omics approaches assist in the elucidation of complex gene functions under various physiological and environmental stress conditions.8 The application of multiple-omics approaches have proven to be successful in breaking down the components of stress response in various economically important crops.9,10\n\nThus, a combination of omics technologies are needed to offer solutions to feed the growing population, through the development of high yielding transgenic crop varieties.11 Application of these technologies would assist in understanding plants response pathways to different abiotic and biotic stresses. Recently various omics strategies have improved and brought transgenic crop varieties that have gained popularity due to their ability to possess various useful agronomic traits.12 Combined omics approaches have proven to complement each other well in analysing complex biological processes. For instance, this was illustrated in halophytes through differential regulation of metabolites, proteins and ions in response to salinity stress.13 For plants to survive both abiotic and biotic stresses they need to adjust their omics profiles accordingly.8 Thus, it is important for a combination of omics techniques to be incorporated in plants response research studies to illuminate molecular pathways that actively control abiotic stresses and provide comprehensive information for biological system analysis.14 This review summarizes the important multiple-omics approaches, their applications, bioinformatics tools and anticipated implementations in agriculture to improve crop yields, productivity and enhanced protection against abiotic and biotic stresses (Figure 1).\n\n\nResearch methodology\n\nThe current review was articulated from high-quality specialized search engines such as Google Scholar, Scopus, ScienceDirect, and PubMed. A body of evidence was collected by screening and selecting the most current and definitive original publications using several keywords (agriculture, biotechnology, genomics, metabolomics, multi-omics, transcriptomics, proteomics) with the goal of narrowing the search to the most relevant studies. The search only looked for peer-reviewed, full-text articles in the English language.\n\n\nGenomics\n\nDuring the previous decades, the conceptualization, development, and utilization of genomic technologies provided a wealth of information that has yet to be discovered.15 The power of first-generation sequencing technologies has revealed the helix to monitor and screen diseases as well as to discover the biosynthetic logic and genetic basis for the synthesis of novel bioactive metabolites in the healthcare and agricultural industries.16 Combining genome mining with synthetic biology has been reported as progressive growth.\n\nGenomics is the study of recombinant DNA, DNA sequencing techniques, bioinformatics, examining the structure, function, evolution, mapping, epigenomic, mutagenomics of genes and genomes.8 DNA sequences and annotations, among other genomic tools, are now deposited in publicly available databases.17 The progressive improvement of computers and networks is required for the storing and handling of these resources. In natural and engineered strains, genomics enables high-throughput DNA sequencing and large-scale bimolecular modelling of metabolic and signalling networks.18 In this era, genomic technologies have been applied to improvement of crop breeding, plant promotion and protection. A body of evidence has shown that bacteria and fungi co-exist with their host plants, thus promoting plant growth and protection.19,20 Tremendous progress has been made in structural genomics indicating morphological and genetic maps to find features of interest.\n\nCountless studies have expressed several genes related to stress tolerance,21 nitrogen fixation,22 for mineral acquisition (Fe, P, etc.),23 phytohormone production (IAA, GA, etc.), adhesion, and other colonization related genes,24 which are significantly important in microbial endophytes existence. The introduction of beneficial microorganisms to crops might lead to plant growth along with antagonistic activity against plant diseases.25 It is worth noting, that the agriculture industries are becoming increasingly involved in finding more sustainable, greener, and environmentally friendly products, which aligns to the 17 Sustainable Development Goals (SDGs). In addition, this is also in response to the worldwide problem of pesticide exploitation and overuse.26 Microbial products can be employed as biofertilizers, plant strengtheners, phyto-stimulators, and biopesticides, depending on their mode of action and effects.27 Tremendous progress has been made in improving the scale-up and bioprocess development of microbial inoculants. The application of genomic technologies, particularly microbial inoculants, have massive benefits.19\n\nSequencing technologies has cemented a “gold standard” for the detection of both known and unknown variants in genomics. Depending on the intended downstream application, different strategies are utilized for each phase. Subsequently, nucleic acid-based sequences will go through cleaning, filtering, assembly, alignment, variant calling, annotation, and functional predictions.27 Shotgun sequencing was one of the most popular techniques utilized with long strands of DNA as well as complete genomes. The conventional chain-termination approach, also known as the “Sanger method,” which is based on the selective incorporation of chain-terminating dideoxynucleosides by DNA polymerase during in vitro DNA replication, was the technology behind shotgun sequencing for a significant portion of its history. In addition, Sanger sequencing is time consuming hence the use of high throughput technologies nowadays, e.g., next generation sequencing (NGS). High-throughput sequencing or microarray hybridization, as well as bioinformatics, are essential genomic analysis methods.28\n\nDespite the massive benefits, microbial inoculants are based on various modes of action, and their interactions are influenced by environmental factors. Another key concern is that microbial inoculants are intended for a wide range of crops globally, they thus might react differently in different species.19 Moreover, to obtain an “ideal” inoculant, culturable microbial species require optimal growth conditions and in-depth understanding of plant/host relationships.29 To address this, fermentation and formulation procedures need to be improved further. Government legislation remains an obstacle in safety aspects of commercialized microbial inoculants.30 Moreover, microbial strains may also act differently in culture than they do in their native habitats. As a result, it is critical to create cultivation-independent methodologies for studying microbial communities.31 Genomic technologies are taking a leap in the right direction by developing innovative biotechnology strategies in plant growth and protection.19 Unfortunately, genomics technologies can only say whether genes are present or not. To address this, transcriptomics can determine when and where each gene is turned on or off. The RNA sequence mirrors the DNA sequence from which it was transcribed, ultimately gene expression is the logical follow-up in research advancements. To gain insight into plant breeding, biotechnological and genetic engineering, a multi-disciplinary approach linking structural, functional, mutational and comparative genomics is required (Figure 2).\n\n\nTranscriptomics\n\nTranscriptomics is basically a collection of RNA single-stranded nucleic acid copies encoded by the genome under stressful physiological environments in a specific cell type/tissue.32,33 Furthermore, transcriptomics measure gene expression at the RNA level and in most cases, it will measure either messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or even non-coding RNA (ncRNA).34 Such innovative technological approaches provide researchers with an advance tool to investigate the microbial populations related to various plants. Gene expression studies provide a magnification into gene structure and function, subsequently expanding on the understanding of biochemical pathways engaged in biological processes.35 A number of studies have reported that the transcriptomics tool provides an innovative approach to improving our knowledge of biology and disease.36,37 In a general sense, transcriptomics is considered as the next step in innovation and it aligns with all the criteria for a true “omics technology”. Despite the impressive breadth of the data collected, challenges such as analysis remain an issue. The creation of appropriate expression measures for expression level comparisons and strategies for identifying differentially expressed genes are two of the most pressing issues (and exons).38\n\nCompounds have an impact on gene expression, which might provide insight into their functional and toxicological features. Expression investigations on disease and disease-free clinical samples may lead to the discovery of new biomarkers. Importantly, gene expression analysis can also be utilized to learn more about the physiological effects of plant genetic manipulation.39 Outdated techniques such as Northern blotting, serial analysis of gene expression have massive drawbacks that make them inappropriate for analysing significant numbers of expression products at the same time. The development of DNA microarray technology has recently resulted in a significant increase in the sensitivity and throughput of expression screening.40 Despite a number of technological and methodological challenges, microarray research has resulted in major improvements in our understanding of biological processes. It can be a low-cost method of identifying the most likely interesting subsets of samples that will produce results in other technological tools.16 High-throughput technologies have overtaken the omics world and transcriptomics is no exception with the introduction of whole-genome and next-generation transcriptome. Hence, transcriptome profiling is easily accomplished as a result of use of deep-sequencing technology.\n\nMicroarrays have now been surpassed as the preferred approach for gene expression profiling by RNA-seq. Furthermore, it also allows for much more exact determination of transcript amounts and isoforms than other approaches. In a recent study conducted by Ahmad and co-workers,41 based on RNA sequencing, the molecular basis of Brassica rapa plant resistance to Hyaloperonospora brassicae (a fungus-like pathogen) was determined. The elevation of PR genes resulted in a physiological response. The loci Bra003774 and Bra025730 in the PR gene family showed a two-fold increase in expression, indicating that WRKY22 and PR1 are overexpressed. PR1 was identified as a delayed response factor to the assaulting pathogen due to its end location in defence responses. PR1, zinc finger protein metabolism was the second most strongly active activity in Brassica cells, with 42% of genes showing increased expression. The plant's transcriptome profile was identified in order to better understand how the host reacts to the pathogenic onslaught and ensure its survival. The authors suggested that the study will be extremely beneficial to global plant protection efforts.41\n\nIn another study, heavy metal particularly cadmium (Cd) tolerance was determined by using transcriptomic profiling in the Calotropis gigantea plant. To deal with Cd, the leaves triggered a number of Cd detoxification pathways, including overexpression of genes involved in Cd transport such as absorption, efflux and enzyme activation. The phytoremediation capacity of C. gigantea in Cd-contaminated soils was investigated using Cd tolerance parameters and molecular mechanisms.42 RNA-sequencing was used to exam the reactions of two varieties inoculated with Alternaria alternata, a resistant type and a susceptible type. A total of 2235 differentially expressed genes (DEGs) in disease-resistant cultivars and 4958 in susceptible cultivars were examined. Furthermore, the expression of DEGs as a defensive response, detection of resistance genes and signal transduction pathways, which will aid in the exploration of resistance mechanisms in response to A. alternata infection, were determined.43 Transcriptomic research has aided in the identification of key functional genes and pathways that may show memory behaviour. Moreover, studies have also been useful in elucidating the function of memory processes in the modulation of ABA-related gene expression in response to drought.44\n\nRNA-Seq, the NGS based technology, has become the main choice to measure gene expression levels and comparing gene expression patterns at unprecedented resolution. Transcriptomics identifies the genes expressed in response to biotic and abiotic stress. A basic reference-based RNA-Seq data analysis pipeline includes pre-processing (removing low-quality sequences to improve alignment), alignment of raw reads with the reference genome, transcript assembly, and detection of differentially expressed genes.45 The new tuxedo suite has been developed because it requires less memory amongst other advantages. It includes three tools for transcript-level analysis of RNA-Seq data, including HISAT2 for spliced alignment of reads to the genome, assembly of transcripts based on mapping to the genome, including novel transcripts, and quantification of these transcripts.46 The final step for the new tuxedo suite is to identify differentially expressed genes and transcripts using Ballgown.47 Pathway approaches analyze databases and gene expression data to identify the significantly impacted pathways in a given condition.48\n\nDespite the potential benefits, there has been records of noticeable downfalls of using transcriptomics. Transcriptomics is not suitable for recognizing adaptive stress response genes. This is due to the massive change in gene expression which may not always translate into a large influence on fitness, enormous gene effects are uncommon, protein activity is most relevant to fitness, and mRNA abundance is an inconsistent predictor of protein activity. It can be computationally difficult to annotate sequences accurately and interpret data, especially when there is no reference genome available.49 Furthermore, biases established during the creation of the cDNA library and sequence alignment can have an impact on the quantification of transcripts. Reproducibility can be hampered by differences in read depth between sequencing platforms. Although RNA-seq has become more economical, many laboratories still cannot afford it. A key component in transcriptomics is that the start-up expenses are high, as well as the cost of each sequencing reaction, which depends on the read depth.50 However, the benefits outweigh the shortfalls, especially when advanced multi-omics is applied. Our better understanding provides new knowledge regarding the relationship between gene expression and fitness in demanding environmental settings.\n\n\nProteomics\n\nProteomics is the study of the total number of proteins expressed in an organism and is divided into several categories, including sequence, structural, functional, and expression proteomics.51 When compared to genomics and transcriptomics with post-transcriptional modifications, this technique is more advanced and reliable; it provides a better understanding of functional molecules (proteins) that carry out various cellular processes (functions), and it can be easily integrated with other omics approaches to elucidate complex plant stress responses.52 There are numerous proteome approaches, and new ones are emerging to address or improve target protein isolation, resolution, chemical and physical properties, and functions.\n\nAmong these, sequence proteomics is concerned with identifying amino acid sequences and characterizing them using high performance liquid chromatography (HPLC).53 Then structural proteomics involves the mapping out of the structure of protein complexes or proteins present in a specific cellular organelle in order to understand their inferred function.54 Thirdly, functional proteomics investigates protein functions using a variety of techniques such as yeast-one or two hybrids and protein microarray profiling.55 Lastly, expression proteomics investigates the quantitative and qualitative expression of proteins using a variety of methods, including separation of proteins on 1-DE sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) based on their molecular mass, and 2-DE based on their net charge in the first dimension and molecular mass in the second dimension.54\n\nPlant proteomics research has advanced and brought improvements in protein extraction and separation to larger scales, moreover becoming a complement technique to transcriptomics and metabolomics.56 Though proteomics could be a powerful approach it requires a series of steps from protein separation and identification to protein functional analysis and bioinformatics inferences involved in crop stress response.57 As opposed to the old conventional proteomics techniques that were mostly chromatography based, recent techniques including SDS-PAGE, 2-DE, and two-dimensional differential gel electrophoresis (2D-DIGE) were developed and are gel based.33 On the same level, protein microarrays/chips utilize minimal sample for protein expression analysis. Additionally, advanced isotope labelling has played a very important role in quantitative proteomic analysis especially in crop improvement studies of abiotic stress adaptation.33 For key molecular weights identification MALDI-TOF, electrospray ionization (ESI), and collision-induced dissociation (CID) are utilized.58\n\nSeveral bioinformatics tools and databases have been developed for various applications including genomics analysis, transcriptomics, proteomics and metabolomics, while a comprehensive list of each approach has been provided separately.59 Proteomics studies generates a large data set using various analytical techniques that are spectrometry or gel based, however such analyses are mainly dependent on several bioinformatic software tools that process, interpret, mine, assess and annotate functions. A bioinformatic tool used for data interpretation obtained from 2-DE gels include the SWISS-2DPAGE. Moreover, multiple databases exist that provide evolutionary and functional annotations of orthologs, predict protein’s subcellular locations such include OrthoDB, OMA orthology, UnitprotKB, WoLF PSORT.60 Various studies have been carried out on the identification of subcellular locations of various proteins to gain guidance for further experimental investigations.61,62 Gene Ontology (GO) term identification provides terminology for biological processes and group associated genes according to various functional terms that describe the “biological process”, “molecular function” or “cellular component”.63 For larger data sets and systematic approaches, tools such as MaxQuant, Proteome Discoverer64 and X!tandem are used for proteomic annotations.65 Furthermore, several bioinformatics databases exist that provide proteomics analysis as indicated in Table 1.\n\nFunctional proteomics has identified different biochemical components such as reactive oxygen species (ROS) including such as quinone reductase, g-glutamylcysteine synthetase, dehydrins, and dehydroascorbate reductase reported in tomato and sunflower crops.73 Important chaperones, such as heat shock proteins, have been identified during proteome functional analyses in wheat and sugarcane.74 Quantitative proteomic studies have also been shown to play a crucial role in various crop responses against abiotic stresses. For example, the use of the iTRAQ method in differential expression of proteins in potato and in two coconut varieties under abiotic stress.75,76 Furthermore, iTRAQ combined with the LC-MS/MS technique has proven to be an effective method widely used for fast identification and quantification of complex protein mixtures in abiotic and biotic stress responses in plants.77 A combination of numerous proteomics approaches including MALDI-TOF, SDS-PAGE, MS, 2-DE, and PMF have been utilized to study response to diverse abiotic stresses in various crops such as rapeseed, soybean, wheat, sugarcane, and cotton.74,78\n\nThere are various limitations that exist in proteomics analysis that normally originate from the initial stage that includes preparation and extraction of samples especially in recalcitrant crops with high content of polyphenols,79 lack of a detailed number of proteins observed and inadequate tools in proteomic data analysis. Furthermore, challenges in peptide separation is a major inhibiting factor to downstream analysis, mass spectrometry-based analysis and bioinformatics tools.80 Another limitation includes lack of a one-step quantification approach that can fully integrate a multi-functional system allowing comprehensive quantification of a wide spectrum of proteins from model and non-model plants.81\n\n\nMetabolomics\n\nMetabolomics deals with the identification and quantification of all metabolites which participate in different cellular events of an organism at a particular time.82 In plant systems metabolites are synthesized through various metabolic pathways.83 In contrast to other omics studies, metabolomic profiling depends on an organism’s cellular location, organ type, tissue and cell types, and the environmental stimuli it’s exposed to or any stress factor.84 Metabolites are divided into two classes, the primary (central) and secondary (specialized) metabolites. The initial class (primary) deals with small molecules used for cell viability, while secondary metabolites are essential for the viability of an organism mainly involved in defence mechanisms.84\n\nSeveral analytical techniques are used to generate metabolic profiles of a given plant sample, including thin layer chromatography (TLC), gas/liquid chromatography-mass spectrometry (GC/LC-MS), liquid chromatography-electrochemistry-mass spectrometry (LCEC-MS), nuclear magnetic resonance (NMR), direct infusion mass spectrometry (DIMS), Fourier-transfer infrared (FT-IR), and capillary electrophoresis.82,85 These methods depend on the approach's sensitivity, selectivity, speed, and accuracy. The methods CE-MS, GC-MS, LC-MS, and NMR are typically employed to profile metabolites from various plant materials.86\n\nNuclear magnetic resonance is considered to be fast and selective with respect to metabolite profiling as opposed to mass spectrometry-based approaches.84 However, a combination of NMR (semi-quantitative) and MS (quantitative) is advisable since it offers a better understanding of metabolome quantification due to the complementarity of these two approaches.18 Metabolomics, combined with other omics approaches such as genomics, transcriptomics, and proteomics serve as a powerful approach that offer solid insights on molecular response pathways, plant biochemical and physiological mechanisms; and their related functions,87 thus ultimately result in its relevance in food security.\n\nSeveral plant metabolomic bioinformatic tools are used to analyse metabolic raw data generated by various analytical tools for data processing, mining, annotation, interpretation and statistical analysis.88 Different web-based programs are used for data pre-processing of metabolites those includes XCMS, METLIN, AMDIS, MeltDB, MetaboAnalys, MetAlign, MZmine 2, and AnalyzerPro for varying analytical techniques. XCMS is a web-based program that assists the user with data processing and statistical analysis,89 while METLIN is mainly used for metabolite annotation.90 MeltDB tool is used for data assessment, processing, and statistical analysis.91 Lastly, AMDIS, KNApSAcK and KOMICS are mainly used for data processing and metabolites present in crop species.92\n\nPlants produce metabolites (secondary) as defence mechanisms against abiotic and pathogen stress. Several studies have reported identification of various metabolites in cereal crops including rice, maize and barley in response to various biotic stressors.93 Rice cultivars demonstrated identification of several metabolites using GC-MS against a pathogen gall midge biotype 1 (GMB1).94 Several metabolite analyses were reported in wheat, maize, tomato, and soybean crops in response to drought, cold and heat stress.95,96 Therefore, metabolomics offers a significant advantage compared to its counter-omics approaches such as transcriptomics and proteomics, in that it reveals the downstream products synthesized by genes and expressed by proteins.83 However, to comprehensively classify metabolites according to their various chemical/physical properties relevant preparation methods and instrumentation need to be utilized for enhancing metabolite performance.97\n\nThere are several disadvantages that impede the full characterization of metabolites from a given organism, including the complex physicochemical properties that cannot be characterized by single or limited analytical techniques, hence combined multiple analysis tools are recommended for a full metabolome analysis.82 Another limitation in creating metabolomics profiles is the consistency of growth conditions and preparation of plant samples due to the instability of metabolites undergoing multiple modifications such as hydrolysis or oxidation. Improper harvesting, handling, extract preparation, and sample storage are the most likely sources of instability in plant metabolite analysis.98 In addition, simple metabolomics analysis requires the use of expensive equipment compared to genetic analysis, hence there are still limited data on these studies. Metabolomic analysis in plant pathology is challenging as it cannot distinguish between plant and microbial metabolites resulting from the interaction between (host and pathogen) plants and microbes as a metabolic response pathway.99 Limited universal metabolite-specific standards and reference compounds to assist in identifying metabolomes.100\n\n\nMetagenomics\n\nMicroorganisms that are effective, play a crucial role in the management of biotic and abiotic stress, reduce the use of chemical fertilizers and increase the plant’s yield by influencing elemental cycling.101,102 Additionally, studying plant pathogens is an effective means for screening the presence of potentially agriculturally important pathogens.103 Substantial understanding of the microbial community structure, annotation of functional genes, as well as metabolic pathways could aid in better understanding the benefits towards plant growth and protection.104\n\nThe introduction of high-throughput sequencing (HTS) has revolutionized the field of microbial ecology.105 Previously, identification of bacteria and fungi was largely done using culture dependent techniques, which are inefficient as some microorganisms cannot be cultured on artificial media. Traditional methods for detecting viruses, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) (PCR), typically target a few viral species and require prior virus knowledge to detect.106 HTS sequencing allows for the study of microbes by sequencing the genetic material directly on the environment,107 avoiding the need for culturing, termed metagenomics.108\n\nThe most exhausted high-throughput sequencing application in microbial ecology is target-gene amplicon sequencing. The 16S rRNA gene is found in all bacteria and has either high or low sequence variability.109 This gene contains nine hypervariable regions (V1–V9) that show significant sequence diversity among bacteria.110 Although no single hypervariable region can distinguish between all bacteria, regions V2, V3, and V6 have the greatest discriminating power and contain the most information in plants. However, Kembel et al.111 argued that the 16S rRNA marker may be inconvenient due to variable copy numbers, with some taxa having up to 15 copies of this gene. As a result, there may be taxa overrepresentation in the final set of sequences, resulting in biased community structure estimations. Nevertheless, the benefits of using 16S rRNA gene markers outweigh the inconvenience, which is why amplicon sequencing is widely used in surveys.\n\nThe internal transcribed spacer (ITS) of fungi is a non-coding region that is highly polymorphic and contains the taxa that can separate sequences into species.112 It is found in the ribosomal RNA operon and ranges in length from 450 to 750 bp.113 The benefit of using ITS for-DNA barcoding is that it has been used in numerous studies and has updated reference sequences in the NCBI database.114 ITS1, ITS2, ITS3, and ITS4 are the most used oligonucleotides for sequence-based fungal classification at the species level.115 Therefore, amplicon sequencing forms the basis in characterizing bacteria and fungi and is an ideal tool to begin studying the microbiome as it is reasonably inexpensive.\n\nShotgun metagenomics has transformed microbiome detection and discovery by enabling untargeted characterisation of entire microbiomes. It has revolutionised plant science by enabling the untargeted, simultaneous detection of multiple viruses, bacteria, archaea and fungi regardless of their genomic nature and can deliver species level identification.116 This includes annotations for metabolic and functional processes. Shotgun-based approaches typically generate large, sequenced data.117 However, their ability to accurately group DNA sequences or assembled sequence contigs into single-species genomes has remained limited.118 Recently a powerful approach has been introduced for metagenomic analysis that can address the challenges with shotgun.119 Hi-C is a technology that provides direct and quantitative measurements of DNA sequences from shotgun sequencing.120 This technology yields higher numbers of high-quality genomes and captures strain-resolution insights. Hi-C eliminates the need for guesswork in metagenomic studies as direct evidence sequences.121 The technique allows for accurate attribution of plasmids, phages, antibiotic resistance genes and other mobile genetic elements to host cells.120\n\nBioinformatic analysis of plant microbiomes demands the use of pipelines that effectively analyze the high throughput data generated by shotgun and target sequencing for microbial profiling and gene annotations. Several pipelines for analysing 16S rRNA gene sequencing data are available, including Quantitative Insights into Microbial Ecology (QIIME2),122 MOTHUR, and Metagenomics - Rapid Annotation using Subsystems Technology (MG-RAST).123 CLC Genomics workbench124 and MEGAN.125 The comparison between the diversity and taxonomic compositions generated by MG-RAST and QIIME using samples was investigated.126 The study concluded that QIIME2 produced compositional assignments that are more accurate. Similarly, Plummer127 found that QIIME2 performance outweighed other platforms. Analysis with the platforms cluster similar sequences into operational taxonomic units (OTUs) or amplicon sequence variants (ASVs) and assign them to a taxonomic reference database, such as GTDB, the most updated bacterial database.128\n\nEssentially the data analysis of metagenome shotgun involves assembly, which includes joining reads into contig sequences that are larger. Newbler, AMOS, MIRA methods were used in assembling metagenomes at an early stage of metagenomics.129 However, due to the low coverage and complexity of metagenomic reads, de novo assemblers, beginning with SOAPdenovo, were preferred for reference mapping. Genovo,130 Meta-IDBA,131 MetaVelvet,132 MAP,133 and Ray Meta are among the improved assemblers now available.134 Following assembly, microbial communities are binned and taxonomically characterized. Functional annotations mainly use BLASTX search against several databases, including SEED, KEGG, EggNOG and COG.57 Further graphical and statistical analysis is carried out using R software in conjunction with RStudio software and various microbiome-related R-packages.135\n\nMetagenomics has been widely applied on different agriculturally important plants and on different microenvironments including the root and leaves. This has also been conducted in response to both abiotic and biotic stresses. For instance, Xu and colleagues136 revealed the role of iron metabolism in drought-induced rhizosphere microbiome dynamics using metagenomic tools. Similarly, patterns in the microbial community and the functional genes associated with salt-tolerant plants and root associated microbiomes of potato grown in Alfisols have been conducted in response to abiotic stress.137–139 Metagenomics studies in response to different bacterial, fungal, and viral diseases have also been conducted in pear fruits, citrus, grapes, tomato, and bananas.138–142\n\nPlant chloroplast and mitochondrial genomes, as well as bacterial 16S rRNA sequences, are similar, making bacterial rRNA gene amplification and sequencing challenging. As a result, the majority of the sequence data is from plant sequences, which are irrelevant to the study. Zarraonaindia et al.141 showed that chloroplast sequences were the most problematic for Vitis vinefera, accounting for up to 98 percent of the total, as opposed to the mitochondrial sequences. It has been proposed that universal peptide nucleic acid (PNA) clamps can obstruct the host plant derived mitochondrial (mPNA) and plastid (pPNA) sequences at the V4 16S rRNA locus.143 However, universal PNA efficacy was tested on Arabidopsis thaliana and the sorghum plant and was discovered to be effective in preventing the amplification of non-targeted sequences.38,144 Additionally, another primer set has also shown to reduce non-targeted plant DNA amplification when performing metagenomic research on microbial endophyte communities.10 The current study has highlighted some shortfalls of using a single omics approach and Table 2 provides a multidisciplinary approach by supplementing some limitations with other omics approaches, hence providing a multi-layered information system.\n\n\n\n• Culture-depended techniques\n\n\n\n• No biological effect of DNA\n\n• No phenotypes data\n\n• No pathway analysis\n\n\n\n• Metagenomics\n\n• Next Generation Sequencing\n\n• Proteomics and Metabolomics\n\n• Metabolomics\n\n\n\n• Provides plant genes information\n\n• Proper classification\n\n• Develop new, specific crop varieties\n\n• Improve food safety\n\n• Resistance to abiotic and biotic stresses\n\n• Improve food security by increasing productivity\n\n• Eliminating toxic compounds such as mycotoxins\n\n\n\n• Ineffective method for identifying genes responsible for environmental stress responses\n\n• Accurate sequence annotation and data interpretation\n\n• No pathway analysis\n\n\n\n• Genomics\n\n• Proteomics\n\n• Metabolomics\n\n\n\n• Gene expression under abiotic stress,\n\n• Improve breeding selection and cultivation\n\n• Examining functional genes and regulatory mechanisms\n\n\n\n• Sample preparation and extraction from recalcitrant crops\n\n• Multiple proteome analysis databases\n\n• One-step quantification\n\n\n\n• Genomics\n\n• Metabolomics\n\n\n\n• Identify proteins that offer promise as biomarkers against biotic and abiotic stressors\n\n• Offers a way to improve crop productivity\n\n• Understanding of the molecular pathways demonstrated by plants faced with various stresses\n\n\n\n• Inconsistency in sample preparation, harvesting and storage\n\n• Metabolites cross-contamination (plant and pathogen)\n\n• Limited metabolite-specific standards\n\n\n\n• Genomics\n\n• Proteomics\n\n\n\n• Explains the biochemical and genetic mechanisms of metabolic variations in plants through targeted and non-targeted methods\n\n• Offer metabolic profiling of genome-edited plants\n\n• Assess food health risk evaluation and regulatory policies related to genetically modified crops\n\n\n\n• Incomplete extraction of DNA\n\n• Data cannot be fully quantified\n\n• Functionality of genes not fully analysed\n\n\n\n• Genomics\n\n• Proteomics\n\n\n\n• Gene expression under abiotic stress\n\n• Examining functional genes and regulatory mechanisms\n\n\nConcluding remarks\n\nA growing body of evidence has cautioned that food safety will be progressively compromised in future due to over population, thus becoming a massive global health and economic crisis. To sustain an ever-growing population, biotechnological based approaches such as “omics” technologies should be implemented as valuable tools for plant production and protection. Omics technologies are a collection of innovative tools such as genomics, proteomics, metabolomics, next-generation sequencing, whole genome sequencing and transcriptomics. Furthermore, they are the key ingredients in the next ground-breaking biotechnology-based products in the agricultural industry. Introduction of other “omics” technologies in relation to genomics as a multi-disciplinary approach have sparked interest in the research community. Most researchers are in agreement with the multi-disciplinary approach of “omics” technologies in order to leverage the benefits and omit the drawbacks. In light of this, the ever-increasing developments in omics technologies provide new opportunities in addressing fundamental issues in food safety, plant production and protection. Thus, opening the door towards eco-friendly sustainable plant production as well as agricultural industry.\n\n\nAuthor contributions\n\nAll authors (Tshegofatso B. Dikobe, Kedibone Masenya and Madira C. Manganyi) have conceptualized, written, reviewed and edited the manuscript. The final manuscript version has been read and agreed upon by all authors.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nAll authors would like to acknowledge their respective universities for their support.\n\n\nReferences\n\nAbberton M, Batley J, Bentley A, et al.: Global agricultural intensification during climate change: a role for genomics. Plant Biotechnol. J. 2016; 14(4): 1095–1098. Publisher Full Text\n\nAgarrwal R, Bentur JS, Nair S: Gas chromatography mass spectrometry based metabolic profiling reveals biomarkers involved in rice-gall midge interactions. J. Integr. Plant Biol. 2014; 56(9): 837–848. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "201458", "date": "20 Sep 2023", "name": "Phetole Mangena", "expertise": [ "Reviewer Expertise Plant Biotechnolgy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reviewed literature on omics-based molecular technologies driving sustainable production and protection in plants. Although, authors clearly and broadly demonstrated the molecular underpinnings and applications of these omics’ technologies, they have paid less attention on their profound role in plant production and protection as captured in the title of this paper. For instance, the section dealing with the “Application based transcriptomics on plant protection” is the only most relevant and more succinct section, reflective of the topic. The entire manuscript should have followed this format, especially on ensuring that the objectives of this paper are addressed. Furthermore, the body somehow appears to consist, in the main, basic literature that is relevant, but not thoroughly discussed, do not provide thorough analysis of the topic and lacks a clear argument capturing the application of these tools for successful plant production and protection. The provided information is too general and should adequately highlight experimental/ practical cases wherein omics was used to improve plant growth and development. It was not necessarily shown how omics have been used to alleviate the effects of biotic and abiotic stresses.\nResearch methodology section is inadequate. Otherwise remove it completely or qualify the number of papers selected, provide a clear criterion for selection and exclusion, including key terms related to plant production and protection used during the selection of papers used, number of records (papers) retrieved using databases as well as other restrictions and relevance criteria. The issues raised above are important and can be improved.\nThe text under “Limitations of Genomics Technologies” completely failed to cover the subheading/topic, please revise the paragraph.\n\nFigure 1, including other illustrations should be positioned just after being cited for the first time in the text.\nUnder Transcriptomics (1st paragraph, line 10) specify the kind of analysis serving as a challenge.\nAvoid using “…including such as…” in the same line throughout the text.\nAny published report/evidence of metabolite profiling in relation to plant protection could have been great.\nLastly, several typos were observed:\nFirst line under Genomics Analysis, there is a typo. Typo on paragraph 2 (Application based transcriptomics on plant protection), line 5, 'exam' should be 'examine'. Gel electrophoresis is correctly referred to as '1-D or 2-D SDS PAGE' not '1-DE or 2-DE'.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "260032", "date": "02 May 2024", "name": "Flavia Vischi Winck", "expertise": [ "Reviewer Expertise Systems biology", "proteomics", "biochemistry", "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\nIn the manuscript \"Molecular technologies ending with ‘omics’: The driving force toward sustainable plant production and protection\", the authors presented a review of the basic concepts of selected omics technologies, with some examples. The authors have reviewed the technologies of four omics applications, including genomics, transcriptomics, metabolomics and metagenomics.\nMajor points: The authors cited the technologies applied to omics studies, but the authors did not cite very important literature where the basics of each technology was coined. The explanations are mostly superficial, and no depth is given to any of the omics technologies presented, specially connecting to studies of plant production or protection. The statements do not include any particularity of the cited references, so the information that it presents is basically general information. (E.g. \"Proteomics is the study of the total number of proteins expressed in an organism and is divided into several categories, including sequence, structural, functional, and expression proteomics.51 ). As a review document, the text should give deep and detailed knowledge about the basic concepts, explore several evidences that drive the topic recently and present an insightful discussion towards the main purpose of the review of exploring omics data generated in studies developed towards sustainable plant production and protection.\n\nFor instance, basic statements such as \"Important chaperones, such as heat shock proteins, have been identified during proteome functional analyses in wheat and sugarcane.74 \") should include some more details about the aspects connecting this study to sustainability or plant protection. A discussion on how important this information is should be discussed. This kind of basic structure of citing the information without deep discussion should be avoided and this pattern should be reviewed thoroughly in the text. It would be important to see not only the citation of omics technologies, and associated tools, but rather which information theses technologies are providing recently that can benefit the sustainable plant production and protection, and how the omics technologies and data generated through theses analysis are providing insightful information that could guide further sustainable applications. Minor points:\nSome citations have no connection with the text (E.G. reference 51, reference 81). It would be important to revise this pattern in the whole text and just cite references directly connected to the information described in the manuscript. Citations dated over 10 years (E.G. Reference number 40) should be avoided if connecting to recent or current applications (E.G. The development of DNA microarray technology has recently resulted in a significant increase in the sensitivity and throughput of expression screening.40 ). The current discussion brings no insights on how the omics technologies can contribute to sustainable plant production and protection\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? No\n\nIs the review written in accessible language? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? No", "responses": [] } ]	1	https://f1000research.com/articles/12-480
https://f1000research.com/articles/12-246/v1	06 Mar 23	{ "type": "Review", "title": "Tale of Two Cities: narrative review of oxygen", "authors": [ "Pranathi Gullapalli", "Nicoletta Fossati", "Dusica Stamenkovic", "Muhammad Haque", "Davide Cattano", "Pranathi Gullapalli", "Nicoletta Fossati", "Dusica Stamenkovic", "Muhammad Haque" ], "abstract": "The human brain contributes 2% of the body weight yet receives 15% of cardiac output and demands a constant supply of oxygen (O2) and nutrients to meet its metabolic needs. Cerebral autoregulation is responsible for maintaining a constant cerebral blood flow that provides the supply of oxygen and maintains the energy storage capacity. We selected oxygen administration-related studies published between 1975–2021 that included meta-analysis, original research, commentaries, editorial, and review articles. In the present narrative review, several important aspects of the oxygen effects on brain tissues and cerebral autoregulation are discussed, as well the role of exogenous O2 administration in patients with chronic ischemic cerebrovascular disease: We aimed to revisit the utility of O2 administration in pathophysiological situations whether or not being advantageous. Indeed, a compelling clinical and experimental body of evidence questions the utility of routine oxygen administration in acute and post-recovery brain ischemia, as evident by studies in neurophysiology imaging. While O2 is still part of common clinical practice, it remains unclear whether its routine use is safe.", "keywords": [ "oxygen therapy", "cerebral autoregulation", "cerebral ischemia", "stroke", "cerebral blood flow" ], "content": "Introduction\n\nThe human brain accounts for only 2% of total body weight, yet receives about 15% of the entire cardiac output: with minimal capacity to store energy. It demands a constant supply of oxygen (O2) and nutrients to meet metabolic needs and maintain cerebral function. Even a short interruption in cerebral blood flow (CBF) can initiate a cascade of pathological events.1,2\n\nIn order to maintain this equilibrium, cerebral autoregulation (CA) and hyperemia are responsible for regulating CBF: while hyperemia maintains localized microscopic blood flow by regulating global CA via constriction and dilation and maintaining CBF between 40 and 60 mL/100 g/min over a wide range of mean arterial pressure (MAP; 50–150 mm Hg).3,4 Outside this range and in the absence of other pathophysiological occurrences, hypotension can technically cause cerebral ischemia, whereas hypertension could lead to a hemorrhagic stroke: both can ultimately initiate a sequence of neuropathological events such as neuroinflammation, synaptic dysfunction, and neuronal death (excitotoxicity, apoptosis, necrosis, etc., also known as programmed or not-programmed cell-death mechanisms).5 In the present narrative review we will summarize some of the general physiological and pathophysiological mechanisms underscoring the role of CA in regulating CBF in various scenarios.\n\nIn a healthy brain, CA—alone and by far—prevents the development of either ischemia or hyperemia caused by MAP changes; however, CA capabilities decline with aging and/or may become impaired, for instance after cerebrovascular accidents. The brain, during these physiological and clinical scenarios is more vulnerable, and it is even further exposed to more damage than it would have been if exposed to the same situations when younger or with better reserves, we could say (hypothetical model in Figure 1).\n\nPanel A. A representation of cardiovascular coupling during normal or stressful events with variation in age, and compensation mechanisms. Panel B. Hypothetical model comparing changes in cerebral blood flow (CBF) in healthy volunteer compared to a patient with chronic ischemic stroke by magnetic resonance imaging.\n\nArbitrary unit=signal intensity; CBF of a young volunteer; CBF: cerebral blood flow; O2: oxygen; 1: pre O2; 2: during O2; 1: post O2.\n\nThe CA can be assessed by measuring CBF response to changes in cerebral perfusion pressure (CPP),6–8 which is a difference between MAP and mean intracranial pressure (ICP).9 However, CBF can also be influenced by combinations of O2 and carbon dioxide (CO2) tensions in the blood (Figure 2).10,11 CBF variations are measured after a ‘steady state’ change in MAP (such as before and after starting a vasopressor infusion) and dynamic CA, which is defined as a response to rapid changes in MAP (such as deflation of a blood pressure cuff).12 Rather than measuring two distinct physiological mechanisms,13 (static and dynamic), the CA expresses the relationship between MAP and CBF velocity over different timeframes.14 In this review, using CA and CBF preservation, we reappraise the risks and benefits of therapeutic O2 administration in patients with brain ischemia and other clinical situations that require supplemental O2 as part of routine clinical care.\n\nA. Cellular interaction (neurons, endothelial cells and glial cells); B. Nutrients/energy (glucose/oxygen); C. Activity/changes of demand, physiological; D. Pathological (seizures, stroke, trauma). Four mechanisms control mechanisms: myogenic, neurogenic, metabolic and endothelial.\n\nCBF: cerebral blood flow; MAP: mean arterial pressure; CMRO2: cerebral metabolic rate of oxygen; CMR: the cerebral metabolic rate; PaO2: O2 partial pressure in arterial blood; PaCO2: CO2 partial pressure in arterial blood; H+: hydrogen ion; pH: potential of hydrogen.\n\n\nMethods\n\nWe searched PubMed for English language abstracts, using searching terms such as “oxygen therapy” AND “cerebral autoregulation” OR “ischemic preconditioning” OR “anesthetic neurotoxicity” OR “ischemic stroke” OR “traumatic brain injury” OR “anesthesia”, from 1975 to October 2021. We chose open and blinded studies, reviews and meta-analyses, and available commentaries and editorials related to MESH terms. Because of the nature of the review (narrative, clinical-experience oriented, translational research motivated), an international criteria/PRISMA is not included, and studies are not graded formally by the level of evidence.\n\n\nOxygen: its history, salubrious properties, other dangerous stories\n\nCarl Wilhelm Scheele was the first scientist to discover O2 in 1771 by heating mercuric oxide, silver carbonate, and magnesium nitrate, but he did not publish his finding till 1777. Meanwhile, in 1774, Joseph Priestley reported that O2 allowed a candle to burn more brightly and has been given credit for O2 discovery.15 The application of O2 in medicine was first reported by Antoine Lavoisier, who described the role of O2 in human respiration. In 1798, the Pneumatic Institute (Bristol, England), started O2 distribution for treating patients with asthma and congestive heart failure.15 In 1880 with the development of O2 cylinders for storage and transport, and in the 1900s with the invention of nasal cannulas and masks, O2 therapy for respiratory pathology became a routine clinical practice.16–18\n\nThe application of hyperbaric O2 utilization is one of the most advanced applications of O2 therapy; it began in 1662 before O2 discovery when a British physician Henshaw compressed air in a closed chamber.19 In the late 1800s, specially designed hyperbaric O2 chambers were built (the first one in North America around 1860).19 In 1972, Takuo Aoyagi invented pulse oximetry, allowing clinicians to measure continuous peripheral O2 saturation and better guide O2 therapy in a clinical context.20\n\nUnder physiological conditions, 98% of inspired O2 is transported in the blood bound to hemoglobin, while the remaining 2% is freely dissolved in the plasma. The bonded O2 to hemoglobin known as oxyhemoglobin increases the partial pressure (PO2) and oxyhemoglobin saturation in the blood.21 Oxygen in the blood comes from lung inhaled air which is transported to organs and cells utilized for energy production essential for organ function.22 Nutrients such as carbohydrates, proteins, and fats are initially broken down into substrates which entered the tricarboxylic acid (TCA) cycle23 and are converted into nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The NADH and FADH2 entered the electron transport chain (ETC), which is composed of several protein complexes located in the inner membrane of the mitochondria. Both NADH and FADH2 donate electrons to the ETC, which carries them down while allowing protons to be pumped into the inner membrane space.23 After electrons reach complex IV, the O2 accepts the electrons and is reduced to water.24\n\nThe protons pumped in the intermembrane space create a mitochondrial membrane potential to convert adenosine diphosphate (ADP) to adenosine triphosphate (ATP) via a synthase enzyme, the main energy molecule in the body.23 Without O2, the ETC would not have a final electron acceptor and the production of ATP would become much less efficient.\n\nOxygen is usually administered under hypoxemia which occurred as a result of a decrease in arterial O2 tension.25 Oxygen therapy is beneficial in pathologies that increased O2 utilization and/or decreased O2 delivery to tissues.24 In patients with acute respiratory failure, supplemental O2 remains an essential treatment component, which has significantly improved with advanced O2 delivery systems.26\n\nGeneral anesthesia (GA) is a typical clinical situation when O2 is commonly administered. GA can indeed decrease O2 partial pressure in arterial blood (PaO2) via multiple mechanisms: functional residual capacity (FRC) decreases after induction of anesthesia due to the diaphragm being cranially displaced in supine position; the decrease in FRC reduces lung compliance and increases airway closure at end-expiration, predisposing to atelectasis and hypoxemia27; moreover, apnea periods during airway manoeuvres can lead to rapid arterial hemoglobin desaturation and hypoxia; therefore, anesthesiologists commonly administer a high inspired O2 fraction (FiO2) before tracheal intubation and extubation.23,24,28\n\nDespite some benefits that O2 supplementation could serve in a variety of clinical scenarios (simple hypoxia, hypoventilation, organ ischemia events, increased O2 demand, toxic hypoxia, to cite few), an indiscriminate administration of therapeutic O2 at high concentrations and/or for a prolonged time may lead to adverse effects: of those, formation of reactive O2 species (ROS) and increased systemic vascular resistance29 could further play a role in generating or contributing for instance to brain injury or other organ injuries. Even a physiological vascular territory where possibly O2 may exert by common sense a salubrious effect, like the pulmonary, may indeed negatively affect hypoxia-induced pulmonary vasoconstriction HIPV, therefore causing ventilation/perfusion (V/Q) mismatching. Overall what we have described as the “oxygen paradox”, is also “The Tale of Two Cities”.30\n\na. Reactive oxygen species (ROS)\n\nPhysiologically, when O2 is utilized in the mitochondrial ETC, only a small amount of ROS is formed by the partial reduction of O2.31 Although ROS such as superoxide anion, hydrogen peroxide, and hydroxyl radical can lead to cell damage, their action is usually counteracted by various intra- and extracellular antioxidants such as superoxide dismutase, glutathione, cytochrome c oxidase.32\n\nWith an increase in O2 supplementation, ROS production is also increased via outstrips the available antioxidant levels. ROS causes damage to nucleic acids, proteins, and lipids leading to cell damage and ultimately cell death.31 An excess of O2 can also lead to inflammation and possible lung damage.29 In lungs, hyperoxia and ROS stimulate nuclear factor kappa B, which releases plasminogen activator inhibitor-1 and tissue factor. The release of these factors causes activation of the coagulation cascade, which may induce further cell damage.21 Importantly, ROS have been shown to play a role in both neuronal death and neurovascular recovery after cerebral ischemia. ROS, interleukin-1β and hydrogen peroxide (H2O2) activate p38-mitogen-activated protein kinase, which is associated with protein oxidation and damage in post-ischemic rodent brains.33 Intriguingly, while ROS mediates neuronal damage during the early phase of ischemia, in later phases it mediates vascular endothelial growth factor (VEGF), angiogenesis, and recovery in the post-ischemic brain.34\n\nb. Oxygen-mediated vasoconstriction and other vaso-mediated mechanisms\n\nROS production linked to excess O2 inhibits cyclooxygenase, decreasing prostaglandins, and thereby lead to vasodilation.35 The ROS superoxide anion inactivates nitrous oxide (NO), a vasodilator, through various mechanisms.36,37 A mechanism by which hyperoxia causes vasoconstriction is by converting arachidonic acid to 20-hydroxy-eicosatetraenoic acid (20-HETE).38 These mechanisms have been described causing systemic vasoconstriction and decreased perfusion to almost all organs,36 supposedly with the exception of the lungs and the placenta: yet HIPV may be disrupted by O2 and similar concerns have been raised for adequate placental flow and the autoregulation of optimal fetal perfusion.39 Hyperoxia-mediated vasoconstriction may affect more the microvasculature rather than larger vessels: for example, the large coronary arteries do not constrict under hyperoxic conditions40; also, a decrease in prostaglandin levels (affected by ROS production) normally cause vasodilation mainly in the microvasculature.41\n\nc. Oxygen therapy post-cardiac arrest and myocardial infarction\n\nSupplemental O2 administration after myocardial infarction (MI) was originally adopted to attenuate tissue ischemia. However, it has been postulated that ROS can in fact cause tissue injury and excess O2 can lead to decreased peripheral coronary perfusion, which in turn may adversely affect patient outcomes.40 A Cochrane review and meta-analysis showed that patients with ST-elevation myocardial infarction (STEMI) or non-STEMI (NSTEMI) treated one hour of inhaled O2 within 24 hours of symptom onset, exhibited an increase in infarct size [measured as increased creatine kinase (CK)] as compared to patients treated with air.42 However, a randomized control trial was unable to show that O2 therapy after MI caused any difference in mortality.43\n\nThe effect of O2 therapy on post-ventricular fibrillation arrest and subsequent return of spontaneous circulation (ROSC) has also been reported.44 In a 28 patients, randomized to receive either 0.3 FiO2 or 1.0 FiO2, their serum samples collected at 24 and 48 hours were analysed for neuron specific enolase (NSE) and protein S-100, showed signs of neuronal injury. Although statistically there was no difference in the NSE and protein S-100 levels between the two groups, a higher NSE levels were reported in patients who received 1.0 FiO2 and did not underwent therapeutic hypothermia.44 There is some evidence suggesting that O2 may not improve outcomes after MI and even worsen the outcome, increasing the risk of neurological injury after cardiac arrest and return of spontaneous circulation (ROSC).\n\nd. Oxygen therapy after ischemic stroke\n\nThe role of hyperoxia in patients with a previous stroke has not been entirely and clearly elucidated. In acute ischemic stroke, the occluded blood vessels disrupt brain O2 delivery causing hypoxia/ischemia and a main infarction area, as well as another area called penumbra, surrounded by healthier tissues amenable of being rescued if cerebral perfusion and oxygenation are restored in a timely fashion. Besides the direct vascular mechanisms of vascular patency though, there are many others which have been discussed so far: those include ROS production and nutrients supply, as well as mechanisms related to the vascular integrity per se or “barriers” integrity, i.e. the blood brain barrier being one of them and discussed later in the paragraph Changes in the Neurovascular Unit after Ischemic Stroke.\n\nAnimal studies reported that an early administration of supplemental O2 after stroke may improve clinical outcomes10,45: however, hyperoxia may physiologically induce cerebral vasoconstriction, which is the other main topic of our narrative review.46 In a similar clinical scenario, the role of O2 and neuronal survival/remodeling has been questioned in other clinical scenarios such as neonatal asphyxia.47\n\n\nCerebral autoregulation\n\nThe neurovascular unit (NVU) includes multiple cell types (neurons, interneurons, astrocytes, smooth muscle cells, pericytes, endothelial cells) and extracellular matrix which are closely associated with cerebral vasculature and their interaction with each other to maintain the blood-brain barrier (BBB) and regulate CBF.48,49 A tight junction between endothelial cells of the blood vessels and BBB’s selective permeability control via NVU pericytes proteins is crucial. The NVU is also involved in vascular tone regulation and an increased in neuronal activity leading to increased CBF and thereby increased oxygenation.50\n\nCerebral autoregulation is also involved in vasoconstriction and vasodilation of cerebral arterioles via myogenic, neurogenic, and metabolic regulation (Figure 2).51,52 Myogenic regulation acts via smooth muscle cell contraction in the wall of blood vessels,53 is the key to maintaining CBF when MAP is outside the autoregulation range, whereas within the autoregulation range neurogenic regulation by the NVU plays a major role.52 As part of the NVU, neurons respond to decreased glucose and hypoxia by generating molecular signals, such as glutamate, to communicate with interneurons and astrocytes.54 Astrocytes on their part can alter vascular tone via prostaglandins, ATP, NO, and lactate.48,54\n\nCerebral blood flow is primarily affected by blood gas concentrations, including O2, CO2, and nitric oxide (NO).55–57 Hypoventilation causes an increase in arterial CO2 tension (hypercapnia) and leads to vasodilation resulting an increase in CBF, whereas inhalation of 100% O2 reduces CBF by 10–15%.58,59 In essence, CBF is directly influenced by blood oxygenation levels and vice versa. Under hypoxic conditions (or hypercapnia), autoregulation initiates activation of mechanisms leading to vasodilation, whereas hyperoxia (or hypocapnia) increases O2 bioavailability as CBF decreases due to vasoconstriction (Figure 2).10,11\n\nThe partial pressure of O2 (PaO2) and partial pressure of carbon dioxide (PaCO2) can also have a combined effect, depending on their respective partial pressures. Ogoh et al. studied the effect of O2 therapy on dynamic CA by exposing nine healthy volunteers to four respiratory interventions: normoxia (0.21 FiO2), isocapnic hyperoxia (0.4 FiO2), isocapnic hypoxia (0.14 FiO2), and hypocapnic hypoxia (0.14 FiO2).60 He used transcranial Doppler to measure middle cerebral artery blood velocity to determine CA. The CA was impaired with normocapnic hypoxia; however, it improved with mild hypocapnic hypoxia. He concluded that hypocapnia-caused vasoconstriction led to improved CA, thus outweighing the negative effect of hypoxia on CA.60\n\nIn the event of CPP reduction such as an acute ischemic stroke, a compensatory mechanism starts to preserve O2 and nutrient supply to the brain. Based on a human study (MRI was used to assess O2 extraction fraction (OEF) in patients with various degrees of middle cerebral artery (MCA) stenosis or acute stroke),61 it was found that the increase in OEF from baseline was higher in the severe MCA stenosis group compared to the mild stenosis group, where collateral circulation allows for some perfusion distal to the lesion. Patients with severe MCA stenosis have a greater reduction in CBF and little collateral circulation, necessitating other mechanisms such as improving OEF to increase brain oxygenation.61\n\nIn animal studies, occluding the MCA caused an increase in CBF and OEF one hour after occlusion; however, at 2–3 hours post-occlusion CBF and OEF had both decreased.62 These findings suggest that soon after MCA occlusion compensatory mechanisms attempt to maintain cerebral perfusion and oxygenation. However, at later stages of MCA occlusion these compensatory mechanisms, and presumably cerebral oxygenation, decrease.62\n\nTo summarize, to maintain O2 and nutrient delivery to the brain during acute stroke, CA causes arterioles to dilate, therefore increasing CBF. Once this compensatory mechanism has been used up, O2 extraction fraction (OEF) can increase significantly to keep O2 metabolism running, but at maximal OEF, continuing or deteriorating CBF reduction may eventually lead to cell death.62\n\nIschemic stroke and subsequent cerebral hypoxia can cause disruption of the NVU and damage to the BBB via the hypoxia-inducible factor 1 (HIF1) transcription factor.63 Additionally, HIF1a and cytokines such as TNF-alpha and IL-1B lead to the activation of matrix metalloproteinases (MMPs) which break down the BBB and increase its permeability.64 A decrease in ATP production and failure of enzymes to maintain normal ion gradients cause endothelial cell swelling and BBB dysfunction.65 Ischemic stroke leads to degradation of extracellular proteins, detachment of pericyte, astrocyte, and microglia activation.49 These changes in the BBB and the NVU allow for peripheral immune cells to enter the brain causing inflammatory stress.66 Loss of BBB integrity also leads to vasogenic edema and increases the risk of hemorrhagic transformation, which can worsen brain damage after stroke.67\n\n\nAnesthesia and autoregulation\n\nGeneral anesthesia (GA) can directly impact cerebral autoregulation, therefore potentially inducing a direct organ damage, in particular brain damage. Besides that, GA could also be cause of neurotoxicity due cell-toxicity, yet anesthesia may also be responsible for molecular mechanisms of neuroprotection therefore modulating neurotoxicity: these mechanisms are encompassed for instance in the same processes responsible for cell and organ protection after ischemic precondition and postconditioning.\n\nA further consideration pertains the use of O2 not simply for the preparation prior to general anesthesia in otherwise healthy subjects, yet in patients during or after cardiac arrest or ischemic stroke that require therapeutic use of O2, and in particular when they require procedures involving GA: at a time when CBF and oxygenation are compromised, GA may pose another threat to organs health or possibly be a reason of organ protection. Here we briefly review the available evidence around some of the potential effects of GA on brain tissue and CA.\n\nVolatile anesthetics can cause neurotoxicity, especially in neonatal rats and non-human primates, although the exact mechanism remains under investigation. They can lead to neuroapoptosis, neurodegeneration, and long-term neurocognitive deficits in animal models through N-methyl-D-aspartate (NMDA) receptor antagonism and GABA receptor activation.68 Ultimately, they can also create oxidative stress in the mitochondria, leading to ROS production which triggers a chain of events causing apoptosis,69 and contribute to neurotoxicity.69–71\n\nBoth in vitro and in vivo studies have shown evidence of propofol-induced cell death.72,73 The mechanism of propofol-induced neurotoxicity is thought to be mediated by multiple pathways including apoptosis, decreased neurogenesis, disruption of dendrite formation, neuroinflammation, Ca2+ signalling, microRNAs, and activation of p75 neurotrophic receptor.72,73\n\nVolatile anesthetics also, seem to play a role in organ protection through ischemic preconditioning and postconditioning, shown by cardiac animal studies a similar mechanism has been found in brain cells.74 Sevoflurane is involved in neuroprotection when administered at specific times before ischemia–reperfusion. Sevoflurane-induced energy preservation decreases both focal and global ischemia and improves outcomes.75 Additionally, sevoflurane and other volatiles inhibit glutamate receptors which are normally stimulated to cause cell injury during ischemia.75\n\nPropofol also induces neuroprotection through ischemic preconditioning and postconditioning via a different mechanism from volatile anesthetics.75 Propofol regulates cytochrome c, Cx43, UCP2, and mitochondrial DNA (mtDNA) transcription, which all play a role in neuroprotection after ischemia. It protects the integrity of the mitochondrial membrane during ischemia, preventing cytochrome c detachment and activation of the apoptosis pathway, thereby preventing neuronal cell death.75\n\nWhile the hemodynamic effects of commonly used anesthetics such as sevoflurane, propofol, and dexmedetomidine are well known, their effect on CA is less extensively studied. Usually the administration of sevoflurane at 1 minimal alveolar concentration (MAC), while it somewhat decreases MAP, overall it maintains CBF: CA is overall preserved as shown by Juhász et al. in 29 patients cohort who underwent GA.76 Using dexmedetomidine with sevoflurane and nitrous oxide anesthesia would also appear to not affect CA.77 McPherson et al. reported fentanyl has no known impact on CA.78\n\nGeneral anesthesia using propofol with target-controlled infusion also maintains CA and CO2 reactivity through a balance between vasodilation and vasoconstriction.73,79 CA was unchanged in patients undergoing GA with propofol-remifentanil infusion but decreased in those receiving high-dose sevoflurane. Additionally, higher CO2 levels did not affect CA in the propofol-remifentanil group, while the high-dose sevoflurane group experienced a further reduction in CA.80 These studies suggest that CA is maintained in patients receiving propofol at doses required for GA.\n\n\nCerebral autoregulation and imaging: a window in the pathophysiology of post-stroke disruption\n\nCurrently, CA can be assessed by raising BP by ~10 mmHg via a pharmaceutical agent while measuring intracranial pressure (ICP), or by continuous monitoring of brain tissue PO2 using an intraparenchymal probe: a decrease, or no change, in ICP indicates an intact CA, while an increase in ICP suggests compromised CA.81 Non-invasive techniques such as transcranial Doppler (TCD) and near-infrared spectroscopy (NIRS) can be used to assess cerebral reactivity, autoregulation, and neurovascular coupling; however, TCD failed to provide more global CBF, whereas NIRS is limited by the infrared penetration power in the deepest parts of the brain. Both methods have accuracy, sensitivity, and reproducibility issues, and lack a complete cerebral hemisphere coverage.82,83 Other methods, such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), and computed tomography (CT) can compute CBF, cerebral blood volume (CBV), mean transit time (MTT), and O2 extraction fraction (OEF); however, they require a radioactive tracer with a short half-life.84–86 Gas-challenge magnetic resonance imaging (MRI) is emerging as a viable option to probe CA by measuring the change in CBF, the cerebral metabolic rate of oxygen (CMRO2), and OEF.87–89\n\nInhalation of pure O2 or carbogen (a mixture of 3–5% CO2 and 95–97% O2) changes blood oxygenation in capillaries and veins by altering oxyhemoglobin versus deoxyhemoglobin content. This manifests as a change in T2-weighted MRI signal, which is the principal blood oxygenation level-dependent (BOLD) imaging contrast (see Figure 1, panel B as a hypothetical model of O2 inhalational effect assessed by MRI).90,91\n\nGas challenge images have been used to compute vessel size.92 Under physiological conditions, the arterial blood is 98% oxygenated resulting in a small difference in magnetic susceptibility between arteries and tissue. With O2 inhalation, an increase in FiO2 from 0.21 to 1.0 leads to a significant change in BOLD contrast. In ischemic stroke patients, Donahue et al. applied carbogen challenge MRI to evaluate cerebrovascular reserve capacity in patients with intracranial stenosis.93 Carbogen increases the fractional concentration of inspired CO2 (FiCO2) and NO-mediated vasodilation, which in turn increase MRI signal intensity. In contrast to O2, carbogen challenge MRI has been extensively applied to study several pathologies such as malignancy, hypoxia, Alzheimer’s, retinopathy, type 2 diabetes, hepatic fibrosis, and chronic kidney disease.94–97\n\nGas-challenging MRI is emerging as a non-invasive mainstream method to study neurophysiology. In a healthy brain, a hypercapnia-induced increase in CBF affects the CMRO2 and OEF. Several studies reported both CMRO2 and OEF are significantly altered in patients with stroke, tumor, and cerebrovascular degenerative disorder.98,99 Both hypercapnia and hyperoxia induce CBF changes leading to BOLD MRI signal by affecting venous blood deoxyhemoglobin concentration has also been applied to compute these two critcal functions.100–102\n\nA localized or global change in oxy- and deoxyhemoglobin concentrations in the brain can alter magnetic susceptibility and blood transverse relaxation time (T2 and T2* relaxation time); this has been recently applied to compute OEF. A change in blood gases can alter magnetic susceptibilities: these changes can be measured by quantitative mapping (QSM), an advanced MRI signal processing method that uses both the magnitude and phase component of a gradient echo MRI signal.\n\nA magnetic susceptibility difference between the veins and the surrounding tissue has been applied to estimate venous O2 saturation. Uwano et al. and Zaitsu et al. applied QSM to measure OEF and validated it with PET imaging, a gold standard to measure OEF.103,104 Previously, Zhang et al. used caffeine and hyperventilation as stimuli to alter susceptibility via a change in oxy- versus deoxyhemoglobin ratio and measured OEF and CMRO2 using QSM.105,106 However, because both caffeine and hyperventilation induce vasoconstriction, the CBF reduction results in a smaller magnetic susceptibility difference between the oxy- versus deoxyhemoglobin creating weaker MRI contrast.105,106 To obviate those limitations, Ma et al. recently applied a carbogen inhalation vasodilator challenge: it increased CBF and resulted in a larger magnetic susceptibility difference between the oxy- versus deoxyhemoglobin, creating stronger MRI contrast.107\n\nTo summarize, CA can be assessed by various methods, all of which have specific limitations. Emerging non-invasive imaging techniques relying on the gas challenge look promising for the study of cerebral pathophysiology.\n\n\nConclusions\n\nWhile exogenous O2 administration is still part of routine clinical management and therapeutic algorithms, it remains unclear at present whether this practice should continue indiscriminately, considering its potential toxic effects. Specifically, despite the clear dependence of the human brain on a constant O2 and nutrient supply, a compelling clinical and experimental body of evidence questions the utility of excess O2 in acute and post-recovery brain ischemia. Many of these findings have been the object of translational research, particularly in the field of neurophysiology imaging. It may be prudent and possibly best practice to limit exogenous O2 administration in the clinical setting to the shortest period required, and consider reducing inspired O2 fractions in patients more predisposed to CA disruption, such as post-stroke recovered patients.\n\n\nAuthors’ contributions\n\nPG: designed the work, contributed to literature search, collected data, interpreted data and wrote the manuscript\n\nNF: designed the work, contributed to literature search, collected data, interpreted data, wrote the manuscript\n\nDS: designed the work, literature search, collected data, interpreted data, edited the manuscript and figures\n\nMH: contributed to literature search, wrote the manuscript, interpreted data, edited the manuscript and figures\n\nDC: initiated this report, designed the work, collected data, interpreted data, edited the manuscript and figures\n\nAll authors read and approved the final manuscript.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nAcknowledgements\n\nThe authors acknowledge Ilija Ilic who graphically designed figures presented in the manuscript.\n\n\nReferences\n\nPatestas M, Gartner L: A Textbook of Neuroanatomy. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "165646", "date": "11 Apr 2023", "name": "Carlo Pancaro", "expertise": [ "Reviewer Expertise Neuroscience", "Obstetric Anesthesiology", "Cardiovascular Physiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 should be commended for the nice, well written review. Here are a few small suggestions:\nPage 3 – I would consider to adopt the Definition of CPP = MAP – ICP or CVP (whichever is highest).\n\nPage 5 - Without O2, the ETC would not have a final electron acceptor and the production of ATP would become much less efficient - Here I would briefly mentioned the Pasteur point where the cell metabolism switches from aerobic to anaerobic leading to lower ATP production. Oxidative metabolism of each glucose molecule results in production of 38 ATP. When the level of oxygen in mitochondria falls to around 1–2 mm Hg (Pasteur point), anaerobic metabolism of glucose results in synthesis of lactic acid with a consequent generation of 2 ATP per glucose only.\n\nPage 5 – Change HIPV to (HPIV).\n\nPage 5 - Intriguingly, while ROS mediates neuronal damage during the early phase of ischemia, in later phases it mediates vascular endothelial growth factor –You might want to add “synthesis” after endothelial growth factor.\n\nBeginning of Page 6 – Change “and thereby lead to” to “and thereby leads to” or “and thereby leading to”.\n\nPage 6 – Change “In a 28 patients” to “In 28 patients”.\n\nPage 6 – FiO2 1.0 - Since in this case 1 represents a fraction and there is no number greater than 1, I would eliminate the \".0\" – I would also do the same for the rest of the review.\n\nPage 6 – change “did not underwent” to “did not undergo”.\n\nPage 6 - (neurons, interneurons, astrocytes, smooth muscle cells, pericytes, endothelial cells) – I would add Microglia to this list.\n\nPage 7- HIF1a – I would spell the alpha here and write HIF1-alpha.\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": "9560", "date": "12 Apr 2023", "name": "Davide Cattano", "role": "Author Response F1000Research Advisory Board Member", "response": "Thank you Dr Pancaro for the valuable comments and suggested revisions. They will improve the reading and understanding of outlined information." } ] }, { "id": "165648", "date": "02 May 2023", "name": "Ivana Budic", "expertise": [ "Reviewer Expertise Preventive", "therapeutical and ethical approach to the preclinical and clinical research of genes and modulators of redox cell signalization in immune", "inflammatory and proliferatory cell response" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 submitted by Gullapalli et al. is a narrative review composed by a team of anesthesiologists, intensivists and neurobiologist, intended for broad audience. It is not an experts review per se, yet the authors organized the different paragraphs accessing several key reference works based on their own experience. A very interesting and novel perspective, is the graphical content, which adds an original aspect to the original approach. The authors started the review emphasizing a general cover on the topic and they search from PubMed of English language abstracts, using adequate searching terms, from 1975 to October 2021.\nThey extracted the essence of several good articles about the use of oxygen in various pathophysiological conditions as well as during anaesthesia, emphasizing the contradictory effects of its application, which justifies the title of the paper \"Tale of Two Cities\". The use of oxygen has been questioned indeed on the beneficial effects in different clinical scenarios as well as in controlled preclinical studies. The authors, even if briefly, address a clinical novel evaluation of the long-term vascular reactivity and cerebral autoregulation after stroke, while exposing patients to administration of oxygen.\nIs the narrative review too general, too long, or even dispersive? On a general knowledge standpoint and trying to offer a scholastic resource for non-experts it is not; it still offers some expert view points on critical topics, like neuroapoptosis and cellular mechanisms of injury as well as neuroimaging and coupling. It does build a case for what it is known and moving in a hypothetical model testing the safety of clinical practice.\nI had the chance to read Dr Pancaro notes about the manuscript and his peer-review of the paper is in line with my own assessment: I agree with the assessment that it is an interesting reading with only few minor changes to be completed. I congratulate the authors for a simple yet unique presentation, and the nice artworks.\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": "9634", "date": "03 May 2023", "name": "Davide Cattano", "role": "Author Response F1000Research Advisory Board Member", "response": "We are grateful to the reviewer for the kind comments and will proceed on changes as recommended." } ] } ]	1	https://f1000research.com/articles/12-246
https://f1000research.com/articles/11-1428/v1	05 Dec 22	{ "type": "Research Article", "title": "Moral judgement among university students in Ica: A view from the perspective of Lawrence Kohlberg", "authors": [ "Rosmery Sabina Pozo Enciso", "Oscar Arbieto Mamani", "Miguel Gerardo Mendoza Vargas", "Oscar Arbieto Mamani", "Miguel Gerardo Mendoza Vargas" ], "abstract": "Background: The aim of this study was to identify moral judgement at the preconventional level, the conventional level, and the post-conventional level in university students in the tenth semester in Ica, in 2022. Method: The research methodology was descriptive-observational, quantitative and cross-sectional. The population consisted of university students in the tenth semester and the sample consisted of 157 university students. A survey was used as a data collection tool and a questionnaire was used to measure the stages of moral judgement according to Lawrence Kholberg.\n\nResults: It was found that 12.75% of the study sample was in the instructional relativism stage, 23.10% were in interpersonal agreement, 35.76% were in social order and authority, 11.95% were in social contract and finally 3.80% were in universal ethical principles. Conclusion: It was concluded and identified that the stages of moral judgement among the study sample indicate that interpersonal agreement, social order and authority are the most developed among university students.", "keywords": [ "Moral judgement", "L. Kholberg", "university students", "moral development" ], "content": "Introduction\n\nWhen discussing moral judgement, it is essential to talk about “ethics”. This term refers to the fact that a person's behavior within society is regulated by rules. From childhood, human beings are immersed in socializing agents such as the family, the school, the environment, among others (Mendizabal et al., 2021). Thus, from an early age, children learn that there are both good and bad behaviors. All these opinions forged based on the different experiences and external agents about the moral problems they experience, and the arguments used in favor of these judgements constitute the development of the cognitive behavior of moral judgement (Noguera, 2018).\n\nAs they pass through stages such as adolescence, youth and adulthood, individuals form their own identity, and become consciously aware of assuming and respecting norms and values. Lawrence Kohlberg (1973) points out that the person will learn and move up in the process of moral judgement in accordance with cognitive development. That is, as the person passes through various stages, he or she will find himself or herself at a different moral level, which will modify his or her reasoning (Mendizabal et al., 2021).\n\nKohlberg (1969) considers that the functioning of moral judgement is organized in cognitive structures from which all types of moral reasoning derive, which is based on universal and abstract principles, therefore, moral judgement is an evaluation and justification of prescriptive values versus what is good and bad (Goenaga, Lopera & Villada, 2021).\n\nKohlberg organized the theory of moral development on the basis of three levels, pre-conventional, conventional and post-conventional. These are characterized by moral problems which, in turn, are divided into two stages representing the individual's criteria for behavior (Cruz Puerto, 2020). The first level, the preconventional level, indicates that people are not guided by society's norms but only by concrete consequences; at the conventional level, the person adheres to society's norms and strives to avoid being punished; finally, at the postconventional level, the person reasons based on ethical principles beyond society's norms (Sandoval, Villega & Vega, 2019).\n\nAt the beginning of university life, cognitive development is reinforced by teachers, who must contribute to the development of students' moral judgement as part of a comprehensive education (Mendizabal et al., 2021). Although programs already include ethics and moral issues in their curricula to guide them during their professional practice, the results are mostly imperceptible. Therefore, it is important that based on a diagnosis of the student's reasoning and moral development, the current educational purposes are evaluated and improved (Contreras et al., 2020).\n\nMoral development has been considered an essential objective in education systems, as it is a progressive and gradual process that requires a combination of academic training, practical skills, and moral competences to be developed (Ranjbar et al., 2017). This is vital as previous studies show that there is an “ethical erosion” in higher education (Reyes et al., 2021), therefore, educational commitment must be addressed in the long term, where a theme of values, democracy, commitment, and justice is instilled as an aspect of lifelong learning (Sandoval, Villega & Vega, 2019).\n\nWe must remember that educational institutions are an important partner in the moral development of individuals and society, facilitating change not only at the personal level, but also contributing to the community and society. Citizens must be educated in democratic values, where they exercise their rights in a responsible manner and fulfil their obligations in solidarity (Pedraza & Pérez, 2019).\n\nIn view of this scenario, it was proposed as a general objective to identify and describe the stages of moral judgment among university students in the tenth semester in the district of Ica, in 2022 in a way that allows to demonstrate and respond to the problem that mobilized the study, that is, how are the levels of moral judgment in university students in the tenth semester in Ica in 2022?\n\nFor which the following specific objectives were set out: to identify moral judgement at the preconventional level, the conventional level, and the post-conventional level in university students in the tenth semester in Ica, in 2022.\n\n\nMethods\n\nThe deductive research approach was used, because a general topic was analyzed and delimited to the particular and descriptive (Ñaupas et al., 2018).\n\nThe study design was quantitative, considering the use of numerical scales for the development of results, non-experimental-observational, because the study was not conducted in a laboratory and the researcher did not intervene in the variables, and cross-sectional (Hernández-Sampieri & Mendoza, 2018).\n\nThe population consisted of university students in the tenth cycle of the administrative sciences career from different universities in the district of Ica, Peru. The type of sampling was non-probabilistic, according to Hernández-Sampieri & Mendoza (2018), indicating that this is done to a “subgroup of the population in which the choice of the elements does not depend on probability but on the characteristics of the research” and in turn by convenience, which, according to Valderrama (2014), indicates that the types of sampling by convenience or intentional “is characterized by a deliberate effort to obtain representative samples through the inclusion in the sample of typical groups”.\n\nGiven the above in the population, a total study sample of 157 university students (men and women), who were in the tenth cycle of the administrative sciences career, was considered, this being a representative number within the study. The total use of data from the proposed sample was made, not discarding, segregating or excluding any particular data.\n\nThe technique used was a survey and the instrument was the questionnaire called “Defining Issues Test” (DIT) which was developed by Rest (1979) and then translated into Spanish by Pérez (1997) as “Cuestionario de Problemas Sociomorales”. Then, Palacios (2003) made a manual called “El uso informatizado del cuestionario de problemas sociomorales (DIT) del Rest” and it was validated by Huanccollucho (2017) with a Cronbach's Alpha reliability of 0.70. The questionnaire was based on the theory of Lawrence Kohlberg (1973), which proposes to examine the moral judgement of a person in each period throughout his or her life and how this may evolve. The full questionnaire can be found in the Extended data.\n\nKholberg proposes six stages of moral judgement divided into three different levels of development (Table 1).\n\nThe instrument is divided into six different stories (see Table 2), which are called “dilemmas”, where a short story on a specific topic is presented. Then, twelve items are posed, including questions and statements, per story.\n\nAt the end of each story, an importance chart is displayed (see Table 3), where the respondent must place the items that he/she considers most important, where 1 is the most important item, according to the student's criteria, and 4 is the item that generates the least importance for the person or the person he/she considers most important.\n\nTable 3 describes what was used to measure the level and development of moral judgement of each university respondent.\n\nThe questionnaire survey was carried out using the Google FormsTM platform, which was distributed to each student in the tenth semester of the administrative sciences course, including instructions for correct completion. All this was done virtually, due to the wave of COVID-19 infection in Peru. The duration of data collection was from June 7 to June 14, 2022.\n\nThe statistical software SPSS version 25 was used for data processing, where the reliability assessment (Cronbach's alpha) of the instrument applied was carried out, obtaining a value of 0.702, and the Microsoft Excel 2019 program was used for all the data processing carried out to create tables with the results found. Regarding the presentation of the results of the article, this was done in a descriptive manner, which allowed us to identify the prevalence of the indicators of the moral judgement variable in the study sample. The data processing was carried out from June 15 to June 30, 2022.\n\nThe project was approved by the president of the ethics committee of the Universidad Autónoma de Ica by means of certificate CO-001-16-2022/CE issued on 15 February 2022.\n\nThe participants in this study were students between the ages of 22 and 25, who were of legal age for the Peruvian state, and they did not need parental consent to complete the survey. Each of them received a consent form which disclosed all information related to the development of the questionnaire, which they were required to read and sign and then return to the researchers, meaning that written informed consent was obtained from all participants. The anonymity of their participation was also guaranteed and that the answers given would be used exclusively for research purposes, all in compliance with ethical guidelines.\n\n\nResults\n\nThe preconventional level of the study sample was analyzed, where a development of instrumental relativism moral judgement of 12.75% is evident in the university students in their tenth semester of the administrative sciences (see Table 4). This result is exclusive of the evaluation of developmental stage II, because the test does not evaluate stage I.\n\nWe analyzed the conventional level where we find a development of moral judgement of interpersonal concordance of 23.10% and social order and authority of 35.76% in the university students in their tenth semester of the administrative sciences corresponding to development stage III and IV respectively (see Table 5).\n\nThe post-conventional level was analyzed, showing a development of moral judgement of social contract of 11.95% and universal ethical principles of 3.80% in the university students in their tenth semester of the administrative sciences corresponding to developmental stage V and VI respectively, determining and reaching a post-conventional level of 15.75% among the study sample (see Table 6).\n\nTable 7 shows the A and M scores, which show the reliability of the data, and whether respondents have lied or had a bias when giving an answer. The scores should be less than 14%. If this is achieved, it means that the responses are truthful and do not need to be invalidated.\n\nIn reference to Figure 1, it is evident that the responses both A (6.90%) and M (5.70%) are less than 14%, so it takes as reliable all the responses collected by the students of the study site. Taking this into account, it is determined that the most predominant moral judgement among the students is the conventional one; being part of this the interpersonal agreement (23.10%) and social order and authority (35.76%): stage III and IV, indicating that the judgement goes with having good behaviors and behaving according to how others expect them to behave, also that they are governed by the regulations and laws determined and that they do not intend to infringe them intentionally, unless they are forced to break some law. Secondly, there is the post-conventional stage; being part of this the social contract (11.95%) and universal ethical principles (3.80%). This group has a judgement empathy, knowing and being aware that all people have different points of view, values, ways of life and so on. They are always being respectful of this, but not preventing that if they see or are in a situation in which they see that the basic and universal principles of a person are being affected; they will put these principles above anything else. And finally, there is the preconventional stage, being part of this the punishment-obedience (0.0%) and instrumental relativism (12.75%), this type of stage would indicate that people act only out of interest and when they know that they will get something beneficial for themselves and for the rest of the people involved, being fairness and justice their predominant ones.\n\n\nDiscussion and conclusion\n\nThe general objective was to identify and describe the stages of moral judgement in university students in the tenth semester in the district of Ica, 2022. Kohlberg (1992) indicates that this stage is based on the individual's sense of morality and is linked to the personal and social relationships that the individual presents. Jaime (2019) found that his study sample also had a prevalence of conventional moral reasoning, with 68.8%, even though the studies were carried out in different educational institutions, time (year of data collection) and academic level of the students; it is evident that the conventional type of judgement is the most deeply rooted within the student in the southern and central area of the Peruvian state.\n\nThe specific objectives were to identify moral judgement at the pre-conventional, conventional, and post-conventional levels in university students in the tenth semester in Ica. The results show a development of moral judgement in the preconventional stage, specifically, in the instrumental relativism stage with 12.75%. Kohlberg (1992) indicates that at this level moral sense and judgement is assumed in such a way that norms are accepted if they favor one's own interests. This is because the individual aims to do what satisfies his or her interests, considering it right that others also pursue theirs. The authority figures come from parents or teachers, and minors qualify their actions by their consequences. Regarding the second specific objective, a development of moral judgement of inter-personal agreement of 23.10% and social order and authority of 35.76% was identified, with a total of 58.86% of conventional level and finally, regarding the third specific objective, a development of moral judgement of social contract of 11.95% and universal ethical principles of 3.80% was identified, with a total post-conventional level of 15.75%. Kohlberg (1992) indicates that at this level the meaning of ethics is defined in terms of more abstract principles and values. The person believes that some regulations are unjust and could be changed or repealed. Huanccollucho (2017) concluded that in the preconventional level he obtained a total value of 11.6%, a total conventional level of 44.9% and a total post-conventional level of 21.6%, where it is evident that, as in the present study, the antecedent shows a higher prevalence in stages II and III (conventional).\n\nIn all the cases reviewed, it is common for the populations and samples studied to be situated at the conventional level. This means that nowadays young people have become aware of abiding by the common rules of coexistence as well as recognizing that all individuals have interests that may not necessarily be the same. It follows that fairness is relative, as it is linked to personal interests, and that an exchange with others is necessary to ensure that one's own interests are satisfied.\n\nIt is important that, in the creation of scenarios for moral development, the educational and teacher's responsibility involves creating a type of conflict that facilitates the development of models of thinking in their students, because, as Kohlberg indicates, models of thinking are not taught, whereas, on the contrary, moral reasoning is self-generated in the environmental exchange and changes progressively.\n\nThe main limitation is the collection of data in a single period and not longitudinally to be able to observe how the stages change according to the knowledge and maturity of each person. Therefore, it is suggested that when replicating the studies, they can be carried out under the type of research mentioned above and thus observe the evolution of moral judgement.\n\nIt was identified that the stages of moral judgement among the study sample indicate that interpersonal agreement (23.10%); and social order and authority (35.76%) are the most developed, instrumental relativism (12.75%) is the least developed and social contract (11.95%) and universal ethical principles (3.80%) are the stages that tend to develop in the future among university students in the tenth semester in the district of Ica.\n\nRegarding the specific objectives, it was possible to identify that the preconventional level was 12.75%, the conventional level was 58.86% and finally the post-conventional level was 15.75% among university students.", "appendix": "Data availability\n\nZenodo. Moral judgement in university students in Ica: a view from the perspective of L. Kohlberg Vr.4. https://zenodo.org/record/7199915#.Y0Nc-nZBy3A (Pozo Enciso et al., 2022).\n\nThis project contains the following underlying data:\n\n• SPSS – Moral judgement database.csv\n\n• SPSS – Moral judgement database.sav\n\n• MORAL JUDGEMENT DATA BASE.xlsx\n\n• MORAL JUDGEMENT DATA KEY.txt\n\n• MORAL JUDGEMENT PART 2.xlsx\n\nZenodo. Moral judgement in university students in Ica: a view from the perspective of L. Kohlberg Vr.4. https://zenodo.org/record/7199915#.Y0Nc-nZBy3A (Pozo Enciso et al., 2022).\n\nThis project contains the following extended data:\n\n• Figure 1 - Moral judgement stage profile among the sample: General\n\n• Social-moral problems questionnaire.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\nAlania Contreras R, Marquez Alvarez G, Quinte Rodriguez S, et al.: Razonamiento moral en estudiantes de Ciencias de la Comunicación de una universidad de Perú. Revista Iberoamericana de Educación. 2020; 3(4). Publisher Full Text\n\nCruz Puerto MS: Desarrollo moral: Tres comprensiones. Revista Iberoamericana De Psicología. 2020; 13(1): 95–103. Publisher Full Text\n\nGoenaga J, Lopera JD, Villada J: Análisis de la evidencia sobre el juicio y la toma de decisiones morales entre el 2005 y 2020. Avances en Psicología Latinoamericana. 2021; 39(3): 1–20. Publisher Full Text\n\nHernández-Sampieri R, Mendoza C: Metodología de la investigación. Las rutas cuantitativa, cualitativa y mixta. Grupo editorial Mc Graw Hill Education;2018.\n\nHuanccollucho N: Juicio moral en mujeres recluidas en el penal de Qenqoro de la ciudad de Cusco, 2017 [Tesis de Pregrado, Universidad Andina del Cusco].2017.Reference Source\n\nJaime D: Clima social familiar y razonamiento moral en estudiantes de nivel secundario en una institución educativa de Huancayo, 2019 [Tesis de Pregrado, Universidad Peruana los Andes].2019.Reference Source\n\nKohlberg L: Stage and sequence: The cognitive-developmental approach to socialization. Handbook of Socialization: Theory and Research.1969; 664–679.\n\nKohlberg L: Continuities in childhood and adult moral development revisited. Life-span Developmental Psychology. 1973; 179–204. Publisher Full Text\n\nKohlberg L: Psicología del desarrollo moral. Editorial Desclée de Brouwer;1992.\n\nNoguera M: Desarrollo moral y sociedad. Revista educación en valores. 2018; 29(1): 39–51.Reference Source\n\nÑaupas H, Valdivia M, Palacios J, Romero H: Metodología de la investigación cuantitativa – cualitativa y redacción de tesis. Ediciones de la U.2018.\n\nPalacios S: El uso informatizado del cuestionario de problemas sociomorales (DIT) del Rest.2003; 20: 5–15.Reference Source\n\nPedraza Chávez J, Pérez Ramírez J: Level of moral judgment in students of the medical career of a public university. Medicina Interna de México. 2019; 35(6): 885–895.Reference Source\n\nPérez E: Cognición y afecto en el desarrollo moral. Valencia, España:Promolibro;1997.\n\nPozo Enciso R, Arbieto Mamani O, et al.:Moral judgement in university students in Ica: a view from the perspective of L. Kohlberg. [Dataset]. Zenodo. 2022. Reference Source\n\nRanjbar H, Joolaee S, Vedadhir A, et al.: Becoming a nurse as a moral journey: A constructivist grounded theory. Nursing Ethics. 2017; 24(5): 583–597. Publisher Full Text\n\nRest J: Development in Judging Moral Issues. Minneapolis, Minnesota, USA:University of Minnesota Press;1979.\n\nReyes Ruiz L, Núñez Ariza A, Núñez Ordóñez A, et al.: Desarrollo moral en contextos educativos: Una revisión sistemática. European Journal of Child Development, Education and Psychopathology. 2021; 9(1): 1–32. Publisher Full Text\n\nRodrigo Mendizabal N, Cordero Altamirano M, Fernández Reyez C, et al.: Medición del Desarrollo Moral en los estudiantes de primer, quinto y séptimo semestre de 5 carreras de la Univalle, Sede La Paz – Bolivia, en la Gestión 1 – 2019: Una aplicación de la prueba Defining Issues Test (DIT), de Kolhberg y Test de Reacción Valor. Revista Compás Empresarial. 2021; 11(32): 120–142. Publisher Full Text\n\nSandoval M, Villegas M, Vega R: Desarrollo moral en los estudiantes mexicanos: Un análisis de la visión de justicia de la teoría de Kohlberg. JURÍDICAS CUC. 2019; 15(1): 69–95. Publisher Full Text\n\nValderrama S: Pasos para elaborar proyectos de investigación científica. Editorial San Marcos;2014." }	[ { "id": "159291", "date": "03 Feb 2023", "name": "Ebtsam Aly Abou Hashish", "expertise": [ "Reviewer Expertise education- research- management - leadership - ethics - quality of care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is clear and comprehensive. I need the authors to describe the target population and their number because the sample is considered a relatively small one. Explain in detail how you ensured the validity and dependability of tools. In addition, how did you maintain the human rights of your participants?\nYour discussion needs to be more direct, and you need to discuss each finding with related supportive or opposing prior studies. Add direct implication to improve students' moral judgment.\nYour references need to be updated, especially in the discussion section, to be within the last five years.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "165032", "date": "07 Mar 2023", "name": "Vered Ne’eman-Haviv", "expertise": [ "Reviewer Expertise morality", "drug use" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well written, clearly and concisely.\nThe main problem with the article is that its theoretical contribution and the rationale for its execution are not clear. What does the knowledge about the moral level of the students contribute to us? The study did not examine the correlation of the level of morality with other variables, but only presents descriptive information about their level of morality, and therefore its contribution to existing research knowledge is not clear.\nBeyond that, there is a lack of data about the subjects: how many men and how many women\", at what age? etc. There is no significant information about them.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "165585", "date": "24 Apr 2023", "name": "Ambrocio Teodoro Esteves Pairazamán", "expertise": [ "Reviewer Expertise Master's degree in Research and Teaching", "and a doctorate in Educational Administration." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe discussion of results should be more direct and concise. Above all, each result found should be discussed with previous studies related to the research topic, showing whether the results are similar or different, either in the same country where the study was conducted or in another part of the world. It is recommended to give a brief analysis of the findings. Preferably the studies intended for discussion should not be older than 5 years.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-1428
https://f1000research.com/articles/11-1429/v1	05 Dec 22	{ "type": "Research Article", "title": "A transit map for micro-scale urban development in Alexandria, Egypt", "authors": [ "Mohamed H. Seoudy", "Adel El Menshawy", "Amr El Adawy", "Mohamed H. Seoudy", "Adel El Menshawy" ], "abstract": "Background: Due to Egypt's strategic location among countries, transportation is one of the most significant development sectors because it plays a major part in today's economy and society and has a large influence on growth and employment. Over the years, the Egyptian General Organisation of Physical Planning (GOPP) has prepared strategic general urban plans in collaboration with local and foreign organisations, including transportation plans. The constant focus of authorities on strategic plans and their inability to implement them on schedule are a major issue. In other words, they always take development from a distant perspective and do not deal with the main problem that exists within cities, as the existing micro-scale transit built environments (MSTBEs) of cities are not ready due to a lack of transit-oriented communities (TOCs), sustainably developed transit supply systems, and mobility hubs. Methods: The \"Enhanced MSTBE Phases\" methodology is used for the key elements of the study design used in this research, depending on data collection, approvals, techniques, and analysis methods. As a case study, these key elements are in the documentation, analysis, and development of the Muharram Bek El Mowkaf El Gedid Mobility Hub (MBMH) and the 800 m radius around it. Results: The results indicate that Enhanced MSTBE Phases led to the establishment of the MBMH and the 800 m radius surrounding it as a sustainable MSTBE in Alexandria, Egypt, which is chosen as the case study. Conclusions: The development of this MSTBE is a catalyst for future effects that will have a long-term impact on meso-scale and, ultimately, macro-scale transit built environments.", "keywords": [ "Micro-Scale", "Transport", "Built Environment", "Transit-Oriented Community", "Transport Supply System", "Sustainable Development", "Mobility Hub" ], "content": "Introduction\n\nA micro-scale transit built environment (MSTBE) refers to neighbourhood-specific urban design that integrates relevant built environment and transportation indicators. It is the smallest scale since it deals with internal trip capture, relative friction, and the pedestrian environment.1 The authors split the sustainable MSTBE into three scopes: transit-oriented communities (TOCs), sustainably developed transit supply systems, and the mobility hubs (including their zones), all of which are supported by transit frameworks, strategies, solutions, and guidelines (see Figure 1-1).\n\nTOCs are walkable, compact, and well-connected neighbourhoods. These neighbourhoods are intended to encourage active transportation by focusing on high-density, mixed-use, and pedestrian-friendly development within the walking distance of frequent transit and utilising mobility management strategies to reduce unnecessary driving. TOCs must be regarded as part of the government's attempt to establish sustainable and innovative means of transportation. The TOC approach aims to aid in developing communities based on transit stations to increase ridership, alleviate traffic congestion, and enhance housing and job opportunities. All of this contributes to the development of entire communities based on solid urban planning and design guidelines. Collaborating to create TOCs has the following primary advantages: the building of complete communities, added value, the creation of a mix of uses, a vibrant public realm, and prominence.2–4\n\nTransit infrastructure and transit services are components of a sustainably developed transit supply system. Transit infrastructure consists of the basic facilities, structures, equipment, technologies, and services that support economic activity and quality of life. It promotes local and regional development by facilitating the flow of goods, connecting production centres with markets, and facilitating people's movements by providing access to work, social opportunities, health and educational facilities, and other services. At the same time, transit services encompass all services (sea, air, land, inland waterways, surveying, and pipelines) that involve the movement of people and products (freight), the rental of carriers with a crew, and related support and auxiliary services – the type and quality of transit services in a neighbourhood influence the establishment of a TOC. Transit service types can be selected based on speed reliability, regardless of infrastructure, and local access attributes are primarily determined by the right-of-way (ROW) type and station or stop spacing.\n\nA mobility hub is a multimodal transportation centre that includes major transit stations and their surrounding areas (approximately 10 min/800 m radius), that connect transit, active transportation, and car commutes and that increase the use of shared modes with an emphasis on employment, living, shopping, and/or recreation. This type of hub is frequently seen as a location where new transportation technology and services can be integrated and utilised to improve user experience and increase transportation alternatives for first- and last-mile travel. As the origin, destination, or transfer point for many trips, a mobility hub plays a vital role in the regional transit system.5–7 When planning the process and understanding the needs and potential in each area, it is common practice to split a mobility hub into zones. The four zones are the primary zone, secondary zone, tertiary zone, and catchment area (see Figure 1-2).8\n\n\nResearch problem\n\nThere have recently been statements that Alexandria's ongoing and future transportation projects will be implemented, such as the high-speed train El Ain El Sokhna-Marsa Matrouh, the Alexandria Metro (Abu Qir-Misr Station), Raml Tram Rehabilitation, and the Establishment of Central Stations on the Express Train Track. These statements are on the official website of the Egyptian National Authority for Tunnels.9–12 From the perspective of the authors, accepting these projects is unfeasible because the existing MSTBEs all over Alexandria are unsustainable owing to their lack of scopes: TOCs, sustainably developed transit supply systems, and mobility hubs. Even if there are one or two scopes, the MSTBE will be incomplete.\n\nTOCs are walkable, compact, and well-connected neighbourhoods that are intended to encourage active transportation by focusing on high-density, mixed-use, and pedestrian-friendly development within the walking distance of frequent transit and utilising mobility management strategies to reduce unnecessary driving. Sustainably developed transit supply systems include transit infrastructure and transit services. Mobility hubs are composed of major transit stations and their surrounding areas, and they play an important role in the regional transportation system by serving as the origin, destination, or transfer point for a considerable share of trips. There are no sustainable guidelines to guide the urban design of qualified transit stations and their surroundings, which results in passenger discomfort. Passengers encounter problems in direct and indirect ways, and therefore, passengers are averse to using public transportation and are forced to use private cars. Lastly, each mode of public transportation operates independently, without integration with other transit systems, and does not facilitate passengers' movement from the start to the end of their trips.\n\n\nStudy aim\n\nThis study aimed to establish a sustainable urban design for MSTBE by gathering, studying data and information, and capturing image survey data on the current state of the case (see Data Availability13). It used SWOT analysis to identify and comprehend key issues impacting the urban design of mobility hub zones. The interaction and balance between transportation, land use, and place-making functions were used throughout to meet the aims of the study, including the following:\n\n• Establishing TOCs, which, by design, encourage people to drive less, walk, cycle, and cross more, with a concentration on high-density mixed-use development on a human scale around frequent transit stops and stations.\n\n• Developing an environmentally friendly transportation supply system.\n\n• Creating a mobility hub to merge several forms of transportation into a single focal point.\n\n\nMethods\n\nOur research paper has no relevance to studies involving humans (individuals, human data, or material) or human participants, including personal genomics studies or clinical trials. Therefore, the authors are not concerned with the Helsinki Declaration. Any individual or attendant from any of the institutions visited during this research does not necessitate any protocol or consent from the individual for the use and publication of data in Egypt. Ethical approval must be obtained only when information is not open access.\n\nThe “Enhanced MSTBE Phases” methodology was created by the authors to establish a sustainable MSTBE, including documentation, analysis, and development. This methodology was the one that was used for the key elements of the study design used in this research, depending on data collection.\n\nApproval requests: Formal requests were made for information and approvals to the transport institutions to obtain approval to use and share the data and not to face any objections throughout the research operation.\n\nTechniques: The techniques involved looking for documents, maps, manuscripts, and references related to transportation in Alexandria and photographing the case study site by using a digital single-lens reflex (DSLR) camera with a professional camera tripod, a padcaster tripod dolly wheel, and a smartphone with a DJI Osmo Mobile 3 Gimbal for the smartphone.\n\nAnalysis Method: SWOT analysis was used as a tool to determine the strengths, weaknesses, opportunities, and threats of the case study site.\n\nThe study focused on the MSTBE in Alexandria and highlighted the Muharram Bek El Mowkaf El Gedid Mobility Hub (MBMH) and the 800 m radius surrounding it as the case study. The authors established a proposal for the MBMH and the 800 m radius surrounding it. This study used the “Enhanced MSTBE Phases” methodology, which was created by the authors, to establish a sustainable MSTBE, including documentation, analysis, and development.\n\nData collection depended completely on the documentation phase, which was the first phase where data and information were collected. From this phase, the authors were able to complete the next phases. The first phase was documentation, which included researching and gathering information and data on the current state of the case. This phase was divided into four aspects, including visits, photography, drawings, and TV shows. The second phase included conducting an overall analysis of the data collected in the first phase, utilising SWOT analysis. Depending on the two prior phases, the third phase came with a proposal for the case study.\n\nAs mentioned earlier, this phase was divided into four aspects:\n\nVisiting relevant transport institutions: Visits were made to the General Authority for Passenger Transport in Alexandria, Public Authority for Planning Transport Projects, National Railway Authority of Egypt, Ministry of Transportation, and Directorate of Housing and Utilities–Alexandria. Appointments were made:\n\n• To view documents, maps, manuscripts, and references related to transportation in Alexandria, such as the Strategic General Urban Plans, which were prepared by the Egyptian General Organisation of Physical Planning (GOPP) in collaboration with other authorised accredited local and foreign organisations: the Maclean Plan 1921 and General Plan (GP) 1959, GP 2005, GP 2017, GP 2025, GP 2032, and GP 2050 (the authors have collected the plans into two figures; see Data Availability,13 “Strategic General Urban Plans (1).jpg” and “Strategic General Urban Plans (2).jpg”).\n\n• To have some available public notes for the transit projects in Alexandria that may be helpful in the study, such as titles of ongoing projects and whether they are on schedule i.e., the high-speed train El Ain El Sokhna-Marsa Matrouh, the Alexandria Metro (Abu Qir-Misr Station), Raml Tram Rehabilitation, and the Establishment of Central Stations on the Express Train Track, as the Egyptian National Authority for Tunnels announced about them; the Alexandria urban transport study is financed by the neighbourhood investment facility from the European Union and managed by the French Development Agency (Agence Française De Développement); the Egyptian Government commissioned EGIS rail, a French advisory office, to prepare a long-term scenario for the Alexandria governorate strategic plan for urban transportation consistent with urban planning for the city; and a partnership between SYSTRA, AECOM, Orascom Construction SAE, The Arab Contractors, Siemens Mobility, and other companies with the Egyptian ministries to consult, design, install, commission, and maintain the systems for the projects.\n\n• To facilitate the operation of capturing image survey data on the current state needed for the case study.\n\nPrior to the visits, the authors submitted “Formal Requests for Information and Approval”, signed by the Vice Dean for Graduate Studies and Research, Prof. Sahar Mahmoud Al-Arnaouti, and stamped by the Faculty of Fine Arts, to the transport institutions and obtained approval to use and share the data.\n\nFor the copies and translation of the formal requests for information and approval, see Data Availability13.\n\nCapturing image survey data: The authors used the following photography and panoramic photography tools to acquire image survey data: (1) a DSLR camera with a professional camera tripod, (2) a padcaster tripod dolly wheel, and (3) a smartphone with a DJI Osmo Mobile 3 Gimbal for the smartphone to capture image survey data on the current state of the case study (see Data Availability,13 “00 Capturing Image Survey Data Tools.jpg”, “01 Primary Zone.jpg”, “02 Secondary Zone.jpg”, and “03 Tertiary Zone.jpg”).\n\nOverlaying, tracing, drawing, and presenting: The authors used the maps obtained from the institutional archives (the authors have collected the plans into two figures; see Data Availability,13 “Strategic General Urban Plans (1).jpg” and “Strategic General Urban Plans (2).jpg”) and the exported images from Google Earth Pro (see the software availability statement for alternatives) to draw the new Alexandria Transit Map by overlaying and tracing with Autodesk AutoCAD (see the software availability statement for alternatives). Then, the authors cropped the case study zone from it and presented the case study zone using Adobe Photoshop (see the software availability statement for alternatives).\n\nTranscribing announcement on TV shows: On the “Al Hekaya” TV talk show, presenter Amr Adeeb and Kamel El-Wazir, the minister of transport of Egypt, announced official transportation news about the transit developments in Egypt and Alexandria (see Data Availability,13 “Official Transportation News through the Media.pdf” provides descriptions of the news).\n\nAfter conducting comprehensive research and data collection, the authors selected the MBMH as the case study zone from among 23 mobility hubs indicated on the new Alexandria Transit Map. The case study zone is located in Alexandria's core area and includes the 800 m radius surrounding the Muharram Bek El Mowkaf El Gedid Bus Terminal. Before the 1952 revolution, the Muharram Bek neighbourhood was considered an elite neighbourhood, with mansions and villas dominating the area. In accordance with GP 2032, an electric high-speed rail terminal will be constructed in front of the Muharram Bek El Mowkaf El Gedid Bus Terminal. Table 1 presents the current profile for the case study zone.\n\n• International Coastal Road\n\n• Alexandria Governorate Bus Route\n\n• Internal Governorate Railway Line\n\n• Martyr Soldier/Muhammad Reda Muhammad Ahmad Bridge\n\n• Stations/stops: Muharram Bek El Mowkaf El Gedid Bus Terminal and Muharram Bek Train Station\n\n• Control systems\n\n• Support\n\n• Guidance\n\n• Propulsion\n\n• Control\n\n• Regional and national buses\n\n• Alexandria Governorate buses\n\n• Private cars\n\n• Taxis\n\nThe Muharram Bek El Mowkaf El Gedid Bus Terminal is the new bus terminal for regional and national bus connections located in Alexandria's Muharram Bek area. Construction of it began in 2001 and was completed in 2003. It is operated by the National Company for Road Construction and Development and serves as the primary departure and arrival point for passengers travelling by land to and from Alexandria. It was considered an alternative to the old bus stop at the Sidi Gaber Train Station before all buses and taxis for the governorates were relocated to the present location. Public transportation runs across the case study zone (see Figure 2). However, there is no integration between two or more public transportation modes. Arriving passengers could use the train, but since the current Muharram Bek railway station is more than 1 kilometre distant, they cannot. An extra stop along the new suburban train line would be beneficial, and getting to the Misr Station Train Station is also difficult. Although the Misr Station is approximately 2 kilometres distant, the railway line prevents pedestrian access to the city centre.\n\nThis figure is the authors’ own work.\n\nThere is currently no mobility hub in the case study. However, the authors created a radius walking distance map to identify and analyse the mobility hub zones (see Figure 3-1). The following table (Table 2) shows the SWOT analysis for each mobility hub zone (see Figure 3-2). SWOT analysis is used to investigate the current and initial conditions of the planned hub site and its surroundings. Analyse existing transport networks, including street connections, cycling, pedestrian infrastructure, and public transport. This review included land use, urban form, and neighbourhood character to fully capture the context of the site. Redevelopment opportunities on and around the site were analysed to understand the potential of mobility hubs to support transit-oriented development. Finally, the site's constraints and opportunities are discussed to determine which elements of the mobility hub are best suited to the site's current issues and build on its strengths. In addition, the view of documents, maps, manuscripts, and references related to transportation in Alexandria, notes taken from visiting relevant transport institutions, and El-Wazir's explanation of official statements from transcribing announcements on TV shows helped the authors build the outline description of the current situation for the SWOT analysis.\n\nThis figure is the authors’ own work. (2) Mobility hub zones – case study zone (present). This figure is the authors’ own work.\n\n• There are lighting units along the road\n\n• Traffic lights control the vehicle and pedestrian passage beside the bus terminal in both directions\n\n• There are street signs\n\n• There are taxicabs\n\n• The Muharram Bek El Mowkaf El Gedid Bus Terminal has an administration building, covered seating areas, bathrooms, and a ticket office\n\n• The bus terminal and its platforms are generally in good condition and clean\n\n• There are paid parking spaces and commercial shops inside the bus terminal\n\n• The Muharram Bek Train Station contains a covered seating area and an administrative building with a manager's office, a bathroom, a computer desk, and a ticket office\n\n• The train station and its platforms are generally in good condition and clean\n\n• There is a parking district – West Alexandria\n\n• There is a car dealership (show and sell)\n\n• There are government buildings and institutions\n\n• There is a pedestrian connection, i.e., the Martyr Soldier/Muhammad Reda Muhammad Ahmad Bridge\n\n• Residential buildings with heights ranging from 4 to 12 floors\n\n• There are development areas; each of them includes a mosque, an event centre, playgrounds, a wedding hall, a swimming pool, and a commercial centre\n\n• There is sporting club\n\n• There are government buildings and institutions\n\n• Entering the Muharram Bek Train Station by an improper entry; on the other hand, the main gate is sealed\n\n• The Muharram Bek El Mowkaf El Gedid Bus Terminal is not visible coming from the El Kabary Express Bridge\n\n• Private vehicles and taxicabs are parking against the bus terminal's wall, causing traffic congestion\n\n• There are no stable taxi stands, which causes traffic problems\n\n• The minibuses are causing traffic congestion at traffic lights by stopping illegally\n\n• No bus lanes\n\n• No safe access to bus stops, which must be provided through sidewalks and proper street crossing sites\n\n• At bus stops, there are no agency logos or visual markers, station names, route maps, or schedules, all of which should be displayed to riders\n\n• The presence of three-wheel tuk-tuks causes traffic problems\n\n• There is no pedestrian infrastructure that ensures pedestrian safety\n\n• There are no crosswalks where the roads would be too unsafe to cross without assistance due to vehicle numbers, vehicle speed, or road widths\n\n• No cycling facilities\n\n• No cycle tracks\n\n• Cyclists cannot ride safely in mixed traffic\n\n• No shared modes, either car or bike\n\n• No alternate parking spaces with trees or rain gardens\n\n• There are no open spaces\n\n• There is no balance in the distribution of services depending on the building types on the site\n\n• There are street vendors in the surroundings\n\n• The lighting units along the road do not usually work at night\n\n• No vehicular infrastructure serves the Muharram Bek Train Station on both sides of the train station\n\n• There is no pedestrian infrastructure that ensures pedestrian safety\n\n• There are no crosswalks where the roads would be too unsafe to cross without assistance due to vehicle numbers, vehicle speed, or road widths\n\n• No cycling facilities\n\n• No cycle tracks\n\n• Cyclists cannot ride safely in mixed traffic\n\n• No shared modes, either car or bike\n\n• No alternate parking spaces with trees or rain gardens\n\n• There are no open spaces\n\n• There is no balance in the distribution of services depending on the building types on the site\n\n• Building heights are inconsistent with street widths, and there are various heights on both sides of the Mahmoudia Canal Road\n\n• There is no pedestrian infrastructure that ensures pedestrian safety\n\n• There are no crosswalks where the roads would be too unsafe to cross without assistance due to vehicle numbers, speed, or road widths\n\n• At intersections, there are no raised crosswalks, which act as speed calming measures and prioritise pedestrians\n\n• No alternate curb extensions or rain gardens with parking spaces to create pinch points on the streets, which help in speed reduction\n\n• No curb extensions to locate street trees, light poles, cycle racks, or other street furniture\n\n• No cycling facilities\n\n• No cycle tracks\n\n• Cyclists cannot ride safely in mixed traffic\n\n• No shared modes, either car or bike\n\n• No alternate parking spaces with trees or rain gardens\n\n• There are no open spaces\n\n• There is no balance in the distribution of services depending on the building types on the site\n\n• The Alexandria High-Speed Electric Rail Terminal will be built on the vacant site in front of the Muharram Bek El Mowkaf El Gedid Bus Terminal; a sign confirms the existence of the Egypt Electric High-Speed Rail Project. The Egyptian Ministry of Transport owns the project, which is supervised by the General Authority for Roads, Bridges, and Land Transport; the general consultant for the project will be SYSTRA, and CASA Construction will handle the contracting; this project will cause a qualitative shift in the realm of transportation, affecting the MSTBE\n\n• There are development services in front of the bus terminal, i.e., petrol and gas stations with commercial amenities located in front of the bus terminal\n\n• The vacant site on which the Alexandria High-Speed Electric Rail Terminal will be established is far too large to be managed alone; the authors expect that further projects will be planned on this land to serve and support the terminal\n\n• There are development services, i.e., petrol and gas stations and a car service centre\n\nFrom the authors' viewpoint, the MBMH will become a mixed-use attraction within the city. The land uses on both sides of the transit corridors will create a distinctive mobility hub location with appealing streetscapes, reinforce the built form and open space transitions to new residential uses. The neighbourhood's developed form will be mainly mid-rise. It will be supported by a mixed-use development optimising connections and views. The authors utilised the prior studies to export a proposal map to create a rich MSTBE. The authors worked on developing certain multi-dimensional aspects, such as land use, the built form, and open space and circulation, so that they could offer a proposal, as presented in the table below (Table 3). Following the table (Table 4), the authors created the proposal profile for the MBMH.\n\n• Concentrate mixed-use development along major transit corridors\n\n• Emphasise mixed-use infill on unoccupied and unused lots\n\n• When employment objectives are met, explore additional applications to create a vibrant hub\n\n• Investigate partnership opportunities for large-format commercial usage\n\n• Concentrate the highest density near transit services\n\n• Provide proper transitions to nearby stable residential neighbourhoods\n\n• Provide necessary services while keeping residential building heights and open areas in mind\n\n• New developments should support improved transit services\n\n• Maintain visible and physical connections to the station while new development occurs\n\n• Provide mid-rise buildings to concentrate the maximum height and density near the rail corridor\n\n• Integrate station amenities on both sides of the transit corridor to provide a walkable environment with direct, weather-protected access\n\n• Consolidate access and servicing to new development\n\n• Implement living streets on the roadways surrounding the transit corridors\n\n• Design buildings on large development blocks to frame outdoor areas (parks, courtyards, gardens, parklets) to give views of the station and to enable continuous access between sites\n\n• Create new open spaces within significant developments\n\n• Create new cycling facilities\n\n• Encourage pedestrian-friendly street design on the roadways near the transit corridors\n\n• Make safe and direct connections across the transit corridors\n\n• ADA accessible design\n\n• Pedestrian access\n\n• Bicycle access\n\n• Parking/park & rides\n\n• Passenger loading zones\n\n• Car-share access\n\n• Bike-share access\n\n• Micro-mobility access\n\n• Real-time transit information\n\n• Integrated trip planning\n\n• Integrated fare payment\n\n• Wayfinding\n\n• Shelters\n\n• Benches\n\n• Lighting\n\n• Services & retail\n\n• Hub placement\n\n• International Coastal Road\n\n• Alexandria Governorate Bus Route\n\n• Internal Governorate Railway Line\n\n• Mahmoudia BRT Route\n\n• Mina El Basal – Muharram Bek BRT Route\n\n• Egypt Electric High-Speed Rail Line\n\n• Walkways/pedestrian connections\n\n• Bridges supported by escalators and elevators\n\n• Stations/stops: Muharram Bek Railway Station (after relocation), Bowalino BRT Stop, Alexandria High-Speed Rail Station BRT Stop, Muharram Bek El Mowkaf El Gedid BRT Stop, Alexandria High-Speed Rail Terminal, Muharram Bek El Mowkaf El Gedid Bus Terminal and Alexandria Governorate Bus Stops\n\n• Control systems\n\n• Support\n\n• Guidance\n\n• Propulsion\n\n• Control\n\n• Intelligent transit systems\n\n• Transportation demand management\n\n• Cycling infrastructure\n\n• Living streets\n\n• Regional and national buses\n\n• Alexandria Governorate buses\n\n• Private cars\n\n• Taxis\n\n• Internal Governorate Railway\n\n• Mahmoudia BRT\n\n• Mina El Basal – Muharram Bek BRT\n\n• Egypt Electric High-Speed Rail\n\n• Pedestrian connections\n\n• Car club bay – electric and conventional\n\n• Bike-share – electric and conventional\n\n• Large-scale cycle parking\n\n• Digital pillar (transport info, ticketing, wayfinding, walking distances, local services)\n\n• EV charging bays\n\n• Covered waiting area\n\n• Improved public realm, safer crossing, road/pavement repairs\n\n• Parklet/community art\n\n• Kiosk for refreshments\n\nThe authors dispersed land uses throughout the case study based on the demands of each mobility hub zone (see Figure 4). In the primary zone, the authors focused on utilising this zone on the MBMH and its amenities on both sides of the transit corridor to offer a walkable environment with direct, weather-protected access. Secondary zone utilisation is mostly mixed-use development along major transit corridors with enhanced transit services and visible and physical links to the mobility hub. Finally, mid-rise buildings are primarily clustered around transit services in the tertiary zone, with the greatest height and density along the railroad track. The needed services are provided within and around the buildings, considering the heights of residential buildings and open spaces. Implementing living streets by installing cycle infrastructure and creating pedestrian-friendly streets along transit corridors and within residential buildings creates safe and direct connections across the transit corridors.\n\nThis figure is the authors’ own work.\n\n\nResults and discussion\n\nThe authors estimated the exact percentages of the land uses for each mobility hub zone in the case study at the current and proposed states (see Figure 5) and compared them to ensure that the balance distribution is suitable and balanced in the table below (Table 5).\n\nThis figure is the authors’ own work. (2) Land uses map – case study zone (proposal). This figure is the authors’ own work.\n\nThe proposal includes the following:\n\n• The new development will provide a direct pedestrian connection to the MBMH.\n\n• The Muharram Bek neighbourhood will be framed by mid-rise construction, providing a dynamic cityscape that transitions to stable residential sections. Mid-rise buildings will provide new areas to live, work, and shop with easy access to public transportation.\n\n• An enhanced streetscape and bike lanes will be installed along with the transit modes.\n\n• Neighbouring residential communities will be protected and enhanced.\n\n• The Muharram Bek El Mowkaf El Gedid Bus Terminal will be integrated with the Muharram Bek Railway Station (after relocation), the Alexandria High-Speed Rail Terminal, and a compact urban street network that includes the Bowalino BRT Stop, Alexandria High-Speed Rail Station BRT Stop, Muharram Bek El Mowkaf El Gedid BRT Stop, and Alexandria Governorate Bus Stops.\n\n• Public and semi-public open spaces will be included in the new development.\n\n• The new mixed-use development will seamlessly link with the multimodal transit hub, creating new housing and employment opportunities.\n\n• In conjunction with new development opportunities, a new bicycle and pedestrian-friendly street character for Muharram Bek will be established.\n\n• The proposed streetscape upgrades in the Muharram Bek neighbourhood will connect all streets with new development.\n\n\nConclusions\n\nTransportation has changed dramatically in the last decade. Demographic changes, increased urbanisation, and changes in employment types and arrangements have put additional demand on existing transportation and transit networks. Alexandria must be ready to accept ongoing/future transportation developments that will complement local transit. This study was concerned with employing new concepts to address community concerns and providing a proposal to optimise the present aspects of the topic under investigation with appropriate adjustments and enhancements.\n\nThe case study is documented, analysed, and developed via the implementation of the Enhanced MSTBE Phases. The Enhanced MSTBE Phases methodology facilitates urban design research by developing MSTBEs to be sustainable based on researching, gathering data and information, and capturing image survey data on the current state of the case study. The SWOT analysis of mobility hub zones provides a platform for analysing internal potential and weaknesses as well as future external opportunities and threats. It considers all positive and negative factors that influence success within and outside the case study. Analysis of the MSTBE in which the authors work assists in anticipating changing trends and applying them to long-term sustainable decision-making.\n\nThis research uses the MBMH as a case study as a starting point for applying Enhanced MSTBE Phases in Alexandria, Egypt. The Enhanced MSTBE Phases methodology develops the case study zone to be a sustainable MSTBE with multi-dimensional aspects such as land use, the built form, open space, and circulation within the 800 m radius of the Muharram Bek El Mowkaf El Gedid Bus Terminal. As a result, a long-term sustainable MSTBE acts as a catalyst for future implications influencing meso-scale and macro-scale transit built environments.\n\n\nSoftware availability\n\n\n\n• Google Earth Pro free alternative: OpenStreetMap.\n\n• Autodesk AutoCAD free alternatives: FreeCAD and LibreCAD (2D).\n\n• Adobe Photoshop free alternatives: GIMP and Krita.", "appendix": "Data availability\n\nMendeley: Comprehensive Database for Mobility Hub Zones, 800m radius surrounding MBBT, Alexandria, Egypt. https://data.mendeley.com/datasets/k8hybkvsb3/5. 13\n\nThis project contains the following underlying data:\n\n• 01 Primary Zone.jpg (A figure shows image survey data in the primary zone= 2.5 minutes walk, 250m. The map and the photos are acquired from the authors’ work).\n\n• 02 Secondary Zone.jpg (A figure shows image survey data in the secondary zone= 5 minutes walk, 500m. The map and the photos are acquired from the authors’ work).\n\n• 03 Tertiary Zone.jpg (A figure shows image survey data in the tertiary zone= 10 minutes walk, 800m. The map and the photos are acquired from the authors’ work).\n\n• Official Transportation News through the Media.pdf (A PDF file provides descriptions for official transportation news through the media).\n\n• Strategic General Urban Plans (1).jpg:\n\n‐ Map 1921: This figure has been reproduced from public domain available content from New York Public Library [URL link: https://digitalcollections.nypl.org/collections/city-of-alexandria-town-planning-scheme#/?tab=about].\n\n‐ Map 1958: This figure has been reproduced from public domain content available from Wikimedia Commons [Licence: US Army Corps of Engineers, 1959 map of Alexandria, Egyptian Region United Arab Republic, CC0 1.0, URL link: https://commons.wikimedia.org/wiki/File:1959_map_of_Alexandria,_Egyptian_Region_United_Arab_Republic.jpg].\n\n‐ Map 2005: This figure has been reproduced and publicly shared with the permission from City’s Municipal of Alexandria.\n\n‐ Map 2017: This figure has been reproduced and publicly shared with the permission from General Authority for Urban Planning.\n\n‐ Map 2025: This figure has been reproduced and publicly shared with the permission from Advisory organisation for implementation of comprehensive planning for Alexandria.\n\n‐ Map 2032: This figure has been reproduced and publicly shared with the permission from General Authority for Urban Planning.\n\n• Strategic General Urban Plans (2).jpg (The authors obtained permission to publicly share this figure from City’s Municipal of Alexandria).\n\nMendeley: Comprehensive Database for Mobility Hub Zones, 800m radius surrounding MBBT, Alexandria, Egypt. https://data.mendeley.com/datasets/k8hybkvsb3/5. 13\n\nThis project contains the following extended data:\n\n• 00 Capturing Image Survey Data Tools.jpg (A figure shows the capturing image survey data tools used in the survey - (1) a DSLR camera with a professional camera tripod, (2) a padcaster tripod dolly wheel, and (3) a smartphone with a DJI Osmo Mobile 3 Gimbal for the smartphone).\n\n• Formal Requests for Information and Approval (1).jpg (A figure shows the formal request, in Arabic, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: General Authority for Passengers Transport in Alexandria).\n\n• Formal Requests for Information and Approval (2).jpg (A figure shows the formal request, in Arabic, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: Public Authority for Planning Transport Projects).\n\n• Formal Requests for Information and Approval (3).jpg (A figure shows the formal request, in Arabic, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: National Railways Authority of Egypt).\n\n• Formal Requests for Information and Approval (4).jpg (A figure shows the formal request, in Arabic, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: Ministry of Transport).\n\n• Formal Requests for Information and Approval (5).jpg (A figure shows the formal request, in Arabic, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: Directorate of Housing and Utilities-Alexandria).\n\n• Translation -Formal Requests for Information and Approval (1).jpg (A figure shows the formal request, in English, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: General Authority for Passengers Transport in Alexandria).\n\n• Translation -Formal Requests for Information and Approval (2).jpg (A figure shows the formal request, in English, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: Public Authority for Planning Transport Projects).\n\n• Translation -Formal Requests for Information and Approval (3).jpg (A figure shows the formal request, in English, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: National Railways Authority of Egypt).\n\n• Translation -Formal Requests for Information and Approval (4).jpg (A figure shows the formal request, in English, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: Ministry of Transport).\n\n• Translation -Formal Requests for Information and Approval (5).jpg (A figure shows the formal request, in English, for information and approval from Graduate Studies & Research, Faculty of Fine Arts to the transport institution: Directorate of Housing and Utilities-Alexandria).\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 want to thank all their colleagues in the Department of Architecture, Faculty of Fine Arts, and Arab Academy for Science and Technology, Alexandria, for being helpful and supportive throughout this research process.\n\n\nReferences\n\nZegras PC 1968: Sustainable urban mobility: exploring the role of the built environment.2005 [cited 2022 Aug 17].https://dspace.mit.edu/handle/1721.1/34170\n\nGuidelines D, Subway FOR, Development IW: TRANSIT ORIENTED COMMUNITIES DESIGN GUIDELINES FOR SUBWAY STATIONS.2022.\n\nMichelle B, Margaret G, Joanne P, et al.: Transit-Oriented Communities Design Guidelines Creating more livable places around transit in Metro Vancouver. TransLink;2012; 164. Reference Source\n\nTransLink: Transit-Oriented Communities - A PRIMER ON KEY CONCEPTS. TransLink;2011; 1–14. Reference Source\n\nGuidelines D, Subway FOR, Development IW, et al.: City of Burlington Offi cial Plan Review: Mobility Hubs Opportunities and Constraints Study. TransLink;2011; (May): 1–13. Reference Source\n\nCoMoUK: An Introduction to Mobility Hubs for Planners and Developers in Scotland.2021. (January).\n\nMetrolinx: Green Paper #2 Mobility Hubs: Development of a Regional Transportation Plan for the Greater Toronto and Hamilton Area (for consultation).2008; 1–54. Reference Source\n\nMetrolinx: Mobility Hub Guidelines.2011;(September); 1–174.\n\nNational Authority for Tunnels:[cited 2022 Nov 1]. Reference Source\n\nNational Authority for Tunnels: [cited 2022 Nov 1]. Reference Source\n\nNational Authority for Tunnels:[cited 2022 Nov 1]. Reference Source\n\nNational Authority for Tunnels:[cited 2022 Nov 1]. Reference Source\n\nSeoudy H, El Menshawy A, El Adawy A:Comprehensive Database for Mobility Hub Zones, 800m radius surrounding MBBT, Alexandria, Egypt. [dataset].2022; Vol. 5. Mendeley Data. Reference Source" }	[ { "id": "162056", "date": "17 Feb 2023", "name": "Ghada Hassan", "expertise": [ "Reviewer Expertise Sustainable urban design", "ecological landscape", "urban ecology", "housing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 tried to study and enhance MSTBE as a key element of the study design used in this research, depending on data collection, approvals, techniques, and analysis methods. A case study, the analysis and development of the Muharram Bek El Mowkaf El Gedid Mobility Hub (MBMH) and the 800 m radius around it.\nThe authors come up with a proposal of replanning the land use of the site.\nMany comments need to be addressed (page numbers refer to the pdf version of the article):\np3: there is a jump to the results and suggestions that are is proven, the author imposes their opinion at the very beginning step of the research, and they might have a concrete reference to support this opinion, but they do not\n\np3: a lot of repetition (sentences ad paragraphs)\n\np3: the methods are mentioned in the paragraph of the study aim, while the 3 mentioned aims are too generic and are aims of project and implementation rather than aims of a research\n\np5: table 1 missing reference\n\np6: fig 2 missing the macroscale map to show location and its relevance, also the fig does not show bus stops as indicated in the key\n\np8-10 important that the SWOT analysis be classified according to the land use, the built form and the open spaces, must be cross referenced with the attributes cited in table 1\n\nFinally the conclusions are based on the result of table 5 where no clear references for its attributes\n\nThe paper seems to be a proposal of site replanning and individual suggestions and not based on evaluation of similar examples and best practice or lesson learned.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9596", "date": "09 May 2023", "name": "Amr El Adawy", "role": "Author Response", "response": "We would like to thank you, Dr. Ghada Hassan, for your comments. In replying to your comments, we took your comments into consideration for the new version of the article: In the introduction, we did not introduce any results or suggestions, but just introduced the micro-scale transit built environment (MSTBE) as being sustainable by dividing it into three scopes, and each scope has been explained individually. As for the concrete reference to support building up sustainable MSTBE, we derived these three scopes from two international case studies in Canada and the United Kingdom (see Data collection, Methods). From these international case studies, we have derived lessons that will be useful for the research's case study development phase. In addition, we supported the scope of mobility hub with Mobility Hub Accreditation, which was developed by Collaborative Mobility UK (CoMoUK) as a tool to evaluate the quality of mobility hub in the case study. We have edited the repetitive sentences in the research problem and explained that the existing MSTBEs all over Alexandria are unsustainable owing to their lack of three scopes, according to many facts including current Alexandrian TOCs, passengers, delays in receiving new transit supply system technologies, and each public transportation mode operating independently. The study aims have been amended to be clearer to the reader, which are to redirect the authorities' focus from the strategic plans to be switched over to the existing MSTBEs in the cities and also to establish a method that includes phases supported by transit frameworks, strategies, solutions, and guidelines to establish an 800m radius micro-scale transit built environment. Lastly, we mentioned the objectives of the study in three points. Table 1, the information in the table is extracted from our work in the case study zone. Figure 2, where the circle represents the case study zone within an 800-meter radius, which equals the micro-scale within neighbourhood borders. Bus stops are clearly marked on the map (3 orange dots); they exist along the Alexandria Governorate Bus Route (magenta curve line). In Table 1, we mentioned general information for the case study zone (present) as well as illustrated it in Figure 2. The information in Table 1 matches the SWOT analysis mentioned in Table 2, where we explained in detail each mobility hub zone (Primary Zone, Secondary Zone, and Tertiary Zone) and translated information of land use, built form, and open spaces in the form of figures captured in the case study. You can check this at Mendeley Data, V5, doi: 10.17632/k8hybkvsb3.5 , entitled \"Comprehensive Database for Mobility Hub Zones, 800m radius surrounding MBBT, Alexandria, Egypt\" (01 Primary Zone.jpg, 02 Secondary Zone.jpg, and 03 Tertiary Zone.jpg). In Addition, the upward part of Figure 5 illustrates clearly the distribution of the current land uses in the case study. The \"Conclusions\" section was rewritten. In the “Results and discussion” section, Table 5 estimated the exact percentages of the land uses for each mobility hub zone of both the current and proposed case study states and compared them, from which we ensured that the distribution of the land uses was fair and acceptable under the guidance of the two international case studies. In the \"Conclusions\" section, we figured out that the matter of the research is that development must start from the micro-scale transit built environment to the meso-scale and then the macro-scale, as well as that people’s behaviour is always affected by the design of the micro-scale transit built environment. We applied the \"Enhanced MSTBE Phases\" methodology to the Muharram Bek El Mowkaf El Gedid Mobility Hub, compared land use percentages, and used the mobility hub’s CoMoUK accreditation assessment. The results were positive, which indicated our success in the methodology's application. Following the successful methodology’s application to the case study, we anticipated that if this method is applied to the other 22 proposed mobility hubs at MSTBEs, the influences of the MSTBEs will reach the meso- and macro-scales. Moreover, Alexandria Strategic General Urban Plans' Transportation Sector Five Goals would be achieved, which would affect the whole governorate’s urban planning and housing, urban and economic development, transportation, and the environment in the future (see Table 7). Referring to the above-mentioned points, it is clear that the proposed case study was based on the evaluation of similar international case studies and lessons learned, not individual suggestions." } ] }, { "id": "168953", "date": "18 Apr 2023", "name": "Atef Ahriz", "expertise": [ "Reviewer Expertise Sustainable architectureSustainable urban designEnergy efficiencySustainabilityClimate", "Microclimate and BioclimateUrban Climatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 entitled “A transit map for micro-scale urban development in Alexandria, Egypt” talks about a very important topic and is in line with the focus of the journal. Aiming to investigate and improve the Muharram Bek El Mowkaf El Gedid Mobility Hub (MBMH) as a crucial component of the research design employed in this study, which involved data collection, approvals, techniques, and analysis methods. The MBMH and its surrounding 800 m radius were analyzed and developed as a case study. The authors put forth a proposal for re-planning the land use of the site. Bellow the detailed review of the paper:\nThe title is suitable and clear\n\nThe abstract is comprehensive, well structured, and well written.\n\nThe paper is excellently structured.\n\nScientifically the manuscript is correct, and uses approved methods and tools to study and enhance MSTBE as a key element of the study design used in this research, depending on data collection, approvals, techniques, and analysis methods.\n\nReferences are sufficient and recent\n\nThe results and discussion part are not comprehensive and detailed regarding the interest of the work. The results should be rewritten to present the results, the highlights and more discussion details.\n\nThe conclusion is too poor regarding the interest of the work; the conclusion must be rewritten to help the reader to understand why your research should matter to them after they have finished reading the paper.\nIn general, the paper is so important but it should be enhanced.\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? No", "responses": [ { "c_id": "9597", "date": "09 May 2023", "name": "Amr El Adawy", "role": "Author Response", "response": "We would like to thank you, Dr. Atef Ahriz, for your comments. In replying to your comments, we took your comments into consideration for the new version of the article: In the “Results and discussion” section: We rewrote it to be comprehensive and detailed regarding the interest of the work. Prior to this section, we included supplementary material in the initial sections to keep the research complete and informative, which would eventually affect the results: We derived the three scopes of the micro-scale transit built environment from two international case studies: the Kennedy Station Mobility Hub in Toronto, Canada, and the London King’s Cross in London, United Kingdom. We have derived lessons, from both of the case studies, about what needs to be done to move towards sustainability and completion in a micro-scale transit built environment. These lessons will be used for the research's case study development phase. We mentioned Collaborative Mobility UK (CoMoUK) and its Mobility Hub Accreditation to provide a set of guidelines for evaluating the quality of the case study’s mobility hub. Out of the above points, in the “Development phase” section, we put the major points for the development of the drastic changes (changes in the land uses, heights, services, open spaces, stops, stations, streets, and urban street elements) in the current state of the whole case study area based on the demands of each mobility hub zone. Finally, in the \"Results and discussion\" section, we discussed the development in the mobility hub zones and the three scopes of the micro-scale transit built environment in detail. We ensured the distribution of the land uses was fair and acceptable under the guidance of the two international case studies. And also, we evaluated the Muharram Bek El Mowkaf El Gedid Mobility Hub using the city centre mobility hub’s CoMoUK accreditation assessment, and the mobility hub was awarded CoMoUK Gold Accreditation. The results were positive. In the opinion of us as authors, if the proposal is implemented as intended, it would be a starting point for a sustainable micro-scale transit built environment in Alexandria, which will affect meso-scale and ultimately macro-scale transit built environments in the long run with positive impacts all over Alexandria. In the \"Conclusions\" section: We figured out that the matter of the research is that development must start from the micro-scale transit built environment to the meso-scale and then the macro-scale, as well as that people’s behaviour is always affected by the design of the micro-scale transit built environment. We applied the \"Enhanced MSTBE Phases\" methodology to the Muharram Bek El Mowkaf El Gedid Mobility Hub, compared land use percentages, and used the mobility hub’s CoMoUK accreditation assessment. The results were positive, which indicated our success in the methodology's application. Following the successful methodology’s application to the case study, we anticipated that if this method is applied to the other 22 proposed mobility hubs at MSTBEs, the influences of the MSTBEs will reach the meso- and macro-scales. Moreover, Alexandria Strategic General Urban Plans' Transportation Sector Five Goals would be achieved, which would affect the whole governorate’s urban planning and housing, urban and economic development, transportation, and the environment in the future (see Table 7)." } ] } ]	1	https://f1000research.com/articles/11-1429
https://f1000research.com/articles/12-132/v1	03 Feb 23	{ "type": "Study Protocol", "title": "A study protocol for investigating the effects of mobile phone-originated electromagnetic waves on thyroid gland and thyroid hormone activities in the brain", "authors": [ "Hendra Zufry", "Achmad Rudijanto", "Djoko Wahono Soeatmadji", "Setyawan Purnomo Sakti", "Khairul Munadi", "Hidayat Sujuti", "Karyono Mintaroem", "Achmad Rudijanto", "Djoko Wahono Soeatmadji", "Setyawan Purnomo Sakti", "Khairul Munadi", "Hidayat Sujuti", "Karyono Mintaroem" ], "abstract": "With widespread users across the globe, topic on the impact of mobile phone in particular regarding its electromagnetic wave radiation deserves a priority. Carcinogenic effects of the electromagnetic wave radiation have been suggested by multiple studies. Effects of the radiation are to involve thermal and non-thermal pathways. Among human organs, thyroid gland is susceptible to the radiation exposure due to its location being superficial and close to the radiation transmitter. Previous studies have indicated the correlation between the mobile phone radiation and thyroid dysfunctions. However, a conclusion that the electromagnetic wave radiation is the causative agent of the thyroid dysfunctions has not been reached. Most of the study are unable to remove the influence of the external electromagnetic wave radiation. Hence, this study aimed to perform an investigation with true experimental design using animal model in an anechoic chamber – an isolated chamber which could minimize the electromagnetic wave noise from the outside. In this article, we present the description of the anechoic chamber along with the research protocols. Parameters affecting the investigation, including signal frequencies inside the chamber and specific absorption rate are also measured.", "keywords": [ "Carcinogenicity", "cellular phone", "radio frequency", "thyroid stimulating hormone", "thyroxine" ], "content": "Introduction\n\nMobile phone is the most used electronic devices working based on the electromagnetic radio frequency. According to the Oceania Radiofrequency Scientific Advisory Association (ORSAA), the cellular phone usage has reached seven billion individuals or 97% of total world’s population.1 In East Asia, the number of mobile phone users is twice as many as its own population (600 million individuals).2 The usage duration ranged widely across countries, from 25 minutes in Japan up to 248 minutes in the Philippines.3 In Switzerland, an individual could spend 30 minutes on their phone.3 In the midst of hyperconnected world, people become more urged of using their phone which is done in at least every 30 minutes.4 With such numbers, the assessment of the effects of mobile phone radiation on human health are urgently needed.\n\nAn epidemiological studies have revealed that 30 min daily use of mobile phone lasted for more than 10 years could increase the risk of glioma in brain cells.5,6 Short-term exposure of the radiation has been reported to be associated with the increased cellular immune activities.7,8 Meanwhile, its long-term exposure could cause irreversible cellular damage.9 Impacts of phone usage is associated with the non-ionic electromagnetic wave radiation transmitted when making or receiving a call.10\n\nIn general, there are two main pathways explaining the biological dysfunctions caused by the exposure of mobile phone-derived electromagnetic wave. The first is through the thermal effects generated by the heat radiation transmitted by the electromagnetic wave, while the latter is through non-thermal effects which have not been completely understood.11 Carcinogenicity of phone radiation has been reported to be associated with increased Ca2+ efflux, oxidative stress imbalance, DNA damage, elevation of ornithine decarboxylase and protein kinases activities, disrupted activity of Na+/K+ phosphatase, melatonin level reduction, disruption in histone kinase activity, and errors in protein synthesis.12–14 Multiple reports have established a correlation between the electromagnetic radiation and impaired estrogen production.15–17 Estrogen shares a similarity with thyroid hormones of being active in nuclear receptors, hence could be affected by the phone radiation in a similar way.18\n\nThyroid gland, a most important endocrine gland, is the most susceptible to the effect of electromagnetic wave radiation from cellular phone owing to its superficial location and close distance with the cellular phone when being used.19,20 A systematic review revealed various effects of the radiation on thyroid function and level of serum thyroid stimulating hormone (TSH).21 Collective evidence by another systematic review also suggested changed levels of triiodothyronine (T3), thyroxine (T4), and TSH occurred in groups receiving cell phone radiation.19 Expression of thyroid-related biomolecules and oxidative stress in rats were found to be significantly correlated with mobile phone-originated electromagnetic radiation.22 A study suggested the significant effect of an 1800 MHz electromagnetic field might take place in hypothalamo-pituitary-thyroid axis.23 By using a Swiss albino mice model, a study reported that the 500–900 MHz electromagnetic field was responsible for the significant reduction of serum thyroid stimulating hormone (TSH). Sub-chronic exposure of 915 MHz (8 h/day for 16 weeks) on Male Sprague–Dawley rats was found to not significantly affect serum T3, T4, and TSH levels.24\n\nDifferent and non-standardized protocols used in investigating the aforementioned effects have made scientist difficult to reach a conclusive finding. Of which, previous studies were unable to exclude the effects of non-ionizing electromagnetic radiation from outside the system. Indeed, a cage modification has been proposed in a study using anechoic chamber allowing whole-body exposure of the electromagnetic field.24 Nonetheless, the possible bias from the external electromagnetic wave influence persists. Therefore, we reported the use of anechoic chamber as a mean to prevent the influence of the external radiation during the investigation.\n\n\nExperimental design\n\nThis study employs a true experimental design by measuring the change of TSH and T4 of the animal subject (Rattus norvegicus) before and after being exposed with mobile phone-originated electromagnetic wave radiation in anechoic chamber. Malondialdehyde (MDA) would also be determined before and after the exposure while nuclear receptor subfamily 3 group C member 1 (NR3C1), monocarboxylate transporter 8 (MCT8), pyruvate kinase, plasma membrane Ca2+ ATPase (PMCA), and thyroid hormone receptor alpha (THRA) would be determined after the exposure. In addition, the histopathological analysis on the thyroid gland will be carried out. All the results would be compared with control group (receiving no radiation exposure). The objective is to reveal the effect of mobile phone-originated electromagnetic wave radiation on thyroid function with minimum influence from the outside environment. The workflow diagram of this study has been presented in Figure 1.\n\nThyroid stimulating hormone TSH; thyroxine, T4; malondialdehyde, MDA; nuclear receptor subfamily 3 group C member 1, NR3C1; monocarboxylate transporter 8, MCT8; pyruvate kinase, plasma membrane Ca2+ ATPase, PMCA; thyroid hormone receptor alpha, THRA.\n\n\n\n• Analytical balance\n\n• TSH and T4 measuring kit (test tube and its rack, micropipette (200 μ), yellow tip, Vidas®)\n\n• Vacutainer red top\n\n• Minor set\n\n• Slide superfrost (Thermoscientific)\n\n• Global System for Mobile Phone (GSM) Sim 900L\n\n• Arduino uno microcontroller\n\n• Real time clock (RTC) module\n\n• Sender—receiver cellular phone module\n\n• Logger data module\n\n• Temperature sensor dht-22\n\n• Spectrum analyzer\n\nThe animals are male rats (Rattus norvegicus) obtained from Faculty of Veterinary, Universitas Syiah Kuala, Banda Aceh, Indonesia. The animals must be three–four months old, weighed 160–250 g, and not showing any abnormalities.\n\n\nAnechoic chamber design\n\nAnechoic chamber has been built at Acoustic, Vibration, and Thermal Laboratory, Faculty of Engineering, Universitas Syiah Kuala, Indonesia. Installation of sound-insulating foam panels on the wall results in a chamber with field-free and sound propagation aligned with ‘inverse square law’. Specific construction design, including the access door and ventilation, allows high sound attenuation and free from structure-borne noise. The chamber has 7.8 m length, 6.3 m width, and 5 m height. It is built with two layers of wall and three layers of iron door to minimize the entrance of signal from the outside. Its interior has a dimension of 6 m × 4.5 m × 3.1 m (length, width, and height, respectively), where the airflow is maintained by a controllable ventilation. The chamber has been installed with a lighting system (6 × 80 Watt lamps) with brightness level of 500 lux. Appearance of the anechoic chamber has been presented in Figure 2.\n\nAs many as three modules of sensor dht-22 are installed surrounding the chamber interior. The sensor is connected to a microcontroller using cable to record the temperature and humidity. Data obtained in the microcontroller are then recorded and displayed through a computer.\n\nSignal strengths from the Global System for Mobile communication (GSM) in the anechoic chamber are measured with spectrum analyzer (max. 3000 MHz). The schematic diagram for signal strength measurement using spectrum analyzer has been presented in Figure 3.\n\nThe signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz (GSM and UMTS signals) with Gaussian Minimum-Shift Keying (GMSK) modulation scheme for GSM, Quadrature Phase Shift Keying (QPSK) – UMTS, and 8PSK – EDGE. In the case of GMSK, the signal is smoothed using low-pass Gaussian filter prior to its modulation into signal carrier and proceeding to frequency modulator. As for QPSK and 8PSK, variants of Phase Shift Keying (PSK), the difference is only located at the code rate capacity. The design and photographed images of the GSM module used in this research have been presented in Figure 4.\n\nThe signal noise in the anechoic chamber is very low, ranged from -79 dBM to -74 dBM (1.2589 × 10-8 mW). The number was obtained from the determination using spectrum analyzer. Several measurement results of the RF/GSM signal frequencies at distance of 10–50 cm has been presented in Table 1.\n\nRF applicator, in a form of a cage, has a dimensional size of 33 × 20 × 7 cm3 with pentagonal shape divided into five chambers. The cage is made from glass where wire mesh is used to cover the top for air circulation during experiment. The distance between the RF signal and the cage is maintained at 10–30 cm.\n\nThe level of electromagnetic wave radiation is represented by Specific Absorption Rate (SAR), an expression to explain the absorbance rate of the electromagnetic radiation (RF) per time unit per kilogram (W/kg). SAR is varied in different parts of the body. The mathematical expression of SAR has been presented in Equation 1.\n\nWhere σ is electrical conductivity of the body tissue (S/m), E is electrical field (V/m), and ρ is the density of body tissue (kg/m3).\n\nIn the real world, the value of ρ is varied, considering each rat has differences in their skin, fat, bones, and so on. Herein, the body density of each rat is assumed to be 1000 kg/m3 with σ = 1 S/m for 2 GHz frequency. The value is applicable for rat with the size of around 20 cm × 4.5 cm × 4.5 cm with an averaged body weight of around 200 g.\n\nTo calculate SARS value, it is of importance to calculate the depth of RF signal penetration using the following equation (Equation 2):\n\nWhere δ is the depth of RF penetration (m), μ is tissue permeability (H/m), and f is averaged RF signal frequencies (mW). In the case of RF penetration into throat tissue, the δ is 1.1 and the averaged uplink frequency for GSM is 9902.5 MHz. Therefore, by using the Equation 2 we obtain the value for δ = 0.0159 m.\n\nIn a GSM 900 system the maximum power is 2 W; hence, the maximum averaged power (P) is 250 mW (1/8 maximum power). If the averaged distance between the phone antenna and rat (A) is 10 cm, the radiation density with maximal penetration (I) could be calculated using Equation 3 resulting in I = 1.481 W/m2. Electrical current is expressed by Equation 4 which can be derived to Equation 5 by subtituting P. With the skin impedance (η) value of 59.094 Ohm, the electrical field strength (E) could be calculated using Equation 5 resulting in E = 9.355 V/m. By inserting all the values required in Equation 1, the SAR is obtained to be 0.0962 W/kg.\n\n\nProcedure\n\nSimple random sampling is employed to randomly assign the animals into four groups, where each group consists of 10 animals. All subjects are considered having the same conditions prior to the treatment. The group assignment has been presented in Table 2.\n\nThe animals, aged two–three months with body weights ranged from 160 to 200 g, are acclimated at room temperature in 12 h dark–12 h light cycle. The animals are fed ad libitum with standardized pellets and water, while being contained in a plastic cage (40 × 25 × 20 cm). The acclimation process lasts for seven days with observation on their behaviors and physical conditions.\n\nRats in intervention group (Group II–IV) are exposed with mobile phone-derived electromagnetic wave radiation for 120, 150, and 180 min/day, respectively. The radiation was exposed in dark condition using two bidirectionally interacting GSM modules, in which their set-up has been presented in Figure 5. RTC is used to control the time of the interaction. The radiation is produced from the stimulation of GSM multiband 4G LTE signal with a frequency of 1800 MHz (Figure 6). The exposure is carried out for 12 consecutive weeks in an anechoic chamber.\n\nGlobal System for Mobile Phone, GSM; real time clock, RTC; Inter Int grated Circuit, I2C.\n\nVenous blood is drawn from the tail of each subject which has been mechanically constrained without anesthesia. The tail is heated for vasolidation and better venous visbiility which could be performed by using heating chamber, heating pad, or heating lamp or by immersing in a warm water. Avoid excessive heating; the animal should be under the lamp no longer than three minutes with a distance of at least 30 cm. All equipment should be sterile, and the skin surface shoul be firstly cleaned and dinsinfected with alcohol 70%. Immobilize the non-dominant tail by hand, and rotate ¼ circle to access the lateral tail vein. The needle is aligned with the tail to 30° for the insertion into the distal vein. Fixation is applied slowly and evenly, and the needle is removed after finished. Observation should be carried out continously during the study period on the subjects’ behavior associated with the ongoing pain.\n\nThe euthanasia should be carried out by professionals. The rats are physically euthanized by cervical dislocation by applying a firm pressure using the thumb and index finger placed on the posterior skull base and spinal cord. Another hand holds the tail part for a quick backward pulling to dislocate the thoracic vertebrae. Check the respiratory arrest and the absence of heartbeat. Thereafter, the organs are harvested by dissection.\n\nThe collected blood sample is stored in an EDTA-containing tube at 2–8°C for maximum 5 days. Blood sample should not be hemolyzed or lipemic. The determinations of serum TSH and T4 were performed on ELISA using commercial kit specialized for Rattus novergicus. Normal ranges for serum TSH and T4 are 0.4–6.0 μIU/mL and 5.0–12.0 μg/dL, respectively. The level of MDA will be measured using ELISA.\n\nBrain tissue, along with the cortex, is collected and rinsed with ice-cold sucrose solution 0.25 M. The tissue is slices and homogenized with 10× operant in a slow setting. The specimen is further treated with Dounce homogenizer as many as 3× operant in a fast speed setting. Dissolution is then carried out to obtain homogenate 6% w/v which is further centrifuged for 10 min at 1.464 × g. The resultant pellet is resuspended in isolating medium and subsequently diluted to yield suspension 6% w/v. As many as 10.4 mL of the suspension is mixed with 1.4 mL Percoll in 15 mL cortex tube and centrifuged for 30 min at 35.540 × g. Two layers are formed on the top side of the type, which are then collected and washed with 5 volumes of sucrose 0.25 M, HEPES-KOH 5 mM, pH 7.1. The obtained pellet is resuspended using the same medium before analyzed using ELISA.\n\nThe whole pituitary gland is collected and frozen in insopentana on dry ice. The sample is melted on slide superfrost (Thermoscientific) and stored at –80°C. The expression is determined based on immunohistochemical method using ELISA.\n\nCryosections are performed on the brain tissue, which is then melted on slide superfrost (Thermoscientific) before undergoing a quick freezing on dry ice and being stored at –80°C. Primary antibodies used for the expression determination are mouse antiβ-tubIII, mouse anticalbindin 1:2000, mouse anti –NeuN 1:500, mouse anti-PV 1:2000. Hybridization is carried out in situ at 25 μm part of the juvenile brain. Riboprobes sense and antisense are synthesized from cDNA RC3 template with spanning nucleotide of 253-486. The expression is based on relative expression in the quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis.\n\nThe cerebral cortex is dissected on an ice-cold glass plate and washed in a buffer (sucrose 0.32 M: EGTA 1 mM: Tris-HCL 10 M; pH 7.4). The sample is sliced and homogenized in the aforementioned buffer (1:20 w/v) with Potter-Elvehjem glass homogenizer. The homogenate is centrifuged 10,000 × g for 15 min at 4°C in a Sorval centrifuge. The obtained mitochondria-free supernatant is collected for the enzymatic activity investigation using ELISA.\n\nAs much as 5 mL venous blood is drawn from each sample, treated with EDTA to prevent the coagulation. DNA genome is extracted from the blood using Gentra Puregene Blood Kit following the manufacturer’s manuals. The DNA sample is stored at –20°C until further use. Indirect determination of the gene expression is carried out using RT-qPCR.\n\nA whole thyroid gland is collected from the subject through dissection, and subsequently fixated using formalin and paraffin for 24 h. Incision is performed on the thyroid gland with a length of 5 μm and the staining is carried out using hematoxylin and eosin following the standardized procedure. Assessment on thyroid follicles is performed under a light microscope. The assessment include:\n\n• Thyroid epithelial cells appeared in a flat shape indicates the hypofunction of TSH.\n\n• Increased form of thyroid epithelial cells indicates the increased function of TSH.\n\n• Transformation of the thyroid epithelial cells into malignant phenotypes.\n\n\nExpected results and discussion\n\nWe expect the reduction of serum TSH and T4 levels and histological alterations on the thyroid epithelial cells upon the radiation exposure during the nighttime which was performed until 12 weeks. A systematic review on 22 studies reporting the effects of cell phone radiation against thyroid functions suggested the detrimental effects of the exposure via thermal and non-thermal pathways.19 Exposure of electromagnetic field (900 MHz, 30 min/day for 5 days/week lasted for 4 weeks) was found to significantly decrease serum TSH, T3, and T4 (p<0.01), in a double-controlled study.25 Exposure of RF radiation (900 MHz, 1 h/day for 50 days) was reported to significantly increase serum T4 and decrease T3 level in Syrian golden hamsters.26 Significant decreases in T3 and T4 were observed in chicken embryos and in the newly hatched chicks, but not in older subjects.23 Enlarged and damaged follicular structures of thyroid was reported in a study exposing a 900 MHz pulse-modulated RF radiation on rats for three weeks (20 min/day).27 No significant effect on thyroid secretory functions, however, is reported from a study investigating sub-chronic whole-body exposure of 915 MHz radiation on rat model.24 In conclusion, T3 levels were consistently reported to be decreased in previous studies using animal model,25,26 but not in the case of T4.26 Furthermore, the effect appeared to be different depending the life stage of the subject (such as embryogenesis).23 Among, the aforementioned study, only one study considered the cage modification to allow even distribution of the exposure throughout the subject’s body.24\n\nFrom this present protocol study, the electromagnetic wave radiation exposure is expected to elevate the plasma MDA as a marker of the oxidation stress. This is as indicator of non-thermal effect of electromagnetic wave radiation. In a previous study, radiofrequency emitted from cellular phone has been reported to induce oxidative DNA damage in a rat model, with significant increase in MDA level.28 Ovarian follicles of mice have been reported to experience oxidative stress induced by cell phone radiation with a significantly elevated level of MDA.29 Since brain is sensitive to electromagnetic wave radiation, we expected that the exposure will reduce the TSH concentration followed by T4. The reduction of T4 activities in the brain will be confirmed by reduction of concentrations of MCT8, pyruvate kinase and PMCA as well as reduction of THRA activities. The reduction of NR3C1 activities will be also measured to prove that there is an additional pathway how electromagnetic wave radiation affects the T4 activities (in circulation or in the brain) apart from thermal and non-thermal pathways. It has been suggested that pathologies of thyroid involve Ca2+ signaling pathways.30 A scoping review has suggested that Ca2+ signaling pathway is disrupted by the cellular phone radiation.31 Increased pyruvate kinase activities are associated with thyroid cancer occurrences.32 These along with dysregulation of NR3C1, MCT8 and THRA expressions are indicators for the carcinogenic effects of the cellular phone radiation.33,34 Finally, we also expected the histology changes of thyroid gland due to thermal or non-thermal effects of electromagnetic wave radiation or reduction of the TSH concentration that potentially have carcinogenic effects.\n\nResults from the previously reported study might be biased from the exposure of other radiations which has polluted the environment. On contrary, we have observed that the anechoic chamber allows the radiation exposure to exclusively originated from the source used during the investigation (by observing the amplitude and frequency of the radiation using spectrum analyzer). Moreover, the anechoic chamber has a significantly lower electromagnetic field as compared to that in the outside. Thus, the study herein would be performed in anechoic chamber preventing the radiation from the outside to influence the investigation. Moreover, our present study would employ 120—180 min/day exposure for 12 consecutive weeks. This exposure duration is relatively longer than reported in previous studies,23,25,26 though it is lower than that in a study investigating the sub-chronic exposure (8 h/day for, 5 day/week, and lasted for 2—16 weeks).24 Taken altogether, this present study could provide strong evidence pertaining to the effect of mobile phone-derived electromagnetic radiation on the thyroid functions.\n\nThe animal study protocol was approved by the Institutional Ethics Committee of Faculty of Veterinary Medicine, Universitas Syiah Kuala (Ref No. 69/KEPH/XII/2020).\n\nThe study has not been completed. The exposure of the electromagnetic wave radiation has been conducted and only some of indicators that have been examined.\n\n\nAuthor contributions\n\nConceptualization, H.Z.; methodology, H.Z.; validation, A.R., D.W.S., S.P.S., K.M., H.S. and K.M.; investigation, H.Z.; resources, H.Z.; writing—original draft preparation, H.Z.; writing—review and editing, A.R., D.W.S., S.P.S., K.M., H.S. and K.M.; visualization, H.Z.; supervision, A.R., D.W.S., S.P.S., K.M., H.S. and K.M.; funding acquisition, H.Z. All authors have read and agreed to the published version of the manuscript.", "appendix": "Acknowledgments\n\nWe would like to thank all the staff at the Faculty of Veterinary Medicine and all the staff at the Thermal, Vibration, and Acoustic Laboratory, Universitas Syiah Kuala for their assistance during the study.\n\n\nReferences\n\nShrestha S, editor. Mobile web browsing: usability study. Proceedings of the 4th international conference on mobile technology, applications, and systems and the 1st international symposium on Computer human interaction in mobile technology.2007.\n\nMubarak A: Child safety issues in cyberspace: A critical theory on trends and challenges in the ASEAN region. Int. J. Comput. Appl. 2015; 129(1): 48–55. Publisher Full Text\n\nRoser K, Schoeni A, Foerster M, et al.: Problematic mobile phone use of Swiss adolescents: is it linked with mental health or behaviour? Int. J. Public Health. 2016; 61(3): 307–315. Publisher Full Text\n\nCoskun S, Karayagız MG: Investigation of problematic mobile phones use and fear of missing out (FoMO) level in adolescents. Community Ment. Health J. 2019; 55(6): 1004–1014. PubMed Abstract \| Publisher Full Text\n\nCarlberg M, Hardell L: On the association between glioma, wireless phones, heredity and ionising radiation. Pathophysiology. 2012; 19(4): 243–252. 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PubMed Abstract \| Publisher Full Text\n\nJargin S: Radiofrequency electromagnetic fields: carcinogenic and other biological effects. Multidiscip. Cancer Investig. 2019; 03(2): 05–13. Publisher Full Text\n\nFadiloglu E, Tapisiz OL, Unsal M, et al.: Non-Ionizing Radiation Created by Mobile Phone Progresses Endometrial Hyperplasia: An Experimental Rat Study. Arch. Med. Res. 2019 2019/02/01/; 50(2): 36–43. PubMed Abstract \| Publisher Full Text\n\nMaluin SM, Osman K, Jaffar FHF, et al.: Effect of Radiation Emitted by Wireless Devices on Male Reproductive Hormones: A Systematic Review. Front. Physiol. 2021; 12: 1568. Publisher Full Text\n\nBalawender K, Orkisz S: The impact of selected modifiable lifestyle factors on male fertility in the modern world. Cent. European J. Urol. 2020; 73(4): 563–568. PubMed Abstract \| Publisher Full Text\n\nMancini A, Di Segni C, Raimondo S, et al.: Thyroid Hormones, Oxidative Stress, and Inflammation. Mediat. Inflamm. 2016 2016/03/08; 2016: 1–12. Publisher Full Text\n\nAsl JF, Larijani B, Zakerkish M, et al.: The possible global hazard of cell phone radiation on thyroid cells and hormones: a systematic review of evidences. Environ. Sci. Pollut. Res. 2019; 26(18): 18017–18031. PubMed Abstract \| Publisher Full Text\n\nLi X, Yu P, Song H, et al.: 1545 Association between endocrine function and radiation exposure. BMJ Publishing Group Ltd;2018.\n\nAlkayyali T, Ochuba O, Srivastava K, et al.: An exploration of the effects of radiofrequency radiation emitted by mobile phones and extremely low frequency radiation on thyroid hormones and thyroid gland histopathology. Cureus. 2021; 13(8). Publisher Full Text\n\nHussien NI, Mousa AM, Shoman AA: Decreased level of plasma nesfatin-1 in rats exposed to cell phone radiation is correlated with thyroid dysfunction, oxidative stress, and apoptosis. Arch. Physiol. Biochem. 2022 2022/11/02; 128(6): 1486–1492. Publisher Full Text\n\nPawlak K, Sechman A, Nieckarz Z: Plasma thyroid hormones and corticosterone levels in blood of chicken embryos and post hatch chickens exposed during incubation to 1800 MHz electromagnetic field. Int. J. Occup. Med. Environ. Health. 2014; 27(1): 114–122. PubMed Abstract \| Publisher Full Text\n\nKim HS, Paik M-J, Kim YJ, et al.: Effects of whole-body exposure to 915 MHz RFID on secretory functions of the thyroid system in rats. Bioelectromagnetics. 2013; 34(7): 521–529. PubMed Abstract \| Publisher Full Text\n\nAdam ANI, Taha ARM: Effects of electromagnetic field on thyroid functions in rats. J. High Inst. Public Health. 2008; 38(3): 568–578. Publisher Full Text\n\nShahryar HA, Lotfi A, Ghodsi MB, et al.: Effects of 900MHz electromagnetic fields emitted from acellular phone on the T3, T4 and cortisol levels in syrian hamsters. Bull. Bet. Inst. Pulawy. 2009; 53: 233–236.\n\nEşmekaya MA, Seyhan N, Ömeroğlu S: Pulse modulated 900 MHz radiation induces hypothyroidism and apoptosis in thyroid cells: a light, electron microscopy and immunohistochemical study. Int. J. Radiat. Biol. 2010; 86(12): 1106–1116. PubMed Abstract \| Publisher Full Text\n\nAlkis ME, Akdag MZ, Dasdag S, et al.: Single-strand DNA breaks and oxidative changes in rat testes exposed to radiofrequency radiation emitted from cellular phones. Biotechnol. Biotechnol. Equip. 2019 2019/01/01; 33(1): 1733–1740. Publisher Full Text\n\nVafere Koohestani N, Zavareh S, Lashkarbolouki T, et al.: Exposure to cell phone induce oxidative stress in mice preantral follicles during in vitro cultivation: An experimental study. International Journal of Reproductive. Biomedicine. 2019; 17(9): 637–646. eng. Publisher Full Text\n\nAsghar MY, Lassila T, Törnquist K: Calcium Signaling in the Thyroid: Friend and Foe. Cancers. 2021; 13(9): 1994. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShİrbandİ K, Rezaeİ Rashnoudİ A, Hassanzadeh G, et al.: Cell phone and wireless radiation hazard on TRPV1 channel activation: A Scoping Review study. J. Cell. Neurosci. Oxidative Stress. 2020; 12(2): 937–946. Publisher Full Text\n\nLiu B, Song M, Qin H, et al.: Phosphoribosyl Pyrophosphate Amidotransferase Promotes the Progression of Thyroid Cancer via Regulating Pyruvate Kinase M2. Onco. Targets Therapy. 2020; 2020(13): 7629–7639.\n\nSohrabi E, Moslemi M, Rezaie E, et al.: The tissue expression of MCT3, MCT8, and MCT9 genes in women with breast cancer. Genes Genom. 2021; 43(9): 1065–1077. PubMed Abstract \| Publisher Full Text\n\nMohamed FEZA, Abdelaziz AO, Kasem AH, et al.: Thyroid hormone receptor α1 acts as a new squamous cell lung cancer diagnostic marker and poor prognosis predictor. Sci. Rep. 2021; 11(1): 7944. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "164254", "date": "31 Mar 2023", "name": "Alicja Bortkiewicz", "expertise": [ "Reviewer Expertise Influence of electromagnetic fields", "noise", "shiftwork on health especially on cardiovascular system and autonomic nervous system", "work-related diseases (mainly cardiovascular diseases)", "evaluation of the fitness to work in drivers", "evaluation of work-fatigue", "physical fitness" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 seems well thought out and described, but I have a few doubts and reservations.\nIn \"Introduction\" it would also be worth quoting meta-analyses of research on head and neck cancers, and not only two selected original papers Carlberg & Hardell (2012) and Hardell L, Carlberg M, Mild KH. (2013)\nMy basic doubts concern the planning of the exposure.\nExposure information is inconsistent. In the \"Intervension\" section, it is stated that the signal frequency will be 1800 MHz, while in the \"Source of radio frequency\" chapter, the authors state that \"The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz\". At what distance from the antenna will the cages with animals be placed? This is not clear if all 10 rats from each group are exposed simultaneously. If so, the EMF generated by the antenna will be disturbed. The same problem occurs due to metal mesh on the top of the cage.\nMoreover the antenna is not fully omnidirectional and its characteristic is unknown, so EMF in close field, inside the cage should be measured, not approximated. Dimensions of the cage do not guarantee uniform field inside, and 40 dB difference in signal within 10 cm distance (Tab. 1) further reinforces doubts of field uniformity. SAR is quantified for various tissues but with one average (?) density and conductivity and independently of animal relative position to antenna. This is totally unacceptable. Likewise depth of penetration is quantified in equation 2 for throat tissue (what kind of tissue is it) but it is strongly dependent on the skin impedance, which on the other hand is used in equation 3. Data on tissue parameters and used equations should be supported by valid literature. The reasons for chosen times of exposure (2, 2.5 and 3 h) is questionable. The reason for independent presentation of the study protocol itself without any results is unclear and suggests artificial invention of topics, unless this protocol is in the project stage. But in this case reviewer strongly recommends correction of the protocol basing of before mentioned comments.\nMinor remarks Where do the mathematical formulas come from, please indicate the source. Below table 1, explain the abbreviations MW and dBM\n\nIs the rationale for, and objectives of, the study clearly described? Yes\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": [ { "c_id": "9639", "date": "09 May 2023", "name": "Hendra Zufry", "role": "Author Response", "response": "Major remarks 1. The protocol seems well thought out and described, but I have a few doubts and reservations. In \"Introduction\" it would also be worth quoting meta-analyses of research on head and neck cancers, and not only two selected original papers Carlberg & Hardell (2012) and Hardell L, Carlberg M, Mild KH. (2013) RESPONSES: Thank you. We have tried our best in addressing your queries. Thank you for this well thought suggestion. We have now cited a meta-analysis on the tumor risks associated with electromagnetic radiation. “A meta-analysis of case-control studies concluded that mobile phone radiation is associated with tumor risks, especially when its usage frequency is > 1000 h.x” Ref: Choi Y-J, Moskowitz JM, Myung S-K, Lee YR, Hong Y-C: Cellular Phone use and risk of tumors: Systematic review and meta-analysis. Int. J. Environ. Res. Public Health. 2020;17(21):8079. 33147845 10.3390/ijerph17218079 2. My basic doubts concern the planning of the exposure. Exposure information is inconsistent. In the \"Intervension\" section, it is stated that the signal frequency will be 1800 MHz, while in the \"Source of radio frequency\" chapter, the authors state that \"The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz\". RESPONSES: Thank you for your concern and sorry for the confusion. Let us clarify. The design was pre-planned, and 1800 MHz is the maximum frequency for 3G spectrum. However, when later tested, we found the frequency was ranged 891-893 MHz. Therefore, we have made revision to resolve this confusion: “The radiation is produced from the stimulation of GSM multiband 4G LTE signal with a frequency of 891—893 MHz (maximum frequency for 3G spectrum is1800 MHz)” 3. At what distance from the antenna will the cages with animals be placed? RESPONSES: Antenna will be placed on the center of the cage. With the cage dimensions being 30 x 19 x 14.4 cm^3 (length x width x height), the furthest distance from the animal to the antenna is therefore 19 cm. In the text we have added the following: “Each cage is divided into five segments with the antenna placed in the center.” 4. This is not clear if all 10 rats from each group are exposed simultaneously. If so, the EMF generated by the antenna will be disturbed. The same problem occurs due to metal mesh on the top of the cage. RESPONSES: Thank you for your concern regarding the EMF disturbance. We placed the antenna in the center inside the cage separated into five segments. The small-diameter metal mesh covering the top of the cage was therefore considered to give insignificant effect to the electromagnetic exposure. And regarding the simultaneous exposure, our apology for the confusion. Please find our revision as follow: “Each cage is divided into five segments with the antenna placed in the center. Each segment will consist of a single rat, and each group will receive simultaneous exposure by employing two separated cages to avoid the disturbance of electromagnetic field.” 4. Moreover, the antenna is not fully omnidirectional and its characteristic is unknown, so EMF in close field, inside the cage should be measured, not approximated. Dimensions of the cage do not guarantee uniform field inside, and 40 dB difference in signal within 10 cm distance (Tab. 1) further reinforces doubts of field uniformity. RESPONSES: Thank you for your concern. As seen in Tab 1, we have measured the radiation power from the antenna in the echoic chamber in various distances. The measurement is performed directly by spectrum analyzer, so it is not mathematically estimated. We believe that the uniformity of the field is achieved since we used real hand-phone concept which allows the omnidirectional property of the antenna – capable of receiving the call from any directions. Regarding the effect of cage design on the field uniformity, we have already explained it above. 5. SAR is quantified for various tissues but with one average (?) density and conductivity and independently of animal relative position to antenna. This is totally unacceptable. Likewise depth of penetration is quantified in equation 2 for throat tissue (what kind of tissue is it) but it is strongly dependent on the skin impedance, which on the other hand is used in equation 3. RESPONSES: It’s true that the value is dependent on the antenna—animal distance. 10-cm distance was taken as the most probable distance of antenna—animal (considering the furthest distance the animal can get from the antenna is 19 cm). Regarding the SAR value, we understand your concern that the value could be different for each tissue. Our aim is to investigate the effect on thyroid gland; unfortunately, relative permeability and conductivity for thyroid gland is not available. Therefore, we assume the values are equal to that in tissues nearby (namely trachea and lung) which have been known. Clarifications have been made in the following paragraph (highlighted in red). Where σ is electrical conductivity in the observed tissue (S/m), E is electrical field (V/m), and ρ is the density of body tissue (kg/m 3). In the real world, the value of ρ is varied, considering each rat has differences in their skin, fat, bones, and so on. Herein, the body density of each rat is assumed to be 1000 kg/m 3. Since the value of σ in thyroid gland is unknown, we assume the value is equal to that in the tissue nearby, namely trachea and lung (σ = 1 S/m for 2 GHz frequency). Since the The value is applicable for rat with the size of around 20 cm × 4.5 cm × 4.5 cm with an averaged body weight of around 200 g. Where δ is the depth of RF penetration (m), μ is permeability of the observed tissue (H/m), and f is averaged RF signal frequencies (mW). Similar to tissue conductivity, the value of permeability for thyroid gland is currently unknown and therefore is assumed to be equal with that of trachea. 6. Data on tissue parameters and used equations should be supported by valid literature. RESPONSES: Thank you. It is true that the calculation protocol should be supported by valid literature. We have now cited it in the paragraph. Please see the amendment: “By inserting all the values required in Equation 1, the SAR is obtained to be 0.0962 W/kg. Equations and biological parameter constants used in calculating the SAR are derived from a previously published report.” Citing: Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verification of Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187 7. The reasons for chosen times of exposure (2, 2.5 and 3 h) is questionable. RESPONSES: It is based on our unpublished preliminary study. The clarification has been included: “The daily exposure duration chosen in this research is based on the results from our preliminary, where changes thyroid functions and other metabolic parameters occurred from 120 to 150 min/day exposure (unpublished). In this study, 180 min/day exposure duration will be employed to observe new findings.” 8. The reason for independent presentation of the study protocol itself without any results is unclear and suggests artificial invention of topics, unless this protocol is in the project stage. But in this case reviewer strongly recommends correction of the protocol basing of before mentioned comments. RESPONSES: With due respect, we do not intend to make the publication as ‘artificial invention of topics’. Our reasons: Publishing this research as a ‘protocol article’ allows its detailed presentation. Further, this is an on-going project where some data have been collected in the preliminary stage. We thank reviewer for their inputs allowing us to perform research with better methodology. Minor remarks 1. Where do the mathematical formulas come from, please indicate the source. RESPONSES: References used: 1. Chakarothai J, Wang J, Fujiwara O, Wake K & Watanabe S (2013).Dosimetri of reverberation chamber for whole-body exposure of small animal.IEEE Transactions on microwave Theory and Techniques, 61(9),3435-3445. 2. Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verificationof Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187 These references have been cited in the text as in the previous response. 2. Below table 1, explain the abbreviations MW and dBM. RESPONSES: Revised. In the Table 1 footnote: mW ; miliwatt, dBm : The power ratio in decibels (dB) of the measured power referenced to one milliwatt (mW)." } ] }, { "id": "164252", "date": "06 Apr 2023", "name": "I Made Pande Dwipayana", "expertise": [ "Reviewer Expertise Endocrinology", "metabolism", "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\nThe introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or thyroid hormone activities in the brain. Please provide also clear research question/aim of the study in the introduction.\n\nThe design of the study protocol is appropriate. Please add statistical analysis that will be used to prove the hypothesis.\n\nPlease provide the reason or the formula for calculating the number of animals in each group in the study to make sure that the number of of sample is adequate to provide the statistical power.\n\nThis study has advantages because the use of anechoic chamber since this setting could reduce unwanted electromagnetic waves. The absorption rate is also measured using clear formula.\n\nMultiple indicators are assessed and determined from TSH, T4, MDA, PMCA, MCT8, pyruvate kinase, mRNA expression of THRA and NR3CI genes. The methods being used are appropriate.\n\nOverall the article is good and only need minor revision.\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": "9638", "date": "09 May 2023", "name": "Hendra Zufry", "role": "Author Response", "response": "1. The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or thyroid hormone activities in the brain. RESPONSES: Thank you for the suggestion. We have made the following amendment: “Herein, other than T 4, and TSH, the effect of electromagnetic wave radiation will be assessed on several biomolecules including malondialdehyde (MDA), nuclear receptor subfamily 3 group C member 1 (NR3C1), monocarboxylate transporter 8 (MCT8), pyruvate kinase, plasma membrane Ca 2+ ATPase (PMCA), and thyroid hormone receptor alpha (THRA) would be determined after the exposure. MDA acts as the biomarker for oxidative stress induced by the electromagnetic wave radiation. Based on our unpublished preliminary study using computational modelling, NR3C1 is suggested to mediate the oxidative damage on cells and morphometric of thyroid gland, thyroid functions (T 4, and TSH), MCT8, non-genetic thyroid hormones-affected proteins (pyruvate kinase and PMCA) and genetic thyroid hormones-affected protein in the brain (THRA).” As mentioned above, gene expression of THRA in the brain is affected by the thyroid hormones. 2. Please provide also clear research question/aim of the study in the introduction. RESPONSES: Amended, please see below: “The aim is to provide holistic evidence on the effect of electromagnetic wave radiation on thyroid gland and thyroid functions with improved study design that is not bias ed from the influence of the external radiation by employing anechoic chamber.” 3. The design of the study protocol is appropriate. Please add statistical analysis that will be used to prove the hypothesis. RESPONSES: Thank you. The following paragraph has been added: Statistical analysis Bivariate analysis will be performed using Spearman test and Chi-square test for continuous and categorical data, respectively. The correlations between the intermediary variables and MDA level or NR3C1 expression will be established using ordinal regression and logistic regression. Statistical significance is considered achieved if p<0.05. All statistical analysis will be performed on IBM SPSS Statistics 23.0 (Armonk, NY, USA). 4. Please provide the reason or the formula for calculating the number of animals in each group in the study to make sure that the number of sample is adequate to provide the statistical power. RESPONSES: Thank you. The following amendment has been made: Sample size To ensure the statistical power will be achieved, the sample size is determined by Federer’s equation ([n – 1][t – 1] ≥15); where n is the number of sample per group and t is the number of groups. In this study, the number of groups is four, so that six animals are at least required for each group 5. This study has advantages because the use of anechoic chamber since this setting could reduce unwanted electromagnetic waves. The absorption rate is also measured using clear formula. RESPONSES: Thank you. 6. Multiple indicators are assessed and determined from TSH, T4, MDA, PMCA, MCT8, pyruvate kinase, mRNA expression of THRA and NR3CI genes. The methods being used are appropriate. RESPONSES: Thank you." } ] } ]	1	https://f1000research.com/articles/12-132
https://f1000research.com/articles/12-472/v1	05 May 23	{ "type": "Research Article", "title": "Comparative evaluation of preventive and interceptive orthodontic treatment need in children aged 6, 9 and 12 years in Central India", "authors": [ "Sumukh Nerurkar", "Ranjit Kamble", "Ranjit Kamble" ], "abstract": "Background: Preventive and interceptive orthodontics are most commonly advocated in patients to reduce the prevalence of malocclusion. The objective assessment of malocclusion is crucial to document the prevalence and complexity of cases. To determine this critical aspect of treating malocclusion, numerous indices have been developed over the years that determine the need for orthodontic treatment. The index for preventive and interceptive orthodontic treatment need (IPION) is one such index that calculates the need of a patient to undergo preventive and interceptive procedures. Due to the paucity of studies that compare this important aspect of preventing malocclusion in different age groups, we have conducted this study. Methods: A total of 384 children aged 6, 9 and 12 years were divided into three groups and evaluated for their need to undergo treatment. The IPION was used to determine this treatment need. The mean IPION score was used to classify the patient into, ‘no need’, ‘moderate need’ and ‘definite need’ of treatment. The children in each group were also divided according to their biological sex. The numbers of children in each group were then compared using Chi-squared test and the mean IPION scores were compared using ANOVA. Results: In the group of 6-year-old children, 63.28% of them needed some type of treatment, in the group of 9-year-old children, 71.88% of them needed treatment and in the group of 12-year-old children, 59.37% of the children needed treatment. Conclusions: The results of the study showed a high proportion of children requiring preventive and interceptive treatment. It was also noted that there was no difference in the treatment need in 6- and 9-year-old children, but it decreased in the 12-year-old group. The study also concluded that the mean IPION scores in boys aged 6 years old was significantly greater than girls aged 6 years old.", "keywords": [ "Interceptive orthodontics", "preventive orthodontics", "IPION", "serial extraction", "IOTN", "DAI" ], "content": "Introduction\n\nSeveral genetic and environmental factors play a part in the development of malocclusion. The long duration taken for the development of occlusion makes it challenging to pin point the involvement of certain factors in the development of malocclusion. Preventive orthodontics is the action taken to preserve the integrity of what appears to be normal at a specific time.1 Ricketts (1976) suggested that in many cases, malocclusion can be prevented or at least intercepted.2 Interceptive orthodontics includes the measures that prevent the progression of a potential malocclusion into a severe type. These procedures are undertaken after the appearance of a malocclusion.\n\nThe objective assessment of malocclusion is crucial to document the prevalence and complexity of the malocclusion. Thus, to determine this critical aspect of treating malocclusion, over the years, there are many indices that determine the need for orthodontic treatment.3 Identifying and determining the problem’s severity are the two crucial aspects of screening patients for early orthodontic treatment. Thus, to determine this critical aspect of treating malocclusion, over the years, there are many indices that determine the need for orthodontic treatment. Older indices like the treatment priority and occlusal index measured the severity of the malocclusion.4 The dental aesthetics index is used to rate the aesthetics of the patient. Index of orthodontic treatment need (IOTN), peer assessment rating (PAR) and dental aesthetic index (DAI) are the most frequently used indices today.5 Although there are many shortcomings to these indices, it is always recommended to use them for categorizing the malocclusion and to determine its severity.\n\nAll these indices can be applied to only permanent dentition or do not help categorize the need for preventive and interceptive orthodontic treatment. Apart from the index for preventive and interceptive orthodontic treatment need (IPION), there is no index that can calculate the need for early orthodontic treatment.6 The IPION was developed for categorizing malocclusion and determining the need to undergo preventive or interceptive orthodontic treatment in children aged 6 and 9 years old and later modified for 12-year-old children.7\n\nThus, with the primary objective of comparing the preventive and interceptive orthodontic treatment needs in children aged 6, 9 and 12 years, we conducted this study.\n\n\nMethods\n\nAfter obtaining clearance from the Institutional Research Ethics Committee of Datta Meghe Institute of Higher Education and Research, the ethical approval letter was obtained on 15th December 2020 (reference number DMIMS (DU)/IEC/2022/345), an analytical cross-sectional study design was followed. Data collection commenced on 27th June 2021.\n\nAfter obtaining written informed consent from the legal guardians of the children for inclusion in the study, a total of 384 patients were selected randomly from the patients coming to the OPD of Orthodontics and Dentofacial Orthopedics at Datta Meghe Institute of Higher Education and Research or in screening camps arranged in schools in and around Wardha.7 The institutes involved in arranging these camps were Sharad Pawar Dental College, Alphonsa School, School of Scholars and New English School. The camps were arranged by the Department of Public Health Dentistry of Datta Meghe Institute of Higher Education and Research. The arrangement of the camps was done after obtaining permission from the head of the institute. An intraoral examination of the patient was done. The patients were categorized into three groups, namely; Group I (6 years old), Group II (9 years old) and Group III (12 years old). Figure 1 depicts the graphical representation of the method followed. Each group was further divided into boys and girls, on the basis of external body characteristics.\n\nIPION, index for preventive and interceptive orthodontic treatment need.\n\nThe patients were screened for the age group of 6-12 years. All the patients presently undergoing or who had undergone orthodontic treatment were excluded. Patients suffering from systemic conditions were also excluded.\n\nFive intra oral standard photographs were taken using cheek retractors and a mirror. The photographs consisted of one frontal, buccal in occlusion and occlusal photographs. The clinical examination was done to evaluate the functional components of the index. A vernier caliper was used to measure the parameters on the dental models, and the presence of caries was detected using an explorer on the patient itself.6\n\nThe final scores of the IPION for Group I and Group II were calculated according to the IPION-6 and IPION-9 index given by Coetzee,6 and that for Group III was calculated according to IPION-12 index by Nerurkar et al.7 Each component had a specific weighing score, which depended on the severity of the malocclusion it caused. The scores of these components are added to get a final score.\n\nThe score of each component was calculated by the product of the component score and the weighting factor.\n\n= Component score × weighing factor\n\nThe patient’s total score was then determined by the summation of the scores of each component.\n\n= ∑ (Component score × weighing factor)\n\nThe results from this study were then statistically analyzed according to the observations.\n\nThe sample size was calculated according to the formula n = N * X/(X + N - 1). Where X = Zα/22 p(1 - p) /MOE2, Zα/22 was taken as 1.96, the MOE was considered as 5%. The population of the region was considered as 100,000 and the sample proportion was 50%. According to the data, the authors considered the sample size to be 383.\n\nThe data are presented as the mean ± SD of the IPION scores and as percentage of children in each group. Statistical analysis was done using descriptive and inferential statistics using ANOVA test (followed by the Bonferroni correction), Chi-squared test and Kappa Statistic. IBM SPSS Statistics (RRID:SCR_016479) version 27 and GraphPad Prism (RRID:SCR_002798) version 7.0 were used for statistical analysis. p < 0.05 was considered to indicate a statistically significant difference.\n\n\nResults\n\nOn comparing the orthodontic treatment need in the 6-, 9- and 12-year-old age groups, the following results were obtained. A total of 36.72% of the children from group I had no need for treatment, 39.06% of the children needed moderate treatment, and 24.22% of the children needed definite orthodontic treatment. In group II, 28.13% of the children did not require any treatment, 43.75% needed moderate treatment, and 28.13% had a definite need for treatment. In group III, 40.63% of the children had no need for any orthodontic treatment, 39.06% needed moderate treatment, and 20.31% needed definite treatment (Table 1 and Figure 2).\n\nData are presented as the number of children in each group. IPION, index for preventive and interceptive orthodontic treatment need; NS, not significant.\n\nIPION, index for preventive and interceptive orthodontic treatment need.\n\nOn comparing the mean IPION scores of different age groups the following results were obtained. The mean IPION score for 6-year-old children was 9.6406, for 9-year-old children it was 10.6484 and for 12-year-old children it was 8.4688. When the mean IPION scores of group I were compared with those of group II, a non-significant association was found. Similarly, no association was found when the IPION scores for group I were compared with that of group III children. When these scores for group I children were compared with that of group III children a significant association was found (Table 2).\n\nData are presented as the mean IPION scores in each group. IPION, index for preventive and interceptive orthodontic treatment need; NS, not significant; S, significant.\n\nOn comparing the mean IPION scores of different sexes the following results were obtained. The mean IPION score for 6-year-old boys was 11.39, whereas that for girls was 8.27. A significant difference was found when these scores were compared. The mean IPION score for 9-year-old boys was 10.85, whereas that for girls was 10.34. The mean IPION score for 12-year-old boys was 9, whereas that for girls was 7.90. No significant difference was found between the sexes in group II and group III (Table 3).\n\nData are presented as the mean IPION scores in each group. IPION, index for preventive and interceptive orthodontic treatment need; NS, not significant; S, significant.\n\n\nDiscussion\n\nA total of 156 children needed moderate treatment, and 93 needed definite treatment. A total of 64.84% of children needed early orthodontic treatment. Subramaniam et al.,8 and Prabhakar et al.,9 showed similar results. According to them, 63% of children required early orthodontic treatment. A similar prevalence of need for treatment was obtained by Tungaraza et al.,10 who concluded that 67.2% of Turkish children needed either moderate or definite treatment. Contradicting results were obtained by Onyeaso,11 who conducted their study in Nigeria and concluded that the need for preventive and interceptive orthodontic treatment was seen in 27% of children. Büyükbayraktar and Doruk,12 also obtained contrasting results. They studied Turkish children and observed that 95.5% of children needed treatment. Rapeepattana et al.,13 gave contrasting results compared to those obtained in this study. They concluded that 96% of the children needed treatment.\n\nA comparison was made between mean IPION scores of 6- and 12-year-old children. The mean score for the 6-year-old children was 9.6406 and for the 12-year-old children it was 8.4688. When compared with each other, there was no significant association between the scores of 6- and 12-year-olds. Because there was no index earlier for evaluating the preventive and interceptive treatment needs in 12-year-old children, there is no study in the literature to compare with the results of the present study.\n\nA comparison was made between the mean IPION scores of 6- and 9-year-old children. The mean IPION score for the 9-year-old group was 10.6484 and that for 6-year-old group was 9.6406. When a comparison was made between the two groups, no significant association was found. Contrasting results were obtained by Galui and Pal,14 who concluded that there is a significant difference between the scores obtained in the 6-year-olds and that in 9-year-old children. They also concluded that IPION-9 scores were greater than the IPION-6 scores.\n\nA comparison between the mean IPION scores of 9- and 12-year-old children. The mean IPION score for the 9-year-old group was 10.6484 and that for the 12-year-old group was 8.4688. When a comparison was made between these groups a significant difference was observed. Because there was no index earlier for evaluating the preventive and interceptive treatment needs in 12-year-old children, there is no study in the literature to compare with the results of the present study.\n\nThe mean IPION score in the 6-year-old boys was 11.39, and that of the girls was 8.27. The scores in the 9-year-old group were 10.85 in boys and 10.34 in girls. In the 12-year-old group, the mean IPION scores were 9.88 in boys and 7.30 in girls. There is no study in the literature to compare with the results of the present study.\n\nThe main limitation of the study is the fact that radiographic analyses were not performed, which could be used for confirming the presence or absence of unerupted or supernumerary teeth.", "appendix": "Data availability\n\nZenodo: F1000. https://doi.org/10.5281/zenodo.7751104. 15\n\nZenodo: STOBE cross sectional checklist. https://doi.org/10.5281/zenodo.7794642. 16\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nChadwick BL: Products for prevention during orthodontics. Br. J. Orthod. 1994; 21(4): 395–398. Publisher Full Text\n\nRicketts RM: Bioprogressive therapy as an answer to orthodontic needs part I. Am. J. Orthod. 1976; 70(3): 241–268. PubMed Abstract \| Publisher Full Text\n\nStein KF, Kelley TJ, Wood E: Influence of heredity in the etiology of malocclusion. Am. J. Orthod. 1956; 42(2): 125–141. Publisher Full Text\n\nÜçüncü N, Ertugay E: The use of the Index of Orthodontic Treatment need (IOTN) in a school population and referred population. J. Orthod. 2001; 28(1): 45–52. PubMed Abstract \| Publisher Full Text\n\nJenny J, Cons NC: Comparing and contrasting two orthodontic indices, the Index of Orthodontic Treatment Need and the Dental Aesthetic Index. Am. J. Orthod. Dentofac. Orthop. 1996; 110(4): 410–416. PubMed Abstract \| Publisher Full Text\n\nCoetzee CE: Development of an index for preventive and interceptive orthodontic needs (IPION) [PhD Thesis]. University of Pretoria.2007.\n\nNerurkar S, Kamble R, Shrivastav S, et al.: Comparative Evaluation of Preventive and Interceptive Orthodontic Treatment Need in Children of 6-9 Years and 9.1-12 Years.\n\nSubramaniam R, Mittal S, Hiregoudar M, et al.: Knowledge of upper primary and secondary school physical education instructors in Davangere city, India, about emergency management of dental trauma. J. Educ. Ethics Dent. 2011; 1(1): 18. Publisher Full Text\n\nPrabhakar RR, Saravanan R, Karthikeyan MK, et al.: Prevalence of malocclusion and need for early orthodontic treatment in children. J. Clin. Diagn. Res. 2014; 8(5): ZC60–ZC61. PubMed Abstract \| Publisher Full Text\n\nTungaraza JP, Mtaya-Mlangwa M, Mugonzibwa AE: Assessment of early orthodontic treatment need and its relationship with sociodemographic characteristics among Tanzanian children using index for preventive and interceptive orthodontic treatment need. Int. J. Orthod. Rehabil. 2019; 10(2): 57. Publisher Full Text\n\nOnyeaso CO: Prevalence of malocclusion among adolescents in Ibadan, Nigeria. Am. J. Orthod. Dentofac. Orthop. 2004; 126(5): 604–607. PubMed Abstract \| Publisher Full Text\n\nBüyükbayraktar ZÇ, Doruk C: Evaluation of the Preventive and Interceptive Orthodontic Treatment Need of an Inner City Group of Anatolian Turkish Children. Cumhur. Dent. J. 2019; 22(3): 318–324. Publisher Full Text\n\nRapeepattana S, Thearmontree A, Suntornlohanakul S: Etiology of Malocclusion and Dominant Orthodontic Problems in Mixed Dentition: A Cross-sectional Study in a Group of Thai Children Aged 8–9 Years. J. Int. Soc. Prev. Community Dent. 2019; 9(4): 383–389. PubMed Abstract \| Publisher Full Text\n\nGalui S, Pal S: Early orthodontic treatment need among 6-9-year-old children of West Bengal. J. Oral Res. Rev. 2021; 13(1): 12. Publisher Full Text\n\nDr. Nerurkar. S : F1000. [Dataset]. Zenodo. 2023. Publisher Full Text\n\nNerurkar S, Kamble R: STOBE cross sectional checklist. [Dataset]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "202532", "date": "14 Nov 2023", "name": "Huang Li", "expertise": [ "Reviewer Expertise Orthodotics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author conduced the study to compare this important aspect of preventing malocclusion in different age groups. Although this study is quite interesting, but still need to address the following questions：\nPlease specify the index for preventive and interceptive orthodontic treatment need (IPION), the reference is from the PhD thesis. Does any published paper used in this index?\n\nWhat is the clinical meaning for this study? Children aged 6, 9 need some type of treatment, it does not mean they need orthodontic treatment at that time. Some malocclusion can be delayed to permanent dentition, considering their treatment duration time and economic burden.\n\nThe reference 7 was not published?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-472
https://f1000research.com/articles/12-175/v1	14 Feb 23	{ "type": "Research Article", "title": "Correlation between salivary immunoglobulin A and interleukin-1beta in smokers with dental caries", "authors": [ "Jaber Al-ibraheem", "Yassir Zyara", "Nibrass Al-Quraine", "Wasna’a M. Abdulridha", "Jaber Al-ibraheem", "Yassir Zyara", "Nibrass Al-Quraine" ], "abstract": "Introduction. Dental caries is one of the most common infectious diseases of the oral cavity, and is an inflammatory disease caused by several factors. Interleukin-1β (IL-1β) is a major mediator of acute inflammation and essential for the development of specific immune responses. The objective of this study was to assess the levels of secretory IgA (s-IgA) and IL-1β in the saliva of smokers with dental caries, and to discover the correlation between these parameters and dental caries.\nMethods. Saliva samples were collected from 30 smokers, age range 21–70 years, with dental caries, in addition to 18 healthy non-smoker volunteers, age range 21–65 years. s-IgA and IL-1β levels in the saliva samples were estimated by enzyme-linked immunosorbent assay (ELISA).\nResults. The mean saliva IgA levels between smokers with dental caries group and healthy subjects were not significantly different (p=0.077), while the saliva levels of IL-1β were higher in the smokers with dental caries group, with a significant difference of p<0.05. No significant associations were found between s-IgA and IL-1β levels, and other parameters such as age, sex, body mass index (BMI) and C-reactive protein (CRP) (p>0.05). There were highly positive associations and significant differences between IL-1β and CRP levels in the two groups under the study (p=0.006).\nConclusions. Our study revealed a significant increase in IL-1β levels in saliva of smokers with dental caries, and a positive association between IL-1β levels and caries disease. There is no significant relationship between elevated IL-1β levels and s-IgA in smokers with dental caries.", "keywords": [ "dental caries", "saliva", "immunoglobulin A", "interleukin-1beta" ], "content": "Introduction\n\nDental caries is a type of chronic oral disease that seriously threatens human beings’ health. Although dentists and researchers have struggled for decades with this oral disease, the incidence and prevalence of dental caries is still very high. Therefore, improving disease management and life cycle management of dental caries is a major issue for all populations. Therefore, a clinical difficulty assessment system for the prevention and management of dental caries was developed based on the diagnosis and classification of dental caries.1 It is a widespread infectious disease, caused by several factors determined by the dynamic balance between pathological and protective factors.2,3 Those agents cause local destruction of sensitive hard tooth tissues by acidic byproducts of bacterial fermentation of dietary carbohydrates.4 Saliva plays an important role in maintaining oral health, helping to build and maintain healthy soft and hard tissues. When saliva flow is reduced, oral health problems such as dental caries and oral infections can develop.3,5 The protective function of saliva can overwhelm bacterial action, so studies indicate the importance of prevention and treatment, as is the case in other infectious diseases.2 Saliva contains antimicrobial proteins, i.e., immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM), as well as antimicrobial agents, i.e., lysozyme, prolene-rich glycoproteins, mucins, lactoferrin, and lactoperoxidases. These parameters are present in low concentrations, but they work by means of a synergistic effect in reducing metabolism and bacterial growth, in addition to being an effective network for oral defense.6\n\nC-reactive protein (CRP) is a protein made in hepatocytes as a result of interleukin-6 (IL-6) and IL-1 stimulation in the acute phase. It belongs to the pentraxin family and is increased in cases of inflammation, injury or infection.7 Cytokines play a major role in modulating the immune response, and are either pro-inflammatory (tumor necrosis factor alpha (TNF-α), IL-1β, transforming growth factor beta (TGF-β), IL-6, and IL-8), or anti-inflammatory (IL-4, IL-10, IL-12, and interferon gamma (IFN-γ)). The nucleus of any cell has the ability to secrete cytokines, but their main source is macrophages and helper T-cells.8 Among the cytokines, interleukins have a crucial function and are implicated in oral cancer. All markers are also important to development the specific immune responses.9 IL-1β and TNF-α have both been considered essential mediators of acute inflammation.5 Our current study aims to assess the levels of secretory IgA (s-IgA) and IL-1β in smokers with dental caries, knowing the correlation between these parameters and caries disease, as well as knowing the relationship between these two parameters in the same patients.\n\n\nMethods\n\nThe present study consisted of 48 volunteers, age range 35–45 years, divided into two groups. The first group included 18 healthy non-smoker volunteers (11 male and seven female), age range 21–65 years, as controls, while the second group included 30 smokers with dental caries (17 male and 13 female), age range 21–70 years, enrolled at the Clinical Center from College of Dentistry at University of Kufa and Al-Kafeel, between October 2021 and March 2022, and they were diagnosed under our supervision. Approval for the study was obtained from the Kufa and Al-Kafeel University Ethics Commission for human subjects. The study protocol was explained to all participants and written informed consent was taken from all before clinical examination or evaluation procedures, e.g. examination of teeth, periodontal, oral mucosal status, assessment of malocclusion, and collection of saliva samples. Also, all patients had a clinical periodontal examination. Individuals with medical disorders such as diabetes mellitus, pregnant or nursing females, and those who had antibiotic drugs or periodontal therapy in the previous three months were excluded from the study. The body mass index (BMI) of each volunteer was calculated in a standard manner.\n\nWhole saliva samples were obtained by expectorating into sterile tubes, during which volunteers were requested not to drink (except water) or to chew gum at least one hour before the assembly. Saliva samples were collected between 09:00 and 11:00 AM. The saliva was collected for 10 min in titration tubes by the drooling method. The saliva samples were clarified by centrifugation (800g) for 10 min at 4°C, then immediately aliquoted and stored at −80°C until required for assays.10\n\nThe concentration of IL-1β in saliva was detected by using human Enzyme-Linked Immunosorbent Assay (ELISA) Kits (Elabscience, USA). The ELISA kit uses a sandwich ELISA in accordance with the manufacturer’s instructions. Absorbance in each well was read on an ELISA reader using 450 nm as the primary wavelength. Concentrations of IL-1β in the saliva were estimated using the standard curve. The level of s-IgA in saliva samples was detected by using ELISA kits (Cayman Chemical, Michigan, USA), according to the manufacturer's instructions. CRP levels were measured by using the human CRP ELISA kit (Diaclone, Besancon, FR). To assess the effect of periodontal disease on the salivary cytokine levels, the community periodontal index of treatment needs (CPITN) was measured in each participant after saliva collection.11 Patients with chronic inflammatory diseases, such as psoriasis, arthritis, Sjögren’s syndrome, and inflammatory bowel disease that may affect levels of salivary cytokines were excluded.\n\nAll statistical analyses were performed by using the SPSS v.24 software program (RRID: SCR_002865, https://scicrunch.org/). Data were expressed as mean ± SD. The significance of the difference between the mean values of the measured parameters between groups were evaluated by the student’s t-test, and chi-square. Correlation was indicated by the Pearson correlation coefficient test, and p<0.05 was considered significant.\n\n\nResults\n\nThis study enrolled 30 smokers with dental caries, in addition to 18 healthy non-smoker volunteers. Statistical analysis of the age of the study subjects showed no significant difference between the mean age of the smokers with dental caries group (41.82 ± 12.459) and healthy group (40.68 ± 12.356; p=0.690), as shown in Table 1. The mean BMI, age, and sex between the experimental and control groups were similar and there was no statistically significant differences (p=0.556, p=0.690, p=0.467, respectively). There was a significant difference in CRP between the two groups (p=0.028). No statistically significant difference in periodontal health assessed by the Community Periodontal Index of Treatment Needs (CPITN) was observed between the two groups (p=0.511), as shown in Table 1. The mean saliva IgA levels among smokers with dental caries group and healthy subjects were not significantly different (p=0.077), while the saliva levels of IL-1β were high in the smokers with dental caries group with significant differences from the healthy group (p<0.05), as shown in Table 2. No significant associations were found between s-IgA and IL-1β levels; with other parameters such as age, sex, BMI and CRP p>0.05. A highly positive correlation and significant differences between IL-1β and CRP levels were observed in the studied groups (p=0.006), as shown in Table 3.\n\n* p<0.05: Differences between patients with dental caries and healthy group.\n\n* p<0.05: Differences between patients with dental caries and healthy group.\n\n\nDiscussion\n\nDental caries is a diet-related disease that continues to be a problem for some dental patients. Saliva is well formulated to protect against dental caries. Saliva’s buffering capability, the ability of saliva to wash the surface of the tooth, clear bacteria, control purification and mineralization, and saliva’s antibacterial activities and other mechanisms all contribute to its primary role in the health of teeth.1,3,12 Several studies have confirmed that knowledge of the functional properties of saliva as well as those of its separate components allows for a better assessment of susceptibility to dental caries.12\n\nCytokines play a large role in regulating many aspects of the immune response, so they are useful tools for diagnosing and monitoring the oral cavity along with other factors.2,5 One of the recent studies indicated the possibility of using saliva as a diagnostic material to measure the biomarkers released during the onset and progression of the disease.5 Immune cell products (IL-1β, TNF-α, IL-6) play an important role in oral mucosal pathology, but the exact role of cytokines in the etiology of dental caries is not well known. In innate responses, the molecular phenotypes associated with the oral pathogen bind to host cell receptors, including dendritic cells that have a role in activating the acute inflammatory response by releasing active pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α.7 On the other hand, in cases where periodontitis develops with potent inflammatory responses, gingivitis will disappear, whereas if infection persists and bacterial proliferation occurs, intensification of the warning response can damage periodontal tissues in the affected patients.13\n\nThe results of our study indicated that the mean BMI, age, and sex between the experimental (dental caries) and control (healthy) groups were similar and not statistically significant (p=0.556, p=0.690, p=0.467, respectively), as shown in Table 1. Conversely, significant differences were observed in the CRP levels between the two groups (p=0.028), but there was no statistically significant difference in periodontal health between the two groups (p=0.511) when assessed by CPITN. These results are in agreement with other studies.13,14 A previous study indicated that high levels of CRP are a risk factor for one type of cancer. Therefore, high levels of CRP before surgery can be considered an undesirable diagnostic indicator.15 Yadav et al.16 noted that the pattern of dental caries varies with BMI, sex, age, race, food habits, oral hygiene practices, socio economic status, and geographical location. Also, they indicated a relationship between CRP levels and disease prognosis. These results are consistent with the results of our current study. Park et al.17 noted the high CRP/albumin ratio in cases of dental caries and its association with disease for long periods, and thus decreased survival rates. A previous study indicated the possibility of using CRP, haemoglobin, and ferritin as vital markers in diagnosing diseases and knowing their development.18 Tampa et al.7 concluded that tumors induce inflammatory responses by increasing CRP levels in serum, and the chronic inflammation contributes to progression of the malignancy. Grimm et al.19 were able to identify a large number of total leukocytes, neutrophils, and high levels of CRP, and also they indicated that a small number of lymphocytes is associated with a lower survival rate in oral cancer.\n\nThe mean salivary IgA between smokers with dental caries and the healthy subjects group was not significant (p=0.077), while the IL-1β rate was statistically different (p<0.05), see Table 2. These results are confirmed by other studies.13,20 Several studies showed an increase in s-IgA levels during the caries process compared with caries-free subjects.2,21 Despite numerous studies, the correlation between s-IgA and dental caries remains unclear. On the other hand, previous studies have shown that elevated salivary IgA levels are associated with decreased dental caries activity, and that s-IgA production provides a local defense with regards to dental infection.22,23 Brandtzaeg24 suggested that elevated IgA levels are a protective response to reduce dental caries, and also indicated that large numbers of carcinogenic bacteria in untreated necrotic lesions could stimulate an immune response and increase salivary IgA levels in the mouth. Salivary cytokine levels were elevated due to disorders of the oral cavity (Table 2), and these results were consistent with previous studies.13,14 In our study, the mean salivary IgA rate was not significant, while the IL-1β rate was statistically different between the dental caries group and healthy subjects. Gomes et al.25 noted that salivary IL-1β and TNF-α levels increase in periodontal disease and are associated with the onset and increased severity of the disease, and that their levels decrease after treatment. Also, they showed that IL-1β and TNF-α are promising biomarkers in detecting periodontal disease. IL-1β plays an important role in the acute inflammatory response, and persists for a relatively long time at the site of inflammation.7 Cogulu et al.9 observed a significant increase in levels of CRP and IL-6 in saliva of the patients diagnosed with apical periodontitis compared with people with normal or healthy teeth. Gomes et al.25 noted the presence of inflammatory cytokines represented by TNF-α and IL-1β in saliva of patients in the early stages, and they indicated their important role in the development of periodontal disease; they concluded that elevated levels of inflammatory cytokines are evidence of disease progression. Several reports indicated the role of acute chronic inflammation in the occurrence and development of carcinogenesis through the modulation of inflammatory cells, and cytokine production,7,26 so it can explain why levels of some cytokines including IL-1β are elevated in patients. These studies support the results of our current study.\n\nThe IL-1β receptor stimulates the activation of signal transducers, Janus kinases (JAK), and activators of transcription (STATs), which then stimulate pathways involving mitogen-activated protein kinase (MAPK), which in turn supports the development of carcinogenesis, and this may explain the reasons for the elevated IL-1β levels in the saliva of the smokers diagnosed with dental caries. The results of this study were validated by27 where they found that higher pro-inflammatory cytokines such as IL-1β in dental caries patients are responsible for the lower number of stony cells and osteoblasts; they also indicated that demineralization of the teeth and the development of cavities occurs in a high incidence in smokers. On the other hand, a tumor can trigger an inflammatory response, causing the release of cytokines i.e., IL-1β and IL-6.28 Gornowicz et al.2 reported increased IL-1β levels in the healthy periodontal group compared with the chronic periodontitis group. Colak et al.29 showed that infants frequently exposed to breast milk, formula milk, sugary liquids, fruit juices and other sweet-tasting liquids for prolonged periods are at risk of early childhood caries (ECC). In contrast to our results,27 noted increased pro-inflammatory cytokines levels in the saliva of smokers with dental caries results in fewer broblasts and osteoblasts. Mclachlan et al.30 concluded that the concentrations of pro-inflammatory cytokine are also increased in both gingival tissues and serum of people with periodontal inflammation. Our study indicated that there was no significant correlation between s-IgA and IL-1β levels, and with other parameters such as age, sex, and BMI p>0.05, but there was a positive association between IL-1β levels and CRP (p=0.006, Table 3).\n\nThe results of our current study are consistent with previous studies.2,13,20 Significant changes in IL-1β levels provide us with great potential for their use as effective biomarkers in saliva. This could open new avenues for treatment plans of targeted leukotriene therapy.30 This conclusion was confirmed by previous reports.5,13 Contrasting with the results of our study,31 reported that dental caries was not significantly associated with IL-1β, interleukin-1 receptor antagonist IL-1Ra), or IL-10 concentrations in the serum and saliva of patients. On the other hand, several studies confirmed that the high level of salivary cytokines occurs as a result of disorders in the oral cavity,2,5,32 and this conclusion is consistent with our current results. However, these results are not consistent with previous studies.5,33,34 On the other hand,35 indicated a significant reversible association between s-IgA and acute activity. A positive association was observed between an elevated IL-1β level and dental caries, while there was no positive association between increased levels of IgA and dental caries. These results are consistent with research highlighting that there is no association between dental caries and increased levels of IgA.13,36\n\nFuture research is necessary to fully characterize the salivary components and their interactions and how these affect the dental caries process. With this knowledge, the use of modified oral molecules as therapeutic agents may become a reality. Also of interest is the potential to influence the secretion of salivary components through increased knowledge and control of the secretory processes responsible for the delivery of those components. The results of our current study suggest a link between the production of inflammatory and immunoregulatory cytokines represented by IL-1β in smokers with dental caries, so this parameter can be used as an important biomarker to indicate the disease severity and progression. Finally, when developing a treatment plan based on the assessment of caries risk and activity of carious lesions, we need to develop a patient-centered and personalized treatment plan to restore the delicate environmental balance of the oral cavity, to control the progression of caries and to restore the structure, and function of the carious teeth.\n\n\nConclusions\n\nIn smokers, a positive association was found between dental caries and elevated IL-1β levels, while not significant differences were found in the level of S-IgA and caries. The present study also indicated no significant correlation between elevated IL-1β levels and s-IgA in smokers with caries disease.\n\n\nEthical approval\n\nApproval for the study was obtained from the Kufa and Al-Kafeel University Ethics Commission for human subjects. Ethical approval committee no. 1730/2022, Kufa University, Iraq\n\n\nConsent\n\nThe study protocol was explained to all participants, and written informed consent was taken from all before clinical examination or evaluation procedures.", "appendix": "Data availability\n\nFigshare. Correlation between Salivary Immuno Globulin A and Interleukin-1beta in Smokers with dental caries. https://doi.org/10.6084/m9.figshare.21909753.v1. 37\n\nThis project contains the following underlying data:\n\nRaw data for comparison between smokers with dental caries and healthy group, according to BMI, age, sex, CRP, and CPITN\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAlbahrani MM, Alyahya A, Qudeimat MA, et al.: Salivary fuoride concentration following tooth brushing with and without rinsing: a randomised controlled trial. J. BMC Oral Health. 2022; 1: 22–53.\n\nGornowicz A, Bielawska A, Bielawski K, et al.: Proinflammatory cytokines in saliva of adolescents with dental cariesdisease. Ann. Agaric. Environ. Med. 2012; 19(4): 711–716.\n\nCheng L, Zhang L, Yue L, et al.: Expert consensus on dental caries management. J. Oral Sci. 2022; 14: 17. Publisher Full Text\n\nThean H, Wong ML, Koh H: The dental awareness of nursing home staff in Singapur- a pilot study. Gerodontology. 2007; 24: 58–63. PubMed Abstract \| Publisher Full Text\n\nWu KJ, Tu CC, Hu JX, et al.: Severity of periodontitis and salivary interleukin-1b are associated with psoriasis involvement. J. Formosan Medical Association. 2022; 1(1): 1–9.\n\nFábián TK, Fejérdy P, Csermely P:Saliva in Health and Disease, Chemical Biology of. Wiley Encyclopedia of Chemical Biology. Hoboken, NJ, USA:John Wiley & Sons Inc.;2008; 1. : 1–9.\n\nTampa M, Mitran MI, Mitran CI, et al.: Mediators of Inflammation- A Potential Source of Biomarkers in Oral Squamous Cell Carcinoma. J. Immunol. Res. 2018; 2018: 1–12. Article ID 1061780. Publisher Full Text\n\nZhang JM, An J: Cytokines, inflammation, and pain. Int. Anesthesiol. Clin. 2007; 45(2): 27–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCogulu D, Onay H, Ozdemir Y, et al.: Associations of interleukin (IL)-1β,IL-1 receptor antagonist, and IL-10 with dental caries. J. Oral Sci. 2015; 57(1): 31–36. PubMed Abstract \| Publisher Full Text\n\nNavazesh M: Methods for collecting saliva. Ann. N. Y. Acad. Sci. 1993; 694(1): 72–77. Publisher Full Text\n\nAinamo J, Barmes D, Beagrie G, et al.: Development of the World Health Organization (WHO) community periodontal index of treatment needs (CPITN). J. Int. Dent. 1982; 32: 281–291.\n\nFrank J: Saliva and Dental Caries. J. Dental Clinics of North America. 1999; 43(4): 579–597. Publisher Full Text\n\nGiudice GL, Nicita F, Militi A, et al.: Correlation of s-IgA and IL-6 Salivary with Caries Disease and Oral Hygiene Parameters in Children. J. Dent. 2020; 8: 3.\n\nPeres CD, Gava RP, Zen IR, et al.: Salivary levels of Interleukin-6 and Tumor Necrosis Factor-alpha in girls’ aged7-17years practicing volleyball. Apunts Med. Esport. 2019; 54(203): 91–101. Publisher Full Text\n\nKhandavilli SD, Ceallaigh PÓ, Lloyd CJ, et al.: Serum C-reactive protein as a prognostic indicator in patients with oral squamous cell carcinoma. Oral Oncol. 2009; 45(10): 912–914. PubMed Abstract \| Publisher Full Text\n\nYadav K, Prakash S: Dental Caries: A Review. Asian J. Biomed. Pharm. Sci. Nepal, Clin Oral Invest. 2016; 8: 91–96.\n\nPark HC, Kim MY, Kim CH: C-reactive protein/albumin ratio as prognostic score in oral squamous cell carcinoma. J. Korean Assoc. Oral Maxillofac. Surg. 2016; 42(5): 243–250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlatt S, Schön H, Sagheb K, et al.: Hemoglobin, C-reactive protein and ferritin in patients with oral carcinoma and their clinical significance a prospective clinical study. J. Craniomaxillofac. Surg. 2018; 46(2): 207–212. PubMed Abstract \| Publisher Full Text\n\nGrimm M, Rieth J, Hoefert S, et al.: Standardized pretreatment inflammatory laboratory markers and calculated ratios in patients with oral squamous cell carcinoma. Eur. Arch. Otorhinolaryngol. 2016; 273(10): 3371–3384. PubMed Abstract \| Publisher Full Text\n\nSeyedmajidi M, Khodadadi E, Maliji G, et al.: Neutrophil Count and Level of Interleukin-1β and Interleukin-8 in the Saliva of Three to Five Year Olds with and without Dental Caries. J Dentistry, Tehran University of Medical Sciences, Tehran, Iran. 2015; 12(9): 662–668.\n\nda Silva Fidalgo TK , Freitas-Fernandes LB, Ammari M, et al.: The Relationship between Unspecific S-IgA and Dental Caries: A Systematic Review and Meta-Analysis. J. Dent. 2014; 42: 1372–1381. PubMed Abstract \| Publisher Full Text\n\nParkash H, Sharma A, Banerjee U, et al.: Humoral Immune Response to Mutans Streptococci Associated with Dental Caries. Natl. Med. J. India. 1994; 7: 263–266. PubMed Abstract\n\nDoifode D, Damle SG: Comparison of Salivary IgA Levels in Caries Free and Caries Active Children. Int. J. Clin. Den. Sci. 2011; 2: 10–14.\n\nBrandtzaeg P: The Oral Secretory Immune System with Special Emphasis on Its Relation to Dental Caries. Proc. Finn. Dent. Soc. 1983; 79: 71–84.\n\nGomes FI, Aragão MG, Barbosa FC, et al.: Inflammatory Cytokines Interleukin-1β and Tumour Necrosis Factor-α - Novel Biomarkers for the Detection of Periodontal Diseases: a Literature Review. J. Oral Maxillofac. Res. 2016; 7(2): e2. PubMed Abstract \| Publisher Full Text\n\nLi X, Wang L, Nunes D, et al.: Pro-inflammatory cytokines up regulate MUC1gene expression in oral epithelial cells. J. Dent. Res. 2003; 82: 883–887. PubMed Abstract \| Publisher Full Text\n\nKurtis B, Tuter G, Serdar M, et al.: Gingival crevicularuid levels of monocyte chemo attractant protein-1 and tumor necrosis factor-α in patients with chronic and aggressive periodontitis. J. Periodontol. 2005; 76(11): 1849–1855. PubMed Abstract \| Publisher Full Text\n\nBrailo V, Vucicevic-Boras V, Lukac J, et al.: Salivary and serum interleukin 1 beta, interleukin 6 and tumor necrosis factor alpha in patients with leukoplakia and oral cancer. Med. Oral Patol. Oral Cir. Bucal. 2012; 7(1): 10–15.\n\nColak H, Dülgergil C, Dalli M, et al.: Early childhood caries update: A review of causes, diagnoses, and treatments. J. Nat. Sci. Biol. Med. 2013; 4: 29–38. PubMed Abstract \| Publisher Full Text\n\nMcLachlan JL, Sloan AJ, Smith AJ, et al.: S100 and Cytokine Expression in Caries. Infect. Immun. 2004; 72(7): 4102–4108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFine DH, Markowitz K, Furgang D, et al.: Macrophage inflammatory protein-1 alpha: a salivary biomarker of bone loss in a longitudinal cohort study of children at risk for aggressive periodontal disease? J. Periodontol. 2009; 80(1): 106–113. PubMed Abstract \| Publisher Full Text\n\nAbdulkareem S, Jasim S: Assessment of IL-17 Level in Biofluids of Periodontopathic Patients and Healthy Individuals. J. Biochem. Cell. Arch. 2021; 21(1): 1405–1412.\n\nLee S, Margolin K: Cytokines in cancer immunotherapy. Cancers. 2011; 3: 3856–3893. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSahibzada HA, Khurshid Z, Khan RS, et al.: Salivary IL-8, IL-6 and TNF-α as Potential Diagnostic Biomarkers for Oral Cancer. Diagnostics. 2017; 7(21): 2–10. Publisher Full Text\n\nChawda JG, Chaduvula N, Patel HR, et al.: Salivary SIgA and Dental Caries Activity. Indian Pediatr. 2011; 48: 719–721. PubMed Abstract \| Publisher Full Text\n\nKoga-Ito CY, Martins CA, Balducci I, et al.: Correlation among mutans streptococci counts, dental caries, and IgA to Streptococcus mutans in saliva. Braz. Oral Res. 2004; 18: 350–355. PubMed Abstract\n\nAl-ibraheem JFA, Zyara YHE, Al-Quraine NT, et al.: Correlation between salivary immunoglobulin A and interleukin-1beta in smokers with dental caries.2023. Publisher Full Text" }	[ { "id": "163658", "date": "21 Feb 2023", "name": "Neihaya Heikmat Zaki", "expertise": [ "Reviewer Expertise Biotechnology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA brief summary of the article, \"Correlation between salivary immunoglobulin A and interleukin-1beta in smokers with dental caries\":\nThere were highly positive associations and significant differences between IL-1β and CRP levels in the two groups under the study. While no significant differences were found in the level of S-IgA and caries. This study also indicated no significant correlation between elevated IL-1β levels and s-IgA in smokers with caries disease.\nThere are some questions that must be answered by authors:\nWhy not include CRP in title and keywords because this parameter has significant differences, as mentioned in results?\n\nThere is a difference in numbers and age range of volunteers between the two groups under study, as mentioned in methods (page 3).\n\nIn the study population, you must mention the doctor who supervised the excluded patients with disorders and disease.\nComments can also be found here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "163659", "date": "27 Mar 2023", "name": "Ghizal Fatima", "expertise": [ "Reviewer Expertise Biomedical sciences" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 tried to explain and assess the levels of secretory IgA (s-IgA) and IL-1β in the saliva of smokers with dental caries, and they reached to a conclusion that there is no significant relationship between elevated IL-1β levels and s-IgA in smokers with dental caries. The whole paper is written in a well presented manner.\nIt is an important topic, especially taking important secretary markers like IgA (s-IgA) and IL-1β in the saliva of smokers with dental caries, and to discover the correlation between these parameters and dental caries. Despite a lot of substantial work has been done in this area, this work will make important contribution in the field of dental caries, as they have taken different markers for assessment.\nThere are just little or no issues with the manuscript that needs to be clarified/addressed. This article is nicely written, it addresses all the scientific questions and answers in the paper, and the statistical analysis is also nicely done. Figures and tables are synchronized in a well manner presentation. Furthermore, the discussion and conclusion parts arewell written, while results are given in an elaborated manner just as they warrant.\nBelow are few specific comments by section:\nAbstract: This section needs to be rechecked again for grammar.\nIntroduction: More information about the correlation in secretary markers with the dental caries has to be explained in detail.\nMethods: This section is well written; it explained all the methods done in the study in a detailed manner.\nReferences: All the recent references are taken 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": [] } ]	1	https://f1000research.com/articles/12-175
https://f1000research.com/articles/11-796/v1	18 Jul 22	{ "type": "Study Protocol", "title": "Protocol for a randomized study of the efficacy of ibandronic acid plus eldecalcitol in patients with gastric cancer after gastrectomy: A comparative study of different routes of administration of ibandronic acid", "authors": [ "Hisaki Aiba", "Nobuyuki Suzuki", "Hiroyuki Sagawa", "Hiroya Hashimoto", "Hiroaki Kimura", "Jumpei Inoue", "Takuya Usami", "Yuta Goto", "Yuichiro Mizuno", "Tomotaka Okubo", "Sunao Ito", "Shuji Takiguchi", "Hideki Murakami", "Nobuyuki Suzuki", "Hiroyuki Sagawa", "Hiroya Hashimoto", "Hiroaki Kimura", "Jumpei Inoue", "Takuya Usami", "Yuta Goto", "Yuichiro Mizuno", "Tomotaka Okubo", "Sunao Ito", "Shuji Takiguchi", "Hideki Murakami" ], "abstract": "Background: Patients who undergo gastrectomy for gastric cancer are susceptible to osteoporosis. To prevent a decrease in bone mineral density, an appropriate prophylaxis is considered important to adjust the post-gastrectomy condition. In this study, we will compare two different routes of administration of ibandronic acid (oral or intravenous) plus eldecalcitol as a potentially more suitable treatment for patients at a high risk of fragile fracture. Protocol: This study protocol describes a randomized, active-controlled, non-blind, single-center, phase II trial. For patients in the investigational arm (Group A), sodium ibandronate hydrate will be administered intravenously once a month with daily oral intake of eldecalcitol; for those in the control arm (Group B), sodium ibandronate hydrate will be administered orally once a month with daily oral intake of eldecalcitol. We will recruit patients aged 20–85 years who have undergone gastrectomy for gastric cancer and are at a risk of fragility fractures. The study will include patients with existing vertebral fractures and/or femoral proximal fractures, or with lumbar and/or proximal femur bone mineral density of less than 80% of the young adult mean. The primary outcome of this study will be the change in lumbar bone mineral density. We will also evaluate the changes in femur bone mineral density, bone metabolism markers, health-related quality of life as evaluated using the EuroQol 5 Dimension (EQ-5D), and digestive symptoms as evaluated using the Gastrointestinal Symptom Rating Scale after 52 weeks of treatment. Conclusions: We believe that appropriate treatments that are adjusted to the condition of patients after gastrectomy are important for the prevention of bone mineral loss. Registration: This study was accepted by the Japan Registry of Clinical Trials (jRCT1041200059, November 6, 2021).", "keywords": [ "osteoporosis", "gastric cancer", "post-gastrectomy", "ibandronic acid", "eldecalcitol" ], "content": "Introduction\n\nPatients who have parts of their stomachs removed due to gastric cancer may suffer from low nutrition, impaired calcium absorption, and bone metabolism disorder, which have been reported as post-gastrectomy complications.1,2 Although oncologic outcomes have improved in terms of long-term survival of patients with gastric cancer,3 more attention should be paid to maintenance and improvement of quality of life (QOL) of patients. Currently, while more than 10 million people in Japan have osteoporosis and the prevalence of osteoporosis after gastrectomy is reportedly 40%,4–6 awareness of how to reduce this post-operative risk remains insufficient. Moreover, it has been reported that osteoporosis is likely to occur due to malnutrition associated with nausea and ulceration of the mucosa as a result of anticancer drugs.7 Thus, the prevention of a possible decrease in bone mineral density, and if necessary, the treatment of osteoporosis, is considered important to prevent future fragile fractures. Moreover, as multi-disciplinary liaison between enterogastric surgeons and physicians who treat osteoporosis does not always occur, some patients do not receive examinations for osteoporosis or appropriate treatments. Better awareness of the risks of osteoporosis is urgently needed for both patients and doctors.8\n\nAdministration of eldecalcitol, an active vitamin D preparation, is assumed to be effective in compensating for the poor intake of vitamin D after gastrectomy.9 Furthermore, bisphosphonate is effective against osteoporosis, by inhibiting osteoclast function,10 and its usefulness has been proven in post-gastrectomy gastric cancer.11 However, the use of oral bisphosphonates (especially nitrogen-containing alendronate) may induce several adverse events.12 On the contrary, ibandronic acid, a type of bisphosphonate, has binary administration routes—oral intake and intravenous infusion13—but it is not clear which route is suitable for patients with gastric cancer after gastrectomy. Therefore, we aim to examine the efficacy of eldecalcitol and ibandronic acid on bone metabolism in patients in the postoperative stage of gastric cancer, focusing on the usefulness of different ibandronic acid administration routes in this randomized, active-controlled, non-blind, single-center (academic hospital in Japan), phase II trial.\n\n\nProtocol\n\nThis study was approved by the Japan Registry of Clinical Trials (jRCT), approval number jRCT1041200059, on November 6, 2021. The aim of this study is to evaluate the efficacy of different routes of ibandronic acid administration (oral tablets and intravenous injection) on bone mineral density, focusing on the digestive symptoms of patients after gastrectomy. The overall study flow chart is shown in Figure 1. This study protocol follows the SPIRIT guidelines.29\n\nWe are conducting this trial in accordance with the principles of the Declaration of Helsinki. The Nagoya City University Certified Review Board (CRB4200003) approved this study (deliberation number 2020B003, November 6, 2021). Written informed consent will be obtained from all trial participants by physicians in charge. Physicians and medical institutions involved in this research will cooperate with the monitoring procedure manual and carry out this study in compliance with clinical research methods, related notices, and good clinical practice.\n\nTo ensure compliance with the protocol treatment, the attending physicians will ask patients about the medication status at each visit and offer guidance if there is any negligence of medication. The attending physicians will confirm the status of compliance with this study.\n\nFor patients in the investigational arm (group A), sodium ibandronate hydrate (Bonviva® intravenous infusion 1 mg syringe; Chugai Pharmaceutical Co., Ltd., Tokyo, Japan) will be administered intravenously once a month with daily oral intake of eldecalcitol (Edirol® Capsule 0.75 μg; Chugai Pharmaceutical Co., Ltd.). For patients in the control arm (Group B), sodium ibandronate hydrate (Bonviva Tablets 100 mg; Chugai Pharmaceutical Co., Ltd.) will be administered orally once a month with a sufficient amount of water (approximately 180 mL) upon waking, with daily oral administration of eldecalcitol. For patients with hypercalcemia (corrected Ca = 10.5 mg/dL or more) identified at the time of registration or administration period, eldecalcitol dose will be reduced to appropriately 0.5 μg daily. Concomitant therapy will be permitted, including palliative and supportive therapies, such as analgesics, antiemetic agents, and anti-vomiting agents, for any symptoms due to primary disease or any adverse events. Additionally, chemotherapy for gastric cancer will be permitted. The usage of bone modulators and calcium/vitamin D supplements will not be permitted.\n\nWe will recruit patients aged 20–85 years who underwent gastrectomy for gastric cancer and are at a risk of fragility fracture. The inclusion criteria will be as follows: i) patients who underwent total or partial gastrectomy for gastric cancer; ii) patients with existing vertebral fractures and/or femoral proximal fractures or with lumbar and/or proximal femur bone mineral density of less than 80% of the young adult mean (YAM); iii) patients with a relatively stable general condition (performance status [PS], 0–2); iv) patients aged ≥20 years and <85 years on the registration date; v) patients with a post-surgery duration of less than six months; and vi) patients with their latest blood test (within 14 days before registration examination) complying with all of the following criteria: white blood cell count ≥ 1,500/μL; platelet count ≥ 100,000/μL; total bilirubin level ≤ 1.5 mg/dL; aspartate aminotransferase level ≤ 100 IU/L; alanine aminotransferase level ≤ 100 IU/L; serum creatinine level ≤ 1.5 mg/dL; and corrected Ca value < 10.5 mg/dL.\n\nThe exclusion criteria will be as follows: i) surgical resection extending to organs other than the stomach (e.g., esophagus and pancreas); ii) patients with long-term immobility; iii) pregnant or breastfeeding women; iv) patients with a history or high risk of osteonecrosis of the jaw; v) patients with a history of hypocalcemia; vi) patients with urolithiasis; vii) patients with a history of allergy to the drugs used in this study; viii) patients with distant metastasis (including ascites cytology positive patients) at the time of registration; ix) patients with significant anorexia; x) patients who are being treated for osteoporosis at the time of registration; and xi) patients who are considered inappropriate by the attending physicians.\n\nThe patient registration center will randomly assign patients to one of two treatment groups. The target number of patients in each group is 35. A computer-generated random sequence, made by Clinical Research Management Center in Nagoya City University Hospital, will be used to allocate a total of 70 patients with no stratification to sequential identification. An independent allocation manager will be responsible for the allocation process, and the allocation list will be concealed an envelope until interventions are assigned.\n\nThe primary outcome of this study is the change in lumbar bone mineral density. Bone mineral density will be measured based on the dual-energy X-ray absorptiometry (DEXA) method. Bone mineral density will be evaluated during the pre-observational period, 24 weeks after the start of medication, and at the time of final observation (52 weeks after the start of medication). In addition, the following secondary assessments will be performed: i) change in femur bone mineral density; ii) change in bone metabolism markers, including tartrate-resistant acid phosphatase 5b (TRACP-5b), total type 1 amino-terminal propeptide (P1NP), and 25OH vitamin D concentration (ELISA method); iii) change in health-related QOL, evaluated using the EuroQol 5 Dimension (EQ-5D)14 at the start date of medication, 12 weeks after the start of medication, 24 weeks later, and at the time of final observation (after 52 weeks); iv) change in digestive symptoms, evaluated using the Gastrointestinal Symptom Rating Scale (GSRS)15 at the start date of medication, 12 weeks after the start of medication, 24 weeks later, and at the time of final observation (after 52 weeks); v) presence or absence of fracture events during the medicated period; and vi) medication compliance rate. A total of 13 ibandronic acid doses will be scheduled in both groups over 52 weeks. The actual number of medications/13 will be calculated as the medication compliance rate.\n\nIn the pre-observation period, medical interviews will be conducted focusing on the patients’ backgrounds including sex, age, height, weight, menopausal age, current medication, medical history (presence or absence of past fractures), drinking/smoking history, presence or absence of femoral fractures in their parents, the area of extension of gastrectomy and the length of the remaining stomach, reconstruction techniques after gastrectomy, surgical intervention (open/laparoscopy/robot-guide), presence or absence of gallbladder resection, and presence or absence of post-operative anticancer drugs. In addition, the mineral density of the lumbar spine and femur will be measured using the DEXA method. Spine radiography will be performed in two directions: front and side in a standing position around the 4th lumbar vertebra and the 9th thoracic vertebra. The alignment of the entire spine and the existing fracture will also be confirmed. After confirming the selection criteria using medical charts and blood tests, the physicians will obtain written informed consent from all trial participants.\n\nOn day one of the trial, vital signs, blood test results, bone metabolism markers (TRACP-5b, P1NP, and 25OH vitamin D concentrations (using ELISA)), and physical function (EQ-5D and GSRS) will be examined.\n\nAfter the start of medication administration, the following will be recorded: vital signs; medication status; the presence of adverse events; presence or absence of new fracture events; blood tests (to determine whether patients comply with the same criteria from the inclusion periods); bone metabolic markers; improvement in physical function evaluation (EQ-5D and GSRS); and bone density measurement (lumbar spine and femur). Subsequently, medical checks for vital signs, compliance with medication, and the presence of adverse events will be performed at 4, 12, 24, and 52 weeks. After the discontinuation of drug administration, observation and investigation will be conducted up to 4 weeks after the date of the last administration of the research drug. If adverse events continue, follow-up and investigation will be continued until recovery from the adverse events (Table 1).\n\na Collection of information on drugs related to the efficacy or safety of the research drug.\n\nb Hematological testing of white blood cell count, red blood cell count, hemoglobin, hematocrit, and platelet count. Testing of alkaline phosphatase, aspartate aminotransferase (glutamate oxalacetate transaminase), alanine aminotransferase (glutamic pyruvic transaminase), lactate dehydrogenase, γ-glutamyl transpeptidase, creatine kinase, total bilirubin, total protein, albumin, urea nitrogen, creatinine, amylase, sodium, potassium, crawl, calcium, and inorganic phosphorus levels, and estimated glomerular filtration rate to confirm the safety of the study.\n\nc Adverse events are all unfavorable events regardless of a causal relationship with drugs.\n\n* Delays in the administration of test and control drugs will be acceptable for up to two weeks. If delayed, the observation date will be changed according to the number of days.\n\nTo evaluate safety, adverse events considered to be related to the research medicine will be monitored. All serious adverse events will be reported from the start of drug administration to the end of the follow-up period, along with the relationship among the type, severity, treatment content, time of expression, and turning point. Adverse events will include all undesirable or unintended symptoms and signs (including abnormalities in laboratory tests). Follow-up investigations will be performed until the disappearance of/or recovery from adverse events. All serious adverse events will be reported to the Certified Review Board by the physicians in charge. The investigators shall promptly and appropriately treat the subjects and record the adverse event, date of onset, severity, and outcome, as well as ascertain the causal relationship of the study drug.\n\nThe data will be collected using a unified Case Report Form, which can be found as Extended data28 and dissemination of the GSRS and EQ-5D charts.\n\nAll individuals involved in this research will properly handle the information of the research subjects and protect the personal information according to the “Act on the Protection of Personal Information.” The information of all patients involved in this study will be confidentially handled using the research specific identification code. Only the principal investigator (H.A.) will handle correspondence table between autonyms and anonyms. During the research, related information will be managed using a highly secured offline computer. Quality of the data will be checked during the data monitoring process.\n\nStatistical analysis will be performed within six months after data collection and enrollment of the final research participant. As it is predicted that the number of events will not be sufficient to make an early cancellation decision in the middle of registration, an intermediate analysis will not be performed.\n\nThe effectiveness of the investigational drugs will be evaluated using the intent-to-treat analysis for all eligible patients. The full analysis set (FAS) will comprise the entire group excluding the following research subjects: patients who do not meet the inclusion criteria, those who meet the exclusion criteria, those who are never administered drugs, and those for whom data are not obtained after the start of research medication. In the FAS, the per-protocol set (PPS) will comprise the entire group excluding the following research subjects: those for whom data on the main evaluation items are not obtained 52 weeks after the start of medication or those with a medication compliance rate of less than 80%. The sample size calculation was based on a previous study,3 which reported that another bisphosphonate preparation (alendronate internal use) and eldecalcitol increased lumbar bone density by approximately 6% at 12 months after administration compared with an increase of approximately 3% with eldecalcitol alone. We expect the difference between the intravenous infusion and oral intake combined with eldecalcitol will show 1.5% higher lumbar bone mineral density at 52 weeks (set to half of the additional effect of alendronate in the above previous study). The number of patients required to detect this with statistical significance is estimated to be 29 in each group with standard deviation 2%, significance level 5% (bilateral sides), and power 80%. Therefore, the target number of patients in this study is 35 (70 patients in total) in each group with the expectation that several patients will drop out.\n\nTo analyze effectiveness in the FAS and PPS, the rate of change in the bone mineral density from before the start of treatment will be calculated with respect to the lumbar bone mineral density at 52 weeks, and a comparison between groups will be performed using a Student’s t-test. In addition, for the analyses of secondary evaluation items in the FAS and PPS, the rate of change from before the start of treatment will be calculated for bone metabolism markers (TRAPC5b, P1NP), femoral bone mineral density, and QOL (EQ-5D, GSRS) at 52 weeks using a Student’s t-test for comparison between groups. The frequency of fragile fracture events in each drug group will be calculated, and an intergroup comparison will be performed using a chi-square test.\n\nOnly the principal investigator (H.A.) will prepare a correspondence table of the research subject identification code set and only the principal investigator and biostatistician (H.H.) can access the final trial dataset.\n\nIn this clinical study, an independent data monitoring officer (Y.G.) will be assigned. On-site monitoring will be conducted to confirm whether the study is carried out safely and according to the research plan and whether the data are collected reliably. Monitoring will be conducted before the start of the research, during the research (every three months), and at the end of the research. In addition, monitoring will be carried out at the time of registration and when a serious adverse event occurs.\n\nThe results of this study will be published in peer-reviewed journals.\n\nWe are currently recruiting patients.\n\n\nDiscussion\n\nThe efficacy of ibandronate in vertebral fractures has been demonstrated in patients with postmenopausal osteoporosis. According to the BONE study, oral daily (2.5 mg) and intermittent ibandronate (between-dose interval of >2 months) intake reduced the risk of new morphometric vertebral fractures by 62%.16 The DIVA study demonstrated the efficacy of intravenous ibandronate as a useful alternative to oral dosing,17 and intravenous ibandronate was approved in Japan in 2013 after a phase II/III clinical study (the MOVER trial18). This study demonstrated the non-inferiority of 1 mg of intravenous ibandronate to risedronate. At three years, the mean relative change in bone mineral density from baseline was 9.0% for the ibandronate group and 7.6% for risedronate groups.18 Moreover, the rate of first new vertebral fractures was 11.6% in the ibandronate group and 13.2% in the risedronate group. Although the rates of acute-phase reaction were higher in the ibandronate group (11.2% in the ibandronate group vs. 4.9% in the risedronate group), severe gastrointestinal adverse events were lower in the ibandronate group (0.5% in the ibandronate group vs. 2.2% in the risedronate group). The efficacy of monthly oral ibandronate was evaluated by the MOBILE study,19 which concluded that the monthly intake was at least as effective and well-tolerated as the daily ibandronate regimen in postmenopausal osteoporosis. The MOVEST study13 involved a randomized trial of 100 mg of monthly oral ibandronate versus 1 mg of monthly intravenous ibandronate in a Japanese population and showed comparable mineral density of the lumbar spine after 12 months of treatment (5.22% [oral] vs. 5.34% [intravenous]). Subsequently, monthly oral ibandronate intake was approved in Japan in 2016. In a real-world setting, a post-marketing observational study of 1,025 Japanese people revealed that 49.7% of the patients were concomitantly prescribed active vitamin D drugs, and preferable results with combined therapy were suggested in terms of bone mineral density at one year (3.54% [ibandronate alone] vs. 5.74%, [ibandronate + eldecalcitol]).20\n\nTo date, studies have reported that the intake of calcium gluconate,21 vitamin D,2 or bisphosphonate22 is useful after gastrectomy. Despite this, a standard treatment has not been established. In Japan, Segawa et al., reported that osteoporosis was recorded in 29.5% of patients after gastrectomy23 and reported that being over 70 years of age, weight loss, and chemotherapy were risk factors for osteoporosis. Sugiyama et al., reported that the rate of increase in lumbar bone mineral density was 9.3% at one year when oral alendronate was administered to patients after gastrectomy.22 Hirota et al., compared minodronate and eldecalcitol in post-operative patients with gastric cancer and the increase in mineral density of the lumbar spine was 4.52% in the minodronate group and 1.72% in the eldecalcitol group.24 Similarly, Ha et al., compared outcomes of administration of a weekly dose of alendronate (70 mg) + daily elemental calcium (500 mg) with cholecalciferol (1,000 IU) with those of administration of daily elemental calcium (500 mg) with cholecalciferol (100 IU) only. They reported a significantly smaller decrease in mineral density of the lumbar spine in patients who received alendronate and those who did not (-0.28 ± 5.20 vs. 8.39 ± 7.41, respectively).25 Although it is difficult to integrate the results of these studies owing to the varied characteristics of patients after gastrectomy, methodology, and types of investigational drugs, there seem to be consistent positive effects on bone metabolism as a result of osteoporosis prophylaxis.\n\nWe should be aware of the disadvantages of bisphosphonate, including acute-phase reaction, general malaise, renal failure, joint pain, gastrointestinal symptoms, hypocalcemia, osteonecrosis of the jaw, and atypical femoral fracture.26 However, according to the results of the MOVEST study,13 there were no significant differences in adverse events between the oral intake and intravenous groups except for gastrointestinal symptoms, and we hypothesize that intravenous injection is appropriate for the management of post-gastrectomy patients. Considering the less variable bioavailability of intravenous ibandronate than the highly variable bioavailability of oral ibandronate,27 especially in patients with malabsorption, a more stable concentration should be assured. In addition, a previous study reported that the completion rate of oral minodronate administration25 was 77.5% in patients who underwent gastrectomy. This relatively low rate of compliance with drug use should be assessed further.\n\nIn conclusion, we plan to conduct a prospective study on the efficacy of ibandronate plus eldecalcitol in patients with gastric cancer after gastrectomy, focusing on different routes of administration. We believe that the results of this trial will conclusively identify a novel strategy for the post-gastrectomy treatment of osteoporosis in patients with gastric cancer.\n\n\nData availability\n\nNo data are associated with this article.\n\nFigshare: case report form.xlsx. https://doi.org/10.6084/m9.figshare.20217278.v3.28\n\n\nReporting guidelines\n\nFigshare: SPIRIT checklist for ‘Protocol for a randomized study of the efficacy of ibandronic acid plus eldecalcitol in patients with gastric cancer after gastrectomy: A comparative study of different routes of administration of ibandronic acid’. https://doi.org/10.6084/m9.figshare.20217338.v1.29\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nZittel TT, Zeeb B, Maier GW, et al.: High prevalence of bone disorders after gastrectomy. Am. J. Surg. 1997; 174(4): 431–438. PubMed Abstract \| Publisher Full Text\n\nGlatzle J, Piert M, Meile T, et al.: Prevalence of vertebral alterations and the effects of calcium and vitamin D supplementation on calcium metabolism and bone mineral density after gastrectomy. Br. J. Surg. 2005; 92(5): 579–585. PubMed Abstract \| Publisher Full Text\n\nVan Cutsem E, Sagaert X, Topal B, et al.: Gastric cancer. Lancet. 2016; 388(10060): 2654–2664. PubMed Abstract \| Publisher Full Text\n\nInoue K, Shiomi K, Higashide S, et al.: Metabolic bone disease following gastrectomy: assessment by dual energy X-ray absorptiometry. Br. J. Surg. 1992; 79(4): 321–324. PubMed Abstract \| Publisher Full Text\n\nYoshimura N, Muraki S, Oka H, et al.: Prevalence of knee osteoarthritis, lumbar spondylosis, and osteoporosis in Japanese men and women: the research on osteoarthritis/osteoporosis against disability study. J. Bone Miner. Metab. 2009; 27(5): 620–628. PubMed Abstract \| Publisher Full Text\n\nHorii C, Iidaka T, Muraki S, et al.: The cumulative incidence of and risk factors for morphometric severe vertebral fractures in Japanese men and women: the ROAD study third and fourth surveys. Osteoporos Int. 2022; 33(4): 889–899. PubMed Abstract \| Publisher Full Text\n\nWissing MD: Chemotherapy- and irradiation-induced bone loss in adults with solid tumors. Curr. Osteoporos. Rep. 2015; 13(3): 140–145. PubMed Abstract \| Publisher Full Text\n\nMcLellan AR, Gallacher SJ, Fraser M, et al.: The fracture liaison service: success of a program for the evaluation and management of patients with osteoporotic fracture. Osteoporos Int. 2003; 14(12): 1028–1034. PubMed Abstract \| Publisher Full Text\n\nNilas L, Christiansen C: Influence of PTH and 1,25(OH)2D on calcium homeostasis and bone mineral content after gastric surgery. Calcif. Tissue Int. 1985; 37(5): 461–466. PubMed Abstract \| Publisher Full Text\n\nRodan GA, Fleisch HA: Bisphosphonates: mechanisms of action. J. Clin. Invest. 1996; 97(12): 2692–2696. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLim JS, Jin SH, Kim SB, et al.: Effect of bisphosphonates on bone mineral density and fracture prevention in gastric cancer patients after gastrectomy. J. Clin. Gastroenterol. 2012; 46(8): 669–674. PubMed Abstract \| Publisher Full Text\n\nJung SY, Sohn HS, Park EJ, et al.: Oral bisphosphonates and upper gastrointestinal cancer risks in Asians with osteoporosis: A nested case-control study using national retrospective cohort sample data from Korea. PLoS One. 2016; 11(3): e0150531. PubMed Abstract \| Publisher Full Text\n\nNakamura T, Ito M, Hashimoto J, et al.: Clinical efficacy and safety of monthly oral ibandronate 100 mg versus monthly intravenous ibandronate 1 mg in Japanese patients with primary osteoporosis. Osteoporos Int. 2015; 26(11): 2685–2693. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEuroQol Group: EuroQol--a new facility for the measurement of health-related quality of life. Health Policy. 1990; 16(3): 199–208. PubMed Abstract \| Publisher Full Text\n\nKulich KR, Malfertheiner P, Madisch A, et al.: Psychometric validation of the German translation of the Gastrointestinal Symptom Rating Scale (GSRS) and Quality of Life in Reflux and Dyspepsia (QOLRAD) questionnaire in patients with reflux disease. Health Qual. Life Outcomes. 2003; 1: 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChesnut CH 3rd, Skag A, Christiansen C, et al.: Oral Ibandronate Osteoporosis Vertebral Fracture Trial in North America and Europe (BONE). Effects of oral ibandronate administered daily or intermittently on fracture risk in postmenopausal osteoporosis. J. Bone Miner. Res. 2004; 19(8): 1241–1249. PubMed Abstract \| Publisher Full Text\n\nEisman JA, Civitelli R, Adami S, et al.: Efficacy and tolerability of intravenous ibandronate injections in postmenopausal osteoporosis: 2-year results from the DIVA study. J. Rheumatol. 2008; 35(3): 488–497. PubMed Abstract\n\nNakamura T, Nakano T, Ito M, et al.: Clinical efficacy on fracture risk and safety of 0.5 mg or 1 mg/month intravenous ibandronate versus 2.5 mg/day oral risedronate in patients with primary osteoporosis. Calcif. Tissue Int. 2013; 93(2): 137–146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller PD, McClung MR, Macovei L, et al.: Monthly oral ibandronate therapy in postmenopausal osteoporosis: 1-year results from the MOBILE study. J. Bone Miner. Res. 2005; 20(8): 1315–1322. PubMed Abstract \| Publisher Full Text\n\nTakeuchi Y, Hashimoto J, Kakihata H, et al.: Effectiveness of monthly intravenous ibandronate injections in a real-world setting: subgroup analysis of a postmarketing observational study. Osteoporos Sarcopenia. 2019; 5(1): 11–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrause M, Keller J, Beil B, et al.: Calcium gluconate supplementation is effective to balance calcium homeostasis in patients with gastrectomy. Osteoporos Int. 2015; 26(3): 987–995. PubMed Abstract \| Publisher Full Text\n\nSugiyama M, Kunisaki C, Kato H, et al.: Utility of alendronate in metabolic bone diseases after gastrectomy. Jpn J. Gastroenterol. Surg. 2011; 44(4): 361–373 [in Japanese]. Publisher Full Text\n\nSegawa T, Koeda K, Amano S, et al.: Bone metabolism in patients with gastric cancer following gastrectomy. The Journal of the Iwate. Medical Association. Communications. 2017; 69(4): 179–192 [in Japanese].\n\nHirota M, Takahashi T, Saito Y, et al.: Utility of monthly minodronate for osteoporosis after gastrectomy: prospective multicenter randomized controlled trials. Ann. Gastroenterol. Surg. 2021; 5(6): 754–766. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHa J, Lee JM, Lim Y, et al.: Effect of bisphosphonate on the prevention of bone loss in patients with gastric cancer after gastrectomy: A randomized controlled trial. Bone. 2020; 130: 115138. PubMed Abstract \| Publisher Full Text\n\nDiel IJ, Bergner R, Grötz KA: Adverse effects of bisphosphonates: current issues. J. Support. Oncol. 2007; 5(10): 475–482. PubMed Abstract\n\nBarrett J, Worth E, Bauss F, et al.: Ibandronate: a clinical pharmacological and pharmacokinetic update. J. Clin. Pharmacol. 2004; 44(9): 951–965. PubMed Abstract \| Publisher Full Text\n\nAiba H: case report form.xlsx. figshare. [Dataset].2022. Publisher Full Text\n\nAiba H: SPIRIT checklist. figshare. [Dataset].2022. Publisher Full Text" }	[ { "id": "168223", "date": "11 Apr 2023", "name": "Carlen Reyes", "expertise": [ "Reviewer Expertise Osteoporosis", "pharmacoepidemiology", "bone fracture" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a protocol of a randomized, active-controlled, nonblind study aimed to examine the efficacy of different ibandronic acid administration routes (oral versus iv) and its effects on bone metabolism in patients that have undergone gastric surgery for gastric cancer in an academic hospital in Japan. This is a very interesting study, well written, which will contribute to generate evidence regarding the best fracture prevention strategy in this population at risk.\nThe authors might wish to address the following suggestions:\nMajor changes:\nThe authors’ state that the age of recruitment is from 20 to 85 years old. This age range is wide, and does not fully match the age of increased risk of gastric cancer or the age of increased risk of bone fracture. Given that usually people with gastric cancer are diagnosed in their 60s’ and that the increased risk of fracture is mostly in people of at least 45 years old (if considering postmenopausal women), including people of 20 years old might underestimate the real effect of ibandronic acid on bone. The authors should consider narrowing the population to focus on those who are at increased risk of the exposure and outcome.\n\nIn the exclusion criteria: “people with a high risk of osteonecrosis of the jaw”, the authors should state how is this going to be addressed or how is it going to be defined.\n\nIn the exclusion criteria: “patients who are considered inappropriate by the attending physician” this exclusion criteria should be qualified as it can include many conditions that could lead to a selection bias; i.e. physicians may be more prone to exclude patients with more comorbid conditions who are also those with an increased risk of bone fracture.\n\nIn the target outcome paragraph, the authors define the lumbar bone mineral density as the primary outcome. The presence of osteophytes and osteoarthritis interfere with the bone densitometry measurements, which makes it not a suitable surrogate of osteoporosis1,2. In fact, osteoporosis is recommended to be assessed only through hip densitometry (i.e. FRAX tool). Authors should consider placing hip densitometry as their primary outcome or assessing osteoarthritis in their evaluation given the increased prevalence of this disease especially in those >50 years old.\n\nSafety paragraph: I am missing a list of adverse events to be assessed. This paragraph is too vague and a predefined list of outcomes (adverse events included and how they are going to be defined) should be clearly detailed.\nMinor changes:\n\nThe inclusion criteria do not specify if the patients need to comply all of the conditions or just any of them.\n\nThe phrase “post-surgery duration of less than six months” is unclear; does it mean that only those that had a surgery in the last 6 months will be included? Please specify.\n\nIn the exclusion criteria: “patients with significant anorexia”, I think the term “significant” is not correct. I would suggest that the authors exclude those with a diagnosis of anorexia.\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": [ { "c_id": "9631", "date": "05 May 2023", "name": "Hisaki Aiba", "role": "Author Response", "response": "Thank you for your advices to this article. Based on your comments and other reviewers’ comments, we will also modify the original protocols. We amended point-by-point according to your suggestions. (Comment of reviewer 1) The authors describe a protocol of a randomized, active-controlled, nonblind study aimed to examine the efficacy of different ibandronic acid administration routes (oral versus iv) and its effects on bone metabolism in patients that have undergone gastric surgery for gastric cancer in an academic hospital in Japan. This is a very interesting study, well written, which will contribute to generate evidence regarding the best fracture prevention strategy in this population at risk. The authors might wish to address the following suggestions: Major changes: (Question1) The authors’ state that the age of recruitment is from 20 to 85 years old. This age range is wide, and does not fully match the age of increased risk of gastric cancer or the age of increased risk of bone fracture. Given that usually people with gastric cancer are diagnosed in their 60s’ and that the increased risk of fracture is mostly in people of at least 45 years old (if considering postmenopausal women), including people of 20 years old might underestimate the real effect of ibandronic acid on bone. The authors should consider narrowing the population to focus on those who are at increased risk of the exposure and outcome. (Response1) This description is based on the PMDA approval of the ibandoronic acid in Japan. We are not actually intended to use it for younger patients. Anyway, the age at least over 45 might be reasonable. So, we changed it (Abstract and Section, “Patient”). (Question 2) In the exclusion criteria: “people with a high risk of osteonecrosis of the jaw”, the authors should state how is this going to be addressed or how is it going to be defined. (Response 2) The risk assessment of the osteonecrosis of the jaw is usually considered by examination of dentists. As local factors for the necrosis, chronic periodontitis, necessity of tooth extractions, poorly-fitting denture use should be checked before administration of bisphosphonate. Also, as the systemic factors, concurrent use of steroids, older age (above 65 years), and coexisting diagnosis of diabetes mellitus should be taken into account. Based on these factors, the dentists and clinicians in charge judge the risk and eligibility (Section, “Patient”). [Bansal H. Medication-related osteonecrosis of the jaw: An update. Natl J Maxillofac Surg. 2022 Jan-Apr;13(1):5-10.] (Question 3) In the exclusion criteria: “patients who are considered inappropriate by the attending physician” this exclusion criteria should be qualified as it can include many conditions that could lead to a selection bias; i.e. physicians may be more prone to exclude patients with more comorbid conditions who are also those with an increased risk of bone fracture. (Response 3) This phrase means the physician’s judgement to permit for the clinical study will be affected several clinical setting of patients. Social problem, economical problem, or the characteristics of the patients will be taken into account. We substitute the information in detail. (Section, “Patient”). (Question 4) In the target outcome paragraph, the authors define the lumbar bone mineral density as the primary outcome. The presence of osteophytes and osteoarthritis interfere with the bone densitometry measurements, which makes it not a suitable surrogate of osteoporosis. In fact, osteoporosis is recommended to be assessed only through hip densitometry (i.e. FRAX tool). Authors should consider placing hip densitometry as their primary outcome or assessing osteoarthritis in their evaluation given the increased prevalence of this disease especially in those >50 years old. (Response 4) Your comment is reasonable. I changed the description (Section, “Target outcome”). (Question 5) Safety paragraph: I am missing a list of adverse events to be assessed. This paragraph is too vague and a predefined list of outcomes (adverse events included and how they are going to be defined) should be clearly detailed. (Response 5) Your comment is reasonable. We inserted several points which should be monitored. Also, we added the criteria for the evaluation of adverse events (Section, “Target outcome”). Minor changes: (Question 6) The inclusion criteria do not specify if the patients need to comply all of the conditions or just any of them. (Response 6) We inserted the description. (Section, “Patients”). (Question 7) The phrase “post-surgery duration of less than six months” is unclear; does it mean that only those that had a surgery in the last 6 months will be included? Please specify. (Response 7) We inserted this criterion with the aim that the early osteoporosis treatment might be effective for the patients after gastrectomy. As you suggested, this phrase is unclear, thus, we changed the phrase as “patients less than six months after gastrectomy.” (Section, “Patients”). (Question 8) In the exclusion criteria: “patients with significant anorexia”, I think the term “significant” is not correct. I would suggest that the authors exclude those with a diagnosis of anorexia. (Response 8) The patients after gastrectomy sometimes experience the change in eating habits and lose weights. This condition might invoke the infringement of regular intake of drugs. Anyway I deleted “significant”. (Section, “Patients”)." } ] }, { "id": "168230", "date": "25 Apr 2023", "name": "Friederike Thomasius", "expertise": [ "Reviewer Expertise Osteology", "diagnosis of osteoporosis", "Osteoporosis therapy", "Osteoporosis Guideline" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Patients after gastrectomy due to Gastric cancer will be randomized into a single center, non blinded trial with two treatment options: Oral Ibandronate monthly plus eldecalcitol, or intravenous ibandronate monthly plus eldecalcitol.\nThe planned patients to be included patients cover the age between 20 and 85 years, patients with existing fractures and lowered BMD, the planned number of patients to be included in this study is 70 (35 per group), duration of the study 52 weeks.\nComments The inculsion criteria should include a sufficient daily calcium intake which should be assessed with a standardized questionnaire, or due to known data of average calcium intake in the examined population, a calcium supplement should be provided\nWith regard to exclusion criteria the contraindications for ibandronic acid therapy are partially missing, and the following need to be added:\ninability to sit or stand in an upgright position stenosis of the esophagus or achalasia hypersensitivity to Ibandronic acid\nTarget outcomes of bone metabolism markers should include CTX as one of the most examined parameters.\nGeneral comment Choosing an oral Bisphosphonate as Osteoporosis therapy after gastrectomy needs more explanation. Due to the low bioavailability of oral Bisphosphonates, a gastrectomy and a change in the passage might negatively influence the absorption of the oral Ibandronate.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "9632", "date": "05 May 2023", "name": "Hisaki Aiba", "role": "Author Response", "response": "Thank you for your advices to this article. Based on your comments and other reviewers’ comments, we will also modify the original protocols. We amended point-by-point according to your suggestions. Summary: Patients after gastrectomy due to Gastric cancer will be randomized into a single center, non blinded trial with two treatment options: Oral Ibandronate monthly plus eldecalcitol, or intravenous ibandronate monthly plus eldecalcitol. The planned patients to be included patients cover the age between 20 and 85 years, patients with existing fractures and lowered BMD, the planned number of patients to be included in this study is 70 (35 per group), duration of the study 52 weeks. (Question 1) The inclusion criteria should include a sufficient daily calcium intake which should be assessed with a standardized questionnaire, or due to known data of average calcium intake in the examined population, a calcium supplement should be provided. (Response 1) Thank you for your comment. I included the questions of behavior of calcium intake and supplement. Also, we checked the baseline calcium values. (Section, “Schedule”) (Question 2) With regard to exclusion criteria the contraindications for ibandronic acid therapy are partially missing, and the following need to be added: inability to sit or stand in an upgright position, stenosis of the esophagus or achalasia, hypersensitivity to Ibandronic acid. (Response 2) We added the following; inability to sit or stand in an upright position, stenosis of the esophagus or achalasia, hypersensitivity to Ibandronic acid on the description about contraindications. (Section, “Patients”) (Question 3) Target outcomes of bone metabolism markers should include CTX as one of the most examined parameters. (Response 3) We depicted it. (Section, “Target outcomes”) General comment Choosing an oral Bisphosphonate as Osteoporosis therapy after gastrectomy needs more explanation. Due to the low bioavailability of oral Bisphosphonates, a gastrectomy and a change in the passage might negatively influence the absorption of the oral Ibandronate." } ] } ]	1	https://f1000research.com/articles/11-796
https://f1000research.com/articles/11-145/v1	04 Feb 22	{ "type": "Research Article", "title": "Situations of work-related diseases and injuries among agriculturists in the upper northeast regions of Thailand", "authors": [ "Sunisa Chaiklieng", "Chuthamas Chagkornburee", "Pornnapa Suggaravetsiri", "Chuthamas Chagkornburee", "Pornnapa Suggaravetsiri" ], "abstract": "Background: Agriculturists exposed to health hazards are affected by increased occupational disease. This retrospective study aimed to investigate situations of work-related diseases and injuries among agriculturists in upper northeast Thailand. Methods: The secondary data of international classification of diseases 10th revision (ICD-10) case reports of occupational disease among farmers for 2014-2016, from the database of the Health Data Center (HDC), were used. We collected the number of registered farmers from the agricultural office and secondary data of ICD-10 from the hospital information system (HIS) of healthcare services in Udon Thani and Roi-Et provinces. The annual morbidity rate of occupational diseases was analyzed and presented at a rate per 100,000 farmers of the studied area. Results: Among farmers who visited healthcare service providers, lung disease, which was not reported as occupational disease of the HDC database, was the highest ranking of all diseases, followed by work-related musculoskeletal disorders (WMSDs), noise- and heat-related diseases, and pesticide toxicity, respectively, while the injury rate was as high as that of WMSDs. The morbidity rate of Udon Thani was higher than that of Roi-Et province, whose rate was closer to the national rate. The number of farmers from the HDC database was not reflective of numbers of registered farmers, even though the case reports of the HDC was lower than that of the HIS with occupational ICD-10 codes, which might explain the underestimation of the morbidity rate of occupational disease. Conclusions: The morbidity rate of lung disease, noise- and heat-related diseases, and pesticide toxicity increased every year (2014-2016) and reflects the health problems among agriculturists in Thailand. The underestimation in the reported disease rate is explained from big data analysis by the rare recording of work-related cases among those agriculturists. Therefore, Thai agriculturists should be supported in registration with occupational diseases surveillance as holistic healthcare.", "keywords": [ "Big data", "occupational disease", "agriculturists", "surveillance", "ICD-10" ], "content": "Introduction\n\nFrom the survey results in 2019, it was found that the total number of employed persons in Thailand was 37.5 million persons. The number of workforce members who had no social security or were informal workers was about 20.4 million, or 54.3%. By region, those in informal employment working in the Northeast made up the largest proportion (34.9%), followed by Central Thailand (23.4%), the North (20.9%), and the South (14.0%).1 When the economic activities of those in informal employment were considered, it was found that more than half of all informally employed persons worked in the agriculture sector (about 11.5 million, or 56.4%).1\n\nAgriculturists exposed to health hazards are affected by increased occupational disease. According to a global report on occupational diseases, the highest prevalence of such disease was work-related musculoskeletal disorders (WMSDs), which ranged between 55.6-97.2%.2–4 The highest reported prevalence was in Korea (97.2%),2 followed by Saskatchewan province of Canada (85.6%),3 and Ireland (55.6%).4 The next most prevalent disease was heat-related disease, which ranged from 35.6-72.3%.5–8 The highest reported prevalence was found in Eastern North Carolina, USA,5 followed by Northeast Italy,6 and North Carolina, USA.7,8 Additionally, prevalence of hearing loss from noise was reported at levels of 16.9-36.1%,9,10 which were 16.9%9 in Korea and 36.1%10 in the USA, while work-related injuries were reported at a prevalence of 69.0% in Nepal.11 Infectious disease was reported in ranges between 0.7-13.5%,12–14 where brucellosis cases in Greek farmers and livestock breeders corresponded to an annual incidence rate of 7.1 per 100,000 population,15 and 8.8-17.0%, which were 8.8% in China,16 8.9% in the Western Cape of South Africa,17 and 17.0% in the Sunsari District of Nepal.18 Chronic lower respiratory tract disease was also reported as chronic obstructive pulmonary disease (COPD) and was found in a systematic review to be from 3.0% to 68.0%,19 as in France, where the rate of COPD was 9.5%,20 and South Africa, where the rate was 3.0% for asthma.21\n\nIn Thailand, the health database of the Health Data Center (HDC) (https://hdcservice.moph.go.th/hdc/main/index.php) showed that the highest prevalence of disease between 2014-2016 was for work-related injuries (about 0.6%), followed by WMSDs (about 0.3%), neurotic hearing loss from noise and heat-related disease (0.2%), and pesticide toxicity (0.04%). There was no statistical report on work-related diseases which included respiratory symptoms or lung disease, and infectious disease on the health database of the HDC. In 2019, the prevalence of chronic lower respiratory tract disease was reported of 15-59 years old population to be 1.8%.22 The previous five-year retrospective study showed a higher morbidity rate of all cases in the research from the Nongbualamphu province of Thailand, which found the highest incidence was for WMSDs (21.7%), followed by pesticide toxicity (0.18-42.9%),23–25 which can be compared to the three-year incidence rate of 48.48 per 100,000 farmers or 0.05%.26 The study also indicated a rate of infectious disease at 0.51%,23 with chronic lower respiratory tract disease (1.11%) and skin disease (1.5%)23 as additional diseases which occurred. The case-control study in agriculturists of Khon Kaen found that noise-induced hearing loss was 49.7%.27 Work-related injuries had a prevalence of 20.1% among the farmers of the Phayao province28 and the prevalence of heat-related disease was also reported as 13.5 per 100,000 farmers, or 0.014%, in agriculturists of the Khon Kaen province.29\n\nThose work-related diseases and injuries in Thailand were identified by the International Statistical Classification of Diseases and Related Health Problems 10th revision (ICD-10)30 (https://icd.who.int/browse10/2019/en) and the specified Y96 external code (work related condition). Nowadays, however, some cases of agriculturists are not practically identified, and this leads to inconsistency in data as above. Hence, healthcare for agricultural workers in Thailand is not covered by labor legislation and as a result they only access services from the National Health Security Office (NHSO) (https://eng.nhso.go.th/view/1/Home/EN-US), without claiming compensation for diseases related to agricultural activities. Although occupational disease case reports are important for enabling the surveillance system to carry out national health policy among agriculturists, there is still no representative morbidity rate of the upper northeast of Thailand. Therefore, this study aimed to investigate situations of work-related diseases and injuries among agriculturists in the upper northeast of Thailand by using the case study of the Udon Thani and Roi-Et provinces.\n\n\nMethods\n\nThis study was approved by the Human Research Ethics Committee of Khon Kaen University (No. HE592154). The underlying data for this study is secondary data collected by the Health Data Centre and agricultural registries and is restricted due to ethical and data protection considerations. Under the ethical approval from the Human Research Ethics Committee of Khon Kaen University (No. HE592154), participants’ data was not allowed to be shared and the secondary data of health dataset and registered farmer dataset were utilized under restriction. The individual information or further reused inclusion data is not permitted under the ethically approved conditions for human research. This was confirmed under the data protection of human/participant privacy as no individuals gave explicit written consent that their identifiable data can be made publicly available.\n\nThis study was a retrospective analytic study that used secondary data from three sources, which were 1) The health database of the Health Data Center (HDC) of Thailand, between 2014 and 2016, for the number of cases of occupational disease among agriculturists (by occupational code) who visited public healthcare service providers as details in HDC data collection and public health region seven (PH region 7) and public health region eight (PH region 8) in Thailand; 2) The secondary data of the list of cultivating farmers who had registered with the provincial agriculture offices of Roi-Et (http://www.roiet.doae.go.th/) and Udon Thani provinces (http://www.udonthani.doae.go.th/) between 2014 and 2016 as in the report Form 04-2; and 3) The secondary data of ICD-10 utilized from the hospital information system (HIS) database as detailed in the HIS data collection that used for routine care services in primary care units, secondary, and tertiary hospitals was selected from the provinces of the pilot study areas (Roi-Et and Udon Thani).\n\nThe sample size of the retrospective descriptive study was the total number of registered agriculturists of the studied area and all cases of occupational diseases who were farmers visiting healthcare units for health services at the studied areas of the upper Northeast of Thailand, during the three years from 1st January, 2014 to 31st December, 2016. The inclusion criteria for farmers from the HDC were those registered under the occupational codes shown in Table 1. The registered agriculturists from a provincial agriculture office of Roi-Et and Udon Thani provinces who were included as the population in this study met the inclusion criteria of cultivated farming classifications according to agricultural activity during 2014-2016.\n\nFor analysis of the morbidity rate of occupational diseases among agriculturists in the upper northeast region, this study used two provinces as representative of the upper northeast of Thailand. The agriculturists from Roi-Et province were representative of PH region 7 and those of Udon Thani province were representative of PH region 8 of Thailand, to compare the healthcare service visits for all cases among farmers in provinces, or PH regions, to those of Thailand as a whole. All registered agriculturists of the two provinces were the representative population of agriculturists in those provinces in Thailand as the nature of the impact from agricultural work was found to be similar across from other areas of Thailand as Roi-Et is more real agriculture among these two provinces.31,32\n\nHDC data collection\n\nThe health database reports for national and PH region 7 and PH region 8 levels in the northeast region of Thailand were collected by using the ICD-10 code of occupational disease used in reports to the HDC22,33–37 over 2014-2016. The data collected were the total number of planting farmers according to occupation code (Table 1) and the number of cases of occupational disease for five occupational diseases classified by ICD-1022,33–37 (Table 2). The total number of registered farmers to the Ministry of Agriculture and Cooperatives and the provincial agriculture office was collected from agricultural database.31 The inclusion criteria for farmers from the HDC were those registered under the occupational codes shown in Table 1.\n\n* additional code consideration or exclusion is correlated with the definition of work-related diseases or injuries.\n\n+ In case of missing codes of the 5th digit (activities, 2) and Y96, they were also countable for the ICD-10 cases from main code consideration of occupational diseases or injuries among registered farmers from provincial agriculture office.\n\nProvincial agriculture offices of Roi-Et and Udon Thani provinces data collection\n\nThose data of the ICD-10 code and the registered agriculturists were also collected as a big dataset for Roi-Et and Udon Thani provinces as representative provinces in the upper northeast regions of Thailand. The secondary data was collected from a provincial agriculture office was as a big dataset with the number and lists of registered agriculturists in the pilot study areas (Roi-Et and Udon provinces) during 2014-2016. The record was explored and copied into an excel file by the information technology personnel who responsible for database of a provincial agriculture office with permission for the use of the data in this study. Reports were included containing the name, gender, house address, farming activities, and planting areas of all registered farmers during 2014-2016 from the Roi-Et and Udon Thani provinces who were cultivated farmers classified according to agricultural activity, i.e., cultivating rice, cassava, corn, soybean, or sugarcane as in the report Form 04-2.\n\nHIS database data collection\n\nAnother big dataset collected from the provincial public health offices was the ICD-10 code record of the health standard data structure (43 health files of file 10, file 15, and file 19) on cases visiting Primary Healthcare Units (PCUs) and the secondary or tertiary hospitals in Roi-Et and Udon provinces for healthcare services in 2014 to 2016). Health-related information of registered agriculturists regarding cases with ICD-10 codes, was listed in the 43 health files of the health standards data structure and standards used for the hospital information systems (HIS) of the hospitals in Roi-Et and Udon Thani provinces: file 10 – diagnosis (opd), file 15 – diagnosis (ipd), and file 19 – surveillance. ICD-10 codes related to occupational disease and the disease data in an excel file was collated using the inclusion and exclusion criteria outlined in Table 2. Those ICD-10 codes were explored and copied from provincial health database by the permission code available only for the information technology personnel of the organization. Raw dataset collected under permission access and restriction use of ICD-10 codes and lists of registered farmers were collated and sorted by the installed hardware to computer including: Processor of CPU @ 1.99 GHz, Installed RAM of 16.0 GB, System type of 64-bit operating system, x64-based processor, and Software including Windows 10 or equal to higher version and Microsoft office (excel program) 2016 licensed by Khon Kaen University or equal to higher version. Duplicated registration of farmers or disease accounts were screened out and removed for duplication and showed that the final number of registered farmers in the period 2014 to 2016 of the Udon Thani province was 154,478, while that of Roi-Et was 207,465 farmers. The ICD-10 codes met the inclusion and exclusion criteria outlined as shown in Table 2 for occupational disease and injuries were considered for analysis of occupational diseases among the registered farmers. The collaborated data of registered farmer and accounted ICD-10 codes among the registered was explored and used for the morbidity rate analysis.\n\nSTATA version 10.0 software (https://www.stata.com/) (StataCorp LLC: College Station, TX) was used for all statistical analyses in this study. A freely accessible alternative software which can be used to complete the statistical analyses in this study would be program R for statistical computing (https://www.r-project.org/). Categorical data were presented as numbers and percentages. To estimate the annual morbidity rate of an occupational disease with a confidence interval (95% confidence interval) among registered agriculturists from 2014-2016. The data of occupational diseases among planting farmers or registered agriculturists was analysed for morbidity rate as per the following formula:\n\nIn case of missing codes of the 5th digit (activities, 2) for work-related injuries or Y96 for work-related musculoskeletal disorders (WMSDs), they were also countable for the ICD-10 cases of occupational diseases or injuries among registered farmers from provincial agriculture office by considering the main ICD-10 code.\n\nThe underlying data (TABLE A B C: Situations of work-related diseases and injuries among agriculturists in the upper northeast regions of Thailand) of this study is available in the underlying data statement.46\n\n\nResults\n\nIn 2014-2016, the number of registered farmers collected from provincial agriculture office in Roi-Et and Udon Thani provinces was 207,465 and 154,478, respectively. There were 53,794 (34.82%) total cases who visited for health services in Udon Thani, and 77,438 (37.32%) total cases in Roi-Et during the three-year period. The number of visiting cases in each year varies upon types of diseases and, among those health problems, we found the highest cases from injuries both in Roi-Et (n=20,341) and Udon-Thani (n=14,025) provinces in 2016.\n\nFor the number of farmers from HDC database, the number of HDC farmers varied upon types of diseases or injuries as well as the number of cases of diseases as shown in Table 3. There were the highest number of farmers accessed to healthcare from cases of noise and heat-related disease in Roi-Et (82,458) and Udon Thani (1,324,168). The number of registered farmers recorded of Ministry of Agriculture and Cooperatives31 were 207,780 and 163,704 for Roi-Et and Udon Thani in 2016, respectively. The morbidity rate of occupational disease in Thailand, i.e., WMSDs, noise- and heat-related diseases, and pesticide toxicity (per 100,000 farmers), increased between 2014 and 2016, according to the HDC database. According to national results, the results of PH region 8 and Udon Thani supported this trend of increasing morbidity rate (as shown in Figure 1). Noise- and heat-related injuries have been increasing every year in all regions, which is also supported by the rates from Roi-Et province and PH region 7.\n\n† the number of planting farmers as per the occupational code from the Health Data Centre (HDC) database.\n\n‡ the number of registered farmers of the Ministry of Agriculture and Cooperatives.36\n\n* HDC morbidity rate per 100,000 HDC farmers.\n\n** There was no report available of occupational disease category by HDC.\n\nThis study analyzed the rate of chronic lower respiratory symptoms (J40-J47) as one category of lung diseases among farmers between 2014 and 2016. This was analysed in this manner following the categories of the top five prevalence of occupational diseases among crop planting farmers23 as in Table 2. The disease was not directly reported as representative of a kind of occupational disease classified in the HDC database, but there was a reported data available as categories by age groups for 5-9 years, 10-14 years, 15-59 years, and 60 years or more, which could be accountable for the cases of workforce ages as agriculturists.22 In 2016, the national rate of lung disease ranged from 943.9-1,129.0 per 100,000 farmers, and the highest rate was found in Roi-Et (1,527.2), which was higher than that of the national total. The rate of lung disease was followed in order by the rates of injuries, WMSDs, noise- and heat-related diseases, and pesticide toxicity, respectively, and there was a higher morbidity rate in Udon Thani compared to Roi-Et. In Roi-Et, injuries were found to be most prevalent at 319.4 per 100,000 farmers in 2014, which was followed by WMSDs (41.0), pesticide toxicity (11.4), and noise- and heat-related diseases (6.8), while the top-ranking rate of Udon Thani was that of WMSDs (405.3), which was followed by those of noise- and heat-related diseases (47.4), and pesticide toxicity (12.8), while the injuries rate was 400.0. In 2015, apart from injuries (352.2), lung disease (1359.3) was the top-ranking rate of diseases among farmers in Roi-Et, the following morbidity rate of diseases were noise- and heat-related diseases (67.3), WMSDs (62.7), and pesticide toxicity (14.5). Meanwhile, in Udon Thani, the morbidity rate of lung disease was the highest rate (831.6), followed by WMSDs (296.8), noise-and heat-related diseases (88.0). A similar trend was found during 2014-2016, in that the morbidity rates of Udon Thani, or PH region 8, were mostly representative of the national rate. In 2016, the morbidity rate of chronic lower respiratory symptoms (lung diseases) was the highest among all diseases and injuries in Roi-Et Province and was also shown to be the highest among all diseases and injuries when compared to other regions and the nation (Figure 1, see underlying data: TABLE A).46\n\nEven though the number of cases of diseases was stable, the number of people who had a recorded occupation of farmer in the HDC database and the number of registered farmers were different. Udon Thani as well as the nation always had a higher recorded number of farmers in comparison to the registered number of farmers, which contrasted with Roi-Et, as shown in Table 3.\n\nIn 2016, all rates of Udon Thani were higher than those of Roi-Et, i.e., with a 5.89, 1.16, 1.89, and 1.20-times higher rate of WMSDs, noise- and heat-related diseases, pesticide toxicity, and injuries, respectively.\n\nAlthough the number of registered farmers of Roi-Et Province was higher than that of Udon Thani, the recorded cases or number of farmers to health service visits were lower compared to Udon Thani, for all diseases and injuries. The morbidity rate of disease was higher in Udon Thani in comparison to Roi-Et. It was found that the morbidity rates of the nation and Udon Thani Province had been increasing similarly and were much higher than the morbidity rate of Roi-Et.\n\nWhen comparing the rate of occupational disease and injuries among registered farmers in 2016 to the rate of farmers who visited health service providers, the higher rate was found in the registered farmers for WMSDs, noise- and heat-related diseases, and injuries (pesticide toxicity was scarcely different), especially in Roi-Et, where the rate was 27.58 times higher for injuries and 16.32 times higher for WMSDs. Regarding those who identified as farmers when visiting Udon Thani health service providers, they had a 1.76 times lower rate of WMSDs and a 15.83 times lower rate of injuries than registered farmers. Similarly, the rates of noise- and heat-related diseases among those who identified as farmers in Udon Thani and Roi-Et provinces were about two times lower in the HDC database, in comparison to the rates of registered farmers.\n\nAccording to the secondary dataset of the health standard data structure (43 health files) and the number of agriculturists registered to the provincial agricultural office during 2014-2016, without duplicated registration of farmers or disease accounts, the analysis showed the number of registered farmers in 2014 to 2016 of Udon Thani Province was 154,478, while that of Roi-Et was 207,465 farmers (see underlying data: TABLE C).46 By utilizing the 43 health files data of the ICD-10 code of occupational cases during 2014 to 2016, there were 53,794 total cases of registered farmers accessed for health services in Udon Thani, and 77,438 total cases in Roi-Et. The number of cases among registered agriculturist varied upon types of diseases or injuries. The results of morbidity rates in 2016 are presented in Table 4. The ranking of morbidity rates of work-related disease among registered farmers was as follows: WMSDs, noise- and heat-related diseases, and pesticide toxicity, respectively. Work-related injuries had the highest rate among all disorders of Udon Thani and Roi-Et (Table 4).\n\nThe significance of diseases among registered farmers changed, in that the rate of WMSDs in Roi-Et became higher than that of Udon Thani, according to the 43 health files data structure, when compared to the HDC database (Figure 2 and underlying data: TABLE B, C).46\n\nFrom the 43 health files data structure, it was found that the morbidity rates among the registered farmers of Roi-Et were highest ranked as follows: WMSDs, noise- and heat-related diseases, and pesticide toxicity, respectively, in every year from 2014 to 2016. The morbidity rate of registered farmers was not consistent with those who identified as farmers, as recorded by the HDC, in every year from 2014 to 2016. In 2016, farmers in Roi-Et had a higher morbidity rate of WMSDs, and the rate of noise- and heat-related diseases was higher than that of Udon Thani. The rates and trends increased every year in this manner, except for pesticide toxicity, which had a lower rate in Roi-Et when compared to that of Udon Thani. In Udon Thani, the morbidity rates from the 43 health files were found to have the same ranking as Roi-Et for every disease. WMSDs had the highest morbidity rate, followed by noise- and heat-related diseases and pesticide toxicity, respectively, and all those rates had increased in the same manner from 2014 to 2016.\n\nThe results from the HDC showed that of all occupational diseases, there was a clear increasing trend for pesticide toxicity and noise- and heat-related diseases, which was not consistent with the decreasing rate of WMSDs in Udon Thani. However, Udon Thani had a higher overall rate than Roi-Et for all occupational diseases.\n\n\nDiscussion\n\nThe numbers of work-related diseases and injuries reported at present among Thai farmers are likely to be lower than the true numbers because the health surveillance among farmers or agricultural workers may not be correct, as can be seen from the following findings and discussion.\n\nBy using registered farmers as the population for the morbidity rate calculation, which was different from using those who used a farmer occupational code as the population to calculate the rate from the HDC database, one could explain the inaccurate number of agriculturists found in healthcare service reports. The number of farmers from the HDC database may sometimes include duplicate recordings of farmers who visited healthcare providers more than once or sometimes be missing the occupational code identification of an agriculturist. That inaccurate number of farmers from the HDC database has led to incorrect results of morbidity rate. Therefore, for the next step of analysis, it was necessary to study registered agriculturists for accounted cases of occupational disease among agriculturists.\n\nThose results of work-related diseases and injuries, excluding lung disease, among registered agriculturists, from the two case studies of Udon Thani and Roi-Et provinces from analysis of the HDC database and the 43 health files data, showed the same ranking of morbidity rate, which were injuries, WMSDs, noise- and heat-related diseases, and pesticide toxicity, respectively. Consequently, both Udon Thani and Roi-Et were possibly representative of national rates. For the case of lung diseases, the number of cases who had lung disease specific to the age group of between 15 to 59 years of age was analysis for the morbidity rate from HDC database, the disease was not further reported in the HDC database as occupational disease as mentioned before. It showed the highest morbidity rate from our analysis for chronic lower respiratory tract disease, or a specified lung disease among occupational diseases. However, when considering the morbidity rates (per 100,000 registers farmers) of diseases and injuries from 2014 to 2016 found in 43 health files data, it was found that the highest morbidity rate from our analysis was not for a specified lung disease which had not been reported before as an occupational disease in the disease surveillance system of the Ministry of Public Health, Thailand. The high morbidity rate of occupational lung disease, which was not yet covered by the HDC database, might be explained by the fact that in disease diagnosis, it was not found to have an external cause, which would require recoding to Y96 to specify an occupational disease group. Some other diseases related to exposure to agrochemical hazards were recommended from the previous studies, e.g., Parkinson's disease38; cancers of specific organs, e.g., lung, pancreas, lymphohematopoietic organs, bladder, prostate gland,39 or thyroid40; gestational diabetes mellitus (GDM)41 or diabetes42; etc. In covering those diseases to build up the surveillance system among agriculturists, some more investigation is still needed to confirm an association between each disease and a specific toxic substance used in pest control. Moreover, occupational lung disease should be considered in health surveillance of disease among farmers as per the previous report done by using a systematic review among Thai agriculturists.43\n\nExcluding lung disease, the three top-ranking rates across all the regions and years were injuries and/or WMSDs, followed by hearing loss and diseases from heat, and pesticide toxicity, respectively. In addition, the rate of injuries was highest among all occupational diseases in Roi-Et, Udon Thani, and the nation. Udon Thani, which was chosen as a good representative province of PH Region 8, had a top-ranking rate which was the same as the nation, namely that of injuries, followed by WMSDs, noise- and heat-related diseases, and pesticide toxicity, respectively. These findings confirmed the similar results found among registered crop farmers in Nongbualamphu, one of the provinces in PH Region 8, Thailand,23 as well as those found in other countries in Asia,2 Canada,3 Europe,4,6 and America.5,7\n\nIn cases of toxicity from pesticides in 2016, the morbidity rate in Roi-Et Province was lower than previously reported among southern Roi-Et farmers during the same period,26 which could be explained by local agricultural behaviors and the fact that pesticide toxicity report cases depended on the disease surveillance of each regional public health service.44 Moreover, it was noteworthy that Roi-Et provincial-level case reports were very low in number compared to those of Udon Thani (Table 3).\n\nAccording to the analysis of ICD-10 utilized from the 43 health files standard among registered farmers, the morbidity rate of WMSDs in Roi-Et was around double that of Udon Thani. In contrast, the morbidity rate of WMSDs in Udon Thani, according to the HDC database, was around 10 times higher than that of Roi-Et. Regarding this comparison of morbidity rates of WMSDs, namely the HDC database versus the 43 health files data records, the trends were found to be unclear. This might be explained by the fact that case reports of WMSDs are not only specific to farming work, or they have multi-factor causes.43 Another explanation might be the wrong or missing diagnostic coding of Y96, which signifies a specified occupational cause, which was confirmed by our findings to support the previous study.44\n\nUdon Thani had a higher rate than Roi-Et with regard to noise- and heat-related diseases and pesticide toxicity, according to HDC database analysis as well as analysis of the 43 health files of the standard data structure. Increasing rates were observed from 2014-2016 among Udon Thani and Roi-Et agriculturists. These two occupational disease groups were confirmed as the major health problems among agriculturists in Thailand.43\n\nInfectious diseases, such as leptospirosis and melioidosis, had the highest prevalence in farmers, but there was no infectious disease group shown for occupational disease in the HDC database. This was not only the case in Thailand; a previous study in Poland showed a significantly higher rate in farmers who had been exposed to repeated tick bites,45 which corresponded to annual reports in Greece from 2004–2015.15 Moreover, brucellosis cases have been found in farmers and livestock breeders, with a high incidence rate of 7.1 per 100,000 population.15\n\nFrom the HDC database, it was found that the top three morbidity rates in Roi-Et, in 2014, were the morbidity rate of WMSDs followed by those of pesticide toxicity and noise- and heat-related diseases, respectively. However, the morbidity rates during 2015 and 2016 changed in order. Meanwhile, the top three morbidity rates of Udon Thani (2014-2016) were the morbidity rate of WMSDs followed those of by noise- and heat-related diseases and pesticide toxicity, respectively. That explains why Roi-Et was unable to be provide good representative data because the province had not reported data as forecast, which contrasted with Udon Thani Province in this study. The morbidity rates from the health data of Roi-Et province showed congruous results among the registered farmers. The highest rate was for work-related injuries, followed by WMSDs, hearing loss and heat-related disease, and pesticide toxicity, respectively. Similar results were found in Udon Thani, where rates of work-related injuries, WMSDs, and noise- and heat-related disease were similar and close to the national rates. Hence, these results reveal that the provincial data of Udon Thani can be representative of the nation. The consistency of the database is shown by the top three highest morbidity rates of disease from the previous study in crop farmers of Nongbualamphu province, which were shown to be WMSDs, noise- and heat-related disease, followed by skin irritation.23 Moreover, the number of reported cases was very different between Udon Thani and Roi-Et, and the morbidity rate of pesticide toxicity in Roi-Et was dramatically lower than that of the previous report from 2016-2018 among registered farmers in Sakon Nakhon, a province in PH Region 8.25 Those cases mentioning skin disease were suspect due to a combined effect in the group of pesticide toxicity, which is that of a reported acute symptom (irritant), and confirmed the previous studies.25,26\n\nThese disputations with strong evidence suggest health surveillance of occupational disease among agriculture workers (informal workers) or Thai farmers by 1) realizing the diagnosis with a coding record of Y96, 2) improving the recording method of occupation to be used practically for agriculturists, and 3) promoting agriculture workers to register on the health database. It was more than interesting that some have mentioned other diseases should be added to ICD-10 for occupational disease, i.e., lung disease, skin disease and infectious disease, for the occupational surveillance system.\n\n\nConclusions\n\nThe morbidity-rate ranking of work-related diseases and injuries among agriculturists in the upper northeast of Thailand was WMSDs, noise- and heat-related disease, and pesticide toxicity, respectively. When comparing the morbidity rates of farmers from the 43 health files data to those of the HDC database, the closest rates were found in the pesticide toxicity rates of Roi-Et and Udon Thani, which contrasted with those of noise- and heat-related diseases. Moreover, in 2016, the rates of WMSDs in Roi-Et from the 43 health files data were more than 10 times higher than those from the HDC data, which was like those of noise- and heat-related diseases. All work-related diseases of the 43 health files data had increasing trends; however, data from the HDC showed rates of WMSDs were in a decreasing trend. Regarding each individual province, Roi-Et had an increasing trend for all diseases, for both data from the 43 health files data and data from the HDC, but a disease of Udon Thani had a clearly decreasing trend, which can be explained by the occupational coding that Udon Thani has developed, thus strongly communicating that WMSDs had not been caused by occupation or only by the agricultural activity of farmers. Database of work-related diseases and injuries among agriculturists in the northeast of Thailand do not reflect reality at the present. There are two major reasons why the morbidity rates are not accurate. Firstly, several cases were completed without identifying as ICD-10 of specifying with extra code Y96 as occupational causes. Secondly, the total number of the population of interest, which was agriculturists, was not the true number. Therefore, recording an occupation when visiting health service providers should be promoted for the future, and those agriculturists should be registered in a healthcare database or surveillance system.\n\n\nData availability\n\nThe underlying data for this study is secondary data collected by the Health Data Centre and agricultural registries and is restricted due to ethical and data protection considerations. Under the ethical approval from the Human Research Ethics Committee of Khon Kaen University (No. HE592154), participants’ data was not allowed to be shared and the secondary data of health dataset and registered farmer dataset were utilized under restriction. The individual information or further reused inclusion data is not permitted under the ethically approved conditions for human research. This was confirmed under the data protection of human/participant privacy as no individuals gave explicit written consent that their identifiable data can be made publicly available. For more information, this research had no personal identified data for accessible because of the ethical issue that does not allow us to hold the personal data of each participant and cannot be shared any data after closing research.\n\nThe raw dataset collected under permission access and restriction use of ICD-10 codes are the 43 health files lists of Roi-Et and Udon Thani provinces in the period 2014 to 2016: file 10 – diagnosis (opd), file 15 – diagnosis (ipd), and file 19 – surveillance and the lists of registered agriculturists in Roi-Et and Udon Thani provinces during 2014-2016. To request access to the underlying data, please email the primary investigator (Sunisa Chaiklieng; csunis@kku.ac.th as the corresponding author. Applications will be reviewed on a case-by-case basis.\n\nThe intermediary data that can be de-identified without compromising anonymity are all presented already in the results and underlying data (TABLE A, B, C) and listed below.\n\nOpen Science Framework (OSF): Situations of work-related diseases and injuries among agriculturists in the upper northeast regions of Thailand. https://doi.org/10.17605/OSF.IO/2JPZM.46\n\nThe project contains the following underlying data:\n\n• TABLE A B C. (Table 1 - data for Figure 1; Table B - data for Figure 2 that is related to HDC database; and Table C - data for Figure 2 that is related to 43 Health files)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nNational Statistical Office of Thailand: Informal employment data in the whole kingdom of Thailand of the third quarter (July-September) in 2019.[cited 2020 Oct 24]. Reference Source\n\nKee D, Haslam R: Prevalence of work-related musculoskeletal disorders in agriculture workers in Korea and preventative interventions. Work. 2019; 64(4): 763–775. PubMed Abstract \| Publisher Full Text\n\nMcMillan M, Trask C, Dosman J, et al.: Prevalence of musculoskeletal disorders among Saskatchewan farmers. J. Agromedicine. 2015; 20(3): 292–301. PubMed Abstract \| Publisher Full Text\n\nOsborne A, Blake C, McNamara J, et al.: Musculoskeletal disorders among Irish farmers. Occup Med (Chic Ill). 2010; 60(8): 598–603. PubMed Abstract \| Publisher Full Text\n\nKearney GD, Hu H, Xu X, et al.: Estimating the prevalence of heat-related symptoms and sun safety–related behavior among Latino farmworkers in Eastern North Carolina. J. Agromedicine. 2016; 21(1): 15–23. PubMed Abstract \| Publisher Full Text\n\nRiccò M, Vezzosi L, Bragazzi NL, et al.: Heat-related illnesses among pesticide applicators in North-Eastern Italy (2017). J. Agromedicine. 2020; 25(1): 52–64. PubMed Abstract \| Publisher Full Text\n\nMirabelli MC, Quandt SA, Crain R, et al.: Symptoms of heat illness among Latino farm workers in North Carolina. Am. J. Prev. Med. 2010; 39(5): 468–471. PubMed Abstract \| Publisher Full Text\n\nArcury TA, Summers P, Talton JW, et al.: Heat illness among North Carolina Latino farmworkers. J. Occup. Environ. Med. 2015; 57(12): 1299–1304. PubMed Abstract \| Publisher Full Text\n\nLee S, Lee K, Lee SJ: Hearing impairment among Korean farmers, based on a 3-year audiometry examination. Ann. Agric. Environ. Med. 2019; 26(1): 148–153. PubMed Abstract \| Publisher Full Text\n\nMasterson EA, Themann CL, Calvert GM: Prevalence of hearing loss among noise-exposed workers within the agriculture, forestry, fishing, and hunting sector, 2003-2012. Am. J. Ind. Med. 2018 Jan 1; 61(1): 42–50. PubMed Abstract \| Publisher Full Text\n\nBhattarai D, Singh SB, Baral D, et al.: Work-related injuries among farmers: a cross-sectional study from rural Nepal. J. Occup. Med. Toxicol. 2016 Oct 26; 11(1): 1–7. Publisher Full Text\n\nAdesiyun A, Rahaman S, Bissessar S, et al.: Seroprevalence of Toxoplasmosis, Leptospirosis and Brucellosis in sugarcane field-workers in trinidad and tobago. West Indian Med. J. 2010; 59(1): 14–19. PubMed Abstract\n\nApsari PIB, Indraningrat AAG, Arwati H, et al.: Short communication: prevalence and risk factors of soil-transmitted helminth infection among farmers in Gelgel Village, Klungkung District, Bali, Indonesia. Biodiversitas. J. Biol. Divers. 2020; 21(4). Publisher Full Text\n\nFiecek B, Grochowalska A, Chmielewski T, et al.: Leptospira spp. and Coxiella burnetii infections occuring in Radomskie District in people of selected professional groups. Przegl. Epidemiol. 2012; 66(4): 605–610.\n\nLytras T, Danis K, Dounias G: Incidence patterns and occupational risk factors of human brucellosis in Greece, 2004–2015. Int. J. Occup. Environ. Med. 2016 Oct 1; 7(4): 221–226. PubMed Abstract \| Publisher Full Text\n\nZhang X, Zhao W, Jing R, et al.: Work-related pesticide poisoning among farmers in two villages of Southern China: A cross-sectional survey. BMC Public Health. 2011; 11: 1–9. Publisher Full Text\n\nLondon L, Nell V, Lou TM, et al.: Health status among farm workers in the Western Cape-collateral evidence from a study of occupational hazards. South African Med. J. 1998; 88(9): 1096–1101.\n\nLamichhane R, Lama N, Subedi S, et al.: Use of pesticides and health risk among farmers in Sunsari District, Nepal. J. Nepal Health Res. Counc. 2019; 17(1): 66–70. PubMed Abstract \| Publisher Full Text\n\nFontana L, Lee SJ, Capitanelli I, et al.: Chronic obstructive pulmonary disease in farmers: A systematic review. J. Occup. Environ. Med. 2017; 59(8): 775–788. Publisher Full Text\n\nJouneau S, Boché A, Brinchault G, et al.: On-site screening of farming-induced chronic obstructive pulmonary disease with the use of an electronic mini-spirometer: Results of a pilot study in Brittany. France. Int. Arch. Occup. Environ. Health. 2012; 85(6): 623–630. PubMed Abstract \| Publisher Full Text\n\nNgajilo D, Singh T, Ratshikhopha E, et al.: Risk factors associated with allergic sensitization and asthma phenotypes among poultry farm workers. Am. J. Ind. Med. 2018; 61(6): 515–523. PubMed Abstract \| Publisher Full Text\n\nMinistry of Public Health Thailand: Health Data Center (HDC). Chronic lower respiratory tract disease. [cited 2020 Oct 18]. Reference Source\n\nChaiklieng S, Phuengsangpaen S, Suggaravetsiri P, et al.: The prevalence and severity of occupational diseases among field crop farmers in Nongbualamphu Province. Journal of the Office of DPC 7 Khon Kaen. 2019; 26(1): 77–86.\n\nGuytingco A, Thepaksorn P, Neitzel RL: Prevalence of abnormal serum cholinesterase and associated symptoms from pesticide exposure among agricultural workers in the South of Thailand. J. Agromedicine. 2018; 23(3): 270–278. PubMed Abstract \| Publisher Full Text\n\nKhamjantarat P, Suggaravetsiri P, Chaiklieng S: Prevalence of illnesses among cultivation farmers in Sakon Nakon province. Journal of the Office of DPC 7 Khon Kaen. 2020; 27(2): 55–66.\n\nThongbu W, Suggaravetsiri P, Chaiklieng S: Prevalence of pesticide poisoning among farmers in Roi Et Province: A study in Southern Roi Et. PHJBUU. 2017; 12(2): 41–52.\n\nChaiyawong P, Suggaravetsiri P, Chaiklieng S: Factors related to hearing loss among planting farmers in Khon Kaen Province. Journal of the Office of DPC 7 Khon Kaen. 2018; 25(3): 88–98.\n\nMolina-Guzmán LP, Ríos-Osorio LA: Occupational health and safety in agriculture. A systematic review. Rev Fac Med. 2020 Oct 1; 68(4): 625–638. Publisher Full Text\n\nMesombat P, Chaiklieng S, Kuster AT: Prevalence of occupational heat related Illness among cultivated farmers in Khon Kaen Province. J. Safety Health. 2020; 13(2): 45–63.\n\nWorld Health Organization: ICD-10 version: 2019.[cited 2020 Oct 15]. Reference Source\n\nAgricultural Extension Information Center of Thailand: Number of registered farmers.[cited 2020 Nov 6]. Reference Source\n\nNational Statistical Office (NSO): The number of industry and industrial product 2014.[Cited 2021 April 22]. Reference Source\n\nMinistry of Public Health Thailand: Health Data Center (HDC). Pesticide toxicity. [cited 2020 Oct 18]. Reference Source\n\nMinistry of Public Health Thailand: Health Data Center (HDC). Occupational injuries.[cited 2020 Oct 18]. Reference Source\n\nMinistry of Public Health Thailand: Health Data Center (HDC). Work-related musculoskeletal disorders.[cited 2020 Oct 18]. Reference Source\n\nMinistry of Public Health Thailand: Health Data Center (HDC). Noise-induced hearing loss.[cited 2020 Oct 18]. Reference Source\n\nMinistry of Public Health Thailand: Health Data Center (HDC). Heat stroke.[cited 2020 Oct 18]. Reference Source\n\nAscherio A, Chen H, Weisskopf MG, et al.: Pesticide exposure and risk for Parkinson’s disease. Ann. Neurol. 2006 Aug; 60(2): 197–203. Publisher Full Text\n\nWeichenthal S, Moase C, Chan P: A review of pesticide exposure and cancer incidence in the agricultural health study cohort. Environ. Health Perspect. 2010; 118(8): 1117–1125. PubMed Abstract \| Publisher Full Text\n\nLerro CC, Beane Freeman LE, DellaValle CT, et al.: Pesticide exposure and incident thyroid cancer among male pesticide applicators in agricultural health study. Environ. Int. 2021; 146: 106187. PubMed Abstract \| Publisher Full Text\n\nSaldana TM, Basso O, Hoppin JA, et al.: Pesticide exposure and self-reported gestational diabetes mellitus in the agricultural health study. Diabetes Care. 2007; 30(3): 529–534. PubMed Abstract \| Publisher Full Text\n\nMontgomery MP, Kamel F, Saldana TM, et al.: Incident diabetes and pesticide exposure among licensed pesticide applicators: Agricultural health study, 1993-2003. Am. J. Epidemiol. 2008; 167(10): 1235–1246. PubMed Abstract \| Publisher Full Text\n\nChaiklieng S, Poochada W, Suggaravetsiri P: Work-related diseases among agriculturists in Thailand: A systematic review. Songklanakarin J. Sci. Technol. 43(3): 638–647.\n\nChaiklieng S, Thongbu W, Suggaravetsiri P, et al.: The pesticide poisoning report among farmers: A case study in Roi Et Province. Journal of the Office of DPC 7 Khon Kaen. 2020; 27(3): 52–64.\n\nZając V, Pinkas J, Wójcik-Fatla A, et al.: Prevalence of serological response to Borrelia burgdorferi in farmers from eastern and central Poland. Eur. J. Clin. Microbiol. Infect. Dis. 2017; 36(3): 437–446. PubMed Abstract \| Publisher Full Text\n\nChaiklieng S: Situations of work-related diseases and injuries among agriculturists in the upper northeast regions of Thailand.2021, December 22. Publisher Full Text" }	[ { "id": "141342", "date": "04 Jul 2022", "name": "Donald Cole", "expertise": [ "Reviewer Expertise Work and environment related conditions and interventions to deal with them. In particular", "WMSD", "pesticide and fungal disease surveillance." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFor a global audience, the first paragraph is less relevant than the 2nd. I would suggest moving it down to a Thailand specific focus before the HDC paragraph.\nIn the literature review paragraph, you might start by citing some of the reviews of the range of agriculture and health in general before going into each disease-disorder-injury type.\nRe the HDC, need some more background about its role in surveillance or health data in Thailand.\n\nA case-control study does not usually result in prevalence estimates. Clarify how this might provide such an estimate.\nGood to have methods of identification of the above. Seems like preliminary work to this paper. Might it be better as an early part of methods?\nThis work is clearly important for Thailand. How might it be important globally, in other MICs for example? How does it relate to the Global Burden of Disease work e.g. on low back pain? Something more generalizable as a scientific objective is needed. For example, it seems that there are two: one around work-related injury and disease surveillance methods, in which the paper makes a contribution; and one around differential burdens in different settings and exploration of reasons.\nGreat use of a registered farmer - farmworker data set. Any sense what the breakdown would be i.e. proportions with different access rights to land? employers or employees?\nNot sure what you mean by \"more real\" agriculture? what is \"less real\" agriculture? How did you decide on the occupational codes in table 1?\nIn some jurisdictions, farmer suicides from pesticides are considered work-related, as reflect the difficult situation of many farmers, with cash and debt squeezes which force them into suicide as one of few options. And pesticides are associated with depression. Please justify your exclusion e.g. more of a narrow safety view of exposure.\nCan you provide some sense of the completeness of coding of health conditions etc. This is often the challenge with regards to occupational disease and occupational injury occurrence.\nData analysis, last two sentences of first paragraph should be edited together. Formula \"of\" or \"in\" rather than \"at\" area of interest.\nI found it confusing to have the number of registered farmers in the same table as number of HDC farmers. the latter is the denominator for table 3 and Figure 1, while the former is the denominator for subsequent analysis shown in table 4, where this denominator of registered farmers should be included.\nNot sure that the interprovincial comparisons are as important globally as they may be internally for national and provincial policy makers. Less emphasis perhaps, unless going to speak to the heterogeneity across regions within countries globally, and some of the reasons for such heterogeneity e.g. industrial sector profiles, meteorological conditions, etc.\nAgree that under-reporting common in surveillance. More reference to this methodological literature, the strengths and weaknesses of different systems, particularly for work-related injuries and diseases is needed.\n\nWhat factors might explain the increasing morbidity trends? Is more awareness, education and diagnosis occurring? more exposure? Need some exploration of reasons. Any data sources that might inform this discussion?\nCurrently much repetition of results here, without linkage to the global literature. How do the rankings here compare with other country rankings of morbidity among agriculturalists? and why, do you think.\nWhy do you think health service providers do not record occupation in Thailand? How does it compare globally?\n\nReally appreciate the data availability statement and your work in this regard. The OSF tables are more other versions of the results, however.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9151", "date": "23 Jan 2023", "name": "Sunisa Chaiklieng", "role": "Author Response", "response": "Dear Prof. Dr. Donald Cole According to your comments, we have much appreciate and have improved paper following your suggestions, as attached version. The clarification for each question as following. 1. For a global audience, the first paragraph is less relevant than the 2nd. I would suggest moving it down to a Thailand specific focus before the HDC paragraph. Answer: It was improved as your suggested, thank you [see Introduction] 2. In the literature review paragraph, you might start by citing some of the reviews of the range of agriculture and health in general before going into each disease-disorder-injury type. Answer: The literature review paragraph was improved following your suggestion [see Introduction]. 3. Re the HDC, need some more background about its role in surveillance or health data in Thailand. Answer: Background of the role in the surveillance of health for the HDC in Thailand was added in Introduction (see Introduction) 4. A case-control study does not usually result in prevalence estimates. Clarify how this might provide such an estimate. Answer: The uncleared sentence as question regarding the case-control study was improved as rewritten as “The case-control study of registered agriculturists in Khon Kaen confirmed that the noise-induced hearing loss cases from the 43 health files data had been diagnosed through the audiometer test of those agriculturists [28],\" thank you for this comment for improvement. 5. Good to have methods of identification of the above. Seems like preliminary work to this paper. Might it be better as an early part of methods? Answer: The preliminary work or our previous investigation to estimate the morbidity rate of occupational diseases by using the big data of ICD-10 codes among registered agriculturists was the retrospective study as the references [24-26, 29]. The case-control study was designed in the on one study of the hearing loss to identify the risk factors among registered farmers [27]. The reference of method was cited in the method section for ICD-10 codes of occupational disease groups and for analysis of the morbidity rate, i.e, reference 24, the study among registered farmers of Nongboulamphu province, thank you for the suggestion. 6. This work is clearly important for Thailand. How might it be important globally, in other MICs for example? How does it relate to the Global Burden of Disease work e.g. on low back pain? Something more generalizable as a scientific objective is needed. For example, it seems that there are two: one around work-related injury and disease surveillance methods, in which the paper makes a contribution; and one around differential burden in different settings and exploration of reasons. Answer: From the big data analysis from the 43 health files is clearly identified cases of occupational disease to estimate the morbidity rate by extracted ICD-10 code of occupational disease, for example, code M545 of the registered farmers holding cultivating activity who was matching national ID to the accessed healthcare farmers. The previous study among crop farmers [reference 24] could show the number of low back pain cases of 4,814 cases in 2016 that reflected the highest morbidity rate of WMSDs (21.73%) among all occupational diseases in registered farmers. The study discovered also lung diseases, which was not reported in HDC database as the occupational disease. The real strong point of the paper is the linking the identified cases to the full farmers population and the outcome is showing the magnitude of risks of the agricultural farmers, which is a large part of the workforce in not only in Thailand but throughout globally. Even in highly developed countries hazards in agriculture are usually under-estimated, and negative outcomes not identified due to compensations mechanisms as we mentioned in the discussion continued from the background of Thai labor protection and compensation among agriculturists who are informal workers in Thailand. This study used two provinces of Roi-Et and Udon Thani which are two of the provinces among the top five provinces engaged in cultivating activities in the Northeast of Thailand and the major of planting area and numbers of farmers in Thailand. The population of agriculturists in those two provinces represented Thai agriculturist as the nature of the impact from agricultural work which was found to be similar across from other areas of Thailand because the main product of all regions in Thailand was rice. Therefore, this paper could make a contribution to the disease surveillance method of work-related injury and diseases as we improved written more focusing in the abstract and discussion and the conclusion. 7. Great use of a registered farmer - farmworker data set. Any sense what the breakdown would be i.e. proportions with different access rights to land? employers or employees? Answer: The most recent agricultural census, conducted every ten years, was conducted in 2013. In each province of the nation, data collection will take place simultaneously by sending around 20,000 officers, 17,000 counting personnel/employees,3,000 academic staff, and farmland holders/heads of each household to conduct interviews to compile fundamental data about agricultural operations. These details will be utilized to choose appropriate agricultural properties and conduct a meeting with qualified details (enumeration). That strongly confirm that the registered farmers in Thailand are representative agricultural farmers as occupation, resulting the most true population at risk on occupational hazards exposure for this study. Roi-Et and Udon Thani which are two of the provinces among the top five provinces engaged in cultivating activities in the Northeast of Thailand were selected as representative provinces. The major of planting area and numbers of farmers in Thailand located in Northeast of Thailand, and the main product of all regions in Thailand was rice as presented in Roi-et and Udon Thani. For accessing of healthcare, this study, by utilizing the 43 health files data of the ICD-10 code of occupational cases and injuries during 2014 to 2016, it was found that there were 53,794 total cases (34.82%) of registered farmers who had accessed healthcare in Udon Thani, and 77,438 total cases (37.32%) in Roi-Et, that was not much different of those provinces. For the cases visited of healthcare as shown as additional results, the characteristics of those cases of registered farmers in Roi-Et and Udon Thani provinces, the largest proportion of cases in Roi-Et were working on rice plantations (98.26%), as well as in Udon thani (89.1%). Those showed only in numbers of the difference because of cultivating activities were different during off-rice season as we discussed. Thai’s agribusiness is clear content with contact farming laws which registered farmers are taken care of by government benefits such as price insurance. This study year have no statistics of agribusinesses number because agricultural census is providing the survey of agricultural holdings and will be report in 2023. The updated agricultural census was reported about statistics of agribusinesses which they provided by survey the registered farmer. At the present, there are no contact farming entrepreneur registered with the government in Roi-Et and Udon Thani. 8. Not sure what you mean by \"more real\" agriculture? what is \"less real\" agriculture? How did you decide on the occupational codes in table 1? Answer: Roi-Et and Udon Thani are two of the provinces among the top five provinces engaged in cultivating activities in the Northeast of Thailand (comprising more than 97.0% of agricultural holdings) as explained before, therefore, the word of “more real” was deleted to be clear from the design of this study. For the occupational codes in table 1 was the recorded code of occupation in HDC database as we already explained in method section; All codes of farmers presented in Table 1 are the cultivating famers or involved activities in cultivated farming as the reference code of farmers from the Health Data Center (HDC) (https://hdcservice.moph.go.th/hdc/main/index.php) and confirmed by the IT personal responsible of coding and the agriculturist expertise. 9. In some jurisdictions, farmer suicides from pesticides are considered work-related, as reflect the difficult situation of many farmers, with cash and debt squeezes which force them into suicide as one of few options. And pesticides are associated with depression. Please justify your exclusion e.g. more of a narrow safety view of exposure. Answer: This study aware of the suicide from pesticide and we already excluded the cases not be accounted to the morbidity rate of pesticide toxicity as shown at notes in Table 2. In addition, the case of patient death during the study year which was accessed from file number 3 of the 43 health files were excluded from the visited cases in this study. 10. Can you provide some sense of the completeness of coding of health conditions etc. This is often the challenge with regards to occupational disease and occupational injury occurrence. Answer: The occupational codes of diseases and injuries used in this study methods are referred to reference code used for Occupational diseases and injuries of HDC database that is the limitation of the secondary analysis. Therefore, those codes were covered as used for the occupational diseases and injuries from the 43 health files data. However, we can provide codes found from the previous studies of 43 health file data and published papers (in Thai) of ICD-10 codes of health conditions of agriculturists, for example; the lower respiratory tract disease (lung diseases): J62, J630-J638, J660-J679, J68, J40-J47, WMSDs: M00-M99, M542, M545, M548, M60, M624, M653, M792, M796, G560, hearing loss and heat illness: H833, H903-H905, T670-T679, T68, and for pesticides toxicity (T600, T601, T602, T603, T604, T608, T609), which recommended more to add specified skin diseases: L01, L02, L230-L239, L240-L249, L250-L259, L303, L502, L503, L55, L560- L569, L904. Those occupational code found and confirmed report from cultivated agriculturists [as Reference no 24, 25,26,29,47] as we discussed. 11. Data analysis, last two sentences of first paragraph should be edited together. Formula \"of\" or \"in\" rather than \"at\" area of interest. Answer: It was corrected as follow; Number of cases reported among registered agriculturists or farmers visited services in an area of interestTotal number of farmers (registered agriculturists or farmers visited services) in an area of interest 12. I found it confusing to have the number of registered farmers in the same table as number of HDC farmers. the latter is the denominator for table 3 and Figure 1, while the former is the denominator for subsequent analysis shown in table 4, where this denominator of registered farmers should be included. Answer: The numbers of registered farmers of Roi-Et and Udon-Thani referred to Table 3 were corrected as presented for table 4. 13. Not sure that the interprovincial comparisons are as important globally as they may be internally for national and provincial policy makers. Less emphasis perhaps, unless going to speak to the heterogeneity across regions within countries globally, and some of the reasons for such heterogeneity e.g., industrial sector profiles, meteorological conditions, etc. Answer: Thank you for this point and that is agree with the design of this study and the results, we have improved the abstract and conclusion to be less emphasis of interprovincial comparison, but representative the rate or trend of morbidity rate of health problems of Thai agriculturist was presented, as shown in the Abstract and Conclusion. For the discussion in the morbidity rate of different, it is mostly pointed for WMSDs which showed the contradictory rate and ranking orders. The heterogeneity across regions within countries, the agricultural productivity while off-rice seasoning was more discussed also for pesticide toxicity. 14. Agree that under-reporting common in surveillance. More reference to this methodological literature, the strengths and weaknesses of different systems, particularly for work-related injuries and diseases is needed. Answer: We confirm the previous studies (24-25) and the preliminary results of the southern Roi-Et (26), of the under- reporting cases from the surveillance system of occupational diseases (44) as discussed. This study showed one pational disease in HDC database, by the method of extracted ICD-10 coded of J40-J47, could be done from the 43 health files data matching to the agriculturists. The strength of Occupational diseases surveillance in the HDC database is the platform cases recorded from the Universal healthcare service which is usually covered for Thai population and farmers in Thailand. However, there were some weakness, 1) at time of this study, is that the recording of occupational disease was not mandatory but depended on the decision of hospital management and resources. Work-related disease effects on examination and treatment were not confirmed, for example, in the case of silicosis; it has symptoms similar to tuberculosis (TB), therefore, the report might be missing because it was not confirmed by silica dust exposure in the workplace from stone and sand, or mining or sculpture, or working in the cement industry. Moreover, for example lung diseases, infectious diseases, had not been included in the HDC database of Thailand as types of occupational disease. 2) The accuracy of the health databases varies based on the hospital level; for instance, data from hospitals at the provincial level are more accurate than those at the district level and all collected data in this study are summations of sources of data at the provincial level, which are sent to the health data centre at the national level. 3) The occupational codes for farmers as shown in Table 1 in HDC database seems to be complicated for recording during routine work for healthcare providers. For this weakness, the second method to use the number of registered agriculturists can be solve the more strength method. The strength of the method utilizing the ICD-10 from the 43 health database are the following; 1) the morbidity rate of lung disease estimation which can be provide among registered farmers by matching personal ID of cases to those farmers in agricultural database. 2) As we found the morbidity rate for WMSDs was the highest concerned diseased among all diseases, WMSDs ICD-10 codes in Table 2 were categorised in the health surveillance system, but they only allow for the knowledge of disease rates and cannot be used to identify the organs that are affected in WMSDs as we have shown from the recent study report [47; the WMSDs: codes of diagnosis were M00-M99, M542, M545, M548, M60, M624, M653, M792, M796, G560], 3) limitation of diagnosis which is depended on the accessed healthcare service level. That is equal from the HDC database and the 43 health files method. 3) Another weakness to be concerned from the disease surveillance system of the use of ICD-10 code with occupational causes, for example, pesticide toxicity, a previous study in Roi-Et province found that ICD-10 identification and reports on the disease surveillance system with external code Y96 (work-related condition) were missing, as were reports in severe cases of pesticide poisoning with T-toxicity coding (44). Therefore, pesticide toxicity was shown to be underestimated in regard to occupation, particularly that of cultivating farmers, and the analysis utilised ICD-10 from the 43 health file data could include more cases without code Y-96 to estimate the rate. Thailand’s public health policy should include ICD-10 code L (skin disease) and code J (lung diseases) to identify the acute and chronic effects of pesticide toxicity among applicators, as recommended in our previous study of cultivating farmers in Sakon Nakhon province (26). By that considered ICD-10 code L, the stronger method of 43 health files data can provide manually extract code ICD-10 of the cases of the registered farmers. 15. What factors might explain the increasing morbidity trends? Is more awareness, education and diagnosis occurring? more exposure? Need some exploration of reasons. Any data sources that might inform this discussion? Answer: From both data source, and we provided more data as shown in underlying data, Table D, and the discussion continued to the conclusions, the trend of increasing rate of diseases was presented. It can be explained from the main factors of health hazards exposure from cultivation activity and types of plantations affecting the different diseases rates of the two province- case study as discussed for pesticides toxicity and WMSDs increasing trend in the Discussion as the follow. “Regarding agricultural productivity in the study area, the Office of Agricultural Economics reported that cassava, rubber, and rice had the highest productivity between 2016 and 2019 (48), as found in the case characteristics of this study. That could support results concerning the significant increase in morbidity rates of all occupational diseases and injuries in Roi-Et from 2014 to 2016; this increase was also found in Udon Thani, particularly in regard to pesticide toxicity. Pesticide toxicity has been shown to be a potential disease of the agricultural sector of Thailand, and as discussed, that problem was exacerbated by the trend of increasing pesticide imports, particularly herbicide imports, during the 5-year period from 2013 to 2017 (49)”. ….”However, from our data of the cases extracted from the 43 files of the most recent year, 2020 [47], it was confirmed that WMSDs had an increasing trend and a higher morbidity rate in Roi-Et than in Udon Thani province. Intriguingly, it was found that between the years 2014 and 2016, the province of Roi-Et had the fourth-highest tonnage of inland rice production in the country (48). In comparison to Udon Thani, Roi-Et's in-season rice yield was approximately 2-3 times higher (50). This study discovered from the 43 health files data that the WMSDs rate in Roi-Et was greater than that in Udon Thani province, presumably because rice farming requires more body exertion in various agricultural activities throughout the year than farming of other crops, e.g., cassava and sugar cane, which is planted more in Udon Thani.” 16. Currently much repetition of results here, without linkage to the global literature. How do the rankings here compare with other country rankings of morbidity among agriculturalists? Answer: The discussion was improved by linking to the global literature and compared with the ranking of morbidity among agriculturist as in section of discussion as the follow; ”For noise and heat related disease, the finding rate was 100 times lower compared to of the America (5, 7), which is similarly lower direction found in the rates of injuries and pesticide toxicity when compared to those found in Napal (10) and China (16). The closest rate to globally was that for the cases of lung disease which is lower than 2 times lower than in the south Africa. This finding too low morbidity rate from the health database of Thai agriculturists when comparing to those global rates might be explain the underestimation of the morbidity of the occupational disease and injuries. As we have a real strong point of the paper from the method from 43 health file big data analysis linking the identified cases to the full farmers population and the outcome is very showing the magnitude of risks of the agricultural farmers, which is a large part of the workforce not only in Thailand but throughout Asia and globally. 17. Why do you think health service providers do not record occupation in Thailand? How does it compare globally? Answer: By HDC, the occupation code is available for case visited for healthcare service as we use the code of farmers involved plantation activities during the period of in this study, however, the many codes available to be selected to be represented farmers occupation as shown in Table 1 which is complicated for the healthcare providers to add in the record. There are also codes of unemployed, general employed, underling person, or no occupation, that could be selected for this filed, which made missing/misclassification in database. The previous study of pesticide poisoning reports among showed incomplete records of occupational cases in 45.14% of all cases, and misclassification case report of agriculturists as occupation code was found in 37.03% of cases in Skon Nakhorn from our previous case-control study [Chaiklieng, S.; Khamjantarat, P.; Suggaravetsiri, P. Factors associated with disease from pesticides toxicity among planting farmers in Sakon Nakhon Province. In: Proceedings of The 9th National Conference in Toxicology (NCT9), Update in toxicology for safe life. 25-26 September 2019, 109-21]. Moreover, there were key concerns regarding public health personnel being responsible for multiple tasks, without any understanding of occupational disease codes, and never having received training for occupational disease recording [44]. As you agreed of under-reporting, and I am sure that even in highly developed countries, hazards and risks in agriculture are usually under-estimated, but negative outcomes not identified due to many reasons, i.e., lack of legal, control and compensations mechanisms. By those reasons, almost no recently update report to compared since 2004 after one study mentioning to that occupational history which was frequently neglect occupational history-taking by the physicians, and the physician had completed an occupational history-taking from only 27.8% of patients (Politi et al. Occupational Medical History Taking. JOEM 2004;46:550-5), which is very low and also lower rate compared to our previous finding in Thailand [44]. 18. Really appreciate the data availability statement and your work in this regard. The OSF tables are more other versions of the results, however. Answer: We added more available results in Table D (underlying data): Characteristics of cases of work-related diseases and injuries among registered agriculturists in Roi-Et and Udon Thani provinces, in the upper northeast of Thailand. All data presented in Table A-D could be useful to learn more in numbers and rates or trend of occupational disease and injuries from this case study. The characteristic of cases of registered farmers, the numbers of cases from 43 health files data and the HDC database and the farmers or registered farmers in each year from our big data analysis during the study period could be useful to understand by other analysis or presentation of the readers or the researchers, because we did not show all rates presentation of the previous years (2014- 2015) in the paper, excepted 2016. Importantly, the data described in this study are accessible from the corresponding author upon request. Due to confidentiality concerns, the data are not publicly available, as mentioned in the paper." } ] }, { "id": "142386", "date": "08 Jul 2022", "name": "Pornchai Sithisarankul", "expertise": [ "Reviewer Expertise Occupational and environmental medicine/health", "preventive medicine", "public health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBackground - ..work-related diseases and injuries.. -- so the result should show or address injuries data\n\nResults - should show key findings in numbers\n\nRegistered farmers may not be the true population at risk, as discussed in Conclusions; moreover, this bias may not be equal in the 2 provinces\n\nOther potential biases are - discrepancy of diagnoses and codings of the 2 provinces and across the 3-year period, different quality of diagnoses and codings of different levels of hospitals, etc. - these should be addressed and discussed\n\nBe aware of the difference between 3-year report/statistics and 3 consecutive-year report/statistics\n\nNeurotic hearing loss - I do not agree to use this term. There are sensorineural, conductive and mixed hearing losses.\n\nRegarding each individual province, Roi-Et had an increasing trend for all diseases, for both data from the 43 health files data and data from the HDC, but a disease of Udon Thani had a clearly decreasing trend, -- I do not understand and am not sure which disease is \" a disease of Udon Thani\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9152", "date": "23 Jan 2023", "name": "Sunisa Chaiklieng", "role": "Author Response", "response": "Dear Prof. Dr. Pornchai Sithisarankul Thank you for your valuable comments, we have improved paper following your suggestions, as the revised version. The clarification for each question is following. 1. Background - work-related diseases and injuries - so the result should show or address Answer: This results and discussion of paper was improved to address injuries data as suggestion. 2. Results - should show key findings in numbers Answer: The results was improved to show key findings in numbers as suggestion. 3. Registered farmers may not be the true population at risk, as discussed in conclusions; moreover, this bias may not be equal in the 2 provinces Answer: The most recent agricultural census, conducted every ten years, was conducted in 2013. In each province of the nation, data collection will take place simultaneously by sending around 20,000 officers, 17,000 counting personnel/employees, 3,000 academic staff, and farmland holders/heads of each household to conduct interviews to compile fundamental data about agricultural operations. These details will be utilized to choose appropriate agricultural properties and conduct a meeting with qualified details (enumeration). By that reason, the registered farmers in Thailand are strongly representative agricultural farmers by an occupation, resulting on occupational hazards exposure and the true population at risk for this study, as we have done in the previous studies and method [24] [25] [28]. In addition, among the registered farmers, the real strong point is the linking of the identified cases to the full farmers population approved by the Provincial Agriculture Office agriculture database, that to show the magnitude of risks of the agricultural farmers. Moreover, the analysis in this study was accurate and have reliability by matching personality with the national ID to the health records of 43 files based on identifiers of individuals to another source of the registered farmers for Bigdata analysis. This method was done for previous studies [24, 25-26] which could show the risks of the Thai agricultural farmers on occupational diseases, particularly WMSDs, heat related illness or pesticide toxicity, that highly risks in agriculture were under-estimated from the previous report accounted 2-10 times of health database as in this study report. Any bias which might not be equal in the 2 provinces, we mentioned in the discussion to this serious and lack or difficulty to get support of recording occupational disease may have caused non-reporting as in the discussion. 4. Other potential biases are - discrepancy of diagnoses and codings of the 2 provinces and across the 3-year period, different quality of diagnoses and codings of different levels of hospitals, etc. these should be addressed and discussed. Answer: The discussion was added for those issues, it was discussed that the accuracy of the health databases varies on the hospital level; for instance, the provincial level is more accurate than the district level and all collecting data in this study are summation at the provincial level sources of data, where sending data to health data center of a country level. Whereas inaccuracies and actual missing data exist in Thailand is the major problem of diagnosing occupational diseases coding of Y96, which is one main problem as we discussed (Discussion section). However, this study method of analysis, we matched case of ICD10 diagnosis code to the registered agriculturist occupation (more by national ID. Both databases are the best representative for presentation the situation of Occupational diseases among agriculturists in Thailand. The only potential bias could be by quality of diagnoses and coding of different levels of hospitals which was addressed in discussion section according to the forecast which might occur and missing diagnostic coding in case of WMSDs, moreover, pesticide toxicity report cases depended on the disease surveillance of each regional public health service.44 5. Be aware of the difference between 3-year report/statistics and 3 consecutive-year report/statistics Answer: We aware of the difference, all parts in this paper and underlying data were rechecked and replaced with the correct term to present in this paper, for example, from 2014 to 2016, in 2016, thank you for this recommendation. 6. Neurotic hearing loss - I do not agree to use this term. There are sensorineural, conductive and mixed hearing losses. Answer: This term was replaced with noise-induced hearing loss or shortly hearing loss following the HDC database and definition. 7. Regarding each individual province, Roi-Et had an increasing trend for all diseases, for both data from the 43 health files data and data from the HDC, but a disease of Udon Thani had a clearly decreasing trend, I do not understand and no sure which disease is a disease of Udon Thani Answer: This sentence was rewritten as; “All work-related diseases and injuries of the 43 health files data and almost all of those of the HDC data had increasing trends from 2014 to 2016.”" } ] } ]	1	https://f1000research.com/articles/11-145
https://f1000research.com/articles/12-3/v1	03 Jan 23	{ "type": "Research Article", "title": "Practice and knowledge of dietary supplement consumption among Indonesian adults post-delta wave of the COVID-19 pandemic", "authors": [ "Annette d'Arqom", "Mhd Zamal Nasution", "Sharifah Zamiah Syed Abdul Kadir", "Junaidah Yusof", "Kayatri Govindaraju", "Mhd Zamal Nasution", "Sharifah Zamiah Syed Abdul Kadir", "Junaidah Yusof", "Kayatri Govindaraju" ], "abstract": "Background: Increasing dietary supplement (DS) consumption was observed during the COVID-19 pandemic, including during the post-Delta wave period. This study aimed to measure the practice of DS consumption and respondents’ knowledge of DS. Methods: An internet-based survey was distributed from October-December 2021 and obtained 541 valid and completed responses. Descriptive analysis was performed to present the practice of DS consumption, including frequency, duration, aim, preferable dosage form etc. Level of knowledge on DS principles, side effects and regulation were also measured. Inferential analyses were conducted to determine the predictors of the respondents’ DS practice and level of knowledge. Results: Data from 541 valid responses showed that 77.63% of respondents consumed DS in the last 3 months, with only 59.52% reporting also consuming DS before the COVID-19 pandemic. One half of the respondents had good knowledge about DS; however, some knowledge regarding side effects and possible drug-supplement interaction needed improvement. Their DS consumption practice was affected by their economic status and history of contracting COVID-19. Nevertheless, the level of knowledge was not affected by the sociodemographic factors and DS supplement experience. Conclusions: Taken together, the practice of self-consumption of DS in Indonesia is increasing; hence, knowledge of DS is necessary to avoid detrimental effects that might occur in the future. Increasing access to information on better labelling and educating consumers about DS are important actions to consider.", "keywords": [ "awareness", "COVID-19", "diet", "healthy lifestyle", "herbal", "mineral", "vitamin", "sociodemographic" ], "content": "Introduction\n\nUnited States Food and Drugs Administration (USFDA) defined dietary supplements (DS) as substances containing vitamins, minerals, amino acids or enzymes, or herb/botanical products that complete the diet's nutritional value. However, DS is not intended for treating, curing, preventing, or diagnosing diseases (FDA, 2022). Despite that definition by the USFDA, much evidence-based research, from fundamental animal studies to randomized clinical trials has shown the effectiveness of DS consumption in preventing disease, improving health, and reducing disease symptoms (Kaviani et al., 2020; Shinto et al., 2014; Sim et al., 2022). Moreover, the global consumption of DS has been steadily increasing for the last decade (Kantor et al., 2016; Kelly et al., 2005). Since 2020, the public's interest in DS use has increased in the wake of COVID-19 worldwide (d'Arqom et al., 2021; Hamulka et al., 2021; Lordan, 2021; Speakman et al., 2021). At the beginning of the pandemic, some DS recorded higher demand and consumption rates due to the perception that DS ingredients might boost immune function and reduce inflammation to help prevent COVID-19 (d'Arqom et al., 2021; Hamulka et al., 2021; Mohsen et al., 2021).\n\nThe COVID-19 outbreak has claimed a total of 6,540,487 lives as of October 2022 (WHO, 2022). More than 90% of COVID-19 deaths involved a pre-existing medical condition, of which 66% of the patients had metabolic syndrome, a pathologic condition due to obesity, insulin resistance, hypertension and hyperlipidemia (Li et al., 2021). This group of patients with one or two of these comorbidities would lead to a low survival rate following COVID-19 transmission due to impaired function via secondary effects on HDL cholesterol (Kitchens et al., 2003), declining respiratory functions due to prolong systemic inflammation indicated by high c-reactive protein (Chen et al., 2014; Kim et al., 2017), and a high risk of cardiac arrest due to the underlying comorbidities of metabolic syndrome (Hess et al., 2017).\n\nAll the mentioned medical complications could be prevented by early detection, being active, and meeting the physiological demands of essential vitamins and minerals. It has been established that vitamin C, B6, D, and E deficiency were associated with an increased risk of metabolic syndrome (Beveridge et al., 2015; Godala et al., 2017; Kodentsova et al., 2019). Since the pandemic hit, healthcare providers and the general public have changed their perceptions of DS, and several countries including Indonesia have been supplying essential dietary supplements to high-risk populations to avoid hospitalization and adverse complications following COVID-19 transmission (Kemenkes, 2022; Weir et al., 2020). A study conducted in the UK on 445,850 participants showed a significant association between the use of multivitamins, omega-3 fatty acids and vitamin D3 supplements with a lower risk for SARS-CoV-2 (Louca et al., 2021). However, uncontrolled and overconsumption of DS might lead to unwanted effects, such as kidney problems, cancer and drug-herbs interactions (Agbabiaka et al., 2018; Asher et al., 2017; d'Arqom et al., 2020; Peng et al., 2004).\n\nIn Indonesia, although the use of DS is high, little published data is available regarding public practice and knowledge towards DS and their safety. As such, more research on public awareness of DS is essential to gain a better understanding of people's beliefs and expectations regarding DS. Therefore, this study aimed to investigate the practice and knowledge of the general public in Indonesia regarding DS during the post-Delta wave of the COVID-19 pandemic. In addition, the predictive factors of DS consumption and level of knowledge were further analyzed.\n\n\nMethods\n\nThis research employed a cross-sectional design. The primary data were collected using an internet-based questionnaire (www.surveyplanet.com). The questionnaire was distributed to the adult population in Indonesia through email and social media using convenience sampling methods from October to December 2021, and multiple responds from single device were prevented based on respondents’ IP address. This study followed the Checklist for Reporting Results of Internet E-Surveys (CHERRIES) and The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines (Eysenbach, 2004; Vandenbroucke et al., 2007). The ethics clearance was issued by the Faculty of Medicine, Universitas Airlangga No. 244/EC/KEPK/FKUA/2021.\n\nThe inclusion criteria were Indonesian adults older than 18 years old and residing in Indonesia during the post-Delta wave of the COVID-19 pandemic. Sample size was calculated using sample size calculator (www.raosoft.com) with 5% margin of error, 95% confidence level, and population number filled with 100,000, resulting in 383 minimum respondents. Respondents were recruited through announcements distributed in social media and email using non-probability sampling.\n\nRespondents independently filled in the online questionnaire using their own device, and the estimated survey length was 12 minutes. To measure respondents' practice and awareness on DS, a set of questionnaires containing three sections was distributed online. The sections comprised basic demographic information, supplement consumption practice, and DS knowledge. The first section consisted of basic demographic factors such as age, sex, domicile, education, occupation, field of study/work, COVID-19 infection status, comorbidity, marital status, and self-claimed economic status. The second section consisted of 17 questions regarding their supplement consumption practice, which included respondents’ consumption of DS, duration, frequency, numbers, place to buy, aims, benefit, and source of information. And the last section comprised 16 questions to measure DS knowledge, including their knowledge on the regulation, side effects, and benefit. The questionnaire was modified from the Prevalence and Awareness Concerning Dietary Supplement Use among Saudi Adolescents (Alfawaz et al., 2020) and Knowledge about dietary supplements and trust in advertising them: Development and validation of the questionnaires and preliminary results of the association between the constructs (Karbownik et al., 2019). The questionnaire was combined and validated by two pharmacologists, a pharmacist, and a psychologist, and was further tested on 20 respondents to ensure that the content and terms used in the questionnaire were relevant and understandable. The reliability measurement on the survey data showed a Cronbach alpha coefficient of 0.750 for DS knowledge. As the reliability value for the preliminary pilot testing is at an acceptable level, the real data collection for this study was proceed. A copy of the survey instrument can be found under Extended data (d’Arqom, 2022).\n\nThis study followed the Helsinki declaration and approved by the Health Research Ethics Committee, Faculty of Medicine, Universitas Airlangga (No. 86/EC/KEPK/FKUA/2021). The aims of the study, the consent, and the permit to publish their responses anonymously were provided in the landing page, prior completing the questionnaire. The respondents provided their consent by clicking the YES button before starting the questionnaire.\n\nOnly the complete responses were processed and analysed. Respondents were grouped based on their sociodemographic factors. Supplement practice and awareness were measured using nominal or ordinal scales. Data were processed and analyzed using Microsoft Excel and SPSS 24.0 (IBM, Chicago, IL, USA). Graphs were visualized using GraphPad Prism 5.0. Descriptive statistical analyses were performed, including the frequency for each categorical variable. The answers to the knowledge survey were measured using correct/wrong/does not know options measured as 1/0/0. The total score was the summation of the 16 question scores and was grouped in four categories based on the total score of each respondent: poor (0-4), moderate (5-8), good (9-12) and excellent (13-16). The Chi-square test and Fisher's Exact test were performed to measure the difference between DS consumption and knowledge on each group. To investigate the predictors of DS consumption, binary logistic regression with 95% confidence intervals (95% CIs) was calculated. Moreover, to investigate the predictors of DS knowledge, ordinal logistic regression with 95% CI was performed. Prior to multivariate regression, univariate regression was performed to determine each sociodemographic factor that might affect DS consumption or level knowledge. Sociodemographic factors with p<0.25 were included to further multivariate regression using the backward elimination method (Bursac et al., 2008). Variables with p<0.05 from the multivariate regression analysis were considered significant predictive factors.\n\n\nResults\n\nFive hundred forty-eight respondents visited the informed consent page, and 541 completed and valid questionnaires used in the final analysis (completion rate was 98.72%). Around two thirds of respondents comprised young adults, females and unmarried individuals. The majority came from the main island, which is the most developed region in Indonesia (82.26%), with self-reported economic status, were average economic status (73.54%) and never had a COVID-19 positive status (78.19%). Table 1 summarizes the respondents’ sociodemographic characteristics. Hundreds (18.48%) were reported to have comorbidities such as obesity (35.65%), hypertension (22.61%), respiratory diseases (21.74%) and others (diabetes mellitus, autoimmune diseases, genetic diseases and cancer) (Figure 1A). The full dataset can be found under Underlying data (d’Arqom, 2022).\n\n(A) types of comorbidities of the respondents, (B) types of DS consumed by the respondents, (C) duration of DS consumption (D) Source of DS, p-value <0.05.\n\nOur study found that 420 (77.63%) respondents consumed DS during the post-Delta wave of the COVID-19 pandemic. Forty percent reported they had never consumed DS before the pandemic. Several sociodemographic factors were associated with DS consumption, for example, middle-aged adults were more likely to consume DS compared with elderly and young adults (p= 0.005). Higher education, higher self-reported economic status, married status, those diagnosed with COVID-19 and working respondents were also more likely to consume DS (p= 0.004, 0.011, 0.000, 0.009, 0.000, respectively). No statistically significant differences were found in DS consumption among sex, domicile and field of work/study. The frequency of supplement consumption based on their sociodemographic factors is summarized in Table 1.\n\nAlmost one half of respondents consumed vitamins (49.28%), minerals (20.19%), natural products (16.71%) and protein or amino acids (13.82%) (Figure 1B). Their aims of DS consumption were mostly for increasing immunity (81.19%), preventing disease (10.71%), and improving physical appearance (8.10%). Almost 40% of the respondents had consumed DS for less than 3 months, while more than one quarter (28.33%) had consumed DS for more than one year. A different pattern of DS consumption emerged between respondents with health-related backgrounds and non-health-related backgrounds, as most of the last group took DS with longer duration than the first group (p= 0.017, Figure 1C).\n\nThe most preferred dosage form was capsule (49.76%), tablet (38.57%), liquid (7.62%) and powder (4.05%). Two thirds of the respondents consumed a single type of DS at one time, two types of DS (23.10%) and only 7.62% consumed three or more types of DS simultaneously. More than 40% of respondents consumed 2 to 5 times/weekly (43.10%), less than twice/weekly (33.33%), and 23.57% consumed it more than 5 times/weekly. Two thirds bought DS from drugs stores (69.29%), e-commerce (18.81%) and any store that sold DS (10.71%), while only 1.19% bought DS from social media. Even though the majority of respondents in both groups were more likely to buy DS from drug stores, the health-related groups were more likely to choose e-commerce as the second option, while the second option for non-health-related respondents was any store that sold DS and e-commerce platforms (p= 0.025, Figure 1D).\n\nThe primary sources of information were family members or friends (49.29%), and health professionals (32.86%), and the rest received information from academic journals, websites and TV/magazine advertisements. Surprisingly, 48.57% consumed DS for more than 3 months without healthcare consultation. Moreover, one half of respondents did not have an exact schedule for consuming DS, while only 30.71% consumed them on a scheduled basis. As expected, more than three quarters of respondents had missed consuming DS (82.62%). Almost three quarters (73.57%) felt the benefit of consuming DS. However, only one third would continue to consume DS if they faced financial difficulty (34.29%). More than one half of respondents consuming DS knew about their side effects and they consumed and endeavored to find information regarding suitable DS (67.38%). Differences in DS consumption between the two groups are summarized in S2 Table. A supplementary material can be found under Extended data (d'Arqom, 2022).\n\nIn a set of questionnaires containing 16 items (S1 Table), respondents’ knowledge on DS was measured. The results showed that 7.21% of respondents had excellent knowledge, 59.52% had good knowledge, 31.24% had fair knowledge and 2.03% had poor knowledge on DS. As expected, based on their field of study or work, the level of knowledge was higher in health-related fields compared with non-health-related fields (p= 0.000, Figure 2A). The majority of respondents of both groups knew DS function was not to replace food but to increase nutritional value of their diet and improve health conditions (Q4, Q5 and Q8). Unfortunately, almost two thirds remained unaware that DS were still needed even though they already consumed a healthy diet (Q16), so DS were necessary for all ages (Q15). Moreover, almost all respondents understood that prior to consuming DS, they needed to consult a doctor or pharmacist and read the instructions or the label (Q2 and Q14). The respondents were also aware that before releasing to the market, DS needed a permit from an authorized organization in Indonesia (Q13). However, they were still unaware that not all traded DS were safe to consume (Q1), because some were illegally traded and not all provided evidence to support their beneficial claims (Q12). Regarding side effects, most respondents understood that consuming too many vitamins was harmful (Q3), but they still had a mindset that natural products were safe to consume because they come from nature (Q9). However, their awareness about DS side effects (Q6, Q10) and possible DS-drug interaction (Q11) needed improvement. The Kruskal-Wallis test showed significant differences in the answers between the two groups (Figure 2B), except for DS function (Q8), side effect DS side effects (Q10), authorization before releasing to the market (Q13) and the necessity of DS for all ages (Q15).\n\n(A) comparison of respondents’ level of knowledge based on their background work/study field, (B) comparison of correct answers based on their background work/study field,\n\n** p-value <0.000\n\nTo measure predictors of DS consumption of this study, binary logistic regression was performed. For supplement consumption, the univariate analysis of binary logistic regression showed respondents with higher education, working, having confirmed COVID-19 status, married status and have higher self-reported economy status were more likely to consume DS. Using those variables, multivariate analysis of a binary logistic regression model showed significant positive predictors for consuming DS were the health-related field of work or study (AOR: 1.733, 95% CI 1.109-2.709). However, respondents reporting no confirmed COVID-19 status were less likely to consume DS (AOR 0.524, 95% CI 0.291-0.944), and as expected, respondents with a lower self-reported economic status were less likely to consume DS post-Delta wave COVID-19 pandemic (AOR 0.309, 95% CI 0.124-0.769) proving the lack of accessibility due to financial constraints. The model estimated the overall accuracy of 77.8% and explained 9.9% of the variation in supplement consumption for preventing COVID-19 (Omnibus tests of model coefficients chi-square: 36.330, p = 0.000). Table 2 showed the predictor factors of DS consumption practice.\n\nOrdinal logistic regression was performed to investigate the respondents’ knowledge of DS. The result of univariate and multivariate analysis showed that sociodemographic factors, including age, sex, domicile, education, work status, COVID-19 experience, marital status, economic status, work/study background, and DS consumption were not significantly affecting the respondents’ knowledge (Table 3).\n\n\nDiscussion\n\nThe COVID-19 pandemic has changed most aspects of human lifestyle including the economy, transportation and of course, the medical field. It has been three years since WHO announced the pandemic, and despite declining cases, the status has not been revoked. Even now, researchers are still actively seeking and revealing new knowledge on COVID-19, including finding preventive measures and definitive drugs for post-COVID complications and long term COVID symptoms. DS as a preventive measure was highly investigated to avoid advanced stages of COVID-19. Several research studies also explored the possibility of DS to reverse the pathophysiology of COVID-19 (Caballero-García et al., 2021), as well as a complementary therapy for patients with COVID-19 and preventive therapy against lung diseases (Zhang & Liu, 2020). Several studies including clinical trials, endeavored to prove that DS effectively reduced disease severity, shortened length of stay and prevented infection by boosting the immune system (Pinnawala et al., 2021). Unfortunately, those clinical trials failed to prove DS ability to do so (Amin & Drenos, 2021; Brunvoll et al., 2022; Murai et al., 2021; Thomas et al., 2021). However, increasing DS consumption and sales have been reported worldwide including in the US, Europe and Asia (d'Arqom et al., 2021; Hamulka et al., 2021; Karlsson et al., 2021; Khabour & Hassanein, 2021; Lordan, 2021) with self-consumption practice, without consultation with healthcare professionals, and still observed in Asia, Africa and America (d’Arqom et al., 2021, (Quispe-Cañari et al., 2021; Sadio et al., 2021; Wegbom et al., 2021; Yasmin et al., 2022). Even though the self-consumption of DS is considered safe, their side effects in high-risk populations, especially individuals with several genetic backgrounds, comorbidities and patients on prescribed medication, could be unpleasant. They might experience unwanted effects due to drug and disease interaction (Agbabiaka et al., 2018; Asher et al., 2017; Ekor, 2014; Peng et al., 2004).\n\nOur study found increasing DS consumption, as more than three quarters of respondents consumed DS during the Delta wave of the COVID-19 pandemic, with only 59.52% consuming DS before the outbreak. Forty percent of the respondents did not consult with healthcare professionals before consuming DS for more than 3 months. This practice was also found before and during the pandemic, in parallel with other similar studies, such as in 1579 US citizens (Blendon et al., 2013), 105 athletes in Saudi Arabia (Aljaloud & Ibrahim, 2013), 651 students in Saudi Arabia (Almegewly et al., 2022) and 48,925 Japanese adults (Chiba & Tanemura, 2022). This consumption was significantly associated with middle age, higher education, higher economic status, marital status, receiving a diagnosis of COVID-19 and employment status. However, our regression model showed that only health-related field of work or study was a positive predictive factor, while never confirmed COVID-19 status and lower self-reported economic status were negative predictors.\n\nYounger respondents might feel healthier and at lower risk of obtaining severe stage of COVID-19 (Libertini et al., 2019; Perrotta et al., 2020), while older respondents might be unaware of the risk and the disease, despite being more prone to transmission (Wolf et al., 2020). Another possible reason was they might be unaware of the risk of interaction between DS and their diseases or drugs consumed (Agbabiaka et al., 2018; Alkhalidi et al., 2019). However, a high possibility existed that respondents having received a diagnosis of COVID-19 would like to prevent re-infection by consuming DS (Ali, 2020; Shahbaz et al., 2022). Married and working respondents might consume DS due to their responsibilities to avoid infection and need to fulfil their duties to their family members. This finding was similar to the study in Indonesia during the first year of the pandemic (d'Arqom et al., 2021), Saudi Arabia (Radwan et al., 2022) and Lebanon (Mohsen et al., 2021). Higher economic status respondents were able to buy the DS and might have a higher responsibility in their workplace; thus, they had consumed DS. Similar phenomena were also reported in Indonesia and Saudi Arabia (d'Arqom et al., 2021; Radwan et al., 2022). A study on 11,240 US adults from 2011 to 2014 also reported similar finding, as consumption of DS was higher among those earning higher incomes (Cowan et al., 2018).\n\nAmong the two groups, respondents with non-health-related background were more likely to consume DS for more than three months compared with respondents with health-related backgrounds. Despite of not being in the medical and healthcare career line, non-health background respondents’ have better long-term compliance. According to Biesalski and team, long term compliance in DS such as multivitamin and mineral supplement brings benefit to general health from developing lifestyle disease and was also documented safe in over 10 years of consumption in the clinical trial (Biesalski & Tinz, 2017). However, this miraculous finding does not apply to all DSs. There are detrimental effects of long-term compliances was also recorded by Stranges et al., whereby individual prolonged used of selenium, an ingredient mainly found in mainstream ingestible beauty supplements are at high risk of developing diabetes type 2 at the later stage of life (Stranges et al., 2007).\n\nDS consumption practice was also associated with the method in acquiring DS. Drug stores were the primary vendor to purchase DS, followed by e-commerce platform and any store selling DS. This finding supported the significant presence of online shopping, due to its convenience and ease during the COVID-19 pandemic (Eger et al., 2021; Gu et al., 2021). Moreover, respondents with health-related background were more likely to buy using e-commerce platforms than the non-health-related respondents. A study involving 34,488 Italians did not investigate the work/study background of the respondents as one of the predictive factors for online shopping; however, they found that younger age, higher education, female, good economic status and extended working hours comprised positive predictors for online shopping (Dominici et al., 2021). This might be one of the reasons, because healthcare professionals and medical students have long working hours (Dyrbye et al., 2017; Shreffler et al., 2020), including during the COVID-19 pandemic (Razu et al., 2021). However, this finding brings us to a concerning question on the regulatory control of e-commerce platform and any store sell DS, mainly due to the no access to expert compared to a drug store whereby there are pharmacist to refer to seek advice or concerns on the supplements and its side effects.\n\nMoreover, our study found that one half of respondents possessed good knowledge levels about DS, with only 2% having poor knowledge. However, our findings might be biased because almost two thirds of our respondents had health-related work/study backgrounds. However, the comparison of DS knowledge in both groups showed respondents with non-health-related backgrounds possessed less knowledge about DS, supporting our above-mentioned concern on the purchasing habits out of drug store. This finding differed from that of 351 students in Saudi Arabia, reporting no difference between the knowledge level of health science students with those of non-health science students (Alowais & Selim, 2019). Unfortunately, most were still partially unaware of the safety and side effects of DS on the market and the necessity of DS consumption. This hurdle was also found among 537 US healthcare professionals (Kemper et al., 2003) and 179 US students pharmacist (Axon et al., 2017). Our study found that their level of knowledge was not determined by sociodemographic factors and DS consumption in the post-delta wave of the COVID-19 pandemic, as no significant predictor factors were found in univariate and multivariate analysis.\n\nEven though sociodemographic factors did not affect the respondents’ knowledge of DS, we need to be aware about health inequality that has been highlighted during the COVID-19 pandemic and has been discussed extensively (McGrail et al., 2022; Mishra et al., 2021). In Indonesia itself, in 2017, the WHO and Ministry of Health Republic of Indonesia reported an existing gap on health status in Indonesia related to economic status, education level, occupation, employment status, age, sex, place of residence and subnational region. These disparities include low consumption of fruits and vegetables, known as the main source of vitamins and minerals from food (WHO & Kemenkes, 2017). These health disparities were also reported in several other studies (Haemmerli et al., 2021; Mulyanto et al., 2019). Due to implementing national coverage insurance, health disparities due to differences in economic status have reportedly decreased (Warsito & Adisasmito, 2020). Unfortunately, during the COVID-19 pandemic, socio-economic disparity was reported to be rising in urban areas, but decreasing in rural areas with the highest COVID-19 cases, even though its effect on health inequalities was unreported (Brata et al., 2021).\n\nEven though the internet-based survey was conducted conveniently and efficiently, the questionnaire might not have reached the respondents in remote areas of Indonesia due to limited internet access. Moreover, limited interaction with the respondents might have created a biased responses to the questionnaire. Furthermore, the sampling methods did not represent the distribution of Indonesian adults. Thus, more respondents and better questionnaire outreach are needed to draw a complete picture of the practices and knowledge level of DS consumption after the pandemic. Despite the limitations, this study brings an important understanding on the practice and knowledge of DSs consumption in Indonesia, which have reconfigured our food and dietary psychology and habits due to the COVID-19 pandemic.\n\nThis study showed the practices and knowledge level of DS consumption in Indonesia after the COVID-19 pandemic. Even though the self-consumption of DS is considered safe, its side effects in high risk populations, namely individuals with several comorbidities and patients on prescribed medication, might experience unwanted effects due to drug and disease interaction. Therefore, collaborative efforts from a multitude of organisations including medical doctors, pharmacists and governmental bodies to uphold their responsibilities to educate and provide essential and easily understandable information available to the general public. Based on this current study, the practice of self-consumption of DS in Indonesia is increasing, and this pattern is expected to increase further in the coming years. Hence, knowledge of DS is necessary to avoid detrimental effects that might occur in the future. This could be done by increasing accessibility of information with better labelling and educating youth, the future consumers to empower users to make wise choices on DS.", "appendix": "Data availability\n\nMendeley Data: Practice and Knowledge DS Indonesian Adult. doi: 10.17632/n2fdtbwrxb.2 (d'Arqom, 2022)\n\nThis project contains the following underlying data:\n\n- Data 514 analysis.xlsx\n\n- Erratum Data 514 analysis.xlsx (correction of coding variables, all started from 0)\n\nThis project contains the following extended data:\n\n- Supplementary file.docx (contains the survey instrument and differences between DS consumption practice of respondents with health-related background and non-health-related background)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAgbabiaka TB, Spencer NH, Khanom S, et al.: Prevalence of drug-herb and drug-supplement interactions in older adults: a cross-sectional survey. 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Publisher Full Text" }	[ { "id": "168163", "date": "30 Mar 2023", "name": "Mikołaj Kamiński", "expertise": [ "Reviewer Expertise Public Health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript presents a study focusing on the practices and knowledge level of dietary supplement (DS) consumption in Indonesia, particularly after the COVID-19 pandemic. The authors have provided a comprehensive background on the topic and presented their findings based on a well-structured questionnaire. However, there are certain aspects that need to be addressed to improve the manuscript's overall quality and clarity.\nMajor Comments:\nThe authors mention that the study was conducted after the COVID-19 pandemic. However, considering that the pandemic is still ongoing and that the WHO has not revoked the pandemic status, it would be better to rephrase the context of the study as \"during the COVID-19 pandemic\" or \"in the post-delta wave of the COVID-19 pandemic\" instead of \"after the COVID-19 pandemic.\"\n\nThe manuscript would benefit from a clear description of the study design, including the sampling method, sample size, and the study period. It is essential to provide these details to assess the study's robustness and reliability.\n\nThe authors should consider providing a demographic breakdown of the respondents, including age, gender, geographic distribution, and other relevant characteristics. This information is crucial to understand the generalizability of the study findings.\n\nThe authors should provide more details about the questionnaire, including how it was developed, its validation process, and how the knowledge levels of respondents were assessed.\n\nThe authors have highlighted several limitations of the study, such as the sampling method, questionnaire outreach, and potential bias in the responses. They should consider discussing potential strategies to address these limitations in future studies to provide a more comprehensive understanding of DS consumption practices and knowledge levels in Indonesia.\n\nThe discussion section could be better organized by separating the key findings and relating them to the existing literature. It is essential to highlight the novel aspects of the study and the implications of the findings for policy and practice.\n\nGiven the study's focus on practices and knowledge levels, it would be helpful to include recommendations for public health interventions to promote safe and informed DS consumption, such as educational campaigns or regulatory measures.\nMinor Comments:\nIn several instances throughout the manuscript, the authors use the term \"DS\" without defining it first. It would be helpful to define the term \"dietary supplements\" before using the abbreviation \"DS\" to improve readability.\n\nSome sentences are lengthy and could be broken down into shorter, more concise statements for better readability.\n\nThe manuscript would benefit from a thorough proofreading to correct grammatical errors, inconsistencies in citations, and formatting issues.\nIn conclusion, the manuscript presents an interesting study examining the practices and knowledge level of dietary supplement consumption in Indonesia during the COVID-19 pandemic. However, several aspects need to be addressed to improve the manuscript's quality and clarity. By addressing these concerns, the authors can present a more robust and reliable study that contributes to the understanding of dietary supplement consumption in the context of the ongoing pandemic.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9617", "date": "04 May 2023", "name": "Annette d'Arqom", "role": "Author Response", "response": "Response to Review and Editor Comments (Manuscript Number: ID 129045) We are very much thankful to the reviewers for their deep review. I have revised the manuscript in the light of their useful suggestions and comments. We hope the revision improves the paper to a level of their satisfaction. Number wise answers to the specific suggestions/comments are as follows: Response to Reviewer: 1 Comments to the Author The manuscript presents a study focusing on the practices and knowledge level of dietary supplement (DS) consumption in Indonesia, particularly after the COVID-19 pandemic. The authors have provided a comprehensive background on the topic and presented their findings based on a well-structured questionnaire. However, there are certain aspects that need to be addressed to improve the manuscript's overall quality and clarity. Major Comments: 1. The authors mention that the study was conducted after the COVID-19 pandemic. However, considering that the pandemic is still ongoing and that the WHO has not revoked the pandemic status, it would be better to rephrase the context of the study as \"during the COVID-19 pandemic\" or \"in the post-delta wave of the COVID-19 pandemic\" instead of \"after the COVID-19 pandemic.\" Response: Thank you very much for bringing up this matter. We change the sentences into post-delta wave of the COVID-19 pandemic as in the line 350 and 354. 2. The manuscript would benefit from a clear description of the study design, including the sampling method, sample size, and the study period. It is essential to provide these details to assess the study's robustness and reliability. Response: Thank you very much for pointing out this issue. We did mention the sampling methods and the study period in the line 52-55 (“The questionnaire was distributed to the adult population in Indonesia through email and social media using convenience sampling methods from October to December 2021, and multiple responds from single device were prevented based on respondents’ IP address.”); and the sample size in the line 62-65 (“Sample size was calculated using sample size calculator ( www.raosoft.com) with 5% margin of error, 95% confidence level, and population number filled with 100,000, resulting in 383 minimum respondents.”). 3. The authors should consider providing a demographic breakdown of the respondents, including age, gender, geographic distribution, and other relevant characteristics. This information is crucial to understand the generalizability of the study findings. Response: Thank you very much for your suggestion. The sociodemographic characteristic of the respondents was provided in table 1. 4. The authors should provide more details about the questionnaire, including how it was developed, its validation process, and how the knowledge levels of respondents were assessed. Response: Thank you very much for bringing up this matter. We did mention about the development of the questionnaire and the validation process as in the line 78-88 (“The questionnaire was modified from the Prevalence and Awareness Concerning Dietary Supplement Use among Saudi Adolescents ( Alfawaz et al., 2020) and Knowledge about dietary supplements and trust in advertising them: Development and validation of the questionnaires and preliminary results of the association between the constructs ( Karbownik et al., 2019). The questionnaire was combined and validated by two pharmacologists, a pharmacist, and a psychologist, and was further tested on 20 respondents to ensure that the content and terms used in the questionnaire were relevant and understandable. The reliability measurement on the survey data showed a Cronbach alpha coefficient of 0.750 for DS knowledge. As the reliability value for the preliminary pilot testing is at an acceptable level, the real data collection for this study was proceed.”). Moreover, the assesment the knowledge level of the respondents was mention in the line 102-105 (“The answers to the knowledge survey were measured using correct/wrong/does not know options measured as 1/0/0. The total score was the summation of the 16 question scores and was grouped in four categories based on the total score of each respondent: poor (0-4), moderate (5-8), good (9-12) and excellent (13-16).”) 5. The authors have highlighted several limitations of the study, such as the sampling method, questionnaire outreach, and potential bias in the responses. They should consider discussing potential strategies to address these limitations in future studies to provide a more comprehensive understanding of DS consumption practices and knowledge levels in Indonesia. Response: Thank you very much for pointing out this matter. We did provide the strategies to address this limitation for better future studies as in the line 348-350 (“Thus, more respondents and better questionnaire outreach are needed to draw a complete picture of the practices and knowledge level of DS consumption post-delta wave.”) 6. The discussion section could be better organized by separating the key findings and relating them to the existing literature. It is essential to highlight the novel aspects of the study and the implications of the findings for policy and practice. Response: Thank you very much for your suggestion. We divide the discussion part into three sections: (1) practice of dietary supplement consumption and its predictive factors, (2) knowledge on dietary supplements and its predictive factors, and (3) limitation and recommendation. 7. Given the study's focus on practices and knowledge levels, it would be helpful to include recommendations for public health interventions to promote safe and informed DS consumption, such as educational campaigns or regulatory measures. Response: Thank you very much for bringing up this matter. We did include the recommendation for promoting the safe and informed DS consumption as in the line 358-361 (“Therefore, collaborative efforts from a multitude of organisations including medical doctors, pharmacists and governmental bodies to uphold their responsibilities to educate and provide essential and easily understandable information available to the general public.”) and 364-365 (“This could be done by increasing accessibility of information with better labelling and educating youth, the future consumers to empower users to make wise choices on DS.”) Minor Comments: 8. In several instances throughout the manuscript, the authors use the term \"DS\" without defining it first. It would be helpful to define the term \"dietary supplements\" before using the abbreviation \"DS\" to improve readability. Response: Thank you very much for pointing out this issue. We did define dietary supplements as DS in the first sentence of the abstract. 9. Some sentences are lengthy and could be broken down into shorter, more concise statements for better readability. Responses: Thank you very much for the suggestion. We re-write some sentences to increase the readability, for example in the line 19-26, 31-36, 222-226, 290-293, 307-311, and 336-342. 10. The manuscript would benefit from thorough proofreading to correct grammatical errors, inconsistencies in citations, and formatting issues. Responses: Thank you very much for the suggestion. Our manuscript has been reviewed by a English native speaker. We do hope it meets your expectation. 11. In conclusion, the manuscript presents an interesting study examining the practices and knowledge level of dietary supplement consumption in Indonesia during the COVID-19 pandemic. However, several aspects need to be addressed to improve the manuscript's quality and clarity. By addressing these concerns, the authors can present a more robust and reliable study that contributes to the understanding of dietary supplement consumption in the context of the ongoing pandemic. Responses: Thank you very much for your review. We do hope that our revision does improve the quality and clarity of the manuscript." } ] } ]	1	https://f1000research.com/articles/12-3
https://f1000research.com/articles/12-467/v1	04 May 23	{ "type": "Study Protocol", "title": "Evaluation and comparison of effects of recycling on the shutter mechanism and shear bond strength (SBS) of various self-ligating brackets: An in vitro study", "authors": [ "Shefali Singh", "Rizwan Gilani", "Rizwan Gilani" ], "abstract": "Introduction\nThe procedures like sandblasting and direct flaming to remove the residual composite from brackets may affect the material properties of brackets, including the shutter mechanism and it’s shear bond strength. This study focuses on the comparison of these properties in new brackets and recycled brackets of various commercially available self-ligating brackets. Objectives To compare the effects of recycling on shutter mechanism and shear bond strength of different manufacturing companies of self-ligating brackets. Methodology Teeth will be mounted on acrylic blocks of different colors and the self-ligating brackets will be bonded on them. These brackets will be debonded by using universal testing machine and the shear bond strength and opening and closing force of shutter will be measured. The debonded brackets will be rebonded after recycling by sandblasting and flaming and the SBS and shutter mechanism force will be measured again and then will be compared with above mentioned group. Expected results\nThe force of closing and opening the shutter mechanism of different commercially available self-ligating brackets will increase. There will be a decrease in the shear bond strength of the different self-ligating brackets available on the market. Conclusion We will be able to determine the quality of commercially available brackets despite recycling by examining the force necessary for opening and closing the shutters of self-ligating brackets, as well as their shear bond strength to ensure that even after recycling, the brackets retain their mechanical integrity and bond strength.", "keywords": [ "Self-ligating brackets", "shear bond strength", "sandblasting", "shutter mechanism." ], "content": "Introduction\n\nIn dentistry, self-ligating brackets (SLBs) have been around for a long time. They have been used since the 1930s as a pioneering technology.1 In the past 30 years there has been a revival of interest in these devices, with various new inventions being developed in this field. Over the years, several advantages have been claimed over conventional bracket systems, such as reduced friction and noise levels. The most convincing prospective advantages of SLBs are a decrease in duration of treatment and a lower level of subjective discomfort associated with the procedure.2 Additionally, poorer biostability is claimed to promote periodontal health and more efficient chairside manipulation. Retrospective studies have suggested that this technique has a definite advantage of reducing the overall treatment time by four to seven months. A reduction is also seen in the number of appointments.3\n\nAs self-ligating orthodontic brackets have gained popularity in orthodontic practices over the past few years, they have become a more frequent choice among patients. As the years have gone on, self-ligating brackets have been increasingly used instead of conventional brackets. As a result of numerous studies depicting their properties and characteristics, such as frictional forces, torque expression, rotation capacity, bond strength, and reconditioning procedures, self-ligating brackets have been demonstrated to be superior in all these areas.4\n\nAfter brackets are bonded on the teeth, it can get debonded due to various factors like inadequate access, moisture contamination, or occlusal stress during masticatory function. Various recycling methods can be used to rebond the debonded brackets, like direct flaming, sandblasting, green stone, and tungsten carbide bur. Studies have reported that recycling of brackets can lead to a degradation of quality, lower bond strength and increased risk of cross-infection.5\n\nThere have been several studies that have compared the shear bond strength (SBS) of orthodontic brackets with varying base surfaces and different types of bases. There have been no studies on whether recycling affects the shutter mechanism of self-ligating brackets. Due to procedures like sandblasting and direct flaming to remove the residual composite from brackets, there is an increase in temperature which may affect the material properties of brackets, including the sliding mechanism.3 The force of closure and opening of the shutter might get affected by this change in temperature during the recycling of self-ligating brackets.\n\nVarious commercially available self-ligating bracket systems will be compared based on the force required to close and open their shutters after the recycling process.5 They are expected to show an increase in the amount of force which may be due to the temperature change due to direct flaming and sandblasting.6\n\nA self-ligating bracket system is designed to provide light forces that are applied to a low-friction surface in order to optimize the physiologic movement of teeth and to ensure a balanced oral environment. Studies have measured the force of shutter mechanism of new SBLs of different commercially available self-ligating brackets but not in recycled brackets. This study will focus on the comparison of change in force of opening and closure of shutter mechanism of new and recycled brackets.\n\nResearch has been done on the SBS of new and recycled conventional brackets and it has been concluded that recycling reduces SBS. This study will be based on comparison of SBS of new and recycled commercially available self-ligating brackets.\n\n\nObjectives\n\n\n\n1. To evaluate the effects of recycling on the shutter mechanism of different self-ligating brackets.\n\n2. To evaluate the effects of recycling on the SBS of different self-ligating brackets.\n\n3. To compare the effects of recycling on shutter mechanism and SBS of different manufacturing companies of self-ligating brackets.\n\nIn vitro study\n\n\nMethods\n\nThis in vitro study will be conducted in the Department of Orthodontics and Dentofacial Orthopaedics, Sharad Pawar Dental College in collaboration with Central Research Laboratory (Centre of Translation Sciences), Sawangi Wardha.\n\nInclusion criteria\n\n• 64 maxillary permanent premolars extracted for orthodontic purpose.\n\n• Non carious teeth with intact enamel and dentine.\n\nExclusion criteria\n\n• Abrasion, attrition, fluorosis or other enamel defects.\n\n• Teeth with developmental anomalies.\n\n• Teeth with no internal resorption.\n\nPumice and rubber prophylactic cups will be used to clean and polish the teeth for 10 seconds followed by washing with water.\n\nTeeth will be mounted on acrylic blocks of different colors to differentiate between the four types of commercially available brackets that are being used.\n\nGroup 1: Selfy\n\nGroup 2: 3M\n\nGroup 3: Damon\n\nGroup 4: AO\n\nAll brackets will be bonded to the teeth using the Transbond XT adhesive system. These mounted teeth will be placed under the universal testing machine for testing:\n\n1. Force of shutter mechanism\n\n2. SBS\n\nThe debonded bracket will be subjected to sandblasting and flaming to remove residual resin and the teeth will be prepared again by removing resin then the brackets will be rebonded on the teeth.\n\nThe force of closure of the shutter of the self-ligating bracket and SBS will be measured again using a universal testing machine.\n\n\nOutcomes\n\nThe force of closing and opening the shutter mechanism, which is the amount of force required to open the slot of different commercially available self-ligating brackets, will be measured by attaching an opening and closing apparatus to universal testing machine. This force is expected to increase after recycling.\n\nSBS is the maximum force which the adhesive joint can tolerate before the bracket is debonded. SBS will be calculated by subjecting them under universal testing machine and it is expected to decrease after recycling.\n\nSample size formula for difference between two means:\n\nWhere\n\nZα = level of significance at 5% i.e., 95%\n\nConfidence interval = 1.96\n\nZβ is the power of test = 80% = 0.84\n\nδ1 = SD of SBS in group 1 = 2.9011\n\nδ2 = SD of SBS in group 2 = 3.8872\n\n∆=Difference betweentwomeans=22.443–19.057=3.386\n\nK=1\n\nn = 16 teeth needed in each group\n\nThe total sample size is 64\n\nStudy reference: Kopal Agarwal et al.7\n\nAll the results will be analysed statistically using SPSS version 27 software. Data for outcomes variables will be tested for normality using the Kolmogorov-Smirnov test. The comparative analysis of the outcomes shutter mechanism force and SBS will be evaluated in Newton for the force of shutter mechanism and Newton/mm2 for SBS respectively. ANOVA will be used to find the significant difference between the mean of the 4 groups. A Tukey test will be used for pairwise comparative evaluation of measurements between two groups. A p-value ≤ 0.05 will be considered as significant at 5% level of significance and 95% confidence of interval.\n\nThe aim of this study will be to assess the efficacy and efficiency of commercially available self-ligating brackets. This study will help determine the quality of commercially available brackets despite recycling in terms of force required to open and close the shutter of self-ligating brackets and its SBS, to ensure good bond strength and mechanical retention even after recycling.\n\nThe study results will be published as an article in a journal.\n\nNot started yet.\n\n\nDiscussion\n\nA self-ligating bracket system is designed to provide light forces that are applied to a low-friction surface in order to optimize the physiologic movement of teeth and to ensure a balanced oral environment. There have been studies that measured the force of shutter mechanism different commercially available self-ligating brackets but not in recycled brackets. This study will focus on the comparison of change in force of opening and closure of shutter mechanism of new and recycled brackets.\n\nResearch has been done on the SBS of new and recycled conventional brackets and it has been concluded that recycling reduces it. This study will be based on the comparison of SBS of new and recycled commercially available self-ligating brackets.\n\nEthical approval was received from Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha (IEC reference number- DMIHER (DU)/IEC/2023/573).", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: Evaluation and comparison of effects of recycling on the shutter mechanism and shear bond strength (SBS) of various self-ligating brackets – An in-vitro study, https://doi.org/10.5281/zenodo.7809604. 8\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 would like to thank my institute and my colleagues.\n\n\nReferences\n\nFleming PS, Johal A: Self-ligating brackets in orthodontics. A systematic review. Angle Orthod. 2010 May; 80(3): 575–584. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDehbi H, Azaroual MF, Zaoui F, et al.: Therapeutic efficacy of self-ligating brackets: A systematic review. Int Orthod. 2017 Sep; 15(3): 297–311. PubMed Abstract \| Publisher Full Text\n\nGandini P, Orsi L, Sfondrini MF, et al.: Opening and closure forces of sliding mechanisms of different self-ligating brackets. J Appl Oral Sci Rev FOB. 2013; 21(3): 231–234. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBadawi HM, Toogood RW, Carey JPR, et al.: Torque expression of self-ligating brackets. Am J Orthod Dentofac Orthop Off Publ Am Assoc Orthod Its Const Soc Am Board Orthod. 2008 May; 133(5): 721–728. Publisher Full Text\n\nChalgren R, Combe EC, Wahl AJ: Effects of etchants and primers on shear bond strength of a self-ligating esthetic orthodontic bracket. Am J Orthod Dentofac Orthop Off Publ Am Assoc Orthod Its Const Soc Am Board Orthod. 2007 Nov; 132(5): 577.e1–577.e5. Publisher Full Text\n\nFotovat F, Shishehian A, Alijani S, et al.: Comparison of shear bond strength of orthodontic stainless-steel brackets on temporary crowns fabricated by three different methods: An in vitro study. Int. Orthod. 2022 Jun; 20(2): 100641. PubMed Abstract \| Publisher Full Text\n\nAgarwal K, Shetty S, Desai A: Effect of Various Mesh Designs on Shear Bond Strength of New and Recycled Brackets – A Comparative, Invitro Study. Indian J Forensic Med Toxicol. 2020 Oct 29; 14(4): 211–218.\n\nDr. Singh S , Dr. Gilani R : Evaluation and comparison of effects of recycling on the shutter mechanism and shear bond strength (SBS) of various self-ligating brackets – An in-vitro study. Zenodo. 2023. Publisher Full Text" }	[ { "id": "203584", "date": "22 Sep 2023", "name": "Saeed AlSamak", "expertise": [ "Reviewer Expertise Orthodontics and bio-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\nThe research protocol \"Evaluation and Comparison of Effects of Recycling on the Shutter Mechanism and Shear Bond Strength (SBS) of Various Self-Ligating Brackets: An In Vitro Study\" explores a significant and relevant topic within orthodontics. This review aims to assess the protocol's strengths and potential areas for improvement based on its content and methodology.\n\nStrengths:\n\nRelevance to Orthodontics: The choice of investigating the effects of recycling on self-ligating brackets is highly relevant in the context of sustainable dentistry and cost-effectiveness. With increasing environmental concerns, understanding the impact of recycling on these critical orthodontic components is essential.\n\nIn Vitro Approach: The decision to conduct an in vitro study allows for precise control over experimental conditions, reducing potential confounding variables. This ensures that any observed effects can be more confidently attributed to recycling, which is appropriate for this research.\n\nComprehensive Methodology: The protocol details the experimental procedures and methods for evaluating the shutter mechanism and shear bond strength. This thorough approach enhances the credibility and reliability of the study's findings.\n\nDiversity of Self-Ligating Brackets: The inclusion of various self-ligating brackets in the study is commendable, as it can provide a more comprehensive understanding of how recycling affects different bracket designs. This diversity enhances the study's applicability to real-world orthodontic practices.\n\nAreas for Improvement:\n\nSample Size Calculation: The protocol should provide a clear rationale for the chosen sample size, including a power analysis to justify the statistical power of the study. Ensuring an adequate sample size is crucial for obtaining meaningful results.\n\nMaterial used: A complete description of the bracket information, including full name, manufacturer, city, and country, should be added. Experimental setup: Details setup using a universal testing machine to measure the shear bond strength and the force of closing and opening the shutter mechanism, like the type of clamp used and point of force application, etc.\n\nRecycling Process: The protocol should provide detailed information on the recycling process used for the brackets. This includes the number of recycling cycles, the method of recycling heat details and sandblasting technique and material that will be used for this purpose, and the criteria for determining when a bracket is considered recycled. In addition, the effect of heat on the dimensional and mechanical properties of self-ligating brackets should be evaluated.\n\nRandomization and Blinding: To minimize bias and increase the study's internal validity, it's essential to outline how randomization and blinding will be implemented during the experiments.\n\nStatistical Analysis: A clear and detailed statistical analysis plan should be included in the protocol, specifying the statistical tests to be used, the significance level, and how the data will be analysed and interpreted.\n\nIn conclusion, the research protocol \"Evaluation and Comparison of Effects of Recycling on the Shutter Mechanism and Shear Bond Strength (SBS) of Various Self-Ligating Brackets: An In Vitro Study\" addresses an important topic within orthodontics. While it exhibits several strengths, such as clear objectives and a comprehensive methodology, it would benefit from further elaboration on sample size calculation, recycling process details, randomization and blinding strategies, and statistical analysis. These improvements will enhance the rigour and credibility of the study, making it a valuable contribution to the field of orthodontics.\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? No\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] }, { "id": "203616", "date": "22 Sep 2023", "name": "Orhan Cicek", "expertise": [ "Reviewer Expertise Orthodontics", "malocclusion", "dentistry" ], "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 to the authors for the study. In order for the study to be published scientifically, it should be re-submitted, which requires re-examination of many issues.\nAt the end of the introduction, the purpose and null hypothesis should be stated. The null hypothesis should be reconsidered in the discussion section according to the results.\n\nThe Method section should be presented in a way that explains the details of all materials used, technical devices, storage conditions, and test methods etc.\n\nResults should be presented clearly with tables and p values.\n\nThe Discussion section is very short. It should be revised, including all findings, the null hypothesis, and past and current literature. The strengths and limitations of the study should also be stated.\n\nThe Conclusion section should be stated.\n\nThe number of references is very few. It should be increased with current studies.\n\nThe rationale and objectives of the research were adequately described. However, a null hypothesis should be added.\n\nThe study design was appropriate to the research question but needs to be elaborated further.\n\nThe methods did not provide sufficient detail to allow replication by others.\n\nThe datasets were clearly presented in a usable and accessible format.\n\nTherefore, the study needs a comprehensive major revision.\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? No", "responses": [] } ]	1	https://f1000research.com/articles/12-467
https://f1000research.com/articles/12-466/v1	04 May 23	{ "type": "Study Protocol", "title": "To assess the efficacy of injection nalbuphine as an adjuvant to intrathecal bupivacaine in endoscopic urological surgeries for prolongation of postoperative analgesia", "authors": [ "Saiesh Raut Dessai", "Dr. Sanjot Ninave", "Amol Bele", "Nitin Alaspukar", "Dr. Sanjot Ninave", "Amol Bele", "Nitin Alaspukar" ], "abstract": "Spinal anesthesia has a quicker onset of action and completely blocks all motor functions. It requires less local anesthetic and has a reduced rate of failed blocks hence the preferred anesthetic method for lower abdomen procedures is spinal anesthesia. Intrathecal local anesthetics benefit from adjuvant medications by extending postoperative analgesia and thus improving the sensory blockade's duration and quality. Intrathecal opioids and local anesthetics work together synergistically to increase the sensory block while decreasing the sympathetic block. Adjuvants to local anesthetics for intrathecal administration have certain benefits, including lowered local anesthetic medication dose, improved hemodynamic stability, and fewer adverse effects. The opioid medication nalbuphine has a kappa agonist and mu antagonist action that reduces pain without having many negative side effects. It acts on opoid receptor as both an agonist and an antagonist to offer visceral nociception with a moderately powerful analgesic. Almost all general anesthesia and regional anesthesia treatments employ it. To cause analgesia, nalbuphine adheres to k receptors. Perioperative analgesia is also improved when administered as an adjuvant to bupivacaine. It is a hybrid which has both agonist and antagonist actions that increases and attenuates the effects of -opioids. Numerous researchers have investigated the properties of intrathecal nalbuphine. This study's objective to determine the efficacy of injectable nalbuphine as an augmentation to intrathecal bupivacaine in endoscopic urological surgeries to extend post operative analgesia.", "keywords": [ "Nalbuphine", "Endoscopic urological Surgeries", "Bupivacaine", "Postoperative pain" ], "content": "Introduction\n\nLower abdomen and lower leg procedures frequently use the anesthetic approach known as a subarachnoid blockade. The quality and length of the sensory blockage are improved by intrathecal local anesthetics combined with adjuvant medicines, which also prolong postoperative analgesia. The most often used spinal adjuvants to extend postsurgical analgesia are intrathecal opioids.1 The spinal adjuvants are the most frequently used to extend postsurgical analgesia.2\n\nTo induce moderately effective analgesia of visceral nociception, nalbuphine, a synthetic opioid analgesic that is extremely lipid-soluble, acts as both an agonist and an antagonist at the -opioid receptor.3 Almost all general anesthesia and regional anesthesia treatments employ it. Nalbuphine causes analgesia by its action on kappa receptors that are present in the central nervous system. Additionally, Adjuvant enhances perioperative analgesia quality while reducing adverse actions.4 It is a hybrid synthetic agonistic antagonist that leads to both increases and attenuation of the actions of opioids.5\n\nNalbuphine can counteract the negative effects of spinal opiates because, when administered systemically, it has a less rate of causing depression in respiratory system.6 When compared to intrathecal morphine, intravenous nalbuphine has fewer adverse effects, which include itching, vomiting,7 and does not appear to have any significant respiratory or hemodynamic side effects.8\n\nThis investigation compares the effectiveness of intrathecal injections of bupivacaine and nalbuphine for the analgesic effects during endoscopic urological procedures. The duration of analgesia will be the main result. Secondary outcomes include 2 dermatome regression, motor block duration, the cephalic extension, changes in hemodynamic parameters, and occurrence of side effects for example shivering, itching, fatigue, nausea/vomiting, and respiratory depression.\n\n\nProtocol\n\nTo compare the efficacy of the addition of Intrathecal Injection Nalbuphine as an adjuvant to Bupivacaine with Intrathecal Injection Bupivacaine alone for analgesia in endoscopic urological surgeries.\n\nPrimary objective\n\nTo compare the duration of postoperative analgesia between Intrathecal Bupivacaine alone and with Injection Nalbuphine as an adjuvant to Injection Bupivacaine.\n\nSecondary objectives\n\n1) To compare the effect on hemodynamic parameters.\n\n2) The commence and duration of sensory and motor block.\n\n3) Adverse consequences (sedation degree, postoperative nausea, vomiting, shivering, urinary retention, and any other)\n\n\nMethods\n\nAfter receiving approval from the Datta Meghe Institute of Education & Research, Sawangi (M), Wardha, DMIMS Institutional Ethical Committee and Screening Committee, the study will be carried out in the Department of Anaesthesiology, JNMC. Before the procedure, written and informed consent will be taken from the participants. Departmental steering committee will be monitoring the progress of study till completion.\n\nResearch design: Comparative Prospective\n\nStudy area: Department of Anaesthesiology JNMC & AVBRH.\n\nStudy period: 2 years\n\nStudy population: Patients undergoing Urological surgeries.\n\nAllocation of patient: Computer generated randomization.\n\nMales and females between the age group of 25-70 years\n\nPatient undergoing Urological operations.\n\nDuration of Surge 1-3 hours\n\nASA I & II\n\nMPC I & II\n\nPatients willing to participate in the study\n\nPatient fulfilling criteria for subarachnoid block\n\nLacking valid informed and written consent\n\nPatients with a history of bleeding disorders\n\nASA grade III and IV\n\nLocalized Infection at the block site\n\nPatient currently on anticoagulants\n\nPatients who have a history of neurological and musculoskeletal disease\n\nThe study will involve 60 participants who meet all inclusion requirements.\n\nTwo study groups will be randomly selected among the study's participants (Table 1):\n\nGroup A (n = 30): Injection Bupivacaine 15 mg (3 ml) + Sterile NS 0.2 ml\n\nGroup B (n = 30): Injection Bupivacaine 15 mg (3 ml) + Injection Nalbhuphine 1.5 mg (0.2 ml)\n\nStatistical analysis will be done using SPSS version 15.0 and a graphical representation of a mean P value of < 0.05 will be considered significant.\n\nThe calculation of sample size is based on:\n\nSample size formula for difference between two means\n\nWhere\n\nZα is the level of significance at 5% i.e., 95% confi1dence interval = 1.96\n\nZβ is the power of the test = 80% = 0.84\n\nσ1 = SD of onset of sensory block in group C = 1.05\n\nσ2 = SD of onset of sensory block in group M = 0.44\n\nΔ = 3.04 – 1.95 = 0.61 and k = 1\n\nn = 30\n\n➢ Reference = VK Chandra sample size determination in health studies, NTI Bulletin, 2006, 42/3 and 4, 55-62.\n\nPre-operative assessment\n\nI. A Day before the surgery, all patients will go through a pre anesthesia check-up.\n\nii. Basic patient information, including demographics, the illness's history, and current manifestations, a general examination, comprehensive evaluation, blood tests, and lab work will be indicated\n\niii) Aim, benefits, and drawbacks will be informed to the patients\n\niv) Each patient who will be a part of the study will give their informed, written consent.\n\nv) They will be required to continue fasting for at least eight hours before the procedure.\n\nv. The study's participants will be split into two groups at random (Table 1).\n\nGroup A (n = 30): Injection Bupivacaine 15 mg (3 ml) + Sterile NS 0.2 ml\n\nGroup B (n = 30): Injection Bupivacaine 15 mg (3ml) + Injection Nalbhuphine 1.5 mg (0.2 ml)\n\n\n\ni. An ECG, pressure monitor, and spo2 probe will be attached intraoperatively.\n\nii. 18g iv cannula will be used to secure an IV line, and fluid @ 10- to 15 ml/kg of Ringer Lactate will be given.\n\niii. Baseline vitals: Lead II's electrocardiogram will be continuously shown, Oxygen saturation, blood pressure, and heart rate will also be recorded for the patient.\n\niv. Premedication will be administered to all patients. 10 minutes before the anesthesia procedure, provide ondansetron 75–100 mcg/kg intravenously.\n\nv. Subarachnoid block using 25-gauge Quinke’s needle with the patient seated or leaning to the left in L3-4/L4-5 intervertebral area in midline will be given\n\nThe patient will be promptly turned into the supine position after the surgery is finished.\n\nOxygen will be given as a supplement to all patients (4-6 litres per minute via Hudson’s mask).\n\nUpcoming values will be noted.\n\n1. The motor and sensory paralysis onset.\n\n2. Highest level of sensory blocking that is reached and the length of time it takes to reach that level will be reported.\n\n3. The sensory blockade's two-segment regression time\n\n4. Sedation level\n\n5. VAS postoperative pain evaluation\n\n6. When to use rescue analgesia\n\n7. Negative outcomes, if any.\n\nUntil surgical anesthesia is obtained at dermatome level T10, the sensory blockage will be assessed using a blunt-tipped needle every 2 minutes. The motor block will be evaluated with modified Bromage scale.\n\n• The method entails checking parameters, two minutes for the first ten minutes, then the next 30 minutes every five minutes, and then every 15 minutes for the remaining time.\n\n• If the heart rate falls below 20% of baseline Inj. Glycopyrrolate 0.2 mg will be administered\n\nIn case there is hypotension (below 20% of baseline) Inj. Mephentermine in titrated boluses will be administered.\n\nAfter the study drug is administered, any adverse effects will be recognized and dealt with accordingly.\n\n• Symptoms such as vomiting, shivering, nausea, and pruritus, will be recorded.\n\n• Ondansetron 4 mg i/v for nausea and vomiting,\n\nInj Tramadol 50 mg i/v for shivering, Inj Hydrocortisone 100mg and injection Pheniramine Maleate for pruritus or any allergic reactions\n\nThe patients will be shifted to the monitoring ward. They will be monitored every 30 minutes for the first 6 hours and thereafter for 24 hours. Rescue analgesia will be given with iv paracetamol will be administered (15-20 mg/kg) when VAS is 4 or more.\n\nSensory block\n\nAt one-minute intervals, the progression of the block was assessed until it reached the T6. The sensory block will be measured using the pinprick method in the midclavicular line with a 27 G needle. The level was then tested every two minutes until the maximum sensory block was reached.\n\nDURATION OF SENSORY BLOCKADE\n\nWhen the highest degree of the sensory blockade has decreased by two segments after the injection of local anesthetic solution, the duration of the sensory blockade will be measured using TWO SEGMENT REGRESSION.\n\nMOTOR BLOCKADE\n\nBromage scale was used to ascess motor block.\n\nThe duration between the administration of the study medication and the point at which the Bromage 3 was detected is known as the onset of total motor blockage.\n\nWhen total anesthesia is established, surgery can begin. Both the sensory and motor levels will be assessed following surgery. Regression to level L1 and two-segment regression time from the maximum level will also be noted. Patients will be routinely monitored in the recovery and postoperative ward following surgery to assess their level of pain using the VAS scale.\n\nAccording to the study, which investigated the efficacy of intrathecal nalbuphine, the medication was found to elevate the potency of SAB and provided better pain control without affecting the patients' hemodynamic profile. Effective analgesia was measured as the time from injection to visual analog scale (VAS) score less than three. No cases of respiratory depression, sedation nausea, pruritus or vomiting were observed. They are typically associated with the activity of the μ receptor, as indicated by past research.8,9 Administering nalbuphine to patients undergoing surgery under spinal anesthesia (SAB) can prolong their pain relief without causing an increase in motor block duration. Our study also found that the inclusion of nalbuphine substantially prolonged the duration of analgesia in patients undergoing surgery but did not have any significant impact on the duration of motor block.\n\nComparisons have been made between nalbuphine and other spinal adjuvants that are frequently used, such as morphine and fentanyl. Study on patients undergoing cesarean section and found that compared to nalbuphine as an intrathecal adjuvant, morphine resulted in a longer duration of analgesia but also led to an adverse consequences such as pruritus, nausea, and vomiting in few cases.7 Another study conducted on patients undergoing hip surgeries also reported similar findings.10 Based on our study's results, it appears that nalbuphine can be a valuable adjuvant for patients undergoing endoscopic urological surgeries using spinal anesthesia. Our findings demonstrated that nalbuphine effectively extended analgesia.\n\nEvaluation of Nalbuphine as an Intrathecal Adjuvant to 0.5% Hyperbaric Bupivacaine at Two Different Doses for Postoperative Pain Management Following Abdominal Hysterectomy it was determined that heavy bupivacaine for sub arachnoid block works well when combined with 1.6 mg of intrathecal nalbuphine. While having no impact on breathing, it potentiated the SAB features and extended the analgesic effect. Nalbuphine with a dosage of 2.4 mg had no further benefits.11 In a study for lower limb anesthesia, it was concluded the addition of 800 mg of nalbuphine and 25 mg of butorphanol as an adjuvant to intrathecal bupivacaine produced better results than the active placebo group. But when it came to extending postoperative analgesia, intrathecal nalbuphine outperformed intrathecal butorphanol.12 Comparing the duration of the sensory block in the buprenorphine group to that in the nalbuphine group after using intrathecal nalbuphine and buprenorphine as an adjuvant in lower limb orthopaedic procedures. But throughout the whole intraoperative and postoperative time, neither group's blood pressure nor heart rate significantly decreased.13\n\nFollowing tubeless percutaneous nephrolithotomy, It was found that caudal bupivacaine can lower the severity of bladder discomfort caused by catheter both by itself and when combined with adjuvant fentanyl and nalbuphine.14\n\nDissemination\n\nIn our investigation, we anticipate that Group BN and Group B experience motor blockades for roughly the same amount of time. Better intra operative anesthetic quality is anticipated in Group BN. Additionally, we anticipate that Group BN will experience longer full and effective analgesia than Group B.\n\nStudy status\n\nControl group data collection ongoing.\n\n\nDiscussion\n\nAccording to the study, which investigated the efficacy of intrathecal nalbuphine, the medication was found to elevate the potency of SAB and provided better pain control without affecting the patients' hemodynamic profile. Effective analgesia was measured as the time from injection to visual analog scale (VAS) score less than three. No cases of respiratory depression, sedation nausea, pruritus or vomiting were observed. They are typically associated with the activity of the μ receptor, as indicated by past research.8,9 Administering nalbuphine to patients undergoing surgery under spinal anesthesia (SAB) can prolong their pain relief without causing an increase in motor block duration. Our study also found that the inclusion of nalbuphine substantially prolonged the duration of analgesia in patients undergoing surgery but did not have any significant impact on the duration of motor block.\n\nComparisons have been made between nalbuphine and other spinal adjuvants that are frequently used, such as morphine and fentanyl. Study on patients undergoing cesarean section and found that compared to nalbuphine as an intrathecal adjuvant, morphine resulted in a longer duration of analgesia but also led to an adverse consequences such as pruritus, nausea, and vomiting in few cases.7 Another study conducted on patients undergoing hip surgeries also reported similar findings.10 Based on our study's results, it appears that nalbuphine can be a valuable adjuvant for patients undergoing endoscopic urological surgeries using spinal anesthesia. Our findings demonstrated that nalbuphine effectively extended analgesia.\n\nEvaluation of Nalbuphine as an Intrathecal Adjuvant to 0.5% Hyperbaric Bupivacaine at Two Different Doses for Postoperative Pain Management Following Abdominal Hysterectomy it was determined that heavy bupivacaine for sub arachnoid block works well when combined with 1.6 mg of intrathecal nalbuphine. While having no impact on breathing, it potentiated the SAB features and extended the analgesic effect. Nalbuphine with a dosage of 2.4 mg had no further benefits.11 In a study for lower limb anesthesia, it was concluded the addition of 800 mg of nalbuphine and 25 mg of butorphanol as an adjuvant to intrathecal bupivacaine produced better results than the active placebo group. But when it came to extending postoperative analgesia, intrathecal nalbuphine outperformed intrathecal butorphanol.12\n\nComparing the duration of the sensory block in the buprenorphine group to that in the nalbuphine group after using intrathecal nalbuphine and buprenorphine as an adjuvant in lower limb orthopaedic procedures. But throughout the whole intraoperative and postoperative time, neither group's blood pressure nor heart rate significantly decreased.13 Following tubeless percutaneous nephrolithotomy, It was found that caudal bupivacaine can lower the severity of bladder discomfort caused by catheter both by itself and when combined with adjuvant fentanyl and nalbuphine.14\n\nINSTITUTIONAL ETHICS committee approved research to be carried out at JAWAHARLAL NEHRU MEDICAL COLLEGE, SAWANGI. Reference number DMIMS (DU)/II/C/2022/93.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nThe author would like to acknowledge the support from staff of Department of Anaesthesia, JNMC, AVBRH, Sawangi, Wardha, India.\n\n\nReferences\n\nBogra J, Arora N, Srivastava P: Synergistic effect of intrathecal fentanyl and bupivacaine in spinal anesthesia for cesarean section. BMC Anesthesiol. 2005 Dec; 5(1): 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwain A, Nag DS, Sahu S, et al.: Adjuvants to local anesthetics: Current understanding and future trends. World J. Clin. Cases. 2017; 5(8): 307–323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta K, Rastogi B, Gupta P, et al.: Intrathecal nalbuphine versus intrathecal fentanyl as adjuvant to 0.5% hyperbaric bupivacaine for orthopedic surgery of lower limbs under subarachnoid block: A comparative evaluation. Indian J. Pain. 2016; 30(2): 90. Publisher Full Text\n\nGunion MW, Marchionne AM, Anderson CTM: Use of the mixed agonist–antagonist nalbuphine in opioid based analgesia. Acute Pain. 2004 Jun; 6(1): 29–39. Publisher Full Text\n\nSchmauss C, Doherty C, Yaksh TL: The analgetic effects of an intrathecally administered partial opiate agonist, nalbuphine hydrochloride. Eur. J. Pharmacol. 1982 Dec; 86(1): 1–7. PubMed Abstract \| Publisher Full Text\n\nPenning JP, Samson B, Baxter AD: Reversal of epidural morphine-induced respiratory depression and pruritus with nalbuphine. Can. J. Anaesth. 1988 Nov; 35(6): 599–604. PubMed Abstract \| Publisher Full Text\n\nCulebras X, Gaggero G, Zatloukal J, et al.: Advantages of Intrathecal Nalbuphine, Compared with Intrathecal Morphine, After Cesarean Delivery: An Evaluation of Postoperative Analgesia and Adverse Effects. Anesth. Analg. 2000 Sep; 91: 601–605. PubMed Abstract\n\nTiwari AK, Tomar GS, Agrawal J: Intrathecal Bupivacaine in Comparison With a Combination of Nalbuphine and Bupivacaine for Subarachnoid Block: A Randomized Prospective Double-Blind Clinical Study. Am. J. Ther. 2013 Nov; 20(6): 592–595. PubMed Abstract \| Publisher Full Text\n\nMukherjee A, Pal A, Agrawal J, et al.: Intrathecal nalbuphine as an adjuvant to subarachnoid block: What is the most effective dose? Anesth. Essays Res. 2011; 5(2): 171–175. PubMed Abstract \| Publisher Full Text\n\nFournier R, Van Gessel E, Macksay M, et al.: Onset and offset of intrathecal morphine versus nalbuphine for postoperative pain relief after total hip replacement: Intrathecal morphine versus nalbuphine. Acta Anaesthesiol. Scand. 2000 Sep; 44(8): 940–945. PubMed Abstract \| Publisher Full Text\n\nShah MS, Masoodi T, Hussain SY, et al.: Nalbuphine as an Intrathecal Adjuvant to 0.5% Hyperbaric Bupivacaine in Two Different Doses for Postoperative Analgesia After Abdominal Hysterectomy: A Prospective, Randomized, Double-Blind Control Study. Cureus. 2022 May 16 [cited 2023 Mar 14]. Reference Source\n\nKumari A, Kullar K, Gupta R: Duration of postoperative analgesia with Nalbuphine vs Butorphanol as an adjunct to spinal anesthesia for lower limb orthopedic surgeries: A randomized double-blind active control trial. J. Anaesthesiol. Clin. Pharmacol. 2021; 37(4): 592–597. PubMed Abstract \| Publisher Full Text\n\nKaushal S, Kamlakar M, Baburao J: Intrathecal nalbuphine vs. buprenorphine as an adjuvant in lower limb orthopedic surgeries: a prospective randomized controlled study. Med. Gas Res. 2021; 11(4): 126–130. PubMed Abstract \| Publisher Full Text\n\nPrajapati D, Patel M, Patel P, et al.: Effect of caudal bupivacaine alone and with adjuvant fentanyl and nalbuphine to minimize the catheter-related bladder discomfort after tubeless percutaneous nephrolithotomy: A prospective randomized study. J. Anaesthesiol. Clin. Pharmacol. 2020; 36(4): 524–530. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "289243", "date": "21 Jun 2024", "name": "Samar Rafik Amin", "expertise": [ "Reviewer Expertise anesthesiology", "pain management", "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\nI acknowledge the authors efforts in conducting this trial which evaluates the effectiveness of nalbuphine as a spinal anesthesia additive to prolong the duration of postoperative analgesia and decrease the incidence of side effects in comparison to Bupivacaine alone. However, I have the following major concerns regarding the study construction; First, The manuscript needs thorough revision before further processing regarding language and grammar, as the authors used three verb tenses (past, present, and future) while describing the study methods. The abstract is not well-structured and doesn't reflect the main features of the study methods, results, and conclusion. It is more like a summary of the introduction. The introduction needs more concentration on the importance of your work and how it was different from the other literature that investigated similar research point. The sample size was not properly calculated. The sample size should be calculated based upon the study 1ry outcome ( duration of analgesia) not onset of sensory block. There was no reference for the prior estimate, also it is improper to use equations anymore in presence of many software that could calculate it more accurately. I think the study was underpowered by the sample size. The study design was not described appropriately as a randomized clinical trial and didn't mention if it was blinded or not. The methods were not clear enough starting from the method of randomization and allocation, the authors need to clarify who was responsible for the process of allocation, anesthesia administration, and outcome collection. Also, The outcomes were not described clearly, for example, What did you mean by duration of analgesia ( the study 1ry outcome) was it the sensory duration of the block or analgesia time or effective analgesia time, define the start and end time points accurately. The hemodynamics were included in your secondary outcomes, however they were not described in the methodology or results. authors should declare The brand name of used nalbuphine and if it was preservative free. The statistical analysis was not sufficiently described. No results section. The discussion section should be rewritten, 1st para of discussion shall be the summary of your key findings, and subsequent paragraphs should be the comparison of your findings with those of previous studies, Last paragraph shall include limitations of your study and then the conclusion ... The previous literature, authors cited in the discussion, should focus on similar study designs with similar conditions as theirs, for example, the present study didn’t include other additives than nalbuphine to hold a comparison with nor include open urological surgeries ( percutaneous nephrolithotomy) to cite its results.\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? No", "responses": [] }, { "id": "289246", "date": "25 Jul 2024", "name": "Manisha S. Kapdi", "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\nDesign, Objectives of the study are perfect. In methodology intervention, grouping ,Randomization are properly defined. Data management& statistical analysis satisfactory. Observations, results & Discussion are in scientific manner Present study evaluates efficacy of opioid Nalbuphine to intrathecal Bupivacaine for\nEndoscopic Urosurgery patients in terms of postoperative analgesia & hemodynamic stability. References are enlisted properly. At last conclusion of research question in very effective manner\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-466
https://f1000research.com/articles/12-464/v1	04 May 23	{ "type": "Research Article", "title": "Transition from transperitoneal to retroperitoneal approach in laparoscopic living donor nephrectomy: team-based and individual learning curve: a cross-sectional study", "authors": [ "Irfan Wahyudi", "Ilham Azka Adriansyah", "Kevin Yonathan", "Fina Widia", "Agus Rizal A Hamid", "Chaidir Arif Mochtar", "Ilham Azka Adriansyah", "Kevin Yonathan", "Fina Widia", "Agus Rizal A Hamid", "Chaidir Arif Mochtar" ], "abstract": "Background: Laparoscopic living donor nephrectomy (LLDN) is a standard practice for kidney donor transplantation due to fewer complications associated with the treatment. Retroperitoneal approach to LLDN is thought to be more advantageous in preventing injuries to various abdominal organs. However, the transition process from transperitoneal to retroperitoneal LLDN is important to ensure the best outcome for the patients. The aim of this study was to investigate the learning curve for retroperitoneal approach in LLDN among urologists in an individual and team-based approach Methods: A retrospective analysis of retroperitoneal LLDN was performed on procedures performed by a kidney donor team consisting of four urologists from January 2019 to January 2022 at Cipto Mangunkusumo National General Hospital, Indonesia. The data were taken from pre-existing medical records. The learning curve for the operation time, warm ischemic time, and estimated blood loss was analyzed using cumulative sum (CUSUM) analysis. Phase 1 represents the initial learning curve, the phase 2 plateau represents the period of proficiency, while phase 3 represents the mastery period. Results: A total of 127 retroperitoneal LLDN procedures were done during the study period by four operators with various experience. The average procedure needed to achieve proficiency was 16.5 procedures. Meanwhile, the average procedure needed to achieve mastery was 28 procedures. Shorter learning curve was achieved by operators with more experience in other laparoscopic procedures. Conclusions: The learning curve for transition from transperitoneal to retroperitoneal approach to LLDN is relatively short and feasible for both individuals and teams of urologists. Therefore, transition to retroperitoneal approach is a feasible option to reduce the complication rate of LLDN.", "keywords": [ "kidney donor", "laparoscopy", "nephrectomy", "transplantation" ], "content": "Introduction\n\nKidney transplantation is the best available option for the management of end stage renal disease (ESRD).1 Transplantation reduces the risk of cardiovascular and mortality events, while also improving quality of life compared to chronic hemodialysis.2 Living donor transplantation is preferred over deceased donor, as it has better survival rates and less delayed graft function.3,4 Laparoscopic living donor nephrectomy (LLDN) is the standard surgical practice due to fewer complications associated with the treatment.5 This technique includes standard laparoscopic, hand-assisted laparoscopic, hand-assisted retroperitoneoscopic, pure retroperitoneoscopic, and robot-assisted live donor nephrectomy.6–9\n\nPrevious studies have shown that the retroperitoneal approach to LLDN has more advantages than the transperitoneal approach; namely shorter access to renal arteries and veins, better visualization during donor nephrectomy, and prevention of injuries of the liver, spleen, and bowel.10–13 However, some adjustments to the techniques require additional training for the operators performing the technique. Evaluation of surgical practice performances could be done using the learning curve.14 However, there has only a few studies regarding the learning curve of the retroperitoneal approach of LLDN. Moreover, a previous study only analyzed learning curve on a single operator, although LLDN is usually performed by a team.15\n\nTherefore, this study aims to investigate the learning curve for retroperitoneal approach in LLDN among urologists in an individual and team-based approach.\n\n\nMethods\n\nA retrospective analysis of laparoscopic living donor nephrectomy (LLDN) procedures was done on four different urologists (operators) with different levels of experience from January 2019 to January 2022 at Cipto Mangunkusumo National General Hospital, Indonesia. All of the operators in our kidney donor team were included in this study. The date was determined as the first LLDN in our center was performed in January 2019 while the endpoint was the most recent date that the yearly review of the procedure was carried out.\n\nEthical Approval to conduct this study was issued by the ethics committee of the Faculty of Medicine Universitas Indonesia–Cipto Mangunkusumo Hospital (KET-720/UN2.F1/ETIK/2018). All patients had received detailed preoperative counselling and their decision to undergo the procedure was taken with written informed consent prior to the surgery. Meanwhile, the data used in this study were collected from the medical records of the patients. The written permission was granted by the ethics committee of the Faculty of Medicine Universitas Indonesia–Cipto Mangunkusumo Hospital for the publication of the study results.\n\nDemographical data was collected from the electronic medical record, including age, sex, and body mass index (BMI, calculated as weight in kilograms divided by square of height in meters). Preoperative data included number of arteries involved and side of the donor kidney. Intraoperative data included surgery duration, warm ischemic time (WIT), and estimated blood loss (EBL). Surgery duration was determined as the length of time from the first incision to renal artery clamping, as the final closure was sometimes done by the residents due to the study location being a teaching hospital. WIT was documented as the length of time from clamping to cold ischemic time. Postoperative complication was documented in accordance to the pre-existing data in the medical records.\n\nThe operative technique used in this study has been reported in other publications and become the routine technique used in our center, including for the subjects in this study.8,16 The LLDN was done under general anesthesia and the donor was positioned in the lateral decubitus position. First incision was then made one finger below the 12th rib with open approach (Hasson technique) to reach access to retroperitoneal space. Retroperitoneal space was created using balloon dilator. Afterward, an 11 mm trocar was then inserted into retroperitoneal space for camera. Other two incisions were made in line: one at the costovertebral angle and one the above anterior superior iliac spine. Pneumoretroperitoneum was created using a carbon dioxide (CO2) insufflation and the pressure maintained at 12 mmHg. The psoas muscle was identified and the Gerota fascia was incised to expose the ureter. Furthermore, the gonadal vein was identified, and its course was followed upward to find the renal vein. Each renal pedicle was identified individually and dissected. Perirenal fat was then dissected from renal parenchyma and the kidney was mobilized. Further dissection was performed to renal artery until its base at aorta and renal vein until below gonadal and adrenal tributaries. The ureter was dissected until the distal part near its crossing to iliac vessel. Retrieval of the kidney was done via suprapubic access created through a modified Pfannenstiel incision.\n\nIn this study, the procedures were performed by four urologists, namely surgeon 1 (S1), surgeon 2 (S2), surgeon 3 (S3) and surgeon 4 (S4). S1 was a urologist with more than 15 years of laparoscopy experience, S2 was a urologist with more than nine years of laparoscopy experience, S3 was a urologist with more than eight years of experience, while S4 was a urologist with more than four years of experience. All of the urology surgeons had performed more than 100 transperitoneal approach LLDN with limited experiences in retroperitoneal approach for other laparoscopic urology procedures, such as partial nephrectomy, adrenalectomy, unroofing renal cyst and proximal ureterolithotomy.\n\nThe learning method for retroperitoneal approaches used was done by viewing the video and visiting urology centers which are familiar with retroperitoneal approach of laparoscopy procedures, either for LLDN or for other nephrectomy procedures. The urology centers visited for the study were The Academic Medical Center, University of Amsterdam (Amsterdam, Netherlands), Radboud University Medical Center (Nijmegen, Netherlands), and SLK Kliniken Heilbronn GmbH (Heilbronn, Germany).\n\nThe average of LLDN surgery in our hospital was between two and three surgeries per week. As per the protocol in our center, all the surgeons were arranged to have fair and similar opportunity to perform retroperitoneal approach LLDN regularly, including in the transition process.\n\nCumulative sum (CUSUM) analysis was performed with SPSS version 20 (RRID:SCR_016479)(IBM Corp, 2011)17 to investigate individual surgeon operative time learning curves to reach optimal performance. Team-based analysis was separately performed to analyze the operative time by all of the four urologists. Three indicators, namely duration of surgery, WIT, and EBL were collected from pre-existing medical records and evaluated to represent the learning process taking place as the surgery repetition occurred. The CUSUM is the running total of differences between the individual data points and the mean of all data points, thus it can be performed recursively. The cases were ordered chronologically from the earliest to the most recent. The surgery duration for each case was defined as xi and the mean surgery duration of all cases was defined as μ. Therefore, the CUSUM at surgery duration n (CUSUMSDn) might be calculated as follow:\n\nThe CUSUMSD1 of the first case was the difference between the duration of surgery for the first case and the μ. The CUSUMSD2 of the second case was the previous case’s CUSUMSD added to the difference between the duration of surgery for the second case and the μ. The process then continued until the CUSUMSD for the last case was calculated.18\n\nAll of the surgeons in this study experienced three learning phases as described in a previous study.18 The three phases of the learning curve were identified by the inflection point of the CUSUM curve. Phase 1 (learning period) showed an expected incline in CUSUM curve which was the initial learning curve, phase 2 (proficiency) showed a decline in CUSUM curve, with a plateau which was the proficient phase with additional experience obtained leading to the achievement of expert competence. Phase 3 was defined as the post-learning (mastery) period, as seen in typical learning curve studies in which there was steeper decline of the CUSUM curve.\n\nTo determine the difference of characteristics and outcomes between phases, the Chi-square test was used to analyze categorical data and Wilcoxon test was used to analyze numerical data. All statistical tests were 2-tailed, and a p-value of < 0.05 was considered to as statistical significance. The statistical analyses were performed with SPSS version 20 (RRID:SCR_016479) (IBM Corp, 2011).\n\n\nResults\n\nA total of 127 patients underwent LLDN during the three years [30], with 31 operations done by S1, 30 operations by S2, 38 operations by S3, and 28 operations by S4. There was no conversion to open nephrectomy during the surgeries performed in this study.\n\nCUSUM analysis of LLDN operative time is shown in Figure 1. Based on the analysis of each surgeons, the average number of procedures needed to achieve proficiency (phase 2) was 16.5. Meanwhile, the average number of procedures needed to achieve mastery (phase 3) was 28. However, S4 did not achieve mastery during the study, thus the average number of procedures needed to achieve mastery was calculated only for S1, S2, and S3.\n\n(a) Surgeon 1 (15+ years of exp), (b) Surgeon 2 (9+ years of exp), (c) Surgeon 3 (8+ years of exp), (d) Surgeon 4 (4+ years of exp).\n\nBased on the analysis, it can be assumed that the number of procedures needed as a team to achieve proficiency (phase 2) was 66 procedures, while the number of procedures needed as a team to achieve mastery (phase 3) was 106 procedures (Figure 2).\n\nThere was a significant reduction in operative time after proficiency and mastery of LLDN (P < 0.001). Meanwhile, intraoperative WIT, EBL, and postoperative complication shows no significant difference between phases (Table 1).\n\n\nDiscussion\n\nMinimally invasive techniques are the preferred technique for donor nephrectomy compared to open nephrectomy. Studies found that a minimally invasive approach is associated with perioperative lower blood loss, lower morbidity,19 reduced analgesia use, shorter length of stay, and faster recovery.5 Retroperitoneal approach or retroperitoneoscopic provides direct access to renal hilum without moving abdominal organs. Therefore, injury or bleeding of the organs could be avoided and bowel function may return rapidly following the surgery.10,20 The crucial disadvantage of this procedure was the difficulty to orient the landmark due to the limited working space of the retroperitoneal approach in LLDN.21 In terms of the learning curve, retroperitoneal approach is more technically demanding and has a steeper learning curve than the transperitoneal approach.22–24\n\nLearning curves represent the relationship between learning repetition and practice performance.25 There are several methods that can be used to analyze the learning curves other than CUSUM analysis, such as graphical visual inspection, split-group method, and regression techniques. CUSUM method is considered the sensitive method adopted by surgical practice for self-and supervisor assessment. This method uses plotted graph and gives accurate progression and level of performance, thus, it can be used to show a process of achieving and maintaining a surgical technique proficiency.14\n\nA comparison study of retroperitoneoscopic donor nephrectomy by a single surgeon between a high volume and a low volume hospital by van der Merwe et al. describes the learning curve differences in surgery time and WIT. This study shows the improvement based on the graph decline.11 Meanwhile, a learning curve study of retroperitoneoscopic donor nephrectomy by Pal et al. analyzes the performance changes between three groups using the split-group method. However, this method has several biases based on its arbitrary group size option and uncertain changes.26\n\nIn our CUSUM analysis study, on an individual level, an average of 16.5 procedures was needed to achieve proficiency and 28 procedures to acquire mastery in the transition from transperitoneal to retroperitoneal approach. There were some differences between the results of this study and other learning curve studies of the retroperitoneal approach of LLDN. Pal et al.26 found a significantly reduced surgery time after 34 pure laparoscopic retroperitoneal nephrectomies. Chin et al.,15 though, observed a substantial difference in surgery time after 150 cases, although this study did not use the CUSUM model analysis. The second reduction of operation time or mastery is found after 300 laparoscopic nephrectomies in a study by Nakajima et al.27 However, the procedures needed to achieve both proficiency and mastery in our study differed on each operator. Operator 1 who had more experience in other surgeries only needed 12 procedures before achieving proficiency, while other operators needed more procedures in accordance with their prior experiences.\n\nBased on the CUSUM analysis, it was found that 66 procedure was needed to achieve proficiency (phase 2) and 106 procedures to acquire mastery (phase 3) for team-based approach of the transition from transperitoneal to retroperitoneal LLDN. Team-based analysis shows a more gradual improvement in operation time compared an individual surgeon-based analysis. A study by van der Merwe et al. shows that the subgroup analysis of a single surgeon shows a more graphically evident improvement in operation time than the team-based analysis.24 However, LLDN is usually approached by several operators, as it is technically demanding and require a significant amount of time.\n\nSeveral variables could affect the learning curve of the retroperitoneal approach of LLDN. The technical skills of each surgeon are related to shorter surgery time and lesser complications. The advantages of the retroperitoneal approach may relate to the surgeon’s experience as the vessel injury, or surgical complications were lower in the more experienced surgeon.10,19 In this study, urologists that have more experience achieve proficiency in operation time in fewer procedures. A study by Zhang et al. showed that senior surgeons had a better learning curve for laparoscopic procedures. Operation time was also significantly faster, with a lower conversion rate to open surgeries.28 Proficiency-based guidance, part-task training, and assigning practice over time might optimize the learning curve.29 There were other problematic factors, such as obese donors prolonged the surgery time then affected the learning curve.6 A few millimeters of perinephric fat would significantly prolong the surgery time.30\n\nA limitation of the study is in the methodology as it is a retrospective study based on pre-existing medical records and done in a single center. Therefore, information bias may occur due to the data input, processing, and collection. Moreover, being a teaching hospital, the performance of urology residents as the operative assistant may create another bias, especially for the operative time. However, we have limited the calculated operative time to only from the first incision to renal artery clamping, as the final closure was sometimes done by the residents.\n\n\nConclusions\n\nTransition from transperitoneal to retroperitoneal approach in laparoscopic living donor nephrectomy has a certain learning curve time to achieve proficiency and mastery, both on the individual and team-based analysis. Prior experience and focused training may improve the learning process. The transition to retroperitoneal approach is a feasible option to improve the outcome of LLDN.\n\n\nAuthors’ contributions\n\nIW involved in conceptualization, protocol development, administration of the study, data collection, funding, manuscript writing, and manuscript finalization.\n\nIAA involved in conceptualization, protocol development, administration of the study, data collection, and manuscript finalization.\n\nKY involved in conceptualization, protocol development, administration of the study, data collection, and manuscript finalization.\n\nFW involved in protocol development, study conceptualization, patient recruitment, and manuscript editing and finalization.\n\nARAH contributed in protocol development, study conceptualization, patient recruitment, and manuscript editing and finalization.\n\nCAM contributed in protocol development, study conceptualization, patient recruitment, and manuscript editing and finalization.\n\nAll authors read and approved the final manuscript.\n\n\nEthics approval and consent to participate\n\nEthical Approval to conduct this study was issued by the ethics committee of the Faculty of Medicine Universitas Indonesia–Cipto Mangunkusumo Hospital (KET-720/UN2.F1/ETIK/2018). Written informed consent was obtained from the patient for the surgery performed in this study. Written informed consent was obtained from a guardian for participants under 18 years old. Written permission was granted by the ethics committee of the Faculty of Medicine Universitas Indonesia–Cipto Mangunkusumo Hospital for the publication of the study results.", "appendix": "Data availability\n\nThe data used in this study were provided from pre-existing medical records of kidney donor patients in Cipto Mangunkusumo National Hospital, Indonesia. The authors obtained the data by submitting the ethical clearance to the research ethics of Faculty of Medicine, University of Indonesia, and requesting the written research permit for the data to the Innovation and Intellectual Property Management Installation of Cipto Mangunkusumo National Hospital, Indonesia.\n\nThe access to the same data will be provided following the formal ethical clearance request to the research ethics of Faculty of Medicine, University of Indonesia, and formal research permit request to the the Innovation and Intellectual Property Management Installation of Cipto Mangunkusumo National Hospital, Indonesia.\n\nDrayad: Transition from transperitoneal to retroperitoneal approach in laparoscopic living donor nephrectomy: Team-based and individual learning curve: A cross-sectional study https://doi.org/10.5061/dryad.3tx95x6kq. 31\n\nThis project contains the following extended data:\n\n- Dataset.sav (SPSS file containing all aggregated data in 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\nAcknowledgments\n\nThe authors would like to express the greatest gratitude for all the medical staffs and patients of Urology Department, Cipto Mangunkusumo National General Hospital, Indonesia who were willing to support the study.\n\n\nReferences\n\nLee L-Y, Pham TA, Melcher ML: Living Kidney Donation: Strategies to Increase the Donor Pool. Surg. Clin. N. Am. 2019; 99: 37–47. PubMed Abstract \| Publisher Full Text\n\nTonelli M, Wiebe N, Knoll G, et al.: Systematic Review: Kidney Transplantation Compared With Dialysis in Clinically Relevant Outcomes. Am. J. Transplant. 2011; 11: 2093–2109. Publisher Full Text\n\nGjertson DW, Cecka JM: Living unrelated donor kidney transplantation. Kidney Int. 2000; 58: 491–499. Publisher Full Text\n\nFakhr Yasseri AM, Namdari F, Gooran S, et al.: Living versus deceased kidney transplantation: Comparison of complications. Urologia. 2021; 88: 185–189. PubMed Abstract \| Publisher Full Text\n\nWilson CH, Sanni A, Rix DA, et al.: Laparoscopic versus open nephrectomy for live kidney donors. Cochrane Database Syst. Rev. 2011;Cd006124. Publisher Full Text\n\nOzturk SA, Yuksel Y, Erbis H, et al.: Laparoscopic Live Donor Nephrectomy: Experience of High-Volume Center with 2,477 Cases. Urol. Int. 2021; 105: 100–107. PubMed Abstract \| Publisher Full Text\n\nJacobs SC, Cho E, Dunkin BJ, et al.: Laparoscopic live donor nephrectomy: the University of Maryland 3-year experience. J. Urol. 2000; 164: 1494–1499. PubMed Abstract \| Publisher Full Text\n\nDols LFC, Kok NFM, Ijzermans JNM: Live donor nephrectomy: a review of evidence for surgical techniques. Transpl. Int. 2010; 23: 121–130. Publisher Full Text\n\nTakagi K, Kimenai H, Terkivatan T, et al.: Learning curves of minimally invasive donor nephrectomy in a high-volume center: A cohort study of 1895 consecutive living donors. Int. J. Surg. 2021; 86: 7–12. Publisher Full Text\n\nHe B, Bremner A, Han Y, et al.: Determining the Superior Technique for Living-donor Nephrectomy: the Laparoscopic Intraperitoneal Versus the Retroperitoneoscopic Approach. Exp. Clin. Transplant. 2016; 14: 129–138. PubMed Abstract\n\nVan der Merwe A, Heyns CF: Retroperitoneoscopic live donor nephrectomy: review of the first 50 cases at Tygerberg Hospital, Cape Town, South Africa. S. Afr. J. Surg. 2014; 52: 53–56. PubMed Abstract \| Publisher Full Text\n\nKohei N, Kazuya O, Hirai T, et al.: Retroperitoneoscopic living donor nephrectomy: experience of 425 cases at a single center. J. Endourol. 2010; 24: 1783–1787. Publisher Full Text\n\nTanabe K, Miyamoto N, Ishida H, et al.: Retroperitoneoscopic live donor nephrectomy (RPLDN): establishment and initial experience of RPLDN at a single center. Am. J. Transplant. 2005; 5: 739–745. Publisher Full Text\n\nValsamis EM, Chouari T, O’Dowd-Booth C, et al.: Learning curves in surgery: variables, analysis and applications. Postgrad. Med. J. 2018; 94: 525–530. PubMed Abstract \| Publisher Full Text\n\nChin EH, Hazzan D, Herron DM, et al.: Laparoscopic donor nephrectomy: intraoperative safety, immediate morbidity, and delayed complications with 500 cases. Surg. Endosc. 2007; 21: 521–526. Publisher Full Text\n\nWadström J: The Higher Rates of Ileus, Readmission and Hernia After Laparoscopic Donor Nephrectomy Reported Can Be Mitigated by Using a Retroperitoneal Approach Via a Pfannenstiel Incision. Transplantation. 2016; 100: e104. PubMed Abstract \| Publisher Full Text\n\nIBM Corp.: IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp; Released 2011.\n\nPark JS, Ahn HK, Na J, et al.: Cumulative sum analysis of learning curve for video-assisted mini-laparotomy partial nephrectomy in renal cell carcinoma. Medicine (Baltimore). 2019; 98: e15367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlowers JL, Jacobs S, Cho E, et al.: Comparison of open and laparoscopic live donor nephrectomy. Ann. Surg. 1997; 226: 483–490. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNg ZQ, Musk G, Rea A, et al.: Transition from laparoscopic to retroperitoneoscopic approach for live donor nephrectomy. Surg. Endosc. 2018; 32: 2793–2799. PubMed Abstract \| Publisher Full Text\n\nTay WK, Kesavan A, Goh YSB, et al.: Right Living Donor Nephrectomies: Retroperitoneoscopic vs Laparoscopic Transperitoneal Approach. Transplant. Proc. 2018; 50: 2333–2337. Publisher Full Text\n\nRuszat R, Wyler SF, Wolff T, et al.: Reluctance over right-sided retroperitoneoscopic living donor nephrectomy: justified or not? Transplant. Proc. 2007; 39: 1381–1385. Publisher Full Text\n\nSulser T, Gürke L, Langer I, et al.: Retroperitoneoscopic Living-Donor Nephrectomy: First Clinical Experiences in 19 Operations. J. Endourol. 2004; 18: 257–262. PubMed Abstract \| Publisher Full Text\n\nvan der Merwe A , Ebinger Mundorf NN, van Heerden H , et al.: Evaluating the differences in the early laparoscopic donor nephrectomy learning curves of a Swiss high volume transplant program and a South African low volume transplant program after knowledge transfer. Afr. J. Urol. 2021; 27: 119. Publisher Full Text\n\nPusic MV, Kessler D, Szyld D, et al.: Experience curves as an organizing framework for deliberate practice in emergency medicine learning. Acad. Emerg. Med. 2012; 19: 1476–1480. PubMed Abstract \| Publisher Full Text\n\nPal BC, Modi PR, Rizvi SJ, et al.: The Learning Curve of Pure Retroperitoneoscopic Donor Nephrectomy. Int. J. Organ Transplant. Med. 2017; 8: 180–185. PubMed Abstract\n\nNakajima I, Iwadoh K, Koyama I, et al.: Nine-yr experience of 700 hand-assisted laparoscopic donor nephrectomies in Japan. Clin. Transpl. 2012; 26: 797–807. PubMed Abstract \| Publisher Full Text\n\nZhang XM, Wang Z, Liang JW, et al.: Seniors have a better learning curve for laparoscopic colorectal cancer resection. Asian Pac. J. Cancer Prev. 2014; 15: 5395–5399. PubMed Abstract \| Publisher Full Text\n\nTakagi K, Kimenai HJAN, Terkivatan T, et al.: A novel difficulty grading system for laparoscopic living donor nephrectomy. Surg. Endosc. 2021; 35: 2889–2895. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchwartz FR, Shaw BI, Lerebours R, et al.: Correlation of preoperative imaging characteristics with donor outcomes and operative difficulty in laparoscopic donor nephrectomy. American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2020; 20: 752–760. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWahyudi I, et al.: Transition from transperitoneal to retroperitoneal approach in laparoscopic living donor nephrectomy: Team-based and individual learning curve: A cross-sectional study, Dryad. Dataset. 2022. Publisher Full Text" }	[ { "id": "178114", "date": "29 Jun 2023", "name": "Mustafa Suat Bolat", "expertise": [ "Reviewer Expertise Urology", "andrology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract Background\nThe authors state that the transition process from transperitoneal to retroperitoneal LLDN is important to ensure the best outcome for the patients. I wonder if there is a transition process in real. According to their experience and educational program, surgeons use their individual techniques. Therefore they only can state that they decided to convert their technique to Retro. Otherwise, it could be misunderstood that nephrectomy should primarily be managed by transperitoneal and it might be converted to retro.\nMethods\nHow did the authors identify Phase 1, 2 and 3?\nIntroduction\nSecond paragraph: Preferentially, l advise the authors to cite paper1. This citation is about to gain experience of laparoscopy. Thus it may improve quality of the manuscript. If authors read it, they will find data supporting their hypothesis.\n\nSixth Paragraph: What were the time period for three centers? Were the authors observer or fellow?\nDiscussion\nIn second paragraph, CUSUM analysis should be given in detail. The authors should remember that many readers might not know about CUSUM analysis.\n\nFifth paragraph - What is the reason for higher procedure experience need for team-based surgery compared to individual one? Please support it in the light of literature.\n\nLast paragraph - I supposed this paper should be a prospective one?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "196411", "date": "12 Oct 2023", "name": "Luis Garcia-Covarrubias", "expertise": [ "Reviewer Expertise Methodology", "surgical techniques" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. Of the references, only 30% are 5 years old or less, recommending that at least 50% of the references be updated.\n2. With respect to the design and in order to allow its reproducibility, I suggest being explicit in the definitions, as well as in the inclusion and exclusion criteria and performing a sample size calculation based on Cohen's D obtained from previous studies.\nI would suggest including the Clavien scale to compare complications or other variables, not just time such as hemorrhage, ischemia time, and whether or not the graft suffered any damage, for example.\n3. Mention the limitations of the study before reaching the conclusions, as it is a retrospective study and is limited by the indication for nephrectomy (right or left) and the assignment of surgeons to the cases, which is not commented on.\nFurthermore, regardless of whether it was transperitoneal or retroperitoneal, the surgeons had extensive experience in laparoscopic surgery.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-464
https://f1000research.com/articles/12-458/v1	02 May 23	{ "type": "Research Article", "title": "The impact of pharmacist behavioral intervention on antibiotics prescribing in pediatric wards", "authors": [ "Jasim Kadhim Abbas", "Basma Zuheir Al-Metwali", "Basma Zuheir Al-Metwali" ], "abstract": "Background: Antibiotics may be prescribed irrationally, which can lead to significant consequences including the emergence of antimicrobial resistance. Pharmacists can play a key role in enhancing the rational antibiotic prescribing. This study aimed to evaluate the current clinical practice of prescribing antimicrobial agents in pediatric wards and to assess the clinical and economic impact of pharmacist interventions on antibiotic prescribing. Methodology: This study was conducted at the pediatric wards of a general hospital, Babel, Iraq. The study population included all pediatric inpatients aged ≤ 12 years and receiving antibiotic. The study consisted of three phases. The first was a pre-intervention phase where data about antibiotic prescription was collected retrospectively. The second phase involved pharmacist intervention where the researcher had provided an oral presentation to the healthcare workers. The third phase involved prospective data collection which was compared with the pre-intervention data. Independent T-test was used to compare the differences in the parameters between the pre-and post-intervention phases. Results: The study included 250 children in each of the pre- and post-intervention phases who received antibiotic treatment. The medical records were reviewed retrospectively. Four antibiotics (amoxicillin, cefotaxime, vancomycin and meropenem) were significantly less frequently used in the post-intervention phase. Single antibiotic treatment was significantly higher in the post-intervention phase (p-value<0.05). Length and days of therapy, and the total cost of the antibiotics used were significantly lower in the post-intervention phase. Culture and sensitivity tests were performed only in 28% of patients in the post-intervention phase where the isolated bacteria were found to be highly resistant to penicillin and cephalosporin. Conclusions: This study demonstrated that pharmacist intervention in general pediatric wards has resulted in favorable clinical and economic outcomes. This emphasizes the essential role pharmacists can play in rational antibiotic use, not only in the pediatric population but also in the adults.", "keywords": [ "Antibiotics", "prescribing", "pediatrics", "pharmacist intervention", "Iraq" ], "content": "Introduction\n\nThe introduction of antibiotics in medicine has resulted in one of the most successful medical advances of the 20th century, i.e. a significant reduction in morbidity and mortality.1 Globally, antibiotics are among the most commonly used medicines, both in the community and hospital settings; however, this has been frequently associated with irrational use (prescription of incorrect doses, self-medication and treatment of non-bacterial illness). This irrational use of antibiotics not only increases the risk of unnecessary adverse effects but also contributes to the development of antimicrobial resistance (AMR) which is a leading cause of serious health and economic problems.2,3 High rates of bacterial resistance to broad spectrum antibiotics have been recorded in Iraq. In a study conducted in Babel, Iraq, all isolated bacteria showed high rates of resistance to the evaluated cephalosporin, and 100% and 87% resistance to cefotaxime and ceftriaxone, respectively.4 Globally, about 1.27 million deaths were directly attributable to AMR in 2019, which was considered the third leading cause of death that year, and it was estimated that AMR may kill 10 million people per year by 2050.5\n\nInfants and children frequently experience infections, which may predispose them to unnecessary antibiotic use due to the viral origin of most of these infections.6 This may predispose children to unnecessary adverse effects, particularly during the first year of life.7 Therefore, rational antibiotic use is very essential in this population.\n\nThe rational use of antibiotics was defined by World Health Organization (WHO) as “the cost-effective use of antimicrobials which maximizes clinical therapeutic effect while minimizing both drug related toxicity and the development of antimicrobial resistance.”8 Another definition by the Center for Disease Control and Prevention (CDC) is “prescribing antibiotics only when they are likely to be beneficial to the patient, selecting agents that will target the likely pathogens and using these agents at the correct dose and for the proper duration.”9\n\nVarious studies have been conducted to assess antibiotic prescribing both in adult and pediatric populations. In a study conducted in a Neonatal Intensive Care Unit, Pune, India, it was shown that 55.9% of neonates received between one and two antibiotics, 37.3% received three to five antibiotics and 6.7% neonates were prescribed more than five antibiotics.10 In Iraq, several studies were conducted to describe the antibiotic prescribing pattern in hospitals. A study conducted in Medical City Teaching Hospitals, Baghdad, has shown that 87% of patients had received the antibiotics at incorrect time and proper antibiotic selection was found only in 11% of patients.11 Additionally, a study conducted in Al-Elwia Pediatric Teaching Hospital in Baghdad has shown that 87.56% of the patients who visited the hospital have received antibiotics, and amoxicillin was the antibiotic preferred by pediatricians.12 Another study that surveyed five teaching hospitals in Baghdad has found that 66.7% of patients were treated with at least one antimicrobial agent and the neonatal intensive care unit had the highest antimicrobial prescription rate (97.2%) where ceftriaxone was the most frequently used antibiotic.13\n\nClinical pharmacist intervention (CPI) is one of the most important interventions used to optimize medication prescription, prevent drug-related issues and lower drug costs for both the patient and the hospital.14 A study conducted in Oman has demonstrated that CPIs on antimicrobial use had positive impact both on the clinical and economic outcomes by increasing the efficacy of the treatment, avoiding unnecessary exposure to an antibiotic, reducing the toxicity of the treatment and the net cost savings was close to $200,000 USD annually.15 Besides, a prospective interventional study conducted in Babel, Iraq showed that pharmacist intervention increased surgeons’ awareness of the proper use of the National Institute of Health and Care Excellence (NICE) guidelines of fluid therapy, subsequently improving prescription practice and decreasing the incidence of fluid-associated complications including fluid overload, weight gain, increased creatinine levels and electrolyte disturbances.16\n\nThis study was the first to evaluate the impact of pharmacist interventions on antimicrobial use in pediatric wards of an Iraqi hospital. This study aimed to evaluate the current clinical practice of antimicrobial agents’ prescription in pediatric wards and to assess the clinical and economic impact of pharmacist interventions on antibiotic use.\n\n\nMethods\n\nThe study was approved by the Ethics and Scientific Committee of Baghdad University college of Pharmacy and the Ethical and Scientific Committee of the Ministry of Health, Babel Health Directorate after obtaining the approval from the administration of the participating hospital. The information was obtained from the patient records. All handled data from medical records were de-identified. No direct interaction was made between the researchers and the patients. Verbal consent was obtained from the participating healthcare providers (physicians, pharmacists, lab staff and nurses) before the seminar. The healthcare providers were invited to the seminar presented by the researchers. There was no risk, so ethical committee did not require written or recorded consent.\n\nThis study was conducted at the pediatric wards of AL-Mahaweel General Hospital, Babel, Iraq between March and September 2022. The study consisted of three phases: a retrospective pre- intervention phase, an intervention and a prospective post-intervention phase.\n\nThe study targeted the healthcare providers in the hospital to improve the antibiotics-related practice (e.g. prescribing). To assess the impact of the pharmacist-led behavioral intervention on patient clinical outcome, the researchers extracted information from the medical charts of pediatric patients. The included medical records were for children (aged ≤12 years) from both sexes, and were admitted to the hospital and received antibiotic treatment. The exclusion criteria were records for pediatric patients who did not receive antibiotic treatment during their hospital stay.\n\nThe pre-intervention phase was performed between March and May 2022. Data about antibiotic prescriptions was retrospectively collected by reviewing the medical records of pediatric patients who were admitted to the hospital in the period between March 2022 and December 2021. The data of this part was analyzed statistically, and the results were used as part of the intervention.\n\nThe intervention included an oral presentation derived by the pharmacist (researcher) introduced to physicians, pharmacists, microbiologists and nurses. The outlines of the oral presentation were: 1) Antimicrobial stewardship(the effort to improve how antibiotics are prescribed), 2) history and benefit from introduction of antibiotics, 3) antibiotic resistance and the global action plan to overcome this problem, 4) results of Iraqi studies about antibiotic prescribing pattern, 5) bacterial susceptibility and resistance to antibiotics, 6) results of the retrospective phase of this study and 7) recommendations to improve antibiotic prescribing.\n\nThe post-intervention phase was performed between July and September 2022 and the data was prospectively collected by reviewing the medical records of pediatric patient who were admitted to the hospital during this period. Data of this phase was analyzed statistically and compared with that of the pre-intervention phase.\n\nData collected during the pre-and post-interventions phases included demographic information (age, gender, weight), diagnosis, antibiotics prescribed, number of antibiotics prescribed, length of therapy (LOT) (defined as the number of days that a patient receives antimicrobial agents irrespective of the number of different drugs), and day of therapy (DOT) (defined as the aggregate sum of days (including admission and discharge days) for which any amount of a specific antimicrobial agent was administered to individual patients regardless of the number of doses administered or dosage strength.17,18 Data about direct cost of antibiotics, and presence of culture and sensitivity tests was also collected.\n\nData was analyzed using the Statistical Package for the Social Sciences (SPSS) software version 25. Descriptive statistics were conducted for all study items. Continuous variables were expressed as means ± standard deviation (SD), whereas categorical variables were expressed as frequencies and percentages. Independent T-test was used to compare the differences in the means of continuous parameters (cost of AB, LOT, DOT) between the two study phases. Chi-square was used to measure the difference in the categorical variables (frequency of AB, number of AB) between the two study phases. A P-value of less than 0.05 was considered statistically significant.\n\n\nResults\n\nThe study recruited 500 children who received antibiotic treatments: 250 patients in the pre-intervention phase and 250 patients in the post-intervention phase. Most of the patients were male in both study phases (56% and 59.6% in the pre- and post-intervention phases, respectively). However, according to Chi-square test, there was no significant (p-value ˂0.05) difference between the two phases in terms of patient sex. The age of the pediatric patients ranged from one to 12 months in both phases. Pneumonia was the most frequent diagnosis in the pre-intervention phase (40.8%) whereas gastroenteritis was the most frequent one in the post-intervention phase (24.4%). These frequencies were comparable between the two study phases (Table 1).\n\nRegarding antibiotic use, four antibiotics (amoxicillin, cefotaxime, vancomycin and meropenem) were significantly more frequently prescribed for children in the pre-intervention phase compared to the post-intervention phase. Two antibiotics (ceftazidime and amikacin) were only prescribed in the post-intervention phase (Table 2).\n\n* Significant according to Pearson Chi-Square.\n\nRegarding the pattern of the antibiotics prescribed, double antibiotic treatment was mostly used in the pre-intervention phase (58.4%), whereas single antibiotic treatment was the most frequent in the post-intervention phase (59.6%). The difference in the number of antibiotics prescribed in the pre- and post-intervention phases was statistically significant (Table 3).\n\n* Significant according to Pearson Chi-Square.\n\nBoth length of therapy and days of therapy were significantly longer in the pre-intervention phase compared to post-intervention phase. The total cost of the antibiotics used in the pre-intervention phase was significantly higher than that in the post-intervention phase, since the cost per single vial/bottle of antibiotic was the same across the two phases (Table 4).\n\n* Significant according to independent T–test.\n\nThe culture and sensitivity (C/S) test were not performed for any patient during the pre-intervention phase. In contrast, 28% of the participating patients in the post-intervention phase did have a C/S test. Three types of samples were tested: urine (35.7%), throat swab (35.7%) and stool (28.6%). Among the seventy C/S tests in the post-intervention phase, only 18 samples had bacterial growth. Urine samples had the highest percent of bacterial growth (40%), while throat swap had the lowest growth rate (8%) (Table 5).\n\nEscherichia coli (E. coli) was the most commonly (61%) identified bacteria in the C/S test, followed by Staphylococcus epidermidis (Staph. epidermidis) (16.6%) (Table 6).\n\nNot all antibiotics were tested in each C/S test. The frequency of antibiotic testing ranged from 13 to 18 times. Four antibiotics had sensitive results in more than half of the conducted C/S tests: nitrofurantoin (100%), meropenem (81.25%), amikacin (70.6%) and gentamicin (53%) (Table 7).\n\n\nDiscussion\n\nIrrational antibiotic use is a global problem that requires special attention to reduce its adverse consequences. Pharmacists, being medication experts, can play a critical role in enhancing antibiotic prescription. To the best of the authors’ knowledge, this is the first study conducted in Iraq that has evaluated the impact of pharmacist intervention on antibiotic prescription in the pediatric population.\n\nMost of the patients were males in both study phases and in the age range of 1-12 months. This may be expected because infants in this age are more prone to infections due to their still-developing immune system. It was estimated that infants during their first year of life may be subjected to five to 11 episodes of infections.19 Pneumonia was the most frequent diagnosis in the pre-intervention phase whereas gastroenteritis was the most frequent one in the post- intervention phase (Table 1). This may be related to the time period during which the data was collected (winter months in the pre-intervention phase versus summer months during the post-intervention phase) where the risk of these kinds of infections increases. Similar results were obtained from other studies in Baghdad, Iraq and India, where gastroenteritis and respiratory tract infections were the most commonly reported diagnoses.12,20\n\nRegarding the type of antibiotics prescribed, amoxicillin, ceftriaxone and gentamicin were the most commonly prescribed drugs (Table 2). These are broad spectrum antibiotics and frequently used as empiric therapy, which could explain the obtained results. In the study conducted in AL- Elwia Pediatrics Hospital in Baghdad, the results have shown that amoxicillin was the most predominant antibiotic prescribed (72.24%) in the emergency departments of the hospital.12 Similarly, results from a study in the United Arab Emirates have shown that amoxicillin was the most frequently prescribed antibiotic (40%).21 In contrast, a study conducted in Saudi Arabia has shown that cephalosporin were more frequently (38%) prescribed in the pediatric intensive care unit (ICU) and aminoglycosides were frequently (45.4%) prescribed in the neonatal ICU.22\n\nIn the current study, results have shown that prescribing broad spectrum antibiotics, including meropenem and vancomycin, significantly decreased after pharmacist intervention (Table 2). Similar results were observed in an interventional study conducted in China in which the prescription of these antibiotics also significantly decreased in the post-intervention phase (p<0.05).23 Similarly, differences in the number of antibiotics prescribed in the two phases was statistically significant. Double antibiotic treatment was mostly used in the pre-intervention phase whereas single antibiotic treatment was the most frequent in the post-intervention phase (Table 3). On the other hand, LOT, DOT and the total cost of the antibiotics prescribed clearly decreased in the post-intervention phase (Table 4). This clearly shows the pivotal role of pharmacists’ intervention in enhancing antibiotic prescribing by the physicians. In Oman, the annual net cost savings from pharmacist interventions on antimicrobial use was close to $200,000 USD.15\n\nIn the current study, antibiotic prescribing was mostly based on the physicians’ experience and the availability of antibiotics at the hospital. C/S test was not done for any patient in the pre-intervention phase of the study. Nearly similar results were obtained in two studies conducted in AL-Elwia Pediatric hospital and five hospitals in Baghdad, where culture and sensitivity tests were only conducted on 0.08% and 1.2% of the patients, respectively.12,13 In comparison, during the post-intervention phase, the C/S test was done for 28% of the participating patients of which only 25.7% had bacterial growth (Table 5). These results show that antibiotics may be frequently prescribed for conditions that may not require their use.\n\nRegarding bacterial sensitivity to antibiotics, four antibiotics had sensitive results in more than half of the conducted C/S tests: nitrofurantoin, meropenem, amikacin and gentamicin. On the other hand, third-generation cephalosporin had a resistance rate of more than 90%, while ampicillin and amoxicillin showed full resistance (Table 7). Highly similar results were obtained from a study conducted in Al-Shomali General Hospital, Babel, Iraq from October 2019 to May 2020. The study showed a high rate of resistance to cefotaxime and ceftriaxone 100% and 87%, respectively, while low resistance was recorded against gentamicin and amikacin; 22% and 19%, respectively.4 In comparison, another study conducted in Bagdad, Iraq in 2012 showed that the rate of resistance to cefotaxime and ceftriaxone was 71.5% and 66.7%, respectively.24 On the other hand, a retrospective study conducted in Al-Diwaniyah Tertiary Hospital from in 2022 found that Staphylococcus species showed high resistance to ampicillin (92.9%) followed by ceftazidime (87.5%), and were highly sensitive to vancomycin. Additionally, E. coli showed high resistance to ampicillin (97.9%) and ceftriaxone (81.3%) while it was highly susceptible to meropenem (97.9%) and amikacin (97.6%).25 This increase in the rate of resistance to these antibiotics can be attributed to their irrational use as they are frequently used as empiric therapy based on their availability and broad spectrum of activity, with no limitation and without relying on culture and sensitivity test results.\n\nThis study had a number of limitations. The study included a relatively small number of patients and was conducted in one hospital. Additionally, this study focused on antimicrobial prescribing practices in the governmental sector without including any data from the private sector.\n\n\nConclusions\n\nThis study demonstrates that the pharmacist intervention on antimicrobial use in pediatrics resulted in favorable clinical and economic outcomes. These results indicate that clinical pharmacists can play an essential role in enhancing the practice of antimicrobial use in hospitals.", "appendix": "Data availability\n\nFigshare: Jasim Annas Data.xlsx, https://doi.org/10.6084/m9.figshare.22348534.v1. 26\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nHutchings M, Truman A, Wilkinson B: Antibiotics: past, present and future. Curr. Opin. Microbiol. 2019; 51(Figure 1): 72–80. Publisher Full Text\n\nLlor C, Bjerrum L: Antimicrobial resistance: Risk associated with antibiotic overuse and initiatives to reduce the problem. Ther. Adv. Drug Saf. 2014; 5(6): 229–241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCostelloe C, Metcalfe C, Lovering A, et al.: Effect of antibiotic prescribing in primary care on antimicrobial resistance in individual patients: Systematic review and meta-analysis. BMJ. 2010; 340(7756): c2096. Publisher Full Text\n\nAL-Khikani FH: Antimicrobial Resistance Profile Among Major Bacterial Pathogens in Southern Babil, Iraq. Galician Med. J. 2020; 27(3): E202036. Publisher Full Text\n\nMurray CJL, Kevin Shunji Ikuta FS: Articles Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022; 399.\n\nCraig JC, Williams GJ, Jones M, et al.: diagnosis of serious bacterial infection in young febrile children: prospective cohort study of 15 781 febrile illnesses.\n\nPrincipi N, Esposito S: Antibiotic-related adverse events in paediatrics: unique characteristics. Expert Opin. Drug Saf. 2019; 18(9): 795–802. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: WHO global strategy for containment of antimicrobial resistance, World Health Organisatin. WHO Glob. Strateg. Contain Antimicrob. Resist. 2001; 1–105. WHO/CDS/CS.\n\nBesser RE: Antimicrobial Prescribing in the United States: Good News, Bad News. Ann. Intern. Med. 2003; 138(7): 605–606. PubMed Abstract \| Publisher Full Text\n\nArticle O, Suryawanshi S, Pandit V, et al.: Antibiotic Prescribing Pattern in a Tertiary Level Neonatal Intensive Care Unit.2015; pp. 21–24.\n\nNagham T, Salih, Kadhim DJ: Adherence to American Society of Health-System Pharmacists Surgical Antibiotic Prophylaxis Guideline in Medical City Teaching Hospitals/Baghdad. Iraqi J. Pharm. Sci. 2016; 3: 62.\n\nObaid AA, Alwan DK, Majeed BA, et al.: Prescribing pattern of antibiotics in AL-Elwia Pediatric Teaching Hospital, Baghdad, 2016. Al-Kindy Coll. Med. J. 2017; 13(January).\n\nKurmanji JM, Hassali A, Versporten A, et al.: Global point prevalence survey in five teaching hospitals in Baghdad, Iraq. Mediterr. J. Infect. Microbes Antimicrob. 2021; 10(April).\n\nAlthomali A, Altowairqi A, Alghamdi A, et al.: Impact of Clinical Pharmacist Intervention on Clinical Outcomes in the Critical Care Unit, Taif City, Saudi Arabia: A Retrospective Study. Pharmacy. 2022; 10(5): 108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalman B, Al-Hashar A, Al-Khirbash A, et al.: Clinical and Cost Implications of Clinical Pharmacist Interventions on Antimicrobial Use at Sultan Qaboos University Hospital in Oman. Int. J. Infect. Dis. 2021; 109: 137–141. PubMed Abstract \| Publisher Full Text\n\nAbbood SK, Assad HC, Al-Jumaili AA: Pharmacist intervention to enhance postoperative fluid prescribing practice in an Iraqi hospital through implementation of NICE guideline. Pharm. Pract (Granada). 2019; 17(3): 1552–1558. Publisher Full Text\n\nPolk RE, Fox C, Mahoney A, et al.: Measurement of adult antibacterial drug use in 130 US hospitals: Comparison of defined daily dose and days of therapy. Clin. Infect. Dis. 2007; 44(5): 664–670. Publisher Full Text\n\nVallès J, Fernández S, Cortés E, et al.: Comparison of the defined daily dose and days of treatment methods for evaluating the consumption of antibiotics and antifungals in the intensive care unit. Med. Intensiva. 2020; 44(5): 294–300. Publisher Full Text\n\nMuller SJ, Zar HJ, Tooke L: Illness episodes in a cohort of preterm infants in their first year of life. SAJCH South African J. Child Heal. 2021; 15(1): 44–49. Publisher Full Text\n\nMohapatra S, Tripathy S, Balaji C, et al.: Prescribing pattern of antimicrobial agents in pediatrics department of a teaching hospital. Int. J. Basic Clin. Pharmacol. 2015; 4(4): 753–756. Publisher Full Text\n\nSharif SI, Nassar AH, Al-hamami FK, et al.: Trends of Pediatric Outpatients Prescribing in Umm Al Quwain, United Arab Emirates. Pharmacol. Pharm. 2015; 06(01): 9–16. Publisher Full Text\n\nBalkhy HH, El-Saed A, Alshehri A, et al.: Antimicrobial consumption in three pediatric and neonatal intensive care units in Saudi Arabia: 33-month surveillance study. Ann. Clin. Microbiol. Antimicrob. 2019; 18(1): 20–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTian J, Wang MM, Jiang X, et al.: Effect of pharmacist interventions on antibiotic use in the general pediatric ward. Pak. J. Pharm. Sci. 2020; 33(3): 1389–1395. PubMed Abstract\n\nAl-nema ZM: Trend of Antibiotics Usage in the Intensive Care Unit in the Medical City in Baghdad. Iraqi J. Pharm. Sci. 2016; 25(1).\n\nAl-Fatlawi BG, Jasim AL: Determining the Prevalence of Upper and Lower Urinary Tract Infections’ Pathogens and Their Antibiotic Susceptibility Profile for Adult Patients in Al-Diwaniya, Iraq. Iraqi J. Pharm. Sci. 2022; 31(4): 86–91.\n\nAbbas JK: Jasim Annas Data.xlsx. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "238435", "date": "16 Feb 2024", "name": "Malcolm Gillies", "expertise": [ "Reviewer Expertise Biostatistics", "pharmacoepidemiology and drug utilisation" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverview Abbas and Al-Metwali report an uncontrolled before-after study of a continuing education intervention to improve antibiotic prescribing in the paediatric wards of a general hospital in Babel, Iraq. They found reduced antibiotic use and uptake of culture and sensitivity tests in a 3-month period after physicians, pharmacists, microbiologists and nurses attended a lecture on antimicrobial stewardship that included feedback of pre-intervention prescribing data. The work is clearly presented, cites largely relevant literature and the statistical methods are appropriate. While these results are encouraging, the intervention and outcome measurement need to be described in greater detail, along with the rationale and context. The deposited study data are incomplete. Also, the study appears to have some significant limitations which need to be noted and discussed. Recommendations along these lines appear below. Reporting The reporting of interventions to reduce antibiotic use often has shortcomings, making them hard to assess and replicate.(1) I recommend that the authors follow the SQUIRE 2.0 guidelines for quality improvement reporting.(2) The article in its current form is missing information for many of the SQUIRE items, e.g. items from sections (9) “study of the intervention(s)”, (10) “measures”, (11) “analysis”, (13) “results”, (16) “limitations” and (17) “conclusions”. Intervention and study design To ensure effectiveness, interventions to change healthcare professionals’ behaviour should be designed systematically, building on knowledge and established practice in knowledge translation.(3) Interventions to improve antibiotic prescribing to hospital inpatients are effective, and there is specific evidence that restriction and enablement can increase compliance with antibiotic policies.(4) More generally, continuing education interventions can improve healthcare professionals’ practice.(5) Nonetheless “passive” approaches to behaviour change are generally ineffective and multifaceted interventions are better than single interventions.(6) Abbas and Al-Metwali incorporated an aspect of audit and review in their education intervention, namely feedback of prescribing data, and this may have contributed to its effect. I recommend the authors describe the rationale for their intervention, including any tradeoffs between effectiveness and cost/feasibility (SQUIRE 2.0 sections 5 and 7). Many interventions to improve antibiotic prescribing practice build on professional, national or institutional clinical practice guidelines, and measure concordance with guidelines as an outcome, taking a recent study by Pillay et al as an example.(7) I recommend that the authors describe any guidelines relevant to their study (SQUIRE 2.0 section 7). If it is meaningful in context and feasible, it would improve the study to analyse the results as % of prescriptions that were consistent with guidelines. If it is impossible to evaluate appropriateness quantitatively, is it possible to infer anything more about appropriateness from the available data, e.g. what can we conclude from the fact that gentamycin use remained the same? The introduction to the article reviews several studies of clinical pharmacist interventions to optimise prescribing, i.e. active review of prescriptions by a clinical pharmacist. As Abbas and Al-Metwali did a different kind of intervention, namely a single educational presentation, this section is a distraction. I recommend that the material on CPIs be replaced by a review of studies that are relevant to the intervention at Al-Mahaweel General Hospital. The effects of this intervention were analysed in an uncontrolled before-and-after design. While this design is commonly used and easy to do, there are serious threats to its validity and more complex designs are preferable.(8) I recommend that the authors describe these limitations in greater depth, for example considering if there were other events during the study period that may have influenced the results. As the authors already noted, the before and after periods covered different seasons and this influenced the case mix. The authors should consider whether the reduction in antibiotic use might be attributed to this case-mix difference. The article omits important information about the process for recruiting participants. I recommend the authors describe how they chose the sample size of 250 patients before and after intervention. The authors should also describe the method for selecting participants among those eligible, e.g. all individuals approached consecutively. I also recommend that they provide a CONSORT-style diagram showing participant flow, detailing the total number of eligible people during each study period, and the number who were approached.\nI have not looked at the study data properly, but I do see that the post-intervention data was not included in the spreadsheet on figshare. Please correct the dataset. Finally, the post-intervention data collection covered a three-month period immediately after the intervention, and therefore describes short-term results. As required in the SQUIRE 2.0 guidelines (section 17), I recommend a comment on the sustainability of the intervention, and on suggested next steps. Other comments There are a few other small errors in the article that should be corrected:\n“the period between March 2022 and December 2021” – please present the dates in order “throat swap” should say “throat swab” Reference 5, author’s surname is “Ikuta” not “Kevin”; title should exclude the word “Articles” Reference 6, title should start with a capital letter; citation details are missing Reference 10, author list should not include “Article O”; citation details are missing\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "307683", "date": "08 Aug 2024", "name": "Laith G. Shareef", "expertise": [ "Reviewer Expertise clinical pharmacy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study aimed to evaluate the current clinical practice of prescribing antimicrobial agents in pediatric wards and to assess the clinical and economic impact of pharmacist interventions on antibiotic prescribing, please refer to the economic impact of the intervention within the study title Regarding the intervention its recommended to add details regarding the source of information. Does the intervention presented to all hospital staff involved in the management of the 250 patients screen for the post intervention phase\nThe P value presented in different form please define the differences between these two forms: 0.000* and <0.0001* Table 5 entitled The number and results of culture and sensitivity tests in the post-intervention group is without significant correlation with aim of this study, if the author seeing its essential to be included at least they must compare the number of ordered test before and after the intervention.\nTable 6. entitled Types of bacteria identified by culture tests (N=18) without relevant information directly related to the core message of this study\nTable 7. entitled The results of antibiotic culture and sensitivity tests within the post-intervention group, without relevant information directly related to the core message of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-458
https://f1000research.com/articles/11-1115/v1	28 Sep 22	{ "type": "Review", "title": "Phytochemical profiling of Piper crocatum and its antifungal activity as Lanosterol 14 alpha demethylase CYP51 inhibitor: a review", "authors": [ "Tessa Siswina", "Mia Miranti Rustama", "Dadan Sumiarsa", "Dikdik Kurnia", "Tessa Siswina", "Mia Miranti Rustama", "Dadan Sumiarsa" ], "abstract": "Mycoses or fungal infections are a general health problem that often occurs in healthy and immunocompromised people in the community. The development of resistant strains in Fungi and the incidence of azole antibiotic resistance in the Asia Pacific which reached 83% become a critical problem nowadays. To control fungal infections, substances and extracts isolated from natural resources, especially in the form of plants as the main sources of drug molecules today, are needed. Especially from Piperaceae, which have long been used in India, China, and Korea to treat human ailments in traditional medicine. The purpose of this review was to describe antifungal activity from Piper crocatum and its phytochemical profiling against lanosterol 14 alpha demethylase CYP51. The methods used search databases from Google Scholar to find the appropriate databases using Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) flow diagram as a clinical information retrieval method. From 1,150,000 results search by database, there were 73 selected articles to review. The review shows that P. crocatum contains flavonoids, tannins, terpenes, saponins, polyphenols, eugenol, alkaloids, quinones, chavibetol acetate, glycosides, triterpenoids or steroids, hydroxychavikol, phenolics, glucosides, isoprenoids, and non-protein amino acids. Its antifungal mechanisms in fungal cells occur due to ergosterol especially lanosterol 14 alpha demethylase CYP51 inhibition as a result of 5,6 desaturase (ERG3) downregulation. P. crocatum has an antifungal activity by its phytochemical profiling that act against fungi by inhibiting the fungal cytochrome P 450 pathway, make damaging cell membranes, fungal growth inhibition, morphological changes, and fungal cell lysis.", "keywords": [ "Piper crocatum", "antifungal", "phytochemical profiling", "lanosterol 14 alpha demethylase", "CYP51" ], "content": "Introduction\n\nMycoses or fungal infections are a general health problem that often occurs in healthy and immunocompromised people in the community (Ramírez et al. 2013). Fungi are divided into four classes: yeasts, filamentous, dimorphic, and dermatophytes; generally, and ubiquitous in the environment, and become pathogenic when immune cells decrease (Howard et al. 2020). Fungal cells have essentially dynamic structure walls for morphogenesis, pathogenesis, and cell viability and act as a dynamic organelle, and need one-fifth of the yeast genome for cell wall biosynthesis (Gow et al. 2017). The development of resistant strain in fungi and the incidence of azole antibiotic resistance in the Asia Pacific which reached of 83% become a critical problem nowadays (Canuto and Rodero 2002; Jianhua and Hai 2009; Whaley and Rogers 2016; Whaley et al. 2017).\n\nThe azoles are commonly used because cheaper and have a broad spectrum of antimicrobials (Lewis 2011; Rosam et al. 2021). To control fungal infections, substances, and extracts isolated from natural resources, especially in the form of plants as the main sources of drug molecules today, are needed (Moghadamtousi et al. 2014; Balouiri et al. 2016). Natural products have limited or no side effects on human and animals antifungal activity (Tabassum and Vidyasagar 2013). Antifungal mechanisms in fungal cells occur due to ergosterol inhibition as a result of 5,6 desaturase (ERG3) downregulation (Neelofar et al. 2011). Ergosterol at the fungal plasma membrane is the most common sterol and binding at lanosterol 14α demethylase, an ergosterol-specific enzyme that can cause lanosterol demethylation (Loeffler and Stevens 2003; Ashley et al. 2006; Cannon et al. 2007; Emami et al. 2017).\n\nPiperaceae plant extracts have long been used in India, China, and Korea to treat human ailments in traditional medicine (Jeon et al. 2019). The part of the Piperaceae family which has large species of up to 1000 is the Piper genus (Durant-Archibold et al. 2018). Piper can be found in temperate regions with tropical and sub-tropical (Lima et al. 2020). Indonesia is located in the equator, which has a tropical climate with high humidity and has many natural and biological resources (Puspita et al. 2018). The seeds and leaves of Piper species are often cultivated and consumed for various diseases treatment such as antifungal, antibacterial, and disinfectant effects (Astuti et al. 2014; Mgbeahuruike et al. 2017).\n\nIsolation of several secondary metabolites of Piper species shows that therapeutically molecules like lignans, flavones, alkaloids, unsaturated amides, long and short-chain esters, aristolactams, monoterpenes, sesquiterpenes, ketones, aldehydes, arylpropanoids, chalcones, propenylphenols, and amide alkaloids as a typical constituent (Gutierrez et al. 2013). However, no tests were found on specific compounds for antibacterial activity from Piper (Barh et al. 2013). Based on the literature, antifungal compounds are classified into flavonoids, amides, acid derivatives, lignans, prenylated benzoic, cyclopentanedione, butenolides, and phenylpropanoids (Xu and Li 2011). Of the various Piper species, the main constituent is amide, which is classified as aristolactams, open-chain alkamides, amides with pyrolidine, 4,5-dioxoaporphines, Piperidine, and Piperidone groups, ceramides, cyclohexanamid, and cyclobutanamide (Do Nascimento et al. 2012). In this review, we summarize the antifungal activity properties, structural studies, and bio-mechanism of P. crocatum, which is commonly found worldwide, against lanosterol 14 alpha demethylase CYP51 in fungi. This review is expected to allow to find new alternative antifungal treatment agents from natural resources based on their active chemical compounds and activities to cure fungal infections, thus reducing the extensive and inappropriate use of antibiotics for antifungal treatment.\n\n\nMethods\n\nThe author searches databases from Google Scholar to find the appropriate databases using the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Flow Diagram as a clinical information retrieval method. The search screening occurred through four stages. The first stage was screening by the keywords, the second stage was screening by the criteria, the third stage was screening by relevance and duplicates, the fourth stage was screening by eligibility. The keyword of this search was ‘Piper’ which yielded 1,150,000 results. The second step was screening by inclusion criteria of the keyword ‘Piper’ that reported articles published from 2003 until 2022, which yielded 326,000 results. For the third step, screening by relevance and duplicate with the keyword of this search is ‘Piper crocatum’ AND ‘antifungal’, which yielded about 498 results. The final screening results included 73 articles to review. This search was conducted from February to May 2022. The criteria for this research was clinical trials in animal testing and human, books, laboratory tests, case studies, article reviews, systematic reviews, narrative reviews, and meta-analyses. The study was conducted with a true-experimental (Double-Blind RCT), quasi-experimental, study protocol, or pilot study. Articles were published in English. The flow chart of the Literature Review showed in Figure 1.\n\n\nResults and discussion\n\nP. crocatum, red betel, a shrub that consists of trunked, 5-10 cm continuous branches, with each segment having roots growing (Safithri and Fahma 2008). The stems of red betel are round, purplish-green heart-shaped leaves, have no flowers, green with grayish-white tapered ends, special aroma, and very bitter (Hermiati et al. 2013).\n\nIsolation of several secondary metabolites of P. crocatum contains of flavonoids, tannins, terpenes, saponins, polyphenols, eugenol (1), alkaloids, quinones (2), chavibetol acetate, glycosides (3), triterpenoids (4) or steroids, hydroxychavikol (5), phenolics, glucosides (6), isoprenoids, and non-protein amino acids (Erviana 2011; Gutierrez et al. 2013; Saputra et al. 2016; Li et al. 2019; Lister et al. 2020; Safithri et al. 2020).\n\nThe bioactive compound of P. crocatum as follows Table 1 and Figure 2.\n\nP. crocatum has been tested to C. albicans with agar diffusion method (Kusuma, Manan, et al. 2017), MIC determination with microdilution method, and MBC determination by the medium surface of Mueller Hinton Agar, and show that extract of ethanol indicates antifungal activity, MIC value 1.25-2.5% w/v, and MBC value 0.75 min (Kusuma, Hendriani, et al. 2017). The study shows about ten (10) known compounds (2,5-dimethoxy-3-glucopyranosylcinnamic alcohol, cimidahurinin, erigeside II, syringin, β-phenylethyl β-D-glucoside, methyl salicylate 2-O-β-D-glucopuranoside, icariside D1, 4-hydroxybenzoic acid β-D-glucosyl ester, benzyl β-D-glucoside, and phenylmethyl 6-O-α-L-arabinofuranosyl-β-D-glucopyranoside), and two new phenolic glucosides (Pipercroside A and B) that isolated from MeOH extract of P. crocatum elucidated by spectroscopy 1D and 2D NMR, HR-ESI-MS analysis also report erigeside II have the best antifungal activity with IC50 value as 58.5 (Li et al. 2019).\n\nPolyphenol inactivates protein and inhibits enzymes on the surface of bacterial cells; flavonoids form complexes that interfere with the function of the bacterial cell wall, inactivating microbial adhesion, enzymes, and cell protein transport by binding to bacterial extracellular proteins through hydrogen and covalent bonds; saponins have hydrophilic molecules and lipid thinning molecules (lipophilic) so that they can make lower cell surface pressure; tannins functions to form complex compounds with enzymes and substrates, thereby disrupting cell membranes, and phenol has hydroxyl and carbonyl groups that can interact with fungal cells through hydrogen bonds, thereby increasing protein coagulation and fungal cell membranes which will cause fungal cells to lyse (Januarti et al. 2019). Inhibition of fungal activity can be done by bother cell membranes, the activity of enzyme and fungi genetic mechanisms (Ejele et al. 2012).\n\nFungal cell walls, fungal-specific, serve as a protection from harmful environments and are aggressive because of its toxic and hydrolytic molecules. Ninety percent consist of polysaccharides, with Saccharomyces and Candida subphylum as the well-characterized fungal adhesins (Figure 3) (Latgé 2007; Gow et al. 2017). The core of the central fungal cell wall consists of chitin that is linked to branched β-1,3-glucan, combined with galactomannan, galactosamynoglycan, and β-1,3-1,4-glucan in A. fumigatus and β-1,6-glucan in C. albicans (Adams 2004; Latgé 2010). Chitin, with a weight of 1-2% from dry cell wall yeast, is the important structure that consists of a homopolymer of β-1,4-linked N-acetylglucosamine that is long and linear, while disrupted chitin synthesis will cause the fungal cell wall to be lysis and unstable in osmotic (Bowman and Free 2006).\n\nErgosterol is a key enzyme in fungal-specific sterols, cytochrome P450 enzyme in fungi derived from S. cerevisiae, belonging to the CYP51 (lanosterol 14-α-demethylase) family, so with inhibition of biosynthesis in ergosterol, will damage cell membranes (Arthington-Skaggs et al. 1999; Flowers et al. 2015). Fungal cell walls that have been damaged can cause fungal growth inhibition, morphological changes, and fungal cell lysis (Buitimea-Cantúa et al. 2013). ERG11 catalyzes C14-demethylation of lanosterol to 4,4'-dimethyl cholesta-8,14,24-triene-3-β-ol, sterol 14-reductase then reductases to 4,4-dimethylzymosterol, which in turn is converted to zymosterol, and with the help of sterol 8-isomerase is converted into ergosterol. Azole inhibits the synthesis of lanosterol 14α-demethylase (CYP51) ergosterol biosynthesis (Figure 4) becomes inhibited and damages cell membranes, leading to fungal lysis (Sanglard et al. 2003; Pemán et al. 2009; Emami et al. 2017; Rosam et al. 2021).\n\nAntifungals that damage cell membrane permeability work by binding to ergosterol in the polyene group, inhibiting the synthesis of ergosterol in squalane monooxygenase or epoxidase in the allylamines group, and inhibiting the synthesis of ergosterol in 14-α-demethylase or fungal cytochrome P450 in the azoles group; antifungal destroying cell walls works by inhibiting the synthesis of 1,3-β-glucan by binding to the glucan synthase enzyme which functions to form glucan in the echinocandin group; and antifungal inhibitors of DNA synthesis from fungal cells by inhibiting the synthesis of thymidilate or pyrimidine analogs in flucytosine (5-Fluorocytosine) and mitotic inhibitors in griseofulvin (Cannon et al. 2007; Lewis 2011).\n\nPhenol\n\nPhenol (carbolic acid) are secondary metabolites that can be found widely in Piper species, but rarely found in algae, fungi, and bacteria; organic compounds with low molecular weight, have one or more substituents hydroxyl group in aromatic phenyl ring, especially benzene, formed from phenylpropanoid or shikimate that produce phenylpropanoids and acetate or malonate polyketide pathway that produce simple phenols or with phenylpropanoids, identified by UV-Vis Spectra and retention times compared with the literature and reference compounds that available (Waniska 2000; Lattanzio 2013; Ferreira et al. 2016).\n\nPhenolic compounds have hydroxyl and carbonyl groups that can interact with fungal cells through hydrogen bonds, thereby increasing protein and cell membranes of pathogen fungal coagulation which will cause the damage and lysis of the fungal cells, and make the next fungal ergosterol growth being anomaly and malformation (Wagner and Donaldson 2014; Mohamed et al. 2017; Silva et al. 2018; Januarti et al. 2019). Phenolic also had antifungal activities by targeting to destroy the fungal pathogenic effects by inhibiting the fungal dimorphic transition, because dimorphic nature is very important for fungal survival in the host, with making a different morphologies in different conditions or temperatures, both as hyphae (pathogenic) or yeast cells (non-pathogenics) (Ansari et al. 2013).\n\nPolyphenol\n\nPolyphenols, known as an antifungal that has been isolated in Piper species, are water-soluble, have at least two phenolic rings, usually have 12-16 groups of phenolic hydroxyl at aromatic rings and have a molecular weight from 500 to 3,000 (Da) (Boulenouar et al. 2011; Cheynier 2012). Polyphenols inactivate protein and inhibit enzymes on the surface of bacterial cells; flavonoids form complexes that interfere with the function of the bacterial cell wall, inactivating microbial adhesion, enzymes, and cell protein transport by binding to bacterial extracellular proteins through hydrogen and covalent bonds (Januarti et al. 2019).\n\nTannin\n\nTannins ([2,3-dihydroxy-5-[[(2R,3R,4S,5R,6S)-3,4,5,6-tetrakis[[3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyl)oxybenzoyl]oxy]oxan-2-yl]methoxycarbonyl]phenyl] 3,4,5-trihydroxybenzoate) are important compounds that have been isolated in the Piper species and have beem classified as natural polyphenol groups, water-soluble, a molecular weight of 500-3000 daltons, condensed and hydrolyzed to polymerization that reaches high degrees and have two or three phenolic hydroxyl and carboxyl functional groups on a phenyl ring, known as antimicrobial, against various types of microorganisms, including bacteria, yeasts, fungi, and virus (Chung et al. 1998; de Jesus et al. 2012; Kardel et al. 2013; Delimont et al. 2017; Zhu et al. 2019; Ge et al. 2020).\n\nTannins inhibit the chitin growth in the fungi cell wall, which will cause fungal growth inhibition and cell metabolism disruption (Ridzuan et al. 2021). With a high affinity to polysaccharides and proteins, tannins function to form complex compounds with enzymes and substrates, thereby disrupting cell membranes (Hoste et al. 2006; Januarti et al. 2019). Plasma membrane and cell wall disruption that being tannin targeted will cause intracellular contents leakage (Zhu et al. 2019). Tannins have the best antifungal activity in C. albicans at a concentration above 7.80 mg/L, similar to nystatin, slightly lower than fluconazole, by shoots increasing, substrate and metal ion reduction, germ tube formation inhibition, and wall ultrastructure changing (Ishida et al. 2006).\n\nSaponins\n\nSaponins (beta-Escin), sapo in Latin, are one of the important secondary metabolites in the plant, insects, and marine organisms. They are amphiphilic, have surfactant, soap-like foams, and are heat-stable (Melzig et al. 2001; Haralampidis et al. 2002; Shi et al. 2004), used as herbs and known as antifungal and antibacterial (Francis et al. 2002; Zhang et al. 2006). Divided into triterpenoid and steroidal saponins based on their aglycones structure and biochemistry (in plants, the core structures-27 carbon atoms-as furostan (16β, 22-epoxy-cholestan) and spirostan (16α, 22:22α, 26-diepoxy-cholestan), they usually have a hydroxyl group at C-3 position for monodesmosidik, and at C-26 at saponin furastanol bidesmosidik or at C-28 at triterpen bidemosidik, sometimes also reported at C-2, C-15, C-16, C-21, lyobipolar so can affect to lower aqueous surface tension and cell membranes.\n\nSaponins destroy the cell membrane by binding in the cell wall with sterol components so that the pores are formed (Ridzuan et al. 2021). Saponins have hydrophilic molecules and lipid thinning molecules (lipophilic) so that they can make lower cell surface pressure (Januarti et al. 2019). It is reported that 64 μg/ml saponin extract can inhibit C. albicans growth and development, by mycelium inhibition, inhibit yeast transition to filamentous, inhibit surface polystyrene adhesion and phospholypase production secretion, and endogenous reactive species oxygen (ROS) induce and the high activity showed by A. minutiflorum saponin (minutoside B) against fungal attack (Barile et al. 2007; Jiang et al. 2015; Yang et al. 2018).\n\nFlavonoid\n\nFlavonoids (bioflavonoids), flavus in Latin, yellow powder, low molecular weight, are secondary metabolites that are very useful as antimicrobials by making bacterial damage, contains diphenylpropane (C6-C3-C6), and have a three-carbon bridge with phenyl groups, as the core structure of 2-phenylbenzopyra, less toxic and low cost (Baum et al. 2001; Galeotti et al. 2008; Can et al. 2015; Kurnia et al. 2018; Khalid et al. 2019; Herdiyati et al. 2020). As an important polyphenol class, flavonoid divided by C-ring oxidation degree, with the three-carbon segment oxidation degree and unsaturation degree, and the major classes are flavonols (3-hydroxy with the different site at OH group of phenolic), flavanones (C-4-keto-group with double-blind of C-2 and C-3), flavanols or flavan-3-ols (C3-hydroxyl group and carbon ring that fully saturated), isoflavones (act like phytoestrogens), aurones, chalcones (two aromatic rings by three-unit carbons to make the group of α,β unsaturated carbonyl), and anthocyanidins (Pourcel et al. 2007; Corradini et al. 2011; Seleem et al. 2017). Flavonoid have activities such as antibacterial, antioxidant, antiinflammation, and antifungal properties by its ability to form complexes with extracellular protein and interfere with the microbial membrane activity because of its lipophilic properties (Candiracci et al. 2011).\n\nResearch showed that flavonoid isolated, like 3,4-dihydroxy-5,6,7-trimethoxyflavone, cirsiliol, cirsimaritin, and hispidulin showed antifungal activity to C. sphaerospermum (Alcerito et al. 2002). Research also showed that flavonoids in honey have an antifungal activity to C. albicans growth inhibition, but not kill the yeasts (Candiracci et al. 2011), prenylated flavanon indicated that high antifungal activity to Trichophyton spp with 1.95 g/ml MIC value (Jin 2019), and flavonoids inhibit the growth of fungal that increased every concentrations level against Aspergillus niger van Tieghem, Aspergillus fumigatus Fresenius, Altenaria alternata (Fr.) Keissler, Penicillium citrii, and Macrophomia phaseolina (Tassi) Goid (Kanwal et al. 2010).\n\nThis literature review summarizes the phytochemicals and antifungal mechanisms of P. crocatum that are commonly found worldwide, and also other important activities of this Piper. Fungal infections are a problem that often occurs and becomes an ongoing and serious threat to public health (Kathiravan et al. 2012; Ramírez et al. 2013). The Polyene amphotericin B is one of the antibiotics that is still often used for the treatment of life-threatening fungal diseases, even though it is known to have toxic side effects (Odds 2003). Fluconazole are also known to have resistance to Candida species due to pressure continuous exposure, drug interactions, and side effects like visual impairment (Canuto and Rodero 2002; Chen and Sorrell 2007). The increasing use of antifungal treatment causes resistance to antibiotics that are used commonly, even in patients who have never used the drug (Arif et al. 2009; Arendrup 2014). The development of strains that are starting to become resistant to antibiotics from fungal species nowadays is a critical problem that must be addressed immediately in therapeutic problems in society by providing new antifungal agents (Johnson et al. 2004; Jianhua and Hai 2009; Moghadamtousi et al. 2014). Natural resources are very important to developing new active molecules and properties, with utilization of natural ingredients as antifungal treatment has a greater level of safety if used appropriately and correctly in terms of dose, time, and method of use (Vengurlekar et al. 2012). Plants have a lot of bioactive secondary metabolites such as flavonoids, terpenoids, alkaloids, tannins, saponins, and other compounds as antifungal agents (Arif et al. 2009). Based on the results of the review, it was found that P. crocatum contain compounds that have activity as an antifungal agent so it is expected to become a new antifungal agent that can slowly replace the extensive use of antibiotics. Therefore, new antifungal agents that safer, few side effects, cheaper, easier to get, and more potent against fungal infections are needed.\n\n\nConclusions\n\nNatural products are an important resource in the discovery and development of new medicinal raw materials. P. crocatum has antifungal activities that against fungal by its compounds and inhibit ergosterol as a key enzyme in fungal-specific sterols. Damaged cell membrane will cause fungal growth inhibition, morphological changes, and fungal cell lysis. Based on the review data, it is hoped that it can be used as a reference regarding information of new potential bioactive compounds as an alternative treatment for fungal infections by their lanosterol 14 α demethylase CYP51 inhibition effect other than the use of antibiotics or currently used drugs.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgments\n\nThe authors are grateful to the Universitas Padjadjaran, Indonesia; Badan Pengembangan dan Pemberdayaan Sumber Daya Manusia (Badan PPSDM) Kementerian Kesehatan, Indonesia, and Poltekkes Kemenkes Pontianak, Indonesia.\n\n\nReferences\n\nAdams DJ: Fungal Cell Wall Chitinases and Glucanases. Microbiology. 2004; 150(7): 2029–2035. Publisher Full Text\n\nAlcerito T, Barbo FE, Negri G, et al.: Foliar epicuticular Wax of Arrabidaea brachypoda: Flavonoids and Antifungal Activity. Biochem. Syst. Ecol. 2002; 30(7): 677–683. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaputra A, Andayani S, Nursyam H: Total Quantity of Phenol and Isolation Methanol Tannin Extract of Red Betel Leaf (Piper crocatum). Int. J. PharmTech. Res. 2016; 9(7).\n\nSeleem D, Pardi V, Murata RM: Review of Flavonoids: A Diverse Group of Natural Compounds with Anti-Candida albicans Activity In Vitro. Arch. Oral Biol. 2017; 76: 76–83. PubMed Abstract \| Publisher Full Text\n\nShi J, Arunasalam K, Yeung D, et al.: Saponins from Edible Legumes: Chemistry, Processing, and Health Benefits. J. Med. Food. 2004; 7(1): 67–78. PubMed Abstract \| Publisher Full Text\n\nSilva V, Igrejas G, Falco V, et al.: Chemical Composition, Antioxidant and Antimicrobial Activity of Phenolic Compounds Extracted from Wine Industry By-Products. Food Control. 2018; 92: 516–522. Publisher Full Text\n\nSuri MA, Azizah Z, Asra R: A Review: Traditional Use, Phytochemical And Pharmacological Review Of Red Betel Leaves (Piper crocatum Ruiz & Pav). Asian J. Pharm. Res. Dev. 2021; 9(1): 159–163. Publisher Full Text\n\nTabassum N, Vidyasagar GM: Antifungal Investigations on Plant Essential Oils. A Review. Int. J. Pharm. Pharm. Sci. 2013; 5(SUPPL. 2).\n\nVengurlekar S, Sharma R, Trivedi P: Efficacy of Some Natural Compounds as Antifungal Agents. Pharmacogn. Rev. 2012; 6(12): 91–99. PubMed Abstract \| Publisher Full Text\n\nWagner A, Donaldson L: Metabolic Engineering of Wood Formation. Appl. Plant Cell Biol. 2014; 22. Publisher Full Text\n\nWaniska RD: Structure, Phenolic Compounds, and Antifungal Proteins of Sorghum caryopses. Int Crop Res Inst Semi-Arid Trop. 2000.\n\nWhaley SG, Berkow EL, Rybak JM, et al.: Azole Antifungal Resistance in Candida albicans and Emerging Non-Albicans Candida Species. Front. Microbiol. 2017; 7(Jan). PubMed Abstract \| Publisher Full Text\n\nWhaley SG, Rogers PD: Azole Resistance in Candida glabrata. Curr. Infect. Dis. Rep. 2016; 18(12). Publisher Full Text\n\nXu W-H, Li X-C: Antifungal Compounds from Piper Species. Curr. Bioact. Compd. 2011; 7(4): 262–267. PubMed Abstract \| Publisher Full Text\n\nYang L, Liu X, Zhuang X, et al.: Antifungal Effects of Saponin Extract from Rhizomes of Dioscorea panthaica Prain et Burk against Candida albicans. Evidence-based Complement Altern. Med. 2018; 2018: 1–13. Publisher Full Text\n\nZhang JD, Xu Z, Cao YB, et al.: Antifungal Activities and Action Mechanisms of Compounds from Tribulus terrestris L. J. Ethnopharmacol. 2006; 103(1): 76–84. PubMed Abstract \| Publisher Full Text\n\nZhu C, Lei M, Andargie M, et al.: Antifungal Activity and Mechanism of Action of Tannic Acid against Penicillium digitatum. Physiol. Mol. Plant Pathol. 2019; 107(January): 46–50. Publisher Full Text" }	[ { "id": "163050", "date": "22 Feb 2023", "name": "Zubaida Yousaf", "expertise": [ "Reviewer Expertise Genetics and genomics of medicinal plants" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 received the following manuscript for review from F1000Research: \"Phytochemical profiling of Piper crocatum and its antifungal activity as Lanosterol 14 alpha demethylase CYP51 inhibitor: a review\". The following sections required revisions and modifications: Title, abstract, introduction, results and discussion and references.\nTitle:\nPlease ensure that you title clearly specifies whether your study focuses on fungal enzyme inhibition assay, mechanism of action or another topic. Or change your title according to the results and discussion. Your abstract and introduction is somewhat related to your title but not according to the results and discussion.\nAbstract: There is no information found on these lines of abstract in your results and discussion:\n\"Its antifungal mechanisms in fungal cells occur due to ergosterol especially lanosterol 14 alpha demethylase CYP51 inhibition as a result of 5,6 desaturase (ERG3) downregulation. P. crocatum has an antifungal activity by its phytochemical profiling that act against fungi by inhibiting the fungal cytochrome P 450 pathway, make damaging cell membranes, fungal growth inhibition, morphological changes, and fungal cell lysis\".\nIntroduction: Difference in statement of study idea in introduction and abstract.\n\nResults and discussion:\nIn heading \"Ethno-botany and ethno-pharmacology of P. crocatum\". Add ethno botanical data related to plant like traditional uses and ethno-pharmacological aspects of plant.\n\nIn heading \"The phytochemical and chemical structures of P. crocatum\". Sentence structure needs improvement. Don't repeat prepositions in sentence making.\n\nGive reasons of bio active compounds (leaf extract and essential oils) you have mentioned in table 1 and figure 2. Do these compounds have anti fungal activities?\n\nIn heading \"Antifungal properties and structure\" these highlighted lines \"Ergosterol is a key enzyme in fungal specific sterols, cytochrome P450 enzyme in fungi derived from S. cerevisiae, belonging to the CYP51 (lanosterol 14-α-demethylase) family, so with inhibition of biosynthesis in ergosterol, will damage cell membranes\". I think these are the exact lines that have reflection of title statement. So, you should provide references of this activity from different published articles here in results and discussion and also in introduction and abstract.\n\nIn figure 3: Spelling mistakes in caption.\nReferences:\nFollowing amendments are required in reference list and citations:\nCheck references according to journal format.\n\nCitations should be according to standard format.\n\nIf more than 3 authors in references use et al after first author name. If 1 or 2 authors then write single or both authors’ name with year like (Mohamed 2006), (Mohammad and Jakupovie 2006), or (Mohamed et al. 2006).\n\nIn case of two or more citations in the text, the citations must be arranged in chronological order.\n\nArrange references alphabetically.\n\nRemove repetition in references.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-1115
https://f1000research.com/articles/12-456/v1	02 May 23	{ "type": "Research Article", "title": "Assessment of work-related stress and burnout among clinical research occupations", "authors": [ "Raul Flores Luna", "Carmen De la Rocha", "Marco Antonio Cid López", "Ivette Venegas López", "Sandra Carolina Gómez Méndez", "Wendy López Romero", "Brenda A. Alvarado García", "Brandon A. Almeida Zarate", "Claudia Mejía Morales", "Raul Flores Luna", "Carmen De la Rocha", "Marco Antonio Cid López", "Ivette Venegas López", "Sandra Carolina Gómez Méndez", "Wendy López Romero", "Brenda A. Alvarado García", "Brandon A. Almeida Zarate" ], "abstract": "Background: Burnout syndrome is included by the World Health Organization (WHO) in the International Classification of Diseases, and it has a direct impact on the health of workers and also on the performance of companies. In this work, we propose a comprehensive methodology for evaluating work stress and burnout in various occupations within a healthcare organization. Methods: The work stress questionnaire and the Maslach Burnout Inventory were administered to employees of 7 different occupations within a company. The effect of the stressors is established using Cohen's d test, as well as the relationship between the main stressors, burnout and occupation. Results: The results of the study indicated that medical service personnel had the highest levels of stress and burnout syndrome, but significant stress levels and burnout scores were also identified among finance, clinical operations, and marketing professionals. Conclusions: Stress and burnout can vary by department, team, or individual in a company. Based on the study, it was concluded that a single approach to assessing stress and burnout may not provide a complete understanding, in fact, a better alternative is to assess each occupation specifically.", "keywords": [ "stress", "occupations", "emotional exhaustion", "depersonalization", "personal fulfillment" ], "content": "Introduction\n\nWork stress is a common issue faced by many employees, and is caused by several factors, including continuous overwork (Panigrahi, 2016), unclear job responsibilities (Kim and Hye-Sun, 2022), and difficulty in adapting to the behaviors of customers, co-workers, or any present health issues (Salama et al., 2022). This stress can result in chronic anxiety, psychosomatic illnesses, and various emotional problems (Gray and Muramatsu, 2011; Selmanovic et al., 2011). Chronic stress can be particularly detrimental to health and well-being, leading to long-term physical and mental health problems. When employees face continuous stress and lack strategies to manage it, they can suffer burnout, a condition characterized by physical, emotional and mental exhaustion (Selmanovic et al., 2011).\n\nOccupations with direct patient or client interaction experience high levels of burnout and stress, including intensive care units, oncology, emergency services, and operating rooms in the medical sector (Lloyd et al., 2002; Piko, 2006; Menéndez & Papasidero, 2019; Chirico et al., 2021; Corona et al., 2022). Burnout syndromes are frequently reported in these high-stress environments (West et al., 2009). Neglecting these issues can have negative consequences, not only for individuals but also for company productivity (Selmanovic et al., 2011; Corona et al., 2022).\n\nThe consequences of ignoring these levels of stress and burnout in a company can be serious. Individuals can suffer from chronic anxiety, psychosomatic illness and emotional problems. This, in turn, can lead to decreased job performance and increased risk of turnover (Selmanovic et al., 2011; Corona et al., 2022). For organizations, the consequences can include decreased productivity, increased healthcare costs and a negative impact on the company’s reputation. To mitigate these risks, it is important for organizations to periodically assess the stress and burnout levels of their employees and implement strategies to support their well-being.\n\nThis paper presents a comprehensive methodology for evaluating work stress and burnout syndrome among the various occupations within an organization dedicated to providing care to people’s health as well as the development of clinical research. The objective of this study is to gain a deeper understanding of the extent and nature of work-related stress and burnout among employees of an organization dedicated to the health care of people, as well as to the development of clinical research. The methodology proposed in this article includes the use of standardized tools, such as surveys and interviews, to gather data from employees across different occupations within the company.\n\n\nMethods\n\nThe study had an exploratory and descriptive scope and involved two cross-sectional surveys conducted at different time points to minimize the potential for any biases or confounding variables that may have arisen if the surveys were conducted simultaneously. The study was conducted using the same set of questions for all participants. This helped to reduce bias, and also made it easy to compare the responses across participants. The participants were assured that their responses would be kept confidential, which was done by providing anonymous surveys or by keeping their personal information confidential. Moreover, all the employees in the company were selected for this study, which helped to reduce bias by ensuring that all members had an equal chance of being selected.\n\nThe study recruited a total of 79 participants from an organization dedicated to providing care to people’s health located in Jalisco, Mexico. All company employees were invited to participate and the sample included people from various departments and positions, ensuring complete coverage. The company provides services related to clinical research, medical testing, and general and specialized consultations, among others.; This company was selected because the CEO planned to create an internal wellness plan within the company. Participants were informed about the study and its purpose, which was to improve the overall well-being and work environment within the company. This transparency helped to build trust and encourage participation. The data collection was paper-based and took place in the month of February 2022 in two different weeks. Sociodemographic background information was collected: sex, age, marital status, number of children and occupation of each participant.\n\nThe first survey used was the Job Stress Survey (Spielberger et al., 2010). It consists of three scales: 1) Job Stress Severity Scale (JS-S) which indicates the average severity score perceived by the individual in 30 stressful situations (ranges from question 2 to 30, question 1 is the standard stressor); 2) Job Stress Frequency Scale (JS-F) which indicates the average frequency with which the 30 stressful situations have occurred in the last 6 months (from question 31 to 60) and 3) Job Stress Index (JS- X) where an overall level of stress is considered. The job stress index is obtained as a result of multiplication between the result of JS-S and JS-F and results between 0 and 81 are obtained (Puteri & Syaebani, 2018); for this case the overall effect size of JS-F index is considered. The effect size was determined using Hedges’ g-test or Cohen’s d-test, according to the nature of the data (Faraone, 2008). The effect was considered small if it was between the values of 0 to 0.49, medium between 0.5 and 0.79, and large between 0.8 and higher (Arnoldo & Víctor, 2015; Faraone, 2008). Cronbach’s α coefficients were 0.97, 0.94, and 0.97 for JS-S, JF-S, and JS-X, respectively.\n\nThe second survey used was the Maslach Burnout Inventory (Maslach et al., 1996). It was used to determine burnout syndrome, being the most used and best validated for the assessment of burnout (West et al., 2009). It consists of 22 items divided into 3 dimensions: 1) emotional exhaustion (EE), with 9 items, the overall score being low if it presents a value from 0 to 18; moderate from 19 to 26 and high > 27; 2) depersonalization (DP), with 5 items, being low from 0 to 5, moderate from 6 to 9 and high > 10; 3) and difficulty in personal accomplishment (PA), with 8 items, being low from 0 to 33 (high level of burnout), moderate from 34 to 39 (medium level of burnout) and high > 40 (low level of burnout) (González & De la Gándara, 2004; Liebenberg et al., 2018; Menéndez & Papasidero, 2019). The items are rated on a 7-point Likert-type scale ranging from 0 (never) to 6 (every day). For the analysis, binary correlations were performed for the scales and the global burnout score, considering all occupations; subsequently, bar graphs were used to observe the occupations where the burnout response and the scales associated with the syndrome predominate (Rumschlag, 2017). Cronbach’s α coefficients were 0.90, 0.60 and 0.81 for EE, DP and PA, respectively. The study by Lee et al. (2019), also showed a lower α value for PA (0.49), so they suggest excluding 3 items from the PA scale to obtain a higher score (0.85), however, when excluding the items, for this sublevel no values higher than 0.67 were reached, so it was decided to continue with the information obtained.\n\nSociodemographic aspects were expressed as frequencies and percentages. Age was recorded considering intervals of < 30 years; 30 to 39 years; 40 to 49 years; 50 to 59 years; and 60 to 80 years (age intervals of the National Vaccination Plan in Mexico). For both types of surveys, normality was assessed using the Shapiro-Wilk test and equality of variances using the Levene test. Sample comparisons were performed using the Mann-Whitney U test for nonparametric data or the t test for parametric data. In comparisons where the sample size was greater than 20, Cohen’s d was used, and when the sample size was less than 20, Hedges’ h was used. In the present study, only stressors with medium to large effects and a significance level ≤0.05% were considered.\n\nStatistical analysis was performed with the IBM SPSS statistical package (version 26, IBM Corporation, Armonk, NY, USA).\n\nThe study was conducted in accordance with the General Law for the Protection of Personal Data. Ethical approval was granted by the Research Ethics Committee of Investigación Biomédica para el Desarrollo de Fármacos, S.A. de C.V. (Folio number: CEI:000001) on December 8 of 2022. The surveys are carried out continuously in the company as part of an internal programme, and ethical approval was sought retrospectively when the authors decided to publish the results. After a complete description of the study to the participants, written informed consent was obtained. The confidentiality of the participants was guaranteed.\n\n\nResults and discussion\n\nThe organization consists of healthcare professionals such as nurses, lab technicians, receptionists, managers, and general practitioners, in addition to personnel who support the development of clinical research, marketing, among others. In the present study, 80 people were contacted to participate, of which 79 participated and completed both surveys. Table 1 describes the number of employees by occupation, all employees who participated in the study and those who participated in both surveys (Flores Luna et al., 2023).\n\nDuring the coronavirus disease 2019 (COVID-19) pandemic, personnel working in this area had variable schedules due to the high demand for COVID tests. The administrative area includes managers, assistants and receptionists who manage the company’s objectives, paperwork and the logistics of company events. Clinical operations, on the other hand, focus on managing the execution of clinical research projects for the pharmaceutical industry and are composed of nurses, clinical research coordinators, pharmacists, specialists, and general managers.\n\nThe sample included 53 women and 26 men, so the sample was mainly represented by women, which may establish a bias in the results. In terms of age, most respondents were under 30 years of age, single, childless, and the most represented occupation corresponded to clinical operations (28%); in this occupation, the main activities were focused on conducting clinical studies sponsored by the pharmaceutical industry, followed by administration and medical services (Table 1), all persons participating in the study. Note that the number of people per occupation is not the same, which may represent a bias in the results.\n\nJob Stress Severity (JS-S)\n\nWhen evaluating the effect of stress severity among all occupations, it was possible to establish that of the 30 stressful situations, 5 showed significant results (Table 2). These are the main causes of stress common to all occupations recorded. According to Santos et al. (2011), the predominant items are part of the subscales of work pressure (4th), lack of support (10th, 17th) and lack of organization (30th).\n\nAlthough the overall severity effect was medium, we continued with the particular evaluation of the JS-S scale in each of the occupations, finding significant results with large effects, mainly in the occupations of medical services with 10 stressful situations (items 1, 2, 3, 6, 11, 13, 17, 21, 27 and 30) and administration with 7 (2, 5, 7, 16, 20, 26, 29). For both occupations, most of the items are associated with the subscale lack of organization, in addition to intense work pressure, predominating in administration (Santos et al., 2011). Both occupations had in common the stressful situation of performing work in free time. On the other hand, clinical operations, financial and commercial presented large effects with 2 (item 4 and 10), 1 (item 10) and 1 (item 11) stressful situations, respectively (data not shown).\n\nFrequency of occupational stress (JS-F). The survey revealed that the most frequent stressful situation is related to insufficient salary, although the effect was medium (Table 3). When analyzing the occupation of medical services and administration, it was observed that the items with large effect that were most frequently reported were 2b (I have to do work in my free time), 16b (In critical situations, decisions are made without careful analysis), 20b (Poorly motivated peers), 26b (Excessive compliance goals) and 29b (Poorly motivated peers). The most predominant items were associated with the lack of organization subscale (Santos et al., 2011).\n\nWork Stress Index (JS-X). The results of the general work stress index (JS-X) indicate a moderate level of stress across all occupations, with a Cohen’s d of 0.46 and Hedges’ g of 0.47.\n\nIn the stress survey, the reliability values of Cronbach’s α coefficients were above 0.85 (JS-S: 0.97, JS-F 0.94 and JS-X: 0.97), so they can be rated as very satisfactory; thus, the results are compatible with the theoretical frame of reference (Holmström et al., 2008).\n\nThe global average burnout shows a low trend of exhaustion (DP: 4.3; PA: 42.8; EE: 18.5), however, the global results of the emotional exhaustion scale (EE: 18.5) provided evidence of an onset of a moderate burnout syndrome among all the occupations evaluated. When performing the correlation analysis, the EE scale presented a strong positive correlation with the general result of burnout, regardless of the type of occupation (Table 4). For the PA scale, despite not being significant, a negative relationship was found with respect to burnout (r = -0.034, p = 0.768), which is consistent with other studies where it is established that this response is strongly determined by organizational processes and structures (Piko, 2006).\n\n* p < 0.05 (2 tales).\n\n** p < 0.01 (2 tales).\n\nSimilarly, it is observed in the scatter diagrams, where the high positive correlation between the EE scale and burnout is evident, and in the case of the DP scale, despite presenting a low R2 value (0.608), it can also indicate its influence in the burnout response (Figure 1).\n\nAbbreviation: A: PD – Burnout ratio; B: EE-Burnout relationship and C: PA-Burnout relationship. R2: coefficient of determination. The 2 confidence bands simultaneously at 95%.\n\nIn Figure 2 we show the relationship between the scales (EE, PA and DP) and the occupations, confirming that the medical services personnel present a response of exhaustion associated with only two (EE and DP) of the 3 scales (Figure 2). West et al. (2009), mention that high values of depersonalization (DP) and emotional exhaustion (EE) and low values of personal accomplishment (PA) indicate exhaustion; however, in the present study, the PA scale for medical services did not show a high value of burnout.\n\nAbbreviations: Mk: Marketing; A: Administration; T: Trade; F: Finance; Co: Clinical Operations; Ms: Medical Services; O: Others.\n\nIn addition, people working in the marketing area presented a high level of burnout associated with the PA scale; it should be noted that the PA score is inversely proportional to the degree of burnout, i.e., the lower the performance or personal accomplishment score, the more affected the person is (González and De la Gándara, 2004). Job satisfaction has an important influence on work-related behaviors such as absenteeism, job performance and turnover intention (Piko, 2006). Other areas that registered moderate burnout scores were finance and clinical operations.\n\nShbeer and Ageel (2022) mention that the main cause of emotional exhaustion (EE) among medical services personnel is related to the nature of their tasks; in this sense, it states that workload, insufficient rewards in the workplace and unfair treatment with employees are the causes associated with EE. Chatterjee et al. (2021); mention in their study on stress in medical service personnel, that physicians tend to present high levels of anxiety due to stress, unlike nurses who develop more irritability; in general, they state that work overload leads to irritability. Piko (2006), suggests that an additional indicator to the burnout and stress survey may be the assessment of psychosomatic symptoms, which have been related to work stress and burnout.\n\nThe lack of evaluation of work stress or the presence of burnout and not having preventive programs can have repercussions on physical, emotional and psychological integrity (Corona et al., 2022) in addition to impacting work efficiency and reducing productivity in the company (Selmanovic et al., 2011).\n\nPrevention activities to reduce stress and the incidence of burnout for workers not only in the medical services sector (Yadav & Sahu, 2022), but also for those who show signs of chronic stress or moderate burnout, contribute to the reorientation of organizational approaches; these activities include modifications of schedules, work processes, and generation of individual-centered strategies (Liebenberg et al., 2018). Shbeer and Ageel (2022) suggest taking breaks, physical activity and medical services promotion programs, to reduce burnout.\n\nThe results of this study allowed the managers of the company where the participants work to support the development of a psychosocial factors prevention plan referred to in the Mexican Official Standard NOM-035-STPS-2018; it included a detailed study of the medical services shift schedules, the creation of integration activities such as: monthly birthday celebrations, active breaks once a week and the teamwork model with dynamics focused on the needs of the community. The company also organizes talks to raise awareness about people with disabilities and against violence. In addition, the quality management system was established, which harmonized the company’s processes and procedures and promoted the development of job descriptions for personnel. These findings highlight the importance of evaluating and implementing customized strategies to overcome stress and burnout syndrome in specific functional areas within organizations. Such an approach can help to localize the main areas where employees are most affected and to tailor interventions that address the unique challenges faced by employees in different roles (Kim & Hye-Sun, 2022; Panigrahi, 2016; Salama et al., 2022).\n\n\nConclusions\n\nBurnout is a state of emotional, physical and mental exhaustion resulting from prolonged stress and exposure to difficult working conditions. The main occupation in which burnout was recorded was in medical services, which is continually noted as the sector most affected by burnout syndrome. In this sense, people working in these fields are likely to experience high levels of mental, emotional and physical stress, which can have a significant impact on their overall well-being.\n\nOther occupations that also recorded burnout according to the scale were finance, clinical operations and marketing. In finance, employees may suffer burnout due to the high pressure they are under to meet performance targets and the fast-paced and changing nature of the financial markets. In clinical operations, employees working in the healthcare sector can suffer burnout due to the demanding nature of their work, which involves treating and caring for patients in often stressful and time-pressured environments. Marketing professionals can suffer burnout due to the constant need to be creative, generate new ideas and meet performance targets. Pressure to meet the high expectations of customers and stakeholders, as well as the fast pace and constantly evolving nature of the marketing industry, can also contribute to burnout in this field.\n\nStress and burnout can manifest differently in different departments, teams, or individuals within a company, depending on factors such as job role, responsibilities, workload, and working conditions. Global assessments that do not take these differences into account may not accurately reflect the true level of stress and burnout in different parts of the company. To overcome this limitation, the companies must carry out partial evaluations that are tailored to specific areas within the company. These evaluations can provide more accurate insights into the specific stressors and burnout risks in different parts of the organization, allowing companies to take targeted and effective action to address them.", "appendix": "Data availability\n\nFigshare: Assessment of work-related stress and burnout among clinical research occupations. https://doi.org.10.6084/m9.figshare.22631374.v1 (Flores Luna et al., 2023).\n\nThis project contains the following underlying data:\n\n- Burnout syndrome.xlsx - Burnout syndrome (1).csv\n\n- Job Stress Survey.xlsx - Job Stress Survey (1).csv\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgements\n\nAuthors want to thank Investigación Biomédica para el Desarrollo de Fármacos S.A. of C.V. for support during the study.\n\n\nReferences\n\nArnoldo T, Víctor CV: Effect size, confidence intervals and statistical power in psychological research. Psychol. 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Res. Public Health. 2022; 19(8): 4428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee CY, Wu JH, Du JK: Work stress and occupational burnout among dental staff in a medical center. J. Dent. Sci. 2019; 14(3): 295–301. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiebenberg AR, Coetzee JF, Conradie HH, et al.: Burnout among rural hospital doctors in the Western Cape: Comparison with previous South African studies. Afr. J. Prim. Health Care Fam. Med. 2018; 10(1): e1–e7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLloyd C, King R, Chenoweth L: Social work, stress and burnout: A review. J. Ment. Health. 2002; 11(3): 255–265. Publisher Full Text\n\nMaslach C, Jackson SE, Leiter MP: The Maslach burnout inventory, Manual. 3rd ed.Mountain View, CA: CPP Inc; 1996; 281.\n\nMenéndez MD, Papasidero M: Stress Factors Associated With Burnout Syndrome in a Hospital Emergency Service: An Observational Study. JACME. 2019; 9(1): 8–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPanigrahi A: Managing Stress at Workplace. J. Manag. Res. Anal. 2016; 3(4): 154–160.\n\nPérez AM: El síndrome de burnout. Evolución conceptual y estado actual de la cuestión. Vivat Academia. 2010; 112: 42–80. Publisher Full Text\n\nPiko BF: Burnout, role conflict, job satisfaction and psychosocial health among Hungarian health care staff: A questionnaire survey. Int. J. Nurs. Stud. 2006; 43(3): 311–318. PubMed Abstract \| Publisher Full Text\n\nPuteri LA, Syaebani MI: Employees work stress level in the hospital. Int. Res. J. Bus. Stud. 2018; 11(3): 231–243. Publisher Full Text\n\nRumschlag KE: Teacher burnout: A quantitative analysis of emotional exhaustion, personal accomplishment, and depersonalization. Int. Manag. Rev. 2017; 13(1): 22–36.\n\nSalama W, Abdou AH, Mohamed SAK, et al.: Impact of Work Stress and Job Burnout on Turnover Intentions among Hotel Employees. Int. J. Environ. Res. Public Health. 2022; 19(15): 9724. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSantos MDLM, Arias ER, Arango MEP, et al.: Elaboración de la versión cubana del cuestionario JSS para la evaluación del estrés psicosocial laboral. Revista Cubana de Salud y Trabajo. 2011; 12(2): 9–18.\n\nSelmanovic S, Ramic E, Brekalo-Lazarevic S, et al.: Stress at work and burnout syndrome in hospital doctors. Med. Arch. 2011; 65(4): 221–224. Publisher Full Text\n\nShbeer A, Ageel M: Assessment of Occupational Burnout among Intensive Care Unit Staff in Jazan, Saudi Arabia, Using the Maslach Burnout Inventory. Crit. Care Res. Prac. 2022; 2022: 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpielberger CD, Vagg PR, Catalina C: JSS, cuestionario de estrés laboral. TEA. 2010.\n\nWest CP, Dyrbye LN, Sloan JA, et al.: Single item measures of emotional exhaustion and depersonalization are useful for assessing burnout in medical professionals. J. Gen. Intern. Med. 2009; 24(12): 1318–1321. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYadav P, Sahu S: Perceived stress, burnout and compassion fatigue among frontline health workers during COVID-19 pandemic. Indian J. Psychiatr. Soc. Work. 2022; 13(1): 32–38. Publisher Full Text" }	[ { "id": "215421", "date": "01 Nov 2023", "name": "Somkene Igboanugo", "expertise": [ "Reviewer Expertise My areas of research include work-related stress", "psychosocial stress in first responders", "and consequences of work-related stress", "including burnout and psychosomatic health conditions." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study explores work-related stress and burnout among occupational groups within healthcare. Since work-related stress and burnout are prevailing issues, this investigation is timely, so I commend the authors.\nI have a mixed feeling towards the objectives of the study. First, I appreciate the effort to measure work-related stress and burnout in different organizational groups, making comparing and identifying groups suffering the most challenging. On the other hand, I believe more could have been done with the data available to the authors. Here are some of my recommendations/comments\nFor example, since work-related stress may lead to burnout, I expected a methodology including correlation or regression analyses that investigates a potential association between both variables (stress and burnout), especially since the data was available. The authors even alluded to it by stating, “When employees face continuous stress and lack strategies to manage it, they can suffer burnout, a condition characterized by physical, emotional and mental exhaustion (Selmanovic et al., 2011). I understand the authors' priorities differ from my expectations; however, I strongly recommend that this analysis be considered to make the study even more beneficial to the readers beyond the organization it was done for.\n\nThe authors mentioned the possibility of bias in their study in their method section. The first sentence stated that the cross-sectional surveys were administered at different times to minimize bias. What bias did they expect would present if the survey was administered once, or what bias did they want to minimize by administering it twice? Secondly, why was the survey administered at different times? Was it to reduce bias or because of the availability of the staff? Or was it part of the study design (Before vs After, time series study), i.e., was the survey administered twice to each participant? Clarity is vital in this case. Thirdly, I will suggest that each time the author mentions bias, it’s helpful to the reader if the author provides an example of bias considered (e.g., selection bias).\n\nThe authors stated, “For the analysis, binary correlations were performed for the scales and the global burnout score, considering all occupations…”. The ‘binary correlations’ are confusing since the variable (burnout dimensions) is not binary. Pearson’s correlation is the better analysis to do in this instance.\n\nIn the result section, the authors spoke briefly about the COVID-19 pandemic, “During the coronavirus disease 2019 (COVID-19) pandemic, personnel working in this area had variable schedules due to the high demand for COVID tests.” This statement may be essential if context is provided. Why did the authors add this statement? Was the sentence to justify data being collected twice or in different weeks because of the staff schedules? Or was it based on study methodology, as I asked in Q2? Please provide the basis for this statement.\n\nUnder the JS-S paragraph, the authors made this comment, “On the other hand, clinical operations, financial and commercial presented large effects with 2 (item 4 and 10), 1 (item 10) and 1 (item 11) stressful situations, respectively (data not shown)”. Except the entire questionnaire was provided as part of supplemental information or the meaning of the item values was provided, this information is not helpful, especially since the data is not shown. I will suggest the authors add the questions that correspond to each item.\n\nThe sentence needs to be shorter and more concise in the third paragraph on page 8. Although it contains valuable information and provides the importance of prevention sentences, its structure may need clarification. I suggest it be broken up into three rather than one long sentence.\n\nCould the authors throw some more light on why the correlation analysis was performed? It's essential to check the associations between the dimensions of burnout and burnout, but I am sure the reader will appreciate some basis on why this is done. I ask this because, based on the correlation analysis, PA was not significantly associated with burnout (table 4). What does this mean when interpreting whether any of the occupational groups were experiencing burnout if one of the dimensions does not meet the significance threshold? Could you tell me what is the implication of this finding? Does it remove the meaning or severity of burnout in this group?\n\nI realize the paper does not have a discussion section with the study limitation. I understand that joining the result and discussion was the best way the authors felt to present their findings, and I also do not know the author submission requirements for F1000 research. Still, it’s always helpful for the readers to see only the analyses in the result section and the author's interpretation of the findings in the discussion. Also, the first two paragraphs in the conclusion section appear to repeat the information in the result section. If it’s helpful, I suggest that the conclusion focuses on the last paragraph, which provides a very insightful and nuanced interpretation from the authors.\nIn conclusion, I commend the authors for their valuable contribution to the extant literature on burnout and work-related stress; however, I recommend a comprehensive and significant revision to address some of my comments. I hope my comments are helpful and lead to a better and improved peer-reviewed version.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "10540", "date": "30 Nov 2023", "name": "Claudia Mejía Morales", "role": "Author Response", "response": "Responses to reviewers. Thank you for taking the time and providing input for the document. They were very enlightening and we are sure that the quality of the document will improve with them. Below you will find the answer to your questions and our reply. Reviewer questions: For example, since work-related stress may lead to burnout, I expected a methodology including correlation or regression analyses that investigates a potential association between both variables (stress and burnout), especially since the data was available. The authors even alluded to it by stating, “When employees face continuous stress and lack strategies to manage it, they can suffer burnout, a condition characterized by physical, emotional and mental exhaustion (Selmanovic et al., 2011). I understand the authors' priorities differ from my expectations; however, I strongly recommend that this analysis be considered to make the study even more beneficial to the readers beyond the organization it was done for. Thank you, it is a good question and we appreciate your input and comments. As a response, the overall mean of work stress and burnout was obtained; subsequently, the means of each of the burnout dimensions were specifically obtained. Linear Regression analyses were performed, considering burnout or its dimensions as the dependent variable and stress as the independent variable. Since the P-value in the ANOVA table is less than 0.05, there is a statistically significant relationship between EE and stress at a confidence level of 95.0%. The correlation coefficient is equal to 0.672, indicating a moderately strong relationship between the variables EE and stress[1]. Since the P-value in the ANOVA table is less than 0.05, there is a statistically significant relationship between Col_1 and Col_2 with a confidence level of 95.0%. The correlation coefficient is equal to -0.318, indicating a relatively weak relationship between the variables PA and stress[2]. He, et al 2020 found correlations between work stress score and two burnout subscale scores (emotional exhaustion and cynicism) were significant (both p < 0.001), but not with professional efficacy. Since the P-value in the ANOVA table is less than 0.05, there is a statistically significant relationship between Col_1 and Col_2 with a confidence level of 95.0%. The correlation coefficient is equal to 0.539, indicating a moderately strong relationship between the PD and stress variables[3]. Since the P-value in the ANOVA table is less than 0.05, there is a statistically significant relationship between Col_1 and Col_2 with a confidence level of 95.0%. The correlation coefficient is equal to 0.623, indicating a moderately strong relationship between the variables stress and burnout[4]. The present study revealed a close association between stress and burnout similar to that established by Mandava, et al., 2018; Jamal & Baba, 2000; Sasa, et al., 2009. References He, S. C., Wu, S., Wang, C., Wang, D. M., Wang, J., Xu, H., ... & Zhang, X. Y. (2020). Interaction between job stress, serum BDNF level and the BDNF rs2049046 polymorphism in job burnout. Journal of Affective Disorders, 266, 671-677. Jamal, M., & Baba, V. V. (2000). Job stress and burnout among Canadian managers and nurses: an empirical examination. Canadian journal of public health, 91, 454-458. Mandava, P., SankarSingaraju, G., Ganugapanta, V. R., & Yelchuri, H. (2018). Comparison of stress, burnout and its association among postgraduate orthodontic and undergraduate students in India. Indian Journal of Dental Sciences, 10(2), 66. Sasaki, M., Kitaoka‐Higashiguchi, K.A.Z.U.Y.O., Morikawa, Y., & Nakagawa, H. (2009). Relationship between stress coping and burnout in Japanese hospital nurses. Journal of nursing management, 17(3), 359-365. 2. The authors mentioned the possibility of bias in their study in their method section. The first sentence stated that the cross-sectional surveys were administered at different times to minimize bias. What bias did they expect would present if the survey was administered once, or what bias did they want to minimize by administering it twice? Secondly, why was the survey administered at different times? Was it to reduce bias or because of the availability of the staff? Or was it part of the study design (Before vs After, time series study), i.e., was the survey administered twice to each participant? Clarity is vital in this case. Thirdly, I will suggest that each time the author mentions bias, it’s helpful to the reader if the author provides an example of bias considered (e.g., selection bias). Good question, we will try to clarify this information as much as possible. The process we followed was closely related to the availability and number of company personnel. In fact, some of them were surveyed one week and others the next week, based on the first study group. This was done to prevent the company's activities from being stopped; Likewise, it has been established that a person's daily situation can influence the responses to the questionnaires (bias), so some of the staff were surveyed the week following the first group. Furthermore, as mentioned in the document, we sought to survey as many of the company's personnel as possible, which was possible by adapting to the time available to respond to the survey, so that the majority of occupations were represented. 3. The authors stated, “For the analysis, binary correlations were performed for the scales and the global burnout score, considering all occupations…”. The ‘binary correlations’ are confusing since the variable (burnout dimensions) is not binary. Pearson’s correlation is the better analysis to do in this instance. Thank you very much for the comment, it is a typing error, actually they are bivariate correlations where the Pearson correlation coefficient was used. I share the correlations obtained: [5] [6] [7] 4. In the result section, the authors spoke briefly about the COVID-19 pandemic, “During the coronavirus disease 2019 (COVID-19) pandemic, personnel working in this area had variable schedules due to the high demand for COVID tests.” This statement may be essential if context is provided. Why did the authors add this statement? Was the sentence to justify data being collected twice or in different weeks because of the staff schedules? Or was it based on study methodology, as I asked in Q2? Please provide the basis for this statement. We tried to add more details in answer 2, the process we followed was related to the personnel available and also to avoid bias among participants. 5. Under the JS-S paragraph, the authors made this comment, “On the other hand, clinical operations, financial and commercial presented large effects with 2 (item 4 and 10), 1 (item 10) and 1 (item 11) stressful situations, respectively (data not shown)”. Except the entire questionnaire was provided as part of supplemental information or the meaning of the item values was provided, this information is not helpful, especially since the data is not shown. I will suggest the authors add the questions that correspond to each item. Thanks for pointing that out, of course, attached is the section of the JSS dimension survey for stress severity assessment. Stress Survey - JSS This type of survey is of continuous use as mentioned in the methodology, it is obtained from Spielberger et al., 2010 and is also reported in works such as Reyes, et al 2016; Acosta Tirado, 2022. These data are mentioned in the methodology of said manuscript. Acosta Tirado, F. M. (2022). Programa de bienestar psicológico para afrontar el estrés laboral en los colaboradores de una Institución Municipal de Otuzco, año 2021: estudio descriptivo propositivo. Reyes Cruz, A. R. (2016). Estrés laboral, resiliencia y consumo de alcohol en trabajadores industriales (Doctoral dissertation, Universidad Autónoma de Nuevo León). Spielberger CD, Vagg PR, Catalina C: JSS, cuestionario de estrés laboral. TEA. 2010. 6. The sentence needs to be shorter and more concise in the third paragraph on page 8. Although it contains valuable information and provides the importance of prevention sentences, its structure may need clarification. I suggest it be broken up into three rather than one long sentence. Thank you for providing us with your comments in manuscript form, we will surely improve this sentence in a future version of the document. 7. Could the authors throw some more light on why the correlation analysis was performed? It's essential to check the associations between the dimensions of burnout and burnout, but I am sure the reader will appreciate some basis on why this is done. I ask this because, based on the correlation analysis, PA was not significantly associated with burnout (table 4). What does this mean when interpreting whether any of the occupational groups were experiencing burnout if one of the dimensions does not meet the significance threshold? Could you tell me what is the implication of this finding? Does it remove the meaning or severity of burnout in this group? We can certainly expand our answer. In fact, the information being called by the reviewer was widely disseminated and justified by the above references, it was made in view of the fact that other publications mention the same thing. It has been established that personal accomplishment (PA) is more a personal resource than a dimension of burnout. Several authors consider that the personal accomplishment dimension has a separate role from both emotional exhaustion and depersonalization, and thus represents perceived professional efficacy. In other words, personal accomplishment would reflect the personal characteristics of workers and not their reactions to stressful situations so it can be considered not as a dimension of burnout but as an individual resource that develops largely independently of emotional exhaustion and depersonalization (López-Núñez, et al., 2020). These negative correlations between the dimensions of burnout and PA have already been evidenced in other publications such as Lee & Ashforth, 1996; showing the meta-correlations between the three dimensions of burnout, emotional exhaustion was strongly related to depersonalization, while both dimensions were moderately negatively related to personal accomplishment (PA) (rcs = -.33 for emotional exhaustion and -.36 for depersonalization). This information was widely reported and is justified by the above-mentioned references, as other publications also confirm this idea. López-Núñez, M. I., Rubio-Valdehita, S., Diaz-Ramiro, E. M., & Aparicio-García, M. E. (2020). Psychological capital, workload, and burnout: what’s new? the impact of personal accomplishment to promote sustainable working conditions. Sustainability, 12(19), 8124. Lee, R. T., & Ashforth, B. E. (1996). A meta-analytic examination of the correlates of the three dimensions of job burnout. Journal of applied Psychology, 81(2), 123. 8. I realize the paper does not have a discussion section with the study limitation. I understand that joining the result and discussion was the best way the authors felt to present their findings, and I also do not know the author submission requirements for F1000 research. Still, it’s always helpful for the readers to see only the analyses in the result section and the author's interpretation of the findings in the discussion. Also, the first two paragraphs in the conclusion section appear to repeat the information in the result section. If it’s helpful, I suggest that the conclusion focuses on the last paragraph, which provides a very insightful and nuanced interpretation from the authors. Thank you. Of course, as you mentioned, that paragraph would be improved. We will keep it in mind for a future version of the manuscript." } ] }, { "id": "267391", "date": "02 May 2024", "name": "Ioannis Pantelis Adamopoulos", "expertise": [ "Reviewer Expertise occupational safety and health", "public health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Authors, Thanks to the authors for sharing their manuscript. I want to point out that the authors have done a lot of good work, but I have some concerns about the manuscript. The article entitled: Assessment of work-related stress and burnout among clinical research occupations 1) Separate the sections results from discussion, make the results clear, and discuses in section discussion the results associated with similar research of all over the world in the manuscript. 2) I believe is important to create a section on manuscript after Discussion section and the Study Limitations and future research . Guidelines: (These are original research manuscripts. The work should report scientifically sound experiments and provide substantial amount of new information. The article should include the most recent and relevant references in the field. The structure should include an Abstract, Keywords, Introduction, Materials and Methods, Results, Discussion, and Conclusions sections. 3) The abstract with section Background , methods and results NEEDS numerical INFORMATIONS ABOUT RESULTS , for example, burnout (r = -0.034, p = 0.768), conclusions. It has the correct specifications and is approved The belongings: 1) Topics. 2) The methods improve the quality of scientific papers, achieve publication goals, and improve consistency and readability. Preserve the flow of expression and professional results, improve structure and presentation; check plagiarism; also, do bibliographic editing, and finally, improve the target of the journal. The study had an exploratory and descriptive scope and involved two cross-sectional surveys conducted at different time points to minimize the potential for any biases or confounding variables that may have arisen if the surveys were conducted simultaneously 3) All content presented in the manuscripts is perfect and has kept the guidelines of the journal, including opinions, methods, statistical analysis and materials, results, and discussions. The article is original research and prototype ideas. 4) Manuscripts using acceptable file formats use the layout options, depending on the length of the research articles, and keep the main text of the body except the abstract, below appropriate words. The authors provide documents during the submission of the abstract. Grammar and punctuation standards were followed in preparing the content. The international system of units followed in the representation of all units in the text, and the recommended structure of the manuscript follows. References: correct method formatting and style guidelines. Contribution to the literature: the uniqueness and importance of this work in terms of research topics, methodologies, and outcomes, enhance the scholarship on the subject. Ideas are presented in a clear, concise, and complete manner. The manuscript is free from any repetitions, irrelevant information, or unjustified generalizations. The theoretical framework is explicitly stated. All claims are backed up with evidence and references. Research problems’ position and significance in the existing literature are emphasized. The chosen methodology is suitable for the problem. The study’s findings are well presented, with sufficient discussions and comparisons to existing literature. Objective and convincing measures exist to support the validity and reliability of the methodology and results. Relevant literature is properly cited, and perfect figures and tables present the results and the findings of this study's especially congrats the authors for the Data scatter diagrams, figure 1 and figure 2.\n\nFinally, the paper have completed the Aims and scope of Journal\nI suggest: accept as it is.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "178478", "date": "21 Jun 2024", "name": "Yupin Aungsuroch", "expertise": [ "Reviewer Expertise Nursing management and leadership", "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\nThe study investigated work-related stress and burnout among clinical research occupations, the results would give a good insight.\nAbstract. Authors need to add important information to the method and results. The number of participants and occupational type are important for the reader. In addition, the result could show a significant number and statistics to emphasize the findings. The results statements need to be revised.\nIntroduction. The study was conducted in February 2022. An explanation of the local situation during that time would give an understanding to the reader, include the pandemic situation, hospital, or others.\nMethods. A strong reason could help the reader understand the consideration of 79 participants. Were they random? Convenience? Or another? A brief explanation of the company could make the reader follow the flow and condition of the study. A short statement could explain the reason why data analysis for bivariate did not explore between work-related stress and burnout. \"The study had an exploratory and descriptive scope and involved two cross-sectional surveys conducted at different time points to minimize the potential for any biases or confounding variables that may have arisen if the surveys were conducted simultaneously.\" Please kindly explain the time problems, biases, and confounding variables.\nResults and discussion could be separated to give a better presentation.\nThere is no study without limitation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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/12-456
https://f1000research.com/articles/11-560/v1	23 May 22	{ "type": "Research Article", "title": "Integration of single-cell RNA-Seq and CyTOF data characterises heterogeneity of rare cell subpopulations", "authors": [ "Emmanouela Repapi", "Devika Agarwal", "Giorgio Napolitani", "David Sims", "Stephen Taylor", "Devika Agarwal", "Giorgio Napolitani", "David Sims", "Stephen Taylor" ], "abstract": "Background: The simultaneous measurement of cellular proteins and transcriptomes of single cell data has become an exciting new possibility with the advent of highly multiplexed multi-omics methodologies. However, mass cytometry (CyTOF) is a well-established, affordable technique for the analysis of proteomic data, which is well suited for the discovery and characterisation of very rare subpopulations of cells with a wealth of publicly available datasets. Methods: We present and evaluate the multimodal integration of single cell RNA-Seq and CyTOF datasets coming from both matched and unmatched samples, using two publicly available datasets. Results: We demonstrate that the integration of well annotated CyTOF data with single cell RNA sequencing can aid in the identification and annotation of cell populations with high accuracy. Furthermore, we show that the integration can provide imputed measurements of protein markers which are comparable to the current gold standard of antibody derived tags (ADT) from CITE-Seq for both matched and unmatched datasets. Using this methodology, we identify and transcriptionally characterise a rare subpopulation of CD11c positive B cells in high resolution using publicly available data and we unravel its heterogeneity in a single cell setting without the need to sort the cells in advance, in a manner which had not been previously possible. Conclusions: This approach provides the framework for using available proteomic and transcriptomic datasets in a unified and unbiased fashion to assist ongoing and future studies of cellular characterisation and biomarker identification.", "keywords": [ "single cell transcriptomics", "single cell proteomics", "CyTOF", "mass cytometry", "single cell integration", "CD11c B cells" ], "content": "Introduction\n\nIn the last decade the analysis of transcriptomes at single cell resolution has revolutionised molecular biology and become one of the most widely used techniques for answering fundamental biological questions of cell identities and functions.1–3 Single-cell RNA sequencing (scRNA-Seq) and mass cytometry (CyTOF) have both contributed immensely in the characterisation of cellular states, the identification of novel populations and the understanding of cellular differentiation trajectories.1,2,4–6 Protein and transcriptomic measurements in single cells, however, have been shown to provide different and complementary information regarding cell states.7,8 Joining the two fields can provide insightful observations on biological systems and functions at a cellular level.\n\nThe simultaneous quantification of transcriptome and proteins of the same cells has now marked the beginning of a new era in which our understanding of cellular systems and processes will be transformed by coupling the phenotypic and transcriptomics landscapes of biology. However, in these early days of multiplex technologies where cost and sample accessibility can be a limiting factor, there is still a lot to be gained from the integration of independent and already available datasets. Mass cytometry can target both intracellular epitopes and epigenetic markers with high precision and also has the advantage of being able to identify novel and very rare populations while profiling up to hundreds of thousands of cells per sample.7,9,10 Most importantly, the large quantity and high quality of single-modality single-cell data that have been already generated in the last few years have the potential to reveal novel biological insights when integrated appropriately.3,11 Therefore, the integration of CyTOF and scRNA-Seq can provide an affordable alternative to a unified view across modalities and aid in the functional characterisation of novel populations.\n\nHere we present the multimodal integration of scRNA-Seq and CyTOF datasets coming from both matched samples (same patients profiled with the two platforms in the same dataset/study) and unmatched samples (different patients from distinct datasets/studies). We use the publicly available datasets from the multi-omic blood atlas of coronavirus disease 2019 (COVID-19) patients (COMBAT Study)12 to present the integration of matched datasets and show how the imputed values projected from the integration compare to the current gold standard measurements of ADT, coming from the available CITE-Seq experiment. Furthermore, we integrate the COMBAT mass cytometry dataset with an unmatched multimodal human PBMC atlas13 as an example of how scRNA-Seq and CyTOF datasets originating from different samples can also be integrated with high accuracy. We demonstrate that the integration of the CyTOF with the scRNA-Seq can guide the difficult task of annotating scRNA-Seq datasets and show that the imputed values generated are correlated to the CITE-seq protein levels. We finally use this integration technique to transcriptionally characterise a rare and heterogeneous subpopulation of CD11c+ B cells originally identified in the COMBAT mass cytometry dataset in a large meta-analysis of 12 COVID-19 scRNA-Seq datasets; thus, demonstrating the potential of this methodology to couple the unique strength of mass cytometry to identify very rare subpopulations with that of scRNA-Seq to functionally describe previously undefined subpopulations with unprecedented granularity.\n\n\nMethods\n\nCOMBAT datasets\n\nFor the integration of matched datasets, we downloaded mass cytometry (CyTOF) and single-cell combined transcriptome (RNA) with surface proteome (ADT) data of peripheral blood mononuclear cells (PBMCs) from the Covid-19 Multi-omics Blood ATlas (COMBAT) Consortium12 (DOI 10.5281/zenodo.5139560). The datasets contained 91 overlapping samples from healthy controls and community COVID-19 cases in the recovery phase (never admitted to hospital) as well as from patients with mild, severe and critical COVID-19.\n\nThe scRNA-Seq and CyTOF datasets had been previously annotated, and the published annotations were retained as gold standard annotations. Both datasets were filtered to exclude cells with unclassified or uncertain annotations. Furthermore, subsets of cells that were extremely rare in the scRNA-Seq dataset (frequency less than 0.05%) and for which there was no overlapping features to identify them (namely reticulocyte and mast cells) were also removed prior to the integration. The scRNA-Seq and CITE-Seq datasets were further filtered to exclude low-quality cells based on i) number of genes, ii) number of features of ADT, iii) number of total UMI (RNA) or iv) number of total UMI of ADT lower than 0.001 of their relevant distributions. As this dataset is a subset of the published dataset (containing only the overlapping samples between scRNA-Seq and CyTOF), the raw RNA-Seq values were downloaded and re-integrated to mitigate batch effects. The batch correction was performed using the Seurat v3 canonical correlation analysis (CCA) anchor based method14 on 89 samples (after the exclusion of 2 samples with less than 80 cells), with the healthy controls as a reference. The published cell annotations which were created from expert immunological knowledge using data from all three modalities (GEX, ADT and VDJ)12 from the COMBAT Consortium were used for comparisons and the data was not re-clustered. The ADT dataset contained normalised values (normalisation using the DSB algorithm,15 details in Ahern et al.12) for 192 ADT tags, of which 39 had an equivalent CyTOF antibody.\n\nThe CyTOF dataset contained batch corrected and transformed values (arcsinh, with cofactor 5), for a panel of 45 markers (details for the batch correction can be found in Ahern et al.12). From those, 40 had an equivalent gene sequenced in the mRNA data (Table 1) and were used for the integration. The IgA and IgG antibodies had very poor staining in the mass cytometry experiment and were not included in any analysis. One channel included a combination of antibodies for IgD and TCRgd that was not used for the integration of the major cell types but was used for the integration of the B cells.\n\nThe gene names that correspond to the proteins from the mass cytometry (CyTOF) dataset and to the antibody derived tags (ADT) names from the COMBAT and refPBMC datasets.\n\nFor the B cell datasets, we extracted all cells from the scRNA-Seq that had been annotated as B cells (32,728 cells) and performed a batch correction with the healthy controls as a reference using Seurat v314 (89 samples after the exclusion of 2 samples with less than 80 cells). For the CyTOF dataset we subsampled 700 B cells per sample (57,159 cells).\n\nHuman PBMC Atlas datasets\n\nThe processed (after cellranger and QC) and annotated cell count matrices and the metadata of the scRNA-Seq was downloaded from the GEO database (GEO accession GS164378). The normalised and processed data object for the 3prime CITE-Seq dataset (ADT) was downloaded from https://atlas.fredhutch.org/data/nygc/multimodal/pbmc_multimodal.h5seurat (from here on named refPBMC). For the scRNA-Seq, each of the 24 samples were individually log normalised and scaled with linear regression of the covariate nUMI. To identify shared cell types across samples, batch correction and sample integration was performed for donor and time across 24 samples using the Seurat v3 Log normalize anchor based reciprocal PCA (RPCA) workflow with the unvaccinated samples (Day 0 samples) designated as the reference dataset, with 50 PCA dimensions and 3000 highly variable genes.\n\nFrom the 161,764 cells, 146,480 cells were retained after filtering of cells annotated as doublets and those with less than 10 UMI counts for the common features between scRNA and CyTOF datasets. Additionally, the celltype.l2 annotations from the publication were renamed and grouped to best match the annotations from the COMBAT CyTOF dataset for visualisation and evaluation of the integration of the two modalities, but still included the cell-types exclusive to the refPBMC Atlas (erythroid cells, hematopoietic stem and progenitor cells-HSPCs, innate lymphoid cells-ILCs and platelets). The renamed annotations were: B (B naïve, B intermediate and B memory); CD4 (including all subclusters of CD4); CD8 (with all subclusters of CD8); dendritic cells (DCs) (ASDC, cDC1, cDC2, pDC); and NK (NK, NK Proliferating and NK CD56 Bright).\n\nFor the Day 0 integration, the refPBMC was filtered to include only the unvaccinated (Day 0) samples (47,962 cells after filtering of cells on the same thresholds as the full dataset). The unvaccinated samples were regressed for the nUMI covariate, batch corrected and integrated across the 8 donors using the same method and parameters as for the full dataset.\n\nCOVID-19 datasets for the B cell CD11c+ subpopulation characterisation\n\nFor the characterisation of the B cell subpopulations, the count matrices, standardised metadata and the predicted celltypes were obtained from the processed HDF5 objects generated as part of the COVID-19 meta-analysis study carried out by Tian et al.16 (downloaded from https://atlas.fredhutch.org/fredhutch/covid/). The RNA count data matrices of all PBMC datasets were extracted for the cells which were predicted to be B cells for the predicted.celltype.l1 and we further removed cells annotated as Plasmablasts in the predicted.celltype.l2. Additionally, to exclude any non-B cells that might have erroneously been predicted as B cells in the original meta-analysis, only cells that had zero UMI counts of CD3E, GNLY, CD14, FCER1A, FCGR3A, LYZ, PPBP and CD8A genes were retained, as previously done by Stewart et al.17 After filtering for non-B cells, datasets with fewer than 60 cells were removed, leaving 13 datasets for downstream analysis.\n\nThe PBMC dataset of Su et al.18 was processed separately in order to use the ADT tags as validation of the CD11c+ subpopulation (25,443 cells). The dataset was split by batch and then individually log-normalised, scaled and linearly regressed for the covariates nUMI and sex. Then it was batch corrected with the Seurat v3 log normalise anchor-based workflow with 1000 common HVG genes identified using the SelectIntegrationFeatures function and 15 CCA dimensions. UMAP dimensional reduction analysis was performed on the integrated object after scaling and regressing out sex with 15 PCA dimensions.\n\nEach of the 12 remaining datasets was first individually log-normalised and scaled with linear regression for the covariates nUMI, sample and sex. The normalised and scaled dataset objects were then integrated and batch corrected with the Seurat v3 log normalise anchor-based workflow with 1000 common HVG genes and 15 CCA dimensions. Subsequently, the integrated assay was filtered to only include samples from COVID-19 patients (77,485 cells) and it was rescaled and regressed for the covariate sex. UMAP dimensional reduction analysis was performed with 15 PCA dimensions on the batch corrected log normalised counts for downstream analyses.\n\nFor all analyses, the scRNA-Seq cells that had very low expression for all common protein-mRNA genes (less than 5 reads in total on all genes) were filtered out. The anchors between the query (scRNA-Seq) and reference (CyTOF) datasets were found based on a canonical correlation analysis (CCA) (with 20 dimensions for COMBAT and 25 dimensions for the reference PBMC dataset) using the FindTransferAnchors function from Seurat v314 (RRID:SCR_016341) and the defaults for the remaining parameters. Alternative options for the reduction were assessed but were found to be suboptimal (data not shown). Subsequently, the anchors were used to project the annotations of the CyTOF to the scRNA-Seq using the TransferData function with the PCA of the scRNA-Seq for the weight reduction and the CyTOF annotations as refdata. Equivalently, the TransferData function was also used to impute the CyTOF markers on the scRNA-Seq cells with the same parameters but using the CyTOF intensity values of the overlapping features as refdata. Finally, the imputed values were merged with the CyTOF data using the merge function and centered in order to run a UMAP analysis to visualize all the cells together. We would like to highlight here that the ADT data is not used in any of the steps of the integration, but at the evaluation stage only.\n\nTo assess the robustness of the integration for various cell numbers, the COMBAT scRNA-Seq and CyTOF datasets were downsampled to 9,100/22,750/45,500/91,000 and 182,000 cells for each modality by randomly subsampling 100/250/500/1000/2000 cells from each sample. The main results were presented for the dataset of 45,500 cells, as the most representative scenario of scRNA-Seq studies.\n\nFor the transcriptional characterisation of the B cell subpopulations, we integrated the CyTOF COMBAT dataset separately with 1) the scRNA-Seq COMBAT dataset, 2) the Su et al. PBMC scRNA-Seq dataset and 3) a meta-analysis of 12 PBMC COVID-19 scRNA-Seq datasets. The first two integrations served in finding a transcriptional signature for the CD11c+ cells, whereas the integration with the meta-analysis of the COVID-19 datasets was done to further define the heterogeneity within the CD11c+ subpopulation. For all integrations, a reduced set of 28 B cell and Plasma markers was utilised based on the features that were used to annotate these subpopulations in Ahern et al.12 The anchors and the projections of annotations and intensity values from the CyTOF were performed using a canonical correlation analysis (CCA) with 15 dimensions and k.weight=30 for the COMBAT and Su et al. integrations and k.weight=20 for the integration with the 12 COVID-19 scRNA-Seq datasets.\n\nIn order to evaluate how well the predicted annotations converged to the true cell types we used the Hubert-Arabie Adjusted Rand Index (ARI) for measuring the correspondence between two partitions of an object set19 and the F1 score. The ARI metric is adjusted for chance and random clusterings are expected to have an ARI equal to 0 and identical clusterings to 1. The metric was calculated using the implementation in the mclust R package20 (v5.4.9). The F1 score between the predicted and published annotations was calculated as the harmonic mean of precision and sensitivity. For F1, the cells for which there was not an equivalent annotation in both datasets were all grouped as Other. Finally, the F1 scores of two extra populations, the predicted Naïve CD4 and Naïve CD8 populations were calculated over T cells only.\n\nTo compare the imputed CyTOF intensities with the ADT normalised values at the pseudobulk level, we summarised the values (using the mean) on the pseudobulk clusters’ annotations (as annotated in the COMBAT study12) and the sample ID, using the function aggregateAcrossCells from the package scuttle21 (v 1.4). Then the values were correlated using Pearson’s correlation.\n\nCD11c+ cells were determined using a threshold defined by the imputed protein values. We reasoned that the distribution of the imputed values for CD11c followed the CyTOF distribution of that marker since the imputation of the markers is a weighted average of the original values. Therefore, we firstly selected the CD11c+ populations of the CyTOF annotations. Then, for those cells, we chose the 1st quartile of the CD11c intensity distribution, which gave an intensity threshold of 1.6 (Supplementary Figure 1, Extended data39) to define the CD11c+ cells. Therefore, all cells with an imputed CD11c value greater than 1.6 in the Su et al. dataset were defined as CD11c+ cells. For the integration with the 12 COVID-19 datasets, the predicted annotations were used to define the subpopulations of interest (the cluster “CLA+ Switched memory B cells” comprised of 23 cells only and was removed from the downstream analysis). The transcriptional identity of the subpopulations was ascertained by finding DEGs using Seurat’s implementation of the Wilcoxon rank-sum test (FindMarkers default parameters, adjusted p value < 0.05). A gene set enrichment analysis (GSEA) was performed using the DGE results generated from FindMarkers (with parameters logfc.threshold=0 and min.pct = 0) and with the R package clusterProfiler22 (v 4.3.3) using the fgsea algorithm with the maximal size of genes annotated for testing set to 800 and minimum gene set size set to 10.\n\nThe R scripts used to run the analyses presented in this article are available from GitHub and archived with Zenodo.40\n\n\nResults\n\nThe ultimate motive of multimodal integrations is to take advantage of complimentary information of the datasets for a holistic understanding of cellular processes of interest. Stuart et al. demonstrated that transferring protein measurements across datasets can be a powerful tool to further our understanding of cell subpopulations. They also showed that this transfer can lead to accurate protein predictions even in the absence of a strong correlation between the RNA and protein of a gene, if alternative combinations of genes exhibit expression patterns that are correlated with cellular immunophenotypes.14 However, these integrations were performed based on RNA features alone. Here we extend this work to show that the integration of transcripts and proteins from RNA and CyTOF datasets based on mRNA-protein correspondences can also yield accurate predictions of cell populations and imputed protein features.\n\nWe used mass cytometry and CITE-Seq data from the COMBAT Consortium12 to showcase and validate the integration of the two modalities. The two datasets were downsampled to 45,500 cells for each modality (sampled as 500 cells per sample) to approximate a balanced real-life scenario and were integrated using Seurat with CyTOF as a reference and scRNA-Seq as a query with the Seurat v3 software.14 After finding the anchors between the two datasets, we used them to transfer the protein intensities to the RNA dataset, creating imputed values of the protein markers for each cell. We then merged the datasets to get a co-embedding of the two datasets on the same space (Figure 1A).\n\nA. UMAP of the integrated COMBAT dataset coloured according to cell type (left) and split by modality (right). Red cells originate from the CyTOF (mass cytometry) dataset and the blue cells from the scRNA-Seq dataset, showing a good overlap between the two modalities.\n\nB. Riverplot of the predicted labels (annotations) for each cell (right) compared to their actual major cell type (published annotations) in the COMBAT dataset (left).\n\nC. Barplot of the F1 scores of the major cell types for different sample sizes of the COMBAT dataset. Each bar shows the F1 score for the integration using a downsampled subset of the data (number of cells according to colour).\n\nD. Frequencies of the observed clusters (per sample cluster frequency based on published annotations) (x axes) compared to the frequencies as predicted by the integration from the CyTOF annotations (y axes) (per sample predicted cluster frequency) in the COMBAT dataset.\n\nWe observed that all major populations of the integrated dataset appear well separated in the uniform manifold and projection (UMAP) visualization and that all common populations are represented equally from both modalities (Figure 1A). Importantly, the T subpopulations are clearly distinguished, separating CD4, CD8 and NK cells. Interestingly, dataset specific subpopulations, such as the basophils from the CyTOF and the platelets from the RNA, form dataset-specific, distinct islands in the UMAP and are not mixed with other populations. The only populations that are not separating clearly and seem to be dispersed across the island of the monocytes are the conventional dendritic cells (cDCs) (cDC1 and cDC2 populations). These observations are in line with the findings in Hao et al. that CD8+ and CD4+ T cells were partially mixed when analysing the transcriptome but separated clearly when clustering on protein data, whereas cDCs formed distinct clusters when analysing RNA but were interspersed with other cell types based on surface protein abundance from the CITE-Seq dataset.13\n\nOne of the hardest tasks in the analysis of scRNA-Seq experiments is the annotation of cell types post clustering. Certain populations, such as CD4/CD8 T and CD8/NK cells are notoriously difficult to disentangle from the transcriptional data alone.13,23,24 Using the anchors provided by Seurat, the cell annotations of CyTOF can be projected to the scRNA dataset to inform the cluster identification of the latter. To validate the accuracy of these predictions, we compared the projected cell types from the CyTOF to the published cell annotations from the COMBAT Consortium which were created using expert immunological knowledge to guide a curated manual integration of the data from the different modalities (GEX, ADT and VDJ)12 (Figure 1B-D). Using the adjusted Rand Index (ARI) as measure of the similarity between two data annotations, we verified a high correspondence of the two label lists, with an ARI of 0.832. Furthermore, we calculated the F1 score of each population to assess the precision and sensitivity of the projections (Figure 1C). B cells and plasmablasts were the cell populations with the best scores, followed by classical monocytes, NK and CD4 T cells, all having a score above 0.9. Non classical monocytes, CD8 and MAIT cells had slightly reduced scores but were still well defined. On the other hand, 40% of gamma delta (γδ) T cells (GDT) were wrongly predicted as CD8 cells, due to the lack of common markers defining this population. TCRgd, the main marker for distinguishing GDT cells, could not be used for the integration because its antibody was added in the same channel as IgD for efficiency, and thus there was no one to one correspondence between the genes and the protein channels anymore. Furthermore, a large proportion of the DC cells were misclassified as classical monocytes, in line with previous observations that cDCs are well defined from transcriptomics signatures, but not easily separated based on surface protein abundance.13 Applying a filter for the prediction score (0.6) markedly improved the F1 scores of DC and GDT to 0.798 and 0.665, respectively, but reduced the number of cells with a prediction by 10.3%. These observations were further validated comparing the observed frequencies of the common cell types in the scRNA to the predicted annotations from CyTOF per sample (Figure 1D).\n\nTo quantify the amount by which the predictions are influenced by the number of cells in the two modalities, alternative scenarios were also examined. Smaller subsets were used to assess the robustness of the projections for lower numbers of cells and two scenarios of larger datasets were used to evaluate whether an increased number of cells further improves the predictions. Therefore, the analysis was repeated for 9,100, 22,750, 91,000 and 182,000 cells per modality. Most predictions remain consistently high even for the lower subsets of cells and increasing the number of cells does not seem to help all the clusters with low scores (Figure 1C). Finally, increasing the number of cells in the CyTOF dataset to 182,000 cells with the same resolution for the scRNA (45,500 cells) also does not provide a significant improvement in the scores (data not shown).\n\nWe next explored the potential of the transcriptomic and proteomic integration to reliably impute the protein markers on the scRNA data. The aim was to assess the accuracy of imputed markers for features that were used in the integration (common genes-proteins between RNA and CyTOF) and for features that did not have an equivalent gene, such as CD45RA and CD45RO. To evaluate the imputation, the transferred protein intensities were compared to the ADT values from the CITE-Seq data, which was considered the “ground truth” of protein measurements.\n\nAs expected, the accuracy of the imputation, measured using Pearson’s correlation of the imputed protein with the ADT, reflected to a certain degree the strength of the correlation between the RNA and real protein measurements (ADT) (Figure 2A). Genes for which the correlation between RNA and ADT was very low in the CITE-Seq data (below 0.2), did not provide a good proxy with the imputed protein values either. This included markers that were not relevant in the dataset (CD66 is a marker for neutrophils, of which there are none in this dataset) and antibodies that did not perform well in the ADT (CTLA4 and CCR7). Notable exceptions were CD141 (a marker for the cDC1 dendritic cells) and CD57 (a marker of cytotoxicity in T and NK populations), for which, even though the correlation between the transcriptome and the protein was poor, the integration was able to recover good proxies for the imputed features.\n\nA. Scatterplot showing the correlation coefficients of the RNA to the ADT for each gene-protein pair (x axes) versus the correlations of the imputed CyTOF (mass cytometry) values to their equivalent ADT (y axes). The three coloured bands depict the strength of the correlations between RNA and ADT values of each gene-protein pair in regions of poor (Pearson’s correlations<0.2, red), medium (0.2<Pearson’s correlations<0.7, orange) and strong (Pearson’s correlations>0.7, green) correlations.\n\nB. UMAP of the integrated dataset (constrained to the cells from the scRNA-Seq alone for all subpanels) showing the RNA expression (1st column) of CD4 (top row) and CD8 (bottom row) versus the ADT (2nd column) and the imputed CyTOF (3rd column).\n\nC. Density plots of the CD4 expression (top 1st column), the CD4 imputed CyTOF intensity (top 2nd column) and the CD4 ADT (top 3rd column) followed by a violin plot of the CD4 imputed intensities according to the published annotations (bottom).\n\nD. Scatterplots of pseudobulk populations per sample and cell type for CD45RO (left) and CD45RA (right). The averaged imputed value of the proteins is depicted on the x axes and the averaged observed ADT value for the same populations on the y axes.\n\nInterestingly, most of the protein markers had a stronger correlation with their ADT counterpart than they did with their respective gene, highlighting that the anchor-based integration was able to recapitulate the protein intensities using the population structure similarities and not directly using the correlations of the proteins with the genes. As a result, we speculated that this integration can be used even for populations for which the correlations between the genes and the proteins is low to moderate. As additional evidence to this and to further assess whether this methodology could define populations which are transcriptionally challenging to disassociate, we focused on the CD4 and CD8 separation. As shown in Figure 2B–C, the imputed values of CD4 provide a very strong signal for the CD4 cells emphasising the differences between positive and negative populations and are strongly correlated with the ADT values (Pearson’s correlation 0.80), even though the gene is very lowly expressed and very poorly correlated with the protein values (Pearson’s correlations of expression to the ADT: 0.34 and to the imputed CyTOF: 0.24). In more detail, only 33.2% of the annotated CD4 cells had any expression of CD4 (more than 0 reads), whereas 90% of them had a high value for the imputed protein (imputed intensity > 2) suggesting that the imputed values could also be used to annotate scRNA-Seq datasets. Intriguingly, 12% of the CD8 cells also appeared to be CD4 positive using the imputed values, representing a CD8 subpopulation that was misaligned by the integration. Most of the CD8 cells with erroneously high imputed CD4 came from the CD8 T central memory (TCM) subpopulation, which has the lowest expression of CD8 amongst the CD8 subpopulations (Supplementary Figure 2, Extended data39). A differential gene expression between the two subpopulations (CD8.TCM vs CD4.TCM) revealed only 4 genes with a significant adjusted p-value and a logFC above 1 (CD8, CD8B, CD4 and CD161), suggesting a very similar transcriptional signature between the two populations, explaining this discordance.\n\nFinally, we examined the correlations between the imputed CD45RA and CD45RO with their respective ADT. Even though these two markers were not used for the integration, weighted projected values could be calculated for each cell based on its anchors. Both CD45RO and CD45RA displayed a moderate Pearson correlation with the equivalent ADT (0.595 and 0.563 respectively, Supplementary Figure 3, Extended data39), but when the correlations were repeated on a pseudobulk level per subpopulation and sample, both markers exhibited a stronger correlation with the ADT, with coefficients of 0.759 and 0.723 respectively (Figure 2D), suggesting that even though the individual single cell predictions can be noisy, the bulk estimates are much more accurate. This was supported by the observation that the imputations were accentuating the differences between the positive and negative populations, creating a smoothing effect on the population imputed antibody intensities (Supplementary Figure 4, Extended data39). CD45RA and CD45RO are primarily markers that are used for the identification of naïve and memory populations of T cells. To provide additional evidence that the T cell subpopulation structure was captured correctly by the integration we calculated the F1 scores of the predicted Naïve CD4 and CD8 populations. Both populations had a high F1 score, with a value of 0.781 for Naïve CD4 and 0.801 for Naïve CD8.\n\nTo further the utility of this method, we hypothesized that the integration could be used to inform transcriptomic datasets coming from unmatched samples and different conditions. The applicability of this would mean that publicly available CyTOF datasets of relevant samples could also help the identification of subpopulations in scRNA-Seq data of independent studies. To evaluate this type of integration, we used a publicly available dataset of twenty-four PBMC samples (hereafter named refPBMC) from eight volunteers collected at three time points: day 0 (immediately before); 3 days and 7 days after the administration of a VSV-vectored human immunodeficiency virus (HIV) vaccine,13,25,26 which we integrated with the CyTOF from the COMBAT Consortium12 (of healthy and COVID-19 samples) (from here on named “Mixed integration”). Consistent with our previous analysis, the integration successfully assigned the correct cell identities to 92.4% of the cells including all the cells which belonged to clusters that could not be resolved using the CyTOF information (erythrocytes, circulating innate lymphoid cells (ILC), HSPCs, platelets) and 92.7% of the cells excluding these populations of cells. The ARI between our predicted clusters and the published clusters (based on the WNN integration of RNA and ADT13) was 0.87, further confirming a good agreement between the annotations.\n\nTo evaluate if the main source of variability between the modalities is the immunological landscape of different individuals or the variety of conditions that these samples are coming from (COVID-19 infection vs HIV vaccination), we repeated the integration on the samples from day 0 (unvaccinated subjects only) with the CyTOF samples of healthy controls from COMBAT (in the following analysis named “Day 0 integration”). Qualitatively the two integrations are distinct, where in the mixed integration the populations of B cells and monocytes form islands where the cells are coming from one modality only, whereas there is a much better overlap of cells in the unvaccinated and healthy controls integration for most major cell types (Figure 3A). This observation suggests that the variability observed in the integration of the mixed datasets is the result of biological variation between infection and vaccination effects, and not the result of the integration of different samples. Interestingly, most of the predicted annotations were equally accurate in both integrations (Figure 3B and Supplementary Figure 5, Extended data39). In the mixed integration, the subpopulation with the lowest score was the plasmablasts (PB), with an F1 score of 0.12. In this instance many monocytes were erroneously predicted to be PBs, which was not observed in any of the previous integrations. The misclassified cells had very low prediction scores (Supplementary Figure 6A, Extended data39) suggesting that on integrations of unmatched datasets and different conditions, applying a prediction score threshold may be necessary. HLA-DR is a known marker for plasmablasts.27 Upon closer inspection the misclassified cells also had upregulated multiple genes of the HLA family, known activation markers for monocytes (Supplementary Figure 6B, Extended data39), suggesting that their activated status could be the reason of their erroneous predictions. Contrary to what we would expect, we observed an equal representation of the misclassified PB on all samples, independent of day of collection (pre/post-vaccination) (Supplementary Figure 5, Extended data39). This hints that the PB signature could not be salvaged for the Day 0 samples either, even though the projected annotation of these cells was accurate when no post-vaccination samples were included.\n\nA. UMAP of the integrated dataset coloured according to cell type (top) and split by modality (bottom). Red cells originate from the CyTOF (mass cytometry) dataset, whereas blue cells from the scRNA-Seq dataset. The 1st column shows the mixed integration of the COMBAT CyTOF and the reference PBMC dataset (unvaccinated and vaccinated individuals) and the 2nd column the Day 0 integration of the COMBAT CyTOF healthy samples with the refPBMC unvaccinated (healthy) samples only.\n\nB. F1 score comparison for the Day 0 integration (light blue) and the Mixed integration (dark blue). The Day 0 and Mixed integration scores are highly concordant for most celltypes apart from the plasmablasts (PB).\n\nC. Scatterplot comparing the correlation coefficients between the imputed CyTOF and the ADT for each gene-protein pair in the COMBAT integration (x axes) versus the same correlations for the unmatched dataset (mixed integration) (y axes). The colour of the dot (and protein name) reflects the strength of the correlation of the RNA with the ADT values in the COMBAT dataset (red for poor correlation, orange for medium and green for strong correlation) and the size of the dot reflects the correlation of the RNA with the ADT values in the refPBMC dataset.\n\nHaving demonstrated the integration’s ability to accurately annotate the cell populations, we next imputed the CyTOF markers on the refPBMC dataset (Mixed integration) to assess the granularity that these integrations can disentangle the underlying biological variation for unmatched datasets. To quantitatively validate the imputations, we calculated the correlations of the imputed protein intensities to the “ground truth” ADT measurements of the reference PBMC dataset. We noted a remarkable similarity of the correlations of the imputed markers with the refPBMC-ADT of the unmatched scenario to their respective correlations with the COMBAT-ADT of the matched integration (Figure 3D). Surprisingly, a number of imputed markers even outperformed their counterparts in the COMBAT integration, such as CD99, CD161 and CD8, suggesting differences in the efficiency of the staining of those antibodies between the two ADT datasets. Va7-2 had much lower correlation in the mixed integration compared to the COMBAT matched integration, but it also had a very low correlation between the RNA and the ADT in the refPBMC (Pearson’s correlation 0.12 vs 0.66 in COMBAT), suggesting a lower quality antibody binding for the ADT of that protein.\n\nOne of the most interesting applications of the integration of proteomic with transcriptomic datasets is the ability to use the former to identify subpopulations of interest and then characterise them in the latter, coupling phenotypic definitions to transcriptomic datasets. CD11c+ B cells are a heterogeneous subset of B cells (usually defined as CD19+, CD21- and CD27-) which is present at low frequency in healthy individuals28,29 but is increased in several conditions including malaria, HIV and autoimmune diseases.27,29–32 These cells have assumed many names in the literature, including ‘atypical (memory) B cells’ (or atBC), ‘Age associated cells’ (ABCs), ‘memory precursors’, ‘exhausted memory cells’27,29,30,33 and more recently have been subdivided into DN2 (double negative 2/extrafollicular ASC precursor B cells) and activated Naïve cells27 but there is no general consensus as to their definition. In the COMBAT Consortium, the authors reported subpopulations of the CD11c+ B cells that were significantly more abundant in critical COVID patients compared to healthy volunteers.12 Atypical B cells along with DN2 and activated naïve CD11c+ cells have also been shown to be increased in COVID-19,34–36 but their transcriptional heterogeneity after COVID-19 infection has not been explored. We therefore set off to find and transcriptionally characterise these cells in the scRNA-Seq dataset.\n\nUsing only the B cells subpopulations from both COMBAT CyTOF and scRNA-Seq datasets (excluding the plasmablasts), we were able to integrate the two modalities using as common features the markers that were selected for the B cells subclustering analysis from the COMBAT Consortium.12 In order to validate the results of the integration, we then downloaded the RNA and CITE-Seq data from an additional dataset from Su et al. of healthy and COVID19 samples18 and repeated the integration using the Su scRNA-Seq and the COMBAT CyTOF data. Even though both scRNA-Seq datasets had a low gene expression of CD11c, both independent integrations revealed a strong presence of CD11c+ subpopulations as defined by the imputed protein marker, which was also validated using the ADT values of the two studies (Figure 4A).\n\nA. UMAP of the COMBAT scRNA-Seq (1st row) and Su et al. scRNA-Seq (2nd row) datasets showing the RNA expression of CD11c (1st column) versus the ADT (2nd column) and the imputed CyTOF (mass cytometry) (3rd column) validating the imputed CD11c predictions.\n\nB. Venn diagram of the differential gene expression of CD11c+ vs CD11c- B cells for the COMBAT and Su et al. datasets. The average logFCs of the differentially expressed genes in both datasets (430 genes) are plotted as a scatterplot.\n\nC. GSEA of differentially expressed CD11c+ genes from COMBAT and Su et al. datasets with two published gene list signatures of atypical memory B cells (MBCs) vs Naïve and vs Classical B cells of malaria infected individuals (Portugal et al.) and of CD11c+ B cells vs CD11c- from healthy donors (Golinksi et al.) showing significant enrichment of these gene sets.\n\nDue to the low frequencies of the CD11c+ subpopulations, the naïve-like CD11c+ and the switched memory CD11c+ subpopulation could only be found in a fraction of the samples in both COMBAT and Su et al. datasets (Supplementary Figure 7, Extended data39). Therefore, we used the imputed values to identify the CD11c+ population of interest (details in the Methods section). Differential gene expression of the CD11c+ cells uncovered a transcriptional signature that had 430 genes in common between the two datasets (99 downregulated and 331 upregulated), all of which had the same direction of effects (Figure 4B). Moreover, we performed a gene set enrichment analysis (GSEA) of the two lists of differentially expressed genes from the COMBAT and Su et al. datasets with two published signatures of CD11c+ cells from healthy controls28 and from atypical BCs of malaria patients.31 Both show a significant enrichment for the CD11c+/atBC signatures (NES>2 and p-values<10-17), further confirming the identity of those cells (Figure 4C).\n\nAmongst the genes with the largest logFC in both studies were FGR, CIB1, EMP3, MPP6 and RHOB (Figure 4B), genes that have been previously associated with the DN2 phenotype.17 Moreover, DN2 cells are known to express high levels of inhibitory receptors, such as those belonging to the family of Fc-receptor-like (FCRL) molecules,17,37 consistent with the upregulation of FCRL3 and FCRL5 in the CD11c+ subpopulation. TCL1A, on the other hand, is a marker of transitional and naïve cells, which is known to be absent in the other B cell populations.17 T-bet (TBX21) is a transcription factor that has been shown to be expressed in CD11c+ B cells, although not all CD11c+ B cells are T-bet+.28 In the COMBAT dataset TBX21 (T-bet) was filtered out due to low expression (expressed in less than 10% of the cells in both subsets) and could not be tested for differential expression between the two subsets. In contrast, TBX21 was significantly differentially expressed between the CD11c+ and CD11c- populations in the Su et al. dataset with a logFC of 0.51 (adjusted p-value<10-14). Additionally, many known transcriptional markers correlating with T-bet expression in B cells, such as ACTB, ALOX5AP, GSTP1 and LAPTM5,17 were enriched in CD11c+ cells for both datasets (Supplementary Figure 8, Extended data39).\n\nTo further explore the transcriptional heterogeneity of the CD11c+ population in COVID-19 patients, we integrated the CyTOF dataset with B cell populations of a meta-analysis of 12 scRNA-Seq datasets of COVID-19 studies.16 The analysis recovered all three CD11c+ subpopulations that had been found in the CyTOF COMBAT dataset. It identified 153 naïve CD11c+ (activated naïve cells/naive-like CD11c B cells, defined as CD27-, IgD+, CD11c+); 400 DN CD11c+ (DN2, defined as CD27-, IgD-, CD11c+) and a less well characterised population of 440 switched memory CD11c+ cells (CD27+, IgD-, CD11c+ cells). All three subpopulations, had a clear signature of genes associating with the CD11c+ phenotype, such as a higher expression of CD19 and CD20 (MSA4A1) as well as CD11c+ hallmark cell surface receptors CD84, CD68, CD8628; inhibitory receptors CD72, FCGR2B, FCRL3, FCRL5, LILRB130 and transcription factors TBX21, ZEB2 and ZBTB3231,32 (Supplementary Figure 9, Extended data39).\n\nPairwise analysis of the 3 subpopulations uncovered 91 differentially expressed genes between the DN CD11c+ cells and the switched memory CD11c+; 44 between the naïve-like CD11c+ and the switched memory CD11c+ (Figure 5A) and a very similar transcriptional profile for the DN2 and the naïve-like CD11c+ cells (no significant differentially expressed genes between the two), in line with the phenotypic similarities of these two populations that have been previously reported.27 The similarity between naïve-like and DN2 CD11c+ cells, and the differences between naïve-like CD11c+ cells and the naïve (CD11c-) cells (529 differentially expressed genes - data not shown), indicates that these naïve-like CD11c+ (or activated naïve) cells are transcriptionally closer to the DN2 than they are to the naïve counterparts. This suggests that these might not be naïve cells, but rather a small subset of CD11c+ cells sharing a limited set of phenotypic markers with classical naïve cells.\n\nA. Volcano plots of DGE between Naïve-like CD11c+ vs Switched memory CD11c+ (left) and DN CD11c+ and Switched memory CD11c+ cells (right) in the meta-analysis of the 12 COVID-19 scRNA-Seq datasets. Significant genes shown in red.\n\nB. Dotplot showing the top genes from A. for the 3 predicted subpopulations of CD11c+ cells in the dataset of Su et al. The genes with an asterisk were also shown to be significant in the comparison of Naïve-like CD11c+ cells vs Switched memory CD11c+ (p-adjusted<0.05).\n\nThe differentially expressed genes between the three subsets uncovered a subset specific expression of CXCR3, which was upregulated in the switched memory subset of CD11c+ cells (Figure 5A). CXCR3, an inflammatory chemokine receptor, has been shown to be upregulated in CD11c+ cells in multiple studies,29,35,37,38 with heterogeneous expression amongst these cells,35,37 but had not previously been associated with a specific subset of CD11c+ cells. Similarly, MPP6 a transcriptional marker of Tbet+ (TBX21) cells31 was downregulated in switched memory CD11c+, providing further evidence for the observed heterogeneity of CD11c+ cells in terms of expression of TBX21.27 Seven of the differentially expressed genes between the naïve-like CD11c+ cells and the switched memory CD11+ cells (CD27, LTB, TSC22D3, ACTG1, FCRL3, FCRL5 and KLF2) were also independently validated in the smaller dataset of Su et al. with significant adjusted p-values (Figure 5B) and many other important markers such as CXCR3 and MPP6 had the same direction of effects.\n\nTo our knowledge, this is the first study to be transcriptionally characterising these very rare subpopulations of cells after COVID-19 infection by leveraging information from two independent single cell technologies and using only publicly available datasets. Importantly, this subpopulation of CD11c+ cells has never been identified without prior sorting of B cells. These results have clearly demonstrated that the multimodal integration substantially improves our ability to resolve cell states, allowing us to identify and characterise previously unreported B cell subpopulations.\n\n\nDiscussion\n\nIn the last decade, single cell methodologies have transformed all fields of biology. Mass cytometry and single cell RNA-Seq have been very widely used in the past, but the integration of the two modalities has not been widely explored so far. In this work, we show the unique advantages that can be gained from formally integrating these two modalities. We demonstrate that CyTOF datasets can be used to annotate and produce high quality imputed proteomics values for both matched and unmatched scRNA datasets. More importantly, we prove that the imputed values can be used to help define rare subpopulations in order to transcriptionally characterise them in depth using publicly available datasets.\n\nThe integration of modalities using imputed or projected values can give us unprecedented power to further our understanding of the transcriptional and proteomic immunological landscapes of health and disease and provide a wealth of interesting hypotheses to be tested, but caution is warranted when using this methodology without validation. We observed great potential in integrating well defined populations of cells, such as CD4 and CD8 T cells, but in cases where the common markers were not sufficient to separate the cellular heterogeneity, the agreement between the projected cellular annotations and the real ones were suboptimal (such as GDT cells; Figure 1B-D). Fundamentally, this integration will always depend on the quality and quantity of the common CyTOF-RNA features.\n\nAn additional limitation of this methodology is that the frequencies of rare subpopulations between different conditions cannot be estimated with high accuracy for medium size scRNA-Seq studies (Supplementary Figure 7, Extended data39). Populations that were less than 1% of the B cells had a high proportion of samples for which no cells could be salvaged for those subpopulations. However, we demonstrated that meta-analyses of RNA datasets can bypass this constraint and managed to unravel the transcriptional heterogeneity of these populations. Exploiting this methodology with the abundance of scRNA-Seq data from the Human Cell Atlas1 could provide endless opportunities to harness the differences between rare subpopulations in health and disease.\n\nWe should note here that the integration of CyTOF with scRNA-Seq datasets is not intended to substitute the invaluable information gained from multi-modal technologies such as CITE-Seq; measurements of antibody derived tags will inherently be more accurate than imputed values. However, there are instances where mass cytometry can provide imputed measures which would not be available in CITE-Seq, such as when rare subpopulations of cells are of interest, as we demonstrate in Figures 4,5. Additionally, intracellular protein modifications of cells, which are not possible to measure in CITE-Seq, could be projected to the RNA datasets, in a similar manner to our imputation of non-overlapping features CD45RA and CD45RO to identify epigenetic states of the cells. Furthermore, extending the integration of scRNA-Seq datasets with Imaging Mass Cytometry (IMC) datasets could facilitate new and exciting discoveries of spatial interactions of healthy and disease states.\n\nIn this article, we have successfully identified and characterised a previously elusive subpopulation of CD11c+ B cells. To our knowledge, this is the first study showing that imputed protein values from a CyTOF and scRNA-Seq integration can lead to an in-depth transcriptional characterisation of a heterogeneous rare subpopulation of cells. Even though the frequencies of these subpopulations vary between datasets, we show that the transcriptional signature that defines those cells can be robustly identified with this integration. To date, there are only a handful of scRNA-Seq experiments that try to describe this CD11c+ population but all are very limited in the numbers of samples and cells used and none of them has managed to isolate all three subpopulations of CD11c+ cells.17,32,37,38 In addition, in these studies the subpopulations of interest had to be sorted prior to sequencing to enrich for rare subtypes. Cell sorting, however, is a laborious process that can introduce bias in the process of selection of cells, especially in the case of less well-defined subpopulations, as is the case with CD11c+ subpopulations, explaining why none of these studies was able to identify all three subpopulations. Managing to couple phenotype-based studies with single cell transcriptomics in an unbiased way can give us unprecedented insights into the cellular machinery of all sections of biology.\n\n\nData availability\n\nThe Ahern et al.12 data was obtained from https://doi.org/10.5281/zenodo.5139560.\n\nThe human PBMC atlas datasets13 were obtained from the GEO database accession GS164378 and from https://atlas.fredhutch.org/data/nygc/multimodal/pbmc_multimodal.h5seurat.\n\nThe Tian et al.16 data was obtained from https://atlas.fredhutch.org/fredhutch/covid/.\n\nZenodo: Supplementary Figures for Repapi et al. https://doi.org/10.5281/zenodo.6513604.39\n\nThis project contains the following extended data:\n\n- supplementary_Repapi_et_al.pdf (PDF file containing Supplementary Figures 1–9)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAnalysis code\n\nAnalysis code available from: https://github.com/emmanuelaaaaa/CyTOF_scRNA_integration\n\nArchived analysis code at time of publication: https://doi.org/10.5281/zenodo.654698240\n\nLicense: MIT", "appendix": "Acknowledgements\n\nWe thank Fabiola Curion from the Institute of Computational Biology, Helmholtz Center Munich in Germany for her critical support throughout this project and for her expert advice on the manuscript.\n\n\nReferences\n\nRegev A, et al.: The human cell atlas. elife. 2017; 6. 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Signal Transduct. Target. Ther. 2021; 6: 1–12.\n\nRepapi E, Agarwal D, Napolitani G, et al.: Supplementary Figures for Repapi et al. 2022. [Dataset]. 2022. Publisher Full Text\n\nRepapi E, Agarwal D: emmanuelaaaaa/CyTOF_scRNA_integration: v1.0 (v1.0). Zenodo. [Analysis code]. 2022. Publisher Full Text" }	[ { "id": "147057", "date": "01 Sep 2022", "name": "Tallulah Andrews", "expertise": [ "Reviewer Expertise Bioinformatics", "Single-cell RNAseq analysis and tool development." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors demonstrate the effective imputation of protein-level expression in single-cell RNAseq data through the integration of CyTOF and the query scRNAseq dataset using the Seurat CCA approach. While they do not expand upon or improve existing methodology, they do effectively demonstrate that their approach is relatively accurate and useful for PBMC datasets from multiple conditions by comparing the imputed values to independent protein measurements from CITE-seq / ADT. The work is technically sound and will be of interest to some aspects of the community - particularly single-cell eQTL analyses which extensively employ PBMC data. I have only a few minor questions and concerns remaining:\nThe authors mention that \"Alternative options for the reduction were assessed but were found to be suboptimal (data not shown).\"\nWhich options were explored and how was it determined that they were suboptimal? Did the authors consider alternative integration methods e.g. LIGER 1\n\nAnnotation and CyTOF intensity values were transferred to the RPCA integrated single-cell object combining many unannotated samples. However, RPCA integration can merge small rare cell populations into bigger clusters particularly if that cell-type is poorly represented / absent from one of the samples. Based on the results for the integration of unmatched samples, it seems that transferring data to individual novel samples rather than integrated maps may be more accurate, could the authors do a systematic comparison of imputing values for un-integrated novel samples vs integrated novel samples for a single biological condition vs integrated novel samples across all biological conditions? This would provide very valuable information for other researchers attempting to apply this approach to their own data.\n\nRe: \"For F1, the cells for which there was not an equivalent annotation in both datasets were all grouped as Other.\"\nWere the \"Other\" cells retained for the ARI & F1 calculations? This could lead to misleading results as one of the known issues with CCA integration is the incorrect merging of cell-types that are present in only one dataset -> thus incorrectly merging Other with Other in this case despite them being two very different cell-types. The authors should ensure ARI and F1 score are calculated after excluding cells labelled as \"Other\".\n\nWhy did the authors calculate F1 scores for Naïve CD4 and Naïve CD8 populations only over T cells? Were there a significant number of non-T cells predicted to be Naïve CD4 / Naïve CD8 cells?\n\nIn the caption of Figure 1D, the authors should specify what is represented by the grey dashed line.\n\nIn Figure 1D the frequencies of cell-types are dependent on each other since they must sum to 1. Thus is it the case that the outlier from the 1:1 relationship in CD4, CD8, and NK plots represent the same sample or are these three different outlier samples?\n\nFigure 1 and Figure 3 both show populations that appear clearly distinct in the UMAP but that have very low accuracy of prediction (Basophils and PB respectively). This seems incongruous but may be the result of known issues with UMAPs creating misleading visualizations. The authors should consider using an approach such as PAGA 2 to visualize the relationships between the cell-types more accurately, as this may clarify that the Basophils and PBs are not as distinct as they appear in the UMAP.\n\nFigure 2 could be improved by including plots imputed CD45RO vs CD45RA and each vs the RNA expression of the common gene (PTPRC) to more clearly illustrate the ability of the imputation to determine protein expression from the overall RNA profile of the cells rather than the respective RNA expression.\n\nThe very high agreement between CyTOF and imputed ADT values in the unmatched dataset, suggests the failure to correctly infer PB cell-type annotations is not a consequence of poor integration but of poor PB annotation in the original dataset. Could the authors confirm this by examining the accuracy of the protein expression imputation (e.g. Figure 3D) specifically for the falsely-predicted PB cells, as well as examining the PB-specific markers from the COMBAT dataset within the falsely-predicted PB cells vs the correctly predicted PB cells?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "8954", "date": "04 Nov 2022", "name": "Εμμανουέλα Ρεπαπή", "role": "Author Response", "response": "Thank you for reviewing our manuscript and for your helpful suggestions. Below are point-by-point responses to the individual comments. We agree with the Reviewer that this needs further explanation and we have added more details in the Methods section along with a Supplementary Table comparing the options tested using the ARI and F1 metrics. We also tried LIGER with both the iNMF (Welch et al., 2019) and the online iNMF(Gao et al., 2021) algorithms. However, as discussed earlier this year in their github repository[1], LIGER does not currently have the option to directly transfer labels from a reference to a query dataset as part of their workflow. The closest equivalent is to cluster the integrated object and assign labels to the clusters via the majority rule from the reference. Therefore, the integrations did not perform well in producing a clustering in high agreement with the published annotation labels (ARI of 0.546 and 0.345 respectively). As a result, we felt that the comparison with the Seurat pipeline would not be on equal terms and could not be formally included in the paper. We thank the Reviewer that highlighted this useful exploration for the benefit of future readers. We agree that this will add value to the paper and provide important information for anyone trying to utilise this approach. We have revised the text to include these important comparisons in the Results and added Table 2 with the ARI and F1 metrics comparing them. For most populations the F1 scores remain relatively consistent between the 3 scenarios, apart from the DC and PB populations which showed higher variability. For these subpopulations the “per sample” integration had the lowest scores, indicating the reduced power to detect rare or low frequency populations for small cell numbers. Furthermore, we have added a Supplementary Figure 11 to show that the imputations of the CyTOF features are very similar between the three scenarios. We apologize that these evaluations were not explained clearly. We have revised the Methods section to reflect more accurately that the non-common (Other) celltypes were only grouped together for the F1 calculations. The ARI metric compared the projected cell types from the CyTOF to the published cell annotations (including both common and dataset specific clusters) without any changes to the clusters’ names. Removing the dataset-specific clusters results in higher estimates of ARI in all scenarios of our study but we felt that including all clusters gives a more representative view of cluster agreement as it considers dataset specific cells which have been misannotated with one of the common labels. For the F1 measure, the cells from the cluster Other were retained to calculate the precision and sensitivity for each cluster (again to ensure that misclassified cells are accurately represented) but the F1 measure was not calculated for this cluster as it was a mix of multiple clusters. Similarly to the ARI, the F1 scores increase with the removal of the dataset-specific clusters but the results are not taking into account the misannotated cells. For this reason we have kept all cells for the calculations of both metrics. The F1 scores of the Naïve CD4 and CD8 were calculated over the T cells to reflect the conventional way of calculating the frequencies of those clusters over T cells alone. No predicted Naïve CD8 T cells had a non-T cell identity and only 15 out of the total 10294 cells that were predicted to be Naïve CD4 T cells were non-T cells. We have added the description in the legend of Figure 1D. Indeed the frequencies of cell types are dependent on each other, and this is the case for the outliers in the subsets of CD4 and CD8 cells which come from the same patient sample. However, the outlier of the NK sample is a distinct sample. Given that this was not a widespread effect, we did not consider removing that sample from the integration. The Reviewer raises an important point regarding known issues with the UMAP algorithm. To address this potential ambiguity, we have added the relevant PAGA figures in the Supplementary Figures. The basophils appear well separated in all graphs, since it is a population with a very distinct signature, even though it is only present in the CyTOF dataset. However, it is obvious that although the plasmablasts (PB) are separating well in the COMBAT integration (Supplementary Figure 2), this is not always the case in the integration with the refPBMC samples (Supplementary Figure 9). Similarly to the results of the F1 score for this population, the Day 0 integration (COMBAT HC and unvaccinated refPBMC) shows the PB separating well and appearing the closest to the B cells. On the other hand, in the Mixed integration (all COMBAT and refPBMC) the PBs appear closer to the monocyte populations, highlighting the inaccuracies of the integration which were also shown in the predicted labels where some monocytes were wrongly predicted as PBs by the model. We agree with the Reviewer that these figures could further enhance our argument of the ability of the imputation to determine protein expression from the overall RNA profile and have added these in our Supplementary Figure 6 and amended the text to highlight this. We apologise that this section of the results was not more comprehensive to further evaluate the issue with the PBs’ misannotations and we thank the Reviewer for highlighting this omission in the comments so that we can address it. We have expanded the results section of the text to include another scenario where all the twenty-four refPBMC samples are integrated only with the CyTOF samples of healthy controls (from COMBAT). In this integration, we observed a much higher F1 score on the predicted PBs (0.68 vs 0.1 for the Mixed integration) highlighting that the reference dataset is key for the proper annotation of the query dataset and that abnormal frequencies in the reference population can create artefacts in the projected populations. However, that doesn’t fully address the question of the discordance of the F1 score of PBs in the mixed integration with the high imputation values that we showed in Figure 3C. The reason of this discordance is that the misannotated PBs are a very low proportion of cells (0.016) out of the total refPBMCs that the correlations are calculated on. Therefore, the effect of this subpopulation is miniscule on the correlations with the ADT values. As we showed in the revised version of our manuscript, the failure to correctly infer PB cell-type annotations is a consequence of the inflated PBs in the reference population (due to the COVID-19 samples) and not of poor PB annotation in the original dataset. References Gao, C., Liu, J., Kriebel, A. R., Preissl, S., Luo, C., Castanon, R., … Welch, J. D. (2021). Iterative single-cell multi-omic integration using online learning. Nature Biotechnology, 1–8. Welch, J. D., Kozareva, V., Ferreira, A., Vanderburg, C., Martin, C., & Macosko, E. Z. (2019). Single-Cell Multi-omic Integration Compares and Contrasts Features of Brain Cell Identity. Cell, 177(7), 1873-1887.e17. [1] https://github.com/welch-lab/liger/issues/262" } ] } ]	1	https://f1000research.com/articles/11-560
https://f1000research.com/articles/11-283/v1	07 Mar 22	{ "type": "Research Article", "title": "In-air Hand Gesture Signature Recognition: An iHGS Database Acquisition Protocol", "authors": [ "Wee How Khoh", "Ying Han Pang", "Hui Yen Yap", "Ying Han Pang", "Hui Yen Yap" ], "abstract": "Background With the advances in current technology, hand gesture recognition has gained considerable attention. It has been extended to recognize more distinctive movements, such as a signature, in human-computer interaction (HCI) which enables the computer to identify a person in a non-contact acquisition environment. This application is known as in-air hand gesture signature recognition. To our knowledge, there are no publicly accessible databases and no detailed descriptions of the acquisitional protocol in this domain. Methods This paper aims to demonstrate the procedure for collecting the in-air hand gesture signature’s database. This database is disseminated as a reference database in the relevant field for evaluation purposes. The database is constructed from the signatures of 100 volunteer participants, who contributed their signatures in two different sessions. Each session provided 10 genuine samples enrolled using a Microsoft Kinect sensor camera to generate a genuine dataset. In addition, a forgery dataset was also collected by imitating the genuine samples. For evaluation, each sample was preprocessed with hand localization and predictive hand segmentation algorithms to extract the hand region. Then, several vector-based features were extracted. Results In this work, classification performance analysis and system robustness analysis were carried out. In the classification analysis, a multiclass Support Vector Machine (SVM) was employed to classify the samples and 97.43% accuracy was achieved; while the system robustness analysis demonstrated low error rates of 2.41% and 5.07% in random forgery and skilled forgery attacks, respectively. Conclusions These findings indicate that hand gesture signature is not only feasible for human classification, but its properties are also robust against forgery attacks.", "keywords": [ "Dynamic Signature", "Hand Gesture Signature", "Gesture Recognition", "Hand Gesture Signature Database", "Image Processing", "Forgeries Attack" ], "content": "Introduction\n\nA conventional dynamic signature recognition usually uses a special digitized device to capture the dynamic properties of a signature. A stylus pen is used to sign the signature on the surface of the digital tablet. This leaves a subtle track, exposing the signature information to others. A forger could learn the pattern from what they obtained from the tablet surface.\n\nNumerous acquisition approaches have been proposed to replace the usage of a tablet for dynamic signatures. For instance, two ballpoint pens with sensors to measure the pen movement during the signing process,1 a wearable device on the wrist (i.e. smartwatches) to capture the hand motion,2 or an on-phone triaxial accelerometer built in a smartphone.3,4\n\nThe introduction of low-cost sensor cameras5 brings up new research opportunities for contactless human-computer interaction (HCI) in various applications such as robotics, healthcare, entertainment, intelligent surveillance, and intelligent environments.6 Human hand gestures and dynamic signature recognition are becoming prevalent. This work proposes a hand gesture signature recognition system with the capability to recognize the identity of a person in a touchless acquisition environment. Additionally, a public database is provided for evaluation purposes.\n\nSome relevant research works have been conducted using their own collected database. Tian et al.7 introduced a Kinect-based password authentication system to explore the feasibility of a Kinect sensor to authenticate user-defined hand gesture passwords. In Ref. 8, the authors proposed a similar hand gesture signature recognition where the hand trajectory was used as the feature. The performance was evaluated on a self-collected database, consisting of 50 different classes. Empirical results demonstrated the feasibility and benefits of depth data in verifying a user’s identity based on a hand gesture signature. Fang et al.9 proposed a fusion-based in-air signature verification. The user’s fingertip was tracked and the signature trajectory was extracted from a video sample captured by a high-speed camera. Malik et al.10 implemented a neural network in recognizing hand gesture signatures for identity authentication. A CNN-based hand pose estimation algorithm was employed to estimate the hand joint position for the index fingertip. Multidimensional dynamic time warping (MD-DTW) was adopted to match the template and test signature data. It was tested on a self-collected dataset with 15 classes. The empirical results exhibited a promising recognition performance with the presence of depth features.\n\nFrom the literature, the existing studies were mainly utilizing their self-collected databases. To the best of our knowledge, there is no publicly available hand gesture signature database. The existence of a publicly available database can provide a freely available source of data to encourage more researchers into the field. For this reason, we present an openly available database, collected by the Microsoft Kinect sensor camera. To protect the privacy of the contributors, only depth information will be shared.\n\n\nDatabase collection\n\nA Microsoft Kinect sensor camera is used as the main acquisition device to collect the samples of in-air hand gesture signature (iHGS) via its built-in IR projector and IR camera. A sample is a video clip that contains a set of image sequences disclosing the hand movement of a signature signing. The Kinect camera is capable of capturing up to 30 depth frames per second (fps). The number of image sequences (frames) of each sample corresponds to the duration of the hand movement and might be varying in each signature. Additionally, other computational factors such as heavy graphical processing and input latency affect the fps in each enrollment. These latencies may lead to a drop in the rate of fps, causing information loss. Thus, to ensure validation, the collected samples that have an fps rate less than 27 are removed and re-captured again. A more detailed data acquisition protocol can be found in Ref. 11.\n\nThe database is named iHGS database. The data collection was conducted in two separate sessions and the entire process took four months to complete. Samples for the second session were collected with a time interval of approximately two to three weeks from the first session. This arrangement is intended to allow the intra-variances in genuine hand gesture signatures, better reflecting real-world situations. Before enrolment, the flow of the entire enrolment process was explained to each participant. They were given ample time to practice and familiarize themselves with the process before data acquisition.\n\nA total of 100 participants were successfully enrolled. Among the participants, 69 were male and 31 female, aged from 18-40 years. 90% of participants were right-handed (signing with their right hand) with only 10% using their left hand (left-handed). Table 1 summarizes the characteristics of the iHGS database.\n\nThere are two subsets of our iHGS database: (1) genuine dataset, and (2) skilled forgery dataset. For genuine dataset, each participant provides 10 genuine samples in each session (session 1 and session 2). A total of 2000 (10×2×100) samples were gathered for this genuine dataset.\n\nA skilled forgery dataset contains forged signature samples. Each forger was provided with one genuine signature sample (signed by the genuine user on a piece of paper) randomly. They were asked to learn the signature with as much time as they needed. Then, each forger was asked to imitate the assigned signature 10 times. A total of 1000 skilled forgery signatures were successfully collected. However, 20 skilled forgery samples from two forgers (10 samples each) were corrupted due to the hardware error. Thus, only 980 skilled forgery samples were obtained. Table 2 summarizes the number of hand gesture signatures for the two subsets in the iHGS database.\n\n\nMethods\n\nHand detection and localization techniques were applied to extract the region of interest (ROI) from each of the depth images of the iHGS database. A predictive hand segmentation technique was performed to precisely extract the hand region from the frames. Refer to Refs. 11, 12 for more information.\n\nAn iHGS sample is a collection of depth image sequences that comprises of n image frames, i.e. n is also the length of the sample. Several basic vector-based features are extracted from the sample. Firstly, a Motion History Image (MHI) process is performed on the preprocessed depth image sequence of each sample along the time. This technique effectively condenses the image sequence into a single grey-scale image (coined as MHI template), while preserving the motion information in a more compact form.13,14 Specifically, MHI template describes the hand location and motion path along the time and generates a spatio-temporal information for the iHGS sample. The MHI image is then transformed into a vector space to produce a vector-based feature. The features explored in this work are as follows:\n\n(a) x-directional summation (VX)\n\nProduced by summing the MHI template in the vertical direction.\n\n(b) y-directional summation (VY)\n\nProduced by summing the MHI template in the horizontal direction.\n\n(c) xy-directional summation (VXY)\n\nThe concatenation of both VX and VY features fora richer one-dimensional summation feature.\n\n(d) Histogram of Oriented Gradient feature (VHOG)\n\nA histogram descriptor is performed on the MHI template to extract the local texture, represented in a distribution of the edge and gradient structure.15 It can discover the shape or the outline of the template image based on the slope or orientation gradient. It is worth noted that each pixel value in the MHI template describes the motion’s temporal information at a particular location. Thus, histogram orientation of the MHI template represents the intensity of motion history which is a useful feature.\n\n(e) Binarized Statistical Image Features (VBSIF)\n\nStatistical-based features are computed and summarized in a single histogram representation. First, the input image is convolved with a set of predefined filters to maximize the statistical independence of the filter responses.16 Then, each response is applied to a nonlinear hashing operator to improve the computational efficiency. Next, the generated code map is regionalized into blocks and recapitulated into a block-wise histogram. These regional histograms are lastly concatenated into a global histogram, representing the underlying distribution of the data. In this work, different BSIF-based features are produced:\n\n• VBSIF-MHI – MHI template is used as input data to the BSIF.\n\n• VBSIF-X –Image sequences of an iHGS sample are projected along the y-axis to generate an X-Profile template. X-Profile template is used as input data to the BSIF.\n\n• VBSIF-Y –Image sequences of an iHGS sample are projected along the x-axis to generate the Y-Profile template. Y-Profile template is used as input data to the BSIF.\n\n• VBSIF-XY – Both X-Profile and Y-Profile templates are used as the data input to the BSIF.\n\n• VBSIF-MHIXY – MHI, X-Profile, and Y-Profile templates are used as the data input to the BSIF.\n\n\nExperimental results\n\nTwo types of performance analyses are conducted: (1) classification performance analysis, and (2) robustness analysis against forgery attacks. A well-known multiclass Support Vector Machine (SVM) is adopted in the classification analysis through a One-versus-One (OVO) approach. The genuine dataset is randomly divided into a training set and a testing set with a ratio of m:n where m is larger than n. The training set is further partitioned into two subsets: validation subset and training subset with the ratio of mp:nq. The training subset is to train the SVM model; while the validation subset is to find the optimal model parameters for a minimal validation error. The model is then tested on the testing set for performance evaluation. The robustness performance analysis measures the security level against impersonation attempts. It demonstrates two attacks: random forgery and skilled forgery. In the former, a testing sample that belongs to a subject i is compared with all the remaining samples of other subjects in the genuine dataset. In the latter, a forged sample of a subject j (from the skilled forgery dataset) is matched with a claimed identity’s sample (i.e., genuine subject i’s sample) from the genuine dataset.\n\nThis analysis is implemented using the multi-class classification feature which is available in a library of SVM (LIBSVM) in MATLAB.17 The samples of the genuine dataset are randomly partitioned into training, validation, and testing subsets, refer to Table 3.\n\nThe data distribution is randomized in five different trials using a polynomial kernel. The optimal hyperparameters for the polynomial kernel are tuned empirically such that the gamma (γ) is set to 20, the degree of the polynomial (d) is set to 2 and the cost (C) is set to 1. The averaged classification measurements including precision, recall, specificity, and F1-score and the standard deviation are reported in Table 4. The accuracies among features are illustrated in Figure 1.\n\nThe classification results show the two BSIF features, VBSIF-XY and VBSIF-MHIXY achieving the best accuracy scores of 97.43% and 93.57%, respectively. It is followed by the HOG feature VHOG with an accuracy of 91.63%. It is noted that the system vaguely classifies the summation features, VX and VY with accuracies of 61.43% and 61.20%. However, there is a boost in performance when concatenating them together, achieving 86.63% classification accuracy.\n\nThe results suggest that some of the proposed vector-based features possess high discriminative information for in air hand gesture signature classification, such as VBSIF-XY and VBSIF-MHIXY. These directly extracted features allow an easy learning process in the SVM model. Apart from that, the small value of standard deviation also implies the stability of the features in predicting the hand gesture signature. In summary, the empirical results substantiate the ability of SVM in escalating the classification performance by using vector-based features.\n\nThis experimental analysis aimed to determine the robustness of the proposed approach against two types of forgery attacks, namely random forgery attacks and skilled forgery attacks.\n\nThe experiments were repeated for five trials. Averaged equal error rate (EER) and standard deviations were recorded. Four distance metrics were examined: Euclidean distance (EucD), Cosine distance (CosD), Chi-Square distance (CSqD), and Manhattan distance (MD).\n\nTables 5 and 6 report the system performances of two forgery attacks. It can be seen that the performances of the four kinds of distance metrics vary with different feature vectors. For the random forgery attack, VHOG with a cosine distance metric yields the lowest EER in random forgery (EER-R) of 2.41% followed by VBSIF-MHIXY with EER-R of 5.18%. Manhattan distance is not able to perform in this context as compared with the other metrics.\n\nUndeniably, distinguishing skilled forgery attacks is more challenging than random forgery attacks since the forgery samples are very similar to the genuine ones. The EERs for the skilled forgery attack is expected to be higher. It is observed that VXY and VHOG with cosine distance achieve the best EER-S of 5.07%, followed by VBSIF-MHIXY with Euclidean distance, obtaining an EER in skilled forgery (EER-S) of 9.45%. It is also observed that most BSIF features could not perform well in verifying skilled forged hand gesture signatures. Similar to the random forgery attack, Manhattan distance obtains the worst performance. These results indicate that the verification performance is not only affected by the extracted features; but is also highly dependent on the employed distance metric.\n\n\nConclusions\n\nIn this paper, we presented a self-collected iHGS database and a detailed description of the acquisition protocol to collect the database. Several basic sets of vector-based features were extracted from the samples. This paper also investigated the effectiveness of classification capability as well as the robustness against forgery attacks. The experimental results for both analyses have shown promising results with the appropriate features extracted from the samples. Our analyses demonstrate the potential of iHGS in both recognition and verification. However, there is room for future exploration in iHGS. The current database was collected in a controlled environment. As a biometric authentication, other external factors such as angles of the camera, the distance between user and acquisition devices, different background complexity, etc should be considered. In particular, it could be further extended by considering those uncontrolled environmental factors to increase the challenge of the database.\n\n\nData availability and materials\n\nFigshare: In-air Hand Gesture Signature Database (iHGS Database) https://doi.org/10.6084/m9.figshare.16643314\n\nThis project contains the following underlying data:\n\n• Genuine dataset (100 contributors labels with ID from 1 to 100)\n\n• Skilled forgery dataset (98 contributors labels with ID from 1 to 100 where ID of 84 and 88 are not included)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nEthics approval and consent to participate\n\nThe experimental analyses were established, according to the ethical guideline and were approved by the Research Ethics Committee (REC) with the ethical approval number EA1452021. Written informed consent was obtained from individual participants.\n\n\nAuthor contributions\n\nW.H. carried out the experiment with support from Y.H. and H.Y. coordinated the data collection and establishment of the database. Besides, W.H. took the lead in writing the manuscript while Y.H. and H.Y. provided critical feedback and helped shape the analysis and manuscript.", "appendix": "References\n\nGriechisch E, Malik MI, Liwicki M: Online Signature Verification using Accelerometer and Gyroscope Proc. 16th Bienn. Conf. Int. Graphonomics Soc. 2013; no. January: pp. 143–146.\n\nLevy A, Nassi BEN, Elovici Y: Handwritten Signature Verification Using Wrist-Worn Devices. ACM Interactive, Mobile, Wearable Ubiquitous Technol. 2018; 2(3): 1–26. Publisher Full Text\n\nBailador G, Sanchez-Avila C, Guerra-Casanova J, et al.: Analysis of Pattern Recognition Techniques for In-air Signature Biometrics. Pattern Recogn. 2011; 44(10–11): 2468–2478. Publisher Full Text\n\nSun Z, Wang Y, Qu G, et al.: A 3-D Hand Gesture Signature Based Biometric Authentication System for Smartphones. Secur. Commun. Networks 2016; 9(11): 1359–1373. Publisher Full Text\n\nZhang Z: Microsoft Kinect Sensor and Its Effect. IEEE Multimed. 2012; 19(2): 4–10. Publisher Full Text\n\nHussain Z, Sheng QZ, Zhang WE: Different Approaches for Human Activity Recognition: A Survey2019; vol. abs/1906.0.\n\nTian J, Qu C, Xu W, et al.: KinWrite: Handwriting-Based Authentication Using Kinect. NDSS’13 2013; 93.\n\nJeon JH, Oh BS, Toh KA: A System for Hand Gesture based Signature Recognition Proceedings of 2012 12th International Conference on Control, Automation, Robotics and Vision, ICARCV 2012 2012; pp. 171–175. Publisher Full Text\n\nFang Y, Kang W, Wu Q, et al.: A Novel Video-based System for In-air Signature Verification. Comput. Electr. Eng. 2017; 57: 1–14. Publisher Full Text\n\nMalik J, Elhayek A, Ahmed S, et al.: 3DAirSig: A Framework for Enabling In-Air Signatures using a Multi-modal Depth Sensor. Sensors (Switzerland) 2018; 18(11): 1–16. PubMed Abstract \| Publisher Full Text\n\nKhoh WH, Pang YH, Teoh AJ: In-air Hand Gesture Signature Recognition System Based on 3-Dimensional Imagery. Multimed. Tools Appl. 2019; 78(6): 6913–6937. Publisher Full Text\n\nKhoh WH, Pang YH, Ooi SY, et al.: Spatiotemporal Spectral Histogramming Analysis in Hand Gesture Signature Recognition. J. Intell. Fuzzy Syst. 2021; 40(3): 4275–4286. Publisher Full Text\n\nDavis JW, Bobick AF: The representation and recognition of human movement using temporal templates. Proc. IEEE Comput. Soc. Conf. Comput. Vis. Pattern Recognit. 1997; 23(402): 928–934. Publisher Full Text\n\nAhad MAR, Tan JK, Kim H, et al.: Motion History Image: Its Variants and Applications. Mach. Vis. Appl. 2012; 23(2): 255–281. Publisher Full Text\n\nDalal N, Triggs B: Histograms of Oriented Gradients for Human Detection. Proceedings - 2005 IEEE Computer Society Conference on Computer Vision and Pattern Recognition, CVPR 2005 2005; vol. I: pp. 886–893. Publisher Full Text\n\nKannala J, Rahtu E: BSIF: Binarized Statistical Image Features. 21st Int. Conf. Pattern Recognit. 2012 no. Icpr: pp. 1363–1366. Publisher Full Text\n\nChang C, Lin C: LIBSVM: A Library for Support Vector Machines. ACM Trans. Intell. Syst. Technol. 2013; 2(3): 1–27. Publisher Full Text" }	[ { "id": "126550", "date": "30 Mar 2022", "name": "Lillian Yee Kiaw Wang", "expertise": [ "Reviewer Expertise IoT", "Education", "Technology Acceptance", "Data Analytics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 discussed how a self-collected iHGS database is built. The acquisition protocol is clearly described. The effectiveness of classification capability and robustness against forgery attacks are also discussed.\n\nSome points for your consideration:\nSome of the threshold values stated in the article should be supported by citation or justification on why that particular value is selected. For example:\n\"Thus, to ensure validation, the collected samples that have an fps rate less than 27 are removed and re-captured again\" Needs citation or justification, why 27?\n\n\"The optimal hyperparameters for the polynomial kernel are tuned empirically such that the gamma (γ) is set to 20, the degree of the polynomial (d) is set to 2 and the cost (C) is set to 1.\" Needs citation or justification, why the parameters were set as such?\n\nThe discussion of results for classification performance analysis is quite brief. Suggest to elaborate further on its impacts, for example, easy learning process and prediction stability -- easy or stable in what way? Also, apparent significance of your results compared to previous research works can be discussed in detail.\n\nSimilar comments for the discussion of results for robustness performance analysis (refer point 2).\n\nMore update-to-date references would be good, particularly references to support the discussions of result should be of recent research works.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9603", "date": "26 May 2023", "name": "Wee How Khoh", "role": "Author Response", "response": "We would like to express our sincerest gratitude to you and to all the reviewers for their constructive comments on our manuscript entitled “In-air Hand Gesture Signature Recognition: An iHGS Database Acquisition Protocol” and thanks for the opportunity to revise the paper. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses below satisfactorily address all the issues and concerns you and the reviewers have noted. 1. \"Some of the threshold values stated in the article should be supported by citation or justification on why that particular value is selected. For example: \"Thus, to ensure validation, the collected samples that have an fps rate less than 27 are removed and re-captured again\" Needs citation or justification, why 27? \"The optimal hyperparameters for the polynomial kernel are tuned empirically such that the gamma (γ) is set to 20, the degree of the polynomial (d) is set to 2 and the cost (C) is set to 1.\" Needs citation or justification, why the parameters were set as such?\" Response: Thank you for the comment. We removed signature samples with a frame-per-second (fps) rate of less than 27 out of 30 in order to maintain the quality of the collected database. This decision was made after careful consideration to ensure that the database is valid and not affected by hardware constraints. For the second point, the hyperparameters of the kernel were determined through empirical testing, and the settings we used were found which yielded optimal and stable performances across multiple experiments. Justification has been added to the manuscript. Revised texts: The hyperparameters for the polynomial kernel are tuned as such that the gamma (γ) is set to 20, the degree of the polynomial (d) is set to 2 and the cost (C) is set to 1. These hyperparameters were determined through empirical testing, and the settings that proposed yielded optimal and stable performance across our multiple experiments were used. 2. \"The discussion of results for classification performance analysis is quite brief. Suggest to elaborate further on its impacts, for example, easy learning process and prediction stability -- easy or stable in what way? Also, apparent significance of your results compared to previous research works can be discussed in detail.\" Response: To resolve the confusion of the use of the “easy learning process”, we have rephrased the paragraph. This paper mainly focuses on the acquisition protocol adapted to record and collect the iHGS samples. A detailed procedure and steps of the acquisition protocol have been outlined. After thorough consideration, we decided not to include the comparison of previous research works as it has already been included in our previous research paper. Revised texts: The results found that certain vector-based features such as VBSIF-XY and VBSIF-MHIXY, possess high levels of discriminative information for classifying in-air hand gesture signatures. Compared to other methods that involve complex preprocessing, the proposed vector-based features are extracted directly from the raw data, without the need for sophisticated techniques. These features can be used directly for classification model training, such as the SVM model, making it more convenient for real-world applications.Furthermore, the small value of standard deviation associated with these features suggests a high degree of stability in predicting hand gesture signatures. This is important in any classification task, as it ensures that the classification algorithm produces consistent and reliable results across a range of input data. The stability of these features is especially valuable in applications where the quality and consistency of the input data may vary. In summary, our findings demonstrate that vector-based features, particularly VBSIF-XY and VBSIF-MHIXY, offer a robust and reliable approach to iHGS classification. These features are easy to use and require minimal preprocessing, making them ideal for real-world applications that require efficient and accurate classification algorithms. 3. \"Similar comments for the discussion of results for robustness performance analysis (refer point 2).\" Response: Thank you for your feedback. We have revised the discussion of results for robustness performance analysis. Revised texts: Distinguishing skilled forgery attacks from genuine signatures is undeniably more challenging than detecting random forgery attacks, due to the high similarity between the forgery and genuine samples. Consequently, the Equal Error Rates (EERs) for skilled forgery attacks are expected to be higher than for random forgery attacks. Our study found that the vector-based features V XY and V HOG, when adopted with the cosine distance metric, achieved the best EER-S of 5.07% for skilled forgery attacks. This is a promising result, and it proves that these features can be effective in distinguishing skilled forgeries from genuine signatures. V BSIF-MHIXY with the Euclidean distance metric, obtained an EER-S of 9.45%, which is also a relatively good result. On the other hand, most BSIF features were found to perform poorly in verifying skilled forged hand gesture signatures, highlighting the importance of carefully selecting the features used for authentication. Similar to random forgery attacks, the Manhattan distance metric achieved the worst performance. Again, it indicates that the selection of the right distance metric is crucial for achieving good verification performance. In summary, these findings demonstrate that the verification performance of iHGS is not solely determined by the extracted features but is also highly dependent on the choice of distance metric. Therefore, careful consideration must be given to both factors in verifying the iHGS. 4. \"More update-to-date references would be good, particularly references to support the discussions of result should be of recent research works.\" Response: Thank you for your comment. This paper focuses on the acquisition protocol of iHGS, which has not been widely researched in recent years, especially regarding the methodology of collecting and establishing the benchmark dataset. Therefore, we have limited our review to similar works related to hand gesture signatures and have incorporated the latest related research into the paper." } ] }, { "id": "126552", "date": "12 Dec 2022", "name": "Cheng-Yaw Low", "expertise": [ "Reviewer Expertise Biometrics", "Pattern Recognition." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 contributes a small-scale in-air hand gesture signature dataset to the research community.\nHowever, this manuscript is overly simplified.\n\nMy concerns are as follows:\nThe authors should compare the new dataset to other repositories, e.g. the ones employed in Refs. 11, 12, etc. Despite of being private, these existing datasets should be at least introduced in the manuscript. Moreover, the main reason why this newly constructed dataset is important to iHGS should further be underlined.\n\nI think some relevant illustrations should be provided?\n\nSome parts in the Method section are not clear, e.g., the data preprocessing section, the feature dimensions for V_HoG and V_BSIF are not disclosed, etc.\n\nThe following statement is problematic? \"The data distribution is randomized in five different trials using a polynomial kernel.\"\nTherefore, a revision is needed prior to indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "9604", "date": "26 May 2023", "name": "Wee How Khoh", "role": "Author Response", "response": "We would like to express our sincerest gratitude to you and to all the reviewers for their constructive comments on our manuscript entitled “In-air Hand Gesture Signature Recognition: An iHGS Database Acquisition Protocol” and thanks for the opportunity to revise the paper. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses below satisfactorily address all the issues and concerns you and the reviewers have noted. 1. \"The authors should compare the new dataset to other repositories, e.g. the ones employed in Refs. 11, 12, etc. Despite of being private, these existing datasets should be at least introduced in the manuscript. Moreover, the main reason why this newly constructed dataset is important to iHGS should further be underlined.\" Response: Thank you for your valuable feedback. We appreciate your comment in our manuscript and would like to clarify that the datasets mentioned in Refs. 11 and 12 are identical to ours in this manuscript. It's important to note that, to date, there are no publicly available in-air hand gesture signature datasets, which makes it challenging to compare our dataset to them. Our manuscript aims to address this gap in the literature by presenting the acquisition protocol used to build the dataset and share the dataset with the public. We hope that this will contribute to further research in this area. Thank you once again for your feedback. 2. \"I think some relevant illustrations should be provided?\" Response: Thank you for your comment. We have updated the manuscript accordingly. Figure 1 and Figure 2 have been included in the revised version to illustrate the iHGS sample acquisition process from both top and side views. We hope these figures will help clarify the methodology and improve the manuscript's overall readability. 3. \"Some parts in the Method section are not clear, e.g., the data preprocessing section, the feature dimensions for V_HoG and V_BSIF are not disclosed, etc.\" Response: We agree that providing more details can enhance the understanding of our research. However, due to the page constraint of the current manuscript, we were unable to include all the necessary information and details. To address this, we have added a statement to the manuscript (in the section “Method”), directing readers to our previous research paper Ref. 12, 13 and 14, where a more comprehensive description of the methodology can be found. 4. \"The following statement is problematic? \"The data distribution is randomized in five different trials using a polynomial kernel.\"\" Response: Thank you for your comment. We apologize for any confusion and misleading in the statement. We have revised the statement as follows: Revised texts: A polynomial kernel of the SVM classifier is utilized as part of our machine learning model. The samples were randomly partitioned into training, validation and testing subsets to evaluate the model’s performance. For cross-validation purposes, we repeated this random partitioning process five times using five different subsets." } ] } ]	1	https://f1000research.com/articles/11-283
https://f1000research.com/articles/12-455/v1	02 May 23	{ "type": "Case Study", "title": "Parks’ sustainability as a method to revive historical parks in Egypt", "authors": [ "Demiana Aiad Ragheb Meshraky", "Adel samy El-menchawy", "Hamad Hassan Moustafa", "Adel samy El-menchawy", "Hamad Hassan Moustafa" ], "abstract": "The role of urban development is to restore the ecological system by making parks more efficient and sustainable. The sustainability of historical parks is concerned with more than just conservation; as it contributes to the pillars of sustainability as well as restoring nature conservation areas. Furthermore, historic parks offer demands regarding how to fulfill modern visitors’ needs, since these needs have not been provided in the previous era, and these parks are frequently susceptible to deterioration. This research aims to apply potential strategies to revive abandoned historical parks in Alexandria, which are considered of a significant value to national and international heritage assets, due to their influence on the residents’ socio-cultural heritage. This aim is achieved through two main dimensions. The first one describes the development of restoring historical parks throughout the ages, identifies parks’ laws and charters, the investigation of parks, the reasons for parks' deterioration in Egypt, and the requirements for sustainable parks. The second one is an application of the strategic criteria to different international parks to measure the parks’ sustainability. This leads to an analysis of how these strategies can be implemented at Antoniadis Park. The research concludes that historical park restoration facilitates eco-friendly development by recognizing the sustainability pillars and their intersection.", "keywords": [ "Eco-friendly Development", "Historical Parks", "Sustainability", "Sustainable Development", "Sustainable Parks" ], "content": "Introduction\n\nHistoric parks and gardens can be found frequently in metropolitan settings. Cities that preserve their cultural history, including old parks and gardens, are more inclusive, diversified, and sustainable. International accords, charters, and standards that have also been included in the national legislation of many nations govern the preservation of historic gardens and parks. Historical gardens result from the efforts of individuals from many backgrounds, including royals, noblemen, gardeners, and lay people. Because of this, these gardens' role in urban life differs across the globe, making it challenging to generalize the interpretations of their design. Their conservation is therefore protected by ongoing observation and the regular assessments of their components. The conditions, opportunities, and challenges in the current socio-economic, environmental, and political context of metropolitan regions have created the consideration of cultural heritage conservation with sustainable development and inspired methods of heritage conservation.1–5\n\nIn historic parks, the participation of visitors in the management of a park can increase awareness and encourage conservation. It also fosters meaningful interaction between park administrators and visitors. The relevance of green space visitor use has increased due to modern trends in sustainability maintenance and resilient urban landscape development, which has led to resilient communities. Where existing, historical public parks can form significant green spaces in the urban fabric.5,6 A good relationship between park elements, including natural elements, places with specific uses, significant structures, small structures, guests, and animals, is considered in park design. Numerous classic public parks across the world started as private gardens. Many sizable historical gardens were converted into public parks while preserving essential design elements by adapting to the needs of society. Converting these private gardens into public parks reflects the historical and cultural advancements of time while preserving the unique landscape character. As a result, historical parks are uniquely defined by their past, which has to be preserved for present and future generations to retain a cultural connection with visitors, that might be lost and forgotten.7\n\n\nMethods\n\nThe research aim can be summarized as an examination of the appropriate criteria to meet sustainability requirements. The methodology adopted is the qualitative approach through a literature review, analytical examples, and a case study. These guide the study to carry out interpretivism research as described below.\n\nFirst, the literature review investigates the development of parks and green areas throughout the ages and the laws created for their development. Second, the Oregon Parks and Recreation Department (OPRD)’s system is examined to understand the criteria for the sustainability of parks and open spaces. The OPRD can guide the landscape design of urban parks to meet the sustainability requirements in terms of environmental, economic and social sustainability by determining specific sustainable indicators. Park examples are analyzed using the OPRD criteria and the strategic framework is developed and evaluated. Then, the framework is applied to the case study of Antoniadis Park in Alexandria, which is further compared to the park examples. Finally, this study concludes with a proposal for Antoniadis Park’s future restoration, which elaborates the design criteria for successful sustainable parks serving local urban environments and enhancing their quality.\n\nAncient Egypt's gardens started as simple fruit orchards and vegetable gardens. They appeared in Egypt just before the Middle Empire (2035–1668). As the nation became wealthier, they gradually became leisure gardens with flowers, ponds, and valleys of fruit and shade trees. Gardens were a common feature of temples, palaces, and private homes, and replicas of gardens were occasionally buried with their owners so they may enjoy them in the afterlife. The gardens' primary function is to adorn the temples; they have a regular geometric pattern and are centered on straight lines. On the axis is a fountain, and among them shrubs that are constantly sprayed consisting of jasmine and daffodils. Rows of sycamore and fig trees surround the fountain, followed by palm trees. A regular distribution of deity statues is made, and a wall surrounds the garden's exterior.8,9 Ashourian and Babylonian Gardens were first created in the Fertile Crescent, where they were regarded as a paradise with the only purpose of enjoyment, rather than food production. As a result, the concept spread throughout the ancient Mediterranean, and by the Hellenistic era, people had cared for their private gardens inside their homes. Gardens were not just about flowers and plants; they also had architectural, sculptural, and aquatic aspects, and ancient landscape gardeners took the surrounding views into account. Therefore, Mesopotamia in the distant past and, especially the spectacular Hanging Gardens of Babylon, are responsible for creating these outdoor relaxing areas. Ashourian and Babylonian Gardens have a typical geometrical style whose primary purpose is to decorate the palaces of princes. The nature of the mountainous country influenced the formation of the gardens, and they were built on the level of the terraced terraces. The king's palace was occupied by the highest position, where he oversaw the entire garden. Below the terraces, streams flow from the water and are distributed around trees, such as walnut, pomegranate, and cedar trees.10\n\nPersian gardens may have originated as early as 4,000 BC. Their airy and delicate characteristics highlighted their natural opposition to the adverse surroundings. In addition to walls and pavilions that are structurally conspicuous in hiding the sun, trees and trellises play a significant role in providing natural shade. Water is crucial to the design and upkeep of the garden because of the heat. Water from a nearby aquifer is transported via a tunnel known as a qanat to facilitate irrigation, which may be necessary. The qanat is then connected to well-like constructions, allowing for water drawing. The Persian architectural style frequently tries to unite the indoors and outdoors by linking an exterior garden with an inside courtyard. Architects frequently insert architectural features, like vaulted arches, to bridge the gap between the external and internal spaces. Persian Gardens have a regular geometric pattern; they created water parks, wall gardens, and sunken gardens. The area of the garden is square or rectangular, divided by two perpendicular roads into four parts. The pond is found at the junction of the roads, and highly decorated walls surround the garden. The trees are carved on the carpets in different shapes.11–13 The Roman civilization has led to highly developed ornamental horticulture and gardens, which flourished between 150 BC and 350 AD. Roman Gardens have a regular geometric pattern. The gardens of the public parks began to appear after they had been limited to temples and palaces only. Monumental fountains and sculptured statues were found. Plants were cultivated in pottery vessels for ease of movement. The space around Roman houses was dedicated to the garden as well as painted walls with paint suited with the garden components.14 The Italian Renaissance Garden is a new type of garden that first appeared in the late 15th-century villas in Rome and Florence. It was designed with the garden views and the surrounding landscape in mind and for contemplating its sights, sounds, and scents. Its inspiration came from classical ideals of order and beauty. Italian Gardens became enormous and more symmetrical, with many statues and water elements. The spread of engineering works related to water, especially in highly steep areas, as well as rare bird cages, spread in the Gardens of the Kings.15\n\nIn the classic, 18th century, Early English Garden, a new landscape garden appeared in England and quickly spread over the continent. It was an ideological representation of nature in an English garden. It is believed that this primitive English garden included symmetrical gravel walkways, carefully-planted short hedges, a park-like open lawn space, and a small kitchen garden with herbs and vegetables. The gardens have a natural style. Lakes take their natural shape, and bridges are built on them with overlapping trees. This garden is considered a painted artboard.16 Japanese Gardens are traditional gardens whose designs are accompanied by Japanese aesthetics and philosophical ideas. This is carried out by avoiding artificial ornamentation and highlighting the natural landscape. Japanese garden designers frequently employ plants and weathered, aged objects to evoke a natural scene and convey the frailty of life and the unrelenting march of time. Previous garden designers were inspired by ancient Japanese art. Many gardens require rocks and gravel as well as water. Although there are many beautiful Japanese flowering plants, herbaceous flowers typically play a much smaller role in Japanese gardens than they do in Western gardens. However, seasonally-flowering shrubs and trees-flowering shrubs and trees are crucial and contrast with the usual predominant green, which make these gardens all the more dramatic. In Japan, evergreen plants are known as “the bones of the garden”.17,18\n\nThe Venice charter, 1964 was the first document drafted internationally to clarify the principles of preserving historical sites. Its most important aspect is developing the concept of a ‘historical monument’ limited to a single architectural work to include all historical areas that reflect a specific historical event and the restoration definition. The document’s purpose is to avoid distracting historical works and complete the work of exploration, and make an accurate documentation of all the preservation steps.19 Florence Charter, the first charter published in 1982 by the International ICOMOS-IFLA, refers specifically to historic gardens and their preservation, and refers to them as “living monuments”. More specifically, according to the Florence Charter, a historic garden is a historically or artistically significant architectural and horticultural combination, and the word ‘historic garden’ applies to tiny gardens and large parks, whether formal or landscape.20 The historical gardens' current use incorporates the materialized aesthetic, ecological, and social qualities of the past.21 Before conservation, studies must include plans and all kinds of different plants. All sculptures should not move from place to place, and priority should be given to restoration work after public use. Once saved, they should be followed up and maintained, and awareness of the importance of historic gardens should be made.20,22 The Charter of New Zealand 1992 emphasizes the purpose of preservation; i.e., taking care of places of historical value and identifying conservation and maintenance methods.23 Conference of Athens 1993 makes aesthetic changes to ancient gardens and advises preserving old buildings using modern methods without change.24\n\nThere are five main reasons for the deterioration of parks in Egypt: planning, social, legal, environmental and economic reasons. Many parks have disappeared due to urban encroachment and population explosion, such as Ibrahim Pasha Park on Al-Rawdah Island. This park consisted of two independent sections, one planned in the English style of free layout and the other in the French style with geometric coordination. These reasons are a lack of full planning on all the city's territory and planning thought towards centralization in providing services and dealing with green areas as little spaces. Social reasons include the absence of a sense of beauty and the prevalence of illiteracy in the people's behavior in their dealings with the parks, the unawareness of the responsible authorities, and the lack of educational curricula on how to preserve plants. The legal reasons are concerned with the articles of the law which are limited to interest in archaeological elements and installations without reference to historical parks and their inclusion in protection and restoration works. Environmental reasons include a lack of interest in plants, which led to the disappearance of rare trees imported from their original countries. Finally, the economic reasons have to do with not allocating a reasonable budget for the maintenance of parks. In addition, their presence in the city center made people greedy to exploit them to make money.25,26\n\nParks offer a variety of environmental, social, and economic advantages, which significantly contribute to sustainable city plans and provide a more comfortable setting for the environment than the parks surroundings. Cooler ambient temperatures may aid in reducing pollutant emissions since ambient temperatures are typically linked to pollutant emissions. Parks that mimic forest conditions can potentially improve carbon storage over time as the net quantity of carbon can accumulate in the soil along with the carbon stored in trees. Also, parks help manage storm water, focusing on higher water runoff rates in green urban areas than in other urban land uses. Parks can significantly reduce environmental health concerns associated with urban life and noise levels.27–30 Parks positively impact physical and mental health as social advantages. They promote and improve physical activity, lessen stress, anxiety and depression, and enhance self-respect, cognitive abilities and constructive social behaviors.31–37 Parks are regarded as an essential component of community growth, rather than merely a place for leisure and relaxation. People from different social and racial backgrounds may be able to connect and interact in an urban park setting.38,39\n\nAdditionally, parks are a teaching tool. Environmental education improves citizens' perceptions, knowledge and behavior to build resilient environments and communities. Urban parks are great places to get hands-on education. By fostering a child's connection to nature through nature-based techniques, future generations will be encouraged and inspired, having gained knowledge and skills through environmental conservation activities. Parks provide a city with economic advantages by enhancing its appeal and making it a popular tourist destination, which generates income and jobs. Urban parks also boost tax income and increase the value of nearby real estate developments.40,41 One of the best methods for resolving environmental issues in metropolitan areas is the sustainable design of parks. Sustainable Park design is the long-term upkeep of diverse ecosystems to ensure that they continue to play various roles in the future. In practice, this should entail adapting to local conditions, utilizing natural processes, continuing park monitoring, and knowledge of recreation management.42\n\nSeveral strategies may be followed for planning a sustainable public park. Social sustainability is essential for community engagement and shared social activity. All people's basic requirements must be satisfied for a community to have a high quality of life and be sustainable. A socially-viable community must be able to construct and maintain park facilities that cater to locals of all ages, abilities and socio-economic backgrounds. Practices for social sustainability involve reaching out to the community's needs, including those of marginalized communities. All community members should be involved in sustainability, not only those who can afford to pay for their own needs.43–46 When discussing economic sustainability in the parks and recreation sector, less emphasis should be placed on deficits, the national debt, and debt-to-gross domestic product (DPG) ratios. Instead, this pillar should be primarily about ensuring a government agency's ability to continue carrying out its public functions while maintaining public assets. Government expenditure and its effects as a whole, including that of the private sector and the local community, are essential factors in economic sustainability. Governmental organizations' public purposes include generating positive value, reducing some adverse effects, and avoiding all adverse effects that are wholly intolerable. Keeping parks and recreation organizations financially-viable prevents unacceptably bad outcomes and ensures the continuation of the agency's public purposes.47,48 Environmental sustainability encourages the ethical and practical use of resources to benefit communities on the long run. It requires slowing down the extraction of renewable resources, pollution, and non-renewable resource depletion to a sustainable level over the long term. A sustainable park should work with the ecosystem and its processes rather than on its own. Communities that surround and depend on these ecosystems gain immediate advantages from healthy ecosystems. Examples include creating clean water, fresh air, groundwater recharge, food, and view sheds.49–51\n\nThe Oregon Parks and Recreation Department (OPRD) is a prominent proponent of environment-friendly parks. The main objectives of sustainable parks are to encourage environment-friendly behavior, extend the useful lives of park structures and infrastructure, and improve the surrounding environment. According to OPRD, Table 1 lists the sustainable (social, economic, and environmental) strategies employed in parks, recreation centers, and open spaces.52\n\nPark examples are selected based on the following criteria to ensure fair comparison and accurate results for the proposal of strategic framework. 1) Geographical location of parks: all the parks are located in the city center and all of them should be located near the city center. 2) Cultural characteristics: the parks have to be categorized as historical parks. 3) Institutional approach: as all selected parks share the same strategy as they all rely on the public use.\n\nFor more than 150 years, Central Park has been the centerpiece of New York City. It is one of America's most important works of art, built between 1858 and 1873. The park is located in Manhattan, in the New York state. Its area is 341ha [843 acres], bounded by Fifth Avenue on the east, 59th Street on the south, and 110th Street on the north. There are 20 gates, and 18 have the same original design. In addition, there are 36 bridges and arches with rural characters. The most famous bridge is the Bow Bridge which crosses over the central park's lake and is linked between the Ramble and Cherry Hill. Cherry Hill is the third-highest area in the park. The Shakespeare Garden was named to recognize the memory of the prominent playwright passing, and includes all kinds of plants mentioned in his writings.53,54\n\nFrom 1858 to 1960, the park faced massive deterioration throughout the century. In 1934, Robert Moss was repairing bridges and collecting donations from people, but he resigned from his job as an official. After that, the park deteriorated again. In 1980, Central Park Conservatory was established to attract volunteers. The Conservatory have developed documents for restoring archaeological sites and replanting tree areas.54,55\n\nIn Table 2, the Central Park Conservation Authority's most significant achievements are reviewed since the Authority has been established till present to preserve its original image on the one hand and improve and develop it on the other hand. The restoration and preservation plan were based on the main objective of the modern field lift for each of the park's topography while studying security aspects and users' needs. Those achievements can be divided into the three pillars of sustainability: social, economic, and environmental.53,56\n\nThere were many sources of funding for the restoration and maintenance of the park. These sources were divided into Government funding by the New York City and Manhattan Park Office, and Individual funding by Alexander Papa Markus who donated half a million dollars to repair the water basin. Also, through the Uris Brothers Foundation, private companies donated $10 million to develop and renovate service centers and $2.5 million to restore the water reservoir's landscape. Mark Haas Foundation donated materially to replace the old irrigation system in the Sheep Meadow.54,57\n\nHandsworth Park was founded in June 1888 by the Handsworth Local Sanitary Board—a body instituted by the government. At that time, the park was less than half its current size. The site is crossed from north to south by the SoHo, Handsworth, and Perry Barr railways. Its area is 25 ha [63 acres]. The site is crossed from north to south by the So-Ho, Handsworth and Perry Barr railways. It lies 15 minutes by bus from the center of Birmingham.58,59\n\nIn the 1940s, the park suffered from the war, as many gates and metal railings were removed. The number of visitors and employees decreased due to the decrease in financial support. In the late 1980s, the park became poor because of the spread of criminal acts and a lack of management. No security system was installed on the gates, so the park became derelict. From the 1980s until now, a group of volunteers called ‘Friends of Handsworth Park’ has improved Handsworth Park. The group’s main goals are preserving the park in favor of the community, raising continuous awareness of the park uses, and the restoration of its historic character.58,60\n\nThe end of a protracted period of neglect of numerous parks was formally marked when Birmingham City Council released its Parks Strategy paper. The strategy guarantees that a network of high-quality parks and other green spaces are provided for Birmingham's residents and tourists. The paper states at the beginning of a lengthy list of objectives.61 The overall social, economic, and environmental quality of urban park regeneration and the allure of neighborhoods influenced by high-quality open space (see Table 3). The redevelopment of parks and open spaces, both new and renovated, has proven to be a particularly effective strategy for revitalizing inner cities. The City Council created park regeneration schemes and funding packages capable of achieving broader urban regeneration goals and objectives regarding environmental improvement, community safety, community use/involvement, and education. The City Council strives to ensure that parks are integral to social re-generation strategies.62 There have been many sources of funding for the restoration and maintenance of the park. These sources are divided into government funding through The Birmingham Park Office, individual funding through collecting donations from the local population, and private funding of about 9.5 million for park restoration led by Consultant Mike Sharpe.62,63\n\nStrong examples of the ambition and complexity of modern landscape design may be found in Mexico City's Xochimilco Ecological Park and the larger landscape it is a part of. The park has won multiple urban design and park planning accolades, including the 1994 ASLA (American Society of Landscape Architects) Merit Award, the Veronica Green Prize in Urban Design, and the Waterfront Center Design Award, among others. Proponents of ecological parks mention it as a tangible model from which to learn. That is not to assume that everyone agrees on the park's natural biological significance. Others, however, contend that the intended purpose is more important than the actual state of the natural environment. Xochimilco is nearly about 12,700 hectares [31,382 acres]; 7,900 hectares [19,521 acres] of them are dedicated to agriculture and forest land, 1200 hectares [2965 acres] are full of canals and lagoons, and 3,600 hectares [8,895 acres] are urbanized.64\n\nThe Xochimilco Ecological Park contains the largest plant market in Latin America, totalling 13 hectares (32 acres). The park and market are located in the southern Mexico City neighborhood of Xochimilco, about 23 km south of the city's historic center. Mario Schjetnan created the park, debuting in 1993 on artificial lake islands known as ‘chinampas’ that were formerly listed as a part of a World Heritage Site.65\n\nThe parkland had been owned privately until 1988. After that, however, the region suffered severe degradation, with lakes and canals drying up and the remaining water becoming salinized and polluted, making farming impossible. The Buenaventura River, which brought contaminated water from other rivers in the city's south, was the primary source of the contamination. As a result, these chinampa farmers could no longer cultivate any food plants on their farms by the 1980s.65\n\nThe local and federal governments created the Xochimilco Park Rescue Plan with the park as its focal point to rescue Mexico City's last chinampas. The project's objective was to recreate the Chinampa ecosystem in the manner it existed under the Aztec Empire to conserve it. The area was refilled with purified water to revitalize and create most surface water today. Biologists and botanists brought in local greenery. Much of the sanitary waste from the earthquake in 1985 and soils excavated from the Metro's construction were brought in to replenish the soils.65\n\nIn 1993, the park finally welcomed visitors after five years of construction. It already had 240,000 trees and 500,000 different types of plants over 210 hectares eight years after it had been founded. However, the proprietors of the chinampa battled the land's expropriation for many years after it had happened. Eight years after it first opened, in 2001, the park saw its worst period. Despite significant investments and attempts at restoration, there were still significant contamination issues, and the courts returned several ejido areas that had been taken over in the 1980s. Numerous neighborhood organizations criticized the project as a failure due to the setback, and demanded that former Mexico City Mayor, Andrés Manuel López Obrador, launch an investigation. Table 4 shows the urban park regeneration's overall social, economic, and environmental quality.64,65\n\nAlexandria’s site on the Mediterranean Sea has made it the home of civilizations. It was founded by Alexander the Great in 331 BC and was planned by the architect Democrats. After Alexander's death, the city was divided into neighborhoods. Alexandria was known for its historical parks, such as Antoniadis Park, Al Shallalat Gardens, and Zoological Gardens, which suffer from extreme neglect.66 There are three main parks in Alexandria (see Figure 1). The exported Figure from Google Earth Pro (OpenStreetMap is a free alternative) to draw Alexandria parks’ Map by editing with Adobe Photoshop (GIMP and Krita are free alternatives).\n\nThis figure is from Google Earth Pro with the authors’ edits.\n\nThe specific choice of Antoniadis Park is made because it is one of the most important parks in Alexandria, founded in the Ptolemaic era of 300 BC. In the 19th century, it was known as the Pasteur Gardens and was owned by a wealthy individual. Then, Muhammad Ali Pasha owned it and built the palace. In 1860, he became King of Antoniadis and asked the French artist Paul Richard to set up a garden as a miniature model of the Versailles Gardens in Paris. In 1918, after his death, his son dedicated this garden to the city of Alexandria, carrying out his father's commandment. The park experienced important events. In 1936, when the Evacuation Agreement was signed, a tree was planted at the palace entrance to commemorate this anniversary. The palace witnessed the preparatory meeting to establish the League of Arab States in 1944. The park's palace hosted kings from various countries, including the King of Albania and his wife. It hosted the daughter of King Fouad I and her husband (Emperor Mohammad Reda) for months.67,68 The park is located in the southern part of Alexandria, bordered by the south and east Shuhada Street, the north of Albert I Street, and the northwest of Mustafa Kamel Street. Its area is 45 acres and there are five gardens.68 Figure 2 shows the relationship between Antoniadis and the surrounding areas. Figure 3 shows the site zoning of Antoniadis Park. Figure 4 shows the components of Antoniadis Park. The three exported Figures from Google Earth Pro to draw the Antoniadis Park Map by editing with Adobe Photoshop.\n\nThis figure is from Google Earth Pro with the authors’ edits.\n\nThis figure is from Google Earth Pro with the authors’ edits.\n\nThis figure is from Google Earth Pro with the authors’ edits.\n\nFlowers Garden was founded by the French engineer Dayton, and supervised by Engineer Richard. It is located on 2 ha [5 acres], designed on the steps of four levels. Each level includes eight basins topped by four symmetrical triangles in shape and an area surrounded by wooden pergolas outside. The finest sunken gardens are finished at the lowest level with a fountain in the center and a statue. Royal Glass is located in the western part. It is built on the body of the occupied ironwork, consisting of several parts, two parts at the front and the others behind, with a square shape and several levels. All sides and roofs are made of glass. The French and English Garden is characterized by the straight lines in the streets, fences, and decorative vases. The trees are trimmed in geometric shapes (conical, pyramidal, square, rectangular and circular). Individual plants are distributed symmetrically, placing ponds of flowers, fountains, or statues as a central element. High walls are not found so as not to block parts of the garden from each other. The Andalusian Garden is characterized by architecture in the al-Arab style, characterized by saving. High walls surround the Andalusian Garden to block the interior view. It has waterfalls, artificial water, fountains, and mosaics beautifying the vases. Roman and Greek styles are characterized by architecture and sculpture in the art of coordinating plants and frequently using vases and fountains amid the green areas. There are seven fountains distributed throughout the garden. The natural style garden has curved lines in which straight lines are used, but without statues, as it simulates nature without decoration.\n\nTrees and shrubs are planted in close or divergent forms without trimming to grow tall in their nature. Pergolas and seats are made of natural wood. The Italian Garden is dominated by architectural art, designed from several different levels, and uses plants with geometric forms, using statues to complement elements of geometric designs. It contains a collection of 17 marble statues representing the legends of the Greek goddess, such as the beauty of Venus and the god of Aratus. Figure 5 shows a collection of world-historical characters' statues. Antoniadis Palace entrance is decorated with two lions made of pure alabaster, and its south side overlooks the tea garden. The palace consists of two floors containing many rooms.68\n\nThis figure is the authors’ own work.\n\nThe lower floor was designated as a reception and dining hall during King Fouad's reign. The upstairs has been divided into two sleeping suites. The first suite includes the King's room and another one for the Queen. It also consists of four rooms for the princesses through which a statue of Venus can be seen, and four marble statues decorate the front elevation. Antoniadis Garden includes an extensive range of palm and semi-palm trees, such as Cycas, and a large group of trees, shrubs, climbers, and perennial plants, such as Bulgarian Fig trees, about 500 years old. Antoniadis Theatre is 6,000 m2 [64,583 Square feet]. It has a green area, rich in plants and rare trees, of 15,000 m2 [161,458 Sq. Ft] overlooked by the palace's terrace and various levels' gardens. The theater has a back of Roman columns, and it can accommodate more than 5,000 spectators.67,69\n\nThe Antoniadis Garden represents an important heritage and recreational value, especially in light of the erosion of green spaces in Alexandria, as it represents an entertainment outlet in addition to the zoo adjacent to it, and that the planning of the garden represents a heritage and not only the statues only aesthetic, as the style of the garden in itself is a heritage that extends hundreds of years And inside Antoniadis, there are a variety of different gardens, such as the rose garden, the statue garden, and the palace garden.67\n\nThe garden has been neglected and deteriorated greatly, as it lacks the most basic services and does not have toilets. The number of heritage statues in the garden was 17 statues belonging to the Greek god of love, which were reduced after the theft over the past years to only 14. The garden was robbed more than once, where an accident occurred and a statue of club marble was stolen, and a similar theft occurred in the nineties and a statue was cut up and smuggled inside the garbage car, and that the Antoniadis Park was a public park that extended to 130 acres, and finally 50 acres, guarded by 12 security personnel On 3 shifts, and that the garden had previously addressed the director of the Basateen Institute a lot to increase the number of security personnel to 60 and arm them, especially since the garden was without fences, and that was to no avail. Its application, in addition to the lack of sufficient lighting at night.66 The garden contains 14 heritage statues, collected by Sir Antoniadis, the founder of the palace and garden, who loved collecting antiques and statues, and that the age of the statues is greater than the age of the garden, calling on the Ministry of Antiquities to consider reconsidering and including them among the antiquities. Addressing the deliberate neglect of the garden, of which the zoo was part and attached to the palace, due to the heritage importance of the garden. Random projects were set up inside the garden before the revolution, with the aim of improving the financial resource, but they contributed to the deterioration of the garden more and the garden is currently suffering from the deterioration of the state of green spaces and only trees remain, due to the exposure of the garden over the past years to administrative problems, weak financial resources and the lack of Sufficient labor to take care of the vast green areas of the garden.69\n\n\nResults\n\nSome essential features can be extracted from the example of Central Park, New York, which are:\n\n1. The park's image and condition before the establishment of the Conservation Authority fluctuated between prosperity sometimes and decline at others, according to the person in charge of its management.\n\n2. The pictures of the park's change before preservation represented the decline of maintenance and cleaning works, which was reflected in the state of its natural elements, like the erosion of grass surfaces and construction like painting on walls.\n\n3. Many of the park's places are associated with well-known historical figures who did successful works for the community, such as Shakespeare's Garden, reflecting the extent to which this community appreciates and respects all the symbols which are part of its history and identity.\n\n4. The presence of a special authority to preserve the park that works under the supervision of the administrative apparatus to which the park belongs helps in taking informed and unified decisions from the authority without conflict.\n\n5. The commission's exclusion of the profit goal from its work and its reliance on the efforts of volunteers from the city's residents contribute to saving a large part of the expenses of maintenance and cleaning work in the park. Some of the saved funds support conservation works in the park as funding.\n\n6. Development is sometimes required in historical elements as long as it meets the needs of users on the one hand and does not negatively affect the historical value of those elements on the other hand.\n\n7. The use of modern methods, such as computer programs, facilitates some tasks related to registration and follow-up of the park elements while implementing preservation, maintenance work, and monitoring of annual reports.\n\nSustainable Handsworth Park Birmingham intends to help the demand for restoring the U.K.'s battered ecosystems:\n\n1. The policy adopted in the park plan's guiding principle was sustainability. As a result, sustainability has been included in as park features as possible through demonstrations, such as wind and solar energy displays, to protect the environment into the twenty-first century.\n\n2. A habitat was established so many animals and plants could live and grow.\n\n3. In exciting surroundings with a wide variety of habitats and sustainability demonstrations, a wide selection of activities is accessible.\n\n4. The park created a program to improve sustainable procurement practices; including reducing waste, impacting the environment and climate, destroying no habitat, and not degrading any soil.\n\nThe sustainable Xochimilco Park summarizes some positive achievements, which are:\n\n1. Restoring the damaged hydrological cycle between the Mexico City region, the Chinampas Zone, and the park zone.\n\n2. Providing migratory birds with a winter refuge.\n\n3. Expanding activities in the park to encompass a more dynamic and valuable engagement with the city.\n\n4. Serving as the city's primary respiratory system.\n\n5. Establishing a solid link and linkage between historic preservation and the protection of natural resources.\n\n6. Creating a park program to improve the landscape's natural and cultural ecologies.\n\nBased on the analysis informed by the OPRD system, and the information illustrated in the park deterioration, it is discovered that Antoniadis Park does not achieve sustainability. There are shortcomings in the environmental, social, and economic criteria (see Table 5). In addition, Antoniadis suffers from several problems:\n\n1. The exploiters' accumulation within the garden leading to indebtedness of millions of pounds,\n\n2. Depreciated facilities (watercourses, stairs, fountains, corridors) and lack security,\n\n3. Apparent negligence in the valuable sculpture of Antoniadis Park,\n\n4. Accumulation of garbage exposing the park to fire hazards,\n\n5. Deteriorated state of the palace, including the Royal Greenhouse,\n\n6. Erosion of irrigation networks and agricultural equipment for the garden,\n\n7. Lack of fertilizers, pesticides, and production requirements for gardening,\n\n8. Tearing down of the park’s walls to bring in drug addicts,\n\n9. Delays in paying financial dues to garden workers, the Antoniadis Palace maintenance, and hence the suspension of all activities since 2004.\n\n\nDiscussion\n\nAntoniadis Park represents an essential aspect of the city's cultural legacy, and these public places hold layers of the city's history. The physical decline of the park is directly tied to the amount of money and focus that municipalities and investors are willing to devote to it. Increasing community contact with the park and the knowledge of the value of the spaces will depend on several circumstances, including the park's status being endangered by deterioration or encroachment.\n\nParks are essential components of the urban ecosystem, serving as both urban core areas and open, mixed-use recreational spaces. Furthermore, as a component of the open space system, it serves an important functional role as a component of the landscape habitat. Therefore, sustainable parks will be a critical component in transforming and improving our lives and cities if historical parks can transition from their current, primarily recreational, role to a new role as a catalyst for community development and enhancement.\n\nAs stated by the framework used in Table 5 and the historical overview, the study shows that Central Park, NYC, achieves all the criteria's sustainability except for the lack of waste recycling, which affects the environmental sustainability of Central Park. On the other hand, Handsworth Park, Birmingham, fulfills all the criteria for sustainability, but safety principles and funding sources are partially achieved. Furthermore, the Xochimilco Park in Mexico achieves all the criteria's sustainability while lacking funding resources, maintenance work and waste recycling. Finally, in Antoniadis Park, four criteria are fulfilled. Still, many sustainability criteria, such as social and environmental indicators, are not entirely achieved, and the others do not exist.\n\n\nConclusion\n\nParks are essential components of the urban environment. Furthermore, they serve crucial functional functions as a component of the landscape. Therefore, sustainable parks are vital in transforming and improving our lives and communities if urban parks can transition from their current, mostly recreational purpose into a new position as a catalyst for community development and enhancement. The goal of transforming Antoniadis Park into a sustainable park is to show how sustainability may help revive neglected parks and the environment and create an educational environment for park visitors by acting as an open classroom to increase awareness.\n\nBased on the analytical studies and the three pillars of sustainability, Table 6 shows the recommended criteria for achieving sustainability in Antoniadis Park, needed to make the parks in Egypt sustainable. In addition, there is another recommendation for funding sources. The budget allocated by the Ministry of Agriculture does not almost cover the cost of care for plants or any development work. Therefore, new funding sources must be considered to ensure the project's continuity, including self-financing in the form of contributions of associations, such as heritage preservation, business people donations, and activities carried out inside (recreational and cultural activities). Private funding can include tickets sold and small indoor projects, exhibition tickets for the palace, and providing booths to sell flowers to be used and returned to the garden.", "appendix": "Data availability\n\nMendeley: Comprehensive Database for Historical Parks.\n\nhttps://data.mendeley.com/datasets/8k6wpx8fvp/1 (Demiana, 2023)\n\nThis project contains the following data:\n\n• Figure 1. png: A figure shows the parks' map of Alexandria.\n\n• Figure 2. jpg: A figure shows the map of the Nouzha complex.\n\n• Figure 3. png: A figure shows the site zoning of Antoniadis Park.\n\n• Figure 4. png: A figure shows the components of Antoniadis Park.\n\n• Figure 5. png: A figure shows the collection of statues in Antoniadis Park.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgement\n\nThe authors want to thank Andrew N. Aziz, School of Engineering, University of Birmingham, Edgbaston, Birmingham, United Kingdom and the City of scientific research and technological applications and Yousab Magdy, a Teaching Assistant at the Arab academy for science, technology, and maritime transport, Alexandria – Egypt for being helpful and supportive throughout the process of this research.\n\n\nReferences\n\nPendlebury J: The Statutory Protection of Historic Parks and Gardens: An Exploration and Analysis of “Structure”, “decoration”, and “Character.”.1997; 2. Publisher Full Text\n\nCarneiro ARS, Menezes JF, Mesquita L: The conservation of historical gardens in a multidisciplinary context: the “Cactário da Madalena,” Recife, Brazil. City Time. 2005; 1(2): 25–35.\n\nFairchild Ruggles D, Silverman H: Intangible Heritage Embodied.2009. 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Heal Place. 2009; 15(2): 586–595. PubMed Abstract \| Publisher Full Text\n\nMoulay A, Ujang N: Legibility of neighborhood parks and its impact on social interaction in a planned residential area. Archnet-IJAR. 2016; 10(1): 184–194. Publisher Full Text\n\nWood L, Hooper P, Foster S, et al.: Public green spaces and positive mental health – investigating the relationship between access, quantity and types of parks and mental well-being. Heal Place. 2017; 48(September): 63–71. PubMed Abstract \| Publisher Full Text\n\nJennings V, Bamkole O: The relationship between social cohesion and urban green space: An avenue for health promotion. Int. J. Environ. Res. Public Health. 2019; 16(3). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTroy A, Grove JM: Property values, parks, and crime: A hedonic analysis in Baltimore, MD. Landsc. Urban Plan. 2008; 87(3): 233–245. Publisher Full Text\n\nRabare RS, Onyango GM: The Role of Urban Parks and Socio-Economic Development: Case Study of Kisumu Kenya. Theor. Empir. Res. Urban. Manag. 2009; 4(3): 22–36.\n\nHaq SMA: Urban Green Spaces and an Integrative Approach to Sustainable Environment. J. Environ. Prot (Irvine, Calif). 2011; 02(05): 601–608. Publisher Full Text\n\nChang CR, Li MH: Effects of urban parks on the local urban thermal environment. Urban For. Urban Green. 2014; 13(4): 672–681. Publisher Full Text\n\nYan H, Dong L: The impacts of land cover types on urban outdoor thermal environment: the case of Beijing. China. J. Environ. Heal Sci. Eng. 2015; 13(1): 43–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGallagher T: Developing Sustainable Park Systems in Oregon.2012;(June 2012); 49. Reference Source\n\nBawatneh S: Central Park, New York City. Sci. Am. 2018; 28(17): 263–264. Publisher Full Text\n\nCentral Park Conservancy: Central Park: A Research Guide.2016; 3–36. 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Thesis (M.C.P.) --Massachusetts Institute of Technology, Dept. of Urban Studies and Planning, 2006.2006. Reference Source\n\nMerlín-Uribe Y, González-Esquivel CE, Contreras-Hernández A, et al.: Environmental and socio-economic sustainability of chinampas (raised beds) in Xochimilco, Mexico City. Int. J. Agric. Sustain. 2013; 11(3): 216–233. Publisher Full Text\n\nHAMDY RS: A STUDY OF PLANT DISTRIBUTION IN NINE HISTORIC GARDENS IN EGYPT. Gard. Hist. 2010; 38(2): 267–314. Reference Source\n\nForster EM: Alexandria: A History and a Guide. 1922. Publish online 2018. Reference Source\n\nMansour H, Abou Dahab T, Ahmed A: STUDIES ON SOME CACTI AND SUCCULENTS, AND THEIR USE IN EGYPTIAN BOTANIC GARDENS. 1. EFFECT OF SALINITY LEVELS AND FERTILIZATION ON VEGETATIVE GROWTH AND LEAF ANATOMICAL Structure OF Agave sisalana PERRINE PLANTS. J. Product. Dev. 2007; 12(2): 367–383. Publisher Full Text\n\nGenady M: حدائق وقصر أنطونيادس بالأسكندرية.2019. Reference Source" }	[ { "id": "175151", "date": "30 May 2023", "name": "Mirza Fuady", "expertise": [ "Reviewer Expertise Architecture and Urban Design" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview comments on article: Parks’ Sustainability as a Method to revive historical parks in Egypt\nThe introduction should reveal the latest developments regarding the historical Antoniadis Park in Egypt, but it is damaged and neglected. Followed by the expected ideal conditions as the results of research on historical parks in other cities that have been restored and are visited by many people. So that it illustrates the importance of this research effort in reviving the excellence of Antoniadis Park as a historical park.\n\nIt is necessary to explain why using criteria from the Oregon Parks and Recreation Department (OPRD) in guiding the landscape design of urban parks so that they meet sustainability requirements in terms of environmental, economic, and social sustainability.\n\nThe arrangement of the results in the article can be made sequentially starting with the investigation of Antoniadis Park, identification of the causes of damage to the park, the park charter and requirements for sustainable parks, analysis of criteria for historic park restoration in other cities, and setting criteria for historic park restoration at Antoniadis Park for park sustainability.\n\nA summary of the development of the world's gardens is presented in a sufficiently complete manner, as an introduction to the development of the various styles of the world's gardens and their characteristics. This description should be linked to the importance of restoring the condition of the park so that it can survive with the times and remain crowded with people.\n\nCorrection for accuracy of mentioning BC time information in the Middle Empire (2035–1668 BC), on page 3.\n\nCorrection for accuracy in the description of Handsworth Park, there are 2 repetitions of the sentence: The site is crossed from north to south by the So-Ho, Handsworth, and Perry Barr railways.\n\nEach table and figure is placed after the introductory narrative paragraph of the table or figure, followed by an explanation of the related table or figure. There are still many tables and figures that are separated by pages with narration.\n\nInformation regarding OpenStreetMap, GIMP, and Krita as free alternatives does not need to be submitted.\n\nExamine the uniformity and neatness in writing titles in references, there are several titles that are dominant in the use of capital letters. There are still references written in Arabic, it is better if the title is also written in English.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [] }, { "id": "181964", "date": "10 Jul 2023", "name": "Riham Nady Faragllah", "expertise": [ "Reviewer Expertise Urban Design and Environmental Design" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper presents an important topic in the field of urban design which is the restoration of historical urban parks and gardens, particularly in Egypt, taking into consideration the sustainability approaches (social, economic and environmental) transforming it to eco-friendly parks. The topic is well discussed and sufficiently presented. However, there are some comments to the author to make the research more solid and coherent.\n\nIntroduction: There are several references in this section (about 7 references). In general, the introduction section is where the author should express his/her background about the research topic, main aim to make this study, methodology adopted, assumptions and the expected findings. Thus, it is recommended to remove the references in this section.\n\nDevelopment of Historical Parks throughout the Ages: This section is explained sufficiently and clearly. However, it is suggested to relate its importance with the research topic about the preservation of historical parks and restoring them to be sustainable.\n\nReasons for Urban Parks' Deterioration in Egypt:\n\nIt is recommended to change the title to \"Reasons for Deterioration of Urban Parks\". That is to say that, the author should state the reasons of deterioration for parks generally, both on the global and local scales and to compare between the two cases.\n\nAnalytical Studies: It is suggested to change the title of this section to \"Materials and Methods\".\nAntondiadis Park: It is suggested to change the title of this section to \"Case Study\". During these days, this park is going under a renovation plan by the government, so it preferable to state the efforts done and to compare it with the Oregon Parks and Recreation Department (OPRD) system.\n\nResults: The results of analyzing the international examples need to be classified according to Oregon Parks and Recreation Department (OPRD) to the social, economic and environmental approaches. Also the sub criteria discussed in Table (1) for sustainable parks should be the same as in Table (5).\n\nDiscussion and Conclusion: Both sections should be expanded to give more details about the results and the recommended criteria for preserving the historical Park of Antoniadis stating the role of each party users, stakeholders, owners, government, ... etc. Also, it is preferable to add future studies.\nTitles Figures and Tables: Main titles should be bold and clear for the reader. It is sufficient to put one map for the location of the park and its relation with the other parks in Alexandria City. Also, it is preferable to add figures that show the deterioration of Antoniadis Park and its restoration.\n\nMost tables are shifted and not in coloration with its place in text.\n\nReferences: There are some references that are very old (before 2000). It is recommended to update them. Also, there are incomplete data for some references such as reference (54,55,56, 59,60,61, ... etc.). It is preferable to complete the information. In addition, there are some references that are written in Arabic. All references should be written in English.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-455
https://f1000research.com/articles/12-453/v1	28 Apr 23	{ "type": "Systematic Review", "title": "Diagnosis of pelvic endometriosis: A systematic review and accuracy meta-analysis of non-invasive tests available in primary care", "authors": [ "Thomas Bainton", "Sobanakumari Jeyapala", "Javier Zamora", "Khalid S Khan", "Amer Raza", "Sobanakumari Jeyapala", "Javier Zamora", "Khalid S Khan", "Amer Raza" ], "abstract": "Background: Endometriosis is a chronic, often debilitating condition with a current significant delay from symptom onset to diagnosis with much of this in primary care. Methods: A systematic review and meta-analysis of the primary literature was conducted to investigate the accuracy of symptoms, clinical history and first-line non-invasive tests to predict pelvic endometriosis (PROSPERO: CRD42020187543). We searched Medline, Embase, Web of Science and Scopus from conception (1966; 1972; 1997; 2004 respectively) to September 2022 for primary test accuracy studies assessing non-invasive tests against reference standard diagnosis for endometriosis. Two authors independently conducted data extraction and quality assessment. Grading of evidence was performed using a novel visual pentagon model. Meta-analyses of test accuracy was estimated using bivariate random effects models. Results: The 125 included studies (250,574 participants) showed mixed quality. Studies applying non-surgical (database/self-reporting) reference standard had a greater risk of bias. In 98 studies applying surgical reference standard, summary diagnostic odds ratios for endometriosis were: dysmenorrhoea 2.56 (95% confidence interval 1.99-3.29); pelvic pain 2.56 (1.73-3.74); dyschezia 2.05 (1.36-3.10); dyspareunia 2.45 (1.71-3.52); family history of endometriosis 6.79 (4.08-11.3); nulligravidity of 2.01 (1.62-2.50); body mass index (BMI) ≥30kg/m2 0.37 (0.19-0.68); trans-vaginal ultrasound scan (TVUSS) endometrioma 91.2 (44.0-189); TVUSS invasive endometriosis 26.1 (9.28-73.5); and cancer antigen-125 (CA-125) >35U/mL 16.0 (8.09-31.7). Sensitivity analysis excluding all high-risk studies found concordant results. Conclusions: This meta-analysis collated the performance of non-invasive tests for endometriosis across a comprehensive and geographically varied population. Study quality was mixed, however results were consistent with high-risk studies excluded. These findings will inform future prediction models for triage in primary care.", "keywords": [ "Endometriosis", "diagnosis", "prediction", "diagnostic laparoscopy", "primary care", "pelvic pain", "dysmenorrhoea" ], "content": "Introduction\n\nEndometriosis is a chronic condition affecting women of reproductive age characterised by extra-uterine deposits of endometrial tissue with a prevalence of up to 10%.1–3 It can be found in peritoneal deposits, ovarian endometriomata, and invasive disease, with sufferers experiencing pain and sub-fertility.2,4–6 There is currently a 7-12 years delay between symptom onset and definitive diagnosis, by laparoscopy and biopsy.7–9 Reducing this has long a research priority.10,11 Up to 58% of sufferers present to primary care 10 or more times before diagnosis, demonstrated across a variety of healthcare systems and geographies.4,7,12–14 A better understanding of the accuracy with which symptoms, clinical history and non-invasive tests diagnose pelvic endometriosis would aid triage and referral.\n\nMany studies have attempted to find replacement tests for diagnostic laparoscopy.15–17 These are often invasive or otherwise not applicable to primary care.16,18 Previous meta-analyses have been restricted by a narrow inclusion criteria yielding a small number of eligible studies, limiting findings.19 Meta-analyses assessing imaging and biomarkers showed that no individual test met their criteria as a replacement or triage test alone, but findings on trans-vaginal ultrasound and serum CA-125 showed a high specificity for disease.20,21 These studies did not include assessment of other clinical factors. A 2019 narrative review demonstrated the importance of clinical factors in prediction of disease, but primary studies were not assessed for quality and absence of meta-analysis limited quantitative assessment of test performance.22\n\nA 2021 case-control study reporting on the accuracy of a simple patient-completed questionnaire identified those at high or low risk of disease with good accuracy reflecting the utility of assessment of patient reported symptoms at a primary care level.23\n\nResearch related to endometriosis is increasing in volume, with 75% of primary studies published in the last decade.24,25 Given this, and due to the limitations of previous reviews a new comprehensive systematic review and meta-analysis is required. We performed such an evidence synthesis and determined the accuracy of symptoms, clinical history, a simple low-cost biomarker and first line ultrasound for the diagnosis of pelvic endometriosis by means of a comprehensive systematic review and accuracy meta-analysis.\n\n\nMethods\n\nThe protocol was designed and registered with PROSPERO (Registration number: CRD42020187543).26 Reporting followed the PRISMA guidelines.27,59\n\nA virtual patient and public involvement meeting involving endometriosis sufferers at Chelsea and Westminster Hospital, London was held following an open invitation on social media to inform them of plans for this study. The aim was to better understand non-invasive predictors of endometriosis and reduce the diagnostic delay, which was well supported and resonated with their personal experience. No specific ethical approval was required for the patient and public involvement, in accordance with Health Research Authority (HRA) guidelines. No specific ethical approval was required as this study retrieved and synthesised data from already published studies. No individual participant data was collected and no participant consent was required.\n\nMedline, Embase, Web of Science and Scopus were searched from conception (1966; 1972; 1997; 2004 respectively) to September 2022 Search strategies are shown in Table 1 (see Extended data).53 Review article references were also screened.\n\nTitles and abstracts were screened independently by two authors (TB; SJ) using EndNote-X9 and duplicate or irrelevant studies removed. Full texts were screened and justification for inclusion or exclusion recorded, differences were resolved by discussion with the senior author (AR).28\n\nWe included published peer reviewed studies reporting accuracy estimates to predict pelvic endometriosis (peritoneal; ovarian; disease >5mm retroperitoneal invasion) for one or more index tests in participants with reported presence/absence of endometriosis. Included tests were dysmenorrhoea; pelvic pain; dyschezia; dyspareunia; nulligravidity; BMI ≥30kg/m2; family history of endometriosis; transvaginal ultrasound finding (TVUSS) of endometrioma; TVUSS finding of invasive disease; serum CA-125 >35U/mL.\n\nTarget population was reproductive age women excluding pregnancy or systemic co-morbidities. Studies with reporting non-reproductive age participants were included only where their data could be excluded from meta-analysis. Studies were included where a 2x2 contingency table for index test(s) could be constructed. We imposed no limits to language of publication, setting, or number of participants. All non-English studies were translated by a medically trained native speaker.\n\nStudies reporting laboratory and ultrasound tests were included only when performed prior to reference standard and using only standard 2D protocols. Definitions for each test for the purposes of study selection are shown in Table 2 (see Extended data).54\n\nWe excluded reviews; case reports; studies where information on recruitment or study population was unavailable; letters; and abstracts. Studies reporting non-pelvic endometriosis were included only where pelvic endometriosis was reported separately. Authors were contacted to obtain full texts only. Failing this, they were obtained through the British Library. Studies with incomplete data preventing determination of inclusion, exclusion or test accuracy were excluded.\n\nFor each included study, two authors (TB; SJ) independently recorded information on study characteristics and data was extracted to form 2x2 tables. Where there was unreliability in data extraction from some non-English language studies, these were excluded.\n\nRisk of bias was assessed independently by two authors (TB; SJ) using the Quality Assessment of Diagnostic Test Accuracy Studies (QUADAS 2) tool.29 For studies regarding serum CA-125 or TVUSS we included the additional questions: ‘was the index test performed by a single operator?’ to assess inter-observer bias; and ‘was timing in the participants’ menstrual cycle controlled for?’. We adjusted the original question ‘if a threshold was used, was it pre-specified?’ to ‘was there a clear definition of what was considered a positive test?’.\n\nDue to differences in design, studies were divided into groups according to application of the reference standard: ‘Complete verification’, all participants received visual inspection of the pelvis at surgery; ‘Partial verification’, all cases received surgical confirmation but controls did not; and ‘Database/self-reporting’, cases confirmed by healthcare coding or self-reporting and controls from healthy populations not known to have endometriosis.\n\nStatistical analysis was conducted using Stata software (version 15)30 to allow exploration of heterogeneity and statistical pooling using a bivariate random effects model and produced summary accuracy measures and summary receiver operative characteristic curves for each index test. A bivariate random effects model was applied for index tests with ≥5 contributing studies, and a univariate fixed effects model for index test with ≤4.\n\nIndex tests were assessed for performance as a ‘rule-in’ or ‘rule-out’ tool with pre-specified threshold summary accuracy of 95% sensitivity/50% specificity or 95% specificity/50% sensitivity respectively.\n\n\nResults\n\nOf 22,016 studies identified 125 met the inclusion criteria involving 250,574 participants (Figure 1). Details of included studies by number of participants and index test(s) are shown Table 2 (see Extended data54). Characteristics of included and excluded studies are shown in Tables 3 and 4 (see Extended data).55,56\n\nMean number of index tests per study was 2 (range 1-6). A total of 241 were assessed across all studies. Included studies were geographically varied: 45 Europe, 34 North America, 19 Asia/Oceania, 13 the Middle East/Africa, 12 South America, and 2 transcontinental. Publication date ranged from 1986 and 2022 with 57% since 2010: 4 before 1989; 22, 1990-1999; 18, 2000-2009; and 71, 2010-2022.\n\nMost studies (75) were ‘single-gate’ design with 50 of ‘two-gate’ design, including all studies in the partial verification and database/self-reporting groups. The mean prevalence of endometriosis in studies of a ‘single-gate’ design was 52% (range 9-93%), due to the selection of matched controls, prevalence in ‘two-gate’ studies was not relevant. There was heterogeneity in population selection, with participants having surgery for a broad range of indications such uterine fibroids or adnexal cysts as well as pelvic pain or sub-fertility.\n\nIn the 61 studies assessing symptom-based tests 20 did so by self-administered questionnaire; 14 by structured interview; 12 by clinical history taking, and 15 were undefined (Table 5 - see Extended data).57\n\nRisk of bias\n\nThe assessment of study quality by QUADAS-2 is presented in Figures 2-6. Overall methodological quality was mixed, with 5 studies presenting a low risk of bias across all domains31–35 and 64 presenting a high risk of bias or applicability in at least one domain.\n\nGraph: review authors’ judgements on each domain presented as percentages across included studies in: T all studies; A Complete verification; B Partial verification; and C Database/self-reporting groups.\n\nBMI, body mass index. CA-125, cancer antigen-125. US, ultrasound. DIE, deep invasive endometriosis.\n\nBMI, body mass index.\n\nBMI, body mass index.\n\nIn patient selection, 22 studies presented a low risk of bias, with 73 and 30 presenting an unclear or high risk respectively. Non-consecutive or non-random selection, two-gate selection for cases and controls, and having a highly selected group of participants (infertility cohort, surgery for a narrow indication etc.) were the main reasons for a high risk of bias.\n\nSymptom based index tests presented an unclear or high risk of bias due to a lack of definition of a positive test and of blinding. Just 9 studies presented a low risk in symptom-based tests across all groups. Index tests applicable to clinical history or investigations performed better, with 66 studies presenting a low risk. Reasons for an unclear or high risk of bias were a lack of pre-specified criteria for a positive test; no blinding to results of the reference standard; and inter-observer variability regarding imaging. Studies in the partial verification group assessed a proportionally higher number of index tests nulligravidity and BMI ≥30kg/m2, which, less subjective to interpretation presented a lower risk of bias.\n\nThe risk of bias regarding the reference standard performed best in the complete and partial verification groups where 74 studies were at low risk of bias. Those with an unclear or high risk lacked information on how likely the surgery was to correctly classify the target condition or operators not blinded to the result of index test(s). In the database/self-reporting group, 5 studies assessed for probable surgical confirmation by means of additional codes at the time of recording and therefore presented a lower risk, all other studies were high risk.\n\nIn flow and timing, the complete verification group presented the lowest risk. An unclear or high risk of bias was attributable to a long (>12 months) or unclear time interval, and a high or unclear withdrawal of participants from analysis. All other studies presented a high risk as not all participants received the same reference standard.\n\nIn patient selection, 40 studies gave low concern, with 63 and 22 giving unclear or high risk respectively. An unclear or high risk was attributable to the two-gate selection of controls, or the study likely to only classify a limited spectrum of disease (tertiary centres or infertility clinics).\n\nIn regard the reference standard, 97 studies showed a low concern. Studies in the database/self-reporting group were deemed high/unclear depending on whether additional coding input for surgery was recorded.\n\nDue to heterogeneity in methodology and study quality, meta-analysis was performed on studies from each group separately.\n\nThe accuracy of index tests to predicting endometriosis was variable, although results across groups were consistent. Each index test gave a positive likelihood for the presence of pelvic endometriosis, apart from a BMI ≥30kg/m2, which decreased the likelihood of disease. The positive likelihood ratio (LR+) for disease was highest in investigation tests and there was a trend towards a greater specificity than sensitivity. The summary results of bi/univariate meta-analysis are shown in Figure 7. An assessment of confidence in individual sensitivity and specificity of each test is displayed by a visual pentagon model, the methodology for this assessment is described in the discussion and legend shown in Figure 8.\n\nBMI, body mass index. TVUSS, transvaginal ultrasound scan. DIE, deep invasive endometriosis. CA-125, cancer antigen-125. DOR, diagnostic odds ratio. LR, likelihood ratio. *univariate analysis where fewer than 4 studies included.\n\nScore of 0 to -2 in each of 5 domains: design (study design type); risk of bias (QUADAS 2 risk of bias); indirectness (QUADAS 2 applicability); inconsistency (visual assessment of inter-study variance in confidence intervals); and imprecision (width of confidence intervals)\n\nInvestigation category tests were the best performing overall and TVUSS finding of endometrioma gave the highest summary LR+ at 21.6, at sensitivity and specificity of 77.2% and 96.4% respectively. Serum CA-125 >35U/mL showed sensitivity and specificity of 55.8% and 92.7% respectively, with LR+ of 7.63. TVUSS finding of DIE had showed sensitivity and specificity of 86.5% and 80.2% with LR+ of 4.39.\n\nSymptom based tests showed LR+ within a similar range: 1.47 (dysmenorrhoea) to 1.93 (dyspareunia). Symptoms showed a generally higher specificity than sensitivity. Dyspareunia showed the highest LR+ at 1.93 with a sensitivity and specificity of 36.3% and 81.1% respectively.\n\nFamily history of endometriosis showed a LR+ of 6.25 with a high specificity (98.5%) but low sensitivity (9.25%). The finding of BMI ≥30kg/m2 showed a decreased likelihood of diagnosis of endometriosis (LR+ 0.44).\n\nHierarchical Summary Receiver Operating Characteristics (HSROC) curves for index tests in each group are shown in Figures 9-11. The HSROC curves show the greatest area under the curve (AUC) for investigation category tests.\n\nBMI, body mass index. TV, transvaginal, DIE, deep invasive endometriosis. CA-125, cancer antigen-125.\n\n(Index tests with fewer than 5 studies omitted). BMI, body mass index.\n\n(Index tests with fewer than 5 studies omitted).\n\nIn the partial verification group, symptom index tests showed a greater LR+ than the complete verification group, range 2.47 (dysmenorrhoea) to 7.13 (dyschezia). Specificity was also higher, range 69% (dysmenorrhoea) to 92% (dyschezia).\n\nIn the database/self-reporting group symptom-based index tests performed similarly to other groups. In partial verification and database/self-reporting groups BMI ≥30 kg/m2 showed no correlation with disease and had 95% CI crossing 1.0. In all other index tests across all groups the 95% CI was >1.0.\n\nThe greatest inter-study variability in confidence intervals was shown in Forest plots for the symptom-based tests, notably pelvic pain. The inter study variance for specificity was generally lower than that for sensitivity, as was the overall width of confidence intervals. Forest plots for each index test in each group are shown in Figures 12-18.\n\nSensitivity analysis performed for studies without any high-risk features is shown in Table 6 (see Extended data58). All studies included are from the complete verification group. Summary accuracy measures are consistent with those in this group for the majority of index tests, although sensitivity for TVUSS finding of endometrioma and DIE reduced to 69.8% and 73.4% respectively.\n\n\nDiscussion\n\nThis meta-analysis presents an up-to-date, large, and geographically varied data set identifying predictive factors for diagnosis of pelvic endometriosis with a high degree of confidence. Index tests showed a positive association with endometriosis and trended towards a greater specificity than sensitivity, excluding elevated BMI, which demonstrated an inverse correlation. TVUSS finding of endometrioma reached a desired threshold for use as a ‘rule-in’ test and none achieved a summary sensitivity of >95%. A family history of endometriosis, dyschezia and serum CA-125 >35U/mL showed summary specificity of >90% although low sensitivity. Sensitivity was poor for symptom and clinical history tests, where the best performing was dysmenorrhoea.\n\nWe undertook a thorough search of the current literature, undergoing analysis by two independent reviewers with strict quality assessment. Attempts were made to mitigate inter-study heterogeneity by division of studies into groups. All index tests are relevant to primary care, are immediately available without novel techniques or additional training and can assist in the triage of those with symptoms that could be attributable to endometriosis.\n\nThere were, however, limitations. Due to difficulties in data extraction from some non-English journals, 15 studies were excluded from the analysis. Some studies, such as Chapron et al 2005, which was seminal in providing a clinical prediction model for moderate/severe endometriosis, were not able to be included due to the inability for construct 2x2 tables.36 We did not contact authors to obtain individual data that was not available in the published text.\n\nOverall, there was significant methodological variance and population heterogeneity in age; presentation; and stage of disease. Variation in selection of cases and controls may not reflect a clinically representative population. Prevalence of disease was higher than seen in the general female population, which may reflect a high degree of surgical accuracy, but also indicates the selective nature of study populations.\n\nThere is the possibility of inappropriate assignment of cases and controls, occurring in both directions due to uneven application of the reference standard, although we attempted to account for this by assigning groups. We included studies that diagnosed endometriosis by visual inspection and there is debate regarding this in the absence of histological confirmation.37–39\n\nThere was variation in the definition of positive symptom index tests. This is common across many reviews and although there is guidance on symptom reporting, it was not clearly followed in all studies.40–42 Assessment of symptoms varied, with most studies using a self-administered questionnaire. Although the use of standardised validated tools would better allow for comparison across studies, the nuance and detail acquired through clinical history taking is likely to better grasp the nature and significance of a symptom and its implications.\n\nIt is likely that imaging and surgical techniques have developed over time. A trend towards recent studies may mitigate this. Imaging modalities such as MRI and advanced sonographic techniques were not assessed due to their lack of universal availability in a primary care setting.\n\nConsidering the balances of strengths and weaknesses, however, we believe that our data synthesis presents an objective summary of the current evidence.\n\nAn understanding of the degree of likelihood associated with various symptoms and features in the clinical history can help assessment of patients with possible endometriosis in primary care.\n\nThe negative association between elevated BMI and endometriosis shown in the complete verification group is consistent with that demonstrated previously.43 This was not replicated across other groups. This may reflect a greater negative correlation between elevated BMI in higher risk populations in all surgical cohorts who may have more severe disease. This possibility is consistent with previous studies, demonstrating a significantly lower BMI in those with severe compared to mild disease and a 12-14% decrease in the likelihood of endometriosis being diagnosed for each unit increase in BMI (kg/m2).31,44 The interplay between BMI and endometriosis pathogenesis, however, remains poorly understood.\n\nThe trend of data from the partial verification and database/self-reporting groups to demonstrate better performing accuracy measures was likely a reflection of the selection of controls. This effect seems to outweigh the possibility of an undiagnosed disease burden in controls not exposed to a surgical reference standard. The accuracy of self-reported diagnosis of endometriosis has been assessed and performs well,45 false attribution of disease in the self-reporting group may therefore only present a small source of bias.\n\nA greater specificity than sensitivity of tests may be associated with their correlation to disease severity. Dyschezia and dyspareunia have been linked to severe disease due to the involvement of a precise anatomical location in cases of invasive disease.46,47 Tests showing a greater sensitivity such as dysmenorrhea were also less specific.\n\nPrevious systematic reviews have similarly highlighted the heterogeneity and poor methodological quality of primary studies, limiting interpretation of findings.17,48 As our methodology allowed a wide inclusion criteria, we applied a novel grading protocol in order to more quantitively assess limitations. Grading of evidence for index tests was performed for sensitivity and specificity by application of a visual pentagon model for grading of test accuracy studies described by Rogozinska and Khan.49 This methodology is described in detail elsewhere but briefly, studies were given a score of 0 to -2 in each of 5 domains: design (study design type); risk of bias (QUADAS 2 risk of bias); indirectness (QUADAS 2 applicability); inconsistency (visual assessment of inter-study variance in confidence intervals); and imprecision (width of confidence intervals). The complete verification group shows the fewest limitations, whist the database/self-reporting studies showed very serious limitations. There was greater limitation in the investigation category tests due to more highly selective populations and a generally higher inter-study inconsistency and imprecision.\n\n\nConclusions\n\nThe need for high-quality studies of predictive factors for endometriosis remains, particularly assessing populations attending primary care. Further multivariate analysis in powerful primary observational studies assessing factors that can be immediately and readily assessed in primary care would be of great value, as we anticipate the index tests assessed in this study to provide a greater degree of accuracy when applied in combination.4,36,50\n\nWe examined serum CA-125 at a cut off >35 U/mL, considered the upper limit of normal range, meta-analysis from 2016 found a cut off of 30 U/mL gave a sensitivity and specificity of 52% and 93% respectively, but sensitivity dropped to just 24% for detection of minimal disease.51 Further research assessing the accuracy of CA-125 at different thresholds and in combination with other tests could help improve accuracy.\n\nTwo recent studies (Fauconnier et al 2021 and Chapron et al 2022) assessed the accuracy of a patient-completed questionnaire and epidemiological data for the early identification of endometriosis and found it could do so with high diagnostic accuracy.23,52 Although these studies were conducted in a high-risk population undergoing surgery, the model maintained accuracy in population with a lower endometriosis prevalence of 10%. We do not anticipate a clinical score replacing laparoscopy due to its added therapeutic advantages and requirement to exclude other pathologies. If, however, disease can be predicted with a high degree of accuracy early on, medical therapy may be instigated and referral made for definitive diagnosis and counselling regarding treatment, prognosis and fertility in a timely manner with the aim of reducing the current extraordinary delay.", "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: Table 1.xlsx. https://doi.org/10.6084/m9.figshare.22419178.v1. 53\n\nThis project contains the following extended data:\n\n- Table 1. xlsx (key word search strategy for all databases to identify primary studies for inclusion).\n\nFigshare: Table 2.xlsx. https://doi.org/10.6084/m9.figshare.22419184.v1. 54\n\nThis project contains the following extended data:\n\n- Table 2. xlsx (index tests and definitions for the purposes of study selection and data extraction and number of included studies and participants reporting on each index test by group according to confirmation of diagnosis of endometriosis. BMI, body mass index; TVUSS, trans-vaginal ultrasound scan; DIE, deep invasive endometriosis; CA-125, cancer antigen-125).\n\nFigshare: Table 3.xlsx. https://doi.org/10.6084/m9.figshare.22419187.v1. 55\n\nThis project contains the following extended data:\n\n- Table 3. xlsx (characteristics of all included studies in the systematic review and meta-analysis).\n\nFigshare: Table 4.xlsx. https://doi.org/10.6084/m9.figshare.22419967.v1. 56\n\nThis project contains the following extended data:\n\n- Table 4. xlsx (excluded studies and reason for exclusion of all screened full texts in the systematic review and meta-analysis).\n\nFigshare: Table 5.xlsx. https://doi.org/10.6084/m9.figshare.22419973.v1. 57\n\nThis project contains the following extended data:\n\n- Table 5. xlsx (method of assessment for all included studies assessing participants for a symptom-based index test (61) in the systematic review and meta-analysis Diagnosis of pelvic endometriosis: a systematic review and accuracy meta-analysis of non-invasive tests available in primary care).\n\nFigshare: Table 6.xlsx. https://doi.org/10.6084/m9.figshare.22419976.v1. 58\n\nThis project contains the following extended data:\n\n- Table 6. xlsx (sensitivity analysis - summary diagnostic results of bivariate meta-analysis of index tests for included studies excluding those with any high-risk feature for bias or applicability. BMI, body mass index. TVUSS, transvaginal ultrasound scan. DIE, deep invasive endometriosis. CA-125, cancer antigen-125. DOR, diagnostic odds ratio. LR, likelihood ratio).\n\nFigshare: PRISMA checklist for ‘Diagnosis of pelvic endometriosis: A systematic review and accuracy meta-analysis of non-invasive tests available in primary care’. https://doi.org/10.6084/m9.figshare.22420591.v1. 59\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nKSK is Distinguished Investigator at University of Granada funded by the Beatriz Galindo (senior modality) program of the Spanish Ministry of Education.\n\n\nReferences\n\nEskenazi B, Warner ML: Epidemiology of endometriosis. Obstet. Gynecol. Clin. N. Am. 1997; 24(2): 235–258. Publisher Full Text\n\nFarquhar C: Endometriosis. BMJ. 2007; 334(7587): 249–253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiudice LC: Clinical practice. Endometriosis. N. 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Publisher Full Text\n\nLier MCI, Vlek SL, Ankersmit M, et al.: Comparison of enhanced laparoscopic imaging techniques in endometriosis surgery: a diagnostic accuracy study. Surg. Endosc. 2020; 34(1): 96–104. Publisher Full Text\n\nChen CX, Kwekkeboom KL, Ward SE: Self-report pain and symptom measures for primary dysmenorrhoea: a critical review. Eur. J. Pain. 2015; 19(3): 377–391. Publisher Full Text\n\nDoggweiler R, Whitmore KE, Meijlink JM, et al.: A standard for terminology in chronic pelvic pain syndromes: A report from the chronic pelvic pain working group of the international continence society. Neurourol. Urodyn. 2017; 36(4): 984–1008. Publisher Full Text\n\nSteingrímsdóttir ÓA, Landmark T, Macfarlane GJ, et al.: Defining chronic pain in epidemiological studies: a systematic review and meta-analysis. Pain. 2017; 158(11): 2092–2107. Publisher Full Text\n\nLiu Y, Zhang W: Association between body mass index and endometriosis risk: a meta-analysis. Oncotarget. 2017; 8(29): 46928–46936. Publisher Full Text\n\nYi KW, Shin JH, Park MS, et al.: Association of body mass index with severity of endometriosis in Korean women. Int. J. Gynaecol. Obstet. 2009; 105(1): 39–42. Publisher Full Text\n\nShafrir AL, Wise LA, Palmer JR, et al.: Validity of self-reported endometriosis: a comparison across four cohorts. Hum. Reprod. 2021; 36(5): 1268–1278. Publisher Full Text\n\nSeracchioli R, Mabrouk M, Guerrini M, et al.: Dyschezia and posterior deep infiltrating endometriosis: analysis of 360 cases. J. Minim. Invasive Gynecol. 2008; 15(6): 695–699. Publisher Full Text\n\nFauconnier A, Chapron C: Endometriosis and pelvic pain: epidemiological evidence of the relationship and implications. Hum. Reprod. Update. 2005; 11(6): 595–606. Publisher Full Text\n\nMay KE, Villar J, Kirtley S, et al.: Endometrial alterations in endometriosis: a systematic review of putative biomarkers. Hum. Reprod. Update. 2011; 17(5): 637–653. Publisher Full Text\n\nRogozińska E, Khan K: Grading evidence from test accuracy studies: what makes it challenging compared with the grading of effectiveness studies? Evid. Based Med. 2017; 22(3): 81–84. Publisher Full Text\n\nEskenazi B, Warner M, Bonsignore L, et al.: Validation study of nonsurgical diagnosis of endometriosis. Fertil. Steril. 2001; 76(5): 929–935. Publisher Full Text\n\nHirsch M, Duffy JMN, Davis CJ, et al.: Diagnostic accuracy of cancer antigen 125 for endometriosis: a systematic review and meta-analysis. BJOG Int. J. Obstet. Gynaecol. 2016; 123(11): 1761–1768. Publisher Full Text\n\nChapron C, Lafay-Pillet MC, Santulli P, et al.: A new validated screening method for endometriosis diagnosis based on patient questionnaires. EClinicalMedicine. 2022; 44: 101263. Publisher Full Text\n\nBainton T, Jeyapala S, Zamora J, et al.: Table 1.xlsx. figshare. Figure. 2023. Publisher Full Text\n\nBainton T: Table 2.xlsx. figshare. Figure. 2023. Publisher Full Text\n\nBainton T: Table 3.xlsx. figshare. Figure. 2023. Publisher Full Text\n\nBainton T: Table 4.xlsx. figshare. Figure. 2023. Publisher Full Text\n\nBainton T: Table 5.xlsx. figshare. Figure. 2023. Publisher Full Text\n\nBainton T: Table 6.xlsx. figshare. Figure. 2023. Publisher Full Text\n\nBainton T: PRISMA checklist. Diagnosis of pelvic endometriosis- A systematic review and accuracy meta-analysis of non-invasive tests available in primary care.docx. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "216534", "date": "27 Oct 2023", "name": "Dan C Martin", "expertise": [ "Reviewer Expertise Endometriosis", "operative laparoscopy", "laparotomy for palpation", "patient advocacy" ], "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\nUpdate 31/10/2023: On the request of the reviewer, a minor language edit has been made to point 5 for clarity. No other edits have been made.\n\nThe authors perform high-level statistical analysis of many poor-quality papers in complex heterogenous populations and do not anticipate a clinical score replacing laparoscopy due to its added therapeutic advantages and the requirement to exclude other pathologies. The useful clinical conclusion is that this is a “watch this space” story.\n\nRegarding if “disease can be predicted with a high degree of accuracy early on, medical therapy may be instigated, and referral made for definitive diagnosis and counseling regarding treatment, prognosis, and fertility in a timely manner with the aim of reducing the current extraordinary delay.” It would be simpler to avoid normalizing pain and treat all pain adequately. The initial treatment of dysmenorrhea and pelvic pain is the same as endometriosis. Good pain treatment is needed; if that also limits endometriosis, that would be a bonus. We spend too much time on statistics and not enough listening to patients.\n\nDiscuss the limitation of endometriosis to minimal and mild stages only with 10.2 years of follow-up of proactive care in Knox B, Ong YC, Bakar MA, Grover SR. A longitudinal study of adolescent dysmenorrhoea into adulthood. Eur J Pediatr. 2019, Sep;178(9):1325-1332. doi: 10.1007/s00431-019-03419-3. PMID: 312927291.\n\nScientists do like statistics more than clinicians, and I will review this, anticipating that the journal will find a statistician who understands the analysis. Although the authors do not need to agree with my comments, they do need to discuss the controversy.\n\nThe title “Diagnosis of pelvic endometriosis: A systematic review and accuracy meta-analysis of non-invasive tests available in primary care” is misleading. No studies are reviewed of testing validated in primary care ObGyn or family practice. As stated in the conclusions, “studies were conducted in a high-risk population undergoing surgery.” There are no studies quoted that were validated in either type of primary care. That is compatible with my reviews. The title is more accurate as, “Diagnosis of pelvic endometriosis: A systematic review and accuracy meta-analysis of non-invasive tests available in tertiary care for recognized endometriosis. Are they useful in tertiary care? Will they be useful in primary care?”\n\nDiscussions are needed for both primary care ObGyn and Family practice where the prevalence of 10% may be an incidence of 0.1% to 0.7% with a mean in diagnosed cases of less than 5 cases yearly in primary care ObGyn and one yearly in family practice. About half of those have pain with or without infertility and need pain and surgical specialists. The half with infertility only needs primary infertility triage and REI referral as needed.\n\nIn the concluding paragraph, Fauconnier et al. 2021 and Chapron et al. 2022 are almost misquoted. “The model maintained accuracy in population with a lower endometriosis prevalence of 10%” suggests this was in a live population, not a computer-generated population. Please clarify that the model was statistical people, not real people. Statisticians may know that, but I had to read the papers to understand your meaning.\n\nThose models are primarily for pain and do not appear to be useful in determining which infertility patients might benefit from GnRH or surgery. Discuss concepts like Likes CE, Cooper LJ, Efird J, Forstein DA, Miller PB, Savaris R, Lessey BA. Medical or surgical treatment before embryo transfer improves outcomes in women with abnormal endometrial BCL6 expression. J Assist Reprod Genet. 2019 Mar;36(3):483-490. doi: 10.1007/s10815-018-1388-x. Epub 2019 Jan 4. PMID: 30610661; PMCID: PMC64390152.\n\nThe sensitivities, etc., do not consider non-recognized endometriosis. Discuss the effect of non-recognized endometriosis on the sensitivity, specificity, PPV, PNV, and accuracy of studies.\n\nDiscuss non-visualized tubal endometriosis and conclusion about salpingectomy in McGuinness B, Nezhat F, Ursillo L, Akerman M, Vintzileos W, White M. Fallopian tube endometriosis in women undergoing operative video laparoscopy and its clinical implications. Fertil Steril. 2020 Nov3.\n\nDiscuss non-visualized bowel endometriosis in Roman H, Merlot B, Forestier D, Noailles M, Magne E, Carteret T, Tuech JT, Martin DC. Nonvisualized palpable bowel endometriotic satellites. Hum Reprod. 2021, 36(3):656-665. doi: 10.1093/humrep/deaa340. PMID: 33432338; PMCID: PMC7891810. Open access: https://academic.oup.com/humrep/article/36/3/656/60858324.\n\nDiscuss non-visualized retrocervical endometriosis in Perry CP, Mena MV. Occult retrocervical endometriosis. A case report. J Reprod Med. 1995 Sep;40(9):652-4. PMID: 85768835.\n\nDiscuss the patients with a normal pelvis at laparoscopy in Koninckx PR, Meuleman C, Oosterlynck D, Cornillie FJ. Diagnosis of deep endometriosis by clinical examination during menstruation and plasma CA-125 concentration. Fertil Steril. 1996 Feb;65(2):280-7. PMID: 8566249. Open Access: https://www.sciencedirect.com/science/article/pii/S0015028216580868?via%3Dihub6.\n\nDiscuss non-visualized sacral endometriosis in Possover M. Laparoscopic morphological aspects and tentative explanation of the aetiopathogenesis of isolated endometriosis of the sciatic nerve: a review based on 267 patients. Facts Views Vis Obgyn. 2021 Dec;13(4):369-375. doi: 10.52054/FVVO.13.4.047. PMID: 35026098. Open Access: https://fvvo.eu/archive/volume-13/number-4/original-articles/laparoscopic-morphological-aspects-and-tentative-explanation-of-aetiopathogenesis-of-isolated-endome/7.\n\nDiscuss the non-visualized ovarian endometriosis in Russell WW. Aberrant portions of the Müllerian duct found in an ovary: Johns Hopkins Hospital Bulletin, 1899, 10(Nos 94-95-96)(Jan-Feb-Mar)):8-10, plates I-III. HathiTrust.org https://babel.hathitrust.org/cgi/pt?id=uva.3470085735&view=1up&seq=20. Google Books Vol 10 Bulletin of the Johns Hopkins Hospital https://www.google.com/books/edition/Bulletin_of_the_Johns_Hopkins_Hospital/nzFHAAAAYAAJ?hl=en&gbpv=0. An easier review to find is Longo LD. Aberrant portions of the müllerian duct found in an ovary: William Wood Russell Johns Hopkins Hospital Bulletin, vol. 10, pp. 8--10, 1899. Am J Obstet Gynecol. 1979, 134:225-6. PMID 3779668.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] }, { "id": "216511", "date": "30 Nov 2023", "name": "Hendy Hendarto", "expertise": [ "Reviewer Expertise Reproductive endocrinology", "endometriosis and 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 of the study should be changed to better match the content of the manuscript and be useful for clinicians for application in the field. Proposed title: “Clinical diagnosis of endometriosis: A systematic review and accuracy of meta-analysis of non-invasive tests to prevent diagnosis delay”.\n\nThe study objectives stated in the abstract and introduction should be answered specifically in the conclusion. In the conclusion, authors do not describe it, but instead write suggestions for future research.\n\nThe author made extraordinary efforts in creating research methods and statistical analysis at several levels, which are described in the written results section in detail and excellence. This should be followed by providing useful recommendations for clinicians to establish a clinical diagnosis of endometriosis.\n\nEndometriosis is a complex disease and involves various factors. Is it sufficient to use bivariate analysis, or is it possible to carry out analysis on more than two variables?\n\nThis study provides an excellent systematic review and meta-analysis, but is less conclusive and should be supplemented with specific recommendations for clinicians.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-453
https://f1000research.com/articles/12-450/v1	27 Apr 23	{ "type": "Systematic Review", "title": "Identifying the most effective Traditional Chinese Medicine treatment modalities for premature ovarian insufficiency: a systematic review and network meta-analysis", "authors": [ "Yen Suan Sin", "Min Chen", "Kim Sia Sng", "Jianwei Zhang", "Yen Suan Sin", "Min Chen", "Kim Sia Sng" ], "abstract": "Background: This study aimed to systematically evaluate and compare the clinical efficacy of different modalities of Traditional Chinese Medicine (TCM) for the treatment of premature ovarian insufficiency (POI) and identify the most effective treatment modality. Methods: Six electronic databases (PubMed, OVID, Scopus, CNKI, VIP and WanFang) were screened from their inception until 20/01/2022. This network meta-analysis (NMA) incorporated hormone replacement therapy (HRT) as the placebo and six different TCM treatment modalities as interventions. The primary outcome was total effective rate; the secondary outcomes were improvement in serum follicle-stimulating hormone (FSH), estradiol (E2), luteinizing hormone (LH) and anti-Müllerian hormone (AMH) levels. The Cochrane risk of bias tool was used to appraise the quality of the included studies. Random effect model was used for pairwise meta-analysis using RevMan 5.4. NMA was performed in STATA. The surface under the cumulative ranking curves (SUCRA) and mean rank (MR) were used as evaluation indexes. Results: Overall, 37 randomized clinical trials involving 2,597 subjects that conformed to the inclusion criteria were included. Herbal medicine combined with catgut-embedding therapy was concluded to achieve the highest total effective rate (MD 4.35; 95% CI 1.32, 14.38) and improvement in AMH level (MD 0.74; 95% CI -0.74, 2.22). HRT combined with herbal medicine and acupuncture was determined to be the most effective treatment model for POI in improving their FSH (MD -13.17; 95% CI -18.17, -8.17), E2 (MD 34.29; 95% CI 14.41, 54.18) and LH (MD -15.01; 95% CI -19.95, -10.06) levels. Conclusions: Combination of HRT, herbal medicine and acupuncture was the most effective treatment modality for patients with POI. We also found that combination of herbal medicine with conventional HRT for patients with POI could effectively improve the clinical effectiveness in all aspects. This result may provide guidance for future treatment guideline implementation for POI. Registration: PROSPERO (CRD42020163873; 28/04/2020).", "keywords": [ "Premature ovarian insufficiency", "Chinese medicine", "Acupuncture", "Network meta-analysis", "Systematic review" ], "content": "Introduction\n\nPremature ovarian insufficiency (POI) is a pathological state of a woman below the age of 40 years suffering from irreversible diminished ovarian function in her reproductive age, it mainly manifests as oligomenorrhoea, hypomenorrhea, amenorrhea, elevated follicle-stimulating hormone (FSH) level (>25 U/L) and short or long term effects of estrogen deficiency.1 The ovarian reserve can be measured by serum anti-Müllerian hormone (AMH) levels. According to the Bologna criteria, a low ovarian reserve is defined as an AMH level below 0.5–1.1 ng/mL (3.6–7.9 pmol/L).2 Review data from 2020 reported that the prevalence of POI is about 10% among women below 40 years old.3 The occurrence of this disease has been increasing among populations living in urban cities. This has captured the attention of public health policy makers and a timely and appropriate preventive measure is urged to be implemented.\n\nHormone replacement therapy (HRT) is the most recommended medical intervention for patients with POI, referring to the European Society of Human Reproduction and Embryology (ESHRE) and International Menopause Society (IMS) guidelines, as well as the Chinese Medical Experts’ Consensus on Clinical Diagnosis and Treatment of POI, stipulated by the Obstetrics and Gynecology Society of Chinese Medical Association.1,4–6 However, there are risks of adverse reactions and associated diseases after HRT application.7,8 Hence, patients often seek out complementary medicine to improve their ovarian functions and increase chances of conception.\n\nVarious Traditional Chinese Medicine (TCM) treatments such as herbal medicine, acupuncture, moxibustion and catgut-embedding therapy have been applied as the treatment for patients with POI. These treatment modalities have been proven to be effective and safe to improve ovarian functions among patients with POI through numerous clinical trials and direct comparison by using meta-analysis.9–11 However, there are inadequate direct and indirect comparisons between different TCM treatment modalities that contribute to clinical health promotion and selection of the optimal therapeutic procedure. Therefore, there is a need to systematically evaluate and analyze the efficacy of these alternatives in order to provide a strong theoretical basis and scientific evidence for clinical practice and research. In this study, we compared the efficacy of different TCM treatment modalities for patients with POI by conducting network meta-analysis (NMA) to rank the therapeutic measures in line with their treatment effect through direct and indirect comparisons of the means, with the prospective to provide an evidence-based guidance for future clinical guidelines.\n\n\nMethods\n\nThe study method is compliant with the Cochrane Handbook for Systematic Reviews of Interventions12 and the methods outlined in Lei Zhang et al.13 It followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) extension statement for network meta-analyses (PRISMA-NMA).14,57 It was registered in the International Prospective Systematic Registration Review (PROSPERO) with registration number CRD42020163873 on 28 April 2020.\n\nRecords identified from six electronic databases, which included three English databases (PubMed (RRID:SCR_004846), OVID, Scopus (RRID:SCR_022559)) and three Chinese databases (CNKI, VIP, WanFang). Reported studies from the inception of each database until 20 January 2022 were screened. Search terms used were a combination of text words and medical subject headings (MeSH), including “Premature Ovarian Insufficiency”, “POI”, “Premature Ovarian Failure” or “Primary Ovarian Insufficiency”. Table 1 shows the search strategy details for each database. Only English and Chinese articles were included. Additionally, the reference lists of all eligible studies were screened to avoid missing eligible studies. The electronic search and literature screening were executed by two researchers (SYS and CM) independently. Any potential disagreement was assessed and discussed with a third reviewer (SKS).\n\nAll relevant TCM randomized controlled trials (RCTs) for POI were included according to the inclusion criteria.\n\nTypes of participant (P): Patients diagnosed with POI according to the European Society of Human Reproduction and Embryology (ESHRE) guideline1 or “Chinese expert consensus on premature ovarian insufficiency”.4\n\nTypes of Intervention (I): The treatment group received TCM treatment modalities, such as herbal medicine, acupuncture, moxibustion and catgut embedding therapy alone or in combination.\n\nComparisons (C): The control group received HRT or one of the aforementioned TCM treatments.\n\nOutcomes (O):\n\n• Primary outcome measurement: Total effective rate.\n\nTotal effective rate = (number of recovery cases + number of effective cases + number of efficient cases)/total number of cases × 100%.\n\n(i) Recovery: clinical manifestations disappeared, menstrual cycle, serum FSH and E2 levels returned to normal, total effective rate ≥95%;\n\n(ii) Effective: clinical manifestations were significantly improved, menstrual cycle, serum FSH and E2 levels were significantly improved, total effective rate ≥70% and <95%;\n\n(iii) Efficient: clinical manifestations were slightly improved, menstrual cycle, serum FSH and E2 levels were slightly improved, total effective rate ≥30% and <70%;\n\n(iv) Invalid: there was no improvement in clinical signs and symptoms, no improvement in menstrual cycle, no improvement in serum FSH and E2 levels, total effective rate <30%.\n\n• Secondary outcome measurements: Serum FSH, E2, luteinizing hormone (LH) and AMH levels.\n\nType of studies (S): RCTs.\n\nStudies were omitted if the studies did not meet the inclusion criteria, for instance animal studies, review articles, case reports, editorials, letters and comments.\n\nThe name of authors, year of publication, subject age group, course of disease, types of TCM treatment modalities, number of subjects in intervention and comparison groups, were extracted into a spreadsheet. Two researchers (SYS and CM) performed the data extraction individually. In case of any discrepancies, another researcher (SKS) were asked to resolve the discrepancies via discussion.\n\nThe Cochrane risk of bias tool was used to appraise the quality and risk of bias of the included studies. The assessment was done independently by two researchers (SYS and CM).15 In this tool, the random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome reporting, incomplete outcome data, selective reporting, and other items were being evaluated in all the included studies. Low, unclear and high risk of bias were used to evaluate each element. In case of discrepancies, discussion among the researchers (SYS, CM and SKS) were done to resolve it.\n\nFor pairwise meta-analysis of each intervention pair, a random effect model was used, with RevMan (RRID:SCR_003581) 5.4 (Nordic Cochrane Centre, Cochrane Collaboration, 2020) being employed. Continuous outcomes between intervention groups can be compared by using standard mean differences (SMDs), while dichotomous outcomes can be analyzed using odds ratios (OR). The relative treatment effectiveness between each intervention pair were estimated using 95% confidence intervals.\n\nA NMA was performed in Stata (RRID:SCR_012763) 14.0 (free alternative, RStudio) and the network evidence graph was generated using the ‘netplot’ command. To compare direct and indirect comparisons of each intervention, I2 and P-value of the global consistency were evaluated, and then the node-splitting analysis method was applied to detect inconsistencies. The inconsistency factor within each closed loop of evidence was used to detect inconsistency among studies. The evaluation indexes included surface under the cumulative ranking curves (SUCRA) and mean rank (MR). The SUCRA value reflected the possibility of the intervention measures, and the intervention was considered more effective if the MR was closer to 0. P<0.05 was considered statistically significant.11\n\nTo determine if there was a small sample effect or publication bias in the network, a comparison-adjusted funnel plot was used.\n\n\nResults\n\nThe selection procedure was recorded in Figure 1 according to the most updated PRISMA 2020 flow diagram for new systematic review.16 A total of 2,602 studies were firstly identified through six databases, including three English databases (PubMed, OVID and Scopus), as well as three Chinese databases (CNKI, VIP and WanFang). Of those, 586 duplicate records were eliminated using EndNote X9 (RRID:SCR_014001) (Clarivate Analytics, USA). By assessing the title and abstract, 1,940 studies were subsequently removed. Full articles of the remaining 76 studies were screened, of which 43 were removed for not reporting relevant outcome data, non-RCT, no full-text access, not relevant to TCM intervention, or intervention only included in one trial, which may lead to small study effects in a NMA. Eventually, 33 RCTs involving 2,597 subjects that conformed to the inclusion criteria were selected into this study and proceed with data extraction and analysis. Among them, all RCTs were Chinese articles. This NMA incorporated HRT as the placebo and six TCM treatment modalities. The characteristics and details of the included 33 RCTs are listed in Table 2.\n\nNote: RCT, randomized controlled trials.\n\nFor the random sequence generation element, 21 studies reported an appropriate method for randomization, thus “low risk”. There were two studies allocated participants to interventions based on participants’ preference and hospital record number therefore “high risk”. The rest only reported random, so it is “unclear”.\n\nIn terms of allocation concealment, 30 studies were judged as having “high risk” of bias as there were inadequate concealment of allocations prior to assignment. Only one study reported clear allocation concealment by using sequentially numbered, opaque and sealed envelope, therefore it was judged as “low risk”. There were two studies reported using assignment envelopes but sufficient details such as whether the envelopes were sequentially numbered, opaque and sealed were not mentioned, thus “unclear”.\n\nIn terms of blinding of participants and personnel, only one study was stated as single-blinded design but did not address the outcome, therefore “unclear”. The rest of the studies were judged as “high risk” as there were no blinding and due to different treatment modalities, participants might have the wrong belief that the intervention was more effective for their treatment.\n\nIn terms of outcome assessment, all studies were judged as “unclear” due to inadequate information for evaluation. In terms of incomplete outcome data, only one study was judged as “high risk” of bias as there were missing outcome data across the intervention groups. The rest were “low risk”. In terms of selective reporting, only one study was judged as “high risk” of bias due to incomplete outcome of interest in the study. The rest were “low risk”. In terms of other bias, all studies were “low risk” except two, in which there were incorrect reported data and discrepancies found in the studies. The graph and summary of the risk of bias are listed in Figure 2.\n\nRisk of bias A) graph and B) summary.\n\nPairwise meta-analysis\n\nTable 3 presents the results of the pairwise meta-analysis and heterogeneity estimates. All 33 studies reported total effective rate, 30 reported FSH, 29 reported E2, 20 reported LH and 13 reported AMH.\n\nThe forest plot of total effective rate indicates that herbal medicine (OR 2.34, 1.46 to 3.76, I2=38%) and HRT + herbal medicine (OR 3.74, 2.61 to 5.35, I2=0%) were more effective than HRT alone. Herbal medicine and acupuncture (OR 9.22, 2.02 to 42.03, I2=0%) was more effective than herbal medicine alone.\n\nThe forest plot of FSH indicates that acupuncture + moxibustion (MD 3.70, 2.18 to 5.23, I2=0%), herbal medicine (MD 2.68, 0.48 to 4.88, I2=79%), herbal medicine + acupuncture (MD 3.29, 0.51 to 6.07, I2=57%) and HRT + herbal medicine (MD 4.82, 3.61 to 6.02, I2=83%) were more effective in improving FSH levels compared to HRT alone.\n\nThe forest plot of E2 indicates that all treatment modalities involved herbal medicine, including HRT + herbal medicine (MD 9.09, 5.49 to 12.70, I2=96%), HRT + herbal medicine + acupuncture (MD 28.43, 25.92 to 30.94, I2=n/a), herbal medicine (MD 8.52, 0.53 to 16.51, I2=96) and herbal medicine + acupuncture (MD 15.04, 1.06 to 29.01, I2=71%) showed higher effectiveness than HRT alone to improve E2 levels among patients with POI.\n\nThe forest plot of LH indicates that therapeutic effects of HRT + herbal medicine (MD 5.20, 2.51 to 7.88, I2=92%), herbal medicine (MD 1.36, 0.36 to 2.36, I2=26%) and herbal medicine + acupuncture (MD 0.87, 0.14 to 1.61, I2=0%) were more superior than HRT alone.\n\nThe forest plot of AMH indicates that herbal medicine (MD 0.68, 0.22 to 1.14, I2=99%) showed better efficacy compared to only HRT in improving AMH levels.\n\nNMA on total effective rate\n\nThe results of the NMA showing comparisons of different treatments based on total effective rate are shown in Figure 3A. The total effective rate are presented in the form of a league table can be found in Figure 4A. A total of 33 RCTs (2,862 participants) with seven different treatment modalities were analyzed. The results of NMA indicate that herbal medicine combined with catgut-embedding therapy have the highest efficiency. According to the SUCRA, which presents treatment ranking to offer the most important benefit outcome (the highest total effective rate), herbal medicine combined with catgut-embedding therapy (87.8%) had the highest effective rate, followed by combination of HRT with herbal medicine and acupuncture (76.8%), HRT combined with herbal medicine (67.1%), acupuncture combined with moxibustion (63.1%), herbal medicine combined with acupuncture (27.3%), HRT alone (16.1%) and herbal medicine only (11.9%). This also showed that combination of TCM treatments with HRT are more effective than HRT alone. Table 4 (part A) and Figure 5A depict the rankings of these seven treatments.\n\nA) Total effective rate. B) FSH, C) E2, D) LH and E) AMH levels. Note: FSH, follicle-stimulating hormone; E2, estradiol; LH, luteinizing hormone; AMH, anti-Müllerian hormone; HRT, hormone replacement therapy; Herb, herbal medicine; Catgut, catgut-embedding therapy; Acu, acupuncture; Moxa, moxibustion.\n\nA) Total effective rate. B) FSH, C) E2, D) LH and E) AMH levels. OR>1 means the top-left treatment is better. Note: FSH, follicle-stimulating hormone; E2, estradiol; LH, luteinizing hormone; AMH, anti-Müllerian hormone; TCM, Traditional Chinese medicine; HRT, hormone replacement therapy; Herb, herbal medicine; Catgut, catgut-embedding therapy; Acu, acupuncture; Moxa, moxibustion.\n\n\n\nA. Total effective rate\n\n\n\nB. FSH\n\n\n\nC. E2\n\n\n\nD. LH\n\n\n\nE. AMH\n\nA) Total effective rate. B) FSH, C) E2, D) LH and E) AMH levels. Note: SUCRA, surface under the cumulative ranking curves; FSH, follicle-stimulating hormone; E2, estradiol; LH, luteinizing hormone; AMH, anti-Müllerian hormone; TCM, Traditional Chinese medicine; HRT, hormone replacement therapy; Herb, herbal medicine; Catgut, catgut-embedding therapy; Acu, acupuncture; Moxa, moxibustion.\n\nNMA on FSH levels\n\nThe results of the NMA showing comparisons of different treatments based on improvements of FSH levels are shown in Figure 3B. The league table results of the NMA on FSH levels are presented in Figure 4B. A total of 28 RCTs (2,384 participants) with seven different treatment modalities were analyzed. The results of NMA indicate that the combination of HRT with herbal medicine and acupuncture achieved the highest efficiency in improving the FSH levels. According to SUCRA, the combination of HRT with herbal medicine and acupuncture (99.9%) had the highest effective rate, followed by HRT combined with herbal medicine (67.4%), acupuncture combined with moxibustion (54.9%), herbal medicine combined with acupuncture (49.6%), herbal medicine combined with catgut-embedding therapy (49.3%), herb alone (23.1%) and HRT only (5.8%). Table 4 (part B) and Figure 5B depict the rankings of these seven treatments.\n\nNMA on E2 levels\n\nThe results of the NMA showing comparisons of different treatments for the changes of E2 levels are shown in Figure 3C. The league table results for the E2 comparison are shown in Figure 4C. A total of 26 RCTs (2,108 participants) with seven different treatment modalities were analyzed. The results of NMA indicate that the combination of HRT with herbal medicine and acupuncture had the highest efficiency. It was also found that the combination of different treatment modalities pairing with herbal medicine were more effective than HRT alone. According to SUCRA, the combination of HRT with herbal medicine and acupuncture (92.6%) had the highest effective rate, followed by herbal medicine combined with catgut-embedding therapy (80.1%), herbal medicine combined with acupuncture (66.7%), HRT combined with herbal medicine (51.6%), herbal medicine alone (40.8%), HRT alone (15.1%) and acupuncture combined with moxibustion (3.2%). Table 4 (part C) and Figure 5C depict the rankings of these seven treatments.\n\nNMA on LH levels\n\nThe results of the NMA showing treatment comparisons based on the difference of LH levels are shown in Figure 3D. The league table results of the NMA for LH levels are shown in Figure 4D. A total of 19 RCTs (1,505 participants) with six different treatment modalities were analyzed. The results of NMA indicate that the combination of HRT with herbal medicine and acupuncture have the highest efficacy. It was also discovered that treatment modalities combined with herbal medicine are more useful than HRT alone. According to SUCRA, the combination of HRT with herbal medicine and acupuncture (99.9%) had the highest effective rate, followed by HRT combined with herbal medicine (71.0%), herbal medicine combined with catgut-embedding therapy (53.9%), herbal medicine alone (40.4%), herbal medicine combined with acupuncture (22.3%) and HRT alone (12.3%). Table 4 (part D) and Figure 5D presents the ranking of these six treatments.\n\nNMA on AMH levels\n\nThe results of the NMA showing treatment comparisons based on the improvement of AMH levels in five treatment modalities are shown in Figure 3E. The league table results regarding the comparison of the AMH levels are presented in Figure 4E. A total of 11 RCTs with reported AMH levels (1,030 participants) were analyzed. The results of NMA indicate that herbal medicine combined with catgut-embedding therapy had the highest efficiency and treatment modalities with the presence of herbal medicine are more effective compared to treatment without herbal medicine. According to SUCRA, herbal medicine combined with catgut-embedding therapy (67.2%) had the highest effective rate, followed by HRT combined with herbal medicine (66.7%), herbal medicine alone (65.8%), acupuncture combined with moxibustion (33.6%) and HRT alone (16.7%). Table 4 (part E) and Figure 5E depict the rankings of these five treatments.\n\nEvaluation of statistical inconsistency\n\nAlthough global inconsistency showed there that were no significant statistical differences for the outcomes, the node-splitting method was employed to further evaluate on the local inconsistency (Table 5). Overall, no statistical inconsistency for the outcomes were seen from the node-splitting outcome, except between direct and indirect comparisons of HRT with combination of HRT and herbal medicine, HRT with herbal medicine, HRT with combination of herbal medicine and acupuncture, herbal medicine with combination of HRT and herbal medicine, herbal medicine with combination of herbal medicine and acupuncture, as P<0.05 indicates for local inconsistency between the local comparisons. These might be due to ineffective sample size for the particular treatment comparisons.\n\n\n\nA. Total effective rate\n\n\n\nB. FSH\n\n\n\nC. E2\n\n\n\nD. LH\n\n\n\nE. AMH\n\nSmall-study effects\n\nThere was a lack of strong evidence on the small-study effects across outcomes based on total effective rate and FSH level (Figure 6A and B).\n\nA) Total effective rate. B) FSH, C) E2, D) LH and E) AMH levels. Note: FSH, follicle-stimulating hormone; E2, estradiol; LH, luteinizing hormone; AMH, anti-Müllerian hormone.\n\nHowever, among the outcome indicators of E2, LH and AMH (Figure 6C, D and E), it could be inferred that heterogeneity between studies were one of the reasons for those studies located on the lateral side of the funnel plot. Comparison-correction funnel plots based on E2, LH and AMH levels were made upon 26, 19 and 11 included studies, respectively (involving seven, six and five interventions, respectively). The asymmetrical funnel plots suggest probability of small-study effects in this study.\n\nSensitivity analysis\n\nFor the paired studies with significant heterogeneity (I2 > 50%), a sensitivity analysis was done by removing any single study one by one. Results showed that there were no significant changes, stipulating that the pooled results were consistent.\n\n\nDiscussion\n\nPrevious studies9,10,17 have demonstrated the efficacy of herbal medicine and acupuncture in treating POI. In this study, we utilized the advantages of NMA method to compare the efficacy of various TCM treatment modalities for this condition. Our findings revealed the comparative efficacy of six treatment modalities: HRT combined with herbal medicine and acupuncture, HRT combined with herbal medicine, herbal medicine combined with acupuncture, herbal medicine combined with catgut-embedding therapy, acupuncture combined with moxibustion and herbal medicine alone. Table 6 shows the compilation of SUCRA rank for each outcome measurement in NMA. Our results indicated that the combination of HRT with herbal medicine and acupuncture was the most effective treatment modality for patients with POI in the present study. These findings suggest that TCM treatment approaches could be considered as a promising therapeutic option for women with POI.\n\nTo the best of our knowledge, this study is the first systematic evaluation covering all treatment modalities in TCM to provide reliable evidence to determine the efficacy and feasibility of TCM treatment for patients with POI. There are a variety of different treatment modalities in TCM and all seem effective in treatment.11,18 However, until now there have been no consensus or standard procedure of TCM treatment recommended for POI. This scientific direct and indirect comparison can provide important guidance on the pros and cons of each intervention and their treatment priority.\n\nThere is no record about POI found in the TCM literature. However, comparing its clinical manifestations, POI can be categorized as “amenorrhoea”, “oligomenorrhoea” and “infertility” based on TCM perspective. The pathogenesis is mainly related to malnourishment of the thoroughfare and conception vessels, as well as the uterus, which might be due to congenital insufficiency, physical and mental overwork, or sexual overstrain. TCM treats a disease based on syndrome differentiation. Different treatment modalities such as oral administration of herbal decoction, acupuncture, catgut-embedding therapy, moxibustion, electroacupuncture, ear acupressure and herbal enema, are suitable for the treatment of POI. In our study, 33 clinical trials using TCM treatment were screened and the therapeutic effect of seven treatment modalities were analyzed. It has been proven that comprehensive treatment models are more effective in improving ovarian function, compared to just using HRT as sole treatment. This would provide clinical practitioners with strong and reliable evidence, as well as a useful alternative during clinical decision-making.\n\nWe also found that the integration of herbal medicine into POI treatment, such as its combination with HRT, acupuncture or catgut-embedding therapy, can significantly improve the ovarian functions by observing on indicators including total effective rate, FSH, E2, LH and AMH levels. Therefore, herbal medicine may be considered as an intervention when HRT alone, the standard procedure recommended by recognized guidelines worldwide,1,4,5 cannot effectively cure POI. This study possesses significance in providing clinical guidance.\n\nAcupuncture has been widely applied on POI treatment particularly in China.10,11,18 It has been demonstrated that acupuncture is safe and effective.19–21 Through early intervention of acupuncture, it has also been found that acupuncture can increase pregnancy rate significantly.22,23 Our study has demonstrated that application of acupuncture in the treatment of POI is also useful to observe clinical improvement. However, it is suggested that acupuncture must be combined with herbal medicine and even HRT in order to optimize the therapeutic effects. This recommended treatment modality not only demonstrates the advantage of TCM treatment, it also provides a potential practical, safe and low-cost treatment option for both the patients and clinicians.\n\nIn this study, we found a shortage of rigorous clinical trials adhering to the international requirements, as well as a paucity of heterogeneity of study candidates, interventions, controls, and outcome measures, which challenge the process of systematic review and NMA. Due to inadequate data from the included studies, the safety of each intervention could not be evaluated. The risk of bias assessment in this study indicates that most of the studies did not clearly state the blinding of outcome assessors, which might impact on the reliability of data analysis outcome.\n\n\nConclusions\n\nIn conclusion, herbal medicine combined with catgut-embedding therapy was shown to be the most effective treatment model to improve total effective rate and AMH levels in the present study. The combination of HRT, herbal medicine and acupuncture was demonstrated to be the most effective treatment model for improving serum FSH, E2 and LH levels. An overview on SUCRA analysis outcomes showed that comprehensive treatment models with the integration of herbal medicine seemed more prone to improve ovarian functions. Monotherapies, such as solely using HRT or herbal medicine treatment, were found to have no obvious advantages on their curative effect for POI. The integration of herbal medicine to conventional HRT may be a favorable approach for enhancing the clinical utility of HRT in the management of POI. RCTs involving multiple centers and different countries are essential to further verify the conclusions of this study, which can also create international awareness on the efficacy of TCM treatments for POI.", "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-NMA checklist for ‘Identifying the most effective Traditional Chinese Medicine treatment modalities for premature ovarian insufficiency: a systematic review and network meta-analysis’. https://doi.org/10.6084/m9.figshare.22567846. 57\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nWebber L, Davies M, Anderson R, et al.: ESHRE Guideline: management of women with premature ovarian insufficiency. Human Reproduction. 2016; 31(5): 926–937. 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Publisher Full Text" }	[ { "id": "196644", "date": "01 Sep 2023", "name": "Muhammad Shahzad Aslam", "expertise": [ "Reviewer Expertise Phytochemistry", "Complementary and alternative medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAccording to authors: Acupuncture has been widely applied on POI treatment particularly in China.10,11,18 It has been demonstrated that acupuncture is safe and effective.19–21 I suggest to broaden the discussion in context to use of Acupuncture in Malaysia, US. We all know its effectiveness on POI treatment in China. Please read literature and include more reference from different geography. If no study in other geography found then given give rational.\n\nProvide rational to use SUCRA Rank for each outcome measurement in NMA..I suggest to compare it with Bayesian Model and Averaging, Rank-Heat Plot.\n\nThe study conclusion must present with future direction. Please include more paragraph.\n\nIs there any comparison given with best TCM TM for POI with pharmacotherapy? The discussion must contain a couple of paragraph to provide rational and treatment comparison.\n\nPlease provide in-depth study limitation in separate paragraph.\n\nPlease provide Prisma Checklist as supplementary file.\n\nPlease suggest to include one heading how to improve the quality reporting of POI. Most of TCM treatment has similar issues. It is mandatory to provide solution. Please elaborate your discussion and specify to POI treatment. Acceptability of TCM treatment is a big issues. I hope you understand my point and add some heading inside discussion.\n\nIf possible i recommend to include proposed Hypothetical mechanism of TCM TM in POI disease.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] }, { "id": "266528", "date": "04 Jun 2024", "name": "Cristina Laguna Benetti-Pinto", "expertise": [ "Reviewer Expertise premature ovarian insufficiency" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI reviewed the manuscript “Identifying the most effective Traditional Chinese Medicine treatment modalities for premature ovarian insufficiency: a systematic review and network meta-analysis” . The authors aimed to evaluate and compare the clinical efficacy of different modalities of Traditional Chinese Medicine (TCM) for the treatment of POI and identify the most effective treatment.\nThe authors registered the systematic review and declared to follow the PRISMA-NMA. They used 6 databases (three English databases and three Chinese databases). Only Traditional Chinese Medicine randomized controlled trials were included, with women treated with herbal medicine, acupuncture, moxibustion and catgut embedding therapy alone or in combination with conventional HT.\nThe effectiveness of treatments was to be evaluated according to clinical manifestation, menstrual cycle, FSH and Estradiol levels, and LH and AMH levels.\nThe issue is interesting and there are gaps in the knowledge of effectiveness of complementary IOF treatment.\nMy major concern is on the outcomes of the treatments. It is not clear who were the POI-women investigated. Why analyze the AMH levels in POI due to gonadal dysgenesis or after an oophorectomy, for example? Is mandatory to identify the causes of POI in each study included. Furthermore, the reduction in FSH, LH are not good outcomes to evaluate the best treatment. The global consensus indicates to evaluate clinical aspects as hot flushes, improvement in sexual function or in vaginal dryness, improvement in bone mass. Theses aspects were not showed. Hence, my suggestion is to change the aim and the title of the reviewed to “Identifying the effectiveness of TCM treatment in changing some hormonal aspects related to POI”\nI have some other concerns that are listed below:\n1.Title – see comments above 2.Abstract – see comments above and change 3.Introduction - -Page 3, first paragraph: the authors included a POI prevalence of about 10%. This rate is not in accordance with the literature or even with the reference cited [3]. Please review and correct (in reference number 3, the prevalence of POI is 1-3%). The authors also cited the use of AMH levels to measure ovarian reserve. AMH measurement is only indicated in specific cases of POI, if the final diagnosis is not completely certain, especially when FSH levels are not stable and above 25 U/L. I suggest a review of these concepts.\nPage 3: “Hence, patients often seek out complementary medicine to improve their ovarian functions and increase chances of conception” – what is the idea of this paragraph? HT is the first and best treatment whenever the woman has no absolute contraindication, which is not frequent. In these cases, complementary treatment or alternative treatment generally aims to minimize the effects of hypoestrogenism. In most POI etiologies, it is unfortunately not possible to improve ovarian function. Please, clarify/rewrite the paragraph.\n4.Methodology and Results Page 6, PRISMA flowchart – please, in the studies included, mention in the figure which database they are from.\nThe authors mentioned that the diagnosis criteria were according to ESRHE guideline OR according Chinese expert consensus on POI. Please, include in the manuscript these criteria (the last article was published in Chinese. These criteria are important to comprehension of systematic review.\nDiscussion:\nPage 19. “Our findings revealed the comparative efficacy of six treatment modalities: HRT combined with herbal medicine and acupuncture, HRT combined with herbal medicine, herbal medicine combined with acupuncture, herbal medicine combined with catgut-embedding therapy, acupuncture combined with moxibustion and herbal medicine alone.” - see “major concern”. And what kind of HT?\nPage 19: “However, comparing its clinical manifestations, POI can be categorized as “amenorrhoea”, “oligomenorrhoea” and “infertility” based on TCM perspective. The pathogenesis is mainly related to malnourishment of the thoroughfare and conception vessels, as well as the uterus, which might be due to congenital insufficiency, physical and mental overwork, or sexual overstrain. TCM treats a disease based on syndrome differentiation.” Please, make clear these concepts. I am not able to understand, according to global consensus of POI.\nPage 19. “Therefore, herbal medicine may be considered as an intervention when HRT alone, the standard procedure recommended by recognized guidelines worldwide,1,4,5 cannot effectively cure POI” – This conclusion is not supported bye the results of this review or by the global consensus. The idea is not clear to me. Please, reviewed.\nAlthough the systematic review was methodologically well done, there are conceptual errors, and conclusions aren’t in line with the recommendations of global consensus. If there is by the authors an extensive revision of the study, with changes in the concepts, aims and conclusions, the manuscript can be submitted to a new analysis.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? No\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/12-450
https://f1000research.com/articles/11-1031/v2	27 Apr 23	{ "type": "Data Note", "title": "Effect of eel and tempe composite flour supplementation on the nutritional status biomarkers of rats with a restricted protein diet: Data from a preclinical trial", "authors": [ "Feny Mentang", "Nurmeilita Taher", "Fahrul Nurkolis", "William Ben Gunawan", "Vincentius Mario Yusuf", "Nindy Sabrina", "Faqrizal Ria Qhabibi", "Nelly Mayulu", "Nurpudji Astuti Taslim", "Happy Kurnia Permatasari", "Nurmeilita Taher", "Fahrul Nurkolis", "William Ben Gunawan", "Vincentius Mario Yusuf", "Nindy Sabrina", "Faqrizal Ria Qhabibi", "Nelly Mayulu", "Nurpudji Astuti Taslim", "Happy Kurnia Permatasari" ], "abstract": "Incorporating eels and tempe can replace and complement the content of proteins, macronutrients, and micronutrients, which may be related to curative effects for malnutrition. In addition, converting the ingredients into a form of flour can increase their shelf life and nutrient concentration. Therefore, an in vivo approach was undertaken to explore further the nutritional status value of biomarkers in malnourished male rats (Rattus norvegicus) after Eel and Tempe Composite (ETC) flour supplementation. Data was collected from blood samples (both plasma and serum) of rats in all groups, and the appropriate biomarkers were analyzed. The final data presented in this study is openly available and can be further analyzed using statistical means to determine the dose of ETC flour as the basis of clinical trials, which other researchers can reproduce. This data may also be valuable for those interested in using different analytical methods to research the same questions or even new preclinical studies focusing solely on nutritional status biomarker analysis methods, including clinical trial prospects.", "keywords": [ "Nutraceuticals", "Nutritional Status", "Protein Diet", "Preclinical Trial", "Eel", "Tempe", "IGF-1" ], "content": "Abbreviations\n\nBW: Body Weight\n\nCIOMS: Council for International Organizations of Medical Sciences\n\nETC: Eel and Tempe Composite\n\nIGF-1: Insulin-Like Growth Factor 1\n\nNP: Normal-Protein\n\nRBP: Retinol-Binding Protein\n\nRP: Restricted-Protein\n\n\nIntroduction\n\nProlonged protein undernutrition can result in malnutrition, anemia, and stunting (Wu, 2016). Malnutrition can manifest through many clinical parameters and biomarkers. The most-reported blood biomarkers of malnutrition are total protein and hemoglobin (Zhang et al., 2017), while serum insulin-like growth factor 1 (IGF-1) have been underreported (Keller, 2019). There is strong evidence that functional meals can help prevent and mitigate the effects of malnutrition (Ahmed et al., 2022), presumably by improving malnutrition biomarkers.\n\nAgricultural products such as soybean (Glycine max L.) tempe is the potential source of protein and other nutrients which may improve malnutrition, anemia, and gut health due to their folate, iron, vitamin B12, probiotics, and isoflavone content (Ahnan-Winarno et al., 2021). In addition to tempe, one of the fishery products that can be used as an alternative protein-rich functional food is eel fish (Monopterus albus) which has immense nutritional contents that is high in vitamin A, vitamin E, and unsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) (Nafsiyah et al., 2018). A recent review also highlighted the role of unsaturated fatty acids on protein metabolism, synthesis, and muscle growth (López-Seoane et al., 2022), which are crucial in a state of malnutrition.\n\nCombining eel and tempe may substitute and complement the protein, macronutrients, and micronutrient content, which may be linked with their malnutrition ameliorating effects. Moreover, turning an ingredient into the form of flour may increase its shelf life and nutrient concentration (Tomasi et al., 2015). Therefore, an in vivo approach was performed to further explore the value of nutritional status biomarkers on malnourished rats after a supplementation of Eel and Tempe Composite (ETC) flour. A review study by Ahmed et al. (2022) stated that only a few of the studies reported a strong link between functional foods and malnutrition.\n\nIn this study, the data was collected from the blood samples (both plasma and serum) of rats in all treatment groups, and the appropriate biomarkers were analyzed, accordingly. The final data presented in this study can be further analyzed using statistical means to define an ETC flour dose as a basis for the clinical trial, which other researchers could reproduce. This data may also be valuable for those interested in using other analytical methods to research the same research questions or even new preclinical studies focusing solely on biomarker nutrition status analysis methods, including clinical trial prospects.\n\n\nMethods\n\nThe protocol for this preclinical study refers to the Animal Research: Reporting in vivo Experiments (ARRIVE) Guidelines by the Institutional Animal Care and Use Committee. See the Reporting guidelines section (Nurkolis and Gunawan, 2022a) for the completed checklist. The study also adheres to the Declaration of Helsinki by The Council for International Organizations of Medical Sciences (CIOMS). This study protocol has been registered at Preclinical Trials Europe preclinicaltrials.eu (PCTE0000271). Ethical approval was obtained from the Ethics Committee at Educational General Hospital, Sam Ratulangi University (approval number 039/EC/KEPK-KANDOU/III/2022). Every effort was taken to keep the experimental animals as pain-free as possible throughout the trial and by maintaining laboratory conditions at room temperature and noise-free.\n\nA total of 1,000 g of fresh Asian swamp eel (Monopterus albus) were purchased live from a local market in Jakarta. The live eels were placed in a bucket measuring 21 cm in diameter with a volume capacity of 5 L (pH 5.5) and acclimatized for 24 hours in a normal light-dark cycle before being euthanized. The Asian swamp eel were euthanised using a slaughter process such as fish processing and cleaned (including removal of the bone) and steamed for 10 minutes. After drying for 12 hours at 60 °C, the meat was grounded using a meat mincer. Then, 1,000 g of soy-based tempe was chopped and steamed for 20 minutes. Before being ground, the tempe sample was steamed and then baked for 12 hours at 60 °C. Both eel and tempe were filtered using a 60-mesh filter. According to the specifications of a previously published preliminary study (Ngadiarti et al., 2022), a formulation with the highest level of folic acid (vitamin B9; 1258.53 ± 1.39 ug/100 g) and unsaturated fatty acids (20.83 ± 0.12%) was prepared by combining eel and tempe flour samples in 1: 3.5 ratio. A complete formulation of ETC flour and the results of proximate amino acids, unsaturated fatty acids, and folate contents were presented by Ngadiarti et al. (2022). The ETC formulations were conducted by Ngadiarti et al. (2022) at the Laboratory of the Ministry of Health Polytechnic Jakarta II (Poltekkes Kemenkes Jakarta II), Jakarta 12540, Indonesia (Ngadiarti et al., 2022).\n\nThirty male Albino Wistar rats (Rattus norvegicus) weighing 40.5 – 5.4 g, aged 3 – 4 weeks, were put in cages under typical laboratory settings (27 ± 2.2 oC) with normal light and dark cycles (12/12 hours). Before the experiment, all rats were acclimatized for a 10-day period. The minimum sample size was determined using the Frederer formula, with the equation = (t-1) (r-1)≥15. t = treatment, r = replication. t in this study is 3, then = (3-1) (r-1) ≥ 15; 2(r-1) ≥ 15; 2r-2 ≥ 15; r ≥ 8.5. So the minimum sample for each group is 8.5. The research team chose 10 (just in case there were rats that needed to be excluded, such as those experiencing moody eating and drinking) (Federer, 1966).\n\nAccording to de Oliveira et al. (2011), all rats were supplemented with restricted-protein diet treatment (RP; 4% w/w protein) for four weeks and separated into three groups, with 10 rats per group. Shuffled blinded envelopes simple randomization (Taken at random from the population). We consider no confounders in this study since they were controlled by choosing the gender of the rats (male, not mixed) and the trials in the laboratory were carried out by a professional veterinary in animal studies. The allocation process and experiment were conducted by NM with the assistance of a professional veterinary. The assessment of the outcomes and data analysis were done by FN and FM. Rats that experienced moody eating and drinking and exhibited stress were excluded. Group C (control) was given an ad libitum diet of normal protein (NP; 23% w/w protein), but did not supplement with ETC. Rats in groups A and B got the same RP diet for 4 weeks but were orally given ETC dosages of 100 and 200 mg/kgBW. These dosages were based on the lower and higher limits for stomach safety in rats (Eichenbaum et al., 2011). Blood samples were taken and analyzed for their protein, hemoglobin, and IGF-1 serum levels. The flowchart of this study is presented in detail in Figure 1.\n\nIGF-1, insulin-like growth factor 1.\n\nPrimary outcomes for this preclinical trial are defined as:\n\n1. Protein (mg/dL)\n\n2. Hemoglobin (mg/dL)\n\n3. IGF-1 (pg/mL)\n\nSecondary outcomes for this preclinical trial define as:\n\n1. Body Weight or BW (g) = Initial Body Weight (g); Final Body Weight (g) and Weight Gain (g/day)\n\n2. Erythrocytes (×106/uL)\n\n3. Haematocrit (%)\n\n4. Retinol-Binding Protein (RBP, ng/mL)\n\nThe details of feed composition are shown in Table 1.\n\n* The combination of salts and vitamins used in diet formulation is referred to AIN 93 recommendation (de Oliveira et al., 2011).\n\nAt the end of the intervention, the rats were fasted overnight and given ketamine anesthesia. The dose used was ketamine at a dose of 75 mg/kg Body Weight and referred to https://www.research.uky.edu/division-laboratory-animal-resources/recommended-anesthesia. To allow clotting at room temperature, a 2.5 mL blood sample is obtained from liver tissue and kept in a dry, clean tube without anticoagulants. Hemoglobin, hematocrit, and erythrocyte levels were determined from the blood plasma samples based on the method introduced by Arnaud et al. (2017) (see also the Biomedical analysis section for a brief description). The sample was then centrifuged for 20 minutes at 3,000 rpm to extract the protein, IGF-1, and Retinol-Binding Protein (RBP) from the blood serum for further analysis.\n\nA fresh blood plasma sample was used to evaluate the hemoglobin levels using the Rat Hemoglobin ELISA Kit (#AB157733). The sample was rinsed in a 1% phosphate buffer salt (PBS, pH 7.4) solution until the liquid cleared. The sample was then concentrated for 20 minutes at 3,000 rpm to produce the supernatant component. Protein levels and IGF-1 were measured with The Rat Protein Assay ELISA Kit (#MBS3808613) and The Rat IGF-1 ELISA Kit (#MBS268050) at a wavelength of 450 nm. Erythrocyte and hematocrit levels are measured using the Rat Erythrocyte Protoporphyrin ELISA Kit (#RTES01121).\n\n\nDataset\n\nThis in vivo research was successfully carried out, and the raw data from the research can be accessed via the Underlying data section (Nurkolis and Gunawan, 2022b).\n\nTable 2 proved that undernourished rats supplemented with a higher dose of ETC flour (group B) would have a higher protein, hemoglobin, and IGF-1 serum than groups A and C. This data suggests that ETC flour may improve nutritional status biomarkers via a dose-dependent manner.\n\nThe data presented in Table 3 revealed a higher increase in weight gain in group B than in group A and C. All secondary outcomes and characteristics, except for food intake, were best in group B. These findings reinforce the previous statement that ETC flour works in a dose-dependent manner.\n\n1 Food Efficiency Ratio (FER, %) = (Body Weight Gain (g/day) /Food Intake (g/day)) × 100.\n\n2 Retinol-Binding Protein (RBP, ng/mL).\n\n\nData availability\n\nFigshare: RAW Data for An Eel and Tempe Composite Flour Supplementation on the Nutritional Status Biomarkers of Rats with A Restricted Protein Diet.xlsx, https://doi.org/10.6084/m9.figshare.19782784.v1 (Nurkolis and Gunawan, 2022b).\n\nThis project contains the following underlying data:\n\n- RAW Data for An Eel and Tempe Composite Flour Supplementation on the Nutritional Status Biomarkers of Rats with A Restricted Protein Diet.xlsx (The measured serum protein, hemoglobin, and IGF-1 levels from each group of rats observed in this study).\n\n\nReporting guidelines\n\nFigshare: ARRIVE Checklist for ‘Effect of eel and tempe composite flour supplementation on the nutritional status biomarkers of rats with a restricted protein diet: Data from a preclinical trial’, https://doi.org/10.6084/m9.figshare.19799731.v2 (Nurkolis and Gunawan, 2022a).\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\nSpecial thanks to Professor Dr. Nurpudji Astuti Taslim, MD., MPH, Sp.GK (K) (Chairman of the Indonesian Association of Clinical Nutrition Physicians), for the motivation and supervision in conducting this research during the pandemic.\n\n\nReferences\n\nAhmed M, Vasas D, Hassan A, et al.: The impact of functional food in prevention of malnutrition. PharmaNutrition. 2022; 19: 100288. Publisher Full Text\n\nAhnan-Winarno AD, Cordeiro L, Winarno FG, et al.: Tempeh: A semi centennial review on its health benefits, fermentation, safety, processing, sustainability, and affordability. Compr. Rev. Food Sci. Food Saf. 2021; 20: 1717–1767. PubMed Abstract \| Publisher Full Text\n\nArnaud F, Higgins A, McCarron R, et al.: Determination of methemoglobin and hemoglobin levels in small volume samples. Artif Cells Nanomed. Biotechnol. 2017 Feb; 45(1): 58–62. PubMed Abstract \| Publisher Full Text\n\nde Oliveira JC , Scomparin DX, Andreazzi AE, et al.: Metabolic Imprinting by Maternal Protein Malnourishment Impairs Vagal Activity in Adult Rats. J. Neuroendocrinol. 2011; 23(2): 148–157. PubMed Abstract \| Publisher Full Text\n\nEichenbaum G, Damsch S, Looszova A, et al.: Impact of gavage dosing procedure and gastric content on adverse respiratory effects and mortality in rat toxicity studies. J. Appl. Toxicol. 2011 May; 31(4): 342–354. PubMed Abstract \| Publisher Full Text\n\nFederer WT: Randomization and sample size in experimentation.1966.Reference Source\n\nKeller U: Nutritional Laboratory Markers in Malnutrition. J. Clin. Med. 2019; 8(6): 775. PubMed Abstract \| Publisher Full Text\n\nLópez-Seoane J, Jiménez SL, Del Coso J, et al.: Muscle hypertrophy induced by N-3 PUFA supplementation in absence of exercise: a systematic review of randomized controlled trials. Crit. Rev. Food Sci. Nutr. 2022 Jan 28; 1–1.\n\nNafsiyah I, Nurilmala M, Abdullah A: Nutrient Composition of Eel Anguilla bicolor bicolor and Anguilla marmorata. J. Pengolah. Hasil Pernikan. Indones. 2018; 21: 504. Publisher Full Text\n\nNgadiarti I, Nurkolis F, Handoko MN, et al.: Physicochemical Properties and Nutrient Content of Tempe Flour Enriched Eel Flour. Open Access Maced. J. Med. Sci. 2022 Jan. 18; 10(A): 552–556. Publisher Full Text\n\nNurkolis F, Gunawan WB: Arrive Checklist for An Eel and Tempe Composite Flour Supplementation on the Nutritional Status Biomarkers of Rats with a Restricted Protein Diet. figshare. Online resource.2022a. Publisher Full Text\n\nNurkolis F, Gunawan WB: RAW Data for An Eel and Tempe Composite Flour Supplementation on the Nutritional Status Biomarkers of Rats with A Restricted Protein Diet.xlsx. figshare [Dataset].2022b. Publisher Full Text\n\nTomasi N, Pinton R, Dalla Costa L, et al.: New ‘solutions’ for floating cultivation system of ready-to-eat salad: A review. Trends Food Sci. Technol. 2015; 46(2): 267–276. Publisher Full Text\n\nWu G: Dietary protein intake and human health. Food Funct. 2016 Mar; 7(3): 1251–1265. Publisher Full Text\n\nZhang Z, Pereira SL, Luo M, et al.: Evaluation of Blood Biomarkers Associated with Risk of Malnutrition in Older Adults: A Systematic Review and Meta-Analysis. Nutrients. 2017; 9(8): 829. PubMed Abstract \| Publisher Full Text" }	[ { "id": "349156", "date": "23 Dec 2024", "name": "Sri Widia Ningsih", "expertise": [ "Reviewer Expertise Biomarker development", "food biochemistry", "biomaterial 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\nSome notes for this study 1. Experimental design. It would be much better if there were a negative control in the in vivo treatment of mice that could be administered as a low-protein diet or food containing no protein at all as a placebo (such as amylum) to reinforce the fact that the positive results of this experiment are due to the test conditions and not external factors. 2. Biomedical Analysis. There was no standard interpretation for the measurement results of hemoglobin, protein, IGF-1, Erythrocytes, hematocrit, or Retinal-Binding Protein (RBP) levels in the blood of mice (whether these are low, normal, or high). This raises the question of whether the measurement results can be applied to all rats.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "342652", "date": "24 Dec 2024", "name": "Bernadette-Emőke Teleky", "expertise": [ "Reviewer Expertise Knowledge in creating and assessing food products for enhanced nutrient delivery", "such as composite flours", "and nutrition science (functional foods)." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 investigates the impact of eel and Tempe composite (ETC) flour supplementation on the nutritional biomarkers of malnourished rats fed a protein-restricted diet. The study highlights ETC flour's potential to improve protein, hemoglobin, and IGF-1 levels, offering a promising nutraceutical approach to combating malnutrition in a dose-dependent manner.\n“in vivo” - Please correct the scientific names to italics (Methods section – Ethics statements – line 1) a statistical analysis could have been done to see the differences between the three groups (Tables 2 and 3). A short discussion would have been welcome\n\nIs the rationale for creating the dataset(s) clearly described? Partly\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": [] } ]	2	https://f1000research.com/articles/11-1031
https://f1000research.com/articles/11-1287/v1	10 Nov 22	{ "type": "Research Article", "title": "Effectiveness of nifedipine, labetalol, and hydralazine as emergency antihypertension in severe preeclampsia", "authors": [ "Donel S", "Dhini Aiyulie Novri", "Yulis Hamidy", "Maya Savira", "Dhini Aiyulie Novri", "Yulis Hamidy", "Maya Savira" ], "abstract": "Background: Preeclampsia is a highly prevalent disease among pregnant women. In the event of hypertensive emergency, nifedipine, labetalol, and hydralazine are assigned as first-line therapies in preeclampsia. Further studies are needed to compare the effectiveness of these drugs to find the most cost-effective drug with minimal side effects. This study aimed to compare the effectiveness of these drugs in lowering blood pressure during hypertensive emergencies in severe preeclampsia. Methods: 60 pregnant women with severe preeclampsia were recruited in this multiple centre double-blind randomized clinical trial from May 2021 to April 2022 in Indonesia. The patients were divided equally into three groups and treated with three doses of nifedipine, labetalol, and hydralazine, respectively within one hour with 20 minutes interval. The effectiveness was measured based on systolic and diastolic blood pressures, and mean arterial pressure (MAP). The observation was carried out until five hours post-third dose administration.\n\nResults: The blood pressure was reduced significantly after the administration of the first to the third dose of each antihypertensive (p<0.05). A single dose administration, four, one, and three patients had 20% MAP reduction in nifedipine, labetalol, and hydralazine group. Three, seven, and one patient had a failure of reaching 20% MAP reduction even after receiving the third dose. The effectiveness of the drugs to achieve 20% reduction of MAP could be ranked as follows: nifedipine>labetalol>hydralazine (57.49%, 42.13%, and 40.87%, respectively) for single dose and hydralazine>nifedipine>labetalol (111.3%, 85.12%, and 90.04%, respectively) for triple dose. Conclusions: Nifedipine is the most effective drug to reduce the blood pressure when single dose administration is used, but requires more doses to further reduce the blood pressure. Hydralazine is the most effective when the drug administration is maxed up to three doses within 60 minutes with 20 minutes interval. Thai Clinical Trials Registry (TCTR): TCTR20221014007 (14/10/2022)", "keywords": [ "Hypertension", "gestation", "systolic blood pressure", "diastolic blood pressures", "mean arterial pressure", "preeclampsia" ], "content": "Introduction\n\nPreeclampsia and eclampsia are major problems in obstetric cares. Preeclampsia is one of the main causes of maternal morbidity and mortality in addition to infection and bleeding with the prevalence rate between 5–8% among pregnant women.1 The main cause of preeclampsia is unknown and the disease is therefore also called the disease of theory. As a result, the guidelines of preeclampsia management are updated and changed from time to time.2 Research continues to be updated to find the best formulation for diagnosis and management with the main aim to provide the management with the most optimal outcome for the mother and foetus. Termination of pregnancy is still considered the gold standard in severe preeclampsia, but this becomes a problem when the pregnancy is still premature as it can increase the infant mortality rate.1\n\nHigh blood pressure is the main indicator in the diagnosis of preeclampsia. Treatment of severe preeclampsia requires anticonvulsant and antihypertensive drugs. The purpose of administering antihypertensive drugs is to prevent cerebral bleeding. The American Congress of Obstetricians and Gynaecologists (ACOG) states that pregnant women with systolic blood pressure 160 mmHg and/or diastolic 110 mmHg that persist for 15 minutes should be considered an emergency condition and receive antihypertensives immediately, no later than 30 minutes.2 To date, ACOG has offered nifedipine, labetalol, or hydralazine as the antihypertensive emergency in preeclampsia.2 In June 2020, ACOG issued a statement that women with gestational hypertension presenting with high blood pressure (severe range) should be treated as same as severe preeclampsia.1\n\nAn indicator of a decrease in blood pressure in patients with severe preeclampsia is the mean arterial pressure (MAP). MAP is obtained by the formula of twice the diastolic pressure plus one systolic pressure, then divided by three.3 The target for MAP reduction in patients with severe preeclampsia is around 20–25%.3 This aims to maintain adequate uteroplacental circulation for the foetus. If blood pressure is reduced too low, there will be a sharp decrease in placental perfusion resulting in foetal distress, while the main goal of the management of preeclampsia is not only centred on the mother, but also on the outcome of the baby.\n\nRecommendations for antihypertensive drugs in preeclampsia are growing rapidly. Multiple drugs are included and excluded from the list of drugs of choice for the treatment of severe preeclampsia. Labetalol and intravenous hydralazine have long been considered as first-line therapy in hypertensive emergencies, severe hypertension in pregnant women, and in the postpartum period.4 Although there is little information regarding the use of calcium channel blockers, several studies have shown that nifedipine can also be considered as first-line therapy, especially when intravenous access is difficult.2 However, further studies are needed to compare the effectiveness of these three drugs with the aim of finding cost-effective drugs with minimal side effects, and right on target.\n\nPrevious studies on the effectiveness of these three drugs showed mixed results.4,5 Hydralazine and labetalol are considered to have a balanced effectiveness and side effect in lowering blood pressure in preeclampsia.4,6 However, labetalol lowered blood pressure more rapidly than hydralazine.4 Another study comparing nifedipine and labetalol showed that these two drugs had the same effectiveness in lowering blood pressure in preeclampsia, but single-use nifedipine was more effective than labetalol.5\n\nIn our preliminary study, the emergency antihypertensive drugs in severe preeclampsia that are often used in the Arifin Achmad Hospital, Riau Province of Indonesia and the affiliated hospitals of Faculty of Medicine, Universitas Riau, are nifedipine and methyldopa. There has never been a standardized study or observation on the safety and effectiveness of antihypertensive drugs in Indonesian population. Therefore, this study was conducted to compare the effectiveness of oral nifedipine, and intravenous labetalol and hydralazine as antihypertensives in severe preeclampsia.\n\n\nMethods\n\nThis study was a double-blind, randomized clinical trial to compare the effectiveness of oral nifedipine, intravenous labetalol, and intravenous hydralazine as antihypertensive emergency in severe preeclampsia. The multicentre study was conducted at four hospitals at Riau Province of Indonesia, Arifin Achmad Hospital, Tengku Rafian Siak Sri Indrapura Hospital, Dumai Hospital, and Bengkalis Hospital, from May 2021 to April 2022.\n\nThe protocol of the study was approved on 28 May 2021 by the Medical and Health Research Ethics Unit, Faculty of Medicine, Universitas Riau with the number B/045/UN19.5.1.1.8/UEPKK/2021. This clinical trial has been registered at Thai Clinical Trials Registry (TCTR) with identification number is TCTR20221014007 (approved on 14 October 2022). This clinical trial was registered retrospectively in TCTR since the protocol of the study has been registered locally and this following the Indonesia law. All patients gave written informed consent before the enrolment. This study is reported in line with the CONSORT guidelines.20\n\nThe severe preeclampsia patients were recruited consecutively from populations that met the inclusion criteria. The minimal sample size of the study was 13 patients. This number based on calculation based on formula:\n\nHowever, a total of 20 severe preeclampsia patients were recruited for each group to prevent underpower due to drop-out and therefore there were 60 patients included in this study. The severe preeclampsia was defined as pregnant women with systolic blood pressure ≥160 mmHg or diastolic ≥110 mmHg that persisted for 15 minutes. Inclusion criteria were: (a) severe preeclampsia patient with 28–34-week gestation; (b) had live foetus; (c) upper arm circumference 23.5–33 cm; and (d) haemoglobin level at least 10.5 g/dL. All severe preeclampsia patients with decreased consciousness; had complications such as eclampsia, HELLP syndrome, kidney failure, or acute pulmonary oedema; in labour; received antihypertensive therapy in the last 12 hours; had an allergy to the trial drugs; and had asthma and heart disease were excluded. In this study the drop-out criteria were: the mother experienced allergy symptoms, had labour, or emergency symptoms such as placental abruption and foetal distress during the study.\n\nAll severe preeclampsia patients who came to the hospitals were subjected to anamnesis, physical and laboratories examination and then selected based on predetermined inclusion criteria. Blood pressure measurements were carried out using a digital sphygmomanometer with the Omron 7600T brand. The patient was asked to sit and rest for 10 minutes, the measurement was conducted twice on the arm with a distance period of 15 minutes, and the data taken was the second measurement. If the patients met the requirements, they were asked to participate in the study and all patients have to sign the written informed consent before the enrolment. The assigned group was computerized randomized using Randomizer software. Simple randomization was employed. In brief, 60 kits were prepared by the authors and were labelled 1 to 60 and each of kit containing three tables and three ampoules with the same sizes and colours. Kits no 1–20, 21–40 and 41–60 contained nifedipine, labetalol and hydralazine, respectively. For example, for one nifedipine kit, it contained 3 nifedipine tablets and 3 ampoules of placebo liquid. An author created the random sequence list consisting who will receive the kit no 1 until 60 based on random sequences provided by Randomizer software. For example, the patient who arrived first and second to the hospital should be given kit no 41 and 23. Each drug was administered 3 doses within 60 minutes (i.e., 20 minutes interval). Each dose of the drug contained: oral nifedipine (20 mg), intravenous labetalol (20 mg in 10 ml of sodium chloride 0.9%) and intravenous hydralazine (10 mg in 10 ml of sodium chloride 0.9%). Since the study consisted multiple healthcare centers, patient enrolment was conducted in one hospital at one time based on allocated time. Therefore, there were no separation of the sequence list between centres.\n\nThe enrolment of the patient was conducted by the healthcare workers in each centre. Health care workers and patients did not know the drugs contained in the kit. Health workers were instructed to administrate one pair of drugs (i.e., one tablet and one ampoule injection; this contained one drug and one placebo) for each time. The blood pressure was assessed 20 minutes after each administration. If the blood pressure did not meet the MAP target, the second pair of drugs (one tablet and one ampoule injection) was given and the blood pressure was reassessed in 20 minutes until the third pair of the drugs. The technique of administering magnesium sulphate and dexamethasone was uniform in all patients. If the MAP was nor reached after the third dose, the patient was treated with other drugs and excluded from the final analysis.\n\nThe end points of this study were: (a) systolic blood pressure; (b) diastolic blood pressure; (c) MAP; and (d) side effects. The blood pressure was measured seven times as follows: (1) minute 0 (pre-treatment); (2) 20 min post-first dose; (3) 20 min post-second dose (i.e., 40 min post-first dose); (4) 20 min post-third dose (i.e., 60 min post-first dose); (5) 2 h post-first dose; (6) 4 h post-first dose; and (7) 6 h post-first dose.\n\nCharacteristics of patients from each group were tested for normality. If the distribution was normal, the data were analysed using one-way ANOVA and followed by post hoc analyses. If the distribution was not normal, the data were analysed using Kruskal-Wallis and Friedmann followed by post hoc analyses. Comparison of the effectiveness of the three drugs was tested using N-Gain analysis. To determine how far the differences exist, Cohen's d effect size analysis was used. Further analysis with Kaplan-Meier survival was conducted. Statistical analysis was performed using SPSS software version 22 (Statistical Package for Social Sciences, Chicago, IL, USA).\n\n\nResults\n\nThe study was conducted on 60 patients with severe preeclampsia and they were divided into 3 groups: 20 patients in the nifedipine group, 20 patients in the labetalol group, and 20 patients in the hydralazine group.19 Patients were recruited from the population that met the inclusion criteria and were willing to participate in the study by signing an informed consent. Patients were then monitored closely and periodically according to the study protocol. The number of patients for each step of enrolment, allocation, follow-up and analysis are presented in Figure 1.\n\nWe recruited 60 severe preeclampsia patients and were divided randomly to receive nifedipine, labetalol and hydralazine. The characteristics of the patients including age, gravida, gestational age (gestation), upper arm circumference (UAC), systolic, diastolic, MAP, pulse, and foetal heart rate of each group are presented in Table 1. Our data indicated that there is uniformity of characteristics between the three groups.\n\na Analysis was conducted using ANOVA.\n\nData regarding the time required for nifedipine, labetalol, and hydralazine to reach the target MAP (20% reduction) are presented in Figure 1. The study found that 4, 1 and 3 patients of nifedipine, labetalol, and hydralazine group achieved the MAP target with only one dose, respectively. There were 3, 7 and 1 patients were unable to achieve the target MAP after three doses of nifedipine, labetalol, and hydralazine, respectively and these patients were excluded from further analyses.\n\nWe assessed the effectiveness of nifedipine, labetalol, and hydralazine to reduce the systolic and diastolic blood pressure where the data were treated as numerically to provided more detailed blood pressure reduction.\n\nNifedipine\n\nThe effectivity of nifedipine to reduce the systolic, diastolic and MAP is presented in Table 2. Our data indicated that nifedipine decreased the systolic pressure the most at 20 min post-first dose (difference from the first measurement to the second measurement) as much as 13.41 mmHg with a p<0.001. Meanwhile for second dose (second third measurement), and the next dose no longer showed a significant difference. Nifedipine also decreased diastolic pressure the most and significantly at 20 min post-first dose as much as 16.71 mmHg with a p<0.001 (Table 2). Nifedipine decreased the MAP 15.61 mmHg, 5.46 mmHg and 12.00 mmHg 20 min post the first, second and the third dose of nifedipine (Table 2). The MAP reduction on 20 min post the first and third doses was significant statistically with p=0.010 and p=0.024, respectively.\n\na Based on estimated marginal means.\n\nb Adjustment for multiple comparisons: Bonferroni.\n\n* The mean difference is significant at p<0.05.\n\n** The mean difference is significant at p<0.01.\n\nThe changes of the mean systolic, diastolic and MAP are presented in Figure 2. The data suggested there was a sharp decrease of systolic and diastolic after the first dose of nifedipine and the blood pressure kept decrease until 20 min after the third dose (after 2 hours of first dose) but it started to bounce again afterward (Figure 2).\n\nLabetalol\n\nLabetalol decreased systolic blood pressure the most and significantly at 20 min post the first and the third dose (13.38 and 12.00 mmHg) (Table 3). The systolic blood pressure continued to decrease until 5 hours post the third dose (Figure 3). The pattern of the of the reduction was similar with diastolic where the reduction mainly occurred after 20 min post the first and third dose (with reduction about 9.69 and 8.30 mmHg, respectively) (Table 3). In contrast, the diastolic blood pressure increased after the 20 min post the third dose (Figure 3). The changes of the MAP were similar with diastolic and the increase of diastolic and MAP were very small after the third dose (Figure 3).\n\na Based on estimated marginal means.\n\nb Adjustment for multiple comparisons: Bonferroni.\n\n* The mean difference is significant at p<0.05.\n\n** The mean difference is significant at p<0.01.\n\nThe blood pressure is measured: (1) minute 0 (pre-treatment); (2) 20 min post-first dose; (3) 20 min post-second dose; (4) 20 min post-third dose; (5) 1 h post-third dose; (6) 3 h post-third dose; and (7) 5 h post-third dose.\n\nHydralazine\n\nEffectivity of hydralazine in lowering the blood pressure has been presented in Table 4. Due to abnormal distribution of the systolic data, Friedman's test was carried out instead. The test yielded p-value of less than 0.001. As for the diastolic blood pressure and MAP, significant decreases was obtained until 20 minutes post-third dose. This can be observed in the curve presented in Figure 4, where sharp decreases of systolic pressure were observed up to 20 minutes post-third administration. However, in the case of systolic blood pressure, a mild rebound occurred at 1 hour post-third administration (Figure 4A).\n\na Based on estimated marginal means.\n\nb Adjustment for multiple comparisons: Bonferroni.\n\n* The mean difference is significant at p<0.05.\n\n** The mean difference is significant at p<0.01.\n\nThe blood pressure is measured: (1) minute 0 (pre-treatment); (2) 20 min post-first dose; (3) 20 min post-second dose; (4) 20 min post-third dose; (5) 1 h post-third dose; (6) 3 h post-third dose; and (7) 5 h post-third dose.\n\nTo compare the effectiveness among the three drugs, N-gain values were calculated based on the MAP reduction and presented in Table 5. The results showed that nifedipine, labetalol, and hydralazine in a single dose had N-gain values of 0.575 (58%); 0.421 (42%); and 0.408 (41%), respectively. This means, according to Melzer criteria,7 these three drugs had medium effectiveness in lowering blood pressure to achieve the target MAP (20%). Meanwhile, if referred to Hake criteria,8 nifedipine is highly effective, while labetalol and hydralazine are less effective in achieving a 20% MAP reduction. We also found significant difference of the MAP among the three drugs when administered in a single dose with p=0.037 based on Kruskal-Wallis test (Figure 5). According to pairwise comparative analysis, only hydralazine and nifedipine appeared to have significant difference with p=0.048 (Table 6). While the effectiveness of nifedipine was not significantly different with that of labetalol (p=0.148), also shown by hydralazine versus labetalol (p=1.000) (Table 6).\n\nThe blood pressure is measured: (1) minute 0 (pre-treatment); (2) 20 min post-first dose; (3) 20 min post-second dose; (4) 20 min post-third dose; (5) 1 h post-third dose; (6) 3 h post-third dose; and (7) 5 h post-third dose.\n\n* The mean difference is significant at p<0.05.\n\n** The mean difference is significant at p<0.01.\n\nAfter the administration of three doses of nifedipine, labetalol, and hydralazine within 60 minutes, the N-gain values were calculated based on the MAP reduction (Table 5). The mean of N-gain were 0.900 (90%); 0.851 (85%); and 1.113 (111%) for nifedipine, labetalol, and hydralazine, respectively. On the basis of both Melzer and Hake criteria,7,8 all the three drugs had highly effective in lowering blood pressure to achieve the target MAP of 20%. Kruskal-Wallis test revealed that the MAP reduction by all drugs were significantly different after three consecutive doses administration (p=0.009) (Figure 5). Based on pairwise comparisons, there were no difference in the effectiveness between labetalol and nifedipine (p=0.919); and between nifedipine and hydralazine (p=0.136) (Table 6). Significantly difference of MAP reduction was observed between labetalol and hydralazine (p=0.007) (Table 6).\n\nThe presence of side effects such as tachycardia, hypotension, foetal heart rate abnormality, nausea, vomit, dizzy, palpitation, headache, chest pain, and tachypnoea was assessed following the administration of all three drugs. Almost all, but 1 patient in nifedipine group, reported no side effects (Table 7). No other side effects were reported by the patient except tachycardia.\n\n\nDiscussion\n\nTo date, ACOG has recommended three types of first-line drugs that can be used as emergency management of hypertension in pregnancy: oral nifedipine, intravenous labetalol, and intravenous hydralazine. The blood pressure targets to be achieved in patients with hypertensive emergencies in pregnancy differ from one guideline to another.9 The latest ACOG bulletin guidelines did not mention the target blood pressure and only mentioned the blood pressure threshold that clinicians should to achieve. This present study was the first to report the direct comparison of the effectiveness of nifedipine, labetalol, and hydralazine together. In our study, a 20-25% decrease in MAP from baseline was considered the target blood pressure to be achieved. We also processed the data numerically so that a decrease of 1 mmHg was calculated and analysed to produce a detailed outcome. The blood pressure and pulse rate of the patients at the baseline are similar to previous studies studying the severe preeclampsia.9–12 No characteristic differences were observed among nifedipine, labetalol, and hydralazine groups in the present study.\n\nOur data suggested that oral nifedipine and intravenous labetalol and hydralazine regimens can be used to treat hypertensive emergencies in pregnancy. If 20-25% reduction of MAP is used as the target, where three doses was set as the maximum, the intravenous hydralazine monotherapy had the lowest failure rate (1 patient). In contrast, intravenous labetalol monotherapy had a failure rate 7 patients (35%, 7/20 patients). As reported previously, labetalol group had relatively low efficacy and required cross-treatment or alternative treatment.10 It is worth noting that the target for MAP reduction may vary across hospitals. Hence, further processing of these ordinal data was not carried out.\n\nBased on the data of systolic pressure following post-administration time, nifedipine had maximum effectiveness by one dose of administration, but experienced a rebound after 4 hours. As in the labetalol group, the blood pressure reduction peaked twice; at post-20 and -60 minutes administration. Patients in hydralazine group had the lowest blood pressures at three points of measurement: at post-20, -40, and -60 minutes administration. These findings suggest that titration dose is required. Comparative analysis among the three drugs using a single dose yielded a statistical significance of p=0.037. When administered with a single dose, nifedipine performed the best, followed by labetalol and hydralazine and no significant difference of efficacy between labetalol and nifedipine. The result is similar to the finding from a previous study10 where labetalol and nifedipine had no significant different in reduction of the blood pressure. Rapid reduction of the blood pressure in nifedipine group in our study also similar with previous study13 where the reduction of the blood pressure was achieved faster by nifedipine compared with hydralazine.13 Both nifedipine and hydralazine work by resulting vasodilating effects.14,15 However, hydralazine has more effect on the arteries than veins15 while nifedipine, as a calcium channel blocker, causes overall vasodilation, thereby leading to a decrease in peripheral vascular resistance.14 Taken altogether, nifedipine could be the most efficacious when administered at a single dose.\n\nAccording to the ACOG guidelines, the maximum dose of emergency antihypertension is 3 times administration within 60 minutes. The comparative analysis revealed that hydralazine yielded the most optimum reduction of blood pressure, followed by nifedipine and labetalol, respectively. This could be observed through the significant reduction of blood pressure on the first until the fourth measurements.\n\nWe did not witness any adverse effects experienced by the mothers or infants following the drug administration, in which our data are similar to that of reported.16–18 Nonetheless, other studies reported the occurrence of nausea and vomiting as adverse effects of labetalol (16%) or nifedipine (8%).10 In another study, patients in labetalol and nifedipine group experienced dizziness and/or headaches (24% vs 12%), palpitations (8% vs 4%), and foetal heart rate abnormalities (4% vs 8%).9 These different findings on side effect might be due to the maximum dose used in both foregoing studies, which was 5 doses.9,10\n\nThe strengths of this study include the multicentred randomized controlled study design, intensive monitoring, and repeated measurements up to seven times. However, this study is limited by the fact that some of the hospitals only used single blinding because labetalol and hydralazine are new to some doctors. We also did not consider the circadian rhythms in our study.\n\n\nConclusions\n\nOur data suggested that nifedipine is the most effective in reducing blood pressure using a single dose administration during hypertensive emergency in severe preeclampsia. The lowering blood pressure effect of nifedipine was not significantly different to labetalol but was significantly superior as compared with hydralazine. Nifedipine had a significant rebound phenomenon in blood pressure after 4 hours of drug administration, hence the necessity of re-administering antihypertensives. In maintaining the low blood pressure, hydralazine was significantly more effective than labetalol and no different effectiveness between hydralazine and nifedipine. All drugs are considered safe after triple dose administration within one hour. Studies assessing laboratory parameters such as soluble fms-like tyrosine kinase 1, endothelin-1, and soluble endoglin post-treatment using those drugs are encouraged in future.", "appendix": "Data availability\n\nFigshare: ‘Effectiveness of nifedipine, labetalol, and hydralazine as emergency antihypertension in severe preeclampsia’. DOI: https://doi.org/10.6084/m9.figshare.20933785. 19\n\nThis project contains the following underlying data:\n\n- Master Data.xlsx [Table containing the raw data of the study].\n\nFigshare: CONSORT checklist for Effectiveness of nifedipine, labetalol, and hydralazine as emergency antihypertension in severe preeclampsia. DOI: https://doi.org/10.6084/m9.figshare.21443028. 20\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 you to all healthcare workers in Arifin Achmad Hospital, Tengku Rafian Siak Sri Indrapura Hospital, Dumai Hospital, and Bengkalis Hospital who assisted in this study.\n\n\nReferences\n\nAmerican College of Obstetricians and Gynecologists: Gestational Hypertension and Preeclampsia. Practice Bulletin No. 222, June 2020.\n\nAmerican College of Obstetricians and Gynecologists: Emergent therapy for acute onset, severe hypertension during pregnancy and the postpartum period. Committee Opinion. Feb 2019; (767).\n\nFogagnolo A: Mean Arterial Pressure and Central Venous Pressure Difference as a Prognostic Tool for Postoperative Acute Kidney Injury. Geneva:Euroanaesthesia;2017; 233.\n\nPatel P, Koli D, Maitra N, et al.: Comparison of Efficacy and Safety of Intravenous Labetalol Versus Hydralazine for Management of Severe Hypertension in Pregnancy. J. Obstet. Gynaecol. India. 2018; 68(5): 376–381. PubMed Abstract \| Publisher Full Text\n\nEasterling T, Mudle S, Bracken H, et al.: Oral antihypertensive regimens (nifedipine retard, labetalol, and methyldopa) for management of severe hypertension in pregnancy: an open-label, randomised controlled trial. Lancet. 2019; 394: 1011–1021. PubMed Abstract \| Publisher Full Text\n\nCunningham FG, Leveno KJ, Bloom SL, et al.: Williams Obstetrics. 25th ed.New York:Mc Graw Hill;2018.\n\nMeltzer DE: The relationship between mathematics preparation and conceptual learning gains in physics: A possible “hidden variable” in diagnostic pretest scores. Am. J. Phys. 2002; 70(12): 1259–1268. Publisher Full Text\n\nHake RR: Analyzing change/gain scores.1999.Reference Source\n\nBiswas SK, Raha SK, Mahbuba.: Oral Nifedipine versus Intravenous Labetalol for Acute Blood Pressure Control in Severe Hypertension of Pregnancy: A Study at Faridpur Medical College Hospital. Faridpur Med. Coll. J. 2021; 16(1): 25–29. Publisher Full Text\n\nRaheem IA, Saaid R, Omar SZ, et al.: Oral nifedipine versus intravenous labetalol for acute blood pressure control in hypertensive emergencies of pregnancy: a randomised trial. BJOG. 2012 Jan; 119(1): 78–85. Epub 2011 Oct 10. PubMed Abstract \| Publisher Full Text\n\nDhali B, Bhattacharya S, Ganguly RP, et al.: A randomized trial of intravenous labetalol & oral nifedipine in severe pregnancy induced hypertension. Int. J. Reprod. Contracept. Obstet. Gynecol. 2012; 1: 42–46.\n\nGarovic VD, Dechend R, Easterling T, et al.: Hypertension in pregnancy: diagnosis, blood pressure goals, and pharmacotherapy: a scientific statement from the American Heart Association. Hypertension. 2022; 79: e21–e41. PubMed Abstract \| Publisher Full Text\n\nRezaei Z, Sharbaf FR, Pourmojieb M, et al.: Comparison of the efficacy of nifedipine and hydralazine in hypertensive crisis in pregnancy. Acta Med. Iran. 2011; 49(11): 701–706. PubMed Abstract\n\nEasterling T, Mundle S, Bracken H, et al.: Oral antihypertensive regimens (nifedipine retard, labetalol, and methyldopa) for management of severe hypertension in pregnancy: an open-label, randomised controlled trial. Lancet. 2019 Sep 21; 394(10203): 1011–1021. Epub 2019 Aug 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAntza C, Dimou C, Doundoulakis I, et al.: The flipside of hydralazine in pregnancy: A systematic review and meta-analysis. Pregnancy Hypertens. 2020 Jan; 19: 177–186. PubMed Abstract \| Publisher Full Text\n\nFiroz T, Magee LA, MacDonell K, et al.: Oral antihypertensive therapy for severe hypertension in pregnancy and postpartum: a systematic review. BJOG. 2014 Sep; 121(10): 1210–1218; discussion 1220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdebayo JA, Nwafor JI, Lawani LO, et al.: Efficacy of nifedipine versus hydralazine in the management of severe hypertension in pregnancy: A randomised controlled trial. Niger. Postgrad. Med. J. 2020 Oct-Dec; 27(4): 317–324. PubMed Abstract \| Publisher Full Text\n\nAali BS, Nejad SS: Nifedipine or hydralazine as a first-line agent to control hypertension in severe preeclampsia. Acta Obstet. Gynecol. Scand. 2002 Jan; 81(1): 25–30. PubMed Abstract \| Publisher Full Text\n\nSuhaimi D:Effectiveness of nifedipine, labetalol, and hydralazine as emergency antihypertension in severe preeclampsia. figshare. [Dataset]. Journal Contribution. 2022. Publisher Full Text\n\nSuhaimi D:CONSORT checklist for Effectiveness of nifedipine, labetalol, and hydralazine as emergency antihypertension in severe preeclampsia. figshare. 2022. [Dataset]. Journal Contribution. 2022. Publisher Full Text" }	[ { "id": "155523", "date": "14 Nov 2022", "name": "Talha Bin Emran", "expertise": [ "Reviewer Expertise Immunology", "Alternative Medicine", "Public Health", "Bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study assessed the effectiveness of nifedipine, labetalol, and hydralazine as emergency antihypertension in severe preeclampsia (SP). SP is a significantly important health problem in both developed and developing countries. My comments:\nThe study was well-designed and conducted with balanced conclusions.\n\nThe title of the study is clear and balanced however the information on RCT is needed such as: “Effectiveness of nifedipine, labetalol, and hydralazine as emergency antihypertension in severe preeclampsia: a randomized control trial”.\n\nThe structured abstract contains all important information, including the registry number.\n\nThe introduction section provides the importance and significance of the problem as well as the scientific gap. The aim of the study is also clearly stated.\n\nThe study setting is clearly stated although the justification for choosing those hospitals might be helpful.\n\nThe sample size is relatively small, but the justification and calculation are provided on how the sample was calculated.\n\nDouble blinding and randomization were conducted to avoid bias.\n\nThe procedures are explained adequately but the information on whether the healthcare workers received the training from the researchers is important to ensure that they worked following the guideline.\n\nThe Results assisted with a flowchart, and this helps the readers to understand each step of enrolment, allocation, follow-up, and analysis.\n\nThe baseline of the data of the patients between the group are relatively homogeneous.\n\nThe comparations between drugs were conducted for each endpoint and should be appreciated.\n\nTable 7. The information in this table can basically be written in two sentences only since there were no side effects in the labetalol and hydralazine groups. Also “Secondary outcome” could be changed to “Side effect.”\n\nDiscussion and conclusion are balanced; the study's strengths and limitations are discussed. However, some suggestions to measure soluble fms-like tyrosine kinase 1, endothelin-1, and soluble endoglin likely do not support the data of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9343", "date": "17 Feb 2023", "name": "Donel S", "role": "Author Response", "response": "The study assessed the effectiveness of nifedipine, labetalol, and hydralazine as emergency antihypertension in severe preeclampsia (SP). SP is a significantly important health problem in both developed and developing countries. My comments: The study was well-designed and conducted with balanced conclusions. RESPONSES: Thank you for your appreciation. The title of the study is clear and balanced however the information on RCT is needed such as: “Effectiveness of nifedipine, labetalol, and hydralazine as emergency antihypertension in severe preeclampsia: a randomized control trial”. RESPONSES: We have revised the tittle by adding: “: a randomized control trial” The structured abstract contains all important information, including the registry number. The introduction section provides the importance and significance of the problem as well as the scientific gap. The aim of the study is also clearly stated. RESPONSES: Thank you for your appreciation. The study setting is clearly stated although the justification for choosing those hospitals might be helpful. RESPONSES: The reasons have been provided: “These hospitals were chosen because they are affiliated with Faculty of Medicine, Universitas Riau.” The sample size is relatively small, but the justification and calculation are provided on how the sample was calculated. Double blinding and randomization were conducted to avoid bias. RESPONSES: Thank you for your appreciation. The procedures are explained adequately but the information on whether the healthcare workers received the training from the researchers is important to ensure that they worked following the guideline. RESPONSES: We have added the information that the healthcare workers have been trained: “The doctors have been informed about the study and have been trained.” The Results assisted with a flowchart, and this helps the readers to understand each step of enrolment, allocation, follow-up, and analysis. The baseline of the data of the patients between the group are relatively homogeneous. The comparations between drugs were conducted for each endpoint and should be appreciated RESPONSES: Thank you for your appreciation. Table 7. The information in this table can basically be written in two sentences only since there were no side effects in the labetalol and hydralazine groups. Also “Secondary outcome” could be changed to “Side effect.” RESPONSES: Thank you for the suggestion. Now Table 7 has been deleted and we have added “side effects” after the secondary outcome. Discussion and conclusion are balanced; the study's strengths and limitations are discussed. However, some suggestions to measure soluble fms-like tyrosine kinase 1, endothelin-1, and soluble endoglin likely do not support the data of this study. RESPONSE: Thank you for the suggestion. This sentence has been deleted." } ] }, { "id": "155526", "date": "02 Dec 2022", "name": "Jon Wantania", "expertise": [ "Reviewer Expertise John Wantania : Maternal Fetal Medicine", "Windy Wariki : Clinical Epidemiologist" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 your hard work. As a reviewer, I appreciate the efforts of the authors. The methods and research results presented are excellent and it hoped that great benefits obtained from this article. However, there are still some minor issues to address, which are listed below, in general:\nThis article is useful, but still there are several weaknesses that need to be considered. The introduction section should describe in more depth what is the novelty and urgency of this study. The methods, and interpretations of the statistical analysis are not carefully presented. Therefore, further consultations with biostatistician or epidemiologist may potentially improve this article. The authors may not be careful enough in writing due to some mistakes in grammar and inconsistency in writing.\n\nIntroduction Paragraph 4: The authors mentioned about the recommendations for antihypertension drugs in preeclampsia and stated, “Multiple drugs are included and excluded from the list of drugs of choice for the treatment of severe preeclampsia”. This sentence is a little unclear. Could you please clarify exactly what you mean about multiple drugs being included and also excluded as well? The authors should modify the sentence so that it is easier for the reader to understand.\nParagraph 4: In the third sentence the authors explained the dosage form of hydralazine, without writing down the dosage form of labetalol.\nParagraph 5: The authors stated: “Hydralazine and labetalol are considered to have a balanced effectiveness and side effect in lowering blood pressure in preeclampsia”, and “Another study comparing nifedipine and labetalol…” Here, the authors should explain if the indicator used to measure “lowering blood pressure” were the same, for example the mean arterial pressure (MAP), and need to confirm quantitatively how much the blood pressure declined so that the reader can judge that they are comparable.\nParagraph 6: In the objective of this study, the authors mentioned the dosage form of nifedipine and labetalol. The authors should consistently explain the dosage form of hydralazine as well, bearing in mind that nifedipine and labetalol have different dosage forms.\nMethods Study setting and registration In line with the subtitle above including registration, the authors should explain matters related to registration in this section instead of explaining it in the ethical considerations section. However, if the authors should explain this in ethical considerations section, can “registration” in this subtitle be eliminated?\nPatient recruitment and criteria In the second sentence the authors stated “The minimal sample size of the study was 13 patients”. This is different from the explanation in the Results section that the total number of samples was 60. Does it mean that the results of the minimum sample size calculation are 13 patients in each group?\nThe minimum sample calculation formula used by the authors is for two groups. Considering that this research was an unpaired numerical comparative study conducted on three groups with measurements of seven times, it would be more appropriate if the authors calculated the number of samples based on the above considerations.\nBlinding and study procedure The authors are suggested to explain in more detail about the following sentence “For example, the patient who arrived first and second to the hospital should be given kit no 41 and 23”. Furthermore, the authors also need to describe who are the “health workers” specifically, mentioned in this sentence “Health workers were instructed to administrate one pair of drug….” with reference to Peraturan Menteri Kesehatan Republik Indonesia Nomor 13 Tahun 2020.\nEndpoints: The authors stated “The blood pressure was measured seven times...” As a suggestion, this sentence should be more suitable when explained in the subtitle “Blinding and study procedure” above.\nStatistical analysis The authors have explained that a normality test has been carried out, therefore it is advised to write down the name of the normality test used, for example by taking into account the number of samples, or other considerations or assumptions.\nResults Figure 1: In the last column of the “Analysis” section, it was written that the total number of respondents was 49 patients, quite different from the explanation in the Results section that 60 patients were analyzed. It is suggested that the authors need to be clearer, for example, when and for what to analyzing 60 patients, or only 49 patients.\nRequired time for nifedipine, labetalol, and hydralazine group achieved the MAP The authors have explained that the results regarding the time required for the three drugs to reach the target MAP are presented in Figure 1. However, the results shown in figure 1 are different from this explanation.\nNifedipine (page 6) In this section, the author has directed the reader for three times to see the results in Table 2, even though all the data in this section are only showed in table 2. As a suggestion, it would be better if Table 2 was written only once, for example at the beginning or end of a paragraph of the explanation. Sentence stating “Nifedipine decrease the MAP 15.61 mmHg, 5.46 mmHg and 12.00 mmHg 20 min post the first…”. However, 5.46 mmHg and 12.00 mmHg are not found in Table 2. Similar with the sentence sixth which is in Table 2 the p=0.010 and p=0.024 are not found.\nTable 2 On the footnote of table 2 was written the mean difference is significant at p<0.005. But in this table, there is no p-value less than 0.05 (p<0.05) which means that this mark () no need to be mentioned in the footnote.\nPage 7: The description below Figure 2 where the author wants to explain the changes of mean systolic, diastolic and MAP. The authors are asked to be more careful in presenting the results where the appearance of Figure 2 inconsistent from this explanation. Figure 2 is a comparison of the three types of drugs in achieving MAP.\nFigure 2: It would be more informative and flowing if the appearance of Figure 2 and its description are in the “Effectiveness comparisons of nifedipine, labetalol, and hydralazine” section compared to the current view which is in the section or explanation of nifedipine.\nLabetalol The authors are advised to revise the figure number that is not in accordance with the explanation about labetalol, which is different from what is presented in Figure 3.\nHydralazine In Table 4, the results of the analysis to explain the decrease in systolic blood pressure were not found. Also, Figure 4 is a description of labetalol, not hydralazine.\nPage 8: Explanation of the results of Kruskal-Wallis test is not present in the Figure 5, but in Figure 6.\nTable 7: Suggestion for the authors regarding reported side effects where there was only one namely tachycardia, it would be better if the result is presented in a narrative manner.\nDiscussion Discussion section is still having major problems, addition information requested on strength and limitation are not clearly explain.\nThe authors are lacking in explanation the agreements and disagreements with findings from other studies.\nLimitations The limitation in methodological should be clearly explained and address as one of the limitations of this study.\nThe authors are suggested to give specific recommendations to the reader as respond to key findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9344", "date": "17 Feb 2023", "name": "Donel S", "role": "Author Response", "response": "Thank you for your hard work. As a reviewer, I appreciate the efforts of the authors. The methods and research results presented are excellent and it hoped that great benefits obtained from this article. However, there are still some minor issues to address, which are listed below, in general: This article is useful, but still there are several weaknesses that need to be considered. The introduction section should describe in more depth what is the novelty and urgency of this study. The methods, and interpretations of the statistical analysis are not carefully presented. Therefore, further consultations with biostatistician or epidemiologist may potentially improve this article. The authors may not be careful enough in writing due to some mistakes in grammar and inconsistency in writing. RESPONSES: Thank you for reviewing our manuscript. The comments and suggestions have improved the quality of our current manuscript significantly. We have added the required information in the methods as requested. Also, we have corrected some typos in the numbering related to the statistics. We have corrected the labels of the figures. The connection between the figures and the explanation texts have been corrected and checked for the correctness. Introduction Paragraph 4: The authors mentioned about the recommendations for antihypertension drugs in preeclampsia and stated, “Multiple drugs are included and excluded from the list of drugs of choice for the treatment of severe preeclampsia”. This sentence is a little unclear. Could you please clarify exactly what you mean about multiple drugs being included and also excluded as well? The authors should modify the sentence so that it is easier for the reader to understand. RESPONSES: We have revised the sentence. Paragraph 4: In the third sentence the authors explained the dosage form of hydralazine, without writing down the dosage form of labetalol. RESPONSES: Have been included: “Intravenous labetalol and intravenous hydralazine have long been considered as first-line therapy in hypertensive emergencies, severe hypertension in pregnant women, and in the postpartum period”. Paragraph 5: The authors stated: “Hydralazine and labetalol are considered to have a balanced effectiveness and side effect in lowering blood pressure in preeclampsia”, and “Another study comparing nifedipine and labetalol…” Here, the authors should explain if the indicator used to measure “lowering blood pressure” were the same, for example the mean arterial pressure (MAP), and need to confirm quantitatively how much the blood pressure declined so that the reader can judge that they are comparable. RESPONSES: We have added more detailed of the information related to the studies. Paragraph 6: In the objective of this study, the authors mentioned the dosage form of nifedipine and labetalol. The authors should consistently explain the dosage form of hydralazine as well, bearing in mind that nifedipine and labetalol have different dosage forms. RESPONSES: Have been included: “Therefore, this study was conducted to compare the effectiveness of oral nifedipine, and intravenous labetalol and intravenous hydralazine as antihypertensives in severe preeclampsia.” Methods Study setting and registration In line with the subtitle above including registration, the authors should explain matters related to registration in this section instead of explaining it in the ethical considerations section. However, if the authors should explain this in ethical considerations section, can “registration” in this subtitle be eliminated? RESPONSES: Thank you for the suggestion. We have changed the subheading to: “Study setting” only and moved the “registration” into: “Ethical considerations and registration” Patient recruitment and criteria In the second sentence the authors stated “The minimal sample size of the study was 13 patients”. This is different from the explanation in the Results section that the total number of samples was 60. Does it mean that the results of the minimum sample size calculation are 13 patients in each group? RESPONSES: 13 patients are minimal sample size for each group. Therefore, we have revised to: “The minimal sample size of the study was 13 patients for each arm.” The minimum sample calculation formula used by the authors is for two groups. Considering that this research was an unpaired numerical comparative study conducted on three groups with measurements of seven times, it would be more appropriate if the authors calculated the number of samples based on the above considerations. RESPONSES: We have checked again and we confirm that this formula also can be used for three groups. Blinding and study procedure The authors are suggested to explain in more detail about the following sentence “For example, the patient who arrived first and second to the hospital should be given kit no 41 and 23”. RESPONSES: We have revised the sentence and we believe it is clear now. “For example, if the list from the software run was 41, 23, 4 and soon, the patient who arrived first, second and third to the hospital should be given kit no 41, 23 and 4, respectively.” Furthermore, the authors also need to describe who are the “health workers” specifically, mentioned in this sentence “Health workers were instructed to administrate one pair of drug….” with reference to Peraturan Menteri Kesehatan Republik Indonesia Nomor 13 Tahun 2020. RESPONSES: They were doctors. And the text has been revised: “The enrolment of the patient was conducted by the doctors in each centre. The doctors have been informed about the study and have been trained. The doctors and patients did not know the drugs contained in the kit.” Endpoints: The authors stated “The blood pressure was measured seven times...” As a suggestion, this sentence should be more suitable when explained in the subtitle “Blinding and study procedure” above. RESPONSES: Thank you for the suggestion. The sentence has been moved within “Blinding and study procedure” subheading. Statistical analysis The authors have explained that a normality test has been carried out, therefore it is advised to write down the name of the normality test used, for example by taking into account the number of samples, or other considerations or assumptions. RESPONSES: We have added the information of the test used to measure the normality of the data. We added: “The normality of data was measured using the Kolmogorov-Smirnov test.” Results Figure 1: In the last column of the “Analysis” section, it was written that the total number of respondents was 49 patients, quite different from the explanation in the Results section that 60 patients were analyzed. It is suggested that the authors need to be clearer, for example, when and for what to analyzing 60 patients, or only 49 patients. RESPONSES: Thank you for questioning this. We have added the information related to this. We add: “Out of 60 patients, 11 patients did not achieve the MAP target after three doses of the tested drugs and therefore were excluded for effectiveness analysis of nifedipine, labetalol, and hydralazine to reduce the blood pressure.” Required time for nifedipine, labetalol, and hydralazine group achieved the MAP The authors have explained that the results regarding the time required for the three drugs to reach the target MAP are presented in Figure 1. However, the results shown in figure 1 are different from this explanation. RESPONSES: Thank you pointing this mistake. It should be Figure 2. Also it has been changed to “Required dose for nifedipine, labetalol, and hydralazine to achieve MAP target” “Data regarding the dose required for nifedipine, labetalol, and hydralazine to reach the target MAP (20% reduction) are presented in Figure 2.” Nifedipine (page 6) In this section, the author has directed the reader for three times to see the results in Table 2, even though all the data in this section are only showed in table 2. As a suggestion, it would be better if Table 2 was written only once, for example at the beginning or end of a paragraph of the explanation. RESPONSES: Now it only mentioned only once in the beginning. Sentence stating “Nifedipine decrease the MAP 15.61 mmHg, 5.46 mmHg and 12.00 mmHg 20 min post the first…”. However, 5.46 mmHg and 12.00 mmHg are not found in Table 2. Similar with the sentence sixth which is in Table 2 the p=0.010 and p=0.024 are not found. RESPONSES: We appreciated with this correction. We have corrected the sentences: “Nifedipine decreased the MAP 15.61 mmHg, 6.37 mmHg and 2.90 mmHg 20 min post the first, second and the third dose of nifedipine. The MAP reduction on 20 min post the first dose was significant statistically with p<0.001.” Table 2 On the footnote of table 2 was written the mean difference is significant at p<0.005. But in this table, there is no p-value less than 0.05 (p<0.05) which means that this mark () no need to be mentioned in the footnote. RESPONSES: Now the * has been deleted. And ** has been changed to “The mean difference is significant at p<0.01.” The in the table also have been deleted. Thank you. Page 7: The description below Figure 2 where the author wants to explain the changes of mean systolic, diastolic and MAP. The authors are asked to be more careful in presenting the results where the appearance of Figure 2 inconsistent from this explanation. Figure 2 is a comparison of the three types of drugs in achieving MAP. RESPONSES: Thank you for catch this mistake. We realized that there was a shift of the labels of the figures. This should be Figure 3 not Figure 2. Now it has been corrected into Figure 3. The revised texts: “The changes of the mean systolic, diastolic and MAP are presented in Figure 3. The data suggested there was a sharp decrease of systolic and diastolic after the first dose of nifedipine and the blood pressure kept decrease until 20 min after the third dose (after 2 hours of first dose) but it started to bounce again afterward (Figure 3).” Figure 2: It would be more informative and flowing if the appearance of Figure 2 and its description are in the “Effectiveness comparisons of nifedipine, labetalol, and hydralazine” section compared to the current view which is in the section or explanation of nifedipine. RESPONSES: We agree with this suggestion because it is hard to follow the texts and the Figure 2. We have re-requested the layout editor to move Figure 2 directly under the subheading: “Required dose for nifedipine, labetalol, and hydralazine to achieve MAP target”. Labetalol The authors are advised to revise the figure number that is not in accordance with the explanation about labetalol, which is different from what is presented in Figure 3. RESPONSES: As we responded previously, we realized that there was a shift of the labels of the figures. Now all have been corrected. Hydralazine In Table 4, the results of the analysis to explain the decrease in systolic blood pressure were not found. Also, Figure 4 is a description of labetalol, not hydralazine. RESPONSES: The systolic data was not distributed normally and therefore the Friedmann test and this analysis produced one p-value only and no 95%CI. To ensure this is clear, we have included the information as a note under the Table 4. “c Analysed using Friedmann test.” As we stated previously, all labels of the figures have been rechecked and corrected. Page 8: Explanation of the results of Kruskal-Wallis test is not present in the Figure 5, but in Figure 6. RESPONSES: All labels of the figures have been rechecked and corrected. Table 7: Suggestion for the authors regarding reported side effects where there was only one namely tachycardia, it would be better if the result is presented in a narrative manner. RESPONSES: Now Table 7 has been deleted as suggested and we have added “side effects” after the secondary outcome. Discussion Discussion section is still having major problems, addition information requested on strength and limitation are not clearly explain. RESPONSES: We have provided the information related to the strengths and limitations of the study. The authors are lacking in explanation the agreements and disagreements with findings from other studies. RESPONSES: Thank you for your suggestions. Unfortunately, available studies are limited and therefore it is not possible to compare with previous studies. Limitations The limitation in methodological should be clearly explained and address as one of the limitations of this study. The authors are suggested to give specific recommendations to the reader as respond to key findings. RESPONSES: We have listed all the limitations of the study. We also have added specific recommendation from the results of the study. We added: “ Based on the present study, in high resource settings, hydralazine is recommended to treat severe preeclampsia cases with hypertensive emergency since it reduces the blood pressure rapidly and has no rebound phenomenon. We recommend to use nifedipine in the low resource settings since it is cheap and easy to use and store while still has good ability to reduce the blood pressure. However, blood pressure maintenance is required because it might rebound after approximately six hours.”" } ] } ]	1	https://f1000research.com/articles/11-1287
https://f1000research.com/articles/11-537/v1	18 May 22	{ "type": "Systematic Review", "title": "The role of genetic factors in microtia: A systematic review", "authors": [ "Indri Lakhsmi Putri", "Alexandria Stephanie", "Rachmaniar Pramanasari", "Moshe Kon", "Citrawati Dyah Kencono Wungu", "Alexandria Stephanie", "Rachmaniar Pramanasari", "Moshe Kon" ], "abstract": "Background: Microtia is a congenital malformation of the outer ears caused by improper embryonic development. The origin of microtia and causes of its variations remain unknown. Because of the lack of clarity regarding the role of genetic variables in microtia, we conducted a systematic review to qualitatively identify the genes most important in the development of microtia to provide an up-to-date review. Methods: Using six search engines, we searched all published studies related to the genetic factors of isolated microtia and syndromic microtia. The identified publications were screened and selected based on inclusion and exclusion criteria by the authors and assessed for methodological quality using the Joanna Briggs Institute (JBI) critical appraisal tools. We found 40 studies, including 22 studies on syndromic microtia and 18 studies on isolated microtia. Data extraction of each study was arranged in tabulation for syndromic and isolated microtia. The extracted data were: first author’s surname, year of publication, country of origin, study design, sample characteristic and gene assessed. Results: After the data were extracted, analyzed, and reviewed, the most common gene suspected to be involved in isolated microtia was Homeobox A2 (HOXA2, 12.1%). Conversely, in syndromic microtia, the two most common genes supposed to play a role were Fibroblast Growth Factor 3 (FGF3, 47.2%) and Treacher–Collins–Franceschetti syndrome 1 (TCOF1, 30.2%). From the studies, the three most prevalent genes associated with microtia were HOXA2 (10%), FGF3 (8.4%), and TCOF1 (5.4%). In syndromic microtia, the most common mutation types were deletion in TCOF1 (46.9%) and missense and deletion in FGF3 (both 38%), and in isolated microtia, the most common mutation type was silent in HOXA2 (54.2%). Conclusions: In summary, genetic factors are involved in microtia; thus, molecular analysis is strongly advised. PROSPERO registration: CRD42021287294 (25/10/21).", "keywords": [ "Microtia", "Genetic Mutation", "isolated", "syndromic", "Genetic Diversity", "FGF3", "HOXA2", "TCOF1" ], "content": "Introduction\n\nMicrotia is a congenital malformation of the outer ears caused by improper embryonic development.1,2 It is distinguished by small, irregularly shaped external ears, and it can occur unilaterally or bilaterally.1 The prevalence of microtia varies among ethnic groups (0.83–17.4 per 10,000 births).2–4 Microtia occurs unilaterally in 80%–90% of cases and bilaterally in 10%–20% of cases.2,4 Boys are approximately twofold more likely than girls to have microtia, and the right–left bilateral ratio is typically 6:3:1.2,3 Roughly one-third to one-half of patients with microtia have craniofacial microsomia.2,3 Microtia is easily misdiagnosed during pregnancy.5,6 If pregnancy ultrasonography suggests microtia, the diagnosis is easily confirmed and diagnosed following birth based on physical examination.5,6\n\nThe etiology of microtia and the causes of its variations remain unknown.4 Although there is compelling evidence that environmental factors such as maternal sociodemographic variables, multiple gestation, diseases (gestational diabetes, cold-like syndrome), and related drug treatments such as isotretinoin use during pregnancy play roles, genetic factors are also believed to influence the embryonic development of microtia.7–9 Estimates of the prevalence of inherited microtia vary greatly, ranging from 3 to 34%.10 Although certain studies discovered candidate genetic variations for microtia, no causal genetic mutation has been identified.11\n\nResearch on animals with isolated microtia as a prominent feature revealed mutations in homeobox A2 (HOXA2), sine oculis homeobox (SIX), eyes absent transcriptional coactivator and phosphatase (EYA), TBX1, IRF6, and CHUK.12 Genes identified to be involved in the development of major syndromic microtia were PLCB4 and GNAI3 for auriculo-condylar syndrome; TFAP2A for branchio-oculo-facial (BOF) syndrome; SIX1 and SIX5 for branchio-oto-renal (BOR) syndrome; CHD7 (SEMA3E) for CHARGE syndrome; FRAS1, FREM2, and GRIP1 for Fraser syndrome; MLL2 and KDM6A for Kabuki syndrome; GDF6 for Klippel–Feil syndrome; fibroblast growth factor 3 (FGF3) for labyrinthine aplasia, microtia and microdontia (LAMM) syndrome; FGFR2, FGFR3, and FGF10 for lacrimo-auriculo-dento-digital syndrome; EFTUD2 for mandibulofacial dysostosis with microcephaly; ORC1, ORC4, ORC6, CDT1, and CDC6 for Meier–Gorlin syndrome; HOXA2 for microtia, hearing impairment, and cleft palate; DHODH for Miller syndrome; SF3B4 for Nager syndrome; H6 family homeobox 1 transcription factor gene (HMX1) for oculo-auricular syndrome (OVAS); SALL1 for Townes–Brocks syndrome; and Treacher–Collins–Franceschetti syndrome 1 (TCOF1), POL1RC, and POL1RDT for Treacher–Collins syndrome (TCS).2\n\nFGF3 mutations are commonly found in LAMM syndrome.13,14 The FGF3 protein regulates a cascade of chemical processes inside cells by binding to its receptor, thereby signaling cells to undergo particular changes, such as proliferating or maturing to perform specialized activities.15 TCOF1 mutations can cause TCS in up to 78% of patients.16 HOXA2 encodes key developmental transcription factors of the second branchial arch, which contributes significantly to the development of the external and middle ear in embryonic development, and it was previously linked to autosomal recessive bilateral microtia.10,17\n\nBecause of the unclear role of genetic factors in microtia, we conducted the first systematic review to qualitatively identify the most important genes in the development of microtia. This may help to improve our understanding of microtia, underline the necessity of gene screening and even make prevention of microtia in the near future possible with the help of gene modification.\n\n\nMethods\n\nWe have registered our protocol with the International Prospective Register of Systematic Reviews (PROSPERO, CRD42021287294 (25/10/21)). We have also screened PROSPERO for similar systematic reviews. No registered protocol reviewing the genetic factors of microtia was identified. The report of this systematic review was formulated according to the recommendations of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statement.18\n\nWe performed an extensive and systematic search of all published studies related to genetic factors implicated in the development or outcome of microtia. Rather than focusing on a single disease, we aimed to provide systematic evidence on all types of microtia, including isolated microtia and syndromic microtia. First, the identified publications were assessed for relevance to the topic of interest using their titles and abstracts. The identified articles were then examined for any duplication using Mendeley. Then, the complete text of all screened papers was reviewed for the inclusion criteria, which were observational studies and case reports/series in the English language that assessed genetic factors in microtia. The exclusion criteria were duplications, reviews, non-English articles, animal studies, and articles in which sufficient details on genetic factors of microtia were not provided.\n\nThree of the four authors (A.S, I.L.P, and R.P) performed the search and study selection, which was supervised by the fourth author (C.D.K.W). We used six electronic bibliographic databases, PubMed, Web of Science, Science Direct, Proquest, Springerlink, and Clinicaltrials.gov, to conduct systematic searches from 1 to 31 October 2021. We checked Medline (PubMed) to identify controlled vocabulary Medical Subject Headings terms related to genetics and microtia. Searching strategies for PubMed are presented in Supplementary Table 1 (see Extended data71) and modified for other electronic databases.\n\nData extraction was conducted independently by three reviewers (A.S., I.L.P., and R.P.) through a standardized form. The methodological quality of studies in this systematic review was assessed using Joanna Briggs Institute (JBI) critical appraisal tools.19 We extracted data once all of the screening and selection steps had been completed. For both syndromic and isolated microtia, two different extraction forms were produced. The following data were extracted: first author’s surname, year of publication, country of origin, study design, sample population, sex, age, type of microtia, analysis method, affected genes, and mutations. Disagreements between the three reviewers were settled by discussion with the fourth reviewer (C.D.K.W.).\n\n\nResults\n\nAll supplementary files can be found in the Extended data.72\n\nWe discovered 3,742 articles after searching six electronic bibliographic databases. In total, 183 publications were removed because they were not available in English, they were animal studies, their full text was not available, or they were duplicated studies. Only 55 articles were eligible for more extensive evaluation after title and abstract screening. Only 40 papers were included in this study after a comprehensive full text analysis (Figure 1). The included studies were reviewed, utilizing a checklist questions form provided by JBI based on the studies’ methodology. Based on the JBI Tools for case reports, case series, and case controls, all publications involved were assessed as low-risk bias (Supplementary Tables 2–4).\n\nFollowing the screening, selection, and data extraction of all included studies, we discovered 22 articles of syndromic microtia involving 106 patients (Table 1 and Supplementary Table 5) and 18 articles of isolated microtia involving 486 patients (Table 2 and Supplementary Table 6). China had the most cases of syndromic microtia (32 subjects [30.2%] in five studies), followed by Turkey (13 subjects [12.3%] in one study); the USA and Belgium (six subjects [5.7%] in three studies each); Poland and Saudi Arabia (four subjects [3.8%] each); and Pakistan, India, Italy, Germany, and Austria (three subjects [2.8%] each). China had the most cases of isolated microtia (415 subjects [85.4%] in 10 studies), followed by Turkey (38 subjects [7.8%] in two studies), the USA (20 subjects [4%]), Belgium (five subjects [1%]), and Iran and Italy (three subjects [0.6%] each). China had the most cases of microtia among all investigations, being the site of 447 of 592 cases (75.5%). Concerning the study design, there were two case reports, 34 case series, and five case control studies included in this analysis.\n\nRegarding studies of syndromic microtia (Table 1), the family history was known for 104 of 106 subjects (98.1%), whereas the family history was not discussed in two subjects (1.9%). Of the 104 subjects, 84 (80.8%) had family histories of microtia. We also discovered that, of the 106 subjects, the gender was known for 105 subjects (99%), which included 62 males (59%). The severity of microtia was known for 158 of the 173 ears (91.3%) involved in this analysis. Of the 158 ears, 98 (62%) had grade I microtia, 44 (27.9%) had grade II microtia, and 16 (10.1%) had grade III microtia. The type of microtia was known in 72 of 106 subjects (67.9%); of these subjects, 15 (20.8%), 4 (5.6%), and 53 (73.6%) had right unilateral, left unilateral, and bilateral microtia, respectively. Based on the gene disorder levels in 22 studies including 106 subjects, 105 subjects (99%) in 21 studies had DNA-level disorders, and one subject (1%) in one study had a chromosomal disorder. We discovered that of the 106 subjects, 32 subjects (30.2%) in seven studies had TCOF1 gene mutations, whereas 50 subjects (47.2%) in five studies had FGF3 gene mutations.\n\nAccording to studies of isolated microtia (Table 2), 274 of 486 subjects (56.4%) had a family history of microtia. The genders of the 486 subjects were known for 461 subjects (94.9%), which included 269 males (58.4%). Based on the severity of microtia, the grading of 551 ears was known for 208 subjects (37.7%), among whom 2 (1%), 67 (32.2%), 135 (64.9%), and 4 subjects (1.9%) had grades I, I, III, and IV microtia, respectively. The type of microtia was specified for 103 subjects (21.2%), being right unilateral, left unilateral, and bilateral in 15 (14.6%), 11 (10.7%), and 77 subjects (74.7%), respectively. Regarding the genetic disorder level, of 486 subjects in 18 studies, 485 subjects (99.8%) in 17 articles had DNA-level disorders, and one subject (0.2%) had a chromosomal disorder. We discovered that 59 subjects (12.1%) in seven studies had mutations in the HOXA2 gene.\n\nWe discovered that of 592 subjects, the family history was known in 590 subjects (99.7%). In total, 358 of the 590 subjects (60.7%) had family histories of microtia. The genders of 566 of the 592 total subjects (95.6%) were known. Among the 566 subjects, 331 (58.5%) were male, and 235 (41.5%) were female. The severity of microtia was known in 366 of the 724 ears (50.5%) examined in this study. Among these ears, 100 (27.3%) had grade I microtia, 111 (30.3%) had grade II microtia, 151 (41.3%) had grade III microtia, and 4 (1.1%) had grade IV microtia. Based on the type of microtia, the type of microtia was known for 175 of 592 subjects (29.6%). Among these 175 subjects, 30 (17.1%) had right unilateral microtia, 15 (8.6%) had left unilateral microtia, and 130 (74.3%) had bilateral microtia. Based on the gene disorder levels described in all 40 studies, 586 subjects (99%) in 38 studies had DNA-level disorders, whereas six subjects (1%) in two studies had chromosomal disorders. We discovered that FGF3 was the most common gene involved in isolated microtia (50 subjects [8.4%]), followed by TCOF1 (32 subjects [5.4%)], and HOXA2 was the most common gene involved in isolated microtia (59 subjects [10%]).\n\nBased on the type of mutation (Table 3), we discovered that in syndromic microtia, the most common type of mutation in TCOF1 was deletion, being detected in 15 of 32 subjects (46.9%). In FGF3, the most common mutation types were missense and deletion, being present in 19 of 50 subjects (38%) each, and in isolated microtia, the most common mutation type in HOXA2 was silent mutation, being present in 32 of 59 subjects (54.2%).\n\n\nDiscussion\n\nMicrotia is a congenital external ear deformity that can range in severity from minor anatomical problems to full ear absence (anotia). Microtia can be a single birth abnormality or part of a broader set of defects or syndrome.8 This systematic review attempted to describe the genes that play important roles in the development of syndromic and non-syndromic microtia. Only 40 studies on genetically linked microtia met our selection criteria. In this study, China had the highest number of microtia cases. The findings of this study contradict epidemiological data reported by Klokars et al., who recorded the highest prevalence of microtia in Quito, Ecuador, South America, (17.4/10,000),3 and Luquetti et al., who stated that the highest prevalence of microtia was in Finland, North Europe (4.3/10,000).2 This could be because China has a higher overall birth rate than other countries2; therefore, even if it has a greater number of microtia cases, the prevalence rate is low.\n\nWe discovered that most patients with microtia had a family history of the disease, including 56.4% of patients with isolated microtia and 80.8% of patients with syndromic microtia. This is consistent with the existing literature, which describes Mendelian hereditary variants of microtia with an autosomal dominant or recessive mode of inheritance.1,4,20 The rates of familial microtia ranged from 3 to 34%.10\n\nBased on the gender classification of each study, we discovered nearly 60% of all patients were male, including 59% of patients with syndromic microtia and 58.4% of patients with isolated microtia. In prior research, microtia was more common in male patients than in female patients, with a sex ratio of 1.5:1 and an estimated 20–40% greater risk in males than in females.4,21\n\nAlthough microtia can arise bilaterally, 77–93% of patients have unilateral involvement.4,21 The most common form of syndromic microtia is bilateral microtia.1 Although bilateral microtia was more common in this study, the type of microtia was only known for 29.6% of subjects. Because of the inadequate data, this could represent a biased outcome for the most prevalent type of microtia.\n\nMost published research on microtia reported the existence or absence of microtia and/or anotia with no further details on severity. Marx (1926), Weerda (1988), Roger (1977), Tanzer (1978), and Hunter (2009) all provided classifications of microtia.5 Their classification system was nearly identical, consisting of four grading levels.4,22 The lobule type, grade III microtia, was the most common microtia type in the literature, accounting for 60% of all cases. The most prevalent severity of syndromic microtia was grade I owing to the high rate of mutations in FGF3, which can cause LAMM syndrome with a grade I microtia presentation.13,14,23,24\n\nFrom all examined studies involved, the most common genes associated with microtia were HOXA2, FGF3, and TCOF1. We discovered that in syndromic microtia, the most common types of mutation were TCOF1 deletion (46.9%), and missense and deletion in FGF3 (38%), whereas in isolated microtia, the most common type of mutation in HOXA2 was silent mutation (54.2%). This was consistent with the literature, which indicated that the most common genes involved in microtia were HOXA2, FGF3, HOXD, ORC1, ORC4, ORC6, CDT1, CDC6, DHODH, HMX1, EYA1, and TCOF1.2\n\nFGF3 encodes a protein member of the FGF family.15 The FGF family has extensive mitogenic and cell survival activities and is involved in various biological processes such as embryonic development, morphogenesis, tissue repair, cell growth and inner ear formation in mice and chicken.15 FGF3 activates a cascade of chemical reactions inside cells that activate certain changes, such as dividing or maturing to take on specialized functions, by attaching to another protein known as a receptor.15 FGF3 haploinsufficiency could also be related with dental and hearing problems. FGF signaling is required for the appropriate development of the otic placode, a thickening of the ectoderm on the outer surface of a developing embryo from which the ear develops.25 FGF3 mutation is often found in syndromic microtia, mainly in LAMM syndrome. Congenital deafness with LAMM syndrome is an autosomal recessive disorder characterized by significant bilateral congenital sensorineural deafness coupled with inner ear defects, grade I bilateral microtia, and microdontia (small teeth) as its major phenotypic features.10,26 The finding of biallelic pathogenic mutations in FGF3 on molecular genetic testing confirms the diagnosis of LAMM syndrome in the proband.13,14,23,24\n\nThe TCOF1 gene, which encodes a suspected nucleolar phosphoprotein known as treacle, has been identified as the cause of TCS,10,27 an autosomal dominant craniofacial development disorder, in up to 78% of patients.16,28,29 Inhibition of mature RNA ribosomal (rRNA) production and gene transcription in neural folds prefusion during the early stage of embryogenesis may cause abnormal development due to treacle haploinsufficiency, caused by mutation in the TCOF1 gene, thus affecting proliferation and proper differentiation of these embryonic cells.30 To date, more than 50 mutations have been identified in the TCOF1 gene, most of which are insertions or deletions. TCS is characterized by cleft palate, hypoplasia of facial bones (particularly the mandible and zygomatic complex), downward slanting of palpebral fissures with colobomas of lower eyelids, external ear deformity, conductive hearing loss, and defects in brain development such as microcephaly and mental retardation.31–36 TCOF1 was the most prevalent gene found in this systematic review. This was because TCOF1 is a causal gene for TCS, which features microtia as one of its clinical symptoms.32 In addition to clinical findings, TCS diagnosis is also confirmed by detection of pathogenic variants of TCOF1, POLR1D, or POLR1B using molecular genetic testing, mainly inherited in an autosomal dominant pattern.37 In accordance with our result, TCOF1 gene deletions were reported to range from a single exon to a whole gene. Despite the fact that >97% of reported cases contained a pathogenic mutation identifiable by sequencing, Bowman et al. (2012) reported 5% of cases (5/92) with a big deletion, suggesting that the rate of large deletions may be higher than current data suggest.38\n\nHomeobox genes participate in the formation of the pharyngeal arches.4,39 They encode transcription factors that determine cell positional identity and morphogenesis during development, as well as switch on cascades of other genes.4,39–41 The Hox gene family was discovered to be grouped inside the genome and ordered on the chromosome in the order of expression during development. This ordered pattern of gene expression could be part of a mechanism that generates morphogenetic specification.4,39 HOXA2 was discovered to be highly expressed in the second brachial arches (BA2), to express critical developmental transcription factors BA2, to play an important role in the development of the external and middle ear during embryonic development, and to be associated with autosomal recessive bilateral microtia as a member of the HOX gene family.10,17,39 In humans, abnormal or lost HOXA2 function, as well as early and late HOXA2 inactivation, results in auditory system malformations, primarily in the external and middle ear, such as a duplicated or absent auricle10,39 Consequently, HOXA2 has been proposed as a key transcriptional regulator of auricle morphogenesis.39 Individuals with a homozygous HOXA2 mutation have far more severe clinical symptoms than those with a heterozygous mutation. In a mouse model, inactivation of Hox2 early in development results in the absence of the pinna, whereas late inactivation results in a hypomorphic auricle.42\n\nAside from HOXA2, FGF3, and TCOF1, other genes linked to microtia include HOXD, ORC1, ORC4, ORC6, CDT1, CDC6, DHODH, HMX1, and EYA1.2 Among these, HMX1 located on chromosome 4p16.1 is prominent. It is involved in the differentiation of the lateral facial mesenchyme downstream of embryonic patterning genes.9,43 In humans, recessive loss-of-function mutations in HMX1 have been linked to OVAS, which is characterized by external ear and eye deformity.9 In a five-generation Chinese family with isolated bilateral microtia, a 10-Mb linkage locus covering 4p16 was discovered.9,43,44\n\nWe hypothesized that a link existed between the types of genes involved and the grade of microtia. For example, FGF3 deletions in LAMM syndrome have been clinically identified as grade I microtia.13,14,23,24 According to the MARX classification,10 the HOXA2 gene was common in the form of microtia type II and was exclusive to isolated microtia.1,17,20,39,45–48 However, no specific type of microtia has been linked to the deletion of TCOF1.10,49\n\nThe field of genetic variables in microtia research is still relatively extensive. More research on genetic variables that contribute to microtia is required, particularly using the next generation sequencing (NGS) and DNA microarray approach. NGS, massively parallel sequencing, or deep sequencing are all terms that refer to DNA sequencing technology that has transformed genomic research.48,49 In comparison to other technologies, NGS can sequence the entire human genome in a single day. Genomes may be examined without prejudice, allowing mosaic mutations to be detected.48 On the other hand, DNA microarray is a revolutionary technique for gene expression profiling.50 Microarrays were created as a method for mapping and sequencing vast amounts of DNA. DNA microarrays offer a far higher throughput and are less time consuming than previous approaches. By applying NGS and microarray for screening of genetic risk factors in microtia, the diagnosis of microtia could be made earlier and the patients could get a more comprehensive treatment.\n\nThis systematic review used recent available evidence and is the first systematic review to describe genetic factors in microtia. All studies included in this review were assessed as being of high quality. However, the limitations of this study included the heterogeneity of studies on the genetic evaluation of microtia in online databases, as well as the absence of information on the details of the subjects; thus, we could not perform a meta-analysis in this study. Gender information was unknown in 4.4% of patients, severity was unknown in 49.5% of subjects, and the type of microtia was unclear in 70.4% of patients. Case reports and case series were the most common study types in this systematic review. More observational research on genetic microtia is required to perform a more comprehensive systematic review and even meta-analysis.\n\n\nConclusions\n\nAccording to this study, most cases of microtia (75.5%) occurred in China, 60.7% of subjects had a family history of microtia, 58.5% of cases occurred in males, 74.2% of cases were bilateral, and 41.3% of cases were grade III microtia. From the studies involved, the three most common genes associated with the development of microtia were HOXA2 (10%), FGF3 (8.4%), and TCOF1 (5.4%). The most prevalent syndromes related to microtia were TCS and LAMM syndrome. Deletion mutations in TCOF1 were found in 15 patients (46.9%), deletion and missense mutations were present in FGF3 in 19 patients (38%), and silent HOXA2 mutations were present in 32 patients (54.2%).\n\nApproximately 76.2% of the genetic factors that contribute to microtia remain unknown. More research on genetic variables in microtia is required, particularly the use of NGS, massively parallel sequencing, or deep sequencing. We recommend that investigations on genetically associated microtia be conducted using observational studies, and the features of patients involved should be described more clearly and comprehensively in the future for better systematic reviews or even meta-analysis.\n\nBy understanding the three most dominant genes associated with microtia (HOXA2, FGF3, and TCOF1), we could promote the early screening and detection of microtia in the next generation, allowing us to provide better education and genetic counseling to patients with microtia regarding the possibility of microtia development in their children, and we hope that this systematic review will serve as a reference for the establishment of a global database of patients with microtia.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nHarvard Dataverse: The Role of Genetic Factors in Microtia: A Systematic Review. https://doi.org/10.7910/DVN/4RRHH0.72\n\nThis project contains the following extended data:\n\n• Supplementary Files.docx (Table 1. Medline (Pubmed) search strategy to identify published literature, tables 2-4 Risk of bias evaluation of studies involved in this systematic review using JBI Checklist, tables 5-6 additional characteristics of studies involved in this systematic review)\n\n• Table Manuscript.docx (Tables 1-2 main characteristics of studies involved in this systematic review, table 3 type of mutation found on analysis)\n\nHarvard Dataverse: PRISMA checklist and flowchart for ‘The role of genetic factors in microtia: A systematic review’. https://doi.org/10.7910/DVN/4RRHH0.72\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAlasti F, Sadeghi A, Sanati MH, et al.: A Mutation in HOXA2 Is Responsible for Autosomal-Recessive Microtia in an Iranian Family. Am. J. Hum. Genet. 2008; 82(4): 982–991. 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PubMed Abstract \| Publisher Full Text\n\nPutri IL, Stephanie A, Pramanasari R, et al.: The Role of Genetic Factors in Microtia: A Systematic Review. Harvard Dataverse. 2022; V5. Publisher Full Text" }	[ { "id": "139395", "date": "08 Jul 2022", "name": "Walid D. Fakhouri", "expertise": [ "Reviewer Expertise Genetics", "Craniofacial development and disorders" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMicrotia and anotia are congenital birth defects of the external ear with a frequency that ranges from 1:2000–1:10,000 live births 1,2. Microtia and anotia can be part of syndromic disorders or isolated events. The causes of isolated and syndromic external ear malformation remain unknown in many cases 2,3,4. However, the genetic components play a major role in causing or increasing the risk for isolated forms of external ear disorders. While microtia is defined as a small malformed external ear, anotia is a lack of external ear. The external ear malformations happen during the first trimester between the fourth and ninth week of pregnancy. The severity can range from mild to complete missing of the external ear with possible inner ear abnormal structures 3.\nIn this systematic review study, Putri et al. analyzed the role of genetic factors in isolated and syndromic forms of microtia. They use six search engines of scientific peer-reviewed publications to screen retrieved articles and identify the relevant articles for their analysis based on a set of inclusion and exclusion criteria established by the reviewers. The authors performed and executed their analysis according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines. The paper provides an excellent rationale for the study with clear objectives of the systematic review. The paper is also well-written and structured and includes the strength and limitations of its analysis of the retrieved articles. However, the description of the prevalence of microtia in the mentioned countries based on the included studies in their systematic review might be misleading or inaccurate. The authors cannot draw a straight line on the microtia prevalence in these countries based only on a few case reports, case-control, observational studies, or a lack of clinical studies in other countries. The authors need to validate the prevalence of microtia in these countries based on other official sources like the CDC or the authors might indicate that the incidences of the cases based on the analyzed studies included in the systematic review might infer the frequency in these countries.\nAlthough 15 articles were relevant to the systematic review based on the titles and abstracts, the authors excluded them from the analysis after the full text was read without providing additional information or justification on why.\nOne point that has not been explained in the systematic review is that the altered genes in syndromic disorders of microtia did not contribute to the isolated form of microtia.\nFinally, the authors also need to explain why their systematic review analysis missed several genes that mutations within their loci can cause microtia as part of syndromic disorders. The other genes that are related to microtia are described below:\nMutations in POLR1B cause Treacher-Collins syndrome 4, an autosomal dominant disorder, characterized by craniofacial dysmorphisms, including downslanting palpebral fissures, malar, and mandibular hypoplasia, and microtia (Sanchez et al., 20205).\n\nMutations within TWIST2 cause Ablepharon-macrostomia syndrome, an autosomal dominant disorder, characterized by congenital ectodermal dysplasia characterized by absent eyelids, macrostomia, microtia, redundant skin, sparse hair, dysmorphic nose and ears, variable abnormalities of the nipples, genitalia, fingers, and hands, largely normal intellectual and motor development, and poor growth (Marchegiani et al., 20156).\n\nHSPA9 mutations cause Even-plus syndrome (AR), which is characterized by prenatal-onset short stature, vertebral and epiphyseal changes, microtia, midface hypoplasia with a flat nose and triangular nares, cardiac malformations, and other findings including anal atresia, hypodontia, and aplasia cutis (Royer-Bertrand et al., 20157)\n\nEFTUD2 mutations cause mandibulofacial dysostosis, Guion-Almeida type (AD), a syndrome characterized by progressive microcephaly, midface and malar hypoplasia, micrognathia, microtia, dysplastic ears, preauricular skin tags, significant developmental delay, and speech delay (Lines et al., 20128)\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "8629", "date": "14 Oct 2022", "name": "Citrawati Wungu", "role": "Author Response", "response": "Thank you for the comments on our manuscript entitled \"The Role of Genetic Factors in Microtia: A Systematic Review\". We appreciate the suggested modifications and have revised the manuscript accordingly. The revised sections are shown in boldface type. The detailed responses to the reviewers’ comments are presented as follows: Reviewer 1 Comment 1: The authors need to validate the prevalence of microtia in these countries based on other official sources like the CDC or the authors might indicate that the incidences of the cases based on the analyzed studies included in the systematic review might infer the frequency in these countries. Comment Response 1: We thank the reviewer for the excellent input, comments, and suggestions, which have helped us to improve our manuscript. We have checked and revised the global prevalence of microtia based on the CDC according to the suggestions. Hopefully, this revision suffices and our manuscript can be considered for indexing (Page 8, last line). Comment 2: Although 15 articles were relevant to the systematic review based on the titles and abstracts, the authors excluded them from the analysis after the full text was read without providing additional information or justification on why. Comment Response 2: Thank you for this comment. We have now added the explanation regarding why we excluded them from the analysis after the full text was read (Figure 1. Prisma Flow diagram). Comment 3: One point that has not been explained in the systematic review is that the altered genes in syndromic disorders of microtia did not contribute to the isolated form of microtia. Comment Response 3: Thank you for this excellent input and comment, which has helped us to improve our manuscript. We have added the explanation as requested (Page 14). Comment 4: Finally, the authors also need to explain why their systematic review analysis missed several genes that mutations within their loci can cause microtia as part of syndromic disorders. The other genes that are related to microtia are described below Comment Response 4: We thank the reviewer for giving us a chance to improve our manuscript. We have repeated the search for articles using the same keywords to get comprehensive results and added missing genes that can cause microtia in our studies." } ] }, { "id": "143637", "date": "09 Aug 2022", "name": "Isabella Monlleó", "expertise": [ "Reviewer Expertise Medical and human genetics", "craniofacial anomalies", "intellectual disability", "disorders of sex 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\nMicrotia is a multidisciplinary and exciting subject. Although easy to recognize clinically, there is a lack of consensus on its definition and terminology, so, over the last 80 years, several classification systems have been launched and coexist. For many dysmorphologists, surgeons, and researchers, microtia is a spectrum of congenital anomalies spanning from minor dysmorphologies of the external ear to anotia plus the absence of the auditory canal. Therefore, it can be referred to as microtia, anotia, or microtia-anotia, involving only the external or the external and middle ear (Luquetti et al. 20111; 20122). Whatever definition is employed, this congenital anomaly may be seen as an isolated defect or as a clinical sign of multiple anomalies collectively named syndromic microtia, several related to recognized syndromes (Luquetti et al. 20111; 20122). The subtle and age-dependent nature of some dysmorphologies and the above-mentioned disagreement may result in inconsistent phenotypic data that lead to misdiagnoses and research selection bias.\nIn the case of syndromic microtia, the etiology encompasses chromosomal abnormalities (aneuploidies, large rearrangements, and submicroscopic disequilibrium), single gene disorders, and teratogenic embryopathies. Different genetic approaches have been used as karyotyping, chromosomal microarray analysis, and DNA sequencing, to extend the list of known genes and causative variants (Gendron et al. 20163, Luquetti et al. 20111, 20122). On the other hand, the etiology of isolated microtia remains poorly understood. Despite this, the higher concordance in monozygotic twins compared to dizygotic twins, the disproportional sexual distribution, the existence of multiplex families, and the prevalence variation across different geographic regions and ethnicities are strong evidence that this is a complex or multifactorial trait. Therefore, the phenotype should be looked at as the result of an imbricate gene-gene and gene-environment interaction instead of the isolated effect of each one (Chen & Zhang 20194, Fan et al. 20205, Gendron et al. 20163, Luquetti et al. 20111, 20122).\nStrategies to investigate the genetic factors underlying multifactorial phenotypes should include linkage analysis, GWAS, exome and genome sequencing, gene-editing technologies, and predictive and functional analysis, with bioinformatics support. The study of familial cases, twins, and large series of patients, and experimental studies using animal models, are equally important (Chen & Zhang 20194, Fan et al. 20205, Gendron et al. 20163, Luquetti et al. 20111, 20122). The enormous volume of data generated by these approaches is available through many genetic databases.\nTherefore, to study microtia (isolated or syndromic) is challenging and should consider the many factors involved in its etiopathogenesis and expressivity, such as genes, environment, exposition, and epigenetic alterations (Chen & Zhang 20194, Fan et al. 20205, Gendron et al. 20163).\nIn this systematic review registered at the PROSPERO platform, the authors aimed to analyze the role of genetic factors in isolated and syndromic forms of microtia. They adhered to the PRISMA guidelines and clearly outlined their inclusion criteria, information sources, search strategies, and details of data collection.\nTaking the above considerations into account and the authors' intent ‘to qualitatively identify the most important genes in the development of microtia’, I have some questions and suggestions:\nWhy did the authors not include human gene and human phenotype databases such as HGMD, ClinVar, MedGen, OMIM, etc., in their search strategy?\n\nWhy did the authors limit their review to observation studies?\n\nWhat are the reasons for excluding 15 articles from the 55 ‘potentially appropriate articles´(Figure 1)?\n\nWhy was the authors' list of genes involved in the microtia phenotype not exhaustive?\n\nWhy did the authors dedicate part of the discussion to epidemiological data since this was not their research question? To do so they would change or widen their searching criteria.\n\nIt would be appreciated if authors make clear the existence of different definitions and classification systems for microtia. Also, they would include information in this regard in Tables 1 and 2. Did the listed papers use the same definitions to grade microtia?\n\nWhat evidence did the authors use to support their hypothesis of a genotype-phenotype correlation between microtia degrees and (alterations in) HOXA2 and FGF3 genes? And for the inexistent G-P correlation involving TCOF1?\n\nReview the use of ‘disorder level’ and ‘gene assessed’ in Tables 1 and 2 since some studies have reported CNVs and chromosomal rearrangements.\n\nReview the inclusion of the 22q11.2 syndrome as an example of isolated microtia in Table 2.\n\nIs the statistical analysis and its interpretation appropriate? Frequency distribution was the single statistical method employed in this study that reviewed 40 case reports/series and a few case-control studies. This method and sources are not suitable to conclude the epidemiology and etiology of microtia. Authors should include national and international registries on birth defects, such as The International Clearinghouse for Birth Defects Surveillance and Research (ICBDSR), the Estudio Colaborativo Latinoamericano de Malformaciones Congénitas (ECLMAC), the European Surveillance of Congenital Anomalies (EUROCAT), South-East Asia Region’s Newborn and Birth Defects Database (SEAR-NBBD), and search for genetic variants associated with microtia in human gene and phenotype databases. They should also employ robust statistics to conclude on prevalence and incidence rates worldwide. Finally, considering that microtia is a heterogenous phenotype, what is the evidence to state that 76.2% of the genetic factors involved in this birth defect remain unknown? Are the authors postulating that 23.8% are already known? I have not seen any statistics to support this inference.\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": [ { "c_id": "8908", "date": "24 Oct 2022", "name": "Citrawati Wungu", "role": "Author Response", "response": "Reviewer 2 Comment 1: Why did the authors not include human gene and human phenotype databases such as HGMD, ClinVar, MedGen, OMIM, etc., in their search strategy? Comment Response 1: We thank the reviewer for the excellent comments to improve our manuscript. We did not include human gene and human phenotype databases such as HGMD, ClinVar, MedGen, OMIM, etc., in our search strategy because we need certain data used in each study to be analyzed in our systematic review. The information provided on these databases is not intended for direct diagnostic use or medical decision-making without review by a genetics professional1,2 and it only collates all known (published) gene lesions responsible for human inherited disease3. This statement was disclaimed on their website. In addition, the systematic review should obtain sources from primary studies, not from genetic databases. Comment 2: Why did the authors limit their review to observation studies? Comment Response 2: We thank the reviewer for giving us an excellent comment to improve our manuscript. We did limit our systematic review to observational studies/non-experimental studies of syndromic microtia and isolated microtia to be analyzed objectively in our study without any intervention from the researcher. Besides, it is impossible to take data from clinical trials/experimental studies as this type of study is not suitable for microtia. Comment 3 What are the reasons for excluding 15 articles from the 55 ‘potentially appropriate articles’ (Figure 1)? Comment Response 3: Thank you for this excellent input and comment, which have helped us to improve our manuscript. We have added the explanation of the excluded articles as requested and additional revision of the PRISMA flow in our study (page 4). Comment 4: Why was the authors' list of genes involved in the microtia phenotype not exhaustive? Comment Response 4: We thank the reviewer for giving us a chance to improve our manuscript. We have repeated the search for articles using the same keywords to get comprehensive results and added missing genes that can cause microtia in our studies (pages 5-9). Comment 5: Why did the authors dedicate part of the discussion to epidemiological data since this was not their research question? To do so they would change or widen their searching criteria. Comment Response 5: Thank you for this excellent input and comment, which have helped us to improve our manuscript. The research question in our study was what genes were most commonly associated with microtia, including syndromic microtia and isolated microtia. In the discussion section, in addition to discussing the most common gene data involved in syndromic microtia and isolated microtia, the gene examination technique used, we also add a discussion section by analyzing patient characteristics data from each study (pages 12-15). Comment 6: It would be appreciated if authors make clear the existence of different definitions and classification systems for microtia. Also, they would include information in this regard in Tables 1 and 2. Did the listed papers use the same definitions to grade microtia? Comment Response 6: We thank the reviewer for giving us excellent input and comments to improve our manuscript. Most of the listed papers used in our study had the same definition to grade microtia, Hunter classification. To homogenize the definition to grade microtia in our study, we use the Hunter classification system for microtia when extracting data from 98 studies and data provided data in Tables 1 and 2 (pages 5-9). Thank you for your suggestion, we would like to make clear the existence of different definitions and classification systems of microtia in our discussion (page 13). Comment 7: What evidence did the authors use to support their hypothesis of a genotype-phenotype correlation between microtia degrees and (alterations in) HOXA2 and FGF3 genes? And for the inexistent G-P correlation involving TCOF1? Comment Response 7: Thank you for this excellent input and comment, which have helped us to improve our manuscript. We have discussed in our discussion about each role of HOXA2, FGF3, and TCOF1 genes in microtia (pages 13-14). Comment 8: Review the use of ‘disorder level’ and ‘gene assessed’ in Tables 1 and 2 since some studies have reported CNVs and chromosomal rearrangements. Comment Response 8 : Thank you for this comment. We use the term “disorder level” in Tables 1 and 2 to describe the types of disorders present and “gene assessed” to describe, while the term \"gene assessed\" refers to the genetic material being examined and whether it is found to be abnormal in genes, chromosomes, or CNV DNA. Comment 9 : Review the inclusion of the 22q11.2 syndrome as an example of isolated microtia in Table 2. Comment Response 9: We thank the reviewer for giving us a chance to improve our manuscript. We have repeated the search for articles using the same keywords to get comprehensive results and added missing genes that can cause microtia in our studies (pages 5-9). Comment 10: Is the statistical analysis and its interpretation appropriate? Frequency distribution was the single statistical method employed in this study that reviewed 40 case reports/series and a few case-control studies. This method and sources are not suitable to conclude the epidemiology and etiology of microtia. Authors should include national and international registries on birth defects, such as The International Clearinghouse for Birth Defects Surveillance and Research (ICBDSR), the Estudio Colaborativo Latinoamericano de Malformaciones Congénitas (ECLMAC), the European Surveillance of Congenital Anomalies (EUROCAT), South-East Asia Region’s Newborn and Birth Defects Database (SEAR-NBBD), and search for genetic variants associated with microtia in human gene and phenotype databases. They should also employ robust statistics to conclude on prevalence and incidence rates worldwide. Comment Response 10: Thank you for this comment. As we know, A systematic review is a summary of the medical literature that uses explicit and reproducible methods to systematically search, critically appraise, and synthesize on a specific issue. It synthesizes the results of multiple primary studies related to each other by using strategies that reduce biases and random errors4,5. BDSR, ECOMAC, EUROCAT, SEARCH-NBBD provide a large database as a result of a collection of various studies, we did not use the data from the ones mentioned above in our systematic review because it would be a secondary big data analysis, not the systematic review we expected from the start. Comment 11: Finally, considering that microtia is a heterogenous phenotype, what is the evidence to state that 76.2% of the genetic factors involved in this birth defect remain unknown? Are the authors postulating that 23.8% are already known? I have not seen any statistics to support this inference. Comment Response 11: Thank you for this excellent input and comment, which have helped us to improve our manuscript. Because of the additional data on missing genes in our systematic review, the percentage of genes that remain unknown increased from 76.2% to 80%. We got this number from the calculation of the number of 100% of genetic factors involved in this birth defect minus 3 most common genes found in our systematic review: %HOXA2 (6.4%) minus %FGF3 (4.9%) and minus %TCOF1 (8.5%) (Page 12)." } ] } ]	1	https://f1000research.com/articles/11-537
https://f1000research.com/articles/12-444/v1	26 Apr 23	{ "type": "Research Article", "title": "Virtual screening of zinc compounds similar to NSAIDS with better pharmacodynamic and pharmacokinetic profiles", "authors": [ "Faith Kamakia", "Stephen Ouma", "Richard Kagia", "Stephen Ouma", "Richard Kagia" ], "abstract": "BACKGROUND: Pain is a common symptom that is managed in both outpatients and inpatients. There are many side effects associated with opioids such as respiratory depression, constipation, hyperalgesia, and tolerance. Non- steroidal anti-inflammatory drugs cause gastrointestinal tract (GIT) irritation and may be a risk factor for developing peptic ulcer disease. This study aimed to generate active analgesic agents from known analgesics, determine the docking scores of these agents to their receptors, determine the pharmacokinetic properties of these agents, and evaluate their toxicity profiles. METHODS: PubChem was used to download smiles for ibuprofen, aspirin and celecoxib. Avogadro optimized the ligands. The smiles were copied to SwissSimilarity and were used as query compounds to generate zinc compounds. DrugBank and Protein Data Bank were used to download cyclooxygenase 1 and 2. Molecular docking was done using Chimera and Autodock Vina. Smiles for both query compounds and generated zinc compounds were pasted onto the Protox II webserver and SwissADME for toxicity and pharmacokinetics properties determination. The data was presented in tabular forms with textual descriptions of the contents in the tables. RESULTS: Aspirin, ibuprofen and celecoxib’s zinc compounds were generated and the first 20 compounds were docked to COX-1 and COX-2 enzymes. Seven, one, and four of the docked compounds showed better binding energies to COX-2 than COX-1. The zinc compounds were analyzed for toxicity profiles. ZINC01680731 and ZINC33823423 were predicted to have LD50 of 1240 mg/kg as compared to aspirin’s 250mg/kg. Ibuprofen and ZINC39120409 showed LD50 of 299mg/kg and they were hepatoactive. Celecoxib and four of its zinc compounds showed LD50 of 1400mg/kg. All compounds had high GIT absorption and they conformed with Lipinski rule of five. CONCLUSIONS: ZINC01680731 0.994 and ZINC00600558 0.988 were identified as the best compounds as they showed better binding affinities, toxicities and pharmacokinetics properties compared to standard compounds.", "keywords": [ "Aspirin", "Ibuprofen", "Celecoxib", "Ligand-based virtual screening", "Structure-based virtual screening", "SwissADME", "Protox II" ], "content": "Introduction\n\nPain is part of normal body protective reflexes. Traditionally, pain has been referred to as a symptom and nowadays, pain is referred as a disease (Clauw et al., 2019). Pain has many different aspects, which encompass both physical and mental aspects. These effects have destructive properties on the body. Pain and related diseases are the leading cause of disabilities and discomfort globally (Fitzmaurice, 2018).\n\nPain is described as irritable, emotional, and sensory activities that could be associated with damage to tissues according to the International Association for the Study of Pain (Cohen et al., 2018). Most patients managed in the outpatient department have chief complaints associated with pain, which decreases the quality of life for many patients. Acute pain occurs in a period of fewer than three months while chronic pain occurs in a period greater than three months (Cohen et al., 2018).\n\nManagement of pain involves both pharmacological and non-pharmacological approaches. Most pharmacological approaches involve the use of analgesics such as NSAIDS and opioids. NSAIDS are used in the management of conditions such as osteoarthritis and lower back p\\in (Machado et al., 2021). These medications involve serious toxicities that may worsen patients’ clinical outcomes and recovery (Bindu et al., 2020).\n\nNon-selective NSAIDS provide relief from pain due to inhibition of both COX-1 and COX-2 (Ngo & Addison, 2018) in the body leading to decreased production of eicosanoids such as prostaglandins. Selective COX-2 agents lead to inhibition of COX-2 causing lack of eicosanoids. Aspirin exerts its pain modulation by blocking synthesis of prostaglandins (Loussert et al., 2020).\n\nAspirin inhibits thromboxane A2 which causes irreversible blocking platelet aggregation, leading to persistent bleeding. Aspirin also causes hypersensitivity in patients which leads to anaphylaxis and bronchospasms, and can cause salicylism that manifests as tinnitus. Additionally, aspirin can cause Reye’s syndrome in children with a past medical history of viral illness (Christiansen et al., 2021).\n\nNSAIDS have ceiling effects where no more analgesia is exerted despite increase in dose. Non-selective COX inhibitors cause renal failure, irritation and bleeding from gastric mucosa. Selective COX-2 inhibitors lead to both renal and liver failure on chronic use (Bindu et al., 2020).\n\nNSAIDS such as ibuprofen, when used during pregnancy, cause closing of the ductus arteriosus. Ductus arteriosus is important in the circulation of blood in neonates, especially those born with low birth weights. Ibuprofen use in neonates causes drug induced neonatal cholestasis which causes jaundice in the infants due to abnormalities in bile acid synthesis and conjugation, as well as disorders in urea cycle of metabolism (Shen et al., 2021).\n\nDue to the many unwanted side effects associated with cyclooxygenase inhibition, there is a need to develop new anti-inflammatory agents devoid of side effects. This study used aspirin, ibuprofen and celecoxib as query compounds to generate new compounds using the SwissSimilarity online tool. The compounds generated were docked to cyclooxygenase 1 and 2 enzymes (COX-1 and COX-2) and the docking scores were compared to those of aspirin, ibuprofen and celecoxib. Compounds with better docking scores to the query compounds were determined for their pharmacokinetics and toxicity profiles using SwissADME and Protox II webservers respectively.\n\n\nStudy Objectives\n\n\n\ni. To determine similar zinc compounds to aspirin, ibuprofen and celecoxib using the SwissSimilarity online tool.\n\nii. To determine zinc compounds with better docking scores to cyclooxygenase 1 and 2 in reference to aspirin, ibuprofen and celecoxib.\n\niii. To determine the pharmacokinetic properties of zinc compounds in reference to aspirin, ibuprofen and celecoxib.\n\niv. To determine the toxicity profiles of zinc compounds in reference to aspirin, ibuprofen and celecoxib.\n\n\nMethods\n\nThe PubChem online tool was opened and aspirin, ibuprofen and celecoxib were searched. The summary button was clicked and this generated the smiles of the compounds. This also allowed the reference compounds (aspirin, ibuprofen and celecoxib) to be downloaded as SDF files. The downloaded files were saved to folders named aspirin, ibuprofen and celecoxib on the desktop of the laptop. Combined Zinc–Drug like was then clicked in SwissSimilarity, which automatically generated a red search button that was clicked on. Zinc compounds were generated.\n\nLigand-based virtual screening uses active ligands for lead identification and optimization rather than the structures of the protein. Using the SwissSimilarity online tool, the known compounds are used as queries in virtual screening to identify new active compounds. There must be similarities between the known ligands to the library of compounds in the form of geometrical measurements. Pharmacophore-based methods use molecular properties and then generate the similarity score between the known compounds and the ligand compounds. The molecular shape determines the overlap of shapes between the compounds and the known agents and creates the similarity scores. The assumptions of the molecular shape are that the overlap in structures of the known compounds and the ligands show activity (Hamza et al., 2012), therefore the ligands with different shapes to the query compounds are missed in virtual screening. The compounds are then scored and differentiated as active or inactive (Hamza et al., 2012).\n\nIn this study, aspirin, celecoxib, and ibuprofen were used as queries to generate zinc compounds based on the structures of the reference compounds (aspirin, celecoxib, and ibuprofen). The SwissSimilarity online tool was used. The SwissSimilarity tool generates agents with high drug-likeness to the agents by use of a vast database like ZINC (Bragina et al., 2022) and this gives a pool of structures to be analysed.\n\nZINC compounds with above 80% similarity scores to the query compounds ibuprofen and aspirin were chosen, while compounds with a similarity score above 70% to celecoxib were chosen. For the compounds that had the same similarity scores, only one compound was included. 20 compounds for ibuprofen, 20 compounds for aspirin and 20 compounds for celecoxib were included giving a total of 60 compounds that were included in this study.\n\nStructure-based virtual screening starts with protein structure identification through NMR spectroscopy or homology modelling. This is followed by binding site identification which considers druggability, stereochemistry, and tautomers as well as protonation of the binding site. Receptor preparation is done by modifying the tautomeric forms of histidine receptors, removing unnecessary side chains, determining flexibility of the compounds as well as solvation of the receptor binding sites. Ligand preparation follows by ensuring they are at as minimal energy state as possible which is done by Chimera software (Pettersen et al., 2004). Docking of the ligands to the receptor is done using AutoDock Vina and scoring is done such that the higher the binding scores of the compounds, the higher the binding affinity (Eberhardt et al., 2021). Post docking analysis of the compounds is completed to show the 2D or 3D interactions of the ligands to their receptors, such as by using Discovery studio (RRID: SCR_015651) (Lionta et al., 2014).\n\nIn this study, cyclooxygenase 1 and 2 enzymes were prepared using Chimera (RRID: SCR_004097) software which eliminated the unwanted residues bound to the receptors. Ligand preparation was done using Avogadro software (RRID: SCR_011958). Molecular docking was done using AutoDock Vina (RRID: SCR_011958). Zinc compounds that showed higher docking scores in reference to aspirin, celecoxib and ibuprofen were further analyzed for pharmacokinetics as well as toxicity profiles.\n\nThe pharmacokinetics properties of the agents were analyzed using SwissADME, which lead to the discovery of agents with improved properties.\n\nTox 21 (The Toxicology in the 21st century) has led to the development of computational methods for the toxicological assessment of compounds from the identified toxicological pathways (Banerjee et al., 2018). ProTox II webserver is commonly used in toxicity predictions and predicts toxicities such as oral toxicity, organ toxicity (hepatoxicity), and toxicological endpoints which include mutagenicity, cytotoxicity, immunotoxicity, and immunogenicity. The molecular mechanism for predicting toxicities in the body is as well generated from the ProTox-II webserver (Banerjee et al., 2018).\n\nProTox II (RRID: SCR_018506) webserver was used in this study to determine toxicity profiles of zinc compounds and were compared to the reference compounds.\n\nDescriptive analysis was chosen since the data involved was numerical. Compounds that showed better docking scores to COX-2 and lower docking scores to COX-1 than aspirin, ibuprofen and celecoxib were identified and were chosen as promising compounds. The highlighted compounds were determined for their toxicity profiles and pharmacokinetics properties. Pharmacokinetic data were analyzed non-numerically since the data generated was qualitative.\n\n\nResults\n\nDocking scores of aspirin and zinc compounds to Cox-1 and cox-2\n\nAspirin downloaded from PubChem was used as the reference against other zinc compounds. The ligands with better docking scores to COX-2 and lower docking scores to COX-1 in reference to aspirin are highlighted in Table 1 below (Faith et al., 2023). ZINC00001050; 0.995, ZINC00409844; 0.992, ZINC01651927; 0.991, ZINC01680731; 0.994, ZINC19405119; 0.986, ZINC33823423; 0.996, and ZINC71771212; 0.994, were analysed further for their pharmacokinetics properties using the SwissADME online webserver and toxicity predictions using the ProTox II webserver.\n\nAll the zinc and aspirin compounds showed high gastrointestinal (GIT) absorption and permeation to the blood-brain barrier (BBB). The compounds were shown not to be P-glycoprotein substrates and to lack enzyme inhibition properties to CYP (cytochrome) 1A2, CYP 2C19, CYP2C9, CYP2D6 and CYP3A4 enzymes. The compounds further comply with the Lipinski rule of five. Exceptions to this were ZINC01651927; 0.991 and ZINC19405119; 0.986, where ZINC01651927; 0.991 showed inhibition of CYP1A2 enzyme while ZINC19405119; 0.986 showed no permeation to the BBB.\n\nDocking scores of ibuprofen and zinc compounds to COX-1 and COX-2\n\nThe reference compound (ibuprofen) had better docking scores to both COX-1 and COX-2 than the zinc compounds except ZINC39120409; 0.996, which showed a higher docking score to COX-2 than ibuprofen and a lower docking score to COX-1 than ibuprofen as illustrated in Table 2. ZINC39120409; 0.996 was further analyzed for its pharmacokinetic properties and toxicity profile.\n\nZINC39120409; 0.996 and ibuprofen showed high GIT absorption and permeation to the blood-brain barrier. The compounds shown not to be P-glycoprotein substrates and lack enzyme inhibition properties to CYP 1A2, CYP 2C19, CYP2C9, CYP2D6 and CYP3A4 enzymes. The compounds further complied with Lipinski rule of five.\n\nCelecoxib and zinc compounds docking scores to COX-2 and 1\n\nThe highlighted compounds in Table 3 showed better docking scores to the COX-2 enzyme than celecoxib and lower docking scores to COX-1 than celecoxib. ZINC00600558; 0.988; ZINC34660892; 0.988; and ZINC43464372; 0.750; were further analyzed for their pharmacokinetics properties as well as toxicity parameters.\n\nCelecoxib showed high GIT absorption and showed inhibition to CYP1A2 and CYP 2C9. Celecoxib lacks inhibition to CYP2C19, CYP2D6, and CYP3A4. Celecoxib is not a substrate to P-glycoprotein and lacks BBB permeation.\n\nZINC00600558; 0.988, ZINC34660892 and ZINC43464372; 0.750 showed high GIT permeability, and a lack of CYP2D6 and CYP3A4 inhibition. These zinc compounds are not substrates of P-glycoprotein, and they lack permeation to the BBB. ZINC00600558; 0.988 and ZINC43464372; 0.750 however, showed enzyme inhibition activity to CYP1A2, CYP2C19, and CYP2C9. ZINC34660892 showed a lack of enzyme inhibition to CYP1A2 and the presence of CYP2C9 and CYP2C19 inhibition. They all complied with Lipinski rule of five.\n\nToxicity\n\nOral toxicity\n\nZINC01680731; 0.994 and ZINC01680731; 0.994 were predicted to be the safest with oral LD50 of 1240 mg/kg as compared to aspirin which was predicted to have oral LD50 of 250 mg/kg. ZINC00409844; 0.992, ZINC01651927; 0.991 and ZINC19405119; 0.986 were predicted to have the same oral LD50 as aspirin of 250 mg/kg. ZINC00001050; 0.995 and ZINC71771212; 0.994 were predicted to have oral LD50 of 650mg/kg (see Table 4).\n\nIbuprofen and ZINC39120409; 0.996 were predicted to have similar oral LD50 of 299 mg/kg.\n\nZINC00600558; 0.988, ZINC34660892; 0.988 and ZINC43464372; 0.750 showed similar oral LD50 of 1400mg/g as celecoxib.\n\nHepatotoxicity and immunotoxicity\n\nZINC00001050; 0.995, ZINC71771212; 0.994 and ZINC33823423; 0.996 were predicted to be hepatotoxic with probability scores of 0.56, 0.56 and 0.55 respectively in relation to aspirin, which is inactive. Ibuprofen and ZINC39120409; 0.996 were predicted to be hepatotoxic with probability scores of 0.66 and 0.56 respectively. ZINC43464372; 0.750 was predicted to be hepatoactive with probability score of 0.58 in relation to celecoxib. None of the compounds was predicted to be immunotoxic (see Table 5).\n\nCelecoxib, ZINC34660892; 0.988 and ZINC43464372; 0.750 were predicted to be carcinogenic with probability scores of 0.56, 0.59 and 0.67 respectively. The rest of the compounds were inactive for carcinogenic, mutagenic and cytogenic properties. ZINC00600558; 0.988 was predicted to be inactive for carcinogenesis as compared to celecoxib (see Table 6).\n\nZINC00409844; 0.992 and ZINC01651927; 0.991 were predicted to have activity on aryl hydrocarbon receptors with probability scores of 0.5 as compared to aspirin, which was predicted to be inactive. Celecoxib, ZINC00600558; 0.988 and were predicted to be active for estrogen receptor with probability scores of 1.0, 0.7 and 0.93 respectively (see Table 7).\n\nZINC00409844; 0.992 and ZINC01651927; 0.991; were predicted to be active for mitochondrial membrane potential with a probability score of 0.67. ZINC43464372; 0.750 was also predicted to be active for mitochondrial membrane potential with a probability score of 0.52,relative to celecoxib that was predicted inactive (see Table 8).\n\nPharmacokinetics profile\n\nBBB permeation\n\nZINC19405119; 0.986, celecoxib, ZINC00600558; 0.988, ZINC34660892; 0.988 and ZINC43464372; 0.750 lack blood brain barrier penetration while aspirin has blood brain barrier penetration (see Table 9).\n\nMetabolism\n\nZINC01651927; 0.991, celecoxib, ZINC00600558; 0.988 and ZINC43464372; 0.750 were all predicted to be inhibitors of CYP1A2. ZINC00600558; 0.988, ZINC34660892; 0.988 and ZINC43464372; 0.750 were predicted to be inhibitors of both CYP2C9 and CYP2C19 in comparison to celecoxib, which lacks CYP2C9 and CYP2C19 inhibition (see Table 10).\n\nPost-docking analysis (Faith et al., 2023)\n\nAspirin and ZINC01680731; 0.994 interactions with COX-2 receptor (Figure 1)\n\nIn both aspirin and ZINC01680731; 0.994, the phenyl ring is involved in pi-pi binding with histidine at chain A of the receptor at position 388. The conventional hydrogen bond in aspirin is at tryptophan of chain A of position 387 while in ZINC01680731; 0.994, three conventional hydrogen bonds are at histidine at positions 207 of chain A, tryptophan at position 387 of chain A and threonine at position 206 of chain A. Van der Waals forces at positions 391 and 390 of leucine, 199 of alanine, 203 of glycine, and 206 and 385 of threonine in chain B of COX-2 receptor in aspirin are different from the van der Waals forces in ZINC01680731; 0.994 at positions 199, 202 and 385. These differences in binding of the compounds to the COX-2 receptor could have been attributed to ZINC01680731; 0.994 having a higher binding score compared to aspirin (Figure 1).\n\nIbuprofen and ZINC39120409; 0.996 2D-interactions with receptors (Figure 2)\n\nBoth ibuprofen and ZINC39120409; 0.996 showed similar modes of interactions in COX-1 as compared to their binding to COX-2. Ibuprofen showed similar interactions to COX-2 as ZINC39120409; 0.996 with the exemptions that ibuprofen’s pharmacophore also bonded with pi-pi binding to active sites in COX-2 which was absent from ZINC39120409; 0.996 binding to COX-2 (Figure 2).\n\nCelecoxib and its zinc compounds 2D-visualization (Figures 3 and 4)\n\nCelecoxib’s pharmacophore binds similarly to active sites in COX-1 and COX-2 with fluorine binding to active sites in both receptors (Figure 3). A halogen bond was noted in all the zinc compounds. Covalent bonding, van der Waals forces, alkyl, and pi-alkyl bonds were noted in all the compounds’ pharmacophores in binding with COX-1 active sites. Celecoxib’s pharmacophore interacted with active sites in COX-2 using van der Waals forces, covalent bonding, and pi-pi bonds, Pi-anion and pi-sigma bonds were noted in ZINC00600558; 0.988, and ZINC01028131; 0.765; respectively (Figure 4).\n\n\nDiscussion\n\nThe drug molecules used as standards in this study were assessed for similar zinc compounds using SwissSimilarity online tool. NSAIDs were analysed for their docking scores with those having better binding energies to COX-2 than the standard known compounds and lower docking energies to COX-1 than the reference compounds being preferred.\n\nCox breaks down arachidonic acid into prostaglandin H2 and prostaglandin G2. Prostaglandin GH2 rapidly decomposes into thromboxane, prostacyclin, and other forms of prostaglandin such as PGE and PGF2a. Thromboxane effects involve platelet aggregation and vasoconstriction, PG12 inhibits platelet aggregation and vasoconstriction, PGE2 regulates renal function and PGF2a is used to indicate oxidative stress (Majed & Khalil, 2012). COX-1 is expressed constitutively in human cells such as somatic cells of the ovary and therefore maintains normal physiological functions (Kirkby et al., 2012) such as normal GIT integrity by maintaining mucus and bicarbonate secretion. COX-2 is highly inducible by factors such as hypoxia (Wang et al., 2019) and inflammation. Conditions such as pre-eclampsia occur due to dysregulation of thromboxane/prostaglandin 12 causing an increase in maternal thromboxane.\n\nIn Table 1, ZINC00001050; 0.995; ZINC00409844; 0.992; ZINC01651927; 0.991; ZINC01680731; 0.994 ZINC19405119; 0.986; ZINC33823423; 0.996; and ZINC71771212; 0.994; showed higher docking scores to COX-1 and COX-2in reference to aspirin. These compounds, therefore, would produce fewer GIT side effects such as ulcers than aspirin. ZINC39120409; 0.996 showed better binding energies to COX-2 than COX-1 than ibuprofen as demonstrated in Table 2. Table 3 demonstrated that ZINC00600558; 0.988; ZINC34660892; 0.988; and ZINC43464372; 0.750; have higher docking scores to COX-2 than COX-1 in reference to celecoxib and therefore, create promising results to the discovery of COX-2 selective agents.\n\nProTox II webserver was used to predict toxicities of the compounds. In Table 4, oral toxicities of the compounds were highlighted. ZINC00409844; 0.992; ZINC01651927; 0.991, ZINC19405119; 0.986 showed LD50 of 250mg/kg similar to aspirin. ZINC00001050; 0.995 and ZINC71771212; 0.994; showed LD50 of 650mg/kg therefore, they were considered safer than aspirin. ZINC01680731; 0.994 and ZINC33823423; 0.996; showed LD50 of 1240mg/kg and therefore, they were considered the safer zinc compounds than aspirin. Celecoxib and its zinc compounds showed LD50 of 1400mg/kg and therefore, they were considered harmful when swallowed as indicated in Table 4. Ibuprofen and its zinc compound showed an LD50 of 299mg/kg.\n\nTable 5 indicated the organ toxicity and immunotoxicity predictions. ZINC00001050; 0.995, ZINC71771212 and ZINC33823423; 0.996; were predicted to be active in causing hepatotoxicity with probability scores of 0.56 and 0.55 as compared to aspirin which was predicted to be inactive. Ibuprofen and its zinc compound ZINC39120409; 0.996 were predicted to be active in causing hepatotoxicity with probabilities of 0.66 and 0.56 respectively. Ibuprofen-induced hepatotoxicity has been demonstrated by other researchers (Zoubek et al., 2020). ZINC43464372; 0.750 was predicted to be hepatotoxic with a probability value of 0.58 as compared to celecoxib which was predicted to be inactive with a probability of 0.60.\n\nCelecoxib and its zinc compounds, namely ZINC00600558; 0.988; and ZINC34660892; were predicted to be active in signalling estrogen receptors with probability scores of 1.0 and 0.70 respectively. These predictions have been supported by other researchers such as (Bafna et al., 2020) where celecoxib was proven to inhibit estrogen receptor alpha positive.\n\nIn Tables 9 and 10, the pharmacokinetics profiles of the NSAIDs were outlined. Compounds which lack cytochrome P450 system inhibition are ideal since they lack the potential to interact with food and other drugs. ZINC01714507; 0.986, celecoxib, ZINC00600558; 0.988; ZINC34660892; 0.988 and ZINC43464372; 0.750 showed CYP2C9 inhibition as outlined in Table 10. Inhibition of CYP2C9 could affect the metabolism of drugs such as S-warfarin and CYP2C19 enzyme inhibition affects the metabolism of drugs such as R-warfarin. ZINC00600558; 0.988; ZINC34660892; 0.988 and ZINC43464372; 0.750 showed CYP2C19 inhibition as indicated in Table 10. ZINC01714506; 0.986; ZINC00600558; 0.988; ZINC43464372; 0.750 and ZINC01714507; 0.986 showed CYP1A2 inhibition as indicated in Table 10. All NSAIDs and their zinc compounds lack CYP2D6 and CYP3A4 inhibition.\n\nAll NSAIDS in Table 9 and their zinc compounds showed permeation to BBB with the exception of ZINC19405119; 0.986; celecoxib, ZINC00600558; 0.988; ZINC34660892; 0.988, ZINC43464372; 0.750, ZINC01557001; 0.987, ZINC19281575; 0.992; and ZINC00294715; 0.980; which showed lack of BBB permeation.\n\nAll the NSAIDs and their zinc compounds were not substrates for the P-glycoprotein efflux system, and this showed promising inferences in terms of influencing the bioavailability of the molecules and resistance. All NSAIDs as well as their zinc compounds showed high GIT absorption, and this is important in the absorption of the molecules and achieving high bioavailability.\n\nConventional hydrogen bonding and van der Waals forces were predominantly between the compounds pharmacophore and cyclooxygenase’s enzyme binding sites. These bonds were manifested by aspirin, ZINC01680731; 0.994, and ibuprofen among other compounds. Other bonding forces were pi-pi stacking of the ligands phenyl ring with the targets such as seen in ibuprofen and celecoxib binding modes in Figures 2 and 3-4 respectively.\n\n\nConclusion\n\nThe objectives of this study were achieved. Compounds with better docking scores to reference compounds were highlighted and their pharmacokinetic profiles were assessed.\n\nThis study gave results that are highly promising in the discovery of drugs with better pharmacokinetic profiles and fewer side effects. Compounds with better docking scores to receptors and their pharmacokinetics profile were identified. ZINC01680731; 0.994 was considered the safest among all ofaspirin’s zinc compounds since it showed the highest LD50 as well as a lack of hepatoactivity. ZINC39120409; 0.996 and ibuprofen showed similar toxicity profiles since they both showed activity in hepatotoxicity. ZINC00600558; 0.988; was predicted to be carcinoinactive as compared to all the zinc compounds of celecoxib and therefore, it was considered the best compound.\n\nIn summary, ZINC01680731; 0.994, ZINC00600558; 0.988 were considered the safest considering their better docking score, better pharmacokinetics and better toxicity profiles. Further studies such as quantitative structure-activity relationship for ZINC01680731; 0.994, ZINC00600558; 0.988, and ZINC39120409; 0.996 should be done to improve on toxicity and pharmacokinetic properties. in vitro studies of these compounds should be done to assess further pharmacological activities.", "appendix": "Data availability\n\nHarvard DataVerse: Virtual screening of Ibuprofen, Celecoxib and Aspirin. https://doi.org/10.7910/DVN/XMFF8N (Faith et al., 2023).\n\nThis project contains the following underlying data:\n\na. ADME for NSAIDs\n\nb. Docking scores\n\nc. Oral toxicity\n\nd. Post Docking analysis\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\nBafna D, Ban F, Rennie PS, et al.: Computer-Aided Ligand Discovery for Estrogen Receptor Alpha. Int. J. Mol. Sci. 2020; 21(12): 4193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBanerjee P, Eckert AO, Schrey AK, et al.: ProTox-II: a webserver for the prediction of toxicity of chemicals. Nucleic Acids Res. 2018; 46(W1): W257–W263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBindu S, Mazumder S, Bandyopadhyay U: Non-steroidal anti-inflammatory drugs (NSAIDs) and organ damage: A current perspective. Biochem. Pharmacol. 2020; 180: 114147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBragina ME, Daina A, Perez MAS, et al.: The SwissSimilarity 2021 Web Tool: Novel Chemical Libraries and Additional Methods for an Enhanced Ligand-Based Virtual Screening Experience. Int. J. Mol. Sci. 2022; 23(2): 811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChristiansen M, Grove EL, Hvas A-M: Contemporary Clinical Use of Aspirin: Mechanisms of Action, Current Concepts, Unresolved Questions, and Future Perspectives. Semin. Thromb. Hemost. 2021; 47 (07): 800–814. PubMed Abstract \| Publisher Full Text\n\nClauw DJ, Essex MN, Pitman V, et al.: Reframing chronic pain as a disease, not a symptom: rationale and implications for pain management. Postgrad. Med. 2019; 131(3): 185–198. Publisher Full Text\n\nCohen M, Quintner J, van Rysewyk S : Reconsidering the International Association for the Study of Pain definition of pain. PAIN Reports. 2018; 3(2): e634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEberhardt J, Santos-Martins D, Tillack AF, et al.: AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. J. Chem. Inf. Model. 2021; 61: 3891–3898. PubMed Abstract \| Publisher Full Text\n\nFaith K, Stephen O, Richard K: Virtual screening of Ibuprofen, Celecoxib and Aspirin.2023. V2. Publisher Full Text\n\nFitzmaurice C: Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 29 cancer groups, 2006 to 2016: A systematic analysis for the Global Burden of Disease study. J. Clin. Oncol. 2018; 36(15_suppl): 1568–1568. Publisher Full Text\n\nHamza A, Wei N-N, Zhan C-G: Ligand-Based Virtual Screening Approach Using a New Scoring Function. J. Chem. Inf. Model. 2012; 52(4): 963–974. Publisher Full Text\n\nKirkby NS, Lundberg MH, Harrington LS, et al.: Cyclooxygenase-1, not cyclooxygenase-2, is responsible for physiological production of prostacyclin in the cardiovascular system. Proc. Natl. Acad. Sci. 2012; 109 (43): 17597–17602. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLionta E, Spyrou G, Vassilatis D, et al.: Structure-Based Virtual Screening for Drug Discovery: Principles, Applications and Recent Advances. Curr. Top. Med. Chem. 2014; 14(16): 1923–1938. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoussert L, Vidal F, Parant O, et al.: Aspirin for prevention of preeclampsia and fetal growth restriction. Prenat. Diagn. 2020; 40: 519–527. Publisher Full Text\n\nMachado GC, Abdel-Shaheed C, Underwood M, et al.: Non-steroidal anti-inflammatory drugs (NSAIDs) for musculoskeletal pain. BMJ. 2021; 372. Publisher Full Text\n\nMajed BH, Khalil RA: Molecular Mechanisms Regulating the Vascular Prostacyclin Pathways and Their Adaptation during Pregnancy and in the Newborn. Pharmacol. Rev. 2012; 64(3): 540–582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNgo SNT, Addison CJ: Are COX-2 Selective NSAIDs Associated with Less GI, Renal, and Cardiovascular Side Effects: Evidence from Animals Treated with NSAIDs. Annu. Res. Rev. Biol. 2018; 29(6): 1–8. Publisher Full Text\n\nPettersen EF, Goddard TD, Huang CC, et al.: UCSF Chimera--A visualization system for exploratory research and analysis. J. Comput. Chem. 2004; 25(13): 1605–1612. PubMed Abstract \| Publisher Full Text\n\nShen X, Huang Y, Guo H, et al.: Oral ibuprofen promoted cholestatic liver disease in very low birth weight infants with patent ductus arteriosus. Clin. Res. Hepatol. Gastroenterol. 2021; 45(2): 101495. PubMed Abstract \| Publisher Full Text\n\nWang T, Fu X, Chen Q, et al.: Arachidonic Acid Metabolism and Kidney Inflammation. Int. J. Mol. Sci. 2019; 20(15). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZoubek ME, Lucena MI, Andrade RJ, et al.: Systematic review: ibuprofen-induced liver injury. Aliment. Pharmacol. Ther. 2020; 51(6): 603–611. PubMed Abstract \| Publisher Full Text" }	[ { "id": "238306", "date": "13 May 2024", "name": "Mohammed Hawash", "expertise": [ "Reviewer Expertise Medicinal Chemistry" ], "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 titled \"Virtual Screening of Zinc Compounds Similar to NSAIDs with Better Pharmacodynamic and Pharmacokinetic Profiles\" addresses a significant concern in pain management by exploring alternatives to opioids and traditional NSAIDs. The methodology employed is commendable, leveraging PubChem, Avogadro, Swiss Similarity, Drug Bank, Protein Data Bank, Chimera, Auto dock Vina, Protox II, and SwissADME for a comprehensive analysis. The generation of zinc compounds based on known analgesics and subsequent docking studies reveal promising candidates with improved binding energies to COX-2, suggesting potential analgesic efficacy.\nThe presentation of results in tabular forms with textual descriptions enhances clarity, facilitating a better understanding of the findings. The toxicity analysis, including LD50 predictions and evaluation of hepatoactivity, adds a crucial dimension to the study, allowing for a comprehensive assessment of safety profiles. However, a more detailed discussion on the limitations and potential challenges of the virtual screening approach, as well as future directions for experimental validation, would strengthen the paper. Overall, this work contributes valuable insights into the development of novel analgesic agents with improved safety profiles, making it a noteworthy contribution to the field. All of these findings seem valuable and suitable contribution to be published in the F1000research journal after justifying mentioned point.\nIn the abstract you wrote “Seven, one, and four of the” it is better to write their cods Considering the aim to generate active analgesic agents with improved pharmacodynamic and pharmacokinetic profiles from known analgesics, what specific evidence from the results supports the assertion that the compounds identified in this study exhibit better pharmacokinetic profiles and fewer side effects compared to the reference compounds (aspirin, ibuprofen, celecoxib)? This point should be clarified more Additionally, could you provide insights into the methodology's reliability in predicting toxicity profiles, especially considering the observed LD50 values and the hepatotoxicity predictions for compounds such as ZINC01680731 and ZINC39120409? The study mentions high gastrointestinal absorption for all NSAIDs and their zinc compounds. How does this characteristic contribute to the overall bioavailability of the compounds, and what role does it play in their therapeutic effectiveness? In the context of pharmacokinetics outlined in Tables 9 and 10, How might these findings guide the selection of zinc compounds for further development? The information regarding the used crystals of COX1 and COX2 should be clear, you have to identify the PDB codes accordingly\n\nBest wishes\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-444
https://f1000research.com/articles/11-1242/v1	01 Nov 22	{ "type": "Research Article", "title": "Ex vivo comparative investigation of suprachiasmatic nucleus excitotoxic resiliency", "authors": [ "Debalina Acharyya", "Joanna Cooper", "Rebecca A. Prosser", "Debalina Acharyya", "Joanna Cooper" ], "abstract": "Background: Glutamate signaling in the brain is regulated by release, reuptake, and receptor responsiveness. In diseased conditions, glutamate signaling can exceed normal regulatory processes, giving rise to a condition called excitotoxicity. Although regional differences in the excitotoxic effects of glutamate in the brain have been reported, the extent and characteristics of these potential differences are not clear. Here we compared the excitotoxic resiliency of the suprachiasmatic nucleus (SCN), anterior hypothalamus (AH) and cortex. Methods: We treated acute brain slices containing either the SCN and AH or the cortex from adult male mice at different times across the diurnal cycle with varying concentrations of N-methyl-D-aspartate (NMDA), NMDA+ α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or control medium. The extent of cell damage was assessed using propidium iodide (PI), a cell death marker. Results: The results indicate that all three brain regions exhibited increasing cell damage/death when treated with increasing concentrations of NMDA. However, higher concentrations of NMDA were needed to significantly increase cell damage in the SCN compared to the cortex and AH. All three brain regions also exhibited greater cell death/damage when treated in the nighttime compared to the daytime, although the SCN exhibited increased cell death during a more restricted time interval compared to the AH and cortex. Conclusions: Together, these data confirm previous studies showing excitotoxic resiliency in the SCN, while extending them in two ways. First, we demonstrate a dose-dependency in excitotoxic susceptibility that differentiates the SCN from the surrounding AH and the cortex using a brain slice preparation. Second, we demonstrate a diurnal rhythm in excitotoxic susceptibility with a broadly similar phase across all three brain regions. These data increase our understanding of the extent and nature of the SCN excitotoxic resiliency, which will inform future studies on the cellular mechanisms underlying this phenomenon.", "keywords": [ "suprachiasmatic", "excitotoxicity", "NMDA", "cortex", "brain slice" ], "content": "Introduction\n\nGlutamate is the primary excitatory neurotransmitter in the brain and as such is critical for proper neural functioning.1 Under physiological conditions, the optimal extracellular glutamate concentration is maintained by a balance between astrocytic reuptake and neural/astrocytic release.2 However, certain disease conditions, including ischemic stroke, traumatic brain injury and neurodegenerative diseases, are accompanied by an excess accumulation of extracellular glutamate. For example, an increase in extracellular glutamate is seen after severe trauma that persists for more than four days.3 This leads to over-activating N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and ultimately to cell damage and death, a phenomenon known as excitotoxicity.4\n\nAlthough excitotoxicity is initiated by glutamate, Ca2+ is the primary mediator of excitotoxic damage. Excessive glutamate-induced influx of Ca2+ disrupts normal metabolic processes, creates oxidative stress, and activates cell death pathways.5 Importantly, the degree of cell death induced by excess glutamate/Ca2+ signaling may differ across the brain. The characteristics and mechanism(s) underlying these regional differences are not clearly understood, but determining intrinsic/extrinsic mechanisms that enhance excitotoxic resiliency could help in developing treatments for this debilitating pathology.\n\nThe suprachiasmatic nucleus (SCN), site of the primary circadian oscillator in mammals, is thought to be resilient to excitotoxic damage. This was first reported in 1980 by Peterson et al.,6 when kainic acid injections into the lateral hypothalamus and ventrolateral geniculate led to widespread death while sparing the SCN. Subsequent studies demonstrated that immortalized rat SCN cells (SCN 2.2 cells) exhibit resiliency to glutamate-induced excitotoxic damage compared to hypothalamic (GT1-7) cells.7,8 Although informative, these latter studies investigated dispersed cells in culture, which lack the normal in situ cellular and extracellular environment. Thus, whether the same resiliency differences and potential underlying mechanism(s) occur under intact tissue conditions is unclear. Moreover, the resiliency of the SCN has not been quantitatively compared to other brain regions, such as the cortex, known to exhibit high excitotoxic susceptibility.8 Lastly, although the SCN exhibits circadian rhythms in many cellular mechanisms, its excitotoxic resiliency has not been investigated across different times of the day. Therefore, we investigated excitotoxic resiliency using acute murine brain slices, comparing the effects across multiple brain regions, different times of the day and using different strengths of excitotoxic stimuli. Our results show 1) the SCN is more resilient to excitotoxic damage compared to the adjacent anterior hypothalamus (AH) and the cortex; 2) the resiliency of all three brain regions is dose-dependent; and 3) the SCN, AH and cortex all exhibit a diurnal rhythm in excitotoxicity resiliency.\n\n\nMethods\n\nAcute coronal brain slices (500 μm) containing both the SCN and adjacent AH or the cortex were prepared from adult male C57Bl/6 mice (Envigo; Indianapolis, IN) housed under a 12:12 light-dark cycle with food and water available ad libitum. All procedures with the animals were approved by the University of Tennessee IACUC committee, protocol #1453, and all efforts were made to minimize suffering of the animals. Slices were prepared between Zeitgeber Time 0-2 (ZT 0-2, where ZT 0=lights on and ZT 12=lights off in the donor animal colony) or ZT 10-12 and placed in Hatton-style brain slice dishes perfused with Earle’s Balanced Salt Solution (EBSS) (MP Biomedicals; OH, USA) supplemented with glucose and sodium bicarbonate, pH 7.4, gassed with 95% O2/5% CO2 and maintained at 37°C as described elsewhere.9 Depending on the experiment, brain slices were maintained for approximately 4 or 10 h prior to experimental treatment.\n\nAt the designated time (ZT 6, 12, 16 or 22), perfusion of the brain slice chamber was paused, and the slices were left untreated or treated by bath application of EBSS supplemented with NMDA (50 μM – 10 mM) (Sigma Aldrich; MO, USA) for 1 h. In some experiments, slices were treated with EBSS supplemented with 500 μM NMDA + 50 μM AMPA (Sigma Aldrich). Following the 1 h treatment, the perfusion medium was replaced with normal EBSS, and perfusion was resumed for an additional 3 h.\n\nAt the end of the 3 h, brain slice perfusion was switched to EBSS containing 4.6 μg/ml propidium iodide (PI) (Invitrogen; OR, USA) for an additional 2 h. At the end of this period, the tissues were placed in 4% paraformaldehyde (Electron Microscopy Sciences; PA, USA) for 10 min, then transferred to a 30% sucrose solution and incubated overnight at 4°C. The following day, the slices were embedded in optimum temperature cutting compound (Tissue Tek; CA, USA), and the resulting tissue blocks were stored in -80°C until sectioning.\n\nThe tissues were sectioned (10 μm) with a cryostat and the sections were collected on gelatin-coated slides and stored in -80οC. For the imaging, slides containing the tissue sections were air-dried and then washed in phosphate buffered saline. After washing, the slides were cover- slipped with Vectashield mounting medium (Thermofisher Scientific; CA, USA) containing 4′,6′-diamidino-2-phenylindole (DAPI) and the edges were sealed with nail-polish. Fluorescence images were acquired using a Leica DM6000B microscope at 10× magnification. Similar settings were maintained for the acquisition of all images.\n\nAll image analyses were performed using ImageJ software (NIH). Given the lack of consensus in the literature for evaluating the extent of cell death/damage in histological images, we compared the utility of three different image analysis protocols: intensity-based cell count (IBCC), particle analysis cell count (PACC) and mean gray value (MGV). For all three protocols, the images acquired were first split into individual channels: blue (DAPI, 405 nm) and red (PI, ~570-610 nM). Using the blue channel, the region of interest (ROI) was marked in each section. The subsequent steps for each procedure are detailed below. In all cases, one image was analyzed per brain slice for each experimental condition and a total of three to eight replicates were analyzed for each experimental condition (replicate numbers varied across experiments). Images were analyzed by at least three individuals blind to the treatment, with greater-than-85% consistency across all individuals. The mean of the individual analyses for each image was used for statistical analysis.\n\nIntensity-based cell count (IBCC)\n\nThe total number of cells in each ROI were manually counted using the blue channel. The red channel exhibited a range of PI intensities, consistent with cells having varying degrees of excitotoxic its induced cell damage and death. Based on a random sampling of these intensities across the entire image, thresholds were determined that separated the cells into three categories: Grade 1 (little or no PI staining; healthy cells), Grade 2 (moderate PI staining; damaged cells) and Grade 3 (intense and sharply delineated PI staining; dead cells).10 The percentage of cells in each category was determined by dividing the number of cells in each category by total number of cells in the ROI.\n\nParticle analysis cell count (PACC)\n\nThe background was subtracted from both the red and the blue channels. Next, the threshold was adjusted in the blue channel to delineate individual cells, using the watershed option on ImageJ to separate clumped cells. The total number of cells in the ROI were counted in the blue channel using the particle analysis option. Using the red channel, the threshold was adjusted twice in ImageJ, once to show only Grade 3 cells and a second time so that Grade 2+3 cells were shown. In both cases, the particle analysis option on ImageJ was used to count the resulting shown cells in the ROI. The number of Grade 3 cells was subtracted to determine the number of Grade 2 cells. The number of Grade 1 cells was calculated by subtracting the number of Grade 2+3 cells from the total number of cells. The percentage of cells in each category was calculated by dividing the number of cells in each category by the total number of cells in the ROI.\n\nMean gray value (MGV)\n\nThe background was subtracted from both the red and the blue channels. Next, the mean gray value in the ROI was determined for each channel. The PI:DAPI ratio was calculated to determine the relative amount of PI staining across the entire ROI without reference to different degrees of staining intensity. The percentage of Grade 2+3 (unhealthy) cells and Grade 1 (healthy) cells were calculated by the formulas:\n\nThe data were graphed, and statistical analysis was performed using GraphPad Prism software (Version 8.0.1). Spearman Pearson correlation was performed to compare the three-imaging analysis (IBCC, PACC and MGV) procedures. Note that the experiments were not designed to test for differences in the percentage of cells across Grades (i.e., whether the percentages of Grade 1, 2 and 3 cells differ from each other). Instead, the experiments were designed to test whether there were significant changes within a particular cell Grade based on experimental condition. Therefore, one-way ANOVA (testing for changes in cell percentages within Grades under different experimental conditions) followed by Tukey test was performed to investigate inter-region differences under control conditions and to compare dose responses to increasing NMDA concentrations within each brain area. Based on our results demonstrating statistical differences across regions under control conditions, subsequent analyses were restricted to within-region comparisons. One-tailed Student’s T-tests (only assessing treatment-induced decreases in healthy cells or increases in damaged/dead cells; Microsoft Excel) were used, as indicated, testing for changes within Grades, within each region of interest, across the various experimental conditions. Differences were considered statistically significant at p<0.05.\n\n\nResults\n\nTo confirm that PI imaging would be useful for assessing excitotoxic damage, tissue slices containing the different regions were left untreated or treated with 50 μM NMDA at ZT6. Figure 1 shows representative images of the different brain areas. The ROIs are marked by the white lines. Clear differences in overall PI staining are apparent, suggesting there are differences in tissue health across the brain regions under both control and NMDA-treated conditions. Based on these apparent differences, we proceeded to compare the extent of regional cell death and damage, beginning with control conditions, using all three of the analysis protocols. The data from these comparisons are summarized in Figure 2.\n\nTop row: SCN (dashed line) and anterior hypothalamus (AH) (solid line) under control and NMDA conditions respectively. Bottom row: cortical sections under control and NMDA conditions respectively. Differences in the intensity of PI staining are evident across the different images based on region and treatment condition.\n\nDifferences under control conditions at ZT6 were analyzed using (A) the IBCC protocol or (B) the PACC protocol. (C) The percentage of healthy vs. unhealthy cells calculated using the MGV analysis protocol. p<0.05; p<0.005; *p<0.001. N=6 for each group (One-way ANOVA).\n\nIBCC: One-way ANOVA determined that there were no statistically significant differences in the percentage of Grade 1 (healthy), 2 (damaged) or 3 (dead) cells (p=0.9) between the SCN and AH under control conditions. However, one-way ANOVA indicated a statistically significant difference between the SCN and cortex (p<0.05). Post-hoc analysis determined that there was a statistically significant difference in the percentage of Grade 1 cells between SCN and cortex (p=0.0001) but not for the percentage of Grade 2 (p=0.0880) or Grade 3 (p=0.2860) cells. One-way ANOVA also indicated a statistically significant difference between the AH and cortex (p<0.05). Post-hoc analysis determined there was a statistically significant difference in Grade 1 cells (p<0.0001) and Grade 2 cells (p=0.0164) but not Grade 3 cells (p=0.2860) between AH and cortex under control conditions.\n\nPACC: One-way ANOVA determined that there were no statistically significant differences in the percentage of Grade 1 (healthy), Grade 2 (damaged) or Grade 3 (dead) cells (p=0.9) between the SCN and AH under control conditions. However, one-way ANOVA showed there was a statistically significant difference between the SCN and cortex (p<0.05). Post-hoc analysis determined that there was a statistically significant difference in the percentage of Grade 1 cells between the SCN and cortex (p=0.0012) but not Grade 2 (p=0.1956) or Grade 3 (p=0.6074) cells. One-way ANOVA also showed a statistically significant difference between the AH and cortex (p<0.05). Post-hoc analysis determined that there was statistically significant difference in the percentage of Grade 1 cells between AH and cortex (p=0.0027) but not Grade 2 (p=0.5235) or Grade 3 (p=0.4377) cells.\n\nMGV: One-way ANOVA determined there were no statistically significant differences in the percentage of Grade 1 (healthy) or Grade 2/3 (unhealthy) cells (p=0.9) between the SCN and AH. However, one-way ANOVAs revealed statistically significant differences in Grade 1 (healthy) cells between the SCN and cortex (p=0.0014) and between the AH and cortex (p=0.0034). However, no significant differences in Grade 2/3 (unhealthy) cells were observed.\n\nThus, overall, we found that under control conditions the SCN and AH exhibit robust health, and that cortical slices exhibit more tissue damage compared to both the AH and SCN regions. The differences were largely restricted to significant decreases in the percentage of Grade 1 (healthy) cells in the cortex and some significant increases in the percentage of Grade 2 (damaged) cells, with no significant differences in the percentage of Grade 3 (dead) cells (based on IBCC and PACC analyses) across the three regions. Given the baseline differences in overall health of the cortex compared to the SCN and AH under control conditions, we determined that the effects of excitotoxic treatments should be assessed separately within each brain region rather than directly comparing across regions.\n\nNext, we sought to compare the three analysis protocols for their ability to assess differences in excitotoxic damage based on the intensity of PI staining in each region under control versus NMDA (50 μM) conditions. The results, based on the three image analysis protocols, are shown in Figure 3. Based on our experimental design, we focused on potential changes within the different cell Grades within each region across the different experimental conditions.\n\nDifferences under control vs. 50 μM NMDA treatment at ZT6 were analyzed using (A) the IBCC protocol or (B) the PACC protocol. (C) The percentage of healthy vs. unhealthy cells calculated using the MGV analysis protocol. p<0.05; p<0.005; p<0.001. N=6 for each group (Student’s t-test).\n\nUsing the IBCC analysis protocol, Student’s t-tests were used to test for changes in Grade 1 (healthy), Grade 2 (damaged) and Grade 3 (dead) cells separately within individual regions. There were no significant differences in the percentage of Grade 1 (p=0.195), Grade 2 (p=0.155) and Grade 3 (p=0.314) cells in the SCN, in the AH [Grade 1 (p=0.100), Grade 2 (p=0.058) and Grade 3 (p=0.059) cells] or in the cortex [Grade 1 (p=0.354), Grade 2 (p=0.337), and Grade 3 (p=0.428) cells] when comparing control versus NMDA treated conditions within each brain region individually. Likewise, based on the PACC analysis procedure, Student’s t-tests determined there were no statistically significant differences in the percentage of Grade 1 (p=0.229), Grade 2 (p=0.067) and Grade 3 (p=0.4613) cells in the SCN, in the AH [Grade 1 (p=0.265), Grade 2 (p=0.361) and Grade 3 (p=0.063) cells] or in the cortex [Grade 1 (p=0.345), Grade 2 (p=0.431) and Grade 3 (p=0.353) cells] when comparing control versus NMDA-treated conditions within each brain region individually.\n\nUsing data from the MGV procedure, Student’s t-tests determined there were no statistically significant differences in the percentage of Grade 2/3 (unhealthy) cells in either the SCN (p=0.072) or the cortex (p=0.200) under control versus-NMDA treated conditions. However, Student’s t-tests determined there was a statistically significant difference in the unhealthy cells in the AH region between control and NMDA conditions (p=0.041). These results, therefore, are somewhat different from those using the previous two procedures.\n\nTo further compare the three analysis procedures, we performed a Pearson Correlation Analysis for each pair of analysis protocols using both the control and 50 μM NMDA treatment data (Figure 4). The results show that the three methodologies were highly correlated, with each pairwise comparison having an R value exceeding 0.98 and with p<0.0001. Thus, all three image analysis protocols appear to generate generally consistent data and are all useful for comparing the extent of PI staining across different tissues/conditions. However, although the MGV procedure is the most straightforward, it provides less detail than the other two methods, which are able to distinguish damaged versus dead cells. The PACC and IBCC methods also appear to be slightly more conservative than the MGV procedure in terms of assessing cell damage/death. Based on these results, for the remainder of the experiments we chose to analyze the images using the IBCC method only.\n\n(A) correlation between the PACC and IBCC protocols (R=0.9908, p<0.0001); (B) correlation between IBCC and MGV protocols (R=0.9825, p<0.0001); (C) correlation between MGV and PACC protocols (R=0.9791, p<0.0001). For B and C, Grade 2 and Grade 3 cell percentages determined using the IBCC and PACC protocols were combined to compare with unhealthy cells based on the MGV protocol, while Grade 1 cell percentages from the IBCC and PACC analyses were compared with the healthy cells based on the MGV protocol.\n\nGiven the minimal effects of 50 μM NMDA treatment at ZT6 across all three brain regions, we proceeded to investigate whether more robust excitotoxic stimuli would increase cell damage when applied at this time point. For this, we treated the brain slices with increasing concentrations of NMDA (up to 10 mM) as well as with a combination of NMDA (500 μM) and AMPA (50 μM). The data are summarized in Figure 5.\n\nOne-way ANOVA followed by Tukey’s post-hoc analysis revealed significant differences in the percentage of healthy (Grade 1), damaged (Grade 2) and/or dead (Grade 3) cells in the (A) SCN, (B) the AH and (C) cortex. p<0.05; p<0.005; p<0.001. N=3-6 for each group.\n\nIn the SCN, one-way ANOVA determined that there were statistically significant differences across the treatment conditions (p<0.05). Post-hoc analysis determined that there was a statistically significant difference in Grade 1 (p=0.0432) and Grade 2 (p=0.0221) cells between control and 10 mM NMDA conditions, but not when comparing control with lower concentrations of NMDA. Thus, at the highest concentration of NMDA we used there was a decrease in Grade 1 (healthy) cells and a concomitant increase in the percentage of Grade 2 (damaged) cells but not Grade 3 (dead) cells in the SCN.\n\nIn the AH, one-way ANOVA also showed statistically significant differences across the treatment conditions (p<0.05). Post-hoc analysis determined there were statistically significant differences in Grade 1 (healthy) cells between control and 1 mM NMDA (p<0.0001) and between control and 10 mM NMDA (p=0.0042). For Grade 2 (damaged) cells there were statistically significant differences between control and 1 mM NMDA (p=0.0001) and between control and 10 mM NMDA (p=0.0024). For Grade 3 (dead) cells there was a statistically significant difference between control and 1 mM NMDA conditions (p=0.0002) but no difference between control and 10 mM. Thus, both 1 mM and 10 mM NMDA treatments decreased the percentage of Grade 1 (healthy) cells and increased the percentage of Grade 2 (damaged)/Grade 3 (dead) and cells in the AH.\n\nWith respect to the cortex, one-way ANOVA indicated that there was a statistically significant difference across the experimental conditions (p<0.05). Post-hoc analysis indicated there were statistically significant differences in the percentage of Grade 1 (healthy) cells between control and 1 mM NMDA (p=0.0251) and between control and 10 mM NMDA (p=0.0157). Thus, similar to the AH, both 1 mM and 10 mM NMDA treatments decreased the percentage of Grade 1 (healthy) cells and in the cortex.\n\nOne-way ANOVA determined that the combined treatment of NMDA (500 μM) + AMPA (50 μM) induced statistically significant changes in the AH compared to control (p<0.05). Post-hoc analysis determined there were statistically significant differences in Grade 1 cells (p<0.0001) and Grade 2 cells (p=0.0003) in the AH region between control versus NMDA+AMPA conditions. Additionally, one-way ANOVA revealed statistically significant differences when comparing the response of the AH to 500 μM NMDA to that of NMDA (500 μM) + AMPA (50 μM). Post-hoc analysis determined that there were statistically significant differences between Grade 1 (p=0.0001), Grade 2 (p=0.0017) and Grade 3 (p=0.0229). In contrast, one-way ANOVA determined that there were no significant differences in the SCN region under control versus NMDA+AMPA conditions (p>0.05). Similarly, NMDA+AMPA did not induce statistically significant changes in the cortex compared to control (p>0.05). There also were no statistically significant differences in the SCN when comparing the response to 500 μM NMDA versus NMDA (500 μM) + AMPA (50 μM). Such a comparison, however, was not possible for the cortex because we did not treat cortical slices with 500 μM NMDA alone.\n\nTo determine whether excitotoxic susceptibility exhibits diurnal variations, tissue slices containing the different brain regions were left untreated or treated with 50 μM NMDA at ZT6, 12, 16 or 22. Because preparing brain slices during the lights-off period can reset the circadian clock,11 we prepared all the slices in the lights-on period. Due to this constraint, treating the slices at four different ZTs required that they be maintained in vitro for different amounts of time. To account for this, slices treated at ZT6 and ZT16 were prepared four to six hours prior to treatment, while slices treated at ZT12 and ZT22 were prepared 10-12 hours prior to treatment. Again, because we were investigating changes in the percentage of cells within each Grade in individual regions in response to treatments at different times of day, we used Student’s t-tests for the statistical analyses. The data are summarized in Figure 6.\n\nNon-paired student t-tests between control and treated samples at different treatment times (ZT6, ZT12, ZT16 and ZT22) revealed significant differences in the percentage of healthy (Grade 1), damaged (Grade 2) and/or dead (Grade 3) cells. p<0.05; p<0.005; * p<0.001, # p approaching significance. N=6 for each group.\n\nIn the SCN treated at ZT6 and ZT12, Student’s t-tests determined there were no statistically significant differences between control and NMDA-treated conditions [Grade 1 (healthy) (p=0.026; not considered significant because the trend was in the opposite direction from the one-tailed test), Grade 2 (damaged) (p=0.096), and Grade 3 (dead) (p=0.94) cells]. However, with the SCN treated at ZT16, Student’s t-tests revealed a statistically significant difference between control and NMDA treated slices in the percentage of Grade 3 cells (p=0.047) but not for Grade 1 (p=0.326) or Grade 2 (p=0.150) cells. For the SCN treated at ZT22, Student’s t-tests revealed statistically significant differences in the percentage of Grade 1 (p=0.009) and Grade 2 (p=0.005) cells between control and NMDA treated conditions but no statistically significant difference between Grade 3 cells under the two conditions (p=0.205). Taken together, these data suggest that SCN is more susceptible to excitotoxic damage during the middle and late night than during the day, with an increase in dead cells only occurring after treatment at ZT 16.\n\nFor the AH treated at ZT6 and ZT12, Student’s t-tests determined that there were no statistically significant differences between control and NMDA conditions [Grade 1 (healthy) (p=0.263), Grade 2 (damaged) (p=0.427) and Grade 3 (dead) (p=0.067) cells]. However, for the AH treated at ZT16, Student’s t-tests indicated statistically significant differences in the percentage of Grade 1 (p=0.00002), Grade 2 (p=0.005) and Grade 3 (p=0.006) cells between control and NMDA treated conditions. A similar pattern was observed for the AH treated at ZT22, where Student’s t-tests indicated statistically significant differences in Grade 1 (p=0.0009), Grade 2 (p=0.005) and Grade 3 (p=0.006) cells between control and NMDA-treated conditions. Thus, the AH is also more susceptible to excitotoxic damage during the night versus the day. Moreover, the degree of excitotoxic damage appears to be more substantial in the AH versus the SCN, based on significant decreases in Grade 1 cells and increases in Grade 2 and Grade 3 cells in the AH after treatment at both ZT 16 and 22.\n\nFor cortical brain slices treated at ZT6, Student’s t-test indicated that there were no statistically significant differences in the percentage of Grade 1 (healthy) (p=0.389), Grade 2 (damaged) (p=0.337) or Grade 3 (dead) (p=0.428) cells between control versus NMDA conditions. For cortical slices treated at ZT12, Student’s t-test indicated that there was a difference in the percentage of Grade 3 cells that did not quite reach significance (p=0.0541) between control vs NMDA conditions. For cortical slices treated at ZT16, Student’s t-test indicated a statistically significant difference in the percentage of Grade 2 cells (p=0.034) under NMDA-treated conditions compared to control conditions but not Grade 1 (p=0.103) or Grade 3 (p=0.395) cells. Lastly for cortical slices treated at ZT22, Student’s t-test indicated statistically significant differences in the percentage of Grade 1 (p=0.00002), Grade 2 (p=0.00007) and Grade 3 (p=0.0039) cells between control and NMDA treated conditions. Thus, the cortex is also more susceptible to excitotoxic damage during the night versus the day. Further, the degree of excitotoxic damage again appears to be more substantial compared to the SCN based on the percentages of healthy, damaged, and dead cells as well as possibly the range of times when it exhibits greater excitotoxic susceptibility.\n\n\nDiscussion\n\nAlthough previous studies have reported that the SCN is resilient to excitotoxic stimuli, neither its degree of resilience compared to other brain areas nor whether there are diurnal variations in its resiliency have been investigated previously. Using acute brain slices, which preserve the in vivo cytoarchitecture, we assessed differences in excitotoxic susceptibility across multiple brain regions and time points and tested varying strengths of excitotoxic stimulation. Together, our data demonstrate that the SCN is more resistant to NMDA-induced excitotoxicity compared to the AH and the cortex. The data also show that there are time-of-day differences in excitotoxic resiliency, with the extent and pattern of excitotoxic susceptibility differing across each region.\n\nWe began with comparing the general health of the cells within brain slices containing each brain region under control conditions. This was done to distinguish differences due intrinsic and/or slice preparation factors versus differences in excitotoxic resiliency. Using three image analysis protocols, we determined that under our control conditions cell damage in cortical brain slices was greater than in either the SCN or the AH. This could be due to differences in neuronal phenotype, the extent of damage to fibers of passage, or other factors. Regardless, the differences under baseline conditions required us to analyze the excitotoxic responses in each brain region separately.\n\nWe initially assessed excitotoxic susceptibility by determining the effect of 50 μM NMDA applied to the brain slices at ZT6, maintaining the tissues for a total of 6 h after the onset of NMDA treatment, and 5 h after the end of the NMDA treatment. This post-treatment interval was implemented to allow time for initial cellular changes to unfold, whether they involved recovery or damage/death processes. We again analyzed the data using three image analysis protocols in order to compare their utility. Using the MGV analysis, which only distinguishes between healthy (Grade 1) versus unhealthy (Grade 2+3) cells, we found a significant increase in unhealthy cells in the AH but not in the SCN or the cortex in response to 50 μM NMDA. These data are consistent with those from previous studies. Peterson et al.6 showed that kainic acid induces more cell damage in hypothalamic areas surrounding the SCN versus the SCN itself, although it is possible that the regional differences were influenced by the extent of kainic acid diffusion through the tissue. Separately, Bottum et al.7 used cultures of immortalized cells to show that glutamate induces more cell death in GT1-7 (hypothalamic) cells than SCN2.2 cells. However, results using immortalized cells in culture could differ from either in vivo or ex vivo preparations due to the loss of the intact tissue environment.12 Importantly, similarly to the MGV analysis protocol, both of these previous studies utilized analyses that would not necessarily distinguish between dead and damaged cells.\n\nOn the other hand, the IBCC and PACC analyses, which distinguish between healthy, damaged, and dead cells, did not show significant increases in excitotoxic damage in any of the three regions in response to 50 μM NMDA. Despite the different results across the three analyses, Pearson Correlation analysis confirmed that the data generated by all three protocols were highly correlated. Nevertheless, comparing the results obtained using the three protocols suggests that the more nuanced analyses (IBCC and PACC) provide a different, albeit perhaps more conservative, assessment of the degree of excitotoxic damage inflicted on cells in our brain slices compared to the MGV analysis. Based on these differences, we decided to use the IBCC analysis protocol for the remainder of the study.\n\nOur dose-response experiments, where we treated the slices with increasing concentrations of NMDA, confirm and build upon the enhanced excitotoxic resiliency of the SCN shown in previous studies. Specifically, we showed that the AH and the cortex exhibited a significant decrease in the percentage of healthy cells in response to both 1 mM and 10 mM NMDA compared to control conditions, with the AH also showing significant increases in damaged and dead cells in response to 1 mM NMDA. The SCN, on the other hand, only exhibits decreases in healthy cells and increases in damaged cells in response to the highest concentration of NMDA used (10 mM). The differences between the SCN and AH are notable given that the data are generated from the same brain slices, and from adjacent areas within the brain slices (e.g., see Figure 1). Thus, differences in slice preparation, tissue handling, or other factors that could vary between individual slices cannot account for these differences.\n\nThe lack of significant increases in dead or damaged cells in the cortex while there were significant decreases in healthy cells could be due to the percentage of both Grade 2 and Grade 3 cells appearing to increase to similar extents (see Figure 5), such that neither change by itself reaches significance. This pattern is different from that seen in both the SCN and AH, where the percentage of dead cells consistently appeared lower than the percentage of damaged cells. Moreover, the percentage of Grade 1 cells in the cortex decreased to the same level as that of Grade 2 and Grade 3 cells in response to both 1 mM and 10 mM NMDA, consistent with substantial cell death/damage in the cortex under these stronger excitotoxic conditions. Again, this differs substantially from the pattern seen in the SCN. Taken together, the results are consistent with the SCN having at least a 10-fold greater resiliency to NMDA-induced excitotoxicity compared to the AH and cortex.\n\nIn addition to treating brain slices with increasing concentrations of NMDA, we also treated them with a combination of NMDA and AMPA. This was done because AMPA receptor activation can enhance NMDA receptor signaling by removing the Mg2+ block.13 We determined that combining 50 μM AMPA with 500 μM NMDA did not exacerbate cell damage in the SCN or cortex beyond that seen in response to control or (for SCN) 500 μM NMDA alone, but it significantly increased the percentage of damaged cells in the AH compared to both control and 500 μM NMDA alone. It is possible that lack of an additive effect of AMPA in the SCN was due to the neurons in this region already having a relatively high resting membrane potential.14 The fact that cell damage did not increase significantly in the cortex in response to NMDA+AMPA compared to control conditions again could be, as seen in response to 1 mM and 10 mM NMDA, because the percentages of both Grade 2 and Grade 3 cells appeared to increase to similar extents to levels comparable to that of Grade 1 cells, so that the changes within each cell Grade did not reach significance.\n\nAlthough excitotoxicity involves multiple cellular process, including mitochondrial dysfunction and increased generation of reactive oxygen species,15 it generally is initiated by excessive glutamate stimulation followed by a surge in intracellular Ca2+. Since, across the different types of glutamate receptors, NMDA receptors typically have the highest Ca2+ permeability,16,17 they are a primary factor driving excitotoxic damage. In this regard, heterogeneity in NMDA receptor expression and subunit composition could contribute to regional differences in the observed excitotoxic susceptibility. In particular, GluN2B subunit expression has been associated with increased excitotoxicity-induced neuronal apoptosis, while GluN2A subunit expression is associated with enhanced neuronal survival in both in vitro and ex vivo models of ischemic stroke.18 Bottum et al. (2010) found in cell culture studies that GluN2A mRNA was expressed at higher levels in SCN2.2 cells compared to GT1-7 cells.7 These data are consistent with an increased resiliency of the SCN; however, it will be important to determine if the differences in mRNA translate to similar differences in the protein levels, and to assess whether these differences are also seen under in vivo and ex vivo conditions. Bottum et al. also found that SCN2.2 cells express GluN2B mRNA which was undetectable in GT1-7 cells.7 This is unexpected, given that GluN2B subunit expression is associated with increased excitotoxicity-induced apoptosis,18 although GluNR2B-containing NMDA receptors may play a neuroprotective role in developing hippocampal neurons.19 These differences regarding a role for GluNR2B in excitotoxicity might be due to different subunit locations (extra-synaptic region versus the synaptic) and their interactions with intracellular proteins.20–22 With respect to cortical excitotoxic susceptibility, both GluN2A and GluN2B mRNA are expressed throughout the forebrain in both mice and rats in levels that vary across age.17,18 However, the relative percentages of these two subunits compared to the hypothalamus have not been studied. Thus, any contribution that NR2B and NR2B subunit expression have on the differential resiliency to excitotoxicity seen in this study remains to be elucidated.\n\nIt is interesting to speculate as to whether the enhanced excitotoxic resiliency of the SCN is important because the SCN receives a large amount of glutamate stimulation from retinal ganglion cells in response to light, and therefore needs compensatory mechanisms.23 As noted above, a key factor in excitotoxicity is a surge in intracellular Ca2+. If not quickly buffered, the increase in cytoplasmic Ca2+ can damage intracellular organelles and activate Ca2+-dependent cell death pathways.24 Cells that can effectively buffer Ca2+ surges might exhibit greater resiliency to excitotoxic damage. In this regard, the SCN contains a dense population of cells expressing the Ca2+ binding protein calbindin.25 It also expresses another Ca2+ binding protein, calreticulin, although to a lesser extent.26 On the other hand, the SCN expresses lower levels of the Ca2+ binding protein parvalbumin, which is more abundant in the cortex.27 The differential expression of these proteins might enhance the Ca2+ buffering capacity of the SCN cells, although this would need to be explored in future studies.\n\nIn addition to showing regional differences in NMDA-induced excitotoxic susceptibility, our study also determined that excitotoxic susceptibility within each region followed a diurnal rhythm. In all three regions, 50 μM NMDA decreased the percentage of healthy cells and/or increased the percentage of damaged/dead cells during the night more than during the day. In line with the resiliency shown in the dose-response experiments, the excitotoxic effects in the SCN again were less than those seen in both the AH and cortex, both in terms of the range of times that excitotoxic effects are seen as well and number of timepoints when significant increases in cell death occur. Although a circadian rhythm in cell death has been shown previously in the hippocampus in response to global ischemia,28 this is the first time a rhythm in excitotoxic susceptibility has been identified in the cortex, SCN and AH under these well-controlled conditions.\n\nIt is not surprising that the SCN exhibits a daily rhythm in excitotoxic resiliency, given that it is the site of the primary mammalian circadian oscillator, and as such it expresses circadian rhythms in numerous cellular processes.29 The rhythm in excitotoxic resiliency could be due to a variety of previously characterized processes, including rhythms in NMDA receptor composition and activity. Studies have shown that GluNR2B mRNA expression in the SCN is higher at ZT10 and ZT16 compared to other time points, and phosphorylation of GluN2B subunits, which increases their activity, is highest at ZT20.30 Additionally, the NMDA component of post-synaptic potentials recorded in the SCN in response to optic nerve stimulation are larger in the night compared to the day, consistent with NMDA activity being required for phase shifting of the clock in the night.31,32 Also, NMDA-induced Ca2+ transients in the SCN are more robust in response to bath application of NMDA at night compared to the day.33 Thus, greater NMDA receptor activity (and increases in intracellular Ca2+) in the SCN at night could contribute to greater nighttime excitotoxic responses in the SCN. Along with this, decreased nighttime calbindin expression, and therefore decreased Ca2+ buffering, could also increase excitotoxic susceptibility at night.34\n\nIt is also possible that time-of-day differences in SCN excitotoxic susceptibility involve glial cells. Astrocytes in the SCN have daily rhythms of glial fibrillary acid protein (GFAP) expression, synapse-associated morphology, ATP release and glutamate release.35–38 Decreased astrocytic arborization and synaptic proximity concurrent with increased astrocytic glutamate release at night could potentially raise the extracellular glutamate concentration, which could increase excitotoxic susceptibility. However, such a possibility would need to be investigated further.\n\nShifting to the AH and cortex, many extra-SCN brain regions have also been shown to exhibit circadian rhythms in cellular activity.39 However, generally their rhythms have a reversed phase compared to the SCN.40,41 Thus, it is surprising that all three areas investigated in this study exhibit a similar phase (more at night versus the middle of the day) in rhythmic excitotoxic susceptibility. Importantly, the staggered times of brain slice preparation we used in these experiments eliminates time in vitro as an explanation for this rhythm in excitotoxic susceptibility. This suggests that exploring aspects of cellular physiology that exhibit similar phases across these three brain regions could prove fruitful in the search for mechanisms contributing to excitotoxic resiliency.\n\nIn summary, we have confirmed and extended previous reports indicating that the SCN is more resilient to excitotoxic damage compared to other brain regions. The excitotoxic susceptibility in all three brain regions investigated is dose-dependent. However, compared to the AH and cortex, a ten-fold higher concentration of NMDA applied at ZT 6 was needed to increase cellular damage in the SCN, and even at the highest dose we tested there were no statistically significant increases in cell death in the SCN. We have also demonstrated, for the first time, that in the SCN (and other brain regions) excitotoxic susceptibility is dependent on the time-of-day when the stimulus is applied, with greater susceptibility occurring during the night period. However, consistent with the greater dose-dependent resiliency seen in the SCN, an increase in cell death was only observed in the SCN after treatment at ZT 16, and an increase in cell damage was only seen in the SCN after treatment at ZT 22, while both the AH and cortex exhibited decreases in healthy cells and increases in cell death and damage across a broader range of treatment times. As discussed, the increased resiliency of the SCN could be due to a variety of factors. Future studies identifying these neuroprotective mechanisms could help in developing therapies aimed at treating conditions linked to excitotoxic damage, including neurodegenerative diseases, stroke, and traumatic brain injury.", "appendix": "Data availability\n\nTennessee Research and Creative Exchange: ex vivo Comparative Investigation of Suprachiasmatic Nucleus Excitotoxic Resiliency - ex vivo Comparative Investigation of Suprachiasmatic Nucleus Excitotoxic Resiliency, https://doi.org/10.7290/xSDiunNFHq. 42\n\nThis project contains the following underlying data:\n\n- Prosser_DATASET_2022-08-15.txt\n\n- Figure 2A_Raw Data_Manuscript.xlsx\n\n- Figure 2B_Raw Data_Manuscript.xlsx\n\n- Figure 2C_Raw Data_Manuscript.xlsx\n\n- Figure 3A_Raw Data_Manuscript.xlsx\n\n- Figure 3B_Raw Data_Manuscript.xlsx\n\n- Figure 3C_Raw Data_Manuscript.xlsx\n\n- Figure 5A_Raw Data_Manuscript.xlsx\n\n- Figure 5B_Raw Data_Manuscript.xlsx\n\n- Figure 5C_Raw Data_Manuscript.xlsx\n\n- Figure 6A_Raw Data_Manuscript.xlsx\n\n- Figure 6C_Raw Data_Manuscript.xlsx\n\n- Figure 6C. xlsx\n\nTennessee Research and Creative Exchange: ex vivo Comparative Investigation of Suprachiasmatic Nucleus Excitotoxic Resiliency - ex vivo Comparative Investigation of Suprachiasmatic Nucleus Excitotoxic Resiliency, https://doi.org/10.7290/xSDiunNFHq. 42\n\nThis project contains the following extended data:\n\n- Tif imaging files for controls and treatments\n\n- Annotation for figures.docx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nZhou Y, Danbolt NC: Glutamate as a Neurotransmitter in the Healthy Brain. J. Neural Transm. 2014; 121(8): 799–817. PubMed Abstract \| Publisher Full Text\n\nMahmoud S, Gharagozloo M, Simard C, et al.: Astrocytes Maintain Glutamate Homeostasis in the CNS by Controlling the Balance between Glutamate Uptake and Release. Cells. 2019; 8(2): 184. PubMed Abstract \| Publisher Full Text\n\nGuerriero RM, Giza CC, Rotenberg A: Glutamate and GABA Imbalance Following Traumatic Brain Injury. Curr. Neurol. Neurosci. Rep. 2015; 15(5): 27. PubMed Abstract \| Publisher Full Text\n\nChoi DW: Excitotoxicity: Still Hammering the Ischemic Brain in 2020. Front. Neurosci. 2020; 14: 14. PubMed Abstract \| Publisher Full Text\n\nWang Y, Qin Z: Molecular and Cellular Mechanisms of Excitotoxic Neuronal Death. Apoptosis. 2010; 15(11): 1382–1402. Publisher Full Text\n\nPeterson GM, Moore RY: Selective Effects of Kainic Acid on Diencephalic Neurons. Brain Res. 1980; 202(1): 165–182. 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PubMed Abstract \| Publisher Full Text\n\nGillette MU: The Suprachiasmatic Nuclei: Circadian Phase-Shifts Induced at the Time of Hypothalamic Slice Preparation Are Preserved in Vitro. Brain Res. 1986; 379(1): 176–181. PubMed Abstract \| Publisher Full Text\n\nHirsch C, Schildknecht S: In Vitro Research Reproducibility: Keeping Up High Standards. Front. Pharmacol. 2019; 10: 10. PubMed Abstract \| Publisher Full Text\n\nBlanke ML, VanDongen AMJ: Activation Mechanisms of the NMDA Receptor.\n\nColwell CS: Linking Neural Activity and Molecular Oscillations in the SCN. Nat. Rev. Neurosci. 2011; 12(10): 553–569. PubMed Abstract \| Publisher Full Text\n\nDong X, Wang Y, Qin Z: Molecular Mechanisms of Excitotoxicity and Their Relevance to Pathogenesis of Neurodegenerative Diseases. Acta Pharmacol. Sin. 2009; 30(4): 379–387. PubMed Abstract \| Publisher Full Text\n\nLau CG, Takeuchi K, Rodenas-Ruano A, et al.: Regulation of NMDA Receptor Ca2+ Signalling and Synaptic Plasticity. Biochem. Soc. 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PubMed Abstract \| Publisher Full Text\n\nPetralia RS: Distribution of Extrasynaptic NMDA Receptors on Neurons. Sci. World J. 2012; 2012: 1–11. PubMed Abstract \| Publisher Full Text\n\nPapouin T, Oliet SHR: Organization, Control and Function of Extrasynaptic NMDA Receptors. Philosophical Transactions of the Royal Society B: Biological Sciences. 2014; 369(1654): 20130601. PubMed Abstract \| Publisher Full Text\n\nFernandez DC, Chang Y-T, Hattar S, et al.: Architecture of Retinal Projections to the Central Circadian Pacemaker. Proc. Natl. Acad. Sci. 2016; 113(21): 6047–6052. PubMed Abstract \| Publisher Full Text\n\nUreshino, Erustes, Bassani, et al.: The Interplay between Ca2+ Signaling Pathways and Neurodegeneration. Int. J. Mol. Sci. 2019; 20(23): 6004. PubMed Abstract \| Publisher Full Text\n\nLeSauter J, Stevens P, Jansen H, et al.: Calbindin Expression in the Hamster SCN Is Influenced by Circadian Genotype and by Photic Conditions. NeuroReport. 1999; 10(15): 3159–3163. PubMed Abstract \| Publisher Full Text\n\nIkeda M, Allen CN: Developmental Changes in Calbindin-D28k and Calretinin Expression in the Mouse Suprachiasmatic Nucleus. Eur. J. Neurosci. 2003; 17(5): 1111–1118. PubMed Abstract \| Publisher Full Text\n\nBjerke IE, Yates SC, Laja A, et al.: Densities and Numbers of Calbindin and Parvalbumin Positive Neurons across the Rat and Mouse Brain. iScience. 2021; 24(1): 101906. PubMed Abstract \| Publisher Full Text\n\nTischkau SA, Cohen JA, Stark JT, et al.: Time-of-Day Affects Expression of Hippocampal Markers for Ischemic Damage Induced by Global Ischemia. Exp. Neurol. 2007; 208(2): 314–322. PubMed Abstract \| Publisher Full Text\n\nWelsh DK, Takahashi JS, Kay SA: Suprachiasmatic Nucleus: Cell Autonomy and Network Properties. Annu. Rev. Physiol. 2010; 72(1): 551–577. PubMed Abstract \| Publisher Full Text\n\nWang LM, Schroeder A, Loh D, et al.: Role for the NR2B Subunit of the N-Methyl-d-Aspartate Receptor in Mediating Light Input to the Circadian System. Eur. J. Neurosci. 2008; 27(7): 1771–1779. PubMed Abstract \| Publisher Full Text\n\nPennartz CMA, Hamstra R, Geurtsen AMS: Enhanced NMDA Receptor Activity in Retinal Inputs to the Rat Suprachiasmatic Nucleus during the Subjective Night. J. Physiol. 2001; 532(1): 181–194. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMintz EM, Marvel CL, Gillespie CF, et al.: Activation of NMDA Receptors in the Suprachiasmatic Nucleus Produces Light-Like Phase Shifts of the Circadian Clock In Vivo. J. Neurosci. 1999; 19(12): 5124–5130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNMDA-Evoked Calcium Transients and Currents in the Suprachiasmatic Nucleus.\n\nHamada T, LeSauter J, Lokshin M, et al.: Calbindin Influences Response to Photic Input in Suprachiasmatic Nucleus. J. Neurosci. 2003; 23(26): 8820–8826. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrancaccio M, Patton AP, Chesham JE, et al.: Astrocytes Control Circadian Timekeeping in the Suprachiasmatic Nucleus via Glutamatergic Signaling. Neuron. 2017; 93(6): 1420–1435.e5. PubMed Abstract \| Publisher Full Text\n\nSantos J, Araújo J, Cunha M, et al.: Circadian Variation in GFAP Immunoreactivity in the Mouse Suprachiasmatic Nucleus. Biol. Rhythm. Res. 2005; 36(1–2): 141–150. Publisher Full Text\n\nViaIle M la, Servière J: Circadian Fluctuations in GFAP Distribution in the Syrian Hamster Suprachiasmatic Nucleus. NeuroReport. 1993; 4(11): 1243–1246. PubMed Abstract \| Publisher Full Text\n\nTso CF, Simon T, Greenlaw AC, et al.: Astrocytes Regulate Daily Rhythms in the Suprachiasmatic Nucleus and Behavior. Curr. Biol. 2017; 27(7): 1055–1061. PubMed Abstract \| Publisher Full Text\n\nAbe M, Herzog ED, Yamazaki S, et al.: Circadian Rhythms in Isolated Brain Regions. J. Neurosci. 2002; 22(1): 350–356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamazaki S, Kerbeshian MC, Hocker CG, et al.: Rhythmic Properties of the Hamster Suprachiasmatic Nucleus In Vivo. J. Neurosci. 1998; 18(24): 10709–10723. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarbour VL, Weigl Y, Robinson B, et al.: Comprehensive Mapping of Regional Expression of the Clock Protein PERIOD2 in Rat Forebrain across the 24-h Day. PLoS One. 2013; 8(10): e76391. PubMed Abstract \| Publisher Full Text\n\nProsser:Ex vivo Comparative Investigation of Suprachiasmatic Nucleus Excitotoxic Resiliency - ex vivo Comparative Investigation of Suprachiasmatic Nucleus Excitotoxic Resiliency. [dataset]. Tennessee Research and Creative Exchange. 2022. Publisher Full Text" }	[ { "id": "155004", "date": "06 Dec 2022", "name": "Luz Navarro", "expertise": [ "Reviewer Expertise Neuroprotection", "diurnal rhythms" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 submitted by Acharyya et al. presents interesting and well-substantiated data.\n\nThe authors compared the excitotoxic resiliency of the suprachiasmatic nucleus, anterior hypothalamus, and cerebral cortex by increasing NMDA concentration in rat brain slices. Also, they analyze this excitotoxic resiliency at different hours of the light-dark cycle.\n\nI only have a few minor comments:\n\nResults: In figure 3C, the colors of the bars do not match those of the legends. It looks like some bars are missing.\n\nDiscussion: Perhaps it could contribute to the discussion; some articles that indicate that the damage caused by an insult to the brain presents diurnal variations in animal models, for example: doi: 10.1007/s12035-017-0524-4; doi: 10.3389/fnins.2020.564992. eCollection 2020; doi: 10.3171/jns.2000.93.1.0082.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9183", "date": "09 Jan 2023", "name": "Rebecca Prosser", "role": "Author Response", "response": "\"Results: In figure 3C, the colors of the bars do not match those of the legends. It looks like some bars are missing.\" We apologize for the oversight and have revised the figure so the legend matches the bars. \"Discussion: Perhaps it could contribute to the discussion; some articles that indicate that the damage caused by an insult to the brain presents diurnal variations in animal models, for example: doi: 10.1007/s12035-017-0524-4; doi: 10.3389/fnins.2020.564992. eCollection 2020; doi: 10.3171/jns.2000.93.1.0082.\" We agree and have included this in the discussion." } ] }, { "id": "157126", "date": "23 Dec 2022", "name": "Malgorzata Beresewicz-Haller", "expertise": [ "Reviewer Expertise 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\nThe manuscript from Acharyya and co-workers addresses the very important topic concerning the phenomenon of differential sensitivity of brain regions to excitotoxic stress, as an important component of brain injuries including stroke.\nIt appears that, under the influence of the same stimulus, some regions of the brain sustain injury, while others possess internal adaptive mechanisms protecting them against harmful conditions. The former regions, described as vulnerable, include the CA1 region of the hippocampus, layers III and V of the cerebral cortex, and striatal cells and Purkinje cells in the cerebellum; while the latter (resistant) regions include the CA2 and CA3 parts of the hippocampus and the suprachiasmatic nucleus (SCN) mentioned in this manuscript. Although the phenomenon in question has been known for a long time, the mechanisms responsible for it remain unclear, implying a need for further research, all the more so as it is believed that the elucidation of adaptive mechanisms might offer a therapeutic approach to stroke and other neurodegenerative diseases.\nHere, the authors use acute murine brain slices to confirm previous observations regarding the resistance of the SCN to excitotoxic injury. They further show that the susceptibility of the SCN, as well as the hypothalamus and cortex, to injury of this kind proves to be dependent on the time of day. Unlike the authors, who deal with circadian rhythms specifically in the SCN, I was very interested in this result showing that the regions’ susceptibility to excitotoxic injury is greater by night than by day. It is thus unfortunate that this result has received so little exposure, especially since the manuscript gains inclusion in the Circadian Clocks in Health and Disease collection.\nMy major concerns\nA major benefit of the work would be to emphasise that intrinsic resistance to excitotoxic stress is not limited to the SCN, but also involves other regions of the brain. This could lead to possible verification (though only through additional experimentation) of whether the resistance in these different regions is manifested at the same level.\n\nSince propidium iodide is not membrane-permeable, it proves useful in differentiating necrotic/apoptotic and healthy cells by reference to membrane integrity. However, it is not useful where differentiation revolves around the degree of damage. I thus have doubts regarding the basis for a distinction to be drawn among PI-positive cells, between those that are damaged or else dead - as was the case for the two image-analysis protocols (IBCC and PACC).\n\nFig. 3 makes clear the rather limited excitotoxic effect of 50 uM NMDA. Thus values in the case of NMDA treatment only differ very slightly from those in the control - to the extent that a question is posed regarding the value of a focus that compares the effectiveness of three different image-analysis protocols, and all the more so since correlations are studied.\n\nComparisons between the SCN and the cortex can prove very misleading. To the best of my knowledge, the cortex has areas within it characterised by vulnerability and resistance to excitotoxic damage, which leaves it crucial for us to have an indication as to which part of the cortex was selected for analysis.\nMinor concerns\nFig. 3C warrants re-examination. The two separate legends here leave this a hard-to-analyse figure. Please also note that the values for the control conditions shown here differ from those noted for control conditions in Fig. 2C - in the face of an obvious expectation that they should be the same. In the current situation, it is difficult to evaluate the results obtained using the mean grey value method, and to relate those to the other two protocols.\n\nIn regard to Fig. 5, an important complication as differences in sensitivity to excitotoxic damage in the brain regions studied are compared is that, unlike the SCN and hypothalamus, the cortex was not treated with 100 and 500 uM NMDA. The consequence is to limit comparisons to the SCN vs. the hypothalamus, but not the cortex.\n\nHere, the effect of excitotoxic stress was analysed 3 hours after the end of NMDA treatment. How was the choice of such a time period justified? Were others also verified? It would be interesting for me to see whether, as the time from the end of NMDA treatment (mainly the 50uM application) extends, the effect of excitotoxic stress between the brain regions studied remains comparable to that observed after 3 h, or is different. In other words, does extending the time from the end of NMDA treatment increase excitotoxic injury?\n\nThere is confusion over the use of the abbreviation ZT, as once this refers to the incubation time with NMDA, while in another case it is the time before NMDA administration that is meant (when brain slices are perfused with EBSS).\n\nThe description of the results is sufficiently difficult to follow that readers may well be discouraged. To meet their needs better, I propose that more emphasis be put on the description of the physiological changes taking place, as opposed to the detailing of the presence or absence of statistical significance.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9184", "date": "09 Jan 2023", "name": "Rebecca Prosser", "role": "Author Response", "response": "\"Here, the authors use acute murine brain slices to confirm previous observations regarding the resistance of the SCN to excitotoxic injury. They further show that the susceptibility of the SCN, as well as the hypothalamus and cortex, to injury of this kind proves to be dependent on the time of day. Unlike the authors, who deal with circadian rhythms specifically in the SCN, I was very interested in this result showing that the regions’ susceptibility to excitotoxic injury is greater by night than by day. It is thus unfortunate that this result has received so little exposure, especially since the manuscript gains inclusion in the Circadian Clocks in Health and Disease collection.\" We agree with the reviewer that the circadian rhythms of susceptibility in the cortex in addition to the SCN and AH are extremely interesting and were a primary reason for submitting our manuscript to this collection. We have added to our existing discussion of these rhythms to further highlight the importance of these findings. \"My major concerns\" \"A major benefit of the work would be to emphasise that intrinsic resistance to excitotoxic stress is not limited to the SCN, but also involves other regions of the brain. This could lead to possible verification (though only through additional experimentation) of whether the resistance in these different regions is manifested at the same level.\" We agree and have added a discussion of other resilient brain regions to the discussion. \"Since propidium iodide is not membrane-permeable, it proves useful in differentiating necrotic/apoptotic and healthy cells by reference to membrane integrity. However, it is not useful where differentiation revolves around the degree of damage. I thus have doubts regarding the basis for a distinction to be drawn among PI-positive cells, between those that are damaged or else dead - as was the case for the two image-analysis protocols (IBCC and PACC).\" We agree and have added references in the discussion section supporting use of PI to assess cell damage and language acknowledge the benefit of using multiple assessment methods in the discussion. \"Fig. 3 makes clear the rather limited excitotoxic effect of 50 uM NMDA. Thus values in the case of NMDA treatment only differ very slightly from those in the control - to the extent that a question is posed regarding the value of a focus that compares the effectiveness of three different image-analysis protocols, and all the more so since correlations are studied.\" We agree and have added language to the discussion section to the effect that additional comparisons of the analytical methods going forward would be useful. \"Comparisons between the SCN and the cortex can prove very misleading. To the best of my knowledge, the cortex has areas within it characterised by vulnerability and resistance to excitotoxic damage, which leaves it crucial for us to have an indication as to which part of the cortex was selected for analysis.\" We agree and have clarified in the methods section the general cortical areas analyzed and added a brief section in the discussion on other brain regions that have been reported to be resistant to excitotoxic damage. \"Minor concerns\" \"Fig. 3C warrants re-examination. The two separate legends here leave this a hard-to-analyse figure. Please also note that the values for the control conditions shown here differ from those noted for control conditions in Fig. 2C - in the face of an obvious expectation that they should be the same. In the current situation, it is difficult to evaluate the results obtained using the mean grey value method, and to relate those to the other two protocols.\" As also suggested by reviewer #1, we have revised Fig 3 so the legend matches the graph, and clarify in the Fig 3 legend that the Y axis is different from that in Fig 2, although the control data are the same in both. \"In regard to Fig. 5, an important complication as differences in sensitivity to excitotoxic damage in the brain regions studied are compared is that, unlike the SCN and hypothalamus, the cortex was not treated with 100 and 500 uM NMDA. The consequence is to limit comparisons to the SCN vs. the hypothalamus, but not the cortex.\" We have added language in the discussion section discussing the lack of treating the cortical tissue with these two concentrations of NMDA and how this affects our comparisons. \"Here, the effect of excitotoxic stress was analysed 3 hours after the end of NMDA treatment. How was the choice of such a time period justified? Were others also verified? It would be interesting for me to see whether, as the time from the end of NMDA treatment (mainly the 50uM application) extends, the effect of excitotoxic stress between the brain regions studied remains comparable to that observed after 3 h, or is different. In other words, does extending the time from the end of NMDA treatment increase excitotoxic injury?\" We agree and have added a discussion of investigating different temporal dynamics in future experiments. \"There is confusion over the use of the abbreviation ZT, as once this refers to the incubation time with NMDA, while in another case it is the time before NMDA administration that is meant (when brain slices are perfused with EBSS).\" We clarify in the methods section that ZT refers to time relative to the animal colony light cycle. \"The description of the results is sufficiently difficult to follow that readers may well be discouraged. To meet their needs better, I propose that more emphasis be put on the description of the physiological changes taking place, as opposed to the detailing of the presence or absence of statistical significance.\" We agree and have added descriptive sentences summarizing each set of data alongside the results of the statistical analyses so that the results section is easier to read." } ] } ]	1	https://f1000research.com/articles/11-1242
https://f1000research.com/articles/12-442/v1	25 Apr 23	{ "type": "Review", "title": "Prevalence and risk factors of posttraumatic stress disorder in COVID-19", "authors": [ "Oscar Medina-Ortiz", "Alejandro Oses-Gil", "Vivian Vanessa Arenas-Villamizar", "Angel Ortega", "Milagros Rojas", "Mervin Chávez-Castillo", "Franlet Araque-Castellanos", "Alejandro Oses-Gil", "Vivian Vanessa Arenas-Villamizar", "Angel Ortega", "Milagros Rojas", "Mervin Chávez-Castillo", "Franlet Araque-Castellanos" ], "abstract": "Posttraumatic stress disorder (PTSD) has a prevalence of 2%–5% in the general population. COVID-19 is regarded as a traumatic agent that can increase the prevalence of this disorder to up to 30%. A documentary review was thus conducted, which included 13 studies on the presence of PTSD in patients who have survived COVID-19 infection and the possible associated factors. Female and young age, as well as other aspects associated with economic losses or living alone, could influence the appearance of this psychological sequela. A preventive mental healthcare program could be implemented during infection in such patients with COVID-19 who show the characteristics described in most studies.", "keywords": [ "COVID-19", "survivor", "posttraumatic stress disorder", "risk factors" ], "content": "Introduction\n\nThe World Health Organization (WHO) reported in 2017 that the prevalence of post-traumatic stress disorder (PTSD) in the non-psychiatric population was between 3.90 and 5.60%.1\n\nIt has been widely described that the fear of the spread of the novel severe acute respiratory syndrome coronavirus 2 and the associated consequences of COVID-19, such as social distancing and the implementation of stay-at-home prevention strategies, can be considered a traumatic stressor.2,3\n\nIn the past, disaster-related experiences of several types (terrorist, natural, and environmental attacks) have been associated with increased depression and anxiety, including PTSD, as well as substance abuse.4 In addition, the fact that people follow 24-h mortality statistics through television news and other news programs may worsen the effects of trauma, with some studies suggesting that repeated exposure to the experience in the media can result in increased anxiety and intensify the stress response.5–7\n\nSome mental health specialists are beginning to draw attention to PTSD as one of the major sequelae of the COVID-19 pandemic.8 The measures to control the virus have possibly been very stressful, which could promote PTSD.9,10 It is essential to remember that the beginning of the pandemic was potentially traumatic because no proper information was known about the virus and its propagation mechanisms;8 therefore, the infection generated anxiety and a sense of threat, which could promote PTSD,11 which usually develops after facing a dangerous situation that can put life at risk.12\n\nRecent research has reported on PTSD related to the coronavirus outbreak, with a finding that the proportion ranges from 12.4% to 31.0% in patients recovering from the illness.9,13–16\n\nThe aim of this review was to report about the prevalence of PTSD in those patients who have survived the COVID-19 infection and to describe which associated factors may influence its occurrence.\n\n\nMethods\n\nPubMed, Science Direct, Scopus and Scielo databases were used to conduct a search for scientific articles using the keywords: posttraumatic stress disorder, coronavirus, COVID-19, survivor, and patients, both in Spanish and English, with a deadline of August 2021. Once the articles were selected, their references were additionally reviewed in search of other important studies related to the main objective of the study. On the first occasion, 38 studies were identified. Those whose sample consisted of physicians, nurses, and other healthcare workers, or in which other mental illnesses were described and in which the prevalence of PTSD was not reported were excluded. In the end, the sample consisted of 13 studies (Table 1).\n\n\nCoronavirus Infection\n\nDifferent viruses have caused the devastation of humanity throughout history; lately, novel diseases caused by Ebola, Zika, Nipah and Coronaviruses viruses have appeared in different parts of the world. Toward the end of 2019, a new coronavirus causing a particular type of pneumonia was identified in the city of Wuhan, China. The property of rapid transmission of this virus led to an epidemic in China, which soon spread to the rest of the world.17\n\nBy the beginning of 2020, WHO defined this pathology as COVID-19 because it was produced by a coronavirus identified in 2019. So far, more than 200 million people have been infected worldwide with this type of pneumonia18; therefore, COVID-19 was declared a pandemic by WHO in March 2020,19 which initially began as a novel coronavirus in China.20\n\nThe incubation period of the disease is similar to that of other viruses. Symptoms usually appear 6 days after contact with another infected person, and the initial symptoms are fatigue, fever, and cough.21,22 Several people would manifest only minor symptoms, but the condition of others will eventually become complicated and they would present major respiratory and cardiovascular diseases, which could lead to death.23\n\n\nPTSD\n\nPTSD is a mental disorder that is triggered after a traumatic experience, such as having an accident or being the victim of a violent act.7 The emotions that were experienced at that moment are experienced again on several occasions and memories frequently appear, which interferes with daily life.24 The coronavirus infection can act as a stressor of such magnitude as to trigger posttraumatic stress. This is because approximately 2%–5% of COVID-19 cases result in death caused by massive alveolar damage and progressive respiratory failure.25\n\nAccording to the 10th WHO classification of mental illnesses,26 PTSD is a pathology that appears as a delayed response to a stressful situation (of short or long duration) of an exceptionally threatening or catastrophic nature, which is likely to cause generalized distress to nearly any person.\n\nAmong its most prominent manifestations are the presence of repeated episodes of fear where the trauma is experienced as if it was happening again, and during sleep nightmares are experienced within a framework of affective flattening that prevents the person from feeling empathy for others, or from leading a normal life under circumstances that could remind him/her of the traumatic event experienced.27 Generally, the person remains in a constant state of alertness before a possible imaginary tragedy such as the incidence that has already happened, which produces autonomic discharges such as hypervigilance and insomnia that lead to anxiety and depression symptoms and even suicidal ideas.28\n\nThe time for the onset of PTSD after experiencing trauma can be anywhere between a few weeks and months. The course is not necessarily continuous, and symptoms can appear and disappear in periods or cycles. Without treatment, the disease may self-limit within two years, but a small group of patients continue to experience symptoms for a longer duration, resulting in a chronic pathology that may even affect the individual’s personality.29\n\nAccording to the DSM-5 classification, PTSD is clinically characterized by avoidance of stimuli reminiscent of the trauma, intrusive thoughts related to the disaster, cognitive and mood changes, as well as constant alertness that makes the person be reactive, all in association with the traumatic events that occurred.29 PTSD causes clinical discomfort and alters the person's work performance and social relationships.30\n\n\nPSTD and COVID-19\n\nIf COVID-19 diagnosis is suspected or confirmed, people may have traumatic psychological symptoms such as experiencing symptoms directly and undergoing invasive treatment (respiratory failure and tracheostomy)10 or witnessing the suffering, struggle, and death of other patients.31 People may fear infection, be hospitalized in isolation, and be rejected by medical staff or their friends and family.32,33 It is likely that having this disease can be compared to a similar burden of that affecting people in any other type of disaster,34 where the symptoms of PTSD are more frequent in those people who suffered the trauma directly.4\n\n\nPrevalence of PTSD\n\nThe prevalence of posttraumatic stress among individuals exposed to COVID-19 varies greatly.35–39\n\nThe study showing the lowest figures is that of Simani Leilla et al.40 who conducted a study in Iran to determine the prevalence of PTSD six months after discharge in 120 patients who had been hospitalized for COVID-19 between February and April 2020. After being assessed with the Posttraumatic Stress Disorder Checklist for DSM-5 (PCL-5) scale, which is based on the DSM-5 criteria for PTSD, 5.8% of the patients were found to have PTSD symptoms. It is likely that the low prevalence was conditioned by the time that had already passed after the infection at the time of applying the measurement instruments.\n\nSince the start of the pandemic in 2019, several review papers have reported data linking PTSD symptoms to the COVID-19 epidemic. In the meta-analysis by Salehi et al.,35 where 35 studies from November 2015 to May 2020 were included under Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), the prevalence of PTSD-related symptoms during coronavirus outbreaks (SARS, Middle East respiratory syndrome, and COVID-19) was estimated at 18%, and out of every 10 patients who survived COVID-19, three had PTSD-related symptoms.\n\nAnother systematic review by Krishnamoorthy et al.,36,38 which includes 50 publications based on the PRISMA model up to April 2020, reported that the prevalence of PTSD during the COVID-19 outbreak ranged from 21.94% to 27.00%.39\n\nThe systematic review by Cooke et al.,38 which includes 14 studies under the PRISMA guidelines, yielded data where the prevalence of post-traumatic stress for people who were exposed to the COVID-19 epidemic was 26.2%, which was higher than that observed in flood (15.74%) and hurricane (17.81%) survivors,41,42 and similar to earthquake survivors (23.66%).43 This may be because natural disasters, such as wildfires or tsunamis, can give people time to adapt, but infectious diseases such as COVID-19 often strike suddenly and unexpectedly, quickly becoming a threat factor that can put people at risk.43 Therefore, outbreaks of infectious diseases can cause more disturbances in the psyche of people than many natural disasters.\n\nThe study that has shown the highest prevalence of PTSD following COVID-19 infection is that conducted by Bo et al.11 at the onset of the pandemic, reporting up to 96.2% of 714 patients with PTSD symptoms assessed using PCL-5. The researchers believed that the rapid transmission of COVID-19 along with distressing news coverage in widely used communication programs (e.g., WeChat and Weibo) and social discrimination toward patients with COVID-19 may have resulted in a higher prevalence of self-perceived COVID-19-associated posttraumatic stress symptoms in this study.\n\nThe perception and coping of COVID-19 may vary depending on where patients are evaluated. There are reports that show a prevalence of PTSD that varies greatly depending on the country where the study was carried out, giving figures ranging from 7% to 53%.44 This could be due to different circumstances and the idiosyncrasy of each of the countries, such as the way in which their citizens perceive stress, the number of people who fell ill and died from the virus, the effectiveness of health programs, the distribution of information that is not true and is threatening, the handling of the epidemic by the health authorities and the trust of the people in the institutions.\n\nPrevious research has made it clear that patients with infectious diseases are often more vulnerable to social discrimination after they are cured,4 and these experiences may result in increased PTSD symptomatology. PTSD levels in those who have dealt with the trauma of having COVID-19 disease have also been found to be highest immediately after the outbreak (0 to 4 weeks).43,45,46\n\nResearch papers report a high prevalence of mental illnesses after an outbreak of an infectious disease occurs.47–49 Generally, psychiatric symptoms lose strength when the epidemic ends, but this is not the same for PTSD symptoms, which can remain present for a long time, generating anxiety and interfering with people's performance of daily life.50 It is therefore important to consider ongoing surveillance and healthcare policies as a preventive strategy for PTSD in both the short and long term.\n\n\nRisk Factors\n\nSeveral risk factors have been identified such as gender, age, and degree of disease complication.10,13,14 In the systematic review conducted by Qiu et al. under PRISMA standards that included 76 articles published up to October 2020, the prevalence of PTSD for people who were in contact with COVID-19 ranged from 23.03% to 34.32%51 with an average of 28.34%, within a 95% confidence interval, much higher than that of the general population, which lies at 3%–5%. Subjects older than 51 years had more PTSD symptoms (62.16%) compared with those who were aged 18–20 years (2.70%) (p < 0.001). A statistically significant difference was observed for post-traumatic stress symptoms according to gender; the results indicated that studies with a higher percentage of male participants (>50%) showed a higher prevalence of PTSD (26.70 vs 41.79%; Q = 5.31; p = 0.021). Regarding regions where the studies were conducted, prevalence of posttraumatic stress symptoms was similar among people from Europe, America, Eastern Mediterranean, Western Pacific, and Southeast Asia (32.13%, 30.48%, 37.74%, 26.34%, and 17.16%, respectively), with no significant differences. Similarly, the prevalence of posttraumatic stress symptoms among people according to their economic income showed no significant differences (high 30.03%, upper middle 27.6% and lower middle 36.07%). A difference was observed between the studies according to the number of subjects included, therefore the articles with a larger sample size had a lower prevalence of PTSD (20.33 vs. 32.08%; Q = 6.61; p = 0.010).\n\nOverall, the risk factors associated with the occurrence of PTSD following COVID-19 infection continue to vary widely. In the study by Lahav,10 976 adults in Israel were included, who were administered the PCL-5 scale for PTSD based on DSM-5 via online.52 Most participants reported experiencing at least one PTSD symptom related to COVID-19 (76.7%). Being younger, female, not being in a formal relationship, belonging to a high-risk group, being diagnosed with the illness, living alone during the outbreak, having a close person in a high-risk group, and negative self-evaluation of their health status were associated with higher distress. An interesting finding of this study was that 90.2% of people who had prior exposure to previous trauma had a higher prevalence of PTSD.\n\nLiu et al.13 conducted a study on 675 patients discharged for COVID-19 who had to remain in quarantine for 28 days once they left Wuham Hospital and who were administered the online PCL-5 during their isolation period. Approximately 12.4% were diagnosed with PTSD and the COVID-19 symptoms that were associated with PTSD presented with cough, fatigue, and chronic chest pain (p < 0.05), corticosteroid treatment (p = 0.016), and severity of illness along with perceived stigma (p < 0.001). Other factors found to be associated with PTSD were living with children (OR: 6.71; p < 0.001) and a family member having died from coronavirus (OR: 7.05; p = 0.005). Sleeping problems, intrusive thoughts, feeling distant or isolated from people, and an inability to concentrate were the most prevalent PTSD symptoms due to COVID-19.\n\nOn the other hand, Cai et al.,14 included in their study a total of 126 COVID-19 surviving patients from Senzhen Hospital, who were quarantined for 14 days after hospital discharge and who were evaluated with the PTSD-22 scale online. The prevalence of PTSD was 31% with a predominance of intrusive thoughts in 41.2% and avoidance in 27.7%. They found higher rates of PTSD among patients younger than 60 years (p = 0.04), those who were not retired (p = 0.005) and those who were female (p = 0.032). Subjects who reported good social support were likely to have lower PTSD scores than those who reported poor social support (p = 0.002). The presence of infected family members or close relatives was significantly related to PTSD (p = 0.033) as was physical distress following COVID-19 infection (p = 0.045).\n\nThe study by Bonsaksen T et al.53 is one that provides more information. It was conducted in Norway between April and May 2020 by means of a nationwide survey on social networks (Facebook, Twitter, LinkedIn and Instagram), which included 4527 subjects over 18 years and without any exclusion criteria, aimed to examine posttraumatic stress reactions due to COVID-19, the prevalence of PTSD and factors associated with posttraumatic stress during the early stages of the COVID-19 outbreak by means of the PCL-5 scale. The prevalence of PTSD was 12.5% for males and 19.5% for females. PTSD was associated with younger age (p < 0.001), female gender (p < 0.001), lack of social support (p < 0.001) and a group of pandemic-related variables, such as economic concerns (OR: 2.23, p < 0.001), expectation of financial loss (OR: 1.39; p < 0.01), having been in quarantine or isolation (OR: 1.31; p < 0.01), being at high risk of complications from COVID-19 infection (OR: 1.77; p < 0,001) and worrying about family and close friends (OR: 1.83; p < 0.001).\n\nAnother study by Eivink G et al.,54 also in a Norwegian population, included subjects discharged before June 1, 2020; it determined the prevalence and risk factors for PTSD among patients who were hospitalized (n=125) and non-hospitalized (n=458) for COVID-19 and who had a positive polymerase chain reaction test for severe acute respiratory syndrome. They were invited to answer the PCL-5 for PTSD electronically or on paper four to eight weeks after discharge and receive a clinic visit at approximately 3 months. Hospitalized patients had higher rate of asthma, diabetes, hypertension, dyspnea, and fever than non-hospitalized patients. PTSD was present in 9.5% of hospitalized patients and 7% of non-hospitalized patients. The disease was more predominant in women, even though no significant differences were found, and PCL-5 score was higher in hospitalized than in non-hospitalized patients (p=0.042). Female sex, being born outside Norway, living alone, and having dyspnea during COVID-19 were associated with higher PCL-5 scores, whereas being hospitalized by COVID-19 was not.\n\nPTSD is a disease that can manifest at a later stage and not necessarily at the time of hospital discharge. A study conducted in Italy by Tarsitani Lorenzo et al.,55 whose aim was to determine the prevalence of PTSD three months after discharge in patients who had survived COVID-19, showed that of 115 subjects included, 10.4% had a diagnosis of PTSD based on the PCL-5 scale. Body mass index greater than 30 (OR:3.51; P=0.03), male sex (P=0.007), having a previous psychiatric diagnosis (OR: 6.30; P<0.001) and suffering from any chronic respiratory disease (OR: 6.03; P=0.053), were the main risk factors.\n\nAge is a relevant factor that should be considered when it comes to applying preventive measures against PTSD. It has been found that the prevalence of PTSD in older adult survivors of road traffic accidents is significantly higher than that of younger people.56 The finding that young people are more likely to have symptoms of PTSD is described in several studies from China,14 but others have found different results.57 However, it is likely that depending on the situation, old age could be a factor that favors facing problems related to death, presenting with less depression58 and anxiety59 among older people. Based on these findings, primary care physicians should be more attentive to early symptoms of trauma among older patients with COVID-19 and implement rapid psychological interventions. Similarly, in this type of accident, women are more likely to develop PTSD.56 However, in the case of COVID-19 infection, some studies have associated men with a higher prevalence of PTSD during the pandemic. It is possible that this is due to the fact that men and older people are more likely to get sick with COVID-19 and have a higher mortality rate,60 and therefore experience increased severity when exposed to trauma.\n\nThe finding that young people are more likely to have symptoms of PTSD is described in several studies from China,14 but others have found different results.57 However, it is likely that depending on the situation, old age could be a factor that favors facing problems related to death, presenting with less depression58 and anxiety59 among older people.\n\nRisk factors for PTSD in COVID-19 survivors are not exclusively generated by the fear of dying or the severity of the illness; they may be triggered by other circumstances. It should be remembered that people were encouraged to take refuge at home, and that educational institutions at all levels stopped working in person, as did as businesses that offered personalized treatment, and tourism, concerts and theater were also affected. This resulted in the economic failure of many companies, wo were forced to lay off their workers.61 Thus, the pandemic and the measures to combat it have raised concerns about public mental health, which can be considered a factor associated with PTSD and other anxiety disorders.62–66\n\nTherefore, quarantine and isolation have also been associated with PTSD.67,68 Isolation triggers negative ideas and thoughts about the disease, and the misfortune it can cause in people, which can result in an increase in post-traumatic stress.69 All the limitations of social interaction also interfere with basic support measures between family members and friends, who are essential to counteract post-traumatic stress reactions.70\n\n\nConclusions\n\nStudies show that prevalence of PTSD in patients surviving coronavirus infection can vary from 5.8% to 96.2% depending on different factors involved in the methodology of each study. However, an average of around 30% could be considered. What all authors do agree on is that it tends to be higher than in the general population, and even in people who have been victims of natural disasters.\n\nThe risk factors are not very clear either since the results of the studies show contradictory aspects. However, in most of them the following risk factors have been established: lack of social support, living alone, being young, being a woman, and facing a series of problems directly related to the coronavirus outbreak, such as economic concerns, fear of infecting family and friends and perceiving oneself as being at high risk. In the same way, a history of mental illness has also been described as an important risk factor.\n\nKnowledge of the prevalence of PTSD and triggering factors is important information to consider for the implementation of prevention strategies both at the beginning and at the end of the infection by COVID-19, and therefore try to avoid or reduce the subsequent development of this mental illness, especially when at the present time government agencies worldwide are echoing the importance of psychological health in citizens with major proposals during the coronavirus pandemic.", "appendix": "Data Availability\n\nNo data are associated with this article.\n\n\nReferences\n\nKoenen KC, Ratanatharathorn A, Ng L, et al.: Posttraumatic stress disorder in the World Mental Health Surveys. Psychol. Med. 2017 Oct; 47(13): 2260–2274. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaslach C, Jackson SE: The measurement of experienced burnout. J. Organ. Behav. 1981; 2(2): 99–113. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaider II, Tiwana F, Tahir SM: Impact of the COVID-19 Pandemic on Adult Mental Health. Pakistan journal of medical sciences. 2020 May; 36(COVID19-S4): S90–S94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMi L, Jiang Y, Xuan H, et al.: Mental health and psychological impact of COVID-19: Potential high-risk factors among different groups. Asian J. Psychiatr. 2020 Oct; 53: 102212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaufman KR, Petkova E, Bhui KS, et al.: A global needs assessment in times of a global crisis: world psychiatry response to the COVID-19 pandemic. BJPsych open. 2020 Apr 6; 6(3): e48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVindegaard N, Benros ME: COVID-19 pandemic and mental health consequences: Systematic review of the current evidence. Brain Behav. Immun. 2020 Oct; 89: 531–542. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHawryluck L, Gold WL, Robinson S, et al.: SARS control and psychological effects of quarantine, Toronto, Canada. Emerg. Infect. Dis. 2004 Jul; 10(7): 1206–1212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReynolds DL, Garay JR, Deamond SL, et al.: Understanding, compliance and psychological impact of the SARS quarantine experience. Epidemiol. Infect. 2008 Jul; 136(7): 997–1007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerntsen D, Rubin DC: The centrality of event scale: a measure of integrating a trauma into one's identity and its relation to post-traumatic stress disorder symptoms. Behav. Res. Ther. 2006 Feb; 44(2): 219–231. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBirkeland MS, Nielsen MB, Hansen MB, et al.: Like a bridge over troubled water? A longitudinal study of general social support, colleague support, and leader support as recovery factors after a traumatic event. Eur. J. Psychotraumatol. 2017; 8(1): 1302692. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "285684", "date": "19 Jun 2024", "name": "Wisdom Obioha", "expertise": [ "Reviewer Expertise Posttraumtic growth", "PTSD", "emotion regulation", "mental health", "cognitive aging and alzheirmer's disease." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is a documentary review of studies on the prevalence of PTSD in patients who have survived COVID-19 infection. In addition to the prevalence, it also provided more context by discussing risk factors that predispose patients to PTSD. The method was adequately described and seems to be methodologically fine, however, the review did not sufficiently discuss protective factors of PTSD or factors that help patients cope with PSTD resulting from COVID-19. In particular, discussing Posttraumatic Growth (PTG) after COVID-19 will provide more practicable insights into how patients progress from PTSD to normal functioning after encounter with COVID-19.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-442
https://f1000research.com/articles/10-806/v1	16 Aug 21	{ "type": "Research Article", "title": "Comparison of cell size, chlorophyll fluorescence and cadmium (Cd2+) bioaccumulation between wild-type and mutant strains of Chlamydomonas reinhardtii upon exposure to Cd2", "authors": [ "Ruby Ynalvez", "Hector Gonzalez-Cantu", "Erick Vazquez-Cano", "Hector Gonzalez-Cantu", "Erick Vazquez-Cano" ], "abstract": "Background: Heavy metal contamination presents a constant threat to biological systems. Simultaneously, heavy metals have become one of the major contaminants in the aquatic ecosystems. In this regard, the investigation of heavy metal-tolerance genes in algae is relevant. Chlamydomonas reinhardtii is a unicellular green alga, and an excellent model organism used in heavy metal studies. In C. reinhardtii, a novel gene designated as Cia7, was hypothesized to play a role in heavy metal homeostasis due to CIA7’s conserved cysteine-residue motif. This study compared two strains of C. reinhardtii, cc4425, the wild-type with the functional CIA7 protein and cc5013, the mutant strain with the disrupted cia7- gene. The hypothesis was that the expression of Cia7 contributes to an increased cadmium (Cd)-tolerance in C. reinhardtii. The Cd-tolerance would be described by physiological markers of microalgae health, and by intracellular accumulation of the metal. Methods: The objectives of this study were (1) to compare chlorophyll fluorescence and cell size in cc4425 and cc5013 exposed to Cd2+, and (2) to compare Cd2+ bioaccumulation in cc4425 and cc5013 strains in different growth media. Flow cytometry, and inductively coupled plasma optical emission spectrometry (ICP-OES) analysis were performed. Results: There was no significant statistical difference in Cd2+ bioaccumulation between the two strains, cc4425 and cc5013, regardless of growth media. However, a statistically significant difference in Cd2+ bioaccumulation (p<0.0001) was determined between the media (with acetate and without acetate). The cia7- mutant, cc5013 was found to be more susceptible to a Cd2+-induced decrease in chlorophyll fluorescence and had a reduced cell size compared to cc4425, the wild-type strain. Conclusions: These observed differences between the strains suggest that CIA7’s biological activity could play a direct or indirect role in increasing Cd tolerance in C. reinhardtii.", "keywords": [ "cadmium tolerance", "flow cytometry", "ICP-OES analysis", "DUF2256 domain", "heavy metal", "microalgae" ], "content": "Introduction\n\nPublic health and environmental concerns from heavy metal contamination have increased in recent times. In fact, among all the pollutants, heavy metals have received particular attention due to their toxic nature, persistence in the environment, and bioaccumulative nature (Ali et al., 2019; Al Osman et al., 2019). Heavy metals are naturally occurring elements that have high atomic weights and densities at least five times greater than that of water. Arsenic, cadmium (Cd), chromium, lead, and mercury are among the priority metals of public health significance; this, in part, is due to their high degree of toxicity. These metals are systemic toxicants known to induce multiple organ damage, even at lower levels of exposure (Sevin et al., 2020; Tchounwou et al., 2012).\n\nHeavy metals are known to be naturally occurring elements; however, anthropogenic activities introduce them in large quantities in the environment (Briffa et al., 2020). The presence of heavy metals in nature can arise from environmental processes like volcanic emissions and rock weathering (Jan et al., 2015; Tuzun et al., 2005). On the other hand, anthropogenic activities include mercury (Hg) contamination from gold mining, lead (Pb) from its use as a gasoline additive, and Cd from fertilizers, batteries, and sewage water (Sevim et al., 2020; Roberts, 2014). Human activities are contaminating the environment and its resources; these activities are discharging heavy metals at concentrations greater than what the environment can handle (Masindi and Muedi, 2018).\n\nThe final deposition points of heavy metal leachate are often aquatic environments, where microalgae are widely distributed and therefore exposed to these metals. Literature evidence suggests that toxic heavy metals may induce deleterious damage to the metabolism and physiology of microalgae (Xie et al., 2019; Abu Bakar et al., 2015). The damage that heavy metals exert at the cellular level is composite and targets different mechanisms. For example, intracellular heavy metal ions induce a variety of biochemical changes that affect the normal growth and metabolic activity of the cell system. These changes include blocking the functional groups of biomolecules, displacing essential metal ions, and modifying the native conformations of biomolecules (Priyadarshini et al., 2019). Heavy metals are also known to interfere and inhibit protein activity. Proteins whose active domains possess sulfhydryl groups are especially susceptible to deleterious effects of heavy metals due to their affinity with them, resulting in irreversible binding (Fein et al., 2019; Hua and Li, 2014). In addition, heavy metals can interfere with metalloproteins or metal-protein complexes by competing with an essential metal cofactor for an enzyme’s binding site (Pinter and Stillman, 2015; Viehweger, 2014). A reported symptom of heavy metal toxicity in a cell is an increase in its oxidative stress, marked by an increase in reactive oxygen species (ROS) (Elbaz et al., 2010; Jiang et al., 2016). ROS, due to their oxidative character, damage cellular structures and macromolecules by interfering with non-covalent interactions.\n\nTo study the deleterious effects of heavy metals and their interactions within biological systems, eukaryotic microalgae have been deemed appropriate subjects (Leong and Chang, 2020; Miazek et al., 2015). Microalgae have evolved resistance to heavy metal stress through their own metabolic pathways, which include metal immobilization by cell walls and extracellular polymeric substances (EPS), and transport through the cell membrane via transporters (Lu et al., 2019; Liu et al., 2016). It is this acclimatization that makes microalgae suitable candidates for heavy metal studies; they can survive relatively high metal concentrations, enabling the observations and analysis of heavy metal-induced effects.\n\nChlamydomonas reinhardtii is a unicellular green alga from the Chlorophyta lineage, and is the best-developed reference organism for studying algal biology, especially with respect to metal metabolism (Blaby-Haas and Merchant, 2012). C. reinhardtii has garnered scientific interest in the context of heavy metal toxicity due to its broad range of heavy metal resistance and tolerance (Zheng et al., 2020; Ibuot et al., 2018; Nowicka et al., 2016). C. reinhardtii is also a potential candidate for phytoremediation, the process of utilizing plants and algae to bioremediate contaminated areas (Leong and Chang, 2020).\n\nIn C. reinhardtii, a primary line of defense against heavy metals is the cell wall. The cell wall displays a high affinity towards metallic cations, and cell wall-less mutants commonly exhibit greater sensitivity towards metals (McFie et al., 1994; Collard and Matagne, 1990). Cell walls contain multiple negatively charged groups that form interactions with metal cations, thus sequestering metals and preventing metal uptake into the cytoplasm (Priyadarshini et al., 2019; Samadani et al., 2018; Hanikenne, 2003). Whereby, intracellularly, heavy metals are compartmentalized in vacuoles, allowing the cell to control the cytoplasmic metallic concentration, and neutralizing the heavy metal’s toxic effect (Perales-Vela et al., 2006). Mechanisms of heavy metal tolerance in C. reinhardtii also include transporters. For example, the mitochondrial half-size ABC (ATP-binding cassette) transporter is implicated in Cd-tolerance (Hanikenne et al., 2005). Heavy metal-binding peptide synthesis has also been shown to be preferentially developed in microalgae and some fungi, thus granting these organisms a higher metal tolerance (Perales-Vela et al., 2006).\n\nC. reinhardtii showed various strategies to cope with Cd stress. Penen et al. (2017) previously reported that most Cd could diffuse in the whole cell, and complexed by thiol ligands. Thiol ligands were also shown to increase with cells’ exposure time to Cd, confirming thiol ligands’ important role in Cd stress regulation. There is also pyrenoidal sequestration of Cd, leading to limited CO2 fixation during Cd exposure (Penen et al., 2019).\n\nMetal tolerance studies including Cd in C. reinhardtii have been extensively reported in the literature. In reported studies of exogenous metallothionein gene expression in C. reinhardtii, Cd was reported as an inducer. The generation and characterization of Cd-resistant and hypersensitive C. reinhardtii mutants have also been reported in studies of heavy metal tolerance (Samadani et al., 2018; Hanikenne, 2003; Hu et al., 2001). In particular, the overexpression of metal transporters in C. reinhardtii for bioremediation is an attractive approach; it may allow both increased metal accumulation into the cell and increased metal tolerance, such as by transfer of a toxic metal out of the cytosol and into an internal compartment (Ibuot et al., 2020; Ibuot et al., 2017). The genetic manipulation of C. reinhardtii has been shown to be successful in creating stable, inherited phenotypes (Pollock et al., 2017). Changes in the heavy metal biosorption capacity of C. reinhardtii have been reported in genetically modified strains (Ibuot et al., 2020; Cheng et al., 2019; Aksmann et al., 2014; Lin et al., 2013; Mayfield et al., 2007; Shimogawara et al., 1998).\n\nA novel gene, Cia7, was isolated from an inorganic carbon-acquiring, and high carbon dioxide-requiring C. reinhardtii mutant, cc5013 (Ynalvez et al., 2008). The gene designated as Cia7 was hypothesized to play a role in heavy metal tolerance or metal detoxification due to its translational product’s amino acid sequence. Sequence alignment utilizing the U.S. National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST, RRID:SCR_004870) revealed homology with many uncharacterized proteins conserved in bacteria; proteins classified as belonging to a family of DUF2256 domain-containing proteins. Members of this family of hypothetical bacterial proteins have domains of unknown functions (DUF). Homologous proteins also included computer modeling predicted proteins, TRAP transporter small permease (92% identity) and an uncharacterized metal binding protein (72% identity). The protein sequence of CIA7 contained a conserved, repeated motif of four cysteine residues whose thiol functional group is hypothesized to chelate heavy metals (Ynalvez and Moroney, 2008). Based on this observation, the aim of this study was to investigate whether Cia7 plays a potential role in metal tolerance in C. reinhardtii.\n\nThis study compared two strains of C. reinhardtii: cc4425, the wild-type with the functional CIA7 protein and cc5013, the strain that has a disruption in the Cia7 gene due to a mutant cia7-. It is hypothesized that CIA7, due to its conserved cysteine residue motif, could play a role in the molecular defense mechanism to attenuate heavy metal toxicity. More specifically, when subjected to metal (Cd2+) stress, C. reinhardtii cc4425 cells that express a functional CIA7 would exhibit higher growth rate and chlorophyll fluorescence, but a decreased metal bioaccumulation when compared to the mutant cc5013. The objectives of this study were to compare cell size, chlorophyll fluorescence, and metal (Cd2+) bioaccumulation between the two strains cc4425 (wild-type) and cc5013 (mutant). Flow cytometry was used in this study. This technique rapidly analyzes single cells or particles as they flow past single or multiple lasers while suspended in a buffered salt-based solution (McKinnon, 2019). Flow cytometers are suitable for microalgae applications as the latter are often a collection of single cells, and microalgae possess endogenous pigments which autofluoresce, making immediate measurements possible, without the need for pre-treatment of cells (Jamers et al., 2009). Flow cytometry analyses in metal studies using C. reinhardtii are relatively scarce. Therefore, this study will also provide a framework for future, related studies. In addition, the results of this study could provide a basis for the further investigation on the role of CIA7 in C. reinhardtii’s heavy metal tolerance or the further investigation on the potential use of Cia7 expression as a biomarker for heavy metal contamination.\n\n\nMethods\n\nC. reinhardtii strains cc4425 (wild-type) and cc5013 (mutant cia7-) were obtained from the Chlamydomonas Resource Center in the University of Minnesota. Cells were maintained as stocks in 1.5% Tris-acetate-phosphate (TAP) agarose solid media, stored at room temperature and subcultured every seven days. Liquid cultures were initiated in 50 mL of TAP medium by using two loopfuls (4 mm spherical scraping) of cells for inoculation. Culture flasks were placed in an orbital shaker (VWR DS2-500-1) at 130 rpm under normal fluorescent room lighting until cultures reached optical density (OD650) of 0.800 +/- 0.050, measured using a Spectronic Genesys 8 spectrophotometer. This OD650 of 0.800 has been calibrated to be equal to 1.0-3.0 × 106 cells/mL, which is log phase in C. reinhardtii.\n\nCells from both strains were cultured in liquid TAP to an OD650 = 0.400 and subjected to their respective metal treatments (CdCl2 at concentrations of 0 μM, 5 μM and 10 μM). Cd treatments in ¼X TAP amounted to a 50 mL final volume. Treatment flasks were prepared and cultured in replicates, one of them serving as the sampling culture flask, and the other as the supplemental culture flask. From the sampling culture flask, 900 μL of cells were taken for analysis, and the sampling flask was replenished with 900 μL of cells from the supplemental culture flask. The replenishment step was done in order to maintain the same volume of cells in the sampling culture flask throughout the 96-hour period. Samples for flow cytometry analysis were diluted 5:1 in saline buffer. Three replicates with three trials were performed per concentration and per strain. Chlorophyll intensity level and cell size were measured using a Cytek DxP8 FACSCalibur Flow Cytometer acquiring 10,000 events. Chlorophyll intensity level was determined by utilizing laser BluFL3 (Cytek) with a wavelength filter 695/40 nm. Cell size was determined as a function of the forward scatter signal (FSC). Cell cultures cc4425 and cc5013 were treated with 0, 5, and 10 μM CdCl2 at midlog phase (OD650 = 0.400). Samples were analyzed at 24-hour time points for a duration of 96 hours via flow cytometry.\n\nAn analysis of variance (ANOVA) associated with a 2 × 3 × 4 factorial experiment in randomized complete block design was performed for data analysis. The factorial arrangement of treatments was the result of two levels of strain (cc5013, cc4425), three levels of Cd concentration (0 μM, 5 μM, and 10 μM), and four levels of time (1, 2, 3, and 4 days). To compare significant main and interaction effects, post-hoc tests in the form of least squares means comparison were performed using the PROC GLM tool of the SAS 9.4 (Statistical Analysis System, RRID:SCR_008567) statistical software. The usual levels of type-I error rates were used (i.e., * if p < 0.05, if p < 0.01 and * if p < 0.001).\n\nC. reinhardtii strains were cultured in liquid media. Cells were kept under constant movement (130 rpm) in an orbital shaker until the cultures reached an OD650 of 0.800 +/- 0.050 as determined by a Spectronic Genesys 8 spectrophotometer. An absorbance of OD650 = 0.800 was calibrated to be equal to 1.0-3.0 × 106 cells/mL (log phase in C. reinhardtii) (Lin et al., 2013; Kirst et al., 2012). Results from preliminary experiments established the sub-lethal Cd concentration of cc4425 and cc5013 at 10 μM. In this regard, cells were harvested and cultured in media treated with CdCl2 at concentrations of 0 μM and 10 μM (0 ppb and 1830 ppb) to a final volume of 150 mL. Two different growth media were used: a minimal (Min) medium and a diluted, by a factor of ¼, TAP medium. Phosphorus has been reported as reducing the bioavailability of free metal ions due to its chelating character; therefore, the TAP medium was diluted (Nowicka et al., 2016). Similarly, the Min medium was used to determine whether a difference existed in C. reinhardtii’s metal bioaccumulation between TAP and the acetate-deprived Min media.\n\nMetal treatment flasks were washed with 1% HNO3 to prevent metal cross-contamination between replicates. The treatment period was 4 days, after which cells reached the logarithmic phase of growth. On the 4th day, cells were equilibrated at OD650 = 0.800 (an absorbance at log phase) and harvested by centrifugation at 3,000 rpm for 10 minutes. The pellets were washed twice with millipore H2O, then resuspended, followed by centrifugation at 3,000 rpm for 5 minutes to rinse pellets of extracellular metals. Washed pellets were desiccated at 100°C, and either frozen at −80°C or used for downstream analysis. Dry pellets were acidified with 5 mL of HNO3, incubated for 20 minutes, and digested in a MARS 6 microwave following the machine’s internal protocol for plant material. Following digestion, samples were diluted 1:10, and analyzed for Cd, by ICP-OES (Agilent Varian 720-ES).\n\nTo analyze the data, an analysis of variance (ANOVA) associated with a 2 × 2 × 2 factorial experiment in randomized complete block design was performed. The factorial arrangement of treatments was the result of two levels of strain (cc5013, cc4425), two levels of media (¼X TAP and Min), and two levels of Cd concentration (0 ppb and 1830 ppb). To compare significant main and interaction effects, a post-hoc test in the form of least squares means comparison was performed using the PROC GLM tool of the SAS 9.4 (Statistical Analysis System, RRID:SCR_008567) statistical software. To further explore the nature of a significant interaction between medium and concentration, a t-test comparing concentration levels was performed for each medium level. The usual levels of type-I error rates were used (i.e., * if p < 0.05, if p < 0.01 and * if p < 0.001).\n\n\nResults\n\nA statistically significant difference in chlorophyll fluorescence was observed between cc4425 and cc5013 (6,127 and 3,858 counts respectively) at 10 μM Cd (Table 1, Figure 1). However, at the control treatment and at 5 μM treatment, cc4425 and cc5013 chlorophyll signals at BluFL3 were not statistically significantly different from each other (Figure 1). It is interesting to note that as the Cd concentration increased, chlorophyll intensity increased for wild-type strain cc4425, while chlorophyll intensity decreased for cia7- mutant cc5013 (Figure 1). This negative trend in chlorophyll intensity level might be indicative of heavy metal toxicity, to which cc5013, whose Cia7 gene has been disrupted, might be more susceptible, including Cd susceptibility, compared to the wild-type cc4425.\n\nData represents pooled data from 0-4 days, whereby sampling was done every 24 h. Error bars represent 95% confidence intervals. Statistical analyses of measurements are displayed in Table 1.\n\nCell size was determined as a function of the forward scatter signal (FSC) (Figure 2). A similar empirical trend in the strains was observed in cell size as measured by the FSC. The effect of Cd on cell size was determined to be statistically significant between strains (p < 0.0001) at all concentrations (Table 2, Figure 2). The mutant, cc5013 was observed to have greater cell size than wild-type cc4425 especially under 0 μM Cd2+. Although not statistically significant (p > 0.05), cc4425 showed an increase in cell size with increase in cadmium concentration, while cc5013 showed a decrease in cell size (Figure 2).\n\nData represents pooled data from 0-4 days, whereby sampling was done every 24 h. Error bars represent 95% confidence intervals. Statistical analyses of measurements are displayed in Table 2.\n\nThis bioaccumulation experiment aimed to investigate the interactions between the in vivo activity of CIA7 in differential Cd2+ concentrations (0 and 1830 ppb [10 μM])), in either a mixotrophic (¼X TAP) or photoautotrophic (Min) growth medium. Although the results are not indicative of a statistically significant correlation, the observations are nevertheless interesting. Due to the lack of cc5013 characterization studies and CIA7’s as-of-yet unknown function, it is difficult to predict what metabolic and physiological changes Cd2+ exposure could cause, and in which range. The factorial arrangement of treatments was the result of two levels of strain (cc5013, cc4425), two levels of medium type (¼X TAP and Min), and two levels of Cd concentration (0 and 1830 ppb). No significant statistical difference (p < 0.5905) was established between Cd2+ bioaccumulation in cc4425 and cc5013, regardless of growth medium (Table 3). In ¼ X TAP, cc4425’s Cd2+ content was measured as 128.63 ppb, and in cc5013 as 155.83 ppb. In Min, cc4425’s Cd2+ content was measured as 28.63 ppb, and cc5013’s as 29.14 ppb.\n\nOn the other hand, a statistically significant difference was determined in the media by concentration effect (p < 0.0001) (Table 4). There was a significant difference in cells’ bioaccumulation of Cd2+ in 10 μM Cd (1830 ppb) treated ¼ X TAP (142.22 ppb) when compared to Cd2+ bioaccumulation in Min (28.88 ppb). On the other hand, as expected, there was no significant difference between ¼ X TAP and Min (14.53 ppb and 14.44 ppb respectively) at 0 ppb Cd (Table 4 and Figure 3). The main difference between the two media is the presence of acetate and phosphate in ¼ X TAP and their absence in Min. The pH of the two media were adjusted to pH 7.0. As previously mentioned, the TAP medium was diluted by a factor of ¼, as phosphorus has been reported as reducing the bioavailability of free metal ions due to its chelating character (Olanarin et al., 2013).\n\nThe “*” denotes a significant difference between two treatments.\n\n\nDiscussion\n\nA statistically significant difference in chlorophyll fluorescence was observed between cc4425 and cc5013 (6,127 and 3,858 counts respectively) at 10 μM Cd (Figure 2). A possible mechanism behind the phenomenon reported in cc5013 is a decrease in chlorophyll intensity level because of Cd substituting magnesium in chlorophyll, leading to the decrease in chlorophyll intensity (Grajek et al., 2020; Rydzyński et al., 2019; Kupper et al., 1996). The apparent absence of this phenomenon in cc4425 at the Cd concentrations used in this study might be, at least in part, contributed to by the biological activity of CIA7. This observed difference in chlorophyll fluorescence between the strains suggests that CIA7’s biological activity could play a direct or indirect role in increasing Cd tolerance in C. reinhardtii.\n\nAlthough not statistically significant, cc4425 showed an increase in cell size with an increase in Cd concentration, while cc5013 showed a decrease in cell size. The trend observed in cell size in cc4425 is partly supported by similar findings in Jamers et al. (2013), in which at 5 μM Cd2+ after 72 hours treatment, cells were bigger than the control group (p < 0.05). Furthermore, the trend reported in this study is likewise partly supported by Franklin et al.’s (2001) findings on the effect of Cu2+ in microalgae: Selenastrum capricornutum and Chlorella sp. In the same study, it was argued that increases in cell size in Cu2+ treatments were due to an increase in Na+ uptake, or the uncoupling of cell growth and cell division. However, whether Cd2+ causes the same effect is not yet known.\n\nCd bioaccumulation differences were analyzed in C. reinhardtii strains cc4425 (wild-type) and cc5013 (mutant). Intracellular metal accumulation has been reported as an indicator of metal tolerance (Collard and Matagne, 1994; Fernandez and Novillo 1995; Perez-Rama et al., 2002; Jamers et al., 2009). The mechanisms of metal tolerance in C. reinhardtii are dependent on metal concentration, availability of nutrients, as well as the general health of the cell (Igiri et al., 2018; Roach et al., 2013). Cd2+ bioaccumulation, for example, occurs once the concentration of the metal reaches critical levels sufficient to both saturate the binding capabilities of the cell wall, and hijack the transport proteins of essential metals with similar redox characteristics (i.e., Zn2+) (Brautigam et al., 2011).\n\nIn this study, cc4425 (wild-type) and cc5013 (mutant strain cia7-) were treated with Cd2+, 10 μM (1830 ppb) final concentration. Following a 96-hour exposure, the intracellular Cd2+ content was less than 10% of the total Cd2+ bioavailable. Due to similarities between this study and that by Fernandez and Novillo 1996, both including similar Cd2+ concentrations, prolonged exposure time and organism, it could be argued that the apparent lack of difference in terms of Cd2+ tolerance amongst cc4425 and cc5013 could be a result of the very well documented phytotoxic effects of Cd2+, reducing the uptake capabilities of both strains at 96 hours. A similar study on the metal accumulation in strains cc4425 and cc5013 was conducted by Vazquez et al. (2018) using Pb2+ in the form of Pb (NO3)2 at concentrations 0 μM and 150 μM, in which they reported no significant differences in the bioaccumulation of Pb2+ as measured analytically by ICP-OES. With both Cd2+ and Pb2+ being well-reported phytotoxic agents, it is possible that both of these metals exert irreversible photosynthetic damage after long-term exposures, and reduce the metal uptake capabilities. These could likely account for the non-significant difference in Cd2+ and Pb2+ bioaccumulation between the wild-type and mutant strains.\n\nHowever, it was initially not expected that there would be a significant difference in cells’ bioaccumulation of Cd2+ caused by medium type. The presence of acetate and phosphate in the diluted TAP medium likely still contributed to a considerably higher buffering capacity compared to Min. Additionally, the pH affects the bioavailability of metals (Glaesener et al., 2013; Olanarin et al., 2013); for example, C. reinhardtii strains showed a higher uptake of Cd at pH 7 and higher Cd tolerance at pH 4 than pH 7 due to the exclusion of Cd at the cell wall surface, which was higher at pH 4 than pH 7 (Samadani et al., 2018). Thus, the choice of medium in this kind of studies is critical and should be given a much careful consideration.\n\nAs a nutritional-rich growth media, the composition of TAP could potentially influence the cell response towards a toxicant (i.e., metals). The acetate present in TAP growth media could potentially shield C. reinhardtii from the phytotoxic effects of Cd, particularly photosynthesis impairment, as it could be integrated into the energy-yielding metabolic pathways of the cells. The findings of this study suggest that acetate supplementation triggers a variation in the response of cc4425 and cc5013 towards Cd stress. These findings are supported by Nagel et al., (1995) who suggested that the biochemical or metabolic adaptations leading to an increase in Cd2+ tolerance resulted in heavy photosynthetic impairments, obligating the cell to upregulate carbohydrate metabolism as a means of energy, resulting in acetate overconsumption.\n\nIn evaluating biological parameters in C. reinhardtii treated with heavy metals, the growth medium’s composition may display an effect in the cells’ responses to heavy metals. Acetate has been described as capable of alleviating photosynthetic stress, with the cell incorporating it into its glyoxylate or citric acid cycle, while under photosynthesis impairment (Roach et al., 2013). The previously reported recovery in growth and increase in reparation in the presence of acetate (Heifetz et al., 2000; Jiang et al., 2016) could have also contributed to the higher Cd bioaccumulation observed in ¼ X TAP.\n\nHeavy metal contamination poses an environmental hazard, and the elucidation of mechanism of tolerance will be relevant in developing techniques and strategies for attenuating this threat. This protein, although of an unknown function, is likely to be significant because it is highly conserved in many organisms. Here we conclude that the results from the comparative analysis in this study indicate that cc5013 (cia7- mutant strain) is more sensitive to Cd2+ than the wild-type strain cc4425, which possesses a functional Cia7 gene. Neither cc4425 nor cc5013 displayed a statistically significant difference in Cd2+ bioaccumulation at 10 μM (1830 ppb) CdCl2.\n\nHowever, a statistically significant interaction between Min media (absence of acetate and phosphate) and Cd2+ was observed, implying that a CIA7-mediated role in metal tolerance in cc4425 could require a supplemental catabolite because of a diminished photosynthetic efficiency, as reported in the literature. Furthermore, the presence of a catabolite could potentially shield the detrimental effects of the metals, as observed in this study. It was established that cc5013 was subjected to an increased photochemical quenching in the presence of Cd2+ as compared to cc4425. This could be attributed, at least partly, to Cd2+ substituting Mg2+ in the porphyrin group of chlorophylls. Cell size differences in the presence and absence of Cd were observed in both cc501 and cc4425. The cell size differences could be attributed, at least partly, to an uncoupling of cell growth and cell division due to heavy metal stress.\n\nFor future studies, the determination and comparison of intracellular Cd2+ bioaccumulation between cc4425 and cc5013 assessed over a shorter period of time (6-12 h) is recommended. This assessment will likely provide a more dynamic analysis of the extent of metal accumulation with fewer generational gaps.\n\n\nData availability\n\nDryad: cell size, chlorophyll fluorescence and cadmium bioaccumulation between wild-type and mutant strains of Chlamydomonas reinhardtii upon exposure to cadmium, https://doi.org/10.5061/dryad.rn8pk0pb4 (Ynalvez et al., 2021).\n\nThis project contains the following underlying data:\n\n- ICP_OES_data_ynalvez_etal.csv (raw ICP-OES data)\n\n- ODValues_ynalvez_etal.csv (raw OD values for each strain, Cd treatment and growth medium)\n\n- raw_flowcytodata_chlorophyll_cellsize_ynalvez_etal.csv (raw forward scatter, flow cytometry and chlorophyll measurements)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAbboud P, Wilkinson KJ: Role of metal mixtures (Ca, Cu and Pb) on Cd bioaccumulation and phytochelatins production by Chlamydomonas reinhardtii. 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PubMed Abstract \| Publisher Full Text\n\nMcKinnon KM: Flow Cytometry: An Overview. Curr. Protoc. Immunol. 2019; 120: 5.1.1–5.1.11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiazek K, Iwanek W, Remacle C, et al.: Effect of metals, metalloids and metallic nanoparticles on microalgae growth and industrial product biosynthesis: A review. Int. J. Mol. Sci. 2015; 16: 23929–23969. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNowicka B, Plucinski B, Kuczynska P, et al.: Physiological characterization of Chlamydomonas reinhardtii acclimated to chronic stress induced by Ag, Cd, Cr, Cu and Hg ions. Ecotoxicol. Environ. Saf. 2016; 130: 133–145. PubMed Abstract \| Publisher Full Text\n\nOlaniran AO, Balgobind A, Pillay B: Bioavailability of heavy metals in soil: impact on microbial biodegradation of organic compounds and possible improvement strategies. Int. J. Mol. Sci. 2013; 14(5): 10197–10228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPenen F, Isaure MP, Dobritzsch D, et al.: Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy. Metallomics. 2017; 9(7): 910–923. PubMed Abstract \| Publisher Full Text\n\nPenen F, Isaure MP, Dobritzsch D, et al.: Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga. Plant Cell Environ . 2019; 43(2): 479–495. PubMed Abstract \| Publisher Full Text\n\nPerales-Vela HV, Peña-Castro JM, Cañizares-Villanueva RO: Heavy metal detoxification in eukaryotic microalgae. Chemosphere. 2006; 64(1): 1–10. PubMed Abstract \| Publisher Full Text\n\nPerez-Rama M, Abalde Alonso J, Herrero Lopez C, et al.: Cadmium removal by living cells of the marine microalga Tetraselmis suecica. Bioresour. Technol. 2002; 84: 265–270. PubMed Abstract \| Publisher Full Text\n\nPinter TB, Stillman MJ: Kinetics of zinc and cadmium exchanges between metallothionein and carbonic anhydrase. Biochemistry-US. 2015; 54: 6284–6293. PubMed Abstract \| Publisher Full Text\n\nPollock SV, Mukherjee B, Bajsa-Hirschel J, et al.: A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii. Plant Methods. 2017; 13: 22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPriyadarshini E, Priyadarshini S, Pradhan N: Heavy metal resistance in algae and its application for metal nanoparticle synthesis. Appl. Microbiol. Biotechnol. 2019; 103: 3297–3316. PubMed Abstract \| Publisher Full Text\n\nRoach T, Sedoud A, Krieger-Liszkay A: Acetate in mixotrophic growth medium affects photosystem II in Chlamydomonas reinhardtii and protects against photoinhibition. Biochim. Biophys. Acta . 2013; 1827: 1183–1190. PubMed Abstract \| Publisher Full Text\n\nRydzyński D, Piotrowicz-Cieślak AI, Grajek H, et al.: Chlorophyll degradation by tetracycline and cadmium in spinach (Spinacia oleracea L.) leaves. Int. J. Environ. Sci. Technol. 2019; 16(10): 6301–6314. Publisher Full Text\n\nSamadani M, Dewez D: Cadmium accumulation and toxicity affect the extracytoplasmic polyphosphate level in Chlamydomonas reinhardtii. Ecotox. Environ. Saf. 2018; 166: 200–206. PubMed Abstract \| Publisher Full Text\n\nSamadani M, Perreault F, Oukarroum A, et al.: Effect of cadmium accumulation on green algae Chlamydomonas reinhardtii and acid-tolerant Chlamydomonas CPCC 121. Chemosphere. 2018; 191: 174–182. PubMed Abstract \| Publisher Full Text\n\nSevim C, Dogan E, Comakli S: Cardiovascular disease and toxic metals. Curr. Opinion Toxicol. 2020; 19: 88–92. Publisher Full Text\n\nShimogawara K, Fujiwara S, Grossman A, et al.: High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics . 1998; 148: 1821–1828. PubMed Abstract \| Free Full Text\n\nTchounwou PB, Yedjou CG, Patlolla AK, et al.: Heavy Metals Toxicity and the Environment. NIH EXS. 2012; 101: 133–164. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVazquez Cano E, Reyes V, Ynalvez RA: Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) analysis of lead bioaccumulation in Chlamydomonas reinhardtii and the role of Cia7 gene. The FASEB J. 2018; 32: 1_supplement, 665.5–665.5.\n\nViehweger K: How plants cope with heavy metals. Bot. Stud. 2014; 55: 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nXie M, Sun Y, Feng J, et al.: Predicting the toxic effects of Cu and Cd on Chlamydomonas reinhardtii with a DEBtox model. Aquatic Toxicol. 2019; 210: 106–116. PubMed Abstract \| Publisher Full Text\n\nYnalvez RA, Moroney JV: Identification and characterization of a novel inorganic carbon acquisition gene, CIA7, from an insertional mutant of Chlamydomonas reinhardtii. Funct. Plant Biol. 2008; 35: 373–381. PubMed Abstract \| Publisher Full Text\n\nYnalvez R, Gonzalez-Cantu H, Vazquez-Cano E: Cell size, chlorophyll fluorescence and cadmium bioaccumulation between wild-type and mutant strains of Chlamydomonas reinhardtii upon exposure to cadmium. Dryad, Dataset . 2021; Publisher Full Text\n\nYu Z, Wei H, Hao R, et al.: Physiological changes in Chlamydomonas reinhardtii after 1000 generations of selection of cadmium exposure at environmentally relevant concentrations. Environ. Sci. Process Impacts . 2018; 20(6): 923–933. PubMed Abstract \| Publisher Full Text\n\nZheng C, Aslam M, Liu X, et al.: Impact of Pb on Chlamydomonas reinhardtii at Physiological and Transcriptional Levels. Front. Microbiol. 2020; 11: 1443. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "115295", "date": "19 Jan 2022", "name": "Jiangxin Wang", "expertise": [ "Reviewer Expertise Algae OMICS toxicology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHeavy metals vs environments and algae is always an important field, and this ms compared cell size, chlorophyll fluorescence and cadmium (Cd2+) bioaccumulation between wild-type and mutant strains of Chlamydomonas reinhardtii upon exposure to Cd ions. Experiments were well designed and statistical analyses were sufficient. However, the ms is rather descriptive, focusing on physiological/cellular levels. The more interesting points such as why and how this mutant is tolerant against Cd, or whether this mutant is also tolerant to other heavy metals. Also, the reviewer is interested to know why this mt has a small cell size even grown in the control medium.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "121675", "date": "21 Feb 2022", "name": "Stefan Schmollinger", "expertise": [ "Reviewer Expertise I work on trace metal metabolism in Chlamydomonas reinhardtii", "regularly conducting elemental analysis of the alga on an Agilent 8900 ICP MS/MS 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 article “Comparison of cell size, chlorophyll fluorescence and cadmium (Cd2+) bioaccumulation between wild-type and mutant strains of Chlamydomonas reinhardtii upon exposure to Cd2” from Ynalvez et al. analyses the effects of Cd on a Chlamydomonas wild-type strain and the cia7 mutant. The authors find a reduction of Chl content, differences in cell size between Wt and mutant, but not to Cd treatment, and Cd accumulation in mixotrophically grown cells\nIn general, I would recommend indexing, but only after some revisions.\nComments:\nReplication. The amount and kinds of replication should be clearly explained for each experiment, at least in the figure legend and in the methods. From the raw data that was deposited, I am assuming that there were either two separate experiments (replicate 1 and 2), where three individual, potentially technical, replicates for each condition were performed. Not completely clear from the methods for Figures 1 and 2, not at all for Figure 3.\nThe decision to combine the different days of Cd incubation I don’t understand. Was there no information in the time series? For the ICP data, it looks like the Cd is accumulating between rep 1, 2, and 3. Were they collected on successive days and Cd slowly accumulated (both Wt and mutant show this tendency)?\nICP-OES data. The authors performed the analysis for both WT and cia7, but chose to only include one of the datasets in the figure, lacking error bars. This might be a consequence from point (2). Additionally, in the text, it is not clear which strain the authors refer to when discussing the results, or present in the figure. From the RawData I think there is no difference between the mutant and WT in terms of Cd accumulation, I do think the interpretation of the authors are correct that the only difference is between TAP and Min.\nFigure quality: The figures could be easily improved by just modifying some of the standard, very basic Excel layout.\nFigures 1 and 2 should not be line graphs, but rather bar plots. You shall not connect what is indeed separate. From the description, I take it that the cultures in 0, 5, and 10 uM Cd are separate flasks, and were done in parallel.\n\nY-axis tick labels are unnecessarily long in Figures 1 and 2, the description could be changed to arbitrary units (which is what they are), and a lot of zeros could be removed. The y-axis itself then could be labeled Chlorophyll fluorescence (a.u.) and Cell size (a.u.) in Fig 1 and 2, respectively. In Fig 3 just Cd2+ (ppb) is fine, the word “measured” is unnecessary.\n\nThe x-axis label in Fig1 and 2 could be CdCl2 (µM) instead of concentration (uM), which would give the reader the luxury to directly identify what concentration the authors are referring to. In Fig 3 the axis should also just be the Cd concentration, CdCl2 (µM), the different media, distinguished by shades of grey, can be added as a legend, either somewhere in the graph or below.\n\nSame for the labels of the strains, instead of just 1 and 2, it could be wild-type (CC-4425) and cia7 (CC-5013) in Fig 1 and 2.\n\nThe white space in the graphs can be significantly reduced by choosing slightly wider bars, which can be done in Excel by reducing the gap width.\nReferences for the minimal media, maybe also a citation for TAP (Harris 1989 for example), should be added to the 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-806
https://f1000research.com/articles/11-363/v1	29 Mar 22	{ "type": "Research Article", "title": "The stock market reaction to COVID-19 vaccination in ASEAN", "authors": [ "Marizsa Herlina", "Ade Yunita Mafruhat", "Eti Kurniati", "Wildan Wildan", "Hilwa Gifty Salsabila", "Ade Yunita Mafruhat", "Eti Kurniati", "Wildan Wildan", "Hilwa Gifty Salsabila" ], "abstract": "Previous studies have shown that the confirmed cases drive investor sentiment, reflecting the stock's return. Based on this, the vaccination growth is also expected to drive the investor’s sentiment, which can be reflected in the return of the stock market in ASEAN. Therefore, this study explores the vaccination impact on stock returns in ASEAN countries. This study contributes to the gap of taking the COVID-19 vaccination impact to the stock return into account by using the panel regression model with HC and Driscoll and Kraay robust covariance matrix estimator, which addresses the cross-dependency and heterogeneity problems. This study is one of the early studies of the topic, especially in ASEAN. The panel regression model with HC and Driscoll and Kraay robust covariance matrix estimator uses three variables: the daily stocks return, vaccine growth, and cases growth. It is a balanced panel data that includes six countries and 117 daily series data, making 702 observations used in the study. The results show conflicting results where daily vaccination growth negatively affects the stock return. This problem can arise for several reasons, such as the uncertainty in the financial market and cross-dependency and heterogeneity detected in the model. We can see that the investors still have a negative sentiment because COVID-19 has resulted in uncertainty on the financial market in ASEAN. This gives us practical implications that the ASEAN country members’ government needs to push vaccination policy more aggressively.", "keywords": [ "Stock reaction", "vaccination", "ASEAN", "panel regression", "heterogeneity", "cross-dependence" ], "content": "Introduction\n\nNo one could predict that the COVID-19 pandemic would be a long-lasting problem facing most of the world. All industries are suffering from the ongoing COVID-19 pandemic, until recently. This was shown from the estimation of the world’s gross output (GO) in 2020, which was −3.5; specifically, all countries also had a negative GO. For Association of Southeast Asian Nations (ASEAN), the economic growth prospect is quite dark due to their strong dependencies on the tourism sector. This sector provided 12% of ASEAN GDP in 2016,1 and it was predicted to rise had there been no pandemic. The pandemic would make it hard to recover the tourism sector because of the travel limitation and quarantine policies applied differently for each country’s borders.2 In the third quartal of 2020, most ASEAN countries suffered from the decrease of GDP except for Vietnam, which increased 2.91% of their GDP.3 While pandemics are still ongoing, ASEAN will face a significant hurdle in its tourism sector, and policymakers should make a new strategy to increase economic growth.\n\nOne of the efforts to overcome the rising cases of COVID-19 is the vaccination program. Since the first quarter of 2021, almost all countries have started a COVID-19 vaccination program; however, the impact of the COVID-19 vaccination on economic growth is still rarely explored because the program is still in the early stages and there are not enough data to analyze. Despite that, the vaccination program is indeed an excellent start to cope with the spread of COVID-19. The program’s outcome is to slow down the spread and reduce positive cases of the COVID-19. The increasing number of vaccinated people can stabilize a saturated condition. With the growing number of people vaccinated, people will feel more secure and think positively about the future, which will help raise a reasonable expectation in the stock market.\n\nMoreover, research in Vietnam4 about the COVID-19 impact on the stock market shows that when Vietnam announced 0 cases of COVID-19 after the lockdown, the stock market in Vietnam raised significantly and became the best performing stock in April and May 2020. Based on this, the impact of the vaccination program is expected to have a similar outcome which is the increase of the stock market performance in other countries. However, it is not always the case that the positive sentiment will always increase the market’s return; otherwise, it can bring down the return because of the abnormal trading volume in the market.5 Especially in a period of uncertainty, the investor’s behavior is hard to predict, reflecting on the stock returns.\n\nEvery country has different conditions and policies; this implies a variety of vaccination impacts to their stock market for each country. Therefore, panel data analysis can be used to see the stock reaction to the vaccination program in ASEAN. Panel data can control individual heterogeneity and identify the impact better than pure cross-section or time-series data only.6,7 Panel data analysis is often used in analyzing the stock market responses study from the COVID-19 outbreak.8–11 They used the stock return to see the market reaction and positive confirmed cases in the respective countries.\n\nSeveral researchers had studied the financial market performance response to the COVID-19 vaccination.12–14 These studies show that vaccination gives a significant effect on the global market. However, the variety of vaccines and each country’s vaccination policies would result in different stock market reactions. Besides, no study considers the vaccination impact in ASEAN countries, to the best of our knowledge.\n\nBased on the previous studies, this study has three significant contributions. First, this study could picture the impact of vaccination on stock returns, and it also shows the benefit or the loss for the future investor. Second, the object of this study is the ASEAN country members. The study’s outcome is expected to help the ASEAN policymakers create a policy that considers the vaccination effect on ASEAN stock returns. Third, this study detected several problems, such as cross-dependency and heterogeneity, potentially leading to biased testing results. Thus, this study used the HC and Driscoll and Kraay robust covariance matrix estimator to address those problems and improve the model results for vaccination on stocks’ return. This paper then explains the whole research process, and it is divided into several sections: introduction, background and literature review, data and methodology, results, discussion, and conclusion.\n\n\nBackground and literature review\n\nThe COVID-19 pandemic affects the stock market.4 In the USA, COVID-19 impacted the US stock market volatility more than any other pandemic since the 1900s.15 The stock market itself is strongly interconnected. The pandemic can negatively change investors' sentiment, which will affect the investment decision where it will be reflected in the stock prices.16 In addition, research involving 64 countries17 showed a negative impact of the COVID-19 pandemic on the stock return. In other words, stock prices reflect the investor’s expectations. When the downfall of the stock market can be seen as a pause in economic activities, it also means that there is price pressure from people’s expectations and fear of the investor’s.10 The research conducted in India about the stock market reaction before and after lockdown showed that the stock return reacted positively to the policy after lockdown announcement.18 The vaccination program and policies may also impact the stock market based on that knowledge.\n\nThe COVID-19 vaccination started on February 18th, 2021, where the high-risk population is the vaccination’s priority target.19 More than 905 million vaccine doses have already been registered worldwide, which means there are 12 doses available for every 100 people. But, the gap between the available dose and the world population still exists.20\n\nThe vaccination program in ASEAN has begun differently in its member countries. The earliest country to start the vaccination program was Indonesia on January 26th, 2021. From Figure 1A, we can see that the three highest vaccinated countries in ASEAN are Singapore, Cambodia, and Brunei, while the lowest three are Indonesia, Philipines, and Myanmar. We also need to consider the size of the population in this case; in terms of the number of vaccinated people, Indonesia, Vietnam, and Thailand are the three highest countries in ASEAN (Figure 1B). This shows that ASEAN countries have been conducting the vaccination program, and this study explored the stock market reaction to the vaccination program in ASEAN countries using panel data analysis.\n\nThe panel data analysis has been used often in analyzing the stock market reaction. The research by8 used the panel regression to see the effect of uncertainty and confirmed cases on the stock market returns in 43 countries; he found that the higher confirmed cases impact greater with the country which also has a higher level of uncertainty. Similar studies were also done by,9 who studied 47 countries on the panel regression to explore the effect of people’s trust in the government and society and also the confirmed cases to the stock market volatility. They found that trust in government and society is significantly crucial to market volatility.\n\nThe stock market reaction study in G-20 countries by10 also used panel data regression and event study to see the impact of the COVID-19 outbreak on the abnormal returns of the stock and found that the COVID-19 outbreak has a negative effect to the stock returns. Moreover, the research of,11 who studied the impact of freedom and growth rate of COVID-19 daily returns, also used a panel regression. He found that the growth of COVID-19 significantly affected the returns negatively, and there is a strong negative relationship between the country’s freedom and the effect of the pandemic in the stock market.\n\nThe link between the vaccine program and the stock market is also mentioned in several studies. The study about the response of the global stock market to vaccine availability had been explored by using five main markets indexes such as Dow Jones, Shanghai, S&P, FTSE, and EURONEXT). The results showed that after the vaccine arrival, the stock prices significantly outweighed before the vaccine arrived.12 Another study using the panel data model of the volatility of the stock market reaction to the vaccination program in the international financial market shows that the stock market volatility significantly dropped by the mass vaccinations.13 The vaccine effectivity on the stock market was analyzed using a wavelet coherence approach in the USA. The COVID-19 vaccination, infection rate, and the case fatality ratio significantly influence the S&P-500 returns at the majority business cycle.14 However, none of them consider the existence of cross-dependency and heterogeneity problems. Therefore, this study incorporated the cases and vaccine of COVID-19 growth to the ASEAN stock returns. We also used the HC estimators to address the issue of heterogeneity and Driscoll and Kraay robust covariance matrix estimator to manage the cross-dependency in the panel data model.\n\n\nMethods\n\nThis study used secondary datasets obtained from investing.com and21 that are available online. Thus, there are no ethical issues in this study.\n\nThe variable used in this study are the daily stock returns,10 which use equation 1:\n\nWhere Rit is the daily return for index i and Pi,t is the closing stock price of the i index stock at the time t and Pi,t−1 is the closing stock price of the i index stock at the time t−1 (a day before). The return was calculated from the available stock indexes in ASEAN (Table 1) available (among the ASEAN members, only six countries had a country stock index) from investing.com. The other variables are the growth of the confirmed positive COVID-19 cases and the growth of the vaccinated people, which follows equations 2 and 3.9 All data are secondary data, originated in the form of total vaccination and confirmed cases of COVID-19 that are gathered from21 and are accessed on July 8th, 2021. All of the analyses on this paper were done using the R software.\n\nThe general model,22 which are estimated in the panel regression and the variables used follow equation 4:\n\nWhere xit=lnCases Growthi,tlnVaccine Growthi,t′; ci=z′iα is the individual effect which contains group-specific variables. The models consist of22,23:\n\n1. Pooled Model which follows the equation 5.\n\nwhere vit≡ci+uit as the composite errors; ci is individual effect; uit is the idiosyncratic errors and t=1, …, T.\n\n2. Fixed Effect Model which follows the equation 6.\n\nwhere ci=z′iα and εit is an error term.\n\n3. Random Effect Model which follows the equation 7.\n\nwhere vit=cijT+ui; jT is the Tx1 vector of ones and ui is a group-spesific random element (for each country). Then, after these models are estimated, several testings were carried out in order to see which model perform best such as i) The Hausman test24 is used for determining the most suitable model between fixed effect and random effect model which the null hypothesis is Random Effect Model is more suitable for the data; ii) The Breusch-Pagan Lagrange Multiplier Test25 in order to see the cross-sectional dependency. In the event of cross-dependency exist, the Driscoll and Kraay robust covariance matrix estimator will be used26; iii) The Breusch-Pagan Test27 in order to see the homoskedasticity assumption. If there is any heteroskedasticity detected, the heteroskedasticity consistent (HC) estimation can be used for the model estimation.28,29\n\n\nResults\n\nThis study used balanced panel data which includes six countries and 117 daily series so the total is 702 observations. From Figure 2, we can see that the growth of confirmed cases and the vaccinated people fluctuate over time. For confirmed cases growth, it fluctuates mostly in Vietnam, Thailand, Malaysia, and the Philippines. Meanwhile, the vaccination growth decreased over time and sometimes it stays flat because the growth did not change that much. If we see the series on Figure 3, the movement of each country evolved around the 0 line, which confirms that the mean of the daily stock return is near 0.\n\nThe model estimation for Pooled Model, Fixed Effect Model, and Random Effect Models are provided in Table 2. From the estimates, vaccine growth is the only one that significantly affects the return of the stock in ASEAN countries. Moreover, both pooled and random effect models produce similar results in the coefficient. All of the estimations for vaccine growth have negative signs, which means that the vaccine growth negatively influences the return of the stock, which is in contrast to the expectation.\n\n* 10%;\n\n 5%;\n\n* 1% significance level.\n\nAfter the model estimation, several tests are conducted to see the most suitable model among all using the Hausman test and Breusch-Pagan Lagrange Multiplier test (Table 3). The Hausman test shows that the fixed effect model is more ideal than the fixed random effect model, which means there is heterogeneity in vaccine growth and case growth on a daily basis. But then the Breusch-Pagan Lagrange Multiplier test shows that there is a cross-sectional dependence problem. This means that there is a dependence on the stock returns among ASEAN countries.\n\n* 10%;\n\n 5%;\n\n* 1% significance level.\n\nIn addition, the Breusch-Pagan test also shows that the variance is not homoskedastic so we estimate the HC estimators for the fixed-effect model which are robust to heteroskedasticity and Driscoll and Kraay robust covariance matrix estimator for cross-dependency problem in Table 4. The HC estimators and the Driscoll and Kraay robust covariance matrix estimators of the fixed effect model shows different results to the previous estimations in Table 2 in terms of the standard error and the p-value. For the HC estimators, all variables significantly affect the daily return of the stock in the ASEAN country. The cases growth indeed negatively influences the return of the stock significantly but the vaccine growth also has a negative coefficient. But, if we use the Driscoll and Kraay robust covariance matrix estimators, all of the variables do not significantly influence the return. The HC and Driscoll and Kraay Robust Covariance Matrix Estimators have different results in testing. Even though we have already addressed each problem, they still have biased results.\n\n* 10%;\n\n 5%;\n\n* 1% significance level.\n\n\nDiscussion\n\nBased on the results above, it is shown that the cases growth negatively impacted the stock returns in ASEAN. This is in line with all of the previous studies.8,9,11 But we found conflicting results in the vaccination growth. The vaccination growth is supposed to impact the stock return positively, but it harms the stock return. This is in line with the theory that the positive sentiment does not necessarily raise the stock returns.5,13 This proves that many things happen in trading. They do not move solely on investors’ sentiment, but many factors interfere with the market, such as government interventions, news, abnormal trading, etc. The goal of the vaccination is herd community which makes a certain proportion of the population have the immunity of a disease. Until now, except for the USA, the world is still far behind the herd immunity threshold.30 If the goal of herd immunity had not been achieved, then the stock market is still in a state of extreme uncertainty; that is why the investor behavior would be hard to predict.5 Future investors must be aware of the risk if they want to invest in this situation. The practical implication of this study is that ASEAN countries need to create strategies that will outweigh the risk of this uncertainty to attract investors such as strengthening the healthcare system to ease the uncertainty. The more advanced countries’ financial markets are proven to be more robust to the pandemic effect because of their advanced technology, communication, and good citizen’s welfare.31 The ASEAN countries should strengthen their citizens’ trust in them to stabilize the situation like The Phillippines did.\n\nSecond, the limitation in this study is the existence of either a cross-dependency problem or heterogeneity left in this model. The HC estimators only addressed the heterogeneity problem but not the cross-dependency problem. Reversely, the Driscoll and Kraay Robust Covariance Matrix Estimators is robust to the cross-dependency problem but not heterogeneity. Both cannot solve those problems simultaneously. The cross-dependency itself can be caused by spatial or spillover effect or unobserved common factors.32 This means that every country has a dependency on each other. The spatial effect of the vaccination on the stock returns need to be explored more.\n\n\nConclusions\n\nThis study explores the vaccination impact on stock returns in ASEAN by using panel regression. It is found that both vaccination growth and the growth cases are impacting the return of stocks in ASEAN. While the growth cases are in line with previous studies, there are conflicting results where the vaccination growth negatively affects the return stock in ASEAN. These results are contrasted with the expectation that vaccination should bring positive sentiment to the investors. The study confirms the research objectives that we found several mixed results in vaccination impact and address the problem of cross-dependency and heterogeneity. These mixed results could be because of the investor’s sentiment, which is in extreme uncertainty because of the non-presence of herd immunity until now. We can see that the investors still have a negative sentiment because COVID-19 has resulted in uncertainty on the financial market in ASEAN. This gives us practical implications that the ASEAN country members’ government needs to push vaccination policy more aggressively. Even though the results showed that the vaccination still negatively influences the stock returns, the vaccination growth has not shown the distribution of the vaccinated people. For example, in Indonesia, only 56,04% of the Indonesian population gets the second vaccination.\n\nMeanwhile, the new variant of COVID-19 keeps evolving and making a new peak on confirmed cases in many countries, including ASEAN. Thus, the economic activities would also be halted if these are not carefully taken care of. So, the ASEAN countries must fasten their second vaccine distribution, and after that, they need to ensure their citizen get the third vaccine. This is a significant move to stabilize the investors’ trust that governments can warranty the citizen’s welfare. This would make the economy slowly recovers, and the investors’ positive sentiment will follow eventually, and next, the financial market can be stabilized in the end.\n\nSecond, there is a cross-dependency and heterogeneity problem in the model which can cause biased test results. Therefore, future studies suggest using another estimator or model to address heterogeneity and the cross-dependency problem simultaneously. For example, consider the spatial panel modeling or another model such as the heterogeneous panel data models with cross-sectional dependence33 that can address this problem to avoid biased test results.\n\n\nData availability\n\nGithub: Data of The Stock Market Reaction to COVID-19 Vaccination in ASEAN. Dataset created for\n\nhttps://github.com/owid/covid-19-data/tree/master/public/data/vaccinations21\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nSundoro HS, Soeprapto VS: The Effect of Tourism Sectors on ASEAN Countries’ Economic Growth: Analysis Panel Regression. Proceedings of the 7th International Conference on Entrepreneurship and Business Management (ICEBM Untar 2018). 2018; p. 180–184.\n\nIMF: World Economic Outlook Update. IMF; 2021.\n\nTaufiqurohman M: Ekonomi Filipina 2020: Terburuk di Asia Tenggara. lokadata.id.2021. Reference Source\n\nHe P, Sun Y, Zhang Y, et al.: COVID–19’s Impact on Stock Prices Across Different Sectors—An Event Study Based on the Chinese Stock Market. Emerg. Mark. Financ. Trade. 2020; 56(10): 2198–2212. Publisher Full Text\n\nSreelakshmi R, Sinha A, Mandal SK: COVID-19 related uncertainty, investor sentiment and stock returns in India. MPRA Pap. 2021; 109549.\n\nHsiao C: Analysis of Panel Data. Analysis of Panel Data.2003.\n\nBaltagi BH: Econometric Analysis of Panel Data. 3rd ed.Chichester: John Wiley & Sons Ltd; 2005.\n\nAshraf BN: Stock markets’ reaction to Covid-19: Moderating role of national culture. Financ. Res. Lett. 2021; 41(November): 101857. Publisher Full Text\n\nEngelhardt N, Krause M, Neukirchen D, et al.: Trust and stock market volatility during the COVID-19 crisis. Financ. Res. Lett. 2021; 38: 101873. Publisher Full Text\n\nSingh B, Dhall R, Narang S, et al.: The Outbreak of COVID-19 and Stock Market Responses: An Event Study and Panel Data Analysis for G-20 Countries. Glob. Bus. Rev. 2020.\n\nErdem O: Freedom and stock market performance during Covid-19 outbreak. Financ. Res. Lett. J. 2020; 36(January): 101671. Publisher Full Text\n\nNgwakwe CC: COVID-Vaccination and Performance in Five Global Stock Market Indexes. ŒCONOMICA. 2021; 17(5): 55–65.\n\nRouatbi W, Demir E, Kizys R, et al.: Immunizing markets against the pandemic: COVID-19 vaccinations and stock volatility around the world. Int. Rev. Financ. Anal. 2021; 77: 101819. Publisher Full Text\n\nKhalfaoui R, Nammouri H, Labidi O, et al.: Is the COVID-19 vaccine effective on the US financial market?. Public Health. 2021; 198: 177–179. PubMed Abstract \| Publisher Full Text\n\nBaker SR, Bloom N, Davis SJ, et al.: The unprecedented stock market reaction to COVID-19. Rev. Asset Pricing Stud. 2020; 10(4): 742–758. Publisher Full Text\n\nLiu H, Manzoor A, Wang C, et al.: The COVID-19 outbreak and affected countries stock markets response. Int. J. Environ. Res. Public Health. 2020; 17(8): 1–19.\n\nAshraf BN: Stock markets’ reaction to COVID-19: Cases or fatalities ?. Res. Int. Bus. Financ. 2020; 54(January): 101249. PubMed Abstract \| Publisher Full Text\n\nAlam MN, Alam MS, Chavali K: Stock market response during COVID-19 lockdown period in India: An event study. J. Asian Financ. Econ. Bus. 2020; 7(7): 131–137. Publisher Full Text\n\nWHO: COVID-19 vaccines. who.int.2021 [cited 2021 Apr 19]. Reference Source\n\nHolder J: Tracking Coronavirus Vaccinations Around the World. The. New York Times. 2021. Reference Source\n\nMathieu E, Ritchie H, Ortiz-Ospina E, et al.: A global database of COVID-19 vaccinations. Nat. Hum. Behav. 2021; 5(7): 947–953. Publisher Full Text\n\nGreene WH: Econometric Analysis. 8th ed.Essex: Pearson Education Limited; 2018. Reference Source\n\nWooldridge JM: Econometric Analysis of Cross Section and Panel Data. Cambridge: The MIT Press; 2002.\n\nHausman JA, Taylor WE: Panel Data and Unobservable Individual Effects. Econometrica. 1981; 49(6): 1377–1398. Publisher Full Text\n\nBreusch TS, Pagan AR: The Lagrange Multiplier Test and its Applications to Model Specification in Econometrics. Rev. Econ. Stud. 1980; 47(1): 239. Publisher Full Text\n\nDriscoll JC, Kraay AC: Consistent Covariance Matrix Estimation with Spatially Dependent Panel Data. Rev. Econ. Stat. 1998 Nov 1; 80(4): 549–560. Publisher Full Text\n\nBreusch TS, Pagan AR: A Simple Test for Heteroscedasticity and Random Coefficient Variation. Econometrica. 1979; 47(5): 1287–1294. Publisher Full Text\n\nWhite H: A Heteroskedasticity-Consistent Covariance Matrix and a Direct Test for Heteroskedasticity. Econometrica. 1980; 48: 817–838. Publisher Full Text\n\nMacKinnon J, White H: Some Heteroskedasticity-Consistent Covariance Matrix Estimators with Improved Finite Sample Properties. J. Econom. 1985; 29: 305–325. Publisher Full Text\n\nCihan P: Forecasting fully vaccinated people against COVID-19 and examining future vaccination rate for herd immunity in the US, Asia, Europe, Africa, South America, and the World. Appl. Soft. Comput. J. 2021; 111: 107708. Publisher Full Text\n\nHuynh N, Dao A, Nguyen D: Openness, economic uncertainty, government responses, and international financial market performance during the coronavirus pandemic. J. Behav. Exp. Financ. 2021; 31(May): 100536. Publisher Full Text\n\nBaltagi BH, Pesaran MH: Heterogeneity and cross section dependence in panel data models: Theory and applications. J. Appl. Econom. 2007; 22: 229–232. Publisher Full Text\n\nGao J, Xia K: Heterogeneous panel data models with cross-sectional dependence. J. Econom. 2020; 219(2): 329–353. Publisher Full Text" }	[ { "id": "134887", "date": "04 May 2022", "name": "Robiyanto Robiyanto", "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\nGood manuscript, however some improvements should be made prior to indexing. This manuscript can contribute to the reference regarding the dynamics of financial markets in the pandemic era. Minor changes required.\nMajor points:\nPlease explain how vaccination could affect stock return? As you know, some stock markets plummeted in the early pandemic event, but then these stock markets then recovered because the pandemic was priced in already. This is not because of merely vaccination, some robustness checking is needed. The awakening of retail investors (i.e. in Indonesia) etc. should be considered.\nMinor points:\nPlease sharpen the research gaps, which need your contributions, especially the third contribution. Please describe some flaws in previous studies which need this solution and how your method could eliminate these flaws.\n\nAdd some recent literature. Some related literature has been published.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9553", "date": "25 Apr 2023", "name": "Marizsa Herlina", "role": "Author Response", "response": "Thank you for your suggestions. We have accommodated all of your requests in the revised papers as follows: 1. Major points: Reviewer: Please explain how vaccination could affect stock return? As you know, some stock markets plummeted in the early pandemic event, but then these stock markets then recovered because the pandemic was priced in already. This is not because of merely vaccination, some robustness checking is needed. The awakening of retail investors (i.e. in Indonesia) etc. should be considered. Answer: In recent studies, the link between vaccination and stock mostly happens in the news about vaccine announcements, resulting in positive sentiment by investors. We already added this in the revised paper in the 1st paragraph in the background and literature review as follows: “In addition, a recent study shows that news about covid help equities in general, whether its positive or negative news(3). Moreover, the Covid-19 vaccine announcement also positively impacted the Chinese stock market. Besides, many studies prove that mass campaign about Covid-19 vaccines receives positive sentiments from investors (3–6). Therefore, the approvals of Covid-19 vaccines lighted the hope of humanity and economic recovery, and this phenomenon is reflected in the stock market (5),” 2. Minor points: Reviewer: Please sharpen the research gaps, which need your contributions, especially the third contribution. Please describe some flaws in previous studies which need this solution and how your method could eliminate these flaws. Answer: This has been addressed in the fifth paragraph of the Introduction in the revised paper as follows: \"However, the Ordinary Least Square (OLS) estimation of the panel regression model requires the assumption of normally distributed and homogenous errors, which are rarely met in many cases in real-life data, especially economic data such as stock prices. If the assumption of homogeneity is violated, the estimator will be biased. Many previous studies used OLS estimation but did not mention diagnostic testing. Hence, we address the issue of where heterogeneity and cross-dependency occur in the panel model error. Therefore, the Heteroskedasticity consistent (HC) estimator can be an alternative estimator to have robustness in heterogeneity. However, heterogeneity can also exist from cross-dependency, and Driscoll and Kraay's robust covariance matrix estimator addresses those problems and improve the model results for vaccination on stocks’ return.\" And in the Background and literature review section, in the last paragraph: “However, none of them consider the existence of cross-dependency and heterogeneity problems. Therefore, this study incorporated the cases and vaccine of COVID-19 growth to the ASEAN stock returns.” Reviewer: Add some recent literature. Some related literature has been published. Answer: We have added recent literatures as you can see in the first point and also from the other reviewer as follows: 1. Yiu MS, Tsang A. Impact of COVID-19 on ASEAN5 stock markets. J Asia Pac Econ. 2021 Aug 21;1–14. 3. Arteaga-Garavito MJ, Croce MM, Farroni P, Wolfskeil I. When the Markets Get CO.V.I.D: Contagion, Viruses, and Information Diffusion. SSRN. 2022; 4. Hartono, Hartono. Covid-19 Vaccine: Global Stock Market “Game Changer.” J Asian Multicult Res Econ Manag Study [Internet]. 2021;2(2). Available from: https://amrsjournals.com/index.php/jamrems/article/view/102 5. Ho KC, Gao Y, Gu Q, Yang D. Covid-19 vaccine approvals and stock market returns: The case of Chinese stocks. Econ Lett. 2022 Jun;215:110466. 6. Oanh TTK. The impact of COVID-19 vaccination on stock market: is there any difference between developed and developing countries? Heliyon [Internet]. 2022 [cited 2023 Apr 2];8(9). Available from: https://linkinghub.elsevier.com/retrieve/pii/S2405844022020060" } ] }, { "id": "139087", "date": "30 May 2022", "name": "Evan Lau", "expertise": [ "Reviewer Expertise Economics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. It is a rather interesting proposition of linking the vaccination and stock market. The authors should be able to provide the theoretical link on how these two were related. At the same time, authors could explore using not just specifically COVID, but also pandemics themselves to find the link.\n2. Why the selected ASEAN countries? How do these 6 countries represent ASEAN?\n3. As the research on the topical issue is on-going, it might be a good idea to expand the sample period at least to 2022. With an expanded sample, I am sure there are more interesting findings. The authors could explore the sub-sample estimation based on the big sampling data.\n4. Driscoll and Kraay Robust Covariance Matrix Estimators - I do not see the relevance of adopting this method. Perhaps the authors could consider other methods or other panel estimates. A non-stationary panel time series perhaps?\n5. Low R2 and negative adjusted R2.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9554", "date": "25 Apr 2023", "name": "Marizsa Herlina", "role": "Author Response", "response": "Thank you for your suggestions. We have accommodated all of your requests in the revised papers as follows: 1. Reviewer: It is a rather interesting proposition of linking the vaccination and stock market. The authors should be able to provide the theoretical link to how these two were related. At the same time, authors could explore using not just specifically COVID, but also pandemics themselves to find the link. Answer: when the previous pandemic hits such as swine flu or influenza, it has been found that it has link to the stock market when it hits massively, and this has been addressed in the 1st paragraph of the Background and literature review of the revised paper as follows: “If we learned from the previous pandemic, such as swine flu, the link from influenza to the stock market could be seen when it began to affect the key individuals massively in the stock market, such as traders, market makers, and overall investing behavior directly or indirectly via decreasing in liquidity due to the decline in information flows and production” 2. Reviewer:Why the selected ASEAN countries? How do these 6 countries represent ASEAN? Answer: The 6 countries, Indonesia, Thailand, Vietnam, Philippines, Singapore, Malaysia are the highest GDP countries in ASEAN, so they significantly drive the ASEAN economies. We have accommodated this in the 6th paragraph of the revised paper. “Second, the object of this study is the six biggest ASEAN country members: Indonesia, Thailand, Philipines, Vietnam, Singapore, and Malaysia. The 6 countries are the highest GDP countries in ASEAN, so We consider these countries to significantly influence the ASEAN stock market (1).” 3. Reviewer: As the research on the topical issue is on-going, it might be a good idea to expand the sample period to at least 2022. With an expanded sample, I am sure there are more interesting findings. The authors could explore the sub-sample estimation based on the big sampling data. Answer: Thank you for the suggestions. This paper limits the time window to 2021 because the sentiment mostly happens when the vaccine is launched. But we will consider your suggestions for future research. 4. Reviewer: Driscoll and Kraay Robust Covariance Matrix Estimators - I do not see the relevance of adopting this method. Perhaps the authors could consider other methods or other panel estimates. A non-stationary panel time series perhaps? Answer: The Driscoll and Kraay Robust Covariance Matrix Estimators can estimate the model when there is heterogeneity from cross-dependency detected, and it is already stated in the paper result section in the last paragraph: “In addition, the Breusch-Pagan test also shows that the variance is not homoskedastic so we estimate the HC estimators for the fixed-effect model, which are robust to heteroskedasticity and Driscoll and Kraay robust covariance matrix in Table 4.” And in the methodology, last paragraph: “In the event of cross-dependency exist, the Driscoll and Kraay robust covariance matrix estimator will be used; iii) The Breusch-Pagan Test in order to see the homoskedasticity assumption. If there is any heteroskedasticity detected, the heteroskedasticity consistent (HC) estimation can be used for the model estimation.” And also in the 5th paragraph on the revised paper we have added more explanation about this: “However, the Ordinary Least Square (OLS) estimation of the panel regression model requires the assumption of normally distributed and homogenous errors, which are rarely met in many cases in real-life data, especially economic data such as stock prices. If the assumption of homogeneity is violated, the estimator will be biased. Many previous studies used OLS estimation but did not mention diagnostic testing. Hence, we address the issue of where heterogeneity and cross-dependency occur in the panel model error. Therefore, the Heteroskedasticity consistent (HC) estimator can be an alternative estimator to have robustness in heterogeneity. However, heterogeneity can also exist from cross-dependency, and Driscoll and Kraay's robust covariance matrix estimator addresses those problems and improve the model results for vaccination on stocks’ return.” A non-stationary panel time series such as VECM panel is not used due to the assumption that both vaccination rate and case growth are independent, so the model has no endogeneity or multicollinearity. Hence, we used the panel data regression model. This has been addressed in the last paragraph of the Background and literature review as follows: “Because the COVID-19 vaccine growth rate and positive cases are unrelated, the panel model regression will be used, considering there is no endogeneity in the variables used.” 5.Reviewer: Low R2 and negative adjusted R2. Answer: The low R2 mainly causes the negative adjusted R2, which means most of the dependent variability was not explained by the independent variables. So it translates that the vaccination rate and case growth do not explain most of the variability of the stock prices in the initial panel model. And there is also a lack of assumption fulfilments in heterogeneity and cross-dependency in the initial panel model, so the next step is we estimate the model again using the HC estimators for the fixed-effect model, which are robust to heteroskedasticity and Driscoll and Kraay robust covariance matrix estimator for the cross-dependency problem. (This has been addressed in the previous question)." } ] } ]	1	https://f1000research.com/articles/11-363
https://f1000research.com/articles/12-440/v1	25 Apr 23	{ "type": "Study Protocol", "title": "Evaluation of effectiveness of advanced platelet rich fibrin (A-PRF) with demineralized freeze-dried bone allograft (DFDBA) placed into fresh extraction sockets with immediate implant placement: A clinical and radiographic study", "authors": [ "Sneha Dare", "Pavan Bajaj", "Pavan Bajaj" ], "abstract": "Background: Dental implant placement has evolved over time with the introduction of numerous novel protocols. Following tooth extraction throughout the healing phase, alveolar ridge resorption is a potential issue that can be mitigated by the utilisation of a variety of techniques, one of which is the placement of immediate implants. The immediate implant treatment protocol offers numerous benefits, such as minimal surgical procedures, which thereby reduce overall treatment time, preserve bone height, and produce better aesthetic results, particularly in the anterior region. Such protocols have made implant insertion so much easier that results can now be anticipated more precisely. The distance between the lateral surface of an implant and surrounding alveolus is known as the jumping gap distance (JGD). JGD of >1.5 mm affects spontaneous bone healing. Research incorporating biomaterials such as membranes produced from blood and bone grafts has significantly contributed to the success of this therapy. Methods: This research will be carried out over the course of a year. Twelve implants will be placed in systemically healthy individuals with one tooth/teeth in the maxilla and/or mandible indicated for extraction. Using a two-stage protocol, implant placement will be done simultaneously after extraction along with augmentation with A-PRF and DFDBA. The second stage surgery will be conducted three months after implant placement. Within 3-6 weeks, abutments will be connected and the final prosthesis will be placed. Expected results: Crestal bone changes and implant stability after using of A-PRF and DFDBA will serve as indicators to determine the implant's success after 6 months. Conclusion: The augmentation procedures have shown to be successful in obtaining bone fill and in resolving bone defects that are present in proximity with implants. An immediate implant placement along with bone augmentation may decrease resorption of horizontal bone. Trial registration: REF/2023/03/064371.", "keywords": [ "Immediate implant", "extraction socket", "jumping gap distance", "A-PRF", "DFDBA" ], "content": "Introduction\n\nDental implant therapy has advanced over time, resulting in predictable, effective, and successful treatment outcomes. During an immediate implant placement procedure, an implant will be placed straight away into an extraction socket after the tooth extraction.1 It reduces the duration of treatment and improves patient comfort. The dimensions of the sockets after extraction are often much larger than the diameters of the implants. While gap can occur at any surface of immediate implant, the buccal bone is of particular importance as it is usually thin and has tendency for resorption and soft tissue recession. The horizontal distance (HD)/jumping gap distance (JGD) is the distance between implant and surrounding alveolus.2 The surface of implants exhibits spontaneous osseointegration and bone healing when possessing HD of ≤1.5 mm. HD of >1.5 mm affects spontaneous bone healing. In order to resolve this issue, combining various materials like bone grafts with growth factor (GF) rich platelet concentrates has the added benefit of promoting faster and more effective healing.\n\nWhen used to treat intraosseous periodontal defects, DFDBA has shown considerable improvements in clinical tissue parameters in both soft and hard tissues.3 Bone morphogenic proteins found in the DFDBA encourage local cell division to produce new bone.4\n\nThe fibrin meshwork structure of A-PRF that results from the polymerisation of the platelets and leukocyte concentration is the factor that influences the release of GF for up to a period of 10 days during the reorganisation of the wound.5,6 PRF production protocol at reduced centrifugation speed (1500 rpm for 14 minutes) produces A-PRF. A-PRF favours the release of higher amount of GFs than PRF, which might affect tissue regeneration directly. There hasn't been research yet to evaluate combined effect of A-PRF and DFDBA placement in peri-implant void on hard and soft tissue alterations followed by immediate implant placement. Therefore, an intent of this study is to evaluate the efficiency of combination of A-PRF in with DFDBA placed simultaneously into an extraction socket followed by placement of immediate implant on the clinical and radiological outcomes.\n\nTo evaluate efficacy of A-PRF and DFDBA in implants placed in fresh extraction socket on the clinical and radiological outcomes.\n\n\n\n1. To evaluate the horizontal and vertical crestal bone changes around immediate implants sockets augmented with A-PRF and DFDBA.\n\n2. To evaluate the changes in the buccolingual dimension of socket around immediate implants augmented with A-PRF and DFDBA.\n\n3. To evaluate the success rate of an immediate implant placed simultaneously with a combination of A-PRF and DFDBA placement.\n\n\nMethods\n\nThe study proposal is approved by “Institutional Ethics Committee” with Ref. No. DMIHER (DU)/IEC/2023/577 on 06/02/2023.\n\nName of registry - Clinical Trial Registry of India\n\nREF No. ‐ REF/2023/03/064371\n\nURL - https://ctri.nic.in/Clinicaltrials/main1.php?EncHid=97508.22015\n\nSubmitted to CTRI on ‐ 04/03/23\n\nSample size calculation\n\nFormula Using Mean difference\n\nPrimary Variable (Buccolingual dimention of socket wall)\n\n(Immediate implant placement, difference after 6 months) Mean ± SD. = 0.10 ± 0.09\n\nAs per reference articles - Bhombe et al (2022)\n\nTotal samples required =13 per Group.\n\nThe calculation gives the result as 13, thus a round figure of 12 samples will be used for the study. This will be a single arm prospective clinical study.\n\nStudy population\n\nIn this study, 12 implants will be placed in systemically healthy individuals who have a need for tooth/teeth replacement through implant from the outpatient “Department of Periodontics”.\n\nInclusion criteria\n\n1. Teeth that require extraction due to fracture of root, residual roots, internal and external resorption, endodontic failures, non-restorable carious lesions, over-retained deciduous teeth.\n\n2. Good oral hygiene, with a full mouth plaque score of less than 25%.\n\n3. Thick gingival biotype.\n\n4. Intact alveolar bony walls appeared both clinically and radiographically.\n\n5. At least 4mm bone must be present below root apex.\n\n6. Bone quality: D1 and D2.\n\n7. The HD should be more than 1.5 mm.\n\nExclusion criteria\n\n1. Altered health conditions that would interfere with bone healing e.g. diabetic patients, blood disorders, osteoporosis, and people allergic to titanium, etc.\n\n2. Space discrepancies between maxilla-mandible.\n\n3. Bone quality: D3 and D4.\n\n4. Implant site having <2mm of width of keratinized gingiva.\n\n5. Para functional habits.\n\n6. Proclined teeth, mal-aligned or rotated anterior teeth, teeth with interdental spacing.\n\n7. Untreated dental diseases.\n\n8. Debilitating tempromandibular (TMJ) joint pathosis.\n\n9. Habit of smoking, alcohol intake or drug abuse.\n\n10. Pregnant woman, lactating mother.\n\nOnly type I extraction sites will be chosen based on the preoperative classification given by Salama and Salama in 1993.7 Before the study begins, details about nutritional status, oral hygiene practises, detailed medical history, and periodontal health status will be recorded in a specially designed case history. Patients will be evaluated under good illumination using William’s graduated probe and mouth mirror. They will be informed about the design and purpose of the clinical trial before the study begins, and their written consent will be obtained.\n\nThis study will be conducted over the course of a year. Twelve implants will be placed in individuals with need of extraction of maxillary and/or mandibular teeth. They will be recruited from the outpatient department of “Department of Periodontics and Implantology, Sharad Pawar Dental College, Sawangi, (Meghe)”. Using a two-stage protocol, implant placement will be done into the fresh extraction socket after the socket has been augmented with A-PRF and DFDBA. Three months after the placement of an implant, a second stage surgery will be performed. Following second stage surgery, abutments will be connected and a prosthesis will be placed within 2 to 4 weeks. Before the procedure, 3 months after implant placement, and 6 months following permanent prosthesis the clinical parameters will be evaluated, which include probing depths around implants, the full mouth modified plaque index (FMPI), the modified papillary bleeding index (FMPBI), and implant mobility using the clinical implant mobility scale (CIMS). Cone Beam Computed Tomography (CBCT) will be obtained for preoperative assessment.8\n\nPrimary outcomes\n\nPrimary outcome will be the vertical and horizontal crestal bone changes around immediately placed implants.\n\nSecondary outcomes\n\nSecondary outcome will the implant mobiliy.\n\nClinical indices\n\nClinical indices will be obtained at three different times: baseline, three months after an implant has been placed, and six months after the final prosthesis. The operator will record the clinical indices for each patient. A mean calculation of these will be obtained for the purpose of assessing the results. The FMPI given by “Turesky – Gilmore – Glickman Modification of Quigley Hein” (1970) will be used to determine oral hygiene status.9 The FMPBI given by “Muhlemann HR.” (1977) will be used to evaluate gingival inflammation.10\n\n\n\na. Width of keratinized gingiva (WKG): Using a University of North Carolina (UNC 15) probe, the WKG on the buccal aspect will be determined which is the distance between the gingival margin and muco-gingival junction.\n\nb. Gingival biotype: A thin or thick gingival biotype will be determined (Muller et al., 2000).11\n\nCBCT will be used for preoperative diagnosis and evaluation of available bone (volumetric) to guide implant placement. The information from these scans can be used to precisely analyse the paranasal sinuses, craniofacial bones, and also the degree of sinus pneumetization.12\n\nFollowing a comprehensive examination and diagnosis, a full mouth scaling will be done, followed by oral hygiene instructions. Until an individual achieves a plaque score of less than 1, the plaque control instructions will be repeated. To determine the maxillo-mandibular relationship, diagnostic casts of each individual will be made prior to the surgical phase. CBCT will be obtained for everyone, and clinical photos will be taken periodically throughout the process.\n\nExtraction of tooth\n\nFollowing complete asepsis and the administration of local anaesthesia, sulcular incisions will be made on the buccal and lingual/palatal surfaces of the tooth to be extracted. In order to visualise the bone plates, full thickness mucoperiosteal flaps reflection will be done on the buccal and palatal/lingual aspects.\n\nAtraumatic extraction will be performed to minimise trauma to the alveolus and achieve minimal socket expansion. An initial incision will be made with a Bard Parker surgical blade of no. 12 or 15, followed by the separation of the supracrestal gingival fibres and periodontal ligament on the mesial and distal aspects of the root. The tooth will then be extracted mainly using periotome, or if there is sufficient tooth structure, the tooth may be carefully extracted with extraction forceps.\n\nAfter the extraction of a tooth, the sockets will be inspected for fractures in the socket walls and then thoroughly debrided. If required, ridge alveoloplasty will be performed to achieve a flat bone surface of sufficient width. The sockets will be curated to induce fresh bleeding. Normal saline will be used to irrigate the extraction sockets, which will then be packed for 5 minutes with gauze soaked in normal saline. To measure the root length of an extracted tooth, UNC 15 probe will be used, while the mesiodistal and buccolingual dimensions of the tooth will be measured with a Vernier Caliper, and these values will be correlated with radiographic findings to determine the diameter and length of an implant.\n\nPlacement of an implant\n\nAfter osteotomy site preparation, implants of appropriate length and diameter will be placed in the recipient site. Implant shoulder would be located at or not more than 1 mm apical to the crestal bone, equating to around 3mm apical to the free gingival margin. The JGD will be measured after the implant placement, and only implants with a gap of 1.5 mm or more will be considered for the study. A-PRF will be prepared by drawing 5 ml of blood from the patient, transferring it to sterile, plain glass-based tubes within 30 seconds, and placing them in a centrifuge machine. Using single-spin centrifugation, it will be centrifuged for 14 minutes at 1500 rpm. Following centrifugation, A-PRF clot will be obtained. DFDBA particles with A-PRF will be placed in the extraction site and will be intimately packed.\n\nAt the time of insertion, the primary stability will be checked by recording the insertion torque value. After stabilisation, the buccal flap will be placed around the implant, and simple interrupted suturing will be done to suture it to the lingual/palatal flap. Immediate postoperative radiographs will be taken as a baseline record and to confirm complete seating of the implants.\n\nSecond stage surgery\n\nThree months after implant placement, the second stage surgery will be performed. The implants will be uncovered by removing the cover screw and replacing it with a gingival former, allowing guided soft tissue healing around 9-10 days. Patients will be given medication after surgery and continue it for at least 3 days post-surgery.\n\nProsthetic reconstruction\n\nAbutment connection followed by definitive prosthesis will be carried out 9-10 days after removal of gingival former.\n\nSix months after the placement of a definitive prosthesis, a complete re-evaluation will be conducted. Clinical parameters will be evaluated, which include probing measurements around implants, FMPBI, FMPI, and implant mobility with the help of CIMS.13,14 Radiographic parameters will be assessed using CBCT. Any biological & technical complications will also be examined.\n\nThe mean and standard deviation (Mean ± SD) values of all the clinical parameters, including FMPI, FMPBI, PPD, buccolingual socket dimension, and radiographic marginal bone level, will be computed. Mean data from the first, third, and sixth months of all patients' treatment will be compared to determine statistical significance. All patients' baseline values will be compared to those at 3 months, and to 6 months after final prosthesis, using Student's paired test. If the probability value (p) is greater than >0.05, the observed difference will be considered statistically insignificant; otherwise, it will be considered significant.\n\nPatient recruitment will begin in June 2023 and is anticipitated to be completed by June 2024.\n\n\nDiscussion\n\nA clinical study assessed the efficacy of “hydroxyapatite” (HA) coated and “titanium plasma-sprayed” implants placed in fresh extraction sockets with respect to its effect on new bone formation and bone-implant contact.15 The investigators came to the conclusion that bone regeneration was significantly more effective when “DFDBA” and a “GTR” barrier material were used together than when a barrier membrane was used alone.\n\nIn another study, investigators compared immediate implant placement with “DFDBA” to that with “modified HA”.16 Here, both groups produced outcomes that were equivalent. Around implants, the mean bone level remained steady and even improved.\n\nPresence of periapical infections may present obstacles for immediate implant placement. However, a study assessed an effectiveness of “platelet concentrates” when combine with an “allograft” (DFDBA)17 on implants placed in extraction sockets with periapical infections. The biomaterials used were meant to fill the JGD. The frequency of outcome measures was assessed at baseline, 3 months, 6 months, and 12 months, which included FMPI, FMBI and gingival aesthetic score. Radiographic parameters were assessed on CBCT at the start of the study and 12 months following implant loading. There were statistically significant differences seen in all periodontal parameter however the gingival aesthetic score greatly improved on the interproximal and midfacial regions. This study also reported an increased implant survival rate in one year.\n\nThe various platelet concentrates when combined with allografts are known for their use in managing JGD. Bhombe et al. assessed the combined effect of “PRF matrix” with “DFDBA” in JGD fill.18 Implant loading was performed three months after implant placement, following two stage protocol. Clinical and radiographic parameters were evaluated at baseline, 3 months, and 6 months. Six months after loading, the implant success was 100%. The authors reported that buccolingual dimensions were preserved, and bone resorption was significantly reduced.\n\nThus, the purpose of this study is to assess the combined efficacy of A-PRF and DFDBA placed in a fresh extraction socket following immediate implant placement using a two-stage approach. This study plan will be reliable to prove advantages pertaining to improvements in the following parameters, such as fewer surgical procedures required, preservation of sufficient bone height, and improved aesthetic results.\n\nThe bone fill and bone defect resolution achieved by the augmentation procedures have been proven to be successful. Reduced horizontal bone resorption may be achieved by simultaneous implant placement with bone augmentation. After using A-PRF and DFDBA, we anticipate faster and more effective healing, which will benefit osseointegration and implant stability. The improvement in bone volume, bone height, and bone density will be confirmed by clinical and radiological parameters.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: SPIRIT checklist for ‘Evaluation of effectiveness of advanced platelet rich fibrin (A-PRF) with demineralized freeze-dried bone allograft (DFDBA) placed into fresh extraction sockets with immediate implant placement: A clinical and radiographic study’, https://doi.org/10.5281/zenodo.7780095.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nHämmerle CH, Chen ST, Wilson TG Jr: Consensus statements and recommended clinical procedures regarding the placement of implants in extraction sockets. Int. J. Oral Maxillofac. Implants. 2004; 19(suppl): 26–28.\n\nBotticelli D, Bergiundh T, Buser D, et al.: The jumping distance revisited: An experimental study in the dog. Clin. Oral Impl. Res. 2003; 14: 35–42. Publisher Full Text\n\nRummelhart JM, Mellonig JT, Gray JL, et al.: A comparison of freeze-dried bone allograft and demineralized freeze-dried bone allograft in human periodontal osseous defects. J. Periodontol. 1989; 60: 655–663. PubMed Abstract \| Publisher Full Text\n\nUrist M, Strates B: Bone morphogenic protein. J. Dent. Res. 1971; 50: 1392–1406. 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\nFujioka-Kobayashi M, Miron RJ, Hernandez M, et al.: Optimized platelet-rich fibrin with the low-speed concept: growth factor release, biocompatibility, and cellular response. J. Periodontol. 2017 Jan; 88(1): 112–121. PubMed Abstract \| Publisher Full Text\n\nSalama H, Salama M: The role of orthodontic extrusive remodeling in the enhancement of soft and hard tissue profiles prior to implant placement: a systematic approach to the management of extraction site defects. Int. J. Periodontics Restorative Dent. 1993 Aug 1; 13(4): 312–333. PubMed Abstract\n\nJacobs R, Salmon B, Codari M, et al.: Cone beam computed tomography in implant dentistry: recommendations for clinical use. BMC Oral Health. 2018; 18: 88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTuresky S: Gilmore, Glickman: Reduced plaque formation by the chloromethyl analogue of vitamin C. J. Periodontol. 1970; 41: 41–43. PubMed Abstract \| Publisher Full Text\n\nMulhemann HR: Psychological and chemical mediators of gingival health. J. Prev. Dent. 1977; 4: 6–17.\n\nMuller HP, Heinecke A, Schaller N, et al.: Masticatory mucosa in subjects with different periodontal phenotypes. J. ClinPeriodontol. 2000; 27: 621–626. Publisher Full Text\n\nDangore-Khasbage, Bhowate R: Utility of the morphometry of the maxillary sinuses for gender determination by using computed tomography. Dent. Med. Probl. 2018; 55(4): 411–417. PubMed Abstract \| Publisher Full Text\n\nMombelli H: Monitoring disease around dental implants during supporative periodontal treatment. Periodontology. 2000; 12(12): 60–68. Publisher Full Text\n\nMisch CE: The implant quality scale: A clinical assessment of the health disease continuum. Oral Health. 1998; 15: 15–25.\n\nGher M, Quintero G, Assad D, et al.: Bone Grafting and Guided Bone Regeneration for Immediate Dental Implants in Humans. J. Periodontol. 1994; 65: 881–891. Publisher Full Text\n\nViswambaran M, Arora V, Tripathi RC, et al.: Clinical evaluation of immediate implants using different types of bone augmentation materials. Med. J. Armed Forces India. 2014 Apr 1; 70(2): 154–162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMedikeri RS, Meharwade V, Wate PM, et al.: Effect of PRF and Allograft Use on Immediate Implants at Extraction Sockets with Periapical Infection—Clinical and Cone Beam CT Findings. Bull. Tokyo Dent. Coll. 2018; 59(2): 97–109. PubMed Abstract \| Publisher Full Text\n\nBhombe KR, Bajaj P, Mundada B, et al.: Combined Effect of Platelet-Rich Fibrin Matrix (PRFM) and Demineralized Freeze-Dried Bone Allograft (DFDBA) in Immediate Implant Placement: A Single-Arm Clinical Trial. Cureus. 2022 Sep 29; 14(9). Publisher Full Text" }	[ { "id": "273933", "date": "12 Jun 2024", "name": "Gustavo Fernandes", "expertise": [ "Reviewer Expertise Periodontics", "Biomaterials", "Dental implants." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear authors, I evaluated the study titled \"Evaluation of effectiveness of advanced platelet rich fibrin (APRF) with demineralized freeze-dried bone allograft (DFDBA) placed into fresh extraction sockets with immediate implant placement: A clinical and radiographic study\".\nMAJOR CONCERNS:\nThis is a study that presented no x-rays and pictures, has a lack of standard and presentation. The type of article was not presented in the title (I suggest to include it in the title, to clarify the understanding for the reader). 18 references were included and only one out of them was published in the last 5 years. I recommend to update all references. The sample size calculation used a wrong variable to calculate the number of individuals to be included; revise and adjust it. Moreover, if the result found was 13, why the authors included 12? \"Study design: This study will be conducted over the course of a year.\" The IRB was approved in \"06/02/2023\". If the results are for 1 year, why the authors presented results for only 6 months? Double-check all points of the guidelines that the authors used, please. I did not find the Results section (there are no results in the article) Why \"Expected outcomes\"?\nAfter raised those points, it is not possible to support the indexed.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\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? Partly", "responses": [] }, { "id": "349264", "date": "06 Jan 2025", "name": "Seong-Gon Kim", "expertise": [ "Reviewer Expertise tissue engineering", "oral and maxillofacial surgery" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article titled \"Evaluation of Effectiveness of Advanced Platelet Rich Fibrin (A-PRF) with Demineralized Freeze-Dried Bone Allograft (DFDBA) Placed into Fresh Extraction Sockets with Immediate Implant Placement: A Clinical and Radiographic Study\" outlines a research plan to assess the combined efficacy of A-PRF and DFDBA in immediate implant placement. While the study presents an intriguing topic, it reflects the characteristics of a preliminary protocol rather than a completed investigation.\nThe abstract provides a broad overview of the study, emphasizing the potential benefits of immediate implant placement with augmentation using A-PRF and DFDBA. However, the background information appears repetitive and overly detailed, lacking conciseness. The inclusion of \"expected results\" highlights the absence of experimental data and reinforces the speculative nature of the claims.\nThe methodology details a one-year clinical study involving 12 implants placed in systemically healthy individuals. It describes the procedural steps, including implant placement into fresh extraction sockets, augmentation, and follow-up surgeries. The outcomes focus on crestal bone changes and implant stability, measured through radiographic and clinical evaluations. Despite this comprehensive plan, the methodology remains unexecuted, as the study lacks results or observational data. While ethical approval and trial registration are mentioned, the absence of implementation diminishes the study’s completeness.\nThe article exhibits inconsistencies in its sample size calculation. Although the formula suggests 13 samples, only 12 are planned, with no rationale provided. Furthermore, the conclusions rely heavily on anticipated results, proposing that augmentation with A-PRF and DFDBA will enhance healing and reduce bone resorption. These assertions are speculative and unsupported by data. The lack of visual evidence, such as radiographic or clinical images, further weakens the study’s claims.\nThe references cited include outdated sources, with only one from the past five years. This limits the study’s relevance in addressing current research gaps. Additionally, the absence of results and visual data hinders the ability to evaluate its potential contributions to implantology.\nTo enhance the article's scientific value, the study must first be conducted, and results presented. The background should be condensed to focus on the research gap and relevance of A-PRF and DFDBA. The sample size calculation requires clarification and consistency. Recent literature should be incorporated to reflect contemporary understanding. Supporting data, including images and detailed results, are necessary for validation. Conclusions should be reframed to reflect findings rather than anticipated outcomes.\nIn its current form, the article serves as a preliminary study protocol with potential for future contributions to implantology. However, the lack of execution and supporting evidence limits its credibility and applicability.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-440
https://f1000research.com/articles/12-434/v1	21 Apr 23	{ "type": "Research Article", "title": "Application of an interrupted time series analysis (ITS) to evaluate the effect of universal dialysis policy from 2006 to 2016 in a province of northeastern Thailand", "authors": [ "Piyalak Pukdeesamai", "Pongdech Sarakarn", "Sirirat Anutrakulchai", "Piyalak Pukdeesamai", "Sirirat Anutrakulchai" ], "abstract": "Background: An interrupted time series (ITS) analysis is a powerful tool for policy evaluation. In Thailand, chronic kidney disease (CKD) is a public health problem that requires a long recovery time and has a high treatment cost. The universal coverage policy for renal replacement therapy (universal dialysis policy), is used to treat this disease but policy evaluation using ITS analysis has rarely been conducted. This study applied ITS analysis to test the effect of such a policy between 2006 and 2016. Methods: Data were retrieved from the electronic database of the health data center in Roi Et Province for the period between January 1, 2006 and December 31, 2016. 15,681 CKD stage 5 patients were included. The intervention under assessment was the universal health coverage system, which has been implemented since 2008. Results: Results showed that before implementation of the universal dialysis policy, the overall trend of access to renal replacement therapy (RRT) slightly increased (0.74; 95% confidence interval (CI): 0.58, 0.90). After implementation of the policy, access sharply increased (6.10; 95%CI: 3.67, 8.54), while the linear trend after policy implementation also slightly increased (0.29; 95%CI: 0.05, 0.14). The stratified analysis showed the same linear directional trend before and immediately after implementing the universal dialysis policy. Conclusions: Implementation of the universal dialysis policy positively impacted the rate of renal replacement therapy in CKD stage 5 patients, while access to health care services also increased.", "keywords": [ "Chronic kidney disease", "Universal health coverage", "Interrupted time series" ], "content": "Introduction\n\nChronic kidney disease (CKD) is a major global public health problem. In high-income countries, the prevalence of CKD stages 1-5 in individuals aged ≥20 years is 8.6% in men and 9.6% in women, and in low- and middle-income countries it is 10.6% and 12.5% in men and women respectively.1 In Asia, the prevalence of CKD stages 3-5 is 11.2%.2 CKD has been linked to morbidity and mortality, for example with cardiovascular disease and anemia.3,4 For CKD stage 3-5 patients, renal replacement therapy is considered to be a clinical practice that can consist of the modalities of hemodialysis, peritoneal dialysis, and kidney transplant. In Thailand from 2007 to 2011, the rate of hemodialysis decreased from 79.2% to 54.3%; the rate of peritoneal dialysis increased from 12.2% to 42.9%; while the rate of kidney transplantation decreased from 8.6% to 2.8%.5 Thailand implemented the universal dialysis policy for helping CKD patients to achieve health care treatment in 2008. Since then, there have been several studies related to CKD in Thailand, for example, an epidemiology study of CKD,5 and analyses looking at the prevalence or trend of glomerular filtration rate (GFR) measurement,6 chronic kidney disease prevention and reduction,6 survival rates and related factors for peritoneal dialysis (PD),7–10 and national peritoneal dialysis.11 However, there has rarely found a study that investigated the effect of the universal treatment policy on the rate of access to health care.\n\nThe study design that we used to evaluate the public health policy/intervention in Thailand was an interrupted time series (ITS).12 Several studies have used an ITS to evaluate policies for non-communicable diseases. Few reviews of ITS analysis have been found in regard to policy evaluation concerning CKD.13–15 Therefore, the aim of this study was to use an ITS analysis to compare the rate of renal replacement therapy in a province of Northern Thailand before and after the universal coverage of renal replacement therapy was implemented in January, 2008.\n\n\nMethods\n\nA secondary data analysis was used in this study. As a first step we retrieved the data for the period between January 1, 2006 and December 31, 2016 from the Health Data Center (HDC) in Roi Et Province. HDC is a health database system that stores monthly health data since 2007 from all health care services belonging to the Ministry of Public Health, Thailand. The health data comprised demographic data, household data, community (village, activity), health facilities in the community, disability details, provider, service, outpatient department (diagnostic date, procedure, drug and laboratory), inpatient department (admission date, diagnostic date, procedure, drug and laboratory) and the prevalence of communicable diseases. The data does not contain identifying information and cannot be linked to individuals. We selected participants with an estimated GFR (eGFR) of less than 15 ml/min/1.73m2 (CKD stage 5), aged ≥18 years and with universal health coverage were included in our study.\n\nThe Khon Kaen University Ethics Committee for Human Research approved this study (permit no.: HE642084).\n\nThe rate of access to renal replacement therapy (RRT) services was calculated by the number of participants accessing treatment for RRT (peritoneal dialysis, hemodialysis) among all dialysis modalities. This data does not include kidney transplant patients. We aggregated the data for each month during the study period. Therefore, the data were created for120 monthly study points between January 1, 2006 and December 31, 2016.\n\nWe also collected information including gender (male, female), age (18 to 44 years, 45 to 60 years, > 60 years), and the primary cause of renal failure (hypertension, diabetes, heart disease).\n\nIn the ITS analysis, segmented regression was used to test the effect of implementation before, and immediately after the intervention. The strength of the model distinguished the effect of the intervention from secular change. Time series data often have many issues that may affect to the robustness of the analysis such as seasonality, time-varying confounders, use of control, and for other more complex ITS designs, over-dispersion and autocorrelation.12 Seasonality refers to some events occurring more frequently than others, which may affect to the results. Time-varying confounders may occur because of the longitudinal data and age variation across time. Finally, autocorrelation is the correlation between the values of the same variables across different observations (period of the study).16\n\nThe dataset for the segmented regression analysis comprised the rate for the event and the time point when the intervention was implemented. An interrupted time series analysis measures trends as slope changes before and after implementation of the intervention, and also the effect seen when the intervention is implemented (the instant effect). Our analysis constructed a series of quarterly rates of access to treatment for renal replacement therapy for CKD stage 5 patients from January 2006 to December 2016. The 24-month pre-intervention period was from January 2006 to December 2007, while the 96-month post-intervention period took place from January 2008 to December 2016. We aggregated the data with 3-month interval. Therefore, there have an 8-time points in the pre-intervention period and 32-time point in post-intervention.\n\nThe equation for the ITS analysis17,18 was as follows:\n\n\nResults\n\nTable 1 shows the characteristics of participants. We included 15,681 patients in the study, which was a total of 1,844 patients before policy (BP) implementation, and 13,837 patients after policy (AP) implementation. 11.76% of the participants were BP and 88.24% of the participants were AP. Most were aged over 60 years and had hypertension conditions, both BP and AP. We also described the rate of treatment for peritoneal dialysis (PD) and hemodialysis that was shown in Table 2.\n\nTable 3 shows that the overall trend of access to RRT increased before policy implementation by 0.74 (95%CI: 0.58, 0.90), while after policy implementation the trend sharply increased by 6.10 (95%CI: 3.67, 8.54). The linear trend after policy implementation slightly increased by 0.29 (95%CI: 0.17, 0.41). The overall trend is shown in Figure 1. For those aged 60 or over, the trend slightly increased before policy implementation, while immediately after policy implementation the trend sharply increased by 5.26 (95%CI: 3.50, 7.01). For those whose primary cause of renal failure was diabetes, hypertension, and heart disease, the trend after policy implementation sharply increased by 9.11 (95%CI: 5.25,12.96), 7.65 (95%CI: 2.22, 13.07), and 11.83 (95%CI: 5.44, 18.22) respectively.\n\n\nDiscussion\n\nResults showed that before implementing the universal dialysis policy, the trend of access to RRT slightly increased, while immediately after implementing the universal dialysis policy the trend sharply increased.\n\nBefore policy implementation in 2008, evidence showed that the rate of access to RRT slightly increased, possible as a result of the universal coverage policy not covering RRT. This meant that patients were responsible for medication payments (approximately 1,500 Thai baht per visit),19 which affected their decision to undergo the therapy. The long-term treatment of RRT and high payments made low-income patients unable to access a clinic and receive appropriate treatment.\n\nBecause of his, the National Health Security Office (NHSO) developed a kidney replacement therapy service system for patients with end-stage chronic renal failure in 2008. This policy was called ‘CAPD first’ and covered renal replacement therapy, kidney transplantation (KT), peritoneal dialysis (PD) and hemodialysis (HD). It provides peritoneal dialysis as the first choice and hemodialysis only for those who are unable to undergo peritoneal dialysis or who have a medical indication prohibiting PD. In the case of an old hemodialysis patient who does not voluntarily undergo peritoneal dialysis, the patient will have to pay 1/3 of the service fee (patients pay no more than 500 baht and NHSO pays 1,000 baht per time).19 This new system resulted in a significantly higher rate of RRT patients with stage 5 renal failure. There are still many patients with renal failure who have not decided to enter the treatment system. Our evidence confirmed that after implementing this policy in 2008, the rate of access to RRT sharply increased and it helped patients to receive treatment because they did not have to bear the expenses themselves but gained benefits allocated by the state.\n\nThe total yearly incidence of RRT increased by an average of 14.8% after the implementation of the universal coverage policy for renal replacement therapy (known first as CAPD) and the yearly incidence of all RRT modalities increased by an average of 34.8% in 2007 to 2009.20 A report by Thai Renal Replacement Therapy (TRT) stated that the number of end-stage renal failure patients receiving renal replacement services increased from 68.34 per million in 2006 to over 181 per million in 2009 (or more than 11,500 new patients per year). The number of people receiving renal replacement services increased significantly from 419 per million to over 639 per million (or more than 40,000 in 2009).21 Our analysis results also showed that the linear trend of RRT after implementing the continuous policy slightly increased. Despite the expected increased volume of patients, the year-by-year growth rate of patients in all RRT modalities seemed to diminish over time.22 due to the CAPD first policy. HD is performed only for those who are unable to undergo peritoneal dialysis or who have a medical indication prohibiting PD. Therefore, HD dialysis will be covered.23 Some patients who meet the criteria for HD decide not to enroll for CAPD due to lack of readiness for the treatment required, such as inappropriate accommodation, lack of caregivers, being unsure of self-cleaning processes at home and fear of infection after the procedure.24\n\nA slight increase in access to RRT services was in line with the assessment of access to services and the provision of renal replacement services under the health insurance system in Thailand. The expected number of patients who accessed the services was more than 35,000 in 2011, but there are only 19,000 cumulative cases under the UHC scheme. At the end of the fiscal year 2012, the number of patients was estimated at more than 48,000 patients, but the cumulative number of cases was only about 23,000.25 In terms of service provision, some hospitals are unable to participate in the universal health insurance program or are unable to provide services. Due to the availability of human resources and location, a health care service might require patients to go to other hospitals that can provide services but are far from their homes, which made it inconvenient and costly for traveling. This might be a consideration when making the decision to select a treatment.\n\nOur study had some limitations. Firstly, data retrieved from other hospitals might have different methods that could have impacted data quality. Secondly, only two hospitals currently incorporate this scheme and one is a private hospital, so some data were incomplete. Finally, our results might not be generalizable to Thailand as a whole because we analyzed data from only one province. However, the strength of our study is an analysis using the interrupted time series model that accurately interpreted the results.\n\n\nConclusions\n\nOur results revealed that after the universal coverage of renal replacement therapy policy implementation, the rate of treatment for RRT slightly increased. Extensive data collection from other health centers would be useful for further research. After the policy implementation, the trends in access to RRT slightly increased. This might be because patients were uncertain about using CAPD at their houses. To further increase the rate of RRT, policy makers should consider this point.", "appendix": "Data availability\n\nfigshare: Application of an interrupted time series analysis (ITS) to evaluate the effect of universal dialysis policy from 2006 to 2016 in a province of northeastern Thailand, https://doi.org/10.6084/m9.figshare.21456261.v5. 26\n\nThis project contains the dataset created during analysis.\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 the Health Data Center officer in Roi Et Province for data preparation.\n\n\nReferences\n\nKovesdy CP: Epidemiology of chronic kidney disease: an update 2022. Kidney Int. Suppl (2011). 2022; 12(1): 7–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuriyong P, Ruengorn C, Shayakul C, et al.: Prevalence of chronic kidney disease stages 3-5 in low- and middle-income countries in Asia: A systematic review and meta-analysis. PLoS One. 2022; 17(2): e0264393. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThomas R, Kanso A, Sedor JR: Chronic kidney disease and its complications. Prim. Care. 2008; 35(2): 329–344. vii. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalantar-Zadeh K, Jafar TH, Nitsch D, et al.: Chronic kidney disease. Lancet. 2021; 398(10302): 786–802. Publisher Full Text\n\nVejakama P, Ingsathit A, Attia J, et al.: Epidemiological study of chronic kidney disease progression: a large-scale population-based cohort study. Medicine (Baltimore). 2015; 94(4): e475. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCha’on U, Wongtrangan K, Thinkhamrop B, et al.: CKDNET, a quality improvement project for prevention and reduction of chronic kidney disease in the Northeast Thailand. BMC Public Health. 2020; 20(1): 1299. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChangsirikulchai S, Sriprach S, Thokanit NS, et al.: Survival Analysis and Associated Factors in Thai Patients on Peritoneal Dialysis Under the PD-First Policy. Perit. Dial. Int. 2018; 38(3): 172–178. PubMed Abstract \| Publisher Full Text\n\nChuengsaman P, Kasemsup V: PD First Policy: Thailand’s Response to the Challenge of Meeting the Needs of Patients With End-Stage Renal Disease. Semin. Nephrol. 2017; 37(3): 287–295. PubMed Abstract \| Publisher Full Text\n\nDhanakijcharoen P, Sirivongs D, Aruyapitipan S, et al.: The “PD First” policy in Thailand: three-years experiences (2008-2011). J. Med. Assoc. Thail. 2011; 94(Suppl 4): S153–S161.\n\nTreamtrakanpon W, Katavetin P, Yimsangyad K, et al.: From the “PD First” policy to the innovation in PD care. J. Med. Assoc. Thail. 2011; 94(Suppl 4): S13–S18.\n\nPongpirul K, Kanjanabuch T, Puapatanakul P, et al.: National feasibility survey of peritoneal dialysis key performance indicators in Thailand from provider perspective. Nephrology (Carlton). 2020; 25(6): 483–490. PubMed Abstract \| Publisher Full Text\n\nBernal JL, Cummins S, Gasparrini A: Interrupted time series regression for the evaluation of public health interventions: a tutorial. Int. J. Epidemiol. 2017; 46(1): 348–355. PubMed Abstract \| Publisher Full Text\n\nZhang Q, Thamer M, Kshirsagar O, et al.: Impact of the End Stage Renal Disease Prospective Payment System on the Use of Peritoneal Dialysis. Kidney Int. Rep. 2017; 2(3): 350–358. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang V, Hammill BG, Maciejewski ML, et al.: Impact of automated reporting of estimated glomerular filtration rate in the veterans health administration. Med. Care. 2015; 53(2): 177–183. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaber DJ, DuBay D, McGillicuddy JW, et al.: Impact of the New Kidney Allocation System on Perioperative Outcomes and Costs in Kidney Transplantation. J. Am. Coll. Surg. 2017; 224(4): 585–592. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaum CF, Schaffer ME: ACTEST: Stata module to perform Cumby-Huizinga general test for autocorrelation in time series.2013.\n\nPenfold RB, Zhang F: Use of interrupted time series analysis in evaluating health care quality improvements. Acad. Pediatr. 2013; 13(6 Suppl): S38–S44. Publisher Full Text\n\nWagner AK, Soumerai SB, Zhang F, et al.: Segmented regression analysis of interrupted time series studies in medication use research. J. Clin. Pharm. Ther. 2002; 27(4): 299–309. Publisher Full Text\n\nNational Health Security Office: NHSO Annual Report. Bangkok: NHSO; 2009.\n\nPraditpornsilpa K, Lekhyananda S, Premasathian N, et al.: Prevalence trend of renal replacement therapy in Thailand: impact of health economics policy. J. Med. Assoc. Thail. 2011; 94(Suppl 4): S1–S6.\n\nKanjanabuch T, Takkavatakarn K: Global Dialysis Perspective: Thailand. Kidney360. 2020; 1(7): 671–675. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThammatacharee N, Suphanchaimat R: Long-Term Projections of Patients Undertaking Renal Replacement Therapy Under the Universal Coverage Scheme in Thailand. Risk Manag. Healthc. Policy. 2020; 13: 27–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Health Security Office: NHSO Annual Report. Bangkok: NHSO; 2008.\n\nWalker RC, Hanson CS, Palmer SC, et al.: Patient and caregiver perspectives on home hemodialysis: a systematic review. Am. J. Kidney Dis. 2015; 65(3): 451–463. PubMed Abstract \| Publisher Full Text\n\nTantivess S, Werayingyong P, Chuengsaman P, et al.: Universal coverage of renal dialysis in Thailand: promise, progress, and prospects. BMJ. 2013; 346: f462. PubMed Abstract \| Publisher Full Text\n\nPukdeesamai P: Application of an interrupted time series analysis (ITS) to evaluate the effect of universal dialysis policy from 2006 to 2016 in a province of northeastern Thailand. Dataset. figshare. 2022. Publisher Full Text" }	[ { "id": "322049", "date": "04 Oct 2024", "name": "Yao Wu", "expertise": [ "Reviewer Expertise Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study assesses the impact of a universal dialysis policy from 2006 to 2016 in a province of north-eastern Thailand using an interrupted time series (ITS) analysis. The authors have made a good start, but the study requires further refinement, particularly in terms of methodological justification and analysis. I provide the following suggestions for improvement:\nThe authors should provide more detailed background information on the universal dialysis policy in the Introduction or Methods sections. Specifically, they should explain the rationale behind this policy, particularly how it aims to increase access to renal replacement therapy (RRT). The authors need to provide a clear rationale for choosing a 3-month interval for their analysis. Is this interval commonly used in similar studies, or does it reflect some meaningful aspect of the policy or clinical practices? In the Methods section, the variable \"X1\" should represent time throughout the entire study period, rather than only the time before the intervention. The authors should refer to more recent studies to justify the use of their ITS analysis equation. I recommend citing literature such as the one linked here: [https://doi.org/10.1186/s12874-021-01306-w].(refer 1) The authors mention that \"only two hospitals currently incorporate this scheme.\" Given this, it would be more appropriate to focus on data from these two hospitals and use the other hospitals as a control group. This would enable the use of a controlled interrupted time series analysis, which would provide a more robust examination of the policy’s impact. Figure 1: It is suggested that the authors include a counterfactual trend line in Figure 1 for the period after 2008. This would help in visualizing what the expected trend would have been without the policy intervention. Authors stated that “Results showed that before implementing the universal dialysis policy, the trend of access to RRT slightly increased, while immediately after implementing the universal dialysis policy the trend sharply increased.” However, this interpretation is incorrect. The change in intercept reflects the immediate effects of the policy introduction, whereas the change in slope reflects the long-term effects. Based on Figure 1, it appears that the slope before 2008 (0.74) is higher than the slope after 2008 (0.29), indicating that the trend of access to RRT actually decreased after the policy was implemented. The results section should be revised to reflect this, and further discussion should be provided to justify and interpret this finding.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-434
https://f1000research.com/articles/12-432/v1	21 Apr 23	{ "type": "Research Article", "title": "Evaluation of serum complement C4 among COVID-19 patients in Khartoum state: a case control-based study", "authors": [ "Mohammed Yahya", "Yousif Ali Rahamtalla", "Nasr Mohammed Nasr", "Babbiker Mohammed Taher Gorish", "Yousif Ali Rahamtalla", "Nasr Mohammed Nasr", "Babbiker Mohammed Taher Gorish" ], "abstract": "Background: The complement system is made up of an abundance of unique plasma proteins that play an important role in innate immunity and inflammation, aiding in the fight against pathogenic microbes and viral diseases. The purpose of this study was to evaluate the serum complement C4 concentration in COVID-19 patients in Khartoum and compare them to healthy controls. Methods: A total of 100 samples were collected, 50 samples from COVID-19 patients who presented as cases and 50 samples from people who were evidently healthy. Overall, 33 (66%) the patient populations in the case group were not in the hospital’s intensive care unit (ICU), compared to 17 (34%) who were. The concentrations of C4 in each serum sample were calculated in milligrams per deciliter. SPSS version (20) was used to analyze the data. Results: The means level of complement C4 (mg/dL) were 37.44±18.618, 23.90±10.229 in the case group and in the control group, respectively. There was a statistically significant difference in complement C4 level between case and control (p-values ≤0.01). In addition, the mean complement C4 level in the ICU and non-ICU case groups was 25.00±17.85 and 43.85±15.712 mg/dL, respectively. There was a statistically significant variance in complement C4 level between ICU and non-ICU (p-values ≤0.01). Furthermore, the cases were divided into four age groups: 20-40, 40-60, 60-80, and over 80 years old. The one-way ANOVA test showed no statistically significant differences between age categories in complement C4 level (P = 0.735) Conclusions: The case group had a higher mean level of complement C4 than the control group, which could be understood by the stimulation of the complement cascade during the COVID-19 illness. Furthermore, the complement C4 level in severe COVID-19 patients was lower than in non-severe COVID-19.", "keywords": [ "Complement C4", "Sudanese", "Covid-19" ], "content": "Introduction\n\nIn the last twenty years, endemic cases of the Middle East respiratory syndrome (MERS)-CoV in 2012 and severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 both occurred, with case mortalities ranging from 14–15% to 35%.1 At the end of December 2019, a new coronavirus was determined to be the root of a cluster of pneumonia cases with an unknown cause in Wuhan, Huanan Fresh fish Supplier Market, the city in China’s Hubei Province that served as the initial site where cases of coronavirus disease 2019 (COVID-19) were identified.2 The novel coronavirus spread rapidly, causing an epidemic in China, and a pandemic with rising incidence in many other nations all over the globe.2 Symptomless infection, mild upper airway disease, severe and highly contagious pneumonia, respiratory distress, and sometimes even fatality are all on the clinical phenotype of SARS-CoV-2 infection.3\n\nInitially, a link to a seafood restaurant in Wuhan that sold live animals and was frequented or worked at by the majority of the previous pneumonia patient populations was noticed. But as the pandemic disease spread, person-to-person transmission took over as the main way it was spread. Large droplets released during sneezing and coughing by symptomatic patients help spread the COVID-19 infectious disease, but transmission can also occur from people who are symptomless before developing symptoms.4 The COVID-19 virus can cause clinical and pathological lung lesions that resemble those seen in other types of acute respiratory distress syndrome (ARDS) because it enters lung tissue after sticking to viral Spike proteins (S) and ACE2 receptors.5\n\nIn order to eliminate invasive pathogens, complement pathways, a crucial part of the innate immune system, are activated by coronaviruses.6 The main complement molecules C3 and C4 are cleaved by the proteolytic process when the complement system is activated, resulting in the cleavage products C3a, C3b, C4a, and C4b, which can cause the recruitment of inflammatory cells and the activation of neutrophils.7 Patients with mild COVID-19 have shown higher levels of C3 and C4 complement, whereas those with severe COVID-19 had lower levels, which might be clarified by higher consumption from the formation of immune complexes.8\n\nIn light of this, the goal of this study was to compare the serum complement C4 levels of COVID-19 patients in Khartoum to those of healthy controls. Additionally, we compared the COVID-19 patients’ levels of C4 based on the severity of their illnesses.\n\n\nMethods\n\nThe Ethics Committee of Sudan University of science and technology approved this research (approval number: 112BNC/12/2021; approval date: 01/12/2021). In addition, written approval was obtained from the Jabra hospital of Emergency and Trauma manager (approval number: JET 36 in December 2021). Respondents provided written informed consent after being guaranteed that only investigators would have access to the data and that it would only be used for investigation. Respondents could fill the questionnaire privately. All procedures in this study were carried out in conformity with the rules and regulations specified in the Helsinki Declaration that were applicable.\n\nDuring the period from January to April 2022 a total of 100 subjects were involved in this study, of which 50 were cases confirmed to have COVID-19 infection and another 50 apparently healthy control were the control group. Cases were matched in sociodemographic characters. Cases and control were randomly selected from patients attending Jabra hospital of Emergency and Trauma. Gender was informed based on the self-administered questionnaire filling.\n\nThe study included patients diagnosed with COVID-19 and apparently healthy controls. Patients who had received any care before being admitted other than antipyretics, had any recorded coinfections, or had immune dysfunction or malignant neoplasms were excluded.\n\nA volume of 3 mL of blood was collected in plain containers from both patients and controls at Jabra Hospital of Emergency and Trauma. Then the samples were centrifuged, serum separated and stored at -80°C until analysis. C4 ELISA kit was obtained from Abcam (United Kingdom). All the protocols were done according to the manufacturer instruction. After that all reagents were brought to 37°C for warming, then 800 μL from R1 (Diluent) were added to 20 μL from the sample or calibrator and 200 μL from R2 (Antibody); finally, the absorbance of calibrator and sample had been read. The concentration of C4 was obtained by calculating the differences between calibrator and sample reading.\n\nThe Statistical Package for Social Science (SPSS) Version 20 was used for data analysis. The data was presented as mean and standard deviation, frequency; Pearson’s correlation, T – test results, and one way Anova have been used as specific tests with a significant threshold value of 0.05 (P-value≤0.05).\n\n\nResults\n\nIn this study, 100 volunteers between the ages of 21 and 92 were enrolled, of whom 50 were COVID-19 patients with a mean age of 60.74±18.044 years, 33 (66%) men, and 17 (34%) women. Additionally, there were 50 individuals who appeared to be in good health; their mean age was 59.68±19.464 years. Of these patients, 33 (66%) were men and 17 (34%) were women (Table 1). Regarding the case group, there were 33 (66%) non-ICU patients and 17 (34%) ICU patients (Table 2). In the case group and control group, the mean levels of complement C4 (mg/dL) were 25.00±17.85 mg/dL and 43.85±15.712 mg/dL respectively. Between the case and control groups, there was a statistically significant difference in complement C4 level (P-value=0.00) (Table 3).\n\nMales and females in the case group had average complement C4 levels of 40.15±20.095 and 32.18±14.466 respectively. There was no discernible gender difference in the complement C4 level among the cases group (P-value=0.153, Table 4). Based on their age, the cases were divided into four groups: the first group was made up of patients that were between the ages of 20 and 40; the second group was made up of patients between the ages of 40 and 60; the third group was made up of cases between the ages of 60 and 80; and the fourth group (80-100 years). The level of complement C4 did not significantly differ between age groups, according to a one-way ANOVA test (P-value=0.735, Table 5). The case group’s average complement C4 levels were 25.00±17.85 mg/dL and 43.85±15.712 mg/dL respectively. There was a statistically significant difference in the complement C4 level between ICU and non-ICU P-value was 0.00 (Table 6).\n\n\nDiscussion\n\nImmunoassays are frequently used in clinical practice to measure complement C4 and determine and track complement activation. A decrease in serum C4 levels brought on by increased immune system C4 consumption. Complement C4 testing in COVID-19 patients may be able to shed light on how clinical risk is balanced between normal and abnormal complement activation.9 In the current study, in serum samples, complement C4 levels were assessed in 50 COVID-19 patients and 50 healthy individuals. The findings showed that there was a significant difference between the case group and control in terms of the mean complement C4 level (P-value=0.00). Zinellu and Mangoni (2021), showed there was a noticeably higher level of complement C4 in the case than in the control, which supports this finding (P-value less than 0.05). In the current study, patients with severe COVID-19 disease had lower complement C4 levels than patients with less severe COVID-19 disease, and this difference was significant (P-value=0.00). This finding concurs with that of Ghazavi and colleagues in Iran, who discovered that complement C4 levels were lower in COVID-19 patients with severe disease (P=0.014) and higher in those without severe disease (P-value 0.012).8 Al-Hakeim and his colleagues also discovered in Iraq that patients who spent longer than two weeks in the hospital had lower complement C4 levels than those who were discharged earlier (P-value less than 0.05).10 This result supported the findings of Al-Hakeim and his colleagues in Iraq, who found no correlation between the mean complement C4 levels across age groups and sex (P=0.681).10 Males and females in the case group had average complement C4 levels of 40.15±20.095 and 32.18±14.466 respectively. There was no discernible gender difference in the complement C4 level among the cases group. Prior clinical evidence has revealed that, while males and females share a comparable illness rate, males are more likely to be hospitalized, experience a more severe illness progression, and die at a greater rate than females.11\n\nA study with a large number of patients in the asymptomatic and ICU groups using COVID-19 is recommended to generate clearer and more significant results. Due to funding limitations, this research was limited to 50 COVID-19 patients. As a result, it is recommended that similar studies be conducted on a greater number of COVID-19 cases. Also, research on the other complement components would be valuable in order to acquire a better understanding of the complements pathway’s involvement. There is considerable debate over how soon complement overuse must be monitored and which components of complement consuming must be evaluated.\n\n\nConclusions\n\nComparing the case group to the control, there was a higher mean level of complement C4, which can be attributed to the activation of the complement system during COVID-19 infection. Furthermore, the complement C4 level was lower in patients with severe COVID-19 particularly in comparison to those with non-severe COVID-19, which could be because the immune system of patients with severe infection consumes more complement C4.\n\n\nAuthors’ contributions\n\nYA performed main experiments, YA, NM, MY, BG collected’ samples and information. MY, designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.", "appendix": "Availability of data and materials\n\nfigshare: Complement C4 in COVID 19 patients in Khartoum case-control study, https://doi.org/10.6084/m9.figshare.22249990.v1. 12\n\nThe project contains the following underlying data:\n\n- Mohammed Yahya data.xlsx (13.57 kB).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgments\n\nThe authors gratefully acknowledge for the great efforts of the staff in Jabra hospital for Emergency and Trauma for their huge contributions in this study.\n\n\nReferences\n\nGao T, Hu M, Zhang X, et al.: Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation. MedRxiv. 2020.\n\nÖzdemir Ö: Coronavirus Disease 2019 (COVID-19): Diagnosis and Management. Erciyes Med. J./Erciyes Tip Dergisi. 2020; 42(3).\n\nZhou F, Yu T, Du R, et al.: Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020; 395(10229): 1054–1062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRothe C, Schunk M, Sothmann P, et al.: Transmission of 2019-nCoV infection from an asymptomatic contact in Germany. N. Engl. J. Med. 2020; 382(10): 970–971. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerdji H, Mayeur S, Schenck M, et al.: Histopathological features in fatal COVID-19 acute respiratory distress syndrome. Medicina Intensiva (English Edition). 2021; 45(5): 261–270. Publisher Full Text\n\nJava A, Apicelli AJ, Liszewski MK, et al.: The complement system in COVID-19: friend and foe? JCI Insight. 2020; 5(15). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJaneway CA Jr, Travers P, Walport M, et al.: Immunobiology: The Immune System in Health and Disease. The complement system and innate immunity. 5th edition.New York: Garland Science; 2001. Reference Source\n\nGhazavi A, Mosayebi G, Keshavarzian N, et al.: Reduction of Inflammatory C3 and C4 Complement Proteins in Severe COVID-19 Patients.2020.\n\nZinellu A, Mangoni AA: Serum complement C3 and C4 and COVID-19 severity and mortality. A systematic review and meta-analysis with meta-regression. Front. Immunol. 2021; 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Hakeim HK, Al-Hamami SA, Almulla AF, et al.: Increased Serum Thromboxane A2 and Prostacyclin but Lower Complement C3 and C4 Levels in COVID-19: Associations with Chest CT Scan Anomalies and Lowered Peripheral Oxygen Saturation. COVID. 2021; 1(2): 489–502. Publisher Full Text\n\nQi S, Ngwa C, Morales Scheihing DA, et al.: Sex differences in the immune response to acute COVID-19 respiratory tract infection. Biol. Sex Differ. 2021; 12(1): 66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYahya M, Gorish B: Complement C4 in COVID 19 patients in Khartoum case-control study. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "182340", "date": "04 Jul 2023", "name": "Adnan Mezher Maysam", "expertise": [ "Reviewer Expertise Clinical immunology", "COVID-19" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article is an important addition to the literature, because the complement is a vital component of innate immunity where it forms the first line of defense against potentially harmful microbes, but its role in COVID-19 is still not clear. In this article, the results showed that case group had a higher level of complement C4 than the control group, which means that the complement plays an important role in the severity of disease, and the C4 complement should be considered as a diagnostic marker.\nI hope the authors intend to evaluate level of anti-pro inflammatory cytokine, such as IL-37 and IL-38, soon in severe and non-severe COVID-19 cases.\nI have carefully read this manuscript and below are my comments:\nAre the authors discussing the role of complement in pathogenesis of COVID-19 patients or in immune system? Clear this point.\n\nIndicate the aim of this paper more clearly and the conclusion also needs improvement.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9863", "date": "05 Jul 2023", "name": "Mohammed Yahya", "role": "Author Response", "response": "Dear Dr. Adnan Mezher Maysam Thank you for your time to review our mansucript. We carefully read your comments and this is our reply please track in a manner that comment followed by the authors response Comment 1: I hope the authors intend to evaluate level of anti-pro inflammatory cytokine, such as IL-37 and IL-38, soon in severe and non-severe COVID-19 cases. Authors response: Thank you for your comment and we will consider it in our near future work to understand the role of these anti - pro inflammatory cytokine in the COVID-19 infection progression Comment 2 Are the authors discussing the role of complement in pathogenesis of COVID-19 patients or in immune system? Clear this point Authors response: Actually this manuscript is discussing the role of complement C4 in the progression of COVID 19 infection and it was based on the evidence that complements are more likely to be consumed among severly ill COVID-19 patient due to development of complication like DIC so the manuscript is more related to pathogenesis of COVID-19 based on immunological activity. Comment 3 Indicate the aim of this paper more clearly and the conclusion also needs improvement. Authors response: Thank you for your comment we will carefully consider it in the revised version of the manuscript Again thank you for your time and efforts" } ] }, { "id": "185092", "date": "31 Jul 2023", "name": "Marwa Hameed Alkhafaji", "expertise": [ "Reviewer Expertise Microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript includes a case-control study that attempted to correlate complement C4 levels with COVID-19 infection pathogenesis and outcome. The complement C4 level in the case group was lower than in the controls. The manuscript material could be viewed as additional support for the previous research highlighting the role of complement in the pathophysiology of COVID-19.\n\nDespite the fact that the manuscript is nicely written, and adds to the current literature, I have some comments and questions that should be addressed by the author:\nThe introduction is excessively brief and lacks critical information about the correlation between complement and COVID-19 pathogenesis, with only one paragraph detailing this crucial part. As a result, authors should cite some recent papers to validate their findings.\n\nSection of the methodology:\n1. How and at what power was the sample size estimated?\n2. The optimal Cases to Control ratio should be 1:4, but the authors used 1:1 why?\n3. Why were patients with immunological dysfunction or malignant neoplasm excluded from the study? Inclusion and exclusion criteria section? It should be clearer or deleted.\n\nResults: This section is well-written, and the statistical analysis and interpretation are precise.\n\nIn the discussion part, ''Prior clinical research has demonstrated that, whereas males and females share a comparable sickness rate, males are more likely to be hospitalized, experience a more severe illness progression, and die at a higher rate than females'' - is this comment a criticism or an interpretation?\n\nConclusions: Well written and accurately reflects the author's findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "10012", "date": "09 Aug 2023", "name": "Mohammed Yahya", "role": "Author Response", "response": "Thanks you for your time to review our manuscript In the introduction part 1. Regarding the introduction part we will make the required modifications which will appear in the second version of the manuscript. In the methodology part 2. Regarding the sample size I would like to inform you that we used a total coverage sampling technique therefore we involved all COVID-19 patients in specific time manner and we use 1:1 case to control ratio because this is the least ratio that can be applied for sampling the case control study population and it's acceptable in many previous investigations. 3. patients with immune dysfunction or malignant neoplasm were excluded be uses such disorders will affect the immune Status of cases and controls and consequently will affect on the complement level including C4 and according this will lead to bias in the results therefore we excluded them from the cases and controls. 5. in the discussion part Prior clinical research has demonstrated that, whereas males and females share a comparable sickness rate, males are more likely to be hospitalised, experience a more severe illness progression, and die at a higher rate than females. This section is interpretative part." } ] }, { "id": "188619", "date": "16 Oct 2023", "name": "Andang Miatmoko", "expertise": [ "Reviewer Expertise Pharmaceutical Sciences" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript provides supporting data regarding the profiles of complement C in healthy subjects and COVID-19 patients with different severity of illnesses. Some information needs to be clarified or confirmed to improve the quality of the manuscript.\n\nPlease give more details about the roles of complement C in the progression of infectious diseases so the manuscript's main idea can be clearly defined. In addition, the authors have cited research that shows the profile of complement c in COVID-19 (Ref.8). So, what is this study's new information or added value? Please define it clearly.\n\nIn this study, the authors did not provide details of medication taken or consumed by the subjects. Please add the details since using different classes of antipyretics may affect the complement c levels.\n\nRegarding mild and severe cases, the authors defined them based on ICU and non-ICU cases. However, ICU patients often have comorbidities, causing poor prognosis of the disease. This study focuses on complement C and the severity level of COVID-19, so it needs specific data regarding the severity of COVID-19, for example, Ct values or any other COVID-19 markers.\n\nPlease describe when the blood sampling was conducted in the method section. In addition, the incubation period of infection may affect the levels of complement c, and it can be analyzed from the day patients suffered from the symptoms and the day they were admitted to the hospital. Please add the data about this. Or, could the author provide the references that the incubation period does not affect the levels of complement c.\n\nSince the profiles of serum complement levels between control and case, and mild case and severe case had different results, which is affected by the immune response, please add the data regarding blood neutrophils so the analysis can be carried out well.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/12-432
https://f1000research.com/articles/12-428/v1	21 Apr 23	{ "type": "Review", "title": "Water-energy-food nexus in the Great Lakes Region of Africa: Current status and prospects", "authors": [ "Willis Awandu", "Micah M. Mukolwe", "Elizabeth Lusweti", "Edwin K. Kanda", "Micah M. Mukolwe", "Elizabeth Lusweti", "Edwin K. Kanda" ], "abstract": "Water-energy-food (WEF) nexus is a concept that aims at integrating these three key economic drivers which are very crucial for the improving the livelihood of the people, general economic sustainable development and social well-being of the world population. The effective utilization and management of these key resources require in-depth planning assessment of symbiosis, competitions and concession with an essential contribution in the nexus. This paper aims at reviewing the WEF in the Great Lakes region of Africa with a focus in Kenya, Democratic Republic of Congo, Rwanda and Uganda. The status and prospects in each country has been assessed and the interconnection between the three fundamental resources together with the effects of climate change towards the security of these resources in the selected countries have been discussed. The review has found out that the selected countries are indeed WEF insecure since the impacts of climate change and poor infrastructure play a vital role in the insecurities highlighted. In the focused countries, it was realized that the existence of weak institutional and legal frameworks, political instability and poor infrastructure indeed hinders the regional attainment of the WEF nexus for sustainable economic development. This therefore calls for a dire need for the adoption of climate change adaptation and mitigation measures in the WEF nexus through an integrated and holistic approach in the Great Lakes Region of Africa.", "keywords": [ "WEF nexus", "Africa Great Lakes region", "WEF security", "climate change" ], "content": "Introduction\n\nThe water-energy-food (WEF) nexus demonstrates clearly an interlink among three interdependent resources in such a way that when one of the resources is affected by a certain action, the effect is felt in one or both of the other areas. These three sectors are very vital and beneficial for the human prosperity, growth in order to reduce poverty and sustainability [Mabhaudhi et al. 2018; El Bassam 2021]. The nexus has drawn the global attention of the scientific research and policy sectors since its inception. Intensive discussions have taken place concerning the WEF nexus during the Bonn 2011 Conference, in the Sixth World Water Forum in Marseille in 2012, during the Rio negotiations in 2012 and the Stockholm Water Week in 2014 [Allouche et al. 2015; Urbinatti et al. 2020]. The concept highlights that there exists an inherent relationship between production and consumption chain of water, energy and food resources [Bijl et al. 2018]. The idea has been intensively conceptualized and debated upon by the experts but not yet incorporated amongst the local communities’ practices.\n\nWEF particularly tends to establish the connectedness such as interlinkages, symbiosis and trade-offs between the components which were considered separately [FAO 2014; Mabhaudhi et al. 2018; Mahlknecht et al. 2020]. For instance, water is required for the generation of hydropower, biofuel production, cooling processes in the nuclear reactor plants, etc. Similarly, water is essential for human survival as it is vital for agricultural production through irrigation, food production, drinking water services. Furthermore, consumption of energy occurs in pumping water for food and irrigation, water extraction and distribution, desalinization, water purification, wastewater treatment and drinking water supply such as in long-distance water delivery from the source [Mahlknecht et al. 2020].\n\nThe WEF existence is subjected to external forces that are geared towards destabilizing the core interactions. The impacts of climatic conditions such as climate change has degenerated the WEF nexus stability such that food, water and energy resources availability is greatly influenced through agricultural output, available water for supply, and industrial cooling and heating demands [Mukhtar et al. 2019]. Already alteration in precipitation patterns is being experienced worldwide as a result of shifts in climatic conditions which is intensified by climate change, with decreased precipitation patterns causing limited access to water in river basins and reduction in groundwater storage capacity thereby leading to severe drought and hunger [Allouche et al. 2015]. Due to the severity of climatic conditions, it was predicted that close to 75-250 million people in Africa would be greatly affected by increased water stress by the year 2020 [IPCC 2007]. This scenario is currently peculiar in East Africa with Kenya’s northern population subjected to extreme drought and hunger in the past three years.\n\nA decrease in rainfall amount reduces the rain-fed agricultural production as the declined rainfall amount results in unavailability of water for food as well as energy production [Lin et al. 2020]. Water stress situations and longer dry periods are a common phenomenon due to less precipitations worldwide with greater effect experienced in the developing countries. Irrigated agriculture and livestock production is negatively impacted by the hitch of prolonged droughts and heat stress. There are individual storms of higher intensity that has resulted to property damages, deaths and loss of biodiversity (recent floods in Nigeria, floods in Germany in July 2021).\n\nWith the exponential human population and economic growth, there is already an intense pressure from the growing demand of satisfying the human needs by means of utilizing the scarce economic resources in supply. There is a disproportionate human utilization of economic resources with continents that are rich and economically stable consuming more resources than the poor continents in the south [UNEP 2016].\n\nDeveloping countries are well endowed with natural resources and their involvement in the international trade contributes significantly to their gross domestic product (GDP). However, due to market control by the western developed countries, these developing countries suffer a great deal from poverty, inequality, conflicts, political instability and insecurity [Ross 1999]. Further challenges such as cross-border and rural-urban migration problems have been on the rise as the population try to look for livelihood in perceived endowed areas. The WEF nexus alignment to sustainable development goals (SDGs), for instance goal number 2 on zero hunger, goal 6 on clean water and sanitation, and goal 7 on affordable and clean energy are likely to benefit the developing countries especially the Great Lakes region of Africa when the resources are well managed using the integrative approach that WEF nexus provides [Mabhaudhi et al. 2018].\n\nThe paper by Kanda et al. [2023], a study review of the Horn of Africa with focus on Djibouti, Somalia, Eritrea and Ethiopia, revealed that the focus countries are indeed WEF insecure. Considering the geographical location, the Horn of Africa has a considerable difference from the Great Lakes Region of Africa in terms of water availability and development. This review aims at compiling documentation about the existing information and knowledge on water, energy and food nexus in the Great Lakes Region of Africa. The study focuses on four countries in the region namely; the Republic of Kenya, The Democratic Republic of Congo (DRC), the Republic of Rwanda and the Republic of Uganda. The general overview of WEF at global scale and narrowing down to the Great Lakes region of Africa with focus countries is discussed.\n\nCurrent status such as the relationship between nexus and WEF securities, the challenges facing WEF in the African Great Lakes region and the prospects are presented. This has been selected to provide an overview aimed at promoting viable interventions in combating the crucial problems associated with WEF sectors using integrated approach other than providing sector specific solutions.\n\n\nMethods\n\nThe review was conducted using evidence-based search on water, energy, and food nexus in the Great Lakes Region of Africa. This involved search conducted on the Web of Science, Scopus and Google Scholar databases. Further databases that are hosted by Organizations such as the World Bank, Food and Agriculture Organizations and World Food Programme were used. The search strategy utilized was adopted from Kanda et al. [2023] in order to screen the relevant literature materials for use in this study.\n\nThis study concentrated mainly on the major aspects of WEF to enhance the understanding of the status and prospects of the topic matter in the focus countries. The review was not exhaustively done considering the many literatures that exists on the subject matter and therefore it was cumbersome to exhaustively utilize the whole literature available. The outcome of the study review is expected to broaden the knowledge base of the study aspect which can form a basis for providing the much-needed solutions to the challenges being faced by the water, energy and food sectors in the region under consideration.\n\nThe Great Lakes Region of Africa (GLRA) has an approximate area coverage of 850,000 km2 surfaces of lakes, rivers and wetlands. This is approximately one-quarter of the earth’s area which is covered by the freshwater surface [Chimatiro et al. 2021]. GLRA has no universally defined boundaries, and it is rich of major lakes that traverses freely through the region [Cowx and Ogutu-Owhayo 2019; Chimatiro et al. 2021]. The region has a population of approximately 287 million people that depends highly on fishing and other agricultural related activities [Lowe-McConnell 2003].\n\nThe Figure 1 shows the map of the GLRA and the major lake basins and the coastlines found within the region.\n\nThe focus countries in this study are Kenya, Uganda, the Democratic Republic of Congo and Rwanda.\n\nThe DRC is located in the GLRA in Central Africa bordered with nine other Sub-Saharan Africa (SSA) countries [Hassan and Tularam 2018]. The country enjoys the fame of being the largest in SSA, having a landmass area coverage of 2.34 million km2 with vast deposits of natural resources for example; oil, diamonds, copper and cobalt [Hassan and Tularam 2018]. The country serves as Africa’s greater water catchment tower having large amount of both groundwater and surface water reservoir respectively [UNEP 2011]. The country has a population of about 96 million people and thus numerated as the third most populated country in SSA behind Nigeria (214 million) and Ethiopia (120 million) people respectively [World Bank 2022a]. Poverty is widespread in DRC with more than 65 % of the population living below the poverty line ($2.15/day), and it was ranked as the world’s poorest countries [UNDP 2013].\n\nThe southern highlands of DRC experiences a cool and dry climate, whereas the alpine climatic conditions are experienced within the Rwenzori Mountains, with a hot and humid climate in the river basin [Kazongo 2016]. The rainfall amount being received in the country lies between 1000-1700 mm, with annual temperature ranges of between 19-31°C [Hassan and Tularam 2018]. There are occurrence of periodic climatic events such as seasonal flooding in the regions of the east and droughts in the south [Samba et al. 2008]. Majority of the population living in the rural areas heavily depends on agriculture and despite the visibly favorable climatic conditions in the DRC, the population is experiencing food insecurity situation as a result of the effects of climate change [Sonwa et al. 2012; Hassan and Tularam 2018; Dove et al. 2021], and efforts to reduce the poverty situation is very minimal due to the severity of the impacts posed by the climatic conditions [Hassan and Tularam 2018].\n\nRwanda is one of the East African countries with no connection to an ocean (i.e., landlocked) and covers an area of approximately 26,000 km2 [Muhire et al. 2015] and having the highest population density in the SSA [World Bank 2017]. Due to the political stability that is being enjoyed in the country after the 1994 genocide incident, the country’s population has exponentially grown to approximately 13 million and the growth is anticipated to continue through 2050 [Republic of Rwanda 2011; World Bank 2022a]. Rwanda experiences a tropical climate that achieves a mean temperature of approximately 20 °C with an average annual rainfall of 1000 millimeter [Ndayisaba et al. 2017; Austin et al. 2020].\n\nThe economy of Rwanda depends intensively on agriculture which is extensively rain-fed, with more than 75% of the population relying on agricultural activities for their livelihood; agriculture also contributes to a third of the country’s Gross Domestic Product (GDP) [Niyitanga et al. 2015; Austin et al. 2020].\n\nUganda is located within East Africa and is found within latitudes 1°30′S and 4°N and longitudes 29°30′ and 35°E [Nema 2006]. The country has a population of 46 million people [World Bank 2022a]. The country has a land coverage area of approximately 242,000 km2 and experiences a diversified climate; and receives an average annual precipitation of 1000-1500 mm in most parts of the country [Obua et al. 2010].\n\nKenya lies within East Africa and has a population of 53 million people [World Bank 2022a] and an area coverage of 580,367 km2. Kenya heavily depends on rainfall performance and distribution for her socio-economic activities [Huho et al. 2012], with over 60% of the activities being weather and climate dependent [Nicholson 2014]. Kenya’s economy is greatly driven by agricultural activities which constitutes about 25% to the real gross domestic product and provides the largest employment opportunity to the population as more than 70% of the population is employed or obtain their daily livelihood within the rural areas from agricultural activities which results to about 50% of the principal export earnings for the country [Kabubo-Mariara and Karanja 2007].\n\nThe climatic and ecological conditions of Kenya are very extreme traversing an altitude varying from sea level to over 5000 m in the highlands [Kabubo-Mariara and Karanja 2007]. The annual rainfall distribution in the country lies between 250 mm in the northern and eastern parts of Kenya also classified as arid and semi-arid land (ASAL) and 2500 mm in the highland and mountainous regions [Kabubo-Mariara and Karanja 2007].\n\nFew publications exist on the studies conducted relating to WEF nexus in the GLRA unlike research articles relating to Southern Africa region with South Africa dominating the studies undertaken. Different countries in GLRA have also conducted varied interrelations with regards to the nexus and thus difficult to connect the progress.\n\nStudy conducted in Rwanda by Johnson and Axelsson [2017] while investigating the role of hydropower for the country’s electrification plans revealed water insufficiency for the anticipated hydropower project plan as its expansion would lead to low water supply to cover the expected water demand in the country by 2026. Also the case study of Kenya explored similar trends observed in the SSA on the main factors contributing towards the deficiency of WEF like exponential population growth, extensive urbanization coupled with booming economy, and climate related vulnerabilities [Wakeford 2017; Okumu et al. 2021; Awandu et al. 2022a; Kanda et al. 2023].\n\nMukuve and Fenner [2015] case study of Uganda analyzed beyond the WEF by incorporating the soil nutrient nexus into the mix for better understanding of the nexus ability in tackling the food insecurity. Their study found out an existence of scarcity throughout the resources considered: water, energy, food and soil nutrients considering the food demand from source to service by incorporating the transformation of resources in the calorific demand analysis. The researchers encouraged the involvement of stakeholders in the process for legitimate and productive outcome.\n\nTrigg and Tshimanga [2020] studied the Congo Basin and outlined its importance as an ecosystem that is not only very large but also having not been well understood by its uniqueness. The researchers pointed out that despite the basin’s potential of supporting millions of livelihoods, very little information of its ecosystem service is known. Similarly, Tshimanga and Hughes [2012] noted that due to the impacts of climate change in the basin which has resulted to rainfall seasonal changes and temperature distributions among others, the vulnerability of the population within the Congo Basin has negatively influenced the subsistence economy of the country.\n\n\nThe WEF nexus securities\n\nThe Great Lakes Region of Africa (GLRA) has an approximate area coverage of 850,000 km2 surfaces of lakes, rivers and wetlands, which is approximately one-quarter of the earth’s area covered by the freshwater surface [Chimatiro et al. 2021]. GLRA water catchment is traversed with major lakes that flow freely through the region [Cowx and Ogutu-Owhayo 2019; Chimatiro et al. 2021], and having the largest diversified freshwater ecosystem rich in aquatic biodiversity [Darwall and Vie 2005]. Millions of people in this region derive their livelihood from this well pronounced and diversified ecosystem [Loiselle et al. 2014].\n\nThe total renewable water resources of the region is about 940 km3/year, out of which the surface renewable water accounts for about 83% of GLRA renewable resource (774.4 km3/year) and the rest represents the renewable ground water resources [FAO 2022].\n\nKenya’s land cover suffers from water stress occasioned by both physical and economic water scarcity and vast proportion of the land mass comprise of arid and semi-arid lands (ASAL) [Ngigi 2002; Koech et al. 2020]. The irregular rainfall distribution both spatially and temporally has greatly accelerated the water stress situation in the country [Ahmed and Ndonye 2022]. The average precipitation in GLRA Is approximately 1200mm/ year, and the distribution ranges from 120 mm/year within Marsabit region in Kenya’s ASAL area to 1500 mm/year in Tshopo, DRC [Ashouri et al. 2015; FAO 2022].\n\nThe focus countries have a production water footprint of 5 billion m3/year of which agricultural activities contributes the most (46%) followed by Municipal water withdrawals (43%) then industrial usage at 11% [FAO 2022]. This production water footprint is more or less on a higher value when compared with the global freshwater withdrawal in similar sectors which amount to 53% (agriculture), 18% (industrial) and 29% (municipal) usage [Mahlknecht et al. 2020].\n\nWith the anticipated world population growth and higher proportion in the African countries, the countries within the GLRA as well as the greater Sub-Saharan Africa should brace for severe water stress situation resulting from additional demands and extreme pollution of the available water sources. Climate change impacts are expected to worsen the situation due to extreme drier conditions and severe variability in the hydrologic cycle [Bates et al. 2008]. Connor [2015]; UNICEF and WHO [2017] advocate for improved universal provision of drinking water and sanitation services through strengthening the water governance and holistic management of the water resources.\n\nAkom et al. [2020] and Boulle [2019] clarify that access to affordable and reliable energy results to an improved living standards of the households and enhanced socio-economic endeavors. Records found within the international organizations mandated with the energy accountability shows that the energy accessibility has improved globally in the recent years with economically stable countries achieving the 100% energy access by households [IEA et al. 2019; World Bank 2022a]. The scenario is different in the developing countries with more than 80% of the population still lack this basic commodity, with the highest proportion found within the Sub-Saharan Africa [IEA et al. 2019; World Bank 2022b]. Production and manufacturing industries and processes require intensive energy input with the industrial sectors representing 50% of the energy consumption. Innovative measures are paramount in the current energy situation to help lower the global temperature to 2 °C [IPCC 2007] as a potential pathways for society to achieve the Paris Agreement goals by striving to achieve the Net Zero Emission by 2050 [IEA 2021].\n\nGenerally power generation requires intensive water use through diversion of flow to create potential energy thereby abstracting water from the main water course and water consumption as a result of evaporation on the surfaces of the dam or during transportation [Falchetta et al. 2019]. Energy generation globally utilizes over 90% of the surface water [UNESCO 2014]. Sub-Saharan Africa is classified as hydropower-dependent continent as majority of the countries majorly rely on 50% of their total energy generation from hydropower and the region hosts close to 45% of the total population who depends entirely on hydropower grid connection [Falchetta et al. 2019]. Over-reliance on hydropower sources have a greater potential to power crisis due to prolonged droughts [IEA 2022a].\n\nKenya’s electricity generation consists of geothermal (44%), hydropower (33%) and diesel-powered (21%) energy sources [Boulle 2019]. The energy mix comprises of 68% biomass, 22% petroleum and 9% electricity [Tashtoush et al. 2019]. There is a greater potential of wind energy generation in Kenya and the exploration of its potential has accelerated in the recent past leading to the development of various wind farms in the country [Masyuko 2022]. Kazimierczuk [2019] review on the wind energy status and policy framework confirms a substantial development of wind power farms in Kenya with Lake Turkana Wind Power plant project being the largest with a potential of 310 MW. Other wind power farms include: Kipeto Wind power (100 MW), Isiolo Wind Power (100 MW), Meru (60 MW), Ngong (52 MW) and Baharini Wind Farm project in Lamu (90 MW).\n\nMajority of the households in the rural Kenya mainly depends on charcoal and firewood for their energy supply [Awandu et al. 2022a], the trend that has led to an extensive deforestation in major parts of the country.\n\nThe Democratic Republic of Congo has a high potential of hydropower concentration located within the Grand Inga River with potential capacity of 44 GW sufficient to supply substantial power demand of the greater SSA region [Taliotis et al. 2014; Green et al. 2015]. Irrespective of the great potential of the Grand Inga River hydropower project and the existing hydropower sources, a greater population in the DRC still depend heavily on wood fuel especially in the rural areas as their main energy source, severely contributing to the detrimental impacts such as deforestation contributing to severe environmental degradation [Kusakana 2016]. The energy consumption in DRC is highest in the residential sector for household uses like cooking and lighting corresponding to 77% of the total energy. The industrial sector comes second with a consumption share of about 20% [Kusakana 2016].\n\nRwanda has in the recent past experienced energy crisis emanating majorly from insufficient investment in energy sector. The ongoing urban industrialization has pushed the energy demands such that the existing energy resources have become insufficient, costly and affected by instability [Safari 2010]. There are massive untapped renewable energy sources in the country such as wind, methane gas, biomass, solar and geothermal which are under exploitation [Lujara and Kaunde 2007; Munyaneza et al. 2016]. All that notwithstanding, the country’s electric power generation capacity is considered lower than her potential and the access level is also limited [Munyaneza et al. 2016; Okumu et al. 2021].\n\nWith the creation of Electrogaz in 1976, the publicly owned company in Rwanda serves only 4.3% of the entire population with accessibility available to only 23% in urban areas and 1% in the rural areas. The energy consumption in Rwanda is about 30kWh/year/inhabitant. This constitutes the use of wood fuel which is approximated to constitute 80% of the total energy consumption. Close to 20% of the total population of Rwanda depends on wood energy for lighting and 99% as source of cooking fuel [Safari 2010].\n\nThere is dire need of sourcing alternative renewable energy sources, which is currently the nation’s top most agenda. The commissioning of the micro hydropower development with a potential capacity of 6.4 MW is a clear indicator of the country’s commitment to reduction of rural energy poverty [Safari 2010].\n\nUganda energy source is mainly derived from hydropower which accounts for 90% of the energy-mix. However, the energy utilization among the population is comprised of 95 % of biomass-based energy sources – wood, charcoal and agricultural residues [Adeyemi and Asere 2014]. This over-reliance on biomass energy sources has greatly contributed to the declining forest cover in the country and hastening the rate of desertification in the dry lands of Uganda [Byakola 2007].\n\nUganda’s per capita electricity consumption is 72 kWh/year/inhabitant and this is due to high energy tariffs compared to the neighbouring countries [Byakola 2007]. Wind, solar and geothermal energy are underdeveloped, with the energy policies in place geared towards the use of modern, clean and energy efficient technologies [Adeyemi and Asere 2014]. The country’s vision 2040 approximates the achievement of 12,700 MW of energy from their available renewable sources [Republic of Uganda 2013].\n\nDue to the high costs of traditional fuel sources in use, energy insecurity together with the need to combat the greenhouse gas emissions due to over-reliance on fossil fuels, the GLRA just like the greater SSA has had paradigm expansion of heavily relying on construction of new dams even though there exists other cost-effective pathways of utilizing the existing large and cheap untapped hydropower potentials [Wu et al. 2017]. However, this paradigm shift need further assessment to enable the creation of alternative pathways that would utilize the alternative energy sources available having a potential to cover close to 866 TWh electricity demand for the vision 2030 [Barasa et al. 2018].\n\nThe adoption of micro and pico hydropower technologies that utilizes the kinetic energy of the flowing rivers [Güney and Kaygusuz 2010] having minimal to no changes to the riverine regime could be adopted for off-grid rural electrification [Vermaak et al. 2014; Awandu et al. 2022b]. Such technologies are compact, reliable, cost-effective and portable and do not require major civil engineering works on the river courses, thereby causing no potential harm to the river ecosystems [Yah et al. 2017]. The technology can be used in hilly areas where the development of conventional hydropower plants might not be feasible [Johnson and Pride 2010].\n\nFigure 2 below summarizes the GLRA total energy supply and their sources. It is evident from the figure that the larger population heavily rely on biofuels as energy supply.\n\nFood security is a measure of the accessibility, availability and quality provision of safe food for consumption by the population [Bernard 2018; Awandu et al. 2022a; Kanda et al. 2023]. The food security status is attained when the population is capable of consuming sufficient safe and nutritious food for active and healthy living [Nkiaka et al. 2021]. Each state has the sole responsibility of ensuring provision of affordable and nutritious food to the citizenry and to ensure national food security for a healthy country and population [FAO 2023b].\n\nSub-Saharan Africa is generally food insecure with close to 300 million people living with severe food insecurity [Roser and Ritchie 2019]. An analysis of GLRA reveals that DRC severely suffers the food insecurity with 33 million people, Kenya (14 million) and Uganda (10 million) having access to one meal a day or none at all when no food aid is provided, while data is lacking for the Rwandan situation [FAO 2023a].\n\nCereals comprise of the most produced crops in the continent of Africa with great variations being observed across the different African countries, with corn, wheat and rice as the main crops doubling as the staple food in most countries [Saleh 2023]. GLRA experiences very low crop yields which has a negative influence on the food security in the region. For instance, Uganda has a crop yield of 2.24 t/ha followed by Kenya (1.81 t/ha), Rwanda (1.46 t/ha) then DRC (0.86 t/ha) [FAO 2021], a scenario that has led to malnutrition in the region as much of the population has no access to sufficient food [Roser and Ritchie 2019].\n\nAgricultural production commands a bigger share in contributing towards the balance of payments for African economies [Giller 2020; OECD 2023]. The agricultural practice is mainly rainfed and therefore susceptible to the adverse effects of climatic conditions [Hjelm et al. 2015]. The availability of water both of good quality and sufficient quantity is critical in agriculture and consequently impacting on the food security [Awandu et al. 2022a].\n\nThe access to food supply in DRC is attributed to the militia conflicts in the region on the local institutions and governance. Due to the constant attacks by the militia group in the Congo forest, there is a general lack of security that prohibits agricultural food production [Alinovi et al. 2007].\n\nKenya’s agricultural production and crop yield severely suffered from drought and extreme dryness that has affected the country for the last three years causing acute shortage of food and water in the country [Ahmed and Ndonye 2022]. Pastoralists are the hard hit with the severe drought as they suffer extreme hunger and loss of lives and animals for three years in a row.\n\n\nChallenges of WEF nexus in Great Lakes Region of Africa\n\nThere is clear understanding within the GLRA of the WEF concept as a tool to understand the interdependencies among the three systems and sustainably manage the resources in meeting the growing demands.\n\na) Concerted efforts have been put in place to address the emerging conflicts in the WEF sectors. However, there is lack of attention particularly the institutions and politics governing the sector [Scott 2017].\n\nb) The conflicting roles of WEF players at all levels in the GLRA negatively influences the coordination and implementation. This results in arrays of confusion and disagreement on the roles and responsibilities of the various institutions involved [Scott 2017].\n\nc) There is lack of concerted efforts on development of policy frameworks that guide on the use of shared water resources. Since each state tends to pursue unilateral policies, this tend to degenerate into conflicts as is the case in of Ethiopian Dam Mega Project [Kanda et al. 2023].\n\n\nProspectus of WEF in the African Great Lakes region\n\nDespite the challenges that hinders regional attainment of the WEF nexus, there are considerable efforts that has been put in place either individually or as collaborations to aid in developing resilience as far as effects of climate change and other associated issues are concerned.\n\na) Kenya’s energy reform policies has enhanced the adoption of friendly policies that has accelerated the transition of clean energy adoption [Kazimierczuk 2019].\n\nb) Through the formulation of policies and sector coordination for the adoption of modernization of agricultural practices such as use of modern irrigation techniques is geared towards sustainable agriculture and resilience in the wave of climate change [Kanda and Lutta 2022].\n\nc) Establishment of comprehensive policies for effective and practical management of disaster, promotes prevention, preparedness, mitigation, responses and recoveries during unforeseen events thereby enhancing resilience to cope with the adverse climatic situations.\n\nd) The urgent call for the increased forest covers as to mitigate the adverse effects of deforestation is of importance as this will reduce the risks of extreme desertification. A good example is the presidential directives and Kenya’s Government commitment to plant about 2 billion trees by the year 2022 [Ngounou 2020].\n\n\nConclusion\n\nThe purpose of this review article was to enhance the understanding of the WEF nexus status and prospects in the Great Lakes Region’s countries of Democratic Republic of Congo, Kenya, Rwanda and Uganda. The study has revealed that there is general insecurity of water, energy and food in the focus countries discussed which has a corresponding trend in the whole region. The challenges are arising from the climate and human induced stresses that significantly affects their usefulness for the well-being of the ecosystem.\n\nThere is general impact of the WEF nexus in the region by climate change resulting from droughts that contributes to reduction of energy, food and water availability and on the other hand excessive precipitation that leads to flooding and destruction of property leading to losses and deaths.\n\nThere is need for concerted efforts in improving the energy access and availability by utilizing low-cost technologies to provide isolated rural areas with electricity. The use of hydrokinetic river turbine is a promising technology considering that these regions have plenty of free-flowing rivers whose kinetic energy can be fully utilized for electricity generation. Due to the irregular rainfall distribution being experienced in the focus countries, there is need for rainwater harvesting and storage options that would enhance water availability for irrigation purposes.\n\nA comparative analysis of the this review paper and with the study by Kanda et al. [2023], though having considered two different regional blocks within the SSA and having different level of development and WEF nexus interlinks reveals that both the regions are severely WEF insecure in their own capacities. However, a holistic view can be drawn in terms of WEF for the further scrutiny and possible concerted efforts to provide solution that will result to a common objective (considering bordering countries).", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAdeyemi K, Asere A: A review of the energy situation in Uganda. Int. J. Sci. Res. Publ. 2014.\n\nAhmed I, Ndonye S: Drought situation in the Kenya ASAL areas now at crisis level. 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Publisher Full Text" }	[ { "id": "181914", "date": "04 Jul 2023", "name": "Hubert Hirwa", "expertise": [ "Reviewer Expertise Water", "energy", "food", "environment", "and health nexus", "Dryland ecosystem management and food security", "Sustainable agriculture" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is conducted based on the concept of the Water-Energy-Food (WEF) nexus and encourages researchers and practitioners on the benefits of using such a concept. It is not clear however to understand what is the nexus about and how it should help the Greater Lake Region of Africa (GLRA) to better analyze the interactions between water, energy, and food components. This study is based on the definition of the WEF nexus in GLRA. The analysis of existing tools to monitor WEF in entire Africa, as well as the water-energy-food-ecosystem-health (WEFBH) nexus in the Greater Horn of Africa, have been conducted. My suggestion, I wish the authors can apply new innovative tools to discover what is hidden from the WEF system. The methodology is not clear. How many papers have the authors selected? How did they go through all the kinds of literature to get tangible results? Therefore, socio-economic and biophysical factors are known. Here, the authors elucidated the current situation and how are they connected. For instance, considering food security (i.e., availability, access, utilization, stability) is a vast topic. It is better to choose one of the four components in terms of the WEF nexus.\nHerein recently published articles that can help the authors to identify the gap and problem statement:\nMuhirwa F., Shen L., Elshkaki A., Chiwuikem C. J., Zhong S., Bonecke E., Hirwa H., Seka M. A., Habiyakare T., Tuyishimire A., Harerimana B., (2023). Alert in the dynamics of water-energy-food production in African countries from a nexus perspective. Resources, Conservation & Recycling 194 (2023). 106990. https://doi.org/10.1016/j.resconrec.2023.106990\n\nImasiku K. and Ntigwirumugara E., (2019). An impact analysis of population growth on energy-water-food-land nexus for ecological sustainable development in Rwanda. https://doi.org/10.1002/fes3.185\n\nHirwa, H., Zhang, Q., Qiao, Y., Peng Y., Leng F., Tian C., Sayidjakhon K., Li F., Kayiranga A., Muhirwa F., Itangishaka A. C., Habiyaremye G., and Ngamiije J., (2021). Insights on water and climate change in the greater horn of Africa: Connecting virtual water and water-energy-food-biodiversity-health nexus. Sustainability 13, 6483. https://doi:10.3390/su13116483\n\nMuhirwa F., Shen L., Elshkaki A., Velempini K., and Hirwa H. (2022). Tracing attribute and scope of research and applied projects in Africa’s water energy food nexus implementation: A review. Environmental Science and Policy 136, 33-45. https://doi.org/10.1016/j.envsci.2022.05.012.\n\nMuhirwa, F.; Shen, L.; Elshkaki, A., Kgosietsile V., Hirwa, H., Zhong S., and Mbandi A. M., (2021). Decoupling Energy, Water, and Food Resources Production from GHG Emissions: A Footprint Perspective Review of Africa from 1990 to 2017. Energies 2021, 14, 6326. https://doi.org/10.3390/en14196326\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] }, { "id": "189069", "date": "25 Jul 2023", "name": "Rodrigo A. Bellezoni", "expertise": [ "Reviewer Expertise Environmental/Energy Engineering", "Environmental/Energy Planning and Policy", "WEF Nexus", "Supply-chains", "Traceability", "Territorial Intelligence", "Sustainability", "Climate Change" ], "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 aims at reviewing the WEF in the Great Lakes region of Africa with a focus in Kenya, Democratic Republic of Congo, Rwanda and Uganda. However, the text is limited to listing the characteristics of the study sites. Thus, the linkages between the systems for water, energy and food (or land use) are not explored by the authors. For example, when listing the main crops grown in a given region, it is crucial to explore the importance of this crop for local food security and, above all, to critically analyse how this crop relates to or impacts water (through irrigation) and energy resources (direct and indirect consumption for food production, e.g. fuels, fertilisers, irrigation, etc). Working with these numbers gives us a dimension of the interdependencies between different systems, in order to identify synergies and trade-offs (e.g. by promoting crop X, we are using a large part of the water from an important water reservoir that also has a function for the energy system - hydropower generation. Thus, greater irrigation and production of this crop, in order to meet food security objectives, could jeopardise hydropower generation objectives (energy transition goals), due to the former reducing inputs for the latter. Similarly, less hydropower could lead to higher thermal energy use and therefore higher associated emissions (jeopardizing climate goals). This would result in electricity-dependent agricultural production (e.g. for irrigation) produced from fossil sources; jeopardising the sustainability of the system and therefore generating undesirable trade-offs).\nTherefore, integrated analysis of the WEF nexus depends on this type of information, as well as information on who is responsible for decision-making in each of these systems and why decisions are being made in isolation, without considering the effects of one system on the others. Such consideration leads us to an analysis of the actors and institutions involved in managing the elements of the nexus.\n\nIn this context, the article needs to be rewritten considering the logic and elements highlighted above, among others, in order to actually contribute to issues concerning the WEF nexus in the region of interest. As it stands, it does not consider interlinkages between systems and actors and, therefore, does not deal with an integrated analysis, a fundamental precept for studies on the WEF nexus.\nAs for the literature review, it is important to describe the review process, presenting the steps and terms used in the queries, in order to limit bias in the choice of reference texts. In addition, I would recommend considering including some interviews with key actors on the WEF nexus topic in the regions of interest, in order to identify working routines in these departments, as well as to hear a little about the main constraints to promoting integrated policies for the WEF nexus in these locations.\n\nFundamental to nexus studies is fostering synergies and reducing trade-offs between the systems of interest. Therefore, it is critical that these elements are a central part of the next version of the manuscript, in guiding discussions and conclusions towards operational, logistical and/or policy recommendations for the WEF nexus in the Great Lakes region of Africa.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? No\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? No", "responses": [] }, { "id": "189051", "date": "14 Aug 2023", "name": "Krishna Mondal", "expertise": [ "Reviewer Expertise Water-energy-food nexus 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\nI have read with interest the paper entitled “Water-energy-food nexus in the Great Lakes Region of Africa: Current status and prospects”. The paper reports upon reviewing the WEF in the Great Lakes region of Africa with a focus on Kenya, the Democratic Republic of Congo, Rwanda, and Uganda. I commend the authors for their hard work.\nUnfortunately, I have several major concerns regarding the relevance and rigor of the scholarly contributions offered by this research. I see significant room for improvement, which will help to enhance clarity, readability, and coherence. Below I articulate my concerns and provide constructive suggestions that I hope will be instruments to develop a clearer paper.\nAbstract\nAlthough there is a lot of review paper in the literature, the authors should briefly explain the necessity of carrying out such a study. However, the abstract fails to demonstrate the novelty of this paper clearly.\nIntroduction\nCitation Usage and Context: Some citations (e.g., Ross 1999) lack context, making it unclear how they relate to the discussion. Ensure that each cited work is properly integrated into the narrative and its relevance is explained.\n\nConciseness and Redundancy: Some parts of the introduction are slightly redundant and could be streamlined to maintain concise and clear communication.\n\nCitations for Specific Claims: Claims such as \"close to 75-250 million people in Africa would be greatly affected by increased water stress by the year 2020\" (IPCC 2007) could benefit from more direct references to the exact page or section in the cited source.\n\nClarity in Objectives: The introduction could be clearer about the specific objectives or research questions the review paper aims to address. Presenting a more explicit research question or purpose statement would enhance the reader's understanding of the paper's focus.\n\nLocal Communities' Practices: The introduction mentions that the WEF nexus has not yet been incorporated among local communities' practices. Consider expanding on this point or providing context for why this gap exists and is relevant.\n\nLocal and Global Focus: While the paper mentions the Horn of Africa, it might be helpful to clarify why the focus shifts to the Great Lakes Region and the specific rationale behind selecting the countries within that region.\n\nOverall Structure: Consider restructuring the introduction to move the discussion of the specific review paper's aims and objectives closer to the beginning, allowing readers to understand the scope and purpose early on.\nThe introduction can be further refined by addressing these suggestions to enhance its clarity, coherence, and impact.\nMethods\nApproach: The approach is not transparent on how you select the research article, how many articles you selected for this research, and the proper methodology is missing.\n\nStatistics: A statistical table needs to show the number of studies in the last ten years in different countries of the Great Lakes Region of Africa.\nBy addressing these suggestions, the methodology can be further robust.\nConclusion\nSynthesis of Main Findings: While the conclusion touches on the challenges and impacts, consider synthesizing these findings more explicitly. Summarize the key challenges and their overarching implications in a concise manner.\n\nQuantitative Information: Whenever possible, consider incorporating quantitative data or statistics to reinforce the severity of the challenges and the potential impact of the proposed solutions.\n\nPolicy and Practical Implications: Discuss the potential policy implications of the findings and recommendations. How might governments, organizations, or communities use this information to make informed decisions? Otherwise, you can separate make a separate section, “Policy and Practical Implications”. You can refer to these two articles:\nhttps://doi.org/10.1016/j.envsci.2022.12.018\n\nhttps://doi.org/10.1002/ird.2800\n\nFuture Research Directions: Mention any potential avenues for future research that have emerged from the current study. This could help guide the reader towards areas that require further investigation.\n\nLanguage and Clarity: Some sentences could be rephrased for enhanced clarity. For example, the sentence \"Due to the irregular rainfall distribution being experienced in the focus countries, there is need for rainwater harvesting and storage options that would enhance water availability for irrigation purposes\" might be clearer if rephrased.\nThe conclusion can be strengthened by addressing these suggestions to provide a more comprehensive and impactful summary of the review's findings, implications, and potential future directions.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-428
https://f1000research.com/articles/12-418/v1	18 Apr 23	{ "type": "Research Article", "title": "Preserved correlation matrices pinpoint extracellular matrix organization as a critical factor in pancreatic ductal adenocarcinoma", "authors": [ "Otília Menyhart", "Áron Bartha", "Balázs Győrffy", "Otília Menyhart", "Áron Bartha" ], "abstract": "Background Gene co-expression correlations frequently signal shared biological functions with coordinated regulation. We hypothesized that maintained correlations might be essential for cellular survival, representing potential vulnerabilities of cancer cells. We aimed to reveal correlations preserved in pancreatic ductal adenocarcinomas (PDAC) across normal and tumor tissues. Methods: We searched the NCBI GEO for raw microarray data and the TCGA project for RNA-seq data. The microarray dataset consisted of 248 tumors and 108 normal samples, allowing the analysis of 12,210 genes. The RNA-seq dataset incorporated 177 tumors, four normal samples from TCGA, and 248 normal samples from GTEx, enabling the analysis of 21,479 genes. Genes with an altered expression were identified with a Mann-Whitney U test at p<0.01, and a Pearson correlation was performed to identify preserved correlations. Results: Altogether 371 significant correlations involving 262 genes were preserved across normal samples and tumors in both RNA-seq and gene chip platforms. The identified close-knit gene network is mainly responsible for extracellular matrix organization. Seven genes (SPARC, COL6A3, MMP2, HTRA1, FN1, PALLD, and COL3A1) were heavily overrepresented in maintained correlations, some of them participating in as many as 58 interactions. High expression of 28 genes was linked to poor disease outcome at FDR ≤ 10%, out of which FN1, an extracellular matrix component, was both overrepresented in maintained correlations and associated with worse overall survival (p = 0.00097, FDR ≤ 5%). The growing expression of two genes, MYL12A and MYL12B, across normal tissues, primary, and metastatic tumors may drive the acquisition of motility by cancer cells. Conclusions: Our results propose novel prognostic biomarkers of PDAC and pinpoint fundamental cellular interactions as potential targets for combination therapies. Furthermore, the presence of significant correlations across different data platforms substantiates the validity of our findings.", "keywords": [ "PDAC", "pancreatic cancer", "FN1", "SPARC", "MYL12A" ], "content": "Introduction\n\nWith one of the lowest survival rates among any cancer, pancreatic cancer (PC) is a gastrointestinal malignancy with exceptional lethality. Despite its relatively low incidence rates, PC is the seventh leading cause of cancer-related death worldwide and the third in the USA after lung and colorectal cancer. The incidence rates are higher in more developed countries1 and display a rising tendency. By 2030, pancreatic cancer is predicted to be the second leading cause of cancer-related mortality in the USA.2 Approximately 90% of pancreatic cancer cases belong to pancreatic ductal adenocarcinomas (PDAC), arising from precursor lesions termed pancreatic intraepithelial neoplasia. The cause of the disease is multifactorial, but cigarette smoking appears to be one of the primary risk factors. However, alcohol consumption, obesity, aging, diabetes mellitus, and family history are significant contributors as well.3–5\n\nUnfortunately, most patients seldom exhibit symptoms at the early stages of the disease. The wide range of non-specific symptoms, including jaundice, back- and abdominal pain, and weight loss, hampers precise diagnosis. In addition, existing screening biomarkers, including serum levels of CA19-9, display relatively low sensitivity and specificity.6 As a result, 80-85% of patients are diagnosed with unresectable tumors characterized by regional spread or metastatic disease. Only 24% of diagnosed patients survive their first year, while five-year survival is as low as 10%. Currently, surgery is the only available potentially curative option.7 Based on the extent of the disease, patients are allocated into one of four categories: resectable, borderline resectable, locally advanced, and metastatic. Altogether, only 15-20% of patients are good candidates for pancreatectomy, and the five-year survival after surgery remains low at 25%. Adjuvant therapy after surgery, including gemcitabine plus capecitabine, or modified FOLFIRINOX (oxaliplatin, irinotecan, leucovorin, and fluorouracil), is the preferred option, as both treatment regimens improve overall survival.8,9 The combination therapies based on modified FOLFIRINOX, or gemcitabine with nab-paclitaxel for metastatic pancreatic cancer, became the gold standard.10–12 In the recent PRODIGE24-trial, a modified FOLFIRINOX regimen resulted in prolonged disease-free (21.6 vs. 12.8 months) and overall survival (54.4 vs. 35.0 months) compared with gemcitabine monotherapy. Nearly 20% of FOLFIRINOX treated patients were alive at 18 months compared to only 6% of those treated with gemcitabine alone.9 Locally advanced disease with vascular involvement is regularly treated with systemic chemotherapies approved in the metastatic setting. Despite standard chemotherapy, the vast majority of resected patients experience recurrence and die within a short period.\n\nDespite extensive molecular characterization, personalized treatment strategies of PDCA are still limited. About 5-9% of pancreatic cancer patients carry germline BRCA1 or BRCA2 mutations. In 2019, the FDA approved olaparib, a PARP-inhibitor, as maintenance therapy for metastatic patients with BRCA1/BRCA2 mutations who had not progressed during first-line platinum-based chemotherapy. Olaparib therapy improved progression-free survival in this setting compared to placebo and became the first approved targeted therapy for pancreatic cancer.13 Other approaches, including the adoption of immune-checkpoint inhibitors, did not yet translate into clinical benefits, as pancreatic tumors are characterized by highly immuno-suppressive microenvironments.14 The diverse response to treatment sparked initiatives for patient classifications, but classical histopathological features lack clinical utility.15 Based on the further genome-wide screening of transcriptional changes, PDCA has been classified into two,16 three,17 or four18 distinct molecular subtypes. Cross-comparison of these studies revealed reproducible biologic subgroups with some prognostic relevance, but the described molecular subtypes have not yet been validated or translated into successful therapeutic decisions.19 Nevertheless, principal drivers of pancreatic tumorigenesis have been identified, including KRAS, SMAD4, CDKN2A, and TP53.1,20,21 Because so few patients can have a successful surgery, and the number of therapies is limited, identifying novel potential biomarkers for early diagnosis and targeted interventions are of utmost importance.\n\nIn this study, our aim was to exploit gene expression alterations between normal and tumor tissue samples. However, instead of searching for genes upregulated in tumors, we approached biomarker selection from a novel perspective. In normal, diploid tissue samples, gene co-expression correlations frequently signal shared biological functions with coordinated regulation. On the other hand, malignant tumors frequently undergo abnormal genetic transformations, but some correlations remain consistent across normal tissues and tumor samples. We speculated that maintained correlations might be vital for cellular proliferation and survival; thus, identifying preserved correlations in gene expression across normal and tumor tissue samples can provide new targets for developing alternate therapies.\n\n\nMethods\n\nWe searched the NCBI GEO database (http://www.pubmed.com/geo) for raw microarray gene chip data focusing only on Affymetrix Human Genome U133A HGU133_2 array platforms. Out of the identified 19 datasets, studies based on cell lines and xenograft models were excluded. We have also removed experiments based on pooled samples. Duplicate samples were filtered by examining the expression profiles of the first 20 genes in each sample. In case of duplications, the first published version was retained. Samples with poor description and missing raw data were rejected.\n\nThe raw CEL files were MAS5 normalized using the Affy Bioconductor library in the R statistical environment (http://www.r-project.org).22 To reduce the batch effect, a second scaling normalization was performed to set the mean expression on each chip to 1000.23 The appropriate probe set for each gene was selected with the JetSet annotation and correction package.24 We integrated the established gene expression dataset into our previously established TNMplot.com platform,25 allowing future exploitation of the database.\n\nWe searched for RNA-seq-based studies from the TCGA project at the Genomic Data Commons (GDC) portal (https://portal.gdc.cancer.gov/) and the Genotype-Tissue Expression (GTEx) portal. Data from primary tumors and normal samples were obtained from the TCGA, while RNA-seq data from normal pancreatic tissue data were obtained from GTEx. Raw counts were normalized by the DESeq2 R package, after which a second scaling normalization was performed.26\n\nStatistical analysis and data processing were performed within the R software package (version 4.0.4). Genes with median expression below the cutoff value of 500 were excluded from subsequent analyses to reduce the chance of noise discoveries. Expression differences for each gene between normal and tumor tissues were compared with the Mann-Whitney U test. Statistical significance was accepted at p < 0.01. We used the Benjamini-Hochberg method to correct for multiple comparisons by calculating the false discovery rate (FDR). Pearson correlation analysis was performed on genes common across the two datasets. A Kruskal-Wallis test was conducted to compare gene expression across normal, tumor, and metastatic tissue samples. The ggplot2 R package was used for data visualization.27 For selected genes, survival analysis was performed by Cox proportional hazards regression using all cutoff values between the lower and upper quartiles of expression. In addition, Kaplan-Meier plots were drawn to visualize survival differences. Only genes performing at FDR ≤ 10% were considered significant for selecting the most robust biomarker candidates.\n\nFor the determination of functionally associated genes, we used the clusterProfiler28 R package and Enrichr, an open-source web-based gene enrichment analysis tool (https://maayanlab.cloud/Enrichr/), that integrates results from multiple libraries.29 Within Enrichr, the list of pathways related to our gene list was determined with the KEGG pathways option. Pathways with a Fisher exact test p-value < 0.05 and high combined score were selected. Biological processes associated with the selected list of genes were determined by GO Biological Process function. Only the top 20 significant terms were considered. The listed q-values are based on adjusted p-values corrected for multiple hypothesis testing using the Benjamini-Hochberg method. We used the CytoScape platform30 and the built-in stringApp31 for network analysis.\n\n\nResults\n\nThe combined microarray (GEO) dataset consists of 248 pancreatic tumors and 108 normal tissue samples, allowing the analysis of 12,210 genes. The RNA-seq dataset incorporates 177 tumors and four normal samples from TCGA, and 248 normal tissue samples from GTEx, enabling the analysis of 21,479 genes.\n\nEach dataset underwent multistep filtering: gene expression differences were assessed between tumor and normal tissue samples based on log2FC values. Genes with log2FC > 0.5 or log2FC < -0.5 from the GEO dataset, and genes with log2FC > 1 or log2FC < -1 from the RNA-seq dataset were incorporated into subsequent analyses. Altogether 6088 genes had altered expression at p < 0.01 across tumor and normal tissue samples in the GEO dataset, while 17,967 genes at p < 0.01 in the RNA-seq dataset. Genes with median expression below the cutoff value of 500 were excluded from subsequent analyses to reduce the chance of noise discoveries; therefore, the number of genes was reduced to n = 2,205 in the GEO dataset and n = 3,773 in the RNA-seq dataset.\n\nWe identified 309 common genes in both RNA-seq and microarray datasets with altered gene expression across normal and tumor tissues (Figure 1A). In general, more overexpressed genes were identified in tumors compared to normal tissues. Out of the 309 genes, 267 had increased expression in primary tumors compared to 42 genes overexpressed in normal samples, both in GEO and RNA-seq datasets. Genes with altered expression across normal and tumor tissues are graphically represented by a VOLCANO plot (Figure 1B).\n\nA Pearson correlation was performed to identify associations between the 309 differentially expressed genes and all genes in the gene list. We analyzed correlations in tumors and normal tissue samples separately, then common correlations were identified. We have filtered out correlations with a correlation coefficient below 0.5.\n\nAltogether, we have identified 371 preserved correlations involving 262 individual genes across tumors and normal tissue samples. These preserved correlations were robust, as were independently identified in both microarray and RNA-seq datasets with R > 0.5 or R < -0.5, and p < 0.01 (Supplemental Table 1). The network of significantly correlated genes is illustrated in Figure 1C. The most significant top 20 correlations in both normal tissues and tumor samples in each dataset are presented in Table 1.\n\nWe observed differences in the strength of preserved correlations between normal tissues and tumor samples. In normal samples, the strongest correlations involved mainly genes encoding collagens both in RNAseq and GEO data. Conversely, in tumors, we observed a shift in the strength of correlations. Genes associated with the regulation of focal adhesion and actin cytoskeleton (ACTR3, MYH11, MYL9, MYLK) along with members of the Heat Shock Protein Family (e.g., HSPA4, HSPH1, HSPA1B), gained momentum and participated in the strongest correlations compared to normal samples (Table 1).\n\nSeventy-nine genes from the identified 309 differentially expressed genes participated in preserved correlations, out of which 73 genes were upregulated in tumors compared to normal tissue samples (Supplemental Table 2).\n\nSome genes overexpressed in tumor samples were strikingly overrepresented among the 371 preserved correlations, including SPARC, COL6A3, MMP2, HTRA1, FN1, PALLD, and COL3A1, some participating in as many as 58 correlations (Figure 2A). Over 70% of all preserved correlations (263 altogether) involved at least one of these seven genes.\n\nOut of the 262 genes participating in 371 preserved correlations, high expression of 28 genes was associated with worse overall survival at FDR ≤ 10%. Out of these, 16 genes (DSG2, FN1, ACTR3, PKM, CAPZB, TSC22D1, COL4A2, ADAM10, ENO1, CLIC1, PARP12, PPP4R1, PSMC4, KRT18, MYL12A, and PSMD2) were upregulated in tumors compared to normal samples (Table 2, Figure 2B).\n\nOut of these 16 genes were upregulated in tumors compared to normal tissue samples. Genes in the boldface were overexpressed in tumors.\n\nAmong the upregulated genes the expression of ENO1 (p = 8.2E-06), DSG2 (p = 0.00021), CLIC1 (p = 0.00031) and ACTR3 (p = 0.00054) showed a particularly strong association with disease outcome (FDR ≤ 2%) (Figure 2B). The heavily overrepresented FN1 gene was also associated with worse overall survival at FDR ≤ 5% (p = 0.00097), providing promising biomarker candidates for subsequent clinical validations. According to a String functional network analysis, the FN1 protein has a central role in the associated protein network of upregulated genes (Figure 2C).\n\nThe 371 preserved correlations incorporated 79 of the initially identified differentially expressed genes, out of which the majority, 73 genes, were overexpressed in tumors compared to normal tissues. With the help of TNMplot.com, we have also compared the expression pattern of these genes between primary tumors and metastases. The expression of MYL12A increased most significantly in metastatic samples compared to primary tumors (Figure 3A). Of note, the expression of both MYL12A and MYL12B gradually intensified across normal samples, primary tumors, and metastatic tissue samples (MYL12A, KW p = 2.57E-11; MYL12B KW p = 1.38E-14). We have observed a similar pattern in seven additional genes, including ENO1 and CLIC1, that were previously identified as robust prognostic biomarkers of survival (Figure 3B).\n\nExpression differences are color-coded: red encodes increasing expression from normal to tumor and from tumors to metastatic samples, while green encodes decreasing expression (A). The expression of MYL12A, MYL12B, CLIC1 and ENO1 genes gradually increased from normal tissue samples to primary tumors and metastatic samples (B).\n\nThe close-knit network of 262 genes involved in preserved correlations is mainly associated with ECM organization and function (C). Gene ontology analysis involving the 79 differentially expressed genes from preserved correlations revealed similar top functions (D).\n\nWe subjected the 262 genes from preserved correlations to gene enrichment analysis. The top biological functions were Extracellular matrix organization, Extracellular structure organization, and External encapsulating structure organization (Figure 3C). The most significantly enriched terms based on KEGG Pathway analysis were Focal adhesion, Protein digestion and absorption, ECM-Receptor interaction, Tight junction, Proteoglycans in cancer, regulation of actin cytoskeleton, and AGE-RAGE signaling pathway in diabetic complications (Table 3A). According to the GO Cellular compartment analysis in Enrichr, 54 genes were significantly associated with the collagen-containing ECM (Supplemental Table 3).\n\nTop functions remained similar in both cases.\n\nThe ITGB1, COL1A2, COL4A1, COL4A2, and FN1 genes were predicted to be involved in multiple cancer-related pathways, such as ECM-receptor interactions, the PI3K-Akt signaling pathway, human papillomavirus infection, and small cell lung cancer.\n\nWhen we narrowed down the gene enrichment analysis to the 73 genes overexpressed in tumors participating in preserved correlations, the most significantly enriched biological functions and KEGG pathways remained similar (Table 3B, Figure 3D).\n\n\nDiscussion\n\nPancreatic cancer is one of the most lethal malignancies with limited options of therapies and biomarkers. In order to pinpoint survival-specific vulnerabilities, we aimed to identify upregulated genes with preserved correlations across normal and tumor samples. Previous research revealed profoundly different biomolecular networks underlying cancer conditions compared to healthy tissues.32 Notwithstanding, we speculated that sustained correlations might offer insight into fundamental biological functions, providing potential novel targets for therapeutic interventions. To increase robustness, we collected data from two independent platforms, including RNA-seq and microarray-based studies. Then, we identified genes with an altered expression between tumors and normal tissues and looked for correlations preserved across both data platforms.\n\nWe have identified 371 preserved correlations comprising 262 genes, including 79 genes with altered expression across tumors and normal samples. These preserved correlations were robust, as were independently identified in both microarray and RNA-seq datasets. Moreover, a few of these differentially expressed genes remarkably dominated preserved correlations, including SPARC, COL6A3, COL3A1, MMP2, HTRA1, FN1, and PALLD. Altogether, 16 genes upregulated in tumor tissues were linked to dismal disease outcomes, out of which the expression of MYL12A gradually increased during disease progression.\n\nUpon close examination, most of these genes are associated with the tumor microenvironment, participating in extracellular matrix (ECM) organization. PDAC has a dense fibrotic stroma, composed of abundant extracellular matrix and other non-neoplastic cell types, responsible for chemo- and radiotherapeutic resistance.33 The abundant ECM deposition, called desmoplastic reaction, plays a crucial role in PDAC. Beyond structural support, the ECM functions as a reservoir of growth factors and a mediator of complex signaling pathways.34,35 The loss of ECM homeostasis and integrity is one of the hallmarks of cancer, resulting in cancer progression36; thus, potential targeting and modifications of the ECM in PDAC are being intensely studied both in the preclinical and clinical settings.37,38\n\nOur results corroborate the importance of ECM organization in disease outcomes. Most importantly, we pinpoint critical interactions as potential targets for combination therapies. Fibronectin (FN1) is a principal constituent of the extracellular matrix that provides the scaffold to build up the entire ECM structure, affecting its architecture and composition. It is overexpressed in PDAC as part of the desmoplastic reaction, is associated with aggressive tumor characteristics,39 and functions as a primary driver of tumor progression by enhancing growth factor signaling.40 FN1 secreted by pancreatic stromal cells promoted gemcitabine resistance by downregulation of ERK1/2 phosphorylation.41 A synthetic FN1 blocking-peptide (Arg-Gly-Asp-Ser) abrogated the gemcitabine resistance in PDAC cell lines.41\n\nOur datasets identified both FN1 and the matricellular protein SPARC remarkably overrepresented in preserved correlations across normal and tumor samples. High stromal expression of SPARC was linked to poor patient prognosis in PDAC,42 and pancreatic stellate–cell-derived SPARC promoted proliferation of PDAC cells in vitro.43 Recent research indicated that the presence of plasma fibronectin determines the specific effect of SPARC on pancreatic cancer cells: depletion of fibronectin switched SPARC from promoting cancer cell proliferation to growth inhibition and induction of apoptosis.43 In our data both FN1 and SPARC expression were correlated with MMP2 in normal and cancer tissue samples. MMP2 is a member of the matrix metalloproteinase (MMP) gene family, primarily located in the tumor stroma, capable of cleaving collagens and other components of the extracellular matrix involved in signal transduction.44 In PDAC, MMP2 is among the most studied gelatinases, with a potential clinical relevance45: MMP2 expression is linked to the activity and the invasive potential of pancreatic cancer cell lines, also associated with the development of the characteristic desmoplastic reaction.46 The potential importance of MMP2 in PDAC progression is supported by studies using selective MMP2, MMP9, and MMP14 inhibitors in mice and Syrian hamsters, such as MMI-166, RO28-2653, OPB-3206, and SB-3CT.45\n\nThe most abundant component of the mammalian ECM is collagen. Collagen Type IV Alpha 2 Chain (COL4A2) encodes one of the six subunits of collagen type IV, a major structural component of basal membranes.47 In our dataset, COL4A2 expression was highly correlated with COL4A1, another type IV collagen alpha protein found in most connective tissues. In pancreatic cell lines and xenograft models of pancreatic cancer, type IV collagen production exceeded that of other ECM proteins examined, including collagen type I.48 The abundantly produced collagen type IV is deposited near the pancreatic tumor cells, creating a basal membrane-like structure.49 The structure colocalizes with integrin receptors on the cancer cell surface, stimulating the proliferation and migration of pancreatic cancer cells. Thus, type IV collagens provide essential survival signals to the pancreatic cancer cells through an autocrine loop.49 High circulating level of postoperative collagen IV has been linked to dramatically reduced survival in PDAC patients undergoing curative surgery.50\n\nSubstantiating previous findings, we found an association between high COL4A2 expression and poor prognosis, supporting the role of COL4A2 as a potential prognostic biomarker of PDAC. The drug candidate T12, a rationally designed inhibitor of collagen IV network formation, reduced both COL4A1 and COL4A2 expression and disrupted EMT-based chemo-resistance in mouse models of breast and lung cancers without apparent toxicity.51 Thus, targeting specific EMC proteins, including COL4A2 and COL4A1, may represent a novel therapeutic opportunity for treating PDAC.\n\nAnother protease, the ADAM Metallopeptidase Domain 10 (ADAM10), has also been heavily investigated by pharmaceutical companies. ADAM10 is ubiquitously expressed and cleaves many substrates involved in cancer signaling, such as PD-L1, NOTCH, EGFR/HER-ligands, among others.52 The evidence for ADAM10 as a suitable cancer biomarker increases: ADAM10 overexpression correlates with poor patient prognosis in cancers of the colon, lung, ovary, uterus, and stomach.53 Moreover, it has a substantial value as a prognostic biomarker in glioblastomas, sacral chordomas, and triple-negative breast cancer and became a potential target.52,54,55 In PDAC, ADAM10 was suggested to be a driver of tumor progression and fibrosis after radiation therapy. Pharmacological inhibition of ADAM10 delayed fibrosis and improved survival in radiation therapy-treated orthotopic and metastatic mouse models.56 Targeting the active ADAM10 with a monoclonal antibody, such as mAb 8C7, that masks the substrate-binding pocket preferentially located in tumors compared to normal tissue may offer targeted inhibition to prevent tumor growth.57\n\nOur results suggest additional insight into the evolving tumor microenvironment during disease progression. The expression of MYL12A was associated with poor prognosis and correlated with MYL12B upregulation within both normal and tumor samples. The expression of both genes increased during tumor progression, from normal tissues to primary tumors to metastatic samples. Myosin Light Chain 12A (MYL12A) and 12B (MYL12B) regulate smooth muscle and non-muscle cell contractile activity. Both genes are implicated in cytokinesis and cell locomotion,58 and intensifying expression levels indicate the acquisition of motility by cancer cells. The biological functions and rising expressions during cancer progression warrant the role of MYL12A both as a potential prognostic biomarker and a therapeutic target of PDAC.\n\nWe observed an increase in the expression of ENO1 and CLIC1 genes between normal samples and tumor tissue. Moreover, in our data, overexpression of both ENO1 and CLIC1 was associated with poor overall survival. Alpha enolase (ENO1) is a glycolytic enzyme localized on the cell surface with a complex role in cancer progression: ENO1 promotes glycolysis and ECM degradation, affects actin remodeling, facilitates tumor invasion, and migration, and activates cancer signaling pathways.59 During inflammation, enolase migrates to the cell surface that promotes the production of pro-inflammatory cytokines, chemokines, reactive oxygen species, and nitric oxide.60\n\nHigher ENO1 expression has been associated with larger tumor size, increased invasion, shorter survival, and poor prognosis in several solid tumors, including hepatocellular carcinomas, breast- and lung cancers,59 and PDAC.61 ENO1 has clinicopathological and diagnostic significance as a tumor-associated antigen as well: combining ENO1 autoantibodies with other biomarkers improved diagnostic sensitivity and accuracy of lung cancer detection.62,63 In PDAC, several fundamental characteristics designate ENO1 to be an ideal biomarker candidate: it is localized on a cell surface, making it targetable; it is upregulated in tumors compared to normal tissues, is associated with clinical outcome, and has vital metabolic functions. Small molecule inhibitors (e.g., ENOblock, ENO1i) are already being investigated in various preclinical settings and disease models, including spinal cord injuries and multiple myelomas.64,65 Additionally, DNA vaccinations targeting ENO1 in combination with chemotherapy may offer promising alternatives in PDAC treatment. In pancreatic cancer-prone mice, treatment with gemcitabine combined with anti-ENO1 vaccine elicited a robust antitumor response and reduced the size of PDAC lesions.66 Administration of an anti-ENO1 mAb inhibited in vitro invasion of human PDAC cells and their metastatic spread in immunocompromised mice; thus, targeting ENO1 may be exploited as a novel therapy to increase the survival of metastatic PDAC patients.67\n\nPDAC presents unique challenges for chemotherapy and immunotherapy with an immunosuppressive microenvironment and a dense stroma, creating a physical barrier to drug delivery; thus, alternative therapeutic targets, such as ion channels, provide an increasingly evaluated option. Ion channels are involved in every aspect of oncogenesis, influencing critical processes within each hallmark of cancer. Chloride Intracellular Channel 1 (CLIC1) plays a vital role in many physiological functions, including ion homeostasis, acidity, and cellular volume, considered to be both the sensor and executor of oxidative stress. It can exist in a soluble form, but in pathological states, CLIC1 is expressed explicitly as a transmembrane chloride channel.68 CLIC1 overexpression has been established in pancreatic cancer tissue and is indicated to be a putative oncogene.69–71\n\nIn summary, our findings provide novel insight into intimate gene-networks corroborating previous knowledge about interactions within the tumor microenvironment. Although our results are based on in silico approaches, maintained correlations across RNA-seq and gene chip platforms substantiate the significance of our findings. Our results may be utilized to develop combination therapies against PDAC and offer novel ideas to be explored in future studies.", "appendix": "Data availability\n\nData have been acquired from public repositories, located at http://www.pubmed.com/geo and https://portal.gdc.cancer.gov/.\n\n\nAcknowledgments\n\nThe authors gratefully acknowledge the support of Dr. Attila Gyenesei and his research group from the University of Pécs, Szentágothai Research Centre. The authors also acknowledge the support of ELIXIR Hungary (www.elixir-hungary.org).\n\n\nReferences\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\nRahib L, Smith BD, Aizenberg R, et al.: Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "281371", "date": "16 Sep 2024", "name": "Stephan Joel Reshkin", "expertise": [ "Reviewer Expertise I work in studying and finding improved treatments/therapies for PDAC" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nConcerning the manuscript entitled “Preserved correlation matrices pinpoint extracellular matrix organization as a critical factor in pancreatic ductal adenocarcinoma” by Menyhart, Bartha and Gyorffy. This is a very interesting study using state-of-the-art statistical methods with various data sets to find correlations between various markers and phenotypes between pancreatic normal tissue (108 samples) and PDAC tissue (248 samples). Interestingly, they find the strongest correlations in genes having to due with the particular Tumor Microenvironment of PDAC. The results of this study will straighten out some of the complexities and arguments concerning the classification of PDAC tissues The study is well presented and the manuscript very well written, especially the Discussion which does an excellent job of clearly putting together a complex study. However, concerning the last paragraph that covered the ion channels, I believe that the authors should study, cite and discuss the results of an older paper that used data-mining analyses of acid-base transporter expression changes to look at the changes in ion channels that regulate acid-base regulation (Kong, SC et al., 2014) [Ref 1]\nFurthermore, I feel that it is necessary to improve a couple of the figures:\nFigure 1C is too small and impossible to read/study, especially in comparison to Figure1B which is too big. Please make 1C big enough to read the details. The same is true of Figure 2C- It is necessary to rearrange or separate the various panels so that 2C becomes readable. This very necessary.\n\nWhile panel of Figure 3A is a bit small; it is readable and, so, can remain as is.\nIf these few questions are taken care out, I feel that the manuscript can be approved.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "353194", "date": "25 Jan 2025", "name": "Howard Crawford", "expertise": [ "Reviewer Expertise Pancreatic cancer biology. Tumor-stromal cell crosstalk." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 have mined publicly available microarray data to explore gene expression associations with patient outcome.\nWhile the computational analysis reported is of very high quality, it is unclear what new has been learned from the study. The vast majority of the most differentially expressed genes are well-known to be genes that are primarily expressed by cancer associated fibroblasts (CAFs). Since the data analyzed comes from the transcriptome of a multicellular, heterogeneous tumor, the overall conclusion of the study could just as easily be that having a large number of CAFs in a tumor is associated with poor outcome, a conclusion that has been explored in more molecular detail over the past decade than what is presented here.\nIt is unclear what additional analysis would be helpful, except possibly to use publicly available single cell data sets to deconvolute the bulk transcriptome data.\nIf this is not useful, the least the authors should do is to discuss their data in the context of the many studies that have been done on the role of CAFs and, in some cases, the specific genes identified (SPARC for instance), to provide context about what this study adds that is unique vs reinforcement of what is already known.\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? No", "responses": [] } ]	1	https://f1000research.com/articles/12-418
https://f1000research.com/articles/12-415/v1	18 Apr 23	{ "type": "Research Article", "title": "Assessment of the level of knowledge about diabetes mellitus among diabetic patients: A cross-sectional study from Nepal", "authors": [ "Divas Adhikari", "Kiran Dhonju", "Egesh Aryal", "Sandip Ghimire", "Suresh Chapagain", "Bishnu Malla", "Prativa Sangroula", "Nimesh Lageju", "Kiran Dhonju", "Egesh Aryal", "Sandip Ghimire", "Suresh Chapagain", "Bishnu Malla", "Prativa Sangroula", "Nimesh Lageju" ], "abstract": "Background\n\nDiabetes mellitus is a chronic non-communicable disease (NCD) often associated with poor psychosocial and health outcomes. Available evidence suggests that patients’ knowledge about the disease is fundamental to its treatment and prevention or delaying of complications. Therefore, this study assessed the level of knowledge of diabetes patients on diabetes mellitus and its associated factors.\n\nMethods\n\nA descriptive cross-sectional study using a translated Nepalese version of the revised Michigan Diabetes Knowledge Scale was conducted among randomly selected patients with diabetes mellitus presenting to the outpatient department (OPD) of Madhyabindu Hospital, Nepal. Data were collected via an interviewer administered questionnaire and analyzed with MS Excel 2016 and IBM SPSS version 25 software.\n\nResults\n\nAmong the 360 participants, 27.2% had good knowledge of diabetes and 72.8% had poor knowledge. Age, level of education, occupation, family history of diabetes, duration of the disease, presence of comorbidity, complications and body mass index (BMI) were significantly associated with knowledge of diabetes mellitus (p-value ≤ 0.05).\n\nConclusions\n\nBased on the study findings, it is important that diabetes self-management education and support (DSMES) programs should be implemented and/or strengthened at the Madhyabindu Hospital and targeted communities to improve diabetes patient’s knowledge on their disease condition and self-care practices.", "keywords": [ "chronic disease", "non-communicable disease", "diabetes mellitus", "knowledge", "Revised Michigan Diabetes Knowledge Scale", "health education", "prevention", "Nepal" ], "content": "Introduction\n\nDiabetes mellitus is a preventable, chronic metabolic, non-communicable disease characterized by hyperglycemia that results from defects in insulin secretion, insulin action, or both.1 In 2019, P Saeedi et al. estimated the global prevalence to be 9.3% and projected it would be 10.2% by 2030 and 10.9% by 2045.2 Recent meta-analysis in Nepal by N Shrestha et al. taking published data dated up to 2020, showed the pooled prevalence of diabetes to be 8.5%.3 So, under the same lifestyle, with an aging population, and unhealthy diet, the prevalence would likely be more unless necessary steps are taken. In addition to this, the meta-analysis also showed that less than half of known diabetic patients were treated with anti-diabetic drugs and only one-third had their blood glucose controlled.3\n\nProper management to control blood glucose can delay the progression of a disease with a significant reduction in diabetes-related complications.4 One of these management methods includes, patient education with appropriate and adequate knowledge of diabetes, which is a key to behavioral change for good glycemic control.5–7 It is imperative for clinicians to know the knowledge status of the patient and counsel them properly, suggest them good educational programs which in turn would assist in the holistic care of the patient. In addition to this, concerned authorities should be aware of the knowledge status of the diabetic patient to change or add policies and programs to meet their needs.\n\nThus, this study aims to understand the knowledge related to diabetes in a diabetic patient using the Revised Michigan Diabetic Assessment tool, which consists of 20 true/false statements that assess a patient’s diabetes related knowledge, including items on diet, blood glucose control, exercise, medication taking, complications and insulin use.8 We also explore the socio-demographic and clinical characteristics of diabetic patients, and assess their relationship with their level of diabetic knowledge.\n\n\nMethods\n\nThis descriptive cross-sectional study was conducted from December 2021 to January 2022 in the outpatient department (OPD) of Madhyabindu Hospital located in the Nawalparasi district of Nepal. All adult patients aged 18 or above attending the outpatient department during the data collection period, with the diagnosis of diabetes mellitus type 1 or 2 based on the American Diabetes Association criteria9 were eligible for the study. Patients with gestational diabetes mellitus were excluded from the study.\n\nThe sample size of the study was 360 which was calculated as follow:\n\nWhere,\n\nn = sample size\n\nZ = 1.96 at 95% confidence interval\n\np = prevalence of knowledge about diabetes = 0.6210\n\nq = 1 – p = 0.38\n\ne = standard error (taking 5%)\n\nA probability simple random sampling technique was used. The average number of diabetic patients visiting OPD per day was fifty. All the diabetic patients who presented to the OPD were marked with a specific number and a lottery method was used to choose the study participants. Fourteen participants were selected each day for data collection.\n\nPermission was obtained from Madhyabindu Hospital and the study was approved by the Nepal Health Research Council (Reg. no. 668/2021). The purpose and benefits of the study were explained to the participants, ensuring no harm or risk. They were made aware that their participation would be voluntary, and the data obtained would be published maintaining privacy. Before data collection, informed written consent was obtained from each participant.\n\nData was collected by one of the researchers via formal interview with each of the participants. The interviewer read the statements present in the questionnaire and the participant responded with whether the statement was true/false or don’t know. The average duration for each interview was 15 minutes. The baseline measurements such as blood pressure, height, body weight, and body mass index (BMI) were obtained at the same visit. Laboratory data were collected from each patient’s medical record. The privacy of the study population was maintained throughout the interview by using serial number instead of their name in questionnaire and written consent form.\n\nWe used the Revised Michigan Diabetes Knowledge Scale True/False version to assess each patient’s knowledge.8 The questionnaire was translated into the Nepali language by the researchers to reduce language barriers among patients. It was then back-translated into English by a bilingual translator. The translation was refined after back translation until agreement was obtained among the researchers. One endocrinologist examined and approved the Nepalese version of the questionnaire.\n\nThe questionnaire consisted of 20 true/false statements that aimed to assess diabetes mellitus knowledge and awareness. Patients who answered more than or equal to 65% of the questions correctly were considered to possess good knowledge about diabetes mellitus.11\n\nSociodemographic variables included age, gender, religion, education level, occupation, and marital status. Clinical characteristics included family history of diabetes, duration since diagnosis of diabetes, types of medications used, comorbidities, body mass index (BMI), blood pressure (BP), fasting blood sugar level, postprandial blood sugar level, and diabetic complications.\n\nMS Excel 2016 and IBM SPSS version 25 were used for data management and analysis. We used frequency for categorical variables, and mean and standard deviation for continuous variables. A Chi-square test was used to study the relationship between variables; a t-test was used to compare means. P value less than or equal to 0.05 was considered statistically significant for all statistical tests.\n\n\nResults\n\nOf the 360 participants, the mean (±SD) age was 58.23±11.83 years (range 24-88 years). A majority of the participants were male (n=217, 60.3%) and had no family history of diabetes mellitus (n=244, 67.8%). Approximately, one-third of the participants were illiterate (n=115, 31.9%) and had diabetes for at least 5 years (n=129, 35.8%). Most of the participants (n=191, 53.1%) were using a combination of oral hypoglycemic agents (OHA) for medical treatment, with 123 (34.2%) patients having complications with neuropathy (n=57,15.8%) being the most common complication.\n\nOther socio-demographic and clinical characteristics of patients are shown in Tables 1 and 2. The full dataset can be found under Underlying data.\n\nThe highest percentage of correct answers (93.9%), (91.7%) and (90.8%) respectively belonged to these questions: “Having regular check-ups with your doctor can help spot the early signs of diabetes complications”, “Exercising regularly can help reduce high blood pressure”, “Eating foods lower in fat decreases your risk for heart disease”. However, the lowest percentage of correct answers (14.7%), (22.2%), (26.9%) were for these questions respectively: “Attending your diabetes appointments stops you from getting diabetes complications”, “Lung problems are usually associated with having diabetes”, “Glycosylated hemoglobin (HbA1c) is a test that measures your average blood glucose level in the past week” as shown in Table 3. Among 30 patients taking insulin, 70% gave the right response to “High blood glucose level can be caused by too much insulin” as shown in Table 3.\n\n98 patients (27.2%) possessed good knowledge about diabetes. Patients with good knowledge of diabetes had a mean age of 53.70±11.81, while patients with poor knowledge of diabetes had a mean age of 59.92±11.41 (p-value < 0.001). 30.45% of males had good knowledge compared to 22.4% of females with a non-significant p-value of 0.094 as shown in Figure 1. Patients living with the disease for more than ten years had poor knowledge of diabetes with a p-value of 0.005.\n\nAccording to this study, poor knowledge increased with age, and decreased with the level of education. Being a farmer, housewife and unemployed was associated with having poor knowledge on diabetes. Having a family member with diabetes was associated with having good knowledge of diabetes. Other variables like duration since diagnosis, co-morbidities, complications and BMI were significantly associated with knowledge status as shown in Table 4.\n\n\nDiscussion\n\nKnowledge of a disease in a patient is a first ladder towards change in behavior. Patient education is of utmost importance in the management of a disease especially when it is a chronic illness like diabetes mellitus.5 This could lead to the empowerment of a patient and thus limit their dependence on clinicians.\n\nThis study showed that 27.2% of patients possessed good knowledge about diabetes. As of investigators’ extensive literature review, two studies done using the same assessment tools were found. One of this study done in a hospital with specialist facility in Saudi Arabia by Almalki et al. showed 21.6% of diabetic patients had good knowledge about diabetes.11 However, another study done in Al-Zahraa Teaching Hospital by Taher et al. showed 78.91% had good knowledge about diabetes.12 Although different studies done at different times in Nepal, the United Arab Emirates and Pakistan had shown poor knowledge status, we cannot compare their findings to this study because of differences in assessment tools and ethnic groups.13–16\n\nThis study showed that there was poor knowledge of diabetes among patients with a low level of education. However, this is in contrast with findings by Taher et al. which showed no significant association between education level and knowledge status.12 This difference in finding could be because the study by Taher et al. was done in the teaching hospital where the participants got advice and treatment from specialists who could give individualized education.12 The fact that this study was conducted at a government hospital where there was a high patient load and a lack of diabetic specialists and trained staff might have contributed to less time for effective individualized counselling and ultimately low knowledge status among the patients. This study also found that being a housewife, and unemployed, was significantly associated with poor knowledge. However, Taher et al. found that there was no significant association between employment and knowledge status.12 This suggests that physicians, while counseling, should focus more on patients with poor education, housewives, and unemployed patients.\n\nA study by Taher et al. showed a significant association between type of medication used and good knowledge, but this study showed no significant association between type of medication used with knowledge status. Younger patients and patients diagnosed with diabetes mellitus recently had good knowledge of diabetes. Similar findings were reported from Almalki et al.11 This may be due to the curiousness about the diagnosis among these patient groups.\n\nThe three most common right responses in this study were similar to that of Taher et al.12 and this might have occurred because these questionnaires are also related to other common chronic diseases like hypertension and obesity. The fact that the complications, co-morbidities, and BMI were associated with poor knowledge suggest that physicians had difficulties in providing the patients with the sufficient information about diabetes mellitus. Clinicians should be aware of the patient’s level of knowledge related to diabetes, and educate them accordingly. In addition to it, different levels of government should go hand in hand to raise awareness regarding diabetes through integration of diabetic programs with other health programs, by implementing dedicated diabetic awareness programs, and diabetes self-management education and support (DSMES) programs. Also, patient knowledge of diabetes mellitus may be increased through government-launched programs for information dissemination about diabetes mellitus using various social media platforms, continuing medical education for doctors on diabetes mellitus, and training female community health volunteers to provide diabetes-related knowledge.\n\nThe findings of this study represents the people with diabetes mellitus in Nawalparasi district and might be generalized to people in other districts in Nepal with similar characteristics. As our study was conducted in a single government hospital, we recommend collective inclusion of primary health care centers, government hospitals and private hospitals for further similar studies.\n\n\nConclusion\n\nThe majority of the patients with diabetes mellitus had poor knowledge about diabetes, and the level of knowledge was affected by these factors; age, level of education, occupation, family history, duration of diabetes mellitus, presence of co-morbidities, complications and BMI. This study shows that there is a need to implement and/or strengthen diabetes awareness and DSMES programs at the Madhyabindu Hospital and targeted communities.", "appendix": "Data availability\n\nFigshare: Assessment of the level of knowledge about diabetes mellitus among diabetic patients: A cross-sectional study from Nepal. DOI: https://doi.org/10.6084/m9.figshare.22039781.v2. 17\n\nThe project contains the following underlying data:\n\n• Working dataset.sav: Data set with quantitative data on age, gender, marital status, education, occupation, family history, duration, co-morbidity, BMI, complications, duration of disease.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nFigshare: Assessment of the level of knowledge about diabetes mellitus among diabetic patients: A cross-sectional study from Nepal. DOI: https://doi.org/10.6084/m9.figshare.22332412.v1. 18\n\nThe project contains the following extended data:\n\n• Data file 1: English and translated Nepali version of questionnaire.docx (Socio-demographics and Revised Michigan Diabetes Knowledge Scale True/False version of questionnaire in English and Nepali format)\n\n• Data file 2: English and Nepali version of informed consent form.docx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAmerican Diabetes Association: Diagnosis and Classification of Diabetes Mellitus. Diabetes Care. 2014 Jan 1; 37(Supplement_1): S81–S90. Publisher Full Text\n\nSaeedi P, Petersohn I, Salpea P, et al.: Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9th edition. Diabetes Res. Clin. Pract. 2019 Nov 1; 157: 107843. PubMed Abstract \| Publisher Full Text\n\nShrestha N, Mishra SR, Ghimire S, et al.: Burden of diabetes and prediabetes in Nepal: a systematic review and meta-analysis. Diabetes Ther. 2020; 11(9): 1935–1946. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUK Prospective Diabetes Study (UKPDS) Group: Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet. 1998; 352(9131): 837–853. Publisher Full Text\n\nPowell CK, Hill EG, Clancy DE: The relationship between health literacy and diabetes knowledge and readiness to take health actions. Diabetes Educ. 2007; 33(1): 144–151. PubMed Abstract \| Publisher Full Text\n\nMbanya JC, Sobngwi E: Diabetes microvascular and macrovascular disease in Africa. Eur. J. Cardiovasc. Prev. Rehabil. 2003; 10(2): 97–102. Publisher Full Text\n\nMacdonald G: Harrison’s Internal Medicine, 17th edition. - by A. S. Fauci, D. L. Kasper, D. L. Longo, E. Braunwald, S. L. Hauser, J. L. Jameson and J. Loscalzo. Intern. Med. J. 2008 Dec 1; 38(12): 932–932. Publisher Full Text\n\nSurvey Instruments\|Diabetes Institute\|Michigan Medicine. Diabetes Institute.2020 [cited 2022 Oct 10]. Reference Source\n\nProfessional Practice Committee: Standards of Medical Care in Diabetes—2018. Diabetes Care. 2018 Jan 1; 41(Supplement_1): S3–S3. Publisher Full Text\n\nNepal C, Vyas P, Bhattarai R, et al.: Knowledge, attitude and practice of type 2 diabetic patients of selected outreach clinic, Dharan, Nepal. Al Ameen J. Med. Sci. 2017; 10(1): 78–85.\n\nAlmalki TM, Almalki NR, Balbaid K, et al.: Assessment of diabetes knowledge using the Michigan brief diabetes knowledge test among patients with type 2 diabetes mellitus. J. Endocrinol. Metab. 2018; 7(6): 185–189. Publisher Full Text\n\nTaher TMJ, Khalil MH, Sarray FTR: Knowledge of Diabetic Patients in Al-Kut City According to Revised Michigan Diabetes Knowledge Test. Electron. J. Med. Dent. Stud. 2022 Jan 15; 12(1): em0093. Publisher Full Text\n\nGautam A, Bhatta DN, Aryal UR: Diabetes related health knowledge, attitude and practice among diabetic patients in Nepal. BMC Endocr. Disord. 2015; 15(1): 1–8. Publisher Full Text\n\nAl-Maskari F, El-Sadig M, Al-Kaabi JM, et al.: Knowledge, attitude and practices of diabetic patients in the United Arab Emirates. PLoS One. 2013; 8(1): e52857. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUpadhyay DK, Palaian S, Shankar PR, et al.: Knowledge, attitude and practice about diabetes among diabetes patients in Western Nepal. Rawal Med. J. 2008; 33(1): 8–11. Publisher Full Text\n\nBadruddin N, Basit A, Hydrie MZI, et al.: Knowledge, attitude and practices of patients visiting a diabetes care unit. Pak. J. Nutr. 2002; 1(2): 99–102. Publisher Full Text\n\nAdhikari D: Assessment of the level of knowledge about diabetes mellitus among diabetic patients: A cross-sectional study from Nepal. figshare.2023 [cited 2023 Apr 2]. Publisher Full Text Reference Source\n\nAdhikari D: Assessment of the level of knowledge about diabetes mellitus among diabetic patients: A cross-sectional study from Nepal. figshare.2023 [cited 2023 Apr 2]. Publisher Full Text Reference Source" }	[ { "id": "176841", "date": "12 Jun 2023", "name": "Bishnu Pathak", "expertise": [ "Reviewer Expertise I am a Primary Care Physician particularly interested in non-communicable disease with special interest in Cardiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a hospital-based cross-sectional study from Nepal that aims to find out the level of knowledge about Diabetes Mellitus among diabetic patients. The researchers found out that the level of knowledge was low (only 27.2% had good knowledge of diabetes) in their setting. The manuscript has been written well and the conclusion of the study is consistent with the results. However, there are a few issues to be addressed:\nMethods:\nIt has been mentioned that this is a descriptive cross-sectional study. But, bivariate analysis has been conducted (chi-square test). So, it is actually an analytical cross-sectional study due to analytical tests being performed to draw conclusions.\n\nThough you have mentioned that simple random sampling was conducted, it looks more like convenience sampling. Otherwise, it needs more elaboration. Why were 14 participants selected each day? Was data collection done on a daily basis? The study period is one year as mentioned.\n\nPlease elaborate in detail on how the anonymity and privacy of patients and their information were maintained in your study.\n\nThe tool named \"Revised Michigan Diabetic Assessment tool\" that has been used is referenced as number 8. But, it is not accessible. Please reference it properly or provide an accessible source.\n\nIn statistical analysis, you have mentioned that mean and standard deviation were calculated for continuous variables. But, did you check the normality of data before opting for means/standard deviation? We generally use the median/interquartile range in case the data is non-normal.\n\nYou have mentioned that a t-test was used to compare means. But, this has not been shown in the results anywhere. If the t-test was not really performed, please omit this sentence from the text.\n\nResults:\nPlease include legends under the table. In table no. 2, mention what co-morbidities were present.\n\nIn table no. 4, please mention in legend that how p-value was calculated (eg. by doing chi-square test in your case).\n\nDiscussion:\nIt would be better if you could write in short about all the limitations of your study, and the measures taken to overcome those limitations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "209512", "date": "23 Oct 2023", "name": "Irene Baroni", "expertise": [ "Reviewer Expertise education and 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\nIntroduction:\n\"Diabetes mellitus is a preventable...disease\" seems incorrect because the most common form of DM, Type-2 diabetes, could often (not always) have been prevented. Please justify or rectify what has been written.\n\nPlease update your references. For example, there is an updated version of IDF Atlas: IDF Diabetes Atlas 2021.\n\nMoreover, the references number 4, 5, 6 and 7 are obsolete. There is a more recent enormous body of scientific literature that can be cited. Please revise.\n\nConsider revising the English writing in the entire manuscript. For example, sentences like \"So, under the same lifestyle, with an aging population, and unhealthy diet, the prevalence would likely be more unless necessary steps are taken\" and \"Knowledge of a disease in a patient\" are unclear.\n\nMethods:\nThe \"Revised Michigan Diabetes Knowledge Scale True/False version\" is meant to be a \"self-report\" tool; the patients must read and answer each question. Why was the researcher chosen to read the questionnaire? Please clarify. I also suggest to add this \"bias\" to the limit section.\n\nThe translation process is somewhat approximative. Please add a reference according to which this process is considered acceptable. This element must be added in the limit section because using a not-validated questionnaire can lead to enormous biases and misjudges.\n\nThe paragraph \"statistical analysis\" is unclear. Please be more specific (i.e. Categorizations and the comparison between groups are not reported).\n\nSome continuous variables have been categorized (i.e. years from diagnosis, age, BMI). Please specify the motives for this choice. Generally, it is the best choice to analyze directly a continuous variable. Following this, by using continuous variables, a correlation could have been used to evaluate the relationship between the variables. Please redo the analysis or justify this choice.\n\nResults:\nI was not able to find the number/rate of T1DM and T2DM patients enrolled. Please add it. What is the statistical test used for the following paragraph? \"according to this study, poor knowledge increased with age and decreased with the level of education. Being a farmer, housewife and unemployed was associated with having poor knowledge on diabetes. Having a family member with diabetes was associated with having good knowledge of diabetes. Other variables like duration since diagnosis, co-morbidities, complications and BMI were significantly associated with knowledge status\". Moreover, results (i.e. p-value) were not reported in the text like in the precedent paragraph. Please be more consistent with the methods section.\n\nDiscussion:\nThe sentence \"This could lead to the empowerment of a patient and thus limit their dependence on clinicians\" needs a reference.\n\nThe authors state that they \"cannot compare their findings to this study because of differences in assessment tools and ethnic groups\", but they used a value from an article - that used another tool to evaluate diabetes knowledge (i.e. KAP questionnaire) - to perform the sample calculation. This is unclear and represents another potential bias of this manuscript. Please clarify.\n\nAlso, even if the authors state what was just reported, they actually compare the \"Taher et al.\" article with this manuscript's findings in the following paragraphs of the discussion. Please clarify.\n\nConsidering the relevant and numerous limits found in this study, there is an extremely urgent need for a \"Limit\" paragraph.\nFinally, in this manuscript, there are various lacks that can lead to important biases. From the introduction and applied methods, passing through the performed analysis and computed data, to the conclusion, it is clear that this manuscript has to undergo a major 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? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-415
https://f1000research.com/articles/11-725/v1	01 Jul 22	{ "type": "Research Article", "title": "Nutritional status, nutrient imbalances, food-related behaviors and dietary supplements use among patients with celiac disease in Lebanon: a national cross-sectional study", "authors": [ "Maha Hoteit", "Zeinab Chamas", "Shaza Assaf", "Malek Michael Bouhairie", "Abbas Bahr", "Romy Daccache", "Rami Matar", "Marwa Hallal", "Michael Maitar", "Mahmoud Hallal", "Samer Hotayt", "Bilal Hotayt", "Zeinab Chamas", "Shaza Assaf", "Malek Michael Bouhairie", "Abbas Bahr", "Romy Daccache", "Rami Matar", "Marwa Hallal", "Michael Maitar", "Mahmoud Hallal", "Samer Hotayt" ], "abstract": "Background: Celiac disease (CD) is an autoimmune disorder triggered by gluten, that occurs in susceptible individuals and is associated with dietary restriction and subsequent nutritional deficiencies. This study investigated the diet quality, nutrition imbalances and nutrition status among young children, adolescents and adults with CD who were referred to several hospitals in Lebanon. Methods: A cross-sectional study in 50 individuals (31.74 ± 15.64 years) with CD was conducted, using biochemical parameters, anthropometric measurements, dietary and physical activity assessments. Results: Of the 50 participants, 38% and 16% were presenting low serum levels of iron and vitamin B12, respectively. The majority of participants were physically inactive and around 40% of them had low muscle mass. A weight loss of 10% to 30% indicating mild to moderate malnutrition was shown in 14% of individuals. The assessment of food-related behaviors shows that 80% of participants were reading nutrition labels and 96% of them were following gluten-free diets (GFD). Some barriers including family ignorance (6%), language of the nutrition labels (20%) and expensive GF products (78%) were limiting the adherence to GFD. The inadequacy of the daily energy intake along with insufficient intakes of calcium and vitamin D were remarked among individuals with CD. However, protein and iron intake were exceeding the recommendations among all age groups, except in males aged 4-8 years and 19-30 years. Half the study participants were using dietary supplements where 38%, 10%, 46%, 18%, 16% and 4% used vitamin D, vitamin B12, iron, calcium, folate and probiotics, respectively. Conclusion: GFD is the key treatment for CD. However, it is not without inadequacies and may cause certain deficiencies such as calcium and vitamin D leading to reduced bone density. This underlines the critical role of dietitians in education and maintenance of healthy GFD among individuals with CD.", "keywords": [ "Celiac disease", "gluten free diet", "anthropometry", "biochemical", "Lebanon" ], "content": "Introduction\n\nCeliac disease (CD) is a chronic inflammatory autoimmune disorder characterized by villous atrophy of the small bowel.1 The exposure to dietary wheat gluten and proteins of rye and barley triggers CD among genetically predisposed persons.2 In certain circumstances, individuals show no symptoms and the diagnosis is only possible through serologic screening.3 According to the European Society for the Study of Celiac Disease (ESsCD) guideline, the diagnostic criteria depend upon the exposure to dietary gluten or related proteins of rye and barley-dependent symptoms, through some histopathologic findings (villous atrophy and crypt hyperplasia) from a biopsy of the duodenum CD, specific antibody levels, HLA-DQ2 and/or HLA-DQ8.4 The prevalence of CD is estimated to be one to two individuals per 100 worldwide5 and roughly one in 99 among children and adolescents.6 Moreover, according to a recent systematic review and meta-analysis, the pooled seroprevalence of CD in the general population is significantly higher in children (2%) compared with adults (1%, p-value=0.01) and significantly greater among females (1.65%) compared to males (0.8%, p-value=0.04).7 The pooled sero-prevalence of CD in the Middle Eastern area (1.47%) is higher than that in South Asia (1.25%) and East-Asia (0.06%).7 The highest prevalence among the Arab regions was reported in Saudi Arabia (3.2%), and the lowest in Tunisia (0.1%).8 In Lebanon, the prevalence of CD was around 0.5% in 2011.9 Furthers, women have shown elevated prevalence compared to men in the Arab countries8 and according to solid evidence, the risk becomes common and high if a first- or second-degree relative has been previously diagnosed.6\n\nCD affects the proximal part of the small intestine, which in turn results in micronutrient malabsorption, particularly iron, folic acid, vitamin B12, calcium and vitamin D, resulting in anemia on the short term and reduced bone density on the long-term.2 Further, beyond inflammation and malabsorption, CD is characterized by recurrent abdominal pain, nausea, vomiting, steatorrhea and loss of weight which also compromise the nutrition status of affected individuals.6 Until today the only therapy for individuals with CD remains the adherence to a gluten-free diet (GFD) along with the avoidance of foods containing wheat, rye, and barley derivatives.10 According to several studies, the adherence to a strict GFD can reduce the risk of health-related complications, for instance: malignancy, osteoporosis, growth retardation in children, liver disorders and reproductive tract disorders.1 According to recent data, the adherence to GFD has remained unchanged over the past twenty years11,12 and a considerable number of individuals with CD do not adhere to the dietary restrictions, which affects the patient’s quality of life and causes compromised nutrition status.10 Moreover, some individuals with CD may look for other approaches to treatments such as complementary and alternative medicine (CAM) which include dietary supplements (DS) use.13 There is scarce data on the DS use in CD. Many studies identified the DS use among CD patients to be within a range of 21.6% to 23.6%.13 This percentage was less than the percentage of DS use among the general population (27%).13 Thus, investigating the nutrition status through dietary assessment of individuals with CD is warranted especially among children, adolescents, and adults to avoid malnutrition and related morbidity. Such an evaluation of GFD adherence should be executed by experienced trained dietitians; however, the access to such dietitians with expertise in the field of CD may be limited. Beyond GFD adherence, many reasons exist for the necessity of nutritional investigation including the poor public awareness about CD, and the lack of regulations for the production and analysis of gluten-free labelled products.14 To the best of our knowledge, there are no published data that address the nutrition situation and dietary supplements use among individuals with CD in Lebanon. Therefore, the aim of the present study was to assess the nutritional status, food-related behaviors, caloric, macro- and micronutrients intake and dietary supplements use among individuals with CD in Lebanon.\n\n\nMethods\n\nSelection procedure\n\nParticipants from all age categories were invited to participate in the current cross-sectional study between February and July 2018. A random selection was performed. Participation in the study was proposed to all patients who came to the pediatrician-gastroenterologists and gastroenterologists in three hospitals in Beirut and Mount Lebanon for a follow-up visit and met the criteria for inclusion, which was a diagnosis of CD based on serological and histological markers according to the international guidelines,15 more than six months prior to study enrolment to guarantee adequate GFD knowledge. All participants with no proven biopsy, pregnant women, history of cancer, eating disorders, malnutrition, renal diseases and any other factors that affect the nutrition situation, were excluded.\n\nQuestionnaires and dietitians’ assessment\n\nThe survey contained 78 questions concerning demographics, disease’s characteristics, history and presentation, food-related behaviors, lifestyle and diet history, weight before and after being diagnosed with CD, adherence to GFD and social and emotional barriers for sustaining the GFD. The questionnaire was derived from the one available from the Canadian Celiac Association website and used previously in the “Canadian Celiac Health Survey”.16 Medical history was evaluated based on the age at diagnosis of CD, family history of CD, symptoms (diarrhea, headache, flatulence and bloating), comorbid diseases (food allergies, food intolerance, other non-communicable disease). Alongside the first questionnaire, additional FFQ for caloric and protein intake, calcium, vitamin D and iron intake was addressed to individuals with CD along with questions on dietary supplement use. This FFQ was derived from two Lebanese-validated questionnaires to address the questions for children, adolescents and adults17,18 in which a substitution of 24 foods with GF foods was executed (see Underlying data69). Dietary supplement use was assessed through patient’s response of “Yes” or “No” to the statement: “I use dietary supplements in addition to GFD”. To investigate the type of supplements used, patients were asked to answer the following question: “Since you were diagnosed, have you used any of the following dietary supplements?” The specific options included vitamin D, probiotics, iron, folate, calcium, and vitamin B12, which are the commonly used DS among individuals with CD.19 The questionnaire used in the present study was reviewed by two national and one international expert in CD and was pre-tested in 14 non-expert individuals as a focus group. The aim of the focus group was to explore a specific set of issues: the questionnaire’s readability, reliability and inter consistency (Cronbach-alpha=0.69). Moderators often commence the focus group by asking broad questions about the topic of interest, before asking the focal questions. Although participants individually answer the facilitator's questions, they are encouraged to talk and interact with each other. In addition, three-day food records were collected and analyzed using Nutritionist Pro software (version 3.2, AXXYA) to assess caloric, micro- and macronutrients intake. The mean value and the standard deviation (SD) were used when interpreting the nutrient’s intake.\n\nWeight and height were measured using automated scale and stadiometer. Body composition including body fat, lean body mass and visceral fat were assessed using Bio-impedancemetry body composition analyzer (BF-511, OMRON). The standard formula of body mass index (BMI) was calculated by dividing weight in kilograms (kg) by height in meters squared (m2).\n\nLaboratory values\n\nAccording to a study by Bledsoe et al. (2019),19 and Marteau et al. (2001)20 albumin and micronutrient deficiencies, including vitamins B12 and D, as well as folate, iron, zinc and copper, are common in adults at the time of diagnosis with CD. Thus, the study participants were tested for three out of six micronutrients (serum iron, folate and vitamin B12) along with albumin levels in the present study. Venous blood was collected after a 12-hour period of fasting, centrifuged and stored at −80°C. The iron level was measured in serum by the in vitro assay method with Roche Diagnostic (Mannheim, Germany) and Hitachi 704 devices (Roche Diagnostics, Switzerland).\n\nAll patients underwent a complete hemogram. However, only albumin, serum iron, folate and vitamin B12 were reported in this study to comply with the outcomes discussed in Bledsoe et al.19 Serum iron was diagnosed according to the criteria followed by the laboratory in which the analysis occurred.21 The reference values for children were: 8.9-21.4 μmol/L (SI units), males (adults): 14-32 μmol/L (SI units) and females (adults): 11-29 μmol/L (SI units) were compared to the values obtained in the current study. Serum folate and vitamin B12 levels were measured using a competitive immunoassay. As for vitamin B12, the reference values for 1–<11 years: 260–1200 pmol/L, 12–<18 years: 200–800 pmol/L, 18–<65 years: 200–600 pmol/L22 were also used to compare the results obtained in this study. Concerning the folate reference values, the normal serum levels were, for children: 5-21 ng/ml and for adults: 2-20 ng/mL.23 As for albumin, it was considered low if the value was <3.5 and high if >5.5 g/dl.24\n\nFood-related behaviors and GFD dietary adherence\n\nSome food patterns including shopping, cooking and eating out habits were assessed among the study participants. The adherence to GFD was assessed by trained dietitians who investigated the infringement regarding dietary recommendations.12 Across the three-day food records, patients presenting a consumption between 3 to 10 g of gluten per day were considered as non-adherent.12,25\n\nFacilitators and barriers\n\nTo search for possible barriers, patients were asked, “what are the major challenges that limit the adherence to a strict gluten-free diet?”. Trained dietitians asked participants to report whatever came to mind concerning this question. The answers were analyzed according to the methodology used by Braun and Clarke.26\n\nIndicators of malnutrition and criteria for the determination of nutritional status\n\nThe determination of the nutritional status was based on anthropometric data (weight, height, BMI), hypoalbuminemia and the percentage of weight loss taking into consideration age and sex factors. A weight loss of 10% to 20% indicated mild malnutrition, a weight loss of 20% to 30% characterized moderate malnutrition, and a weight loss of more than 30% indicates severe malnutrition.25\n\nPhysical activity\n\nSeveral types of physical activity such as aerobic (fitness), swimming, running, strength training gym, walking, cycling and yoga were investigated. The frequency of physical activity was based on frequency per week: one time per week, two-three times per week, four or more per week, daily or no physical activity.\n\nStatistical analysis of the study was performed using SPSS 28.0 (IBM Statistical Package for Social Sciences software version 28). The descriptive data were presented as percentages for categorical variables and mean ± SD for quantitative variables. Categorical variables were compared with the Chi-square test and continuous variables were compared with the student-T test. P<0.05 was considered a statistically significant difference with a 95% confidence interval.\n\nAt the beginning of the study, a consent form was signed by participants. Moreover, this study received the approval of the ethics committees at the Lebanese university (CU#18-46). In addition, an assent form was signed by the parents of children and adolescents under 18 years old.\n\n\nResults\n\nA total of 50 participants (78% females; mean age=31.74 ± 15.64 years) was enrolled in the present study. Out of 50, eight participants were aged below 18 years. Age categories were classified according to the dietary reference intake classes27 of which 46% of the population’s age ranged between 31 and 50 years. In the current study, females were younger than males (p-value=0.03). Moreover, around 66% of all the participants had university degrees and more than 70% were remunerated for more than 1,500 million Lebanese pounds (LBP) per months. Half the study’s participants were married. Overall, in almost all the characteristics, there were no significant differences between females and males in this study (p>0.05). The sociodemographic characteristics of the study participants are displayed in Table 1.\n\nLBP: Lebanese Pounds.\n\na SD=standard deviation.\n\nb classification according to the DRIs values.27\n\nTable 2 includes the medical history, anthropometric measures and lifestyle characteristics of the study participants. In the current study, males were taller than females (p-value=0.042). It was observed that the weight of all participants increased in a range of 4 to 5.6 kg since being diagnosed with CD. This was significantly higher among males compared to females (p-value=0.023). Moreover, 45% of females were overweight compared to 37% males. The assessment of malnutrition was based on Kotze et al.25; a weight loss of 10% to 30% indicating mild to moderate malnutrition were shown in 14% of the study population. The body composition analysis conducted for the study participants revealed that 40% of them had low muscle mass (5.0 to 32.8% for males and 5.0 to 25.8% for females).28 Further, the mean muscle mass was higher in males compared to females (p-value=0.034). In total, 60% of participants were having high fat mass (more than 20% in males and more than 30% in females). In addition, the mean value of fat mass among women where higher than that among men (p-value=0.014). Furthermore, the majority of the study participants had low visceral fat and there were no significant differences regarding the mean and SD of visceral fat among both genders (p-value=0.25).\n\nCD: celiac disease; BMI: body mass index.\n\na Gluten free diet.\n\nb Weight change is calculated by subtracting weight after from the weight before and the significance is determined by paired sample t-test.\n\nc Subjects aged under 20 years were excluded from this analysis.\n\nd p≤0.05 indicates the significance of values of independent tests between the means of female and males.\n\nAs for the medical history, around half the study participants had a family history of CD. The mean age at diagnosis was 22.28±13.55 (range=1-57 years). Symptoms related to CD such as abdominal bloating, diarrhea, headache, and flatulence appeared in most participants (94%) and 6% were asymptomatic. Around 66% were facing moderate to severe symptoms. The participants were asked about comorbidities along with CD; only 24% of them had CD with other diseases of which dermatitis, arthritis, lactose intolerance and food allergy. As for the medications, 78% of the study participants reported being on medical treatments for CD symptoms.\n\nAs for the laboratory values, hypoalbuminemia was observed in 14% of the study population. Women were having significantly higher mean and SD levels of albumin compared to men (p-value=0.02). Out of 50 participants, 38% and 16% were presenting low serum levels of iron and vitamin B12, respectively. None of the study participants presented with folate deficiency. As for the lifestyle behaviors, the majority of participants were non-smokers, non-drinkers and physically inactive (Table 2).\n\nThe food-related behaviors assessed among the study participants are presented in Table 3. Around 80% of the study participants reported reading nutrition labels when shopping groceries. Moreover, almost 96% of them were following a nutritional diet of which was the GFD. To assess their adherence to GFD, the examination of 96% of the participant’s three-day records reported low intake of gluten (<3g per day). Only 4% weren’t adhering to the GFD recommendations. As reported by the participants, some barriers including family ignorance (among 6% of the population), language on the nutrition labels (among 20% of the population) and expensive GF products (among 78% of the population) were limiting the adherence to GFD. It was observed that 72% of the study population had good cooking experience and 64% of the study participants were involved in preparing meals for themselves. On the other hand, almost all the subjects were unaware of a possible cross-contamination during cooking. Despite that, 82% of the subjects with CD preferred fast foods, although they consumed their meals frequently at home (92%). It was reported that the adherence to GFD by these participants induced a hunger feeling during the day. Most of our study population necessitated extra care by family members regarding cooking and eating. No significant differences were observed between genders.\n\nGF: gluten-free; GFD: gluten-free diet; F: female; M: male.\n\na Patients presenting an ingestion of approximately 3–10 g of gluten per day were considered as non-adherent.12,25\n\nTable 4 shows the mean energy and nutrients intake calculated through the three-day food records reported by the study participants or their families. Among female children aged four to eight years, the mean daily energy (E) and protein (P) intake were estimated as 1396 ±424.15 Kcal/d and 56.41 ±18.31 g/d, respectively. In comparison with the reference values of the recommended daily allowances (RDA), the daily intake of E and P was 130% and 268%, respectively. On the other hand, it was observed that female children aged 9-13 years had insufficient daily E intake (75% vs RDA) but surpassed by 317% the daily protein requirements. Similarly, among the female participant’s age groups 19-30, 31-50 and 50-70, there were inadequacies in the daily E intake along with overconsumption of protein per day. Otherwise, inadequacies in the daily E and P intake were observed among the male children (four to eight years) and those aged 19 to 30 years. Moreover, among those aged 9-13 and 31-50, the daily E intake was inadequate, but the P intake was high. No significant differences regarding the daily E and P intakes were observed between females and males, except in the 19-30 age group, where the daily energetic and protein ratios were high in females compared to males (p-value=0.024 and p=0.047, respectively).\n\nEI: energy intake; PI: protein intake.\n\n* The analysis couldn’t be generated due to the low sample size; the missing values in the table are due to the absence of males above the age of 50 years.\n\nThe average daily nutrient intake of calcium, iron and vitamin D is reported in Table 5. Among females and males, none of the study participants showed adherence to the daily recommendations for calcium and vitamin D intake. On the other hand, almost all the study participants from both genders showed a high daily consumption of iron.\n\na In each age category, the age groups (13-14) were removed due to the limited number of patients.\n\nb The missing values in the table are due to the absence of males above the age of 50 years.\n\nDietary supplements were used by 54% of the study population of which 38%, 10%, 46%, 18%, 16% and 4% used vitamin D, vitamin B12, iron, calcium, folate and probiotics, respectively. No difference was observed between genders regarding DS use (Table 6). According to Figure 1, it appears that the usage of DS among individuals with CD was not excessive.\n\n\nDiscussion\n\nThis study, the first of its kind in Lebanon, highlights the nutrition imbalances, P-E malnutrition, adherence to GFD, food-related behaviors and dietary supplements use among participants with CD. Our findings show that 78% of individuals with CD were women. This finding was higher than the data reported by Jansson-Knodell et al.,30 and Ashtari et al.7 which showed that the prevalence of women diagnosed with CD was 65% between 1990 and 2015 and the sero-prevalence of CD reported among women was 165 women out of 2000, respectively. On the other hand, our finding aligned the data reported by Singh et al. which showed that the prevalence of CD was higher among women compared to men.31 In addition, according to Bai et al., there are physiological differentials among genders in CD32: “men have indirect evidence of greater malabsorption than females and have female-predominant associated diseases when they present with celiac disease”.32 This study supported our finding concerning the remarked P-E deficiency among males compared to females in our study. Further, almost all the participants had symptoms related to CD such as abdominal bloating, diarrhea, headache, and flatulence; around 66% were facing moderate to severe symptoms and 78% of them reported being on a medical treatment for CD symptoms. These findings were two times higher than the prevalence reported by Stasi et al., who showed that 22% of individuals with CD faced persistent abdominal symptoms.33 In the present study, 14% presented hypoalbuminemia and mild to moderate malnutrition (10%-30% weight loss in the previous six months). This data agreed with Wierdsma et al.34 where 17% of individuals with CD were malnourished (>10% undesired weight loss). On the other hand, the percentage of underweight women (5%) in our study wasn’t in accordance with Wierdsma et al.34 in which 22% of the women were underweight and 29% of the patients were overweight (BMI > 25 kg/m2). Our study showed that around 40% of individuals with CD had low muscle mass and 60% had high fat mass. This result was in contradiction with the results reported in a national study which showed that subjects with CD had a lower fat mass (17.8±2.0 kg)35 but in concordance with two recent systematic reviews on children and adults that showed that after one year follow-up to GFD’ adherence, fat mass of CD patients significantly increased compared to that at baseline.36,37 According to Agarwal et al., patients with CD are more susceptible to develop metabolic syndromes and fatty liver diseases.38 Thus, these patients should be regularly counseled by experienced dietitians for nutritional and metabolic factors about healthy diets and physical activity. However, the majority of participants in our study were physically inactive (72%). This prevalence of sedentary patterns among our study population was higher than that reported by Nestares et al. (37%) among children and adolescents39 but lower than the prevalence of physical activity among women with CD in Poland (70%).40\n\nOut of 50 participants, 38% and 16% were presenting low serum levels of iron and vitamin B12, respectively. None of the study participants presented folate deficiency. These results showed consistency with a recent systematic review that showed high occurrence of hypoalbuminemia and anemia among individuals with CD.41 Furthermore, micronutrient deficiencies are common features among individuals with CD. This was demonstrated in the study of Bledsoe et al.19 where weight loss was seen in 25.2%, albumin was low in 19.7%, vitamin B12 was low in 5.3% and folate was low in 3.6% of cases. Moreover, it was shown that 87% of patients with CD in the study of Wierdsma, et al.34 had low vitamin B6 (14.5%), folic acid (20%), and vitamin B12 (19%). Additionally, 46% of patients with CD had low serum iron, and 32% had anemia.34\n\nIn our study, around 80% of participants read nutrition labels when shopping groceries. Reading and understanding food label’s topics have been discussed widely and have been associated with strict GFD adherence.42 Our finding was in accordance with Butterworth et al., who showed that amongst 74% of individuals with CD, some factors were in compliance with adherence to GFD: being a member of a celiac society, understanding food label’s language, and having access to gluten-free products with reasonable prices, regular dietetic and medical follow-up.43 Causes of adherence/non-adherence to a GFD are multifactorial and differed according to their nature and magnitude.44–47\n\nAlmost 96% of our population were following a GFD. To assess their adherence to GFD, the examination of the participant’s three-day records reported low intake of gluten (<3g per day) among 96% of the study participants. Only 4% weren’t adhering to the GFD recommendations. This finding was consistent with a study conducted in individuals with CD in Spain (92.5%).48 According to a Spanish study, 72% of individuals with CD showed acceptable adherence which was associated with higher levels of self-efficacy.49 Three studies from the UK showed that white patients were adhering to GFD in a range from 53 to 81%.50 Non-adherence to GFD was also associated with diagnosis at younger ages and smoking.50 This was not shown in our study. In Canada, GFD adherence was assessed and revealed that the percentage of strict adherence was only 56%.51 The assessment of GFD adherence of 70 Swedish adolescents with CD showed that 86% of them were adherent to GFD five years after screening.52 Moreover, of 5310 adult and adolescent Australians and New Zealanders with CD, 61% ashered to a GFD.53 Three studies on adolescents from Brazil identified adherence rates from 36 to 86% taking into consideration that younger children (up to 12 years) were more likely to comply with the diet54 while teenagers interrupted their diet purposefully. Parents of pediatric patients with CD from the Slovak Republic, reported a GFD adherence of 69% by children.55 In addition, among 38 studies, the adherence was ranging from 42 and 91%.49 According to a recent systematic review of 49 studies, the adherence rates were ranging from 23 to 98%.56 This wide variability in adherence rates may be explained by the different populations examined (e.g., adults, adolescents, children, ethnic minorities), but also by the different tools used for evaluating adherence.\n\nAs reported by the participants, some barriers including family ignorance (among 6% of the population), language on the nutrition labels (among 20% of the population) and expensive GF products (among 78% of the population) were limiting the adherence to GFD. These findings were in concordance with Muhammad et al.,44 who showed that not understanding food labelling was associated with lower GF dietary adherence scores. A higher proportion of individuals in the United Kingdom (UK) reported difficulties in acquiring knowledge about GF products (ranging between 5% and 76%) and understanding of food labels (4%-53%).57 It was observed that 72% of the study population had good cooking experience and 64% of the study participants prepared their meals. On the other hand, almost all the subjects were unaware of a possible cross-contamination during cooking food. Despite 82% of the subjects with CD preferring fast foods, they frequently consumed their meals at home (92%). Beyond cooking meals at home and cross-contamination, our findings were consistent with the findings reported in a recent systematic review indicating that the most significant barriers limiting the adherence to GFD included: “lower knowledge of CD” (35%); “restaurant/supermarket shopping” (30%); “poor patient education from practitioner” (17.5%); and “low intention/motivation to adhere to a GFD (17.5%)”.45\n\nIn our study, patients with CD had an inadequate total daily energy intake. This finding was concordant with several studies from Poland, Germany and Spain.58–60 As for nutrients intake, among females and males, none of the study participants showed adequacy to the daily recommendations for calcium and vitamin D intake. These results, altogether, showed an unbalanced diet in terms of micronutrients, that were in accordance with those shown in several studies.14,61–63 On the other hand, almost all the study participants from both genders showed a high daily consumption of iron. This finding contradicts the results reported in other studies.60,64–66\n\nDS were used by 54% of the study population of which 38%, 10%, 46%, 18%, 16% and 4% used vitamin D, vitamin B12, iron, calcium, folate and probiotics, respectively. It appears that the usage of DS among individuals with CD wasn’t in excess of that reported in the general population in Lebanon.67 In our study, the most commonly used DS was vitamin D and the least used one was prebiotics. According to the literature, gastrointestinal symptoms can be alleviated through prebiotics intake. It has been shown that Bifidobacterium infantis can alleviate gastrointestinal symptoms if administered to CD patients before meals for three weeks.68 This can explain the occurrence and persistence of gastrointestinal manifestations among the majority of participants in the current study.\n\nThe crucial strength of this study is that it fulfills a comprehensive assessment of dietary intake and food-related patterns, biochemical parameters, body composition, and physical activity. However, this study had some limitations: 1) the selected group of patients doesn’t represent the general population with CD in Lebanon; 2) it was a cross-sectional study and, thereby, our findings must be interpreted with caution; 3), physical activity levels were self-reported; 4) there is an important limitation of data on caloric, macro- and micronutrient composition of the recalled three-day record. The low sample size must also be considered.\n\n\nConclusion\n\nMonitoring nutritional status using dietary, anthropometric, blood tests and assessing physical activity are the key components in the management of CD. The ideal GFD should be balanced with macronutrients and micronutrients, with reasonable access and prices. Fortification/enrichment of GF foods that are frequently consumed should be encouraged to avoid deficiencies. This underlines the critical role of dietitians in education and maintenance of healthy GFD among individuals with CD.\n\n\nData availability\n\nOpen Science Framework: Celiac disease_Nutrition_Lebanon, https://osf.io/qcfb3.69\n\nThis project contains the following underlying data:\n\n‐ SPSS Celiac.sav (raw questionnaire, morphometric and serology data)\n\nOpen Science Framework: Celiac disease_Nutrition_Lebanon, https://osf.io/qcfb3.69\n\nThis project contains the following extended data:\n\n‐ Questionnaire.pdf\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nTherrien A, Kelly CP, Silvester JA: Celiac Disease: Extraintestinal Manifestations and Associated Conditions. J. Clin. Gastroenterol. 2020; 54(1): 8–21. PubMed Abstract \| Publisher Full Text\n\nLebwohl B, Rubio-Tapia A: Epidemiology, Presentation, and Diagnosis of Celiac Disease. 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PubMed Abstract \| Publisher Full Text\n\nRodrigues M, Yonamine GH, Fernandes Satiro CA: Rate and determinants of non-adherence to a gluten-free diet and nutritional status assessment in children and adolescents with celiac disease in a tertiary Brazilian referral center: A cross-sectional and retrospective study. BMC Gastroenterol. 2018; 18(1). PubMed Abstract \| Publisher Full Text\n\nRimárová K, Dorko E, Diabelková J, et al.: Compliance with gluten-free diet in a selected group of celiac children in the Slovak Republic. Cent. Eur. J. Public Health. 2018; 26(Supplement): S19–S24. Publisher Full Text\n\nMyléus A, Reilly NR, Green PHR: Rate, risk factors, and outcomes of nonadherence in pediatric patients with celiac disease: A systematic review. Clin. Gastroenterol. Hepatol. 2020; 18(3): 562–573. PubMed Abstract \| Publisher Full Text\n\nMuhammad H, Reeves S, Ishaq S, et al.: Adherence to a Gluten Free Diet Is Associated with Receiving Gluten Free Foods on Prescription and Understanding Food Labelling. Nutrients. 2017; 9(7): 705. PubMed Abstract \| Publisher Full Text\n\nKikut J, Konecka N, Szczuko M: Quantitative assessment of nutrition and nutritional status of patients with celiac disease aged 13–18. Rocz. Panstw. Zakl. Hig. 2019; 70(4): 359–367. PubMed Abstract \| Publisher Full Text\n\nMartin J, Geisel T, Maresch C, et al.: Inadequate nutrient intake in patients with celiac disease: results from a German dietary survey. Digestion. 2013; 87(4): 240–246. PubMed Abstract \| Publisher Full Text\n\nBallestero-Fernández C, Varela-Moreiras G, Úbeda N, et al.: Nutritional Status in Spanish Adults with Celiac Disease Following a Long-Term Gluten-Free Diet Is Similar to Non-Celiac. Nutrients. 2021; 13(5): 1626. PubMed Abstract \| Publisher Full Text\n\nThompson T, Dennis M, Higgins LA, et al.: Gluten-free diet survey: Are Americans with celiac disease consuming recommended amounts of fibre, iron, calcium and grain foods?. J. Hum. Nutr. Diet. 2005; 18(3): 163–169. PubMed Abstract \| Publisher Full Text\n\nHopman EGD, le Cessie S , von Blomberg BM , et al.: Nutritional Management of the gluten-free diet in young people with celiac disease in the Netherlands. J. Pediatr. Gastroenterol. Nutr. 2006; 43(1): 102–108. PubMed Abstract \| Publisher Full Text\n\nAl-Raee MB, El-Sakka MA, Al-Wahaidi AA: In depth analysis of risk factors for celiac disease amongst children under 18 years old in the Gaza strip. A cross sectional study. Nutr. J. 2012; 11: 97. PubMed Abstract \| Publisher Full Text\n\nScricciolo A, Elli L, Doneda L, et al.: Efficacy of a High-Iron Dietary Intervention in Women with Celiac Disease and Iron Deficiency without Anemia: A Clinical Trial. Nutrients. 2020; 12(7): 2122. PubMed Abstract \| Publisher Full Text\n\nUnalp-Arida A, Liu R, Ruhl CE: Nutrient intake differs among persons with celiac disease and gluten-related disorders in the United States. Sci. Rep. 2022; 12(1): 5566. PubMed Abstract \| Publisher Full Text\n\nNestares T, Martín-Masot R, Labella A, et al.: Is a Gluten-Free Diet Enough to Maintain Correct Micronutrients Status in Young Patients with Celiac Disease?. Nutrients. 2020; 12(3): 844. PubMed Abstract \| Publisher Full Text\n\nMohsen H, Yazbeck N, Al-Jawaldeh A, et al.: Knowledge, Attitudes, and Practices Related to Dietary Supplementation, before and during the COVID-19 Pandemic: Findings from a Cross-Sectional Survey in the Lebanese Population. Int. J. Environ. Res. Public Health. 2021; 18(16): 8856. PubMed Abstract \| Publisher Full Text\n\nSmecuol E, Hwang HJ, Sugai E, et al.: Exploratory, randomized, double-blind, placebo-controlled study on the effects of Bifidobacterium Infantis Natren life start strain super strain in active celiac disease. J. Clin. Gastroenterol. 2013; 47(2): 139–147. PubMed Abstract \| Publisher Full Text\n\nHoteit M: Celiac disease_Nutrition_Lebanon.2022, May 10. Publisher Full Text" }	[ { "id": "143011", "date": "22 Jul 2022", "name": "Sherine Khater", "expertise": [ "Reviewer Expertise Gastroenterology", "celiac disease and malignant complications of celiac disease." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a very interesting study about nutritional status in CD (celiac disease) patients on GFD (gluten-free diets) in Lebanon, with many parameters studied. However, I have 2 remarks:\n\nInclusion criteria include celiac disease patients attempting a GFD for at least 6 months. This should appear in the title of the article (the population is not CD patients at diagnosis but CD patients on GFD or attempting a GFD). This point should also appear in the abstract (Background section and Methods section). A very positive point was to evaluate the adherence to GFD of the patients included, which was high and thus reducing bias.\n\nIn the discussion part, some references are inadequate because they studied micronutrient deficiencies in CD patients at diagnosis, and not in CD patients following a GFD (example: references 32, 34, 41). These references should be replaced by others, for example: Cardo A et al, Nutritional Imbalances in Adult Celiac Patients Following a Gluten-Free Diet, Nutrients 2021, 13, 2877.\n\nConcerning use of probiotics in CD, there is no strong evidence to date. For this reason, I think it is better to remove this section in the discussion: \"According to the literature, gastrointestinal symptoms can be alleviated through prebiotics intake. It has been shown that Bifidobacterium infantis can alleviate gastrointestinal symptoms if administered to CD patients before meals for three weeks. This can explain the occurrence and persistence of gastrointestinal manifestations among the majority of participants in the current study\". Persistence of symptoms might more probably be explained by associated irritable bowel syndrome, or by inadvertent gluten ingestion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "8581", "date": "21 Nov 2022", "name": "Maha Hoteit", "role": "Author Response", "response": "The authors would like to thank Reviewer 1 for reviewing the manuscript. All comments were taken into consideration. Changes in the manuscript are highlighted in track changes." }, { "c_id": "8969", "date": "21 Nov 2022", "name": "Maha Hoteit", "role": "Author Response", "response": "Dear Reviewer 1 All the comments raised are edited in the amended version. Many thanks" } ] }, { "id": "151835", "date": "05 Oct 2022", "name": "Hamed R. Takruri", "expertise": [ "Reviewer Expertise Human 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\nGenerally, the paper is good and informative, although there are some writing and structural mistakes; these are highlighted and commented on in the attached PDF article. Some long sentences should be rewritten and separated into short sentences.\n\nIt is not clear how the intake of gluten was estimated.\n\nIt is unclear how the weight loss was evaluated; is it in reference to ideal body weight or to initial weight at the beginning of the study?\n\nThe paper can be indexed after considering the notes mentioned above.\n\nPlease find further comments in the annotated pdf hyperlinked above or at the following link: https://f1000researchdata.s3.amazonaws.com/linked/455693.Maha_Hoteit_article_121859_-_with_referees%27_%28Hamed_Takruri%29_notes.pdf\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "8970", "date": "21 Nov 2022", "name": "Maha Hoteit", "role": "Author Response", "response": "Dear Reviewer 2 Thank you for your comments. We took into consideration all the raised comments and are available in the modified version. We provide below a reply point by point to your comments: \"Generally, the paper is good and informative, although there are some writing and structural mistakes; these are highlighted and commented on in the attached PDF article. Some long sentences should be rewritten and separated into short sentences.\" \"It is not clear how the intake of gluten was estimated:\" For dietary compliance, patients were accurately interviewed by a trained physician and dietitian to assess the habitual intake of serving of gluten-containing food per week (e.g. pasta, pizza, bread, or cookies). Compliance to gluten-free diet was defined as follows: very low = more than one serving of gluten containing food/week; low = up to one serving of gluten-containing food/week; good = no gluten-containing food/week. Then based on the three days record the g/d data of gluten was investigated. We followed the same methodologies adopted in the below 2 studies: Ciacci, C., Cirillo, M., Cavallaro, R., & Mazzacca, G. (2002). Long-term follow-up of celiac adults on gluten-free diet: prevalence and correlates of intestinal damage. Digestion, 66(3), 178–185. https://doi.org/10.1159/000066757 Kotze L. M. (2004). Gynecologic and obstetric findings related to nutritional status and adherence to a gluten-free diet in Brazilian patients with celiac disease. Journal of clinical gastroenterology, 38(7), 567–574. https://doi.org/10.1097/01.mcg.0000131720.90598.6a \"It is unclear how the weight loss was evaluated; is it in reference to ideal body weight or to initial weight at the beginning of the study?\" In the last 6 months prior to the interview. We followed this study : Collins N. (2003). Protein-energy malnutrition and involuntary weight loss: nutritional and pharmacological strategies to enhance wound healing. Expert opinion on pharmacotherapy, 4(7), 1121–1140. https://doi.org/10.1517/14656566.4.7.1121 \"The paper can be indexed after considering the notes mentioned above. Please find further comments in the annotated pdf hyperlinked above or at the following link: https://f1000researchdata.s3.amazonaws.com/linked/455693.Maha_Hoteit_article_121859_-_with_referees%27_%28Hamed_Takruri%29_notes.pdf\" All the comments were taken into consideration." } ] }, { "id": "151836", "date": "10 Oct 2022", "name": "Anil K. Verma", "expertise": [ "Reviewer Expertise celiac disease", "Gluten related disorders", "HLA-DQ distribution", "Non-celiac gluten sensitivity" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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: Nutritional status, nutrient imbalances, food-related behaviors, and dietary supplements use among patients with celiac disease in Lebanon: a national cross-sectional study\nOpinion In this manuscript, The authors have investigated the nutritional status, food-related behaviors, caloric, macro-, and micronutrient intake and dietary supplements use among individuals with CD in Lebanon. The authors claim, there is no data related to this issue in Lebanon. On a general search on the web, I did not find any related articles from Lebanon. Hence, I appreciate the authors for paying attention to the nutritional status of celiac patients living in Lebanon. However, I have multiple doubts that I am summarising below:\nMethod\n\nIn the Selection procedure part: In the ‘exclusion criteria’ the authors mentioned that they eliminated patients with malnutrition. But in Table 2, they assessed the risk of malnutrition in the patients with malnutrition. Please clarify it.\n\nThe line of measurements: \"Weight and height were measured using automated scale and stadiometer. Body composition including body fat, lean body mass, and visceral fat was assessed using Bio-impedancemetry body composition analyzer (BF-511, OMRON)\" Please explain the standardization of measurements.\n\nLaboratory value: The authors mentioned the collected data from three different centers. So, I think, the normal range of parameters could be changed. How did the authors standardized the laboratory results?\n\nFood-related behaviors and GFD dietary adherence: \"Across the three-day food records, patients presenting a consumption between 3 to 10 g of gluten per day were considered as non-adherent\" In this line, please explain how you calculated gluten intake from the diet record? Which method did you use?\n\nFood record calculation: The authors showed the energy and nutrient intake in Tables 4, and 5, but they don't explain how they calculated this parameter from the diet? Which program did they use for the calculation?\n\nStatistical analysis: The authors did not mention the normality of the data set. How did they decide the normality test like (t-Test) without checking normality?\n\nPlease also mention the full form of the SPSS first then give its abbreviation in the brackets e.g., XXX (SPSS)\nResults:\n\nIn data with a statistically significant difference, please indicate under tables that: 1) Which test did you use and 2) The cut of point of significant P value (like p<0,05 or 0,001).\nDiscussion:\n\nThe discussion part contains multiple repeats of the results. On the other hand, The authors haven't interpreted the result (Like How, and why ..). For example: What are the factors that influenced adherence to GFD in Lebanon? Please after comparing the results with other studies, also give original comments related to your data.\n\nPlease first write the whole explanation of this term \"P-E malnutrition\" (Protein-energy malnutrition)\n\n\"In our study, the most commonly used DS was vitamin D and the least used one was prebiotics\" The authors mentioned prebiotics, I think it was written by mistake. Please clarify this term whether probiotic or prebiotic?\n\nVillous Atrophy takes place in the small bowel only, no need to specify its location small bowel. Alternatively, the authors may write, partial to total villous atrophy. (First line of the main introduction)\n\nThe first line of the Selection procedure contains results (February to July 2018). Please shift this in the results section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "8971", "date": "21 Nov 2022", "name": "Maha Hoteit", "role": "Author Response", "response": "Dear Reviewer 3, we modified the original manuscript according to your comments: Method \"In the Selection procedure part: In the ‘exclusion criteria’ the authors mentioned that they eliminated patients with malnutrition. But in Table 2, they assessed the risk of malnutrition in the patients with malnutrition. Please clarify it.\" All the patients collected in this study were referred to us by their physicians. According to their medical files, these patients weren’t malnourished. We excluded those who were diagnosed by physicians as malnourished patients because this interferes with our objectives. However, in our study, we were assessing the risk of malnutrition according to many specific criteria of which weight loss, albumin…. \"The line of measurements: \"Weight and height were measured using automated scale and stadiometer. Body composition including body fat, lean body mass, and visceral fat was assessed using Bio-impedancemetry body composition analyzer (BF-511, OMRON)\" Please explain the standardization of measurements.\" Percentage body fat (%BF) was measured by whole-body BIA to the nearest 0.1% using a digital scale/body composition monitor (Omron BF 511, 50 kHz, 500 µA, Kyoto, Japan), which includes an 8-sensor technology using both hands and feet. The participants stood with bare feet on electrodes on the scale with their knees and back straight while grasping a handle that also includes electrodes with both their hands horizontally raised, elbows extended straight, and maintaining a 90°-angle to the body. A previous study that compared body composition estimates using BIA devices with DXA and whole body magnetic resonance imaging indicated that the use of devices with additional hand electrodes provides a more accurate prediction of body composition and are suitable for public use. In order to determine percentage body fat, the device uses electrical impedance, along with the participant’s height, weight, age, and gender to generate results. The readings were obtained in duplicates and the average recorded. According to the manufacturer’s instructions, percentage body fat was measured two hours or more after breakfast. Omron Healthcare references for age and gender were used to classify the study participants into low, normal, high, and very high %BF. We followed these articles: A. Bosy-Westphal, W. Later, B. Hitze et al., “Accuracy of Bioelectrical Impedance Consumer Devices for Measurement of Body Composition in Comparison to Whole Body Magnetic Resonance Imaging and Dual X-Ray Absorptiometry,” Obesity Facts, vol. 1, no. 6, pp. 319–324, 2008. Healthcare Omron, Omron Body Composition Monitor. Instruction Manual BF 511, Omron Healthcare Cooperation Ltd., Kyoto, Japan, 2008. \"Laboratory value: The authors mentioned the collected data from three different centers. So, I think, the normal range of parameters could be changed. How did the authors standardized the laboratory results?\" The three centers were using same equipment and no issues were presented in the laboratory results. \"Food-related behaviors and GFD dietary adherence: \"Across the three-day food records, patients presenting a consumption between 3 to 10 g of gluten per day were considered as non-adherent\" In this line, please explain how you calculated gluten intake from the diet record? Which method did you use?\" For dietary compliance, patients were accurately interviewed by a trained physician and dietitian to assess the habitual intake of serving of gluten-containing food per week (e.g. pasta, pizza, bread, or cookies). Compliance to gluten-free diet was defined as follows: very low = more than one serving of gluten containing food/week; low = up to one serving of gluten-containing food/week; good = no gluten-containing food/week. Then based on the three days record the g/d data of gluten was investigated. We followed the same methodologies adopted in the below 2 studies: Ciacci, C., Cirillo, M., Cavallaro, R., & Mazzacca, G. (2002). Long-term follow-up of celiac adults on gluten-free diet: prevalence and correlates of intestinal damage. Digestion, 66(3), 178–185. https://doi.org/10.1159/000066757 Kotze L. M. (2004). Gynecologic and obstetric findings related to nutritional status and adherence to a gluten-free diet in Brazilian patients with celiac disease. Journal of clinical gastroenterology, 38(7), 567–574. https://doi.org/10.1097/01.mcg.0000131720.90598.6a \"Food record calculation: The authors showed the energy and nutrient intake in Tables 4, and 5, but they don't explain how they calculated this parameter from the diet? Which program did they use for the calculation?\" This was already available in text. The sentence is “In addition, three-day food records were collected and analyzed using Nutritionist Pro software (version 3.2, AXXYA) to assess caloric, micro- and macronutrients intake. The mean value and the standard deviation (SD) were used when interpreting the nutrient’s intake.” \"Statistical analysis: The authors did not mention the normality of the data set. How did they decide the normality test like (t-Test) without checking normality?\" The normality was tested for sure. The test used was Shapiro test. \"Please also mention the full form of the SPSS first then give its abbreviation in the brackets e.g., XXX (SPSS)\" Done Results: \"In data with a statistically significant difference, please indicate under tables that: 1) Which test did you use and 2) The cut of point of significant P value (like p<0,05 or 0,001).\" Done Discussion: \"The discussion part contains multiple repeats of the results. On the other hand, The authors haven't interpreted the result (Like How, and why ..). For example: What are the factors that influenced adherence to GFD in Lebanon? Please after comparing the results with other studies, also give original comments related to your data.\" Done \"Please first write the whole explanation of this term \"P-E malnutrition\" (Protein-energy malnutrition)\" Done \"\"In our study, the most commonly used DS was vitamin D and the least used one was prebiotics\" The authors mentioned prebiotics, I think it was written by mistake. Please clarify this term whether probiotic or prebiotic?\" Probiotics \"Villous Atrophy takes place in the small bowel only, no need to specify its location small bowel. Alternatively, the authors may write, partial to total villous atrophy. (First line of the main introduction)\" Done \"The first line of the Selection procedure contains results (February to July 2018). Please shift this in the results section.\" Done" } ] } ]	1	https://f1000research.com/articles/11-725
https://f1000research.com/articles/11-1440/v1	06 Dec 22	{ "type": "Research Article", "title": "Community review: a robust and scalable selection system for resource allocation within open science and innovation communities", "authors": [ "Chris L.B. Graham", "Thomas E. Landrain", "Amber Vjestica", "Camille Masselot", "Elliot Lawton", "Leo Blondel", "Luca Haenal", "Bastian Greshake Tzovaras", "Marc Santolini", "Thomas E. Landrain", "Amber Vjestica", "Camille Masselot", "Elliot Lawton", "Leo Blondel", "Luca Haenal", "Bastian Greshake Tzovaras" ], "abstract": "Resource allocation is essential to selection and implementation of innovative projects in science and technology. Current “winner-take-all” models for grant applications require significant researcher time in writing extensive project proposals, and rely on the availability of a few time-saturated volunteer experts. Such processes usually carry over several months, resulting in high effective costs compared to expected benefits. We devised an agile “community review” system to allocate micro-grants for the fast prototyping of innovative solutions. Here we describe and evaluate the implementation of this community review across 147 projects from the “Just One Giant Lab’s OpenCOVID19 initiative” and “Helpful Engineering” open research communities. The community review process uses granular review forms and requires the participation of grant applicants in the review process. Within a year, we organised 7 rounds of review, resulting in 614 reviews from 201 reviewers, and the attribution of 48 micro-grants of up to 4,000 euros. The system is fast, with a median process duration of 10 days, scalable, with a median of 4 reviewers per project independent of the total number of projects, and fair, with project rankings highly preserved after the synthetic removal of reviewers. Regarding potential bias introduced by involving applicants in the process, we find that review scores from both applicants and non-applicants have a similar correlation of r=0.28 with other reviews within a project, matching traditional approaches. Finally, we find that the ability of projects to apply to several rounds allows to foster the further implementation of successful early prototypes, as well as provide a pathway to constructively improve an initially failing proposal in an agile manner. Overall, this study quantitatively highlights the benefits of a frugal, community review system acting as a due diligence for rapid and agile resource allocation in open research and innovation programs, with implications for decentralised communities.", "keywords": [ "community science", "decision making" ], "content": "Introduction\n\nThe distribution of scientific funding through grants requires the identification of novel, feasible and potentially impactful projects. However, the traditional scientific grant allocation system involving a closed panel of experts in the field, or in similar fields,1 is notoriously slow,2 time consuming and expensive, often taking months and occurring in timescales of yearly rounds or grant calls. In extreme cases, the grant review program can be more costly than simply allocating small grants to each applicant, as in the case of the NSERC grant system of 2008.3 In addition, the allocation of grants has shown to suffer from various biases, such as the composition of the grant panel,4 gender and geographical location,5 group based dynamics personality triumphing over other qualitative factors,6–8 and socio-psychological factors such as group dynamics and personality traits triumphing over other qualitative factors.8,9 Overall, selection results are only weakly predictive of future performance.10\n\nOften, the reason to conduct grant allocations in a ‘closed’ setting is to protect the intellectual property of the grant applicants. As a result, the majority of unsuccessful grant applications, which contain a large amount of research effort, are inevitably lost, unavailable to the public after the fact.11 The recent emergence of the open science movement12–14 has reversed this incentive, with open access practices and early sharing of results such as pre-registration now becoming normalised by institutions and journals.15\n\nBeyond the allocation of funding, the review of early-stage, unpublished work by community peers has been leveraged to allocate other types of resources. For example, conferences often need to allocate time for their participants to showcase their work to other members of the community during a usually short amount of time, thereby providing a platform for promoting the work, building novel collaborations, and getting feedback to improve a manuscript. In such cases, peer reviewing is needed to decide in a collegial fashion whether a work is worth a full oral presentation, a shorter lightning talk, a poster, or is not of a high enough standard to be showcased to participants. For example, the EasyChair online platform has been used by close to 100k conferences for handling such review processes.16 Often, participants to a conference are also part of the “program committee” reviewing the proposed abstracts and papers of peer applicants, alongside external members of the scientific community. This allows for a rapid process usually lasting less than a few weeks.\n\nThis suggests there is a potential for a new, more agile route for community-driven grant allocation bypassing pre-selected grant panels that handle funds and introduce barriers,8 and relying instead on peer applicants to handle a large-scale application process in a short timescale. In this study, we present the design, implementation, and results of a community-driven, open peer-review system to support two open research communities during the coronavirus disease 2019 (COVID-19) pandemic across seven selection rounds (Figure 1): the “OpenCOVID19” initiative from Just One Giant Lab (JOGL)14,17 and the COVID relief charity Helpful Engineering.18 We show that this system is robust (unaffected by reviewer removal), agile (fast timeline), iterative (covering multiple grant rounds), decentralised (driven by the community), and scalable. Finally, we discuss these results and the perspectives they offer for the design of future community-driven review systems.\n\n(a) Stages of the open peer review process JOGL rounds 3-5. The online review forms and templates are found in supplementary data. (b) community review method JOGL rounds 3-5 (c) distribution of project type to expertise across rounds.\n\n\nMethods\n\nThe implementation of a crowd-based, open peer-review system followed the need to support two nascent community efforts, first by allocating volunteers to projects in the COVID relief charity Helpful Engineering,18 then by allocating funding to projects in the JOGL “OpenCOVID19” initiative.17 The method was developed as an open access grant review and funding allocation system, meaning that it was open to anyone willing to review. It was implemented using the Just One Giant Lab platform (app.jogl.io) as the project proposal host, and free-to-use tools and forms to conduct the review process (Extended Data:FigS219). The implementation was applied and refined over 7 rounds across 1 year.\n\nThe peer review system was conducted on early phase projects within both JOGL and Helpful Engineering. These projects were submitted by project leaders to a grant review process in order to allocate volunteers in the case of Helpful Engineering, and funding in the context of OpenCovid19. Reviews of these projects (see Figure 1b) were initially conducted by members of the community and included members of other projects who also submitted their project for review.\n\nAs a consequence of the process being experimental and serving an urgent need, the process was altered over time. However, it followed the same general pattern (Figure 1, Extended Data:FigS119). First, a template for the grant proposal was created by the community and was iteratively edited (Extended Data19) template followed typical grant application templates,20 with sections on team composition, the project general hypothesis and its timeline. The proposal was then submitted using a google form, which requested an email address and required only one application per project (Extended Data:FigS2a19). In Helpful Engineering rounds this included a link to their proposal hosted in editable google documents, while in JOGL rounds this included instead a link to their open access JOGL page proposal. The project links were manually formatted into a google sheet with a link to a review form for convenience, along with descriptions of desirable reviewer skills by the applicants in the proposal submission form to help reviewers find relevant projects (Extended Data: FigS2B19). A technical evaluation form scoring various criteria (eg: proposal efficacy, team composition, impact) on a scale from 1-5 (Extended Data19) was created by the designers of the program and iteratively changed following feedback from the community (Extended Data:FigS2c19). This form separated questions on projects into two areas centred around Impact and Feasibility for ease of identifying the problems and/or strengths in their grant application. A message with a link to the reviewer form for use in review, along with a nested google sheet containing project proposal links was spread among the community through announcements and email. In later rounds (JOGL 3-5) all applicants were asked to review at least three other projects and the process was randomised, removing the need for a sheet. The review process was given between 4 days HE 1, 8 days HE 2, 7 Days - JOGL 1, 10 days - JOGL 2, 16 days - JOGL 3, 21 days - JOGL 4 and 28 days JOGL 5, (Extended Data:FigS1b19) to allow reviews to occur and be collected via a google form into a google sheet automatically (Extended Data:FigS2d19). No reviewer selection was performed, however usernames (Slack handles or JOGL user names depending on the round) and emails were collected for conducting further analyses. The average reviewer scores were then composed into a presentation to the community, and those projects with a score above a given impact/feasibility threshold (Extended Data:FigS2e19) were chosen for grant funding. Due to the community aspect of our study, members from the JOGL HQ participated in the process, and their removal from the analysis does not change the observations (Extended Data:FigS1019), we therefore retain these in our analysis.\n\nAs mentioned in the previous section, the method of review was iteratively changed throughout the programme, elongating from an initial “emergency style” four day period of review and allocation (HE round 1) to 21 and 28 days in JOGL rounds 4 and 5 as the need for rapid response reduced, with an overall median average of 10 days per round (Extended Data:FigS1b19). As such, the design of the general process described in Figure 1 and Extended Data:FigS119 had some variations. For example, initially applicants were not required to review applications (Figure 1b). Upon scaling up of the programme, the process was adapted to be less dependent on volunteer reviewers, (Extended Data:Fig S1b,A-D19) and more dependent on the applicant’s reviews of their competing peers (Figure 1c). In JOGL rounds 3, 4 and 5 (Extended Data:FigS1b19) teams depositing a proposal could only be eligible after having reviewed at least three other teams. The changes in the process and differences in the rounds are summarised in Extended Data:FigS1c.19 The major changes between Helpful Engineering (HE) and JOGL rounds (Extended Data:FigS1c19) occurred through changes in the nature of proposal submission from google document links to an online project repository. In addition, HE rounds offered no grants, but instead publicity and allocation of members to projects, while JOGL offered microgrants worth up to 4000 euros per team (Extended Data:FigS2c19).\n\nIn Helpful Engineering, this review method allowed 54 projects to be reviewed and ranked by score for community recruitment purposes, with no official threshold, but instead an arbitrary set of “Highlighted projects”. Within JOGL this grant system reviewed 96 eligible applications (Figure 2) and allocated requested funds to 36 of these. Once the review process had taken place, the cut-off threshold of scores given by reviewers to projects for funding by JOGL was decided by an absolute threshold (above 3.5/5 average reviewed score) rather than a rejection rate. The absolute 3.5/5 threshold was chosen due to the gap in project scores in the first JOGL round, and maintained at this standard for consistency. Those with a score above the threshold were funded.\n\n(a) Number of Reviewers and projects during each round of peer/grant review. HE- Helpful Engineering Crowd reviews, JOGL- Just One Giant Lab funded projects. (b) Number of reviews per individual reviewer. (c) Number of reviewers per project. Despite a scale-up in the number of projects, the number of reviews per round scales linearly with the number of projects applying.\n\nThe results of each round, and number of reviews per reviewer were closely monitored through simple email handle tracking by a data handling administrator. If a number of emails were found to be grading a particular project and not others this was suggestive of fraudulent behaviour and self-grading. These reviews were then removed, and teams that were found responsible for this bad behaviour were removed from the review process, as described in grant round participation rules. This was performed only one time across all rounds prior to the rule of each reviewer having a minimum review count for their scores to be counted, which was created after this event.\n\nIn order to compute the correlation between reviews within a project, we first proceeded with data cleaning. Indeed, in several rounds, reviewers had to answer only a subset of questions from the review form that corresponded to the topic of the project (e.g. data project vs bio project). However, in some cases, projects were assigned to one or the other category by the different reviewers, leading them to answer to different sets of questions, making the correlation only partial. To mitigate this effect, for each project we kept only the reviews that corresponded to the choice of topic that was most expressed among reviewers. If no majority could be found, the project was removed from analysis. We then converted review scores into vectors of length the number of grades in the form. A Spearman’s rho correlation was then computed between all pairs of reviews within a project. Finally, for each review we computed the average correlation with the other reviews in the project. This number was then associated with the features of the reviewer who produced the review (Figure 4 and Extended Data:FigS719).\n\nFor JOGL rounds 1-5, we categorised the 23 to 29 questions from the review forms into either impact or feasibility related questions (see Underlying Data Review forms). The feasibility and impact categories were used to provide two dimensional projections of project scores during the result presentation.\n\nFor all JOGL rounds, reviewer responses of the \"What is your expertise relevant to this project\" question were manually coded into simple categories per review (see Table S1 in the Extended Data19). This data was then used as a proxy for expertise distribution across rounds (Figure 1b).\n\nIn addition, reviewer responses to the \"Which category would you say the project falls under?\" question were manually coded into a set of simple categories, representing a summary of the project types across rounds per review (see Extended Data conversion table21). The data, due to suggested categories provided by the form, needed little manual coding, but was formatted into a list, then concatenated into similar project types for simplicity. This data was used to assess project type distribution across rounds (Figure 1b).\n\nIn order to perform the bootstrap analysis of Figure 3d, we first ranked all projects using their average review score across reviewers. We then selected a review at random. If the corresponding project had at least another review, we removed the selected review and recomputed the average scores and final ranking. We then computed the Spearman correlation between the obtained scores and the original scores. This process was repeated until each project had only one review. Finally, we reiterated this analysis 50 times. The analysis code can be found as Extended Data.19\n\n(a) Heatmap showing review scores (rows) across questions (columns) for the JOGL round 4. Row and column clustering was performed using correlation distance and average linkage. (b) We show for PC1 (53% variance) the weights of the questions from the original question space. PC1 has near uniform weights across dimensions, indicating that it corresponds to an average score. (c) Project average score across reviewers as a function of number of reviewers. (d) Bootstrap analysis showing the Spearman correlation between the final project ranking and simulated project rankings with increasing proportion of reviews removed from the analysis (see Methods).\n\nWe confirm all ethical guidelines have been followed, using the same ethical procedures described in Commission Nationale de l'Informatique et des Libertes registration number “2221728” for user research and “2227764” for grant administration. Consent was granted by a user agreement on JOGL’s website upon signup (https://app.jogl.io/data), and on the google forms used during the study.\n\n\nResults\n\nWe describe in Figure 2 the reviewing activity across the seven rounds implemented. Despite the large differences in number of projects between rounds, we find that the number of reviews per round scales linearly with the number of projects applying (Figure 2a). In addition, the number of reviews per individual and number of reviewers per project have relatively stable distributions across rounds, independent of scale (Figure 2b-a). For example, despite the substantial growth in reviewers and projects in JOGL round 5, we find that the distributions of number of reviews per reviewer and number of reviewers per project are comparable to those observed in the previous rounds, highlighting the scalability of this review system to different systems. Finally, we note that the number of reviewers per project show a sustained increase from JOGL round 3 onwards, corresponding to the change in review process, where applicants were required to review at least 3 other projects (see Methods). This highlights the benefits of this requirement in promoting sustained engagement.\n\nIn order to obtain a granular score for each project, the reviewers had to grade between 23 (JOGL 1-2) and 29 (JOGL 3-5) criteria in the review form.21 We first investigate whether these questions would cover different dimensions of project quality. We show in Figure 3a a heatmap of reviewer scores in JOGL round 4 across 20 questions (removing questions only representing a minority of projects), visually showing a greater inter-review variability (rows) than inter-questions variability (columns). As such, respondents seem to assign a project with either low scores or high scores throughout their review. To quantify the number of dimensions of variation across grades, we conduct a Principal Component Analysis (PCA) on the questions correlation matrix, i.e correlations between pairs of questions across reviews (see Extended Data:Fig S2a19). We find that the first principal component (PC1) explains most of the variance (53%), with the next largest PC explaining less than 6% of the variance (Extended Data:Fig S319). When examining the weights of the various questions in PC1, we find that they all contribute to a similar level (Fig 3b), meaning that the PC1 is close to the average over all questions, confirming the visual insight from Fig 3a. This shows that scores are highly correlated, and that the average score across the review form is a reasonable operationalisation of project quality. In addition, we find that the top 10 PCs explain ~90% of the variance, indicating that review forms could be reduced in complexity using only half of the number of questions to obtain a similar outcome.\n\nWe next investigate the reliability of the review scores obtained across reviewers. As suggested by the previous section, for each review we compute the average score across all criteria from the review form. In the following, we refer to this average score as the review score. We observe a generally good discrimination of review scores between projects, with intra-project variation smaller than inter-project variation (Extended Data:FigS419).\n\nFinally, we investigate the robustness of the final project ranking as a function of the number of reviews performed using a bootstrap analysis (see Methods). For each project, a project score is computed by averaging its review scores, and projects are then ranked by decreasing score. We show in Figure 3d the Spearman correlation between the original project ranking and the ranking obtained when removing a certain proportion of reviews. We find that even with only one review per project, the final ranking is strongly conserved (rho=0.75 and see Extended Data:FigS519), confirming that intra-project variability is much smaller than the range of inter-project variability. This supports our design strategy, showing that the use of a granular form allows us to differentiate between projects whilst minimising the impact of individual reviewers’ variability.\n\nThe previous results show the existence of variability between reviews from different reviewers, yet with limited impact on final rankings (Figure 3d). Here we investigate the source of review variability: is it due to inherent grading variability between individuals, or can it be attributed to other factors? To evaluate this question, we analyse how review score varies with reviewer attributes. We explore in particular two possible sources of bias for which we could gather data: expertise and application status. First, reviewer expertise might be important in determining an accurate project score. This feature is operationalised using the self-reported expertise grade (1 to 5) present in the review forms of JOGL rounds. Second, a majority of reviewers (65%) were applicants of other competing projects, which could lead to a negative bias when reviewing other competing projects.\n\nWe show in Figure 4 how the review score varies as a function of these reviewer characteristics. We find that review score increases slightly with expertise (Figure 4a, Spearman’s rho=0.1, p=0.039). However, the strongest effect is found when looking at applicant bias: review scores from applicants are significantly lower than those from non-applicants (Figure 4b, p=1.4e-7). Given the fact that in JOGL rounds 3-5, applicants were required to score at least 3 projects, they are found to have a lower expertise towards other projects (Extended Data:Fig S619), which could explain the lower scores as suggested by Figure 4a. Yet, when controlling for review expertise, we find that application status is the main contributing factor, with a score difference between applicants and non-applicants of -0.52 points (p=1.61e-6, Extended Data:Supplementary Table 119). This supports that application status is a significant source of bias in the final score.\n\nBreakdown of project score as a function of (a) self-assessed expertise, (b) applicant status (i.e. the reviewer is also an applicant in the round). See Fig S4 for a breakdown by review round. (c) For each project, we compute the ratio between the proportion of applicant reviewers to the average proportion of applicant reviewers observed in the round. The boxplot compares the computed enrichments to the ones obtained for randomly assigned reviewers to projects, showing that applicants are evenly distributed across projects. (d) For each project, we compute the ratio between the proportion of applicant reviewers to the average proportion of applicant reviewers observed in the round. The boxplot compares the computed enrichments to the ones obtained for randomly assigned reviewers to projects, showing that applicants are evenly distributed across projects.\n\nSuch differences could be due to unfair grading, with reviewers from a certain category (applicants or non-applicants) grading more “randomly” than others. To analyse this effect, we need to look beyond average score into correlations. Indeed, two similar average scores could stem from highly different fine-grain score vectors. Imagine two reviewers grading 3 questions from 1 to 5. The first reviewer gives the grades 1, 2, and 5, while the second gives 5, 1, and 2. These reviews produce the same average score (2.67). However, their fine-grain structure is anti-correlated, with a Pearson correlation r = -0.69. In our context, we find that review scores are positively correlated, with a median Pearson correlation between their reviews of r = 0.28 across rounds (Figure 4d), in line with previous observations in traditional funding schemes [35]. More importantly, we find no difference between applicants and non-applicants in their correlation with other project reviews (Figure 4c). This indicates that the variability between grades within a review form are conserved across reviewer characteristics (see Fig S7 and Extended Data:Fig S9 for the other characteristics19). As such, if applicants are uniformly distributed across projects, one will not expect a difference in the final rankings.\n\nIn the JOGL implementation of the community review system, projects can apply to any number of rounds, irrespective of whether or not they have already successfully obtained funding in a previous round. We found 9 projects that applied to multiple rounds. On average, the relative performance of the projects in a grant round increases as a function of the number of participations (Figure 5a). We find that this effect is explained by re-participation being associated with early success, with initially lower performing projects eventually dropping out (Figure 5b-c). As such, the multiple round scheme supports projects with a high initial potential in the long-term through repeated micro-funding allocations. We also note that in the case of 2 projects, re-participation after an initial failure allowed them to pass the acceptance threshold. This highlights how constructive feedback allows for a rapid improvement of a project and its successful re-application in the process.\n\n(a) Project score percentile as a function of participation count. For each project, a score percentile is computed to quantify their relative rank within a specific application round, allowing to compare multiple projects across rounds. Participation count refers to the successive number of rounds a project has applied to. The black line denotes the average across projects, error bars represent standard error. Dots correspond to projects with only one participation, and lines to re-participating projects. Finally, the color gradient indicates relative score at first participation, from red (low) to green (high). (b) Same as a., after subtracting the percentile at first participation. (c) Score percentile at first participation as a function of whether or not a project has re-participated.\n\n\nDiscussion\n\nIn this manuscript we describe the “community review” method for the identification of novel, feasible and potentially impactful projects within two communities of open innovation: Helpful Engineering and OpenCovid19. This process was leveraged for the attribution of volunteers as well as micro-grants to projects over a year, in an agile and iterative framework.\n\nKey to the system is the requirement of applicants to take part in the reviewing process, ensuring its scalability. As such, the number of reviews is proportional to the number of projects applying (Figure 2), with a fast median process duration of 10 days. This requirement comes at a risk, since applicants might be negatively biased towards other projects they are competing against. Accordingly, we found that applicants consistently give a lower score to projects when compared to non-applicants (-0.52 points). This bias cannot be explained solely by the lower expertise of applicants towards the randomly assigned projects. Indeed, we found that self-reported expertise has only a limited impact on the final score (Figure 4c). The effect is most stringent for rare cases of self-reported expertise of 1 and 2 out of 5, suggesting that a threshold of 3 might be implemented to remove non-expert bias. It is on the other hand possible that non-applicants are positively biased towards projects from which they might have personally been invited to review. We however noted no such report in the conflict of interest question in the review form.\n\nDespite these biases, we found that applicants and non-applicants have a similar behaviour when grading questions in the form, with a stable Pearson correlation between their reviews of r = 0.28 (Figure 4/Extended Data:Fig S819). This is slightly higher than the correlation of 0.2 observed in an analysis of the ESRC’s existing peer review metrics,22 suggesting comparable outcomes when compared to existing institutional methods. The similarity of their correlation profiles means that such biases contribute a similar “noise” to the system: they might change the overall average scores, but not their ranking as long as applicants are well distributed across projects. Accordingly, we found that the community review system is robust to the removal of reviewers, with an average ranking Spearman correlation of 0.7 in the extreme case of one reviewer per project.\n\nFinally, we showed that some projects apply multiple times to the application rounds. While the number of such projects of this type is small (9 projects), we find that it had two benefits. First, we found two projects that re-applied after an unsuccessful application, allowing them to pass the acceptance threshold on the second application. This showcases the ability of the feedback system to benefit projects in constructively improving their application. Furthermore, we found that the number of applications of a project is strongly dependent on its performance on the first application. This means that the iterative process allows to select highly promising projects and sustain their implementation in the mid- to long-term. This is of particular importance when considering traditional hackathon systems, where promising projects are usually not supported over longer periods of time.\n\nThe speed and cost-efficiency of the community review process has allowed for a reactive response to the high-pressure environment created by the pandemic. This agility has meant that within the short time frame given, projects have been able to produce literature, methods and hardware and put them to use.23–28 Overall, the community review system allows for a rapid, agile, iterative, distributed and scalable review process for volunteer action and micro-grant attribution. It is particularly suited for open research and innovation communities collaborating in a decentralized manner and looking for ways to distribute common resources fairly and swiftly. Finally, community review offers a robust alternative to institutional frameworks for building trust within a network and paves the way for the installation of community-driven decentralized laboratories.", "appendix": "Data availability\n\nOpen Science Framework: DATA FOR: Community review: a robust and scalable selection system for resource allocation within open science and innovation communities. https://doi.org/10.17605/OSF.IO/CAZ4N. 21\n\nThis project contains the following underlying data:\n\n- Review Data (the raw responses of the review rounds analysed by the paper, and the raw data used in the study.)\n\n- Project round progress.csv (aggregated data and is based on data post analysis for our final figure, and the scores of each project over time, however we have aggregated this for ease of viewing.)\n\n- Grant Review forms (the forms used to assess each proposal)\n\n- Peer Review protocol (the protocol used to analyse the raw data, giving the correlation values we refer to in the paper)\n\n- Coded expertise (the simplified version of project and reviewer type collected during review)\n\nOpen Science Framework: EXTENDED DATA FOR: Community review: a robust and scalable selection system for resource allocation within open science and innovation communities, https://doi.org/10.17605/OSF.IO/W5Q9B. 19\n\nThis project contains the following extended data:\n\n- Supplementary figures 1-10\n\n- Supplementary table 1\n\n- Analysis code\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nAn earlier version of this article can be found on biorxiv at doi (https://doi.org/10.1101/2022.04.25.489391). We acknowledge the work of the projects involved and Helpful Engineering as well as Just One Giant Lab volunteers for their logistical and communications help throughout the pandemic.\n\n\nReferences\n\nLangfeldt L: The Decision-Making Constraints and Processes of Grant Peer Review, and Their Effects on the Review Outcome. Soc. Stud. Sci. 2001 Dec 1; 31(6): 820–841. Publisher Full Text\n\nHerbert DL, Barnett AG, Graves N: Australia’s grant system wastes time. Nature. 2013 Mar; 495(7441): 314–314. PubMed Abstract \| Publisher Full Text\n\nGordon R, Poulin BJ: Cost of the NSERC Science Grant Peer Review System Exceeds the Cost of Giving Every Qualified Researcher a Baseline Grant. Account. Res. 2009 Feb 27; 16(1): 13–40. PubMed Abstract \| Publisher Full Text\n\nRoumbanis L: Peer Review or Lottery? A Critical Analysis of Two Different Forms of Decision-making Mechanisms for Allocation of Research Grants. Sci. Technol. Hum. Values. 2019 Nov 1; 44(6): 994–1019. Publisher Full Text\n\nSeverin A, Martins J, Delavy F, et al.: Gender and other potential biases in peer review: Analysis of 38,250 external peer review reports. PeerJ. Inc. 2019 Jun [cited 2021 Jul 9]. Report No.: e27587v3.Reference Source\n\nCoveney J, Herbert DL, Hill K, et al.: ‘Are you siding with a personality or the grant proposal?’: observations on how peer review panels function. Res. Integr. Peer. Rev. 2017 Dec 4; 2(1): 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPier EL, Raclaw J, Kaatz A, et al.: ‘Your comments are meaner than your score’: score calibration talk influences intra- and inter-panel variability during scientific grant peer review. Res. Eval. 2017 Jan; 26(1): 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFogelholm M, Leppinen S, Auvinen A, et al.: Panel discussion does not improve reliability of peer review for medical research grant proposals. J. Clin. Epidemiol. 2012 Jan; 65(1): 47–52. Publisher Full Text\n\nCoveney J, Herbert DL, Hill K, et al.: ‘Are you siding with a personality or the grant proposal?’: observations on how peer review panels function. Res. Integr. Peer. Rev. 2017; 2: 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFang FC, Casadevall A: NIH peer review reform--change we need, or lipstick on a pig? Infect. Immun. 2009 Jan 21;2009 Mar; 77(3): 929–932. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Hippel T , von Hippel C : To apply or not to apply: a survey analysis of grant writing costs and benefits. PLoS One. 2015; 10(3): e0118494. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLandrain T, Meyer M, Perez AM, et al.: Do-it-yourself biology: challenges and promises for an open science and technology movement. Syst. Synth. Biol. 2013 Sep; 7(3): 115–126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMasselot C, Tzovaras BG, Graham CLB, et al.: Implementing the Co-Immune Open Innovation Program to Address Vaccination Hesitancy and Access to Vaccines: Retrospective Study. J. Particip. Med. 2022 Jan 21; 14(1): e32125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSantolini M: Covid-19: the rise of a global collective intelligence? The Conversation;2020 [cited 2021 Apr 2].Reference Source\n\neLife Journal Policy: eLife.2022.Reference Source\n\nEasy Chair:2021.Reference Source\n\nKokshagina O: Open Covid-19: Organizing an extreme crowdsourcing campaign to tackle grand challenges. RD Manag. 2021 Mar 23; 52: 206–219. Publisher Full Text\n\nHelpful Engineering: Helpful.[cited 2021 Jul 9].Reference Source\n\nGraham CLB: EXTENDED DATA FOR: Community review: a robust and scalable selection system for resource allocation within open science and innovation communities.2022, November 26. Publisher Full Text\n\nJe-S electronic applications - Economic and Social Research Council:[cited 2021 Jul 9].Reference Source\n\nGraham CLB:DATA FOR: Community review: a robust and scalable selection system for resource allocation within open science and innovation communities. [Dataset].2022, November 26. Publisher Full Text\n\nJerrim J, de Vries R : Are peer-reviews of grant proposals reliable? An analysis of Economic and Social Research Council (ESRC) funding applications. Soc. Sci. J. 2020 Mar 6; 1–19. Publisher Full Text\n\nAidelberg G, Aranoff R, Javier Quero F, et al.: Corona Detective: a simple, scalable, and robust SARS-CoV-2 detection method based on reverse transcription loop-mediated isothermal amplification. ABRF;2021.Reference Source\n\nBektas A: Accessible LAMP-Enabled Rapid Test (ALERT) for detecting SARS-CoV-2. Viruses. 2021. Publisher Full Text Reference Source\n\nCheng C: COVID-19 government response event dataset (CoronaNet v. 1.0). Nat. Hum. Behav. 2020; 4(7): 756–768. PubMed Abstract \| Publisher Full Text\n\nGreshake Tzovaras B, Rera M, Wintermute EH, et al.: Empowering grassroots innovation to accelerate biomedical research. PLoS Biol. 2021 Aug 9; 19(8): e3001349. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMonaco C, Jorgensen E, Ware S: The One Hour COVID Test: A Rapid Colorimetric Reverse-Transcription LAMP–Based COVID-19 Test Requiring Minimal Equipment. ABRF;2021 Sep.Reference Source\n\nTzovaras BG: Quantified Flu: an individual-centered approach to gaining sickness-related insights from wearable data. medRxiv. 2021." }	[ { "id": "161990", "date": "17 Feb 2023", "name": "Ferdinando Patat", "expertise": [ "Reviewer Expertise Astrophysics: Supernovae. Peer-review: proposal selection and time allocation 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\nThe paper reports on the design, deployment and evaluation of what the authors describe as an 'agile community review system' for allocating micro-grants. As opposed to classical peer-review performed by pre-selected expert panels, the community review involves the participation of applicants themselves. It therefore provides as scalable solution, in the sense that the number of reviewers increases with the number of applications, hence keeping the review load manageable and the duty-cycle faster than in the classical panel case. The data show that the inter-reviewer correlation is similar to what has been reported for the classical paradigm. Also, the final rankings are shown to be robust against the randomised removal for reviewers.\nThe paper presents the case very clearly and accurately. One aspect which is weak (honestly this is probably the only weakness of the paper) is that the casual reader is left under the impression that the idea of a community review is new and proposed here for the first time. And that is not the case. This concept, sometimes indicated as distributed peer review (DPR), has been around for many years. As far as I know the idea appeared first in 2009 in a paper by Saari & Merrifield (2009)1. Although it was referring to applications for telescope time, the concept was of general interest. This mechanism has been implemented at least in three major, ground-based astronomical facilities (GEMINI, ALMA, ESO). Also, a similar distributed process has been deployed in the field of computer sciences for the selection of conference papers5. Other interesting examples are those of the US National Science Foundation2,3 and the US National Institute of Food and Agriculture (NIFA), which has deployed DPR in 20166,7.\nThe authors should therefore provide a short recap of these initiatives and cite the applicable references. In addition to the papers published by the above organisations, there is an article on Physics Today4 which gives a nice summary for the specific cases in astronomy.\nOther minor points, which the authors should consider, are listed here:\nScalability. The authors correctly state that the approach is scalable, in the sense that increasing the number of projects linearly increases the number of reviewers. They show this in Fig. 2b. At a first read it looks like this is an unexpected results and, somehow, the data show that this is the case. However, I argue that this is completely expected, since the number of potential reviewers, on average, is directly proportional to the number of submitted projects. This would not be necessarily the case if the system would not force the applicants to review proposals. Maybe the authors should be more explicit on this.\n\nIn the section about fraudulent behaviour, the authors state that this was removed from the review process. However, they do not explain what this means in practice in terms of actions taken against those reviewers. Is their project simply rejected? Are they warned? Or is just their evaluation removed from the data?\n\nThe caption of Figure 4 for panels c) and d) does not match what is presented in the figure and also its description in the main text section Measuring reviewer biases:\n\"For each project, we compute the ratio between the proportion of applicant reviewers to the average proportion of applicant reviewers observed in the round. The boxplot compares the computed enrichments to the ones obtained for randomly assigned reviewers to projects, showing that applicants are evenly distributed across projects. (d) For each project, we compute the ratio between the proportion of applicant reviewers to the average proportion of applicant reviewers observed in the round. The boxplot compares the computed enrichments to the ones obtained for randomly assigned reviewers to projects, showing that applicants are evenly distributed across projects.\"\nThe authors never explain what they mean by \"enrichment\". Also, while panel c) may be showing a ratio, this is not the case for panel d), which on the y-axis has the label \"inter-review correlation\", which sounds like a Pearson correlation, and not a ratio. In general, I am rather confused about panels c) and d) in Fig. 4, which I reckon requires a better explanation also in the main text (which, by the way, never includes the word \"enrichment\").\n\nAs shown in Fig. 3a, there are two evaluation criteria related to the Team composition. Given that many organisations are now moving to a dual anonymisation of the applications, it would be interesting to see how the scores change if one removes these two indicators from the final score. I guess that, given the large number of indicators (and the results shown by the authors about the slight change when 50% of them are removed), no measurable effect is going to be seen. Also because, since the team's identity is known to the reviewers, there is certainly a cross-talk between this indicator and all others. It is probably therefore impossible to disentangle its effect from the available data. Nevertheless, I suggest the authors mention this aspect.\nThe study is well designed, the analysis was conducted in an appropriate way and included a satisfactory level of checks and controls. The methods are well explained and the necessary data are made available so that the analysis can be repeated and validated. The statistical analysis and its presentation meet the required standards.\nThe conclusions are well supported, interesting and useful for other organisations which may consider adopting a similar schema. I therefore recommend the article for indexing after the point on the missing citations and short description of existing cases is 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? Yes\n\nAre 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": "9437", "date": "10 Mar 2023", "name": "Chris Graham", "role": "Author Response", "response": "Dear Ferdinando, Thank you for a thorough and well-rounded review of our article. We are glad that you found the article interesting and our methods to be robust and the data well described. You are correct, our method is very similar to distributed peer review (DPR) and we should have included citations and mentions of this in our literature review. Its use in astronomy, and especially by the NSF are particularly interesting, as are the articles which cite its drawbacks. Thank you for improving the paper with mention of this. We have incorporated the mention of our use of DPR throughout the paper. We have now expanded the introduction to speak about distributed peer review, and throughout conclusions in the article have also made reference to distributed peer review, although we did not initially use DPR, and were instead adopting a crowd-sourcing approach, and then combined the two methods. These changes have been made especially to the Introduction, where we have incorporated your suggested references, as well as others not mentioned In terms of your specific comments, for Figure 2d, the wording has now been changed to reflect the reality that the community review method was expected to be scalable. The use of language was through the assumption of a null hypothesis and was innappropriate. We have also changed the Figure legend of Figure 4c, as pointed out to be reflective of the figure. This was a copy-editing error. The use of the word ' enrichment' has also been specified for Figure 4d. Your final point, on removing team composition questions is very interesting especially when thinking about double anonymisation of reviewers. We hope, based on the data of the PCAs which separated the questions by their power and difference to other questions (Figure 3b), that this was not a big influence on the final scores therefore would agree with your own assumption that this wouldn't effect the rankings greatly. Unfortunately although your hypothesis for the inclusion of a further figure to analyse this is sound, our team would prefer to keep the paper in its current state in terms of new hypotheses, but think this is an excellent question and in future publications will ask this very question if given the chance. Overall your review has identified some important literature and potential issues, as well as a new hypothesis and we are very grateful for it. Thank you for taking the time to analyse our article in the way you have, you've enlightened us specifically on the astronomy field's use of DPR and shown how there is precedent for such methods, with the example of the National Science Foundation's pilot study. Hopefully there will be further attempts with such large organisations in the future, perhaps with safeguarding measures to counteract any collusion, as well as controls studies for grant review with existing techniques." } ] } ]	1	https://f1000research.com/articles/11-1440
https://f1000research.com/articles/12-414/v1	18 Apr 23	{ "type": "Policy Brief", "title": "Developing bird friendly transmission power lines in Kenya", "authors": [ "Peggy Ngila", "Chiawo David", "Margaret Owuor", "Oliver Wasonga", "Elizabeth Ellwood", "Jane Mugo", "Nelly Masayi", "Timothy Ikime", "Stanley Chelelgo", "Dominic Mugo", "Chiawo David", "Margaret Owuor", "Oliver Wasonga", "Elizabeth Ellwood", "Jane Mugo", "Nelly Masayi", "Timothy Ikime", "Stanley Chelelgo", "Dominic Mugo" ], "abstract": "Background: Kenya's rich wildlife is an important natural resource and is vital to the country's economy. However, rapid infrastructure development in key wildlife areas has threatened the survival of numerous species including raptors. Policy: The growing distribution of electricity grids in Kenya poses a significant risk to raptors and populations of other large birds. This research aimed to review the policy gaps in Kenya’s National Energy Policy (2018) examining the strategies for developing bird friendly transmission power lines in Kenya, with a goal to reduce the negative impact on raptor populations and improve their conservation. The findings indicate a high overlap between the ecological niche of raptors with medium and high voltage transmission power lines, with indication of high-risk hotspots for electrocution and collisions. Recommendations: This policy brief therefore proposes the adoption of bird friendly power lines in new transmission infrastructure projects and retrofitting existing power lines with bird friendly devices. This can be achieved through strengthening the legal and regulatory frameworks in the National Energy Policy. This will help prioritize avian conservation in power lines infrastructural development projects including the development of specific policies and guidelines for the placement, orientation, and marking of power lines to reduce the risk of electrocution and collisions of raptor species. Conclusions: Kenya committed to protecting migratory bird species and their habitats by signing the Convention on the Conservation of Migratory Species of Wild Animals in 1999. However, the National Energy Policy of 2018 lacks guidelines for bird-friendly power lines. Addressing this requires collaboration from the government, power companies, research institutions, conservation groups, and local communities. A concerted effort involving public education, targeted funding for research, and stakeholder collaboration can help Kenya meet its conservation obligations and safeguard vital habitats for migratory birds.", "keywords": [ "Green technology", "Raptors Electrocution and Collision", "Raptor conservation", "Bird Electrocution", "Bird friendly power lines", "Avian electrocution" ], "content": "Introduction\n\nKenya Wildlife Sector is guided by the Constitution of Kenya (The Constitution of Kenya, 2010), the Wildlife Conservation and Management Act of 2013 (The Wildlife Conservation and Management Act, 2013) and Vision 2030 (Government of Kenya Vision 2030, 2008) and aims to provide an overarching framework that prioritizes, co-ordinates and inspires participation for the transformation of the wildlife sector in Kenya (Ministry of Tourism and Wildlife (2018). Kenya ranks among the world’s top wildlife destinations with its wildlife being an important economic resource (Ministry of Tourism and Wildlife, 2018). Human activities including habitat fragmentation and degradation, overexploitation, and rapid development have threatened the survival of several species, including raptors through loss of wildlife connectivity areas (Ogada & Keesing, 2010). Among the 1,100 bird species in Kenya, 102, nearly 10%, are raptors which play a crucial role in controlling prey populations, carcass decomposition, disease transmission (Birdlife International, 2022), and maintaining the ecological balance (Donázar et al., 2016).\n\nOverhead power lines pose a significant threat to bird populations worldwide, particularly to endangered species such as raptors that have a high incidence of electrocution (Angelov et al., 2013; Harness et al., 2008; Real et al., 2001). Collisions with electrical wires alone are responsible for the deaths of over one billion birds (Figure 1) annually (Loss et al., 2014). The problem of raptor mortality due to energy infrastructure is pervasive in Africa, with reports of electrocutions in countries such as Kenya, Sudan, and South Africa, where at least 14 diurnal raptor species have been affected (Boshoff et al., 2011; Smallie & Virani, 2010). While larger birds are at greater risk due to their size, smaller birds are also vulnerable to electrocution because transformers contain numerous closely-spaced energized parts (Prinsen et al., 2011). The electricity expansion projects in many African countries e.g. Kenya's “Last Mile Connectivity Project” aimed at providing electricity access to every rural home by 2020 (Kenya Power, 2016), further exacerbates the threat to avian populations.\n\nImage by Peter Olsoy.\n\nLicensed under http://creativecommons.org/licenses/by-nc/4.0/.\n\nPower poles and power lines pose a significant threat to birds, with the extent of harm depending on various factors, including the environmental conditions of the site, the morphology and ecology of the bird species, and the technical parameters of the power line construction (Bernardino et al., 2018). The threat is especially pronounced for large birds, such as storks, vultures, herons, and raptors (Barrientos et al., 2012), which frequently migrate long distances and rely on open spaces where energy infrastructure is often developed. As a result, power lines often intersect soaring bird corridors, resulting in significant fatalities. Above-ground power lines pose three major threats to soaring birds: the risk of electrocution, as birds perched on power poles or cables can be electrocuted if they cause short circuits; the risk of collision, as birds may collide with power line cables that are difficult to perceive as obstacles; and the loss of habitat, as power lines may cut through suitable habitats and landscapes for birds, causing avoidance behaviours. To protect bird populations, it is essential to consider these threats when planning and constructing power lines.\n\nPower lines are classified into three categories based on voltage levels, namely high voltage, medium voltage, and low voltage. High voltage or transmission lines (60 kV to 700 kV+) carry electricity at high voltages from power plants to substations (APLIC, 2006). Medium voltage or distribution lines (2.4 kV to 60 kV) deliver low-voltage electricity to individual consumers. Distribution line pylons are typically 8-12 meters high, much smaller than those used on transmission lines. Low voltage lines (120 V to 600 V) are commonly used to connect residential or small commercial customers to utilities. The likelihood of bird collisions with power lines depends on the voltage and construction characteristics of the line (Bevanger, 1994). Birds are more likely to collide with transmission lines carrying a voltage of 110 kV or higher, due to the greater number of conductors, the presence of undetectable earth wires, higher tower heights, and larger distances between towers (Bevanger, 1994; Janss & Ferrer, 1998). Therefore, measures should be taken to reduce the risks of bird collisions, particularly for high-voltage transmission lines.\n\n\nPolicy outcomes and implications\n\nThis paragraph describes the methodology used for mapping raptors’ niche distribution and high collision and electrocution risk areas in Kenya. Raptor data was obtained from the Global Biodiversity Information Facility (GBIF, (RRID:SCR_005904)) for the entire Kenya and R software (RRID:SCR_001905) was used to filter and clean the data using the “tidyr” package (Ngila, 2023a). After filtering and cleaning, 11,529 records were used in the modelling. We chose a combination of 19 bioclimatic variables, elevation data from the Shuttle Radar Topographic Mission DEM digital elevation model (SRTM DEM), slope and aspect data calculated from the DEM, and topographic roughness index. We also obtained normalized difference vegetation index (NDVI) data from the National Oceanic Atmospheric Administration (NOAA) website and human influence index (HII) data from Last of the Wild (found here). A variance inflation factor procedure under the “usdm” package in R was used to eliminate variables with a variance inflation factor higher than 10. From an original set of 25 variables, we preserved just the 12 most important factors based on raptors’ ecological needs. The study obtained raptor data from GBIF for the period 2013-2020 (https://doi.org/10.15468/dl.hzyp6v) (Ngila, 2023). Table 1 shows the 12 most important factors that were preserved. To model the distribution of raptors in Kenya, we utilized an ensemble forecasting method from the R package (biomod2 (RRID:SCR_018246)) (Thuiller et al., 2009). This method produces an average of the projections from the ensemble model to obtain the final potential distribution of raptors. The resulting map was binarized into presence-absence values at approximately 1km resolution (Figure 2). To identify areas of high impact of power lines on raptor populations, we obtained power line transmission data from the World Bank and used it to generate collision risk map. The main objective of this policy brief was to propose strategies for bird-friendly transmission power lines in Kenya to mitigate raptor mortality due to collision and electrocution. The results demonstrate significant hotspots of raptor electrocution and collisions in Kenya, particularly in areas where transmission lines overlap with raptor ecological niches (see Figure 3). Furthermore, Table 2 presents the suitable areas for raptor distribution in Kenya, organized by county. The policy brief also highlights the policy gaps in the power transmission infrastructure development Kenya to be addressed in order to achieve bird-friendly transmission power lines.\n\na Isothermality = (Mean diurnal range/Temperature annual range)(×100).\n\nb Temperature annual range = (Maximum temperature of warmest month – Minimum temperature of coldest month).\n\nScales show suitability of raptors with 0 showing absence of raptors and 1 showing presence.\n\nThese transmission lines of >60 kV pose a huge threat to raptors as they are at risk of electrocution and collision.\n\nSuitable habitat amounted to 90, 729 km2, which corresponds to 16% of the total Kenya territory. Narok (13512 km2), Samburu (11,500 km2), Laikipia (8427 km2), Kajiado (7181 km2) and Taita/Taveta (6615 km2) are the regions encompassing the largest potentially suitable surface for Raptors while Mandera (0 km2), Wajir (0 km2), Garissa (0 km2), Lamu (0 km2) and Mombasa (0.9 km2) were those including none or the smallest amount of suitable areas (Table 1).\n\nKenya had a transmission network of 4766 circuit km at 132 kV and above voltage levels by June 2017, which included 585 km of 400 kV lines, 374.59 km of 220 kV lines, and 839.11 km of 132 kV lines (National Energy Policy, 2018). The Kenya Electricity Transmission Company (KETRACO) is currently constructing 4500 new lines to double the transmission network, introducing high voltage 400kV and 500kV direct current (DC) lines and three major regional interconnectors to Ethiopia, Uganda, and Tanzania. By 2031, it is projected that 16,000 km of transmission lines and associated substations will have been constructed (National Energy Policy, 2018). KETRACO is also planning an additional 4200 km of lines to expand and strengthen the grid. The existing and proposed electric distribution and transmissions in Kenya pass through major suitable raptor habitat in the country (Figure 4). Kenya is a signatory of the Convention of Conservation of migratory species of wild animals, a treaty signed in 1999, to conserve migratory species of birds in migratory routes and preserve habitats commonly used by migratory birds. However, with the proposed electricity transmission network, we see a gap in addressing dangers of electrocution and collision to migratory birds including raptors. The (National Energy Policy, 2018) lacks policy guidelines on bird friendly power lines despite Kenya’s ratification of the convention of conservation of migratory species of wild animals.\n\nThese wire markers are very important as they make power lines more visible to raptors and pilots especially near an air-strip.\n\nURL: https://uploadwikimedia.org/wikipedia/commons/2/2c/Wire_marker.jpg.\n\nLicence: Creative Commons CC 1.0 Universal Public Domain Dedication.\n\nSeveral studies (Alonso et al., 1994; Eccleston & Harness, 2018; Erickson et al., 2005; Janss & Ferrer, 2000; Rioux et al., 2013) have shown that power lines can be particularly dangerous when located along migratory corridors. We therefore suggest the following;\n\n1) Use of bird-friendly power lines in new transmission infrastructure projects, as well as upgrading existing power lines with bird-friendly equipment such as insulation and perch discouragers and deflectors when possible. Insulating electricity lines, which may entail either live wires or burying them underground, is one example. A before-and-after research in urban surroundings in Tucson, Arizona, USA, conducted in 2003, provided proof of this (Dwyer & Mannan, 2007). Insulating potentially harmful parts of electrical pylons resulted in fewer electrocutions (0.2 electrocutions/nest) compared to before insulation (1.4 electrocutions/nest). A 2010 research assessment (Jenkins et al., 2010) discovered significant decreases in avian mortality following power line labeling in the United States and South Africa. Following the marking of four sections of the line in the winters of 1989-90 and 1990-1 in Extremadura, Spain (Alonso et al., 1994), there were fewer collisions (60% reduction in fatalities: 18 vs. 45; 61% decrease in birds flying between lines: 357 birds/day vs. 124 birds/day).\n\n2) Strengthening legal and regulatory frameworks to prioritize avian conservation in power line infrastructure development projects, including the development of specific policies and guidelines for power line placement, orientation, and marking to reduce the risk of electrocution and raptor collisions. A before-and-after study in the western Pyrenees, Spain (Donázar & Fernández, 1990), found that the population of griffon vultures Gyps fulvus increased from 282 pairs (in 23 colonies) in 1969-75 to 1,097 pairs (46 colonies) in 1989 following the initiation of multiple conservation interventions including the creation of reserves at nine breeding colonies (one in 1976, eight in 1987). This study is also discussed in more detail in ‘Use legislative regulation to protect wild populations’, ‘Restrict certain pesticides or other agricultural chemicals’ and ‘Provide supplementary food to increase adult survival’.\n\n3) Public education programs to raise awareness of the importance of raptors to ecosystems and impacts of power lines on their population, as well as the encouragement of community development in conservation efforts. A 1998 literature analysis on crane Grus spp. conservation (Davis, 1998) shows how whooping cranes G. americana continued to decrease in the United States notwithstanding legislative protection, until aggressive public education programs were implemented. Prior to schooling, natural causes accounted for less than half of all whooping crane deaths, while just four whooping cranes were shot between 1968 and 1998.\n\n4) Provide funding and support for research on raptor populations and developing new technologies to reduce the negative impact of power lines on wildlife.\n\n\nActionable recommendations\n\nElectricity is a daily necessity though the requisite power lines have a negative impact on the lives of birds. To reduce collisions and electrocution proper measures recommend the following risk reductions:\n\n1) Line placement that takes into account the migratory patterns and high-bird use areas. This can be done using: regular bird surveys and monitoring to map high-bird use areas and migratory patterns in potential areas to inform line placement decisions; consulting with bird conservation groups and experts to gather information about bird habitat and behavior in the hotspot areas as well as using Geographic information systems (GIS) technology to analyze bird distribution and behavior patterns for integration in environmental assessment reports at before electricity line placement project inception. The line placements to consider biological and environmental factors such as bird flight paths, prevailing winds, and topographical features, to reduce the rate of collisions and electrocutions. The expected outcome will be reduced cases of collisions and electrocutions due to friendly migratory routes, corridors and breeding areas. The limitation for this recommendation would likely be limited support towards long-term research on birds survey at the potential project implementation sites. Lack of long-term plans for electricity expansion projects to inform bird survey activities and spontaneous projects due to political declarations and interests could pose a challenge.\n\n2) Put regulations on power line markings to increase the visibility of the line (Figure 4). Line markers are safety instruments applied to overhead power transmission lines. Use of high visibility markers or reflective materials to increase line visibility for birds, especially in low-light or foggy conditions. Also, use of bird diverters or markers to increase the visibility of the lines. The outcome will be reduced cases of collisions because the lines will be more visible to the birds. Implementing bird-friendly power line measures such as line markings, bird diverters, and underground lines to be implemented by power line transmission contractors to reduce bird mortality due to collisions and electrocution with power lines. Implementing these recommendations may require significant financial and human resources. Electricity companies, in particular, may be hesitant to invest in bird-friendly power line design and implementation due to cost concerns, due to the contractual nature of such projects in Kenya, most companies are keen to optimize profit margins.\n\n3) Burying lines where feasible and warranted. A feasibility study can be conducted to determine whether burying lines is a viable option in the collision risk hotspot areas, taking into account factors such as cost, soil conditions and terrain. This has a likelihood of totally eliminating bird collisions and electrocution however burying power lines may be expensive and difficult to implement in certain areas, such as rocky or mountainous terrain and areas yet to be marked for development. The expected outcome will be reduced cases of collision and electrocution due to overhead lines in the flying routes.\n\n4) Conducting regular monitoring of cases of electrocution and collision of birds. The expected outcome will long-term data to inform the expansion of transmission lines, areas to improve to achieve to reduce the cases of collision and electrocution. Limited research funding towards such highly specific research and limited collaboration between development, government agencies and research institutions may pose a challenge to the implementation.\n\n5) Provide training and capacity building for relevant stakeholders, including electricity company staff, conservationists, and government officials, on bird friendly power line design and implementation. Also, through collaborating with local bird conservation groups and experts to provide specialized training on bird behavior and habitat. Integrating bird-friendly power line engineering into the training programmed for implementers is recommended.\n\n6) Establishing partnerships and collaborations with electricity companies, conservation organizations, and relevant government agencies to implement bird friendly power line measures. This can be done through collaborating with local conservation groups and experts to develop and implement bird-friendly power line measure and by building partnerships with electricity companies and relevant government agencies to promote and implement bird-friendly power line measures. However, if key stakeholders such as electricity companies, government agencies, and local communities are not fully engaged in the implementation process, the recommendations may not be effectively implemented.\n\n7) Bird electrocution risk should form part of environmental impact assessment associated with planned electricity distribution infrastructure. This can be achieved by conducting environmental impact assessments that include a thorough assessment of bird electrocution risks in the risk hotspot areas. Clear mitigation measures to reduce bird electrocution risks in project proposals and designs should form the basis of approval of such projects. The challenge could be the high cost of such projects against the funds allocated. Electricity transmission project to take into consideration of collision and electrocution mitigation in the allocation of project funds.\n\nDespite the limitations mentioned above, the implementations of these recommendations is likely to have several positive impacts on bird populations and the environment. Here are some potential impacts:\n\n1. Habitat conservation: bird-friendly power line design and implementation can help conserve important bird habitats, including migratory corridors and breeding areas.\n\n2. Enhanced biodiversity: by reducing bird mortality, these measures can help maintain and enhance biodiversity in the project area. This is particularly important for threatened and endangered bird species.\n\n3. Improved stakeholder relations: collaborating with local communities, conservation groups, and relevant government agencies can improve stakeholder relations and promote awareness about bird conservation issues.\n\n4. Reduced liability: implementing bird-friendly power line measures can reduce the risk of liability for electricity companies in cases where bird collisions or electrocution lead to property damage or human injury.\n\n5. Improved corporate social responsibility: implementing these measures can help electricity companies fulfill their social responsibility and demonstrate their commitment to environmental conservation.\n\n\nMaking the skies friendly\n\nThere are many types of effective insulation for poles of medium-voltage power lines such as plastic hoods, silicon tubes, long rod insulators, and plastic insulators covering the metal consoles. Some of the top solutions are those which allow the birds to securely perch on poles. For example, line markings to increase the visibility of electric lines has shown to reduce the risk of collisions (Figure 5). A wide range of potential line marking devices has evolved over the years, including spheres, swinging plates, spiral vibration dampers, strips, swan-flight diverters, aerial marker spheres, and tapes. A review of 21 on wire markings shows a reduced bird mortality by 55-94% (Barrientos et al., 2012). Some deterrents, such as rotating mirrors, are aimed at deterring birds from perching nearby, while others such as spikes, act as physical barriers to prevent birds perching close to live cables.\n\nThey are mostly installed near airstrips to make them visible to pilots.\n\nURL: https://upload.wikimedia.org/wikipedia/commons/2/26/Einebenenleitung.jpg.\n\nLicense: Ikar.us, CC BY 3.0 DE <https://creativecommons.org/licenses/by/3.0/de/deed.en> via Wikimedia Commons.\n\n\nDiscussion\n\nMany elements within a bird's surroundings, such as electric pole type and layout, topography, and habitat, can impact electrocution probability (Lehman et al., 2007). On a large scale, landscape characteristics such as composition of vegetation, the quantity of prey, and the availability of perches can attract raptors or concentrate birds near power lines (Hunting, 2002). At the scale of an individual pole, variables like as geography and relative pole height influence its usefulness as a perch. At the lowest scale (the perch itself), variables such as pole-top layout, clearances between electrical components, physical dimensions of raptors, and raptor behavior all work together to induce electrocutions.\n\nThe distribution of raptor habitats in Kenya overlaps with both current and proposed electric infrastructure (Figure 1). Areas with the highest habitat suitability for raptors are primarily located in Narok, Samburu, Laikipia, and Taita-Taveta counties. These areas have a relatively high presence of major national reserves and parks, and conservancies such as Maasai Mara National Reserve, Samburu National Park, Laikipia Conservancy, and Tsavo East and West National Parks. However, the proposed and existing electrical infrastructure extensively overlap with the niche of raptors in these highly suitable areas for raptor species, causing (potential) collisions and electrocutions. Therefore, developing bird-friendly power lines in Kenya can be an effective strategy for reducing the collisions and electrocutions of raptor populations and improving their conservation. Due to interruptions in power supply and maintenance expenses, as well as financial costs and inconveniences to the consumers, bird collisions and electrocutions can also have a negative financial impact on energy firms (Küfeoǧlu & Lehtonen, 2015).\n\n\nConclusions\n\nWe recommend mapping of important raptor habitats at the electrocution and collision hotspots, retrofitting of dangerous power lines, and engaging with key stakeholders to raise awareness for support of bird-friendly power lines. It is also critical that, following the retrofitting of the power lines, continuous monitoring of the power lines is performed to determine whether the power pole structures used as perches and associated mortalities are correctly identified, and whether the pole modifications prevented or reduced mortalities. Environmental assessments to ensure that new power lines are safe for birds, taking into account the various species in the proximity that could be electrocuted; eagles and vultures have large wingspans, which must be taken into account when specifying spacing distances between phase cables on power lines. Environmental evaluations should also guarantee that the path a power line follows through ecosystems and landscapes is least likely to attract perching birds, e.g. by avoiding, whenever practicably and economically possible wide, flat plains. It is critical that these environmental evaluations have some kind of legal backing to ensure compliance. The successful implementation of these recommendations will require collaboration among various stakeholders, including the government, power companies, conservation organizations, and local communities. By working together, we can create a sustainable energy infrastructure that supports both human development and biodiversity conservation.\n\nIn conclusion, this research highlights the critical need for further exploration and development of policies and infrastructure in Kenya to align with the conservation needs of endangered and migratory species. The impact of power line infrastructure on wildlife, particularly raptors and bird species, is a growing concern, and new technologies must be developed to minimize this impact. Additionally, understanding the economic costs and benefits of implementing bird-friendly power line designs and measures is essential to balance them against other societal priorities. Conservation organizations and local communities must be engaged and empowered to participate in efforts to protect raptors and their habitats from the impact of power lines. Finally, power transmission companies need to be incentivized to adopt bird-friendly power line designs and implement measures to reduce the risks of raptor electrocution and collision to ensure a sustainable future for wildlife and their habitats. By conducting further research in these areas, we can develop a more comprehensive understanding of the challenges and opportunities associated with protecting raptors and other wildlife from the impact of power lines and identifying more effective strategies for achieving this goal.", "appendix": "Data availability\n\nRepository: Global biodiversity information facility (GBIF)\n\nRaptors occurrence data, https://doi.org/10.15468/dl.hzyp6v\n\nThis project contains the source data used to create:\n\n• Raptors file.csv (File contains raptors’ data in Kenya including the scientific name, geographical location (longitude and latitude), taxonomic information (Kingdom, phylum, class, order, family genus and specific epithet), taxonomic rank, vernacular name, accepted scientific name, level1, level 2 and level 3 geographic information and the IUCN category.\n\nRepository: The World Bank\n\nTransmission power lines distribution in Kenya, https://datacatalog.worldbank.org/search/dataset/0040440\n\nThis project contains the following source data:\n\n• Kenya’s electricity transmission network (shapefile)\n\nLicense: Data are available under the terms of the Creative Commons Attribution 4.0\n\nZenodo: Raptors data, https://doi.org/10.5281/zenodo.7788198 (Ngila, 2023).\n\nThis project contains the following extended data:\n\n- Raptors.csv.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgements\n\nWe acknowledge the Center for Biodiversity Information Development at Strathmore University for providing the research fellowship and institutional support for this study. We would like to acknowledge GBIF and the Peregrine Fund for providing Raptors’ data through the GBIF site. The National Museums of Kenya, iDigBio, the African Conservation Center, South Eastern Kenya University, and Kenyatta University provided institutional support for niche modelling training. We would like to extend our gratitude to Veronica Muniu, Strathmore University, for administrative support during the project and Lucy Waruinge of African Conservation Centre for training on development of policy briefs.\n\n\nReferences\n\nAlonso JC, Alonso JA, Mufioz-Pulido R: Mitigation of bird collisions with transmission lines through groundwire marking. Biol. Conserv. 1994; 67: 129–134. Publisher Full Text\n\nAngelov I, Hashim I, Oppel S: Persistent electrocution mortality of Egyptian Vultures Neophron percnopterus over 28 years in East Africa. Bird Conserv. Int. 2013; 23(1): 1–6. 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Report No. P500-02-071F. 2002.\n\nJanss GFE, Ferrer M: Rate of Bird Collision with Power Lines: Effects of Conductor-Marking and Static Wire-Marking (Tasa de Choques por Parte de Aves con Líneas del Tendido Eléctrico: Efecto de Marcadores de Conducción y Marcadores de Estática). J. Field Ornithol. 1998; 69(1): 8–17.\n\nJanss GFE, Ferrer M: Common Crane and Great Bustard Collision with Power Lines: Collision Rate and Risk Exposure. Wildl. Soc. Bull. 2000; 28(3): 675–680. Reference Source\n\nJenkins AR, Smallie JJ, Diamond M: Avian collisions with power lines: A global review of causes and mitigation with a South African perspective. J. Raptor Res. 2010; 20(3): 263–278. Publisher Full Text\n\nKenya Power: Environmental and Social Screening Project Report for Last Mile Connectivity Project. Kenya Power. 2016. Reference Source\n\nKüfeoǧlu S, Lehtonen M: Interruption costs of service sector electricity customers, a hybrid approach. Int. J. Electr. Power Energy Syst. 2015; 64: 588–595. Publisher Full Text\n\nLehman RN, Kennedy PL, Savidge JA: The state of the art in raptor electrocution research: A global review. Biol. Conserv. 2007; 136(2): 159–174. Publisher Full Text\n\nLoss SR, Will T, Marra PP: Refining estimates of bird collision and electrocution mortality at power lines in the United States. PLoS One. 2014; 9(7): e101565. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinistry of Tourism and Wildlife: National Wildlife Strategy 2030. A bridge Version. Blueprint to transform wildlife conservation in Kenya. (n.d.).2018.\n\nNational Energy Policy: 2018.\n\nNgila P: Raptors data (Version 1). [Data]. Zenodo. 2023. Publisher Full Text\n\nNgila P: Species distribution modeling in R (SDM). [Code]. Zenodo. 2023a. Publisher Full Text\n\nOgada DL, Keesing F: DECLINE OF RAPTORS OVER A THREE-YEAR PERIOD IN LAIKIPIA, CENTRAL KENYA. J. Raptor. Res. 2010; 44(2): 129–135. Publisher Full Text\n\nPrinsen H, Boere G, Píres N, et al.: REVIEW OF THE CONFLICT BETWEEN MIGRATORY BIRDS AND ELECTRICITY POWER GRIDS IN THE AFRICAN-EURASIAN REGION. 2011. Reference Source\n\nReal J, Grande JM, Mañosa S, et al.: Causes of death in different areas for bonelli’s eagle hieraaetus fasciatus in spain. Bird Study. 2001; 48(2): 221–228. Publisher Full Text\n\nRioux S, Savard JPL, Gerick AA: Avian mortalities due to transmission line collisions: a review of current estimates and field methods with an emphasis on applications to the Canadian electric network. Avian Conserv. Ecol. 2013; 8(2). Publisher Full Text\n\nSmallie J, Virani MZ: A preliminary assessment of the potential risks from electrical infrastructure to large birds in Kenya. Scopus. 2010; 30: 32–39.\n\nThe constitution of Kenya: 2010. Reference Source\n\nThe Wildlife Conservation and Management Act: 2013.\n\nThuiller W, Lafourcade B, Engler R, et al.: BIOMOD - A platform for ensemble forecasting of species distributions. Ecography. 2009; 32(3): 369–373. Publisher Full Text" }	[ { "id": "189044", "date": "14 Aug 2023", "name": "Natalia Rebolo-Ifrán", "expertise": [ "Reviewer Expertise Human infrastructures impact on birds" ], "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\nAlthough I consider that the article has the potential to be indexed, as it has interesting results, as it is currently presented it is confusing. The authors state that their primary objective is to review the regulations governing the development of electric transmission lines, but they do not detail the methodology used to compile this information. Nor do they cite the references of the studies from which the recommendations were made. In addition, they describe the methodology of how they made the overlay map, but then they do not discuss the results, and the use that could be made of such cartography remains inconclusive. I think the authors should focus on presenting the results (the map of the overlap between power lines in Kenya and the distribution of raptor species) in more detail and discuss them in terms of the conservation of bird species. The paper is confusing from the start, as the results do not quite match what the authors propose in the introduction. This is my main criticism, which can be solved by adequately detailing the main objectives and results of the paper.\nHere are my suggestions in detail\nIntroduction\nPlace the reference for the following statement: “Distribution line pylons are typically 8-12 meters high, much smaller than those used on transmission lines”\nThe introduction should include a paragraph demonstrating the importance of this type of study and making it clear what the objective of the work is. The same goes for the abstract.\nIn the introduction, it is not so clear whether the article refers to electrocution or collision and whether it is about raptors or migratory birds. I suggest directing the reader to the subject that will be addressed in the article. In this case, collision of birds of prey with power transmission lines. I also suggest looking for exclusive references to this topic to cite the background.\nPolicy outcomes and implications\nThe subtitle does not correspond to the content of the paragraph. In this section, the authors describe the methodology for the creation of the map where they show the distribution of birds of prey together with the power lines in Kenya (which I consider to be the main result of the work).\nThe following result is the most relevant of the work: “The results demonstrate significant hotspots of raptor electrocution and collisions in Kenya, particularly in areas where transmission lines overlap with raptor ecological niches (see Figure 3).” The authors should explain this result in more detail.\nIt is necessary to mention the bird species represented on the map.\nTable 2 would be greatly enriched by placing the linear meters of current power lines in each county of Kenya. This would help to understand which locations are priorities for mitigation measures according to the amount of habitat available and occupied by the power lines.\nThis statement does not correspond to figure 4, but to figure 3: “The existing and proposed electric distribution and transmissions in Kenya pass through major suitable raptor habitat in the country (Figure 4).”\nI suggest placing titles according to the text developed. For example, \"Mitigation measures studied for impacts of power lines on birds\" or “Measures to protect birds from power line impacts” before the paragraph that begins with: \"Several studies (Alonso et al., 1994; Eccleston&Harness, 2018; Erickson et al., 2005; Janss & Ferrer, 2000; Rioux et al., 2013) have shown…”\nThe “Actionable recommendations” section lacks references. It is necessary that the authors provide such citations or explain whether such recommendations arise from their work. This section could be linked to the previous section where they cite previous work on best management practices for power line management to protect birds.\nThe recommendations made deal with collisions and electrocutions, but the title of the article only refers to collisions with transmission lines.\nThe paragraph beginning with \"The distribution of raptor…\" and ending with \"…causing (potential) collisions and electrocutions\" corresponds to the main result of the study and should be placed with a subtitle beforehand and developed. The discussion section should discuss how the implications of these interesting results for the conservation of bird species in Kenya.\nFigure 4 and 5 show the same collision mitigation measures. I suggest choosing just one of the two.\nIn the conclusions of the study the authors say that it is important to map important raptor habitats in electrocution and collision hotspots, but they do not mention how this action can help bird conservation. This is the most important thing to discuss beforehand as it is the result of their study.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Partly\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Partly\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? No", "responses": [] }, { "id": "184296", "date": "21 Aug 2023", "name": "Francisco Guil Celada", "expertise": [ "Reviewer Expertise Power lines and biodiversity." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is clearly written and it deals with a very interesting topic, and the most relevant and recent research is cited (I suggest 4 complementary references that might be useful for the authors). But, to my view, a link is missing between the analysis developed and the suggested practices for minimising the effects of the transmission power lines.\n\nThis is my main concern, the suggested practices (pages 9-10, of the pdf version of the article). These practices should follow the IA hierarchy (mitigate-correct-compensate), and some CBD Decisions might be helpful for defining the most relevant aspects (see for example COP 6 Decision VI/7 at https://www.cbd.int/decision/cop/?id=7181). Thus, I would recommend the authors an in-depth review of the suggested practices, as some are not very technically feasible (like burying or isolating transmission lines), and an explicit vinculation with the IA hierarchy. Authors may only take a look at this report (CMS's Raptors MoU, https://www.cms.int/en/publication/position-statement-birds-and-power-lines-risks-birds-electricity-transmission-facilities)\nThere are also some minor aspects:\nPage 3. 1st paragraph. Human activities may involve, but not include habitat fragmentation.\nPage 3, 2nd paragraph. Loss et al. (2014) estimate is for the US, not global.\nPage 3, 2nd paragraph. Small birds are (mainly) vulnerable to electrocution because of overhead wires, in power lines with grounded crossarms. Transformers are not very frequent (see Guil et al. 2011).\nPage 3, 3rd paragraph. I would remove construction (3rd line), because these aspects are part of the power line, not for its construction.\nPage 3, 3rd paragraph, 4th line. Old world vultures are part of the raptors.\nPage 4, 1st par, 1st line. This classification varies among countries. Authors should highlight the countries were it applies.\nPage 4, 1st par, 6th line. This sentence has been said previously.\nPage 4, 1st par, 8th line. Earth wires may have low detectatibility, but they are not undetectable.\nPage 4, 2nd par, 6th line, The 19 bioclimatic variables shpuld be referenced.\nPage 4, 2nd par, 8th line, usdm package should be referenced.\nPages 5 and 6. Figures 2 and 3. As there is no much difference among those 2 figures, I should suggest the authors to remove (figure 2?).\nPages 6 and 7. These table should be supplementary material.\nPage 8, 1st paragraph, lines 3-6. This has already been said.\nPage 8, 3rd paragraph, line 3. I would prefer the use of covering instead of Insulating.\nPage 8, 3rd paragraph. In certain Spanish regions, the authorities have agreed an amount per collided bird (depending on the species) with power companies (for both power lines and wind power facilities) and the power companies have to pay this amount to the authority for developing managing actions.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-414
https://f1000research.com/articles/12-412/v1	17 Apr 23	{ "type": "Research Article", "title": "Role of health system strengthening (HSS) in reducing neonatal mortality in Bangladesh: A document review", "authors": [ "Md. Ziaul Matin", "Shamina Sharmin", "Minjoon Kim", "ASM Sayem", "Fazlur Rahman", "Abu Sayeed MD Abdullah", "Sifat Parveen Sheikh", "Moonmoon Islam Khan", "MA Halim", "Md. Ziaul Matin", "Shamina Sharmin", "Minjoon Kim", "ASM Sayem", "Fazlur Rahman", "Sifat Parveen Sheikh", "Moonmoon Islam Khan", "MA Halim" ], "abstract": "Background: The majority of neonatal deaths occur in low- and middle-income countries like Bangladesh. This study explored the role of health system strengthening (HSS) in reducing neonatal mortality. Methods: The document review was the method to conduct this study. The secondary data, including the reports and protocols, were reviewed to find out the success, challenges and scope of the improvement of HSS interventions in reducing neonatal deaths. The analysis was performed descriptively. Results: The National Neonatal Health Strategy was found adopted in Bangladesh including the policies and strategies for newborn health and newborn health service delivery. The evidence-based newborn care initiatives were found to cover the essential newborn care, application of chlorohexidine in newborn care, role of post-natal care in reducing child death, management of birth asphyxia, use of Antenatal Corticosteroid (ACS) for the prevention of complication related to preterm and Kangaroo Mother Care. Conclusions: The health system strengthening with improving the quality of newborn care is very much essential at facilities both at urban and rural settings to reduce neonatal mortality in Bangladesh.", "keywords": [ "newborn health", "kangaroo mother care", "antenatal corticosteroid", "LMIC", "Bangladesh" ], "content": "Introduction\n\nGlobally about 2.3 million neonatal deaths occurred in the year 2021, where 80% of these deaths are preventable.1 The neonatal death is a key obstacle to improve the survival of children under-five. Neonatal fatalities now account for about two-thirds of all deaths in the first year of life and roughly half of all deaths in children under the age of five.2\n\nAccording to the Bangladesh Demographic and Health Survey (BDHS), under-five mortality is 46 per 1,000 live births, while newborn mortality is 38 per 1,000 live births.3 These figures show a significant improvement over the early 1990s, when they were 133 and 87 percent, respectively. The neonatal mortality rate, has improved more slowly, falling from around 52 deaths per 1,000 births in the early 1990s to 28 fatalities per 1,000 in the mid-2010s.4\n\nThe World Health Organization (WHO) defined the neonatal death as the deaths among live births during the first 28 completed days of life.5 The majority of newborn deaths happen during the first week of life. Prematurity problems, infections, and intrapartum difficulties are the leading causes of neonatal deaths in Bangladesh. The majority of babies are delivered at home without the assistance of a qualified attendant, which makes it difficult to prevent these deaths. Within two days following delivery, only 32% of babies receive postnatal care from a medically qualified practitioner. Essential newborn healthcare services have improved, but only 6% of babies receive all of the components of necessary newborn care.4\n\nBangladesh has made significant progress in improving newborn health through development of a National Newborn Health Strategy, conducting operational research, improving community-based care, and establishing a National Technical Working Committee, and evidence-based interventions. Every Newborn Action Plan and the creation of a Comprehensive Newborn Care Package has already been initiated in Bangladesh.3 Still there are some gaps for which we are lagging behind to reach our desired goal, putting emphasis on strengthening the health system is the ultimate solutions to recover these gaps.\n\nThis study aims to find out the role effective health system in reducing neonatal mortality. Very few studies have been conducted specially in Bangladesh that solely give importance to improve our health system for the reduction of neonatal death.\n\n\nMethods\n\nIn this study the relevant documents were reviewed A thorough search of national policy and strategy papers, program implementation plans, survey reports and statistics, program reports, journal articles, and academic research yielded a lot of information on Bangladeshi neonatal health. This material was categorized, studied, reviewed, and summarized in order to do this analysis. The bibliography at the conclusion of this paper contains a list of the sources used.\n\nWe focused on the initiatives in our health system that has been taken to reduce neonatal mortality. We tried to figure out the success these existing initiatives. We figured out how these interventions are playing role to reduce neonatal deaths. We tried to find out the challenges of these interventions that are creating hindrance to reach the goal. Besides we also tried to find out the way so that our health system can overcome these challenges through these documents. Steps of methodology is shown in Figure 1.\n\nThe documents were primarily gathered from ministry of health and family welfare (MOHFW) directorates, such as the Directorate General of Health Services (DGHS), the Directorate General of Family Planning (DGFP), and the National Institute of Population Research and Training (NIPORT); and United Nations (UN) agencies such as the UN Development Program (UNDP), the UN Children's Fund (UNICEF), Save the Children and World Health Organizations (WHO). The domain of newborn care was selected which were relevant in strengthening the health system (Table 1).\n\n\nResults\n\nThe results of the document review include the review findings of the Policies and strategies for newborn health, newborn health service delivery and evidence-based newborn care initiatives.\n\nA brief summary of the documents that we have used in this study are also mentioned in Table 2.\n\nThe Government of Bangladesh recently implemented measures that place a high priority on improving neonatal health. Bangladesh adopted a National Neonatal Health Strategy in 2009, which marked a significant step forward in the country's newborn health. Since then, newborn health measures have been taken through a series of discussions with stakeholders, experts, and development partners, with the National Technical Working Committee for Newborn Health playing a key role. Bangladesh issued a “call to action” in July 2013, pledging to eliminate child deaths from preventable causes by 2035. This declaration aimed to improve existing neonatal therapies while also introducing new evidence-based, internationally acceptable newborn health interventions. Bangladesh has begun developing its national strategy, BENAP, as part of the global Every Newborn Action Plan (ENAP).6\n\nTraditional healers, neighborhood clinics, and hospitals are among the health-care professionals available to Bangladeshis. Government agencies, non-governmental organizations (NGOs), and the private sector may all be involved in providing services.\n\nGovernment sector\n\nThe Ministry of Health and Family Welfare (MOHFW) is solely responsible for providing health services and formulating national policies, guidelines, and action plans within the government. The Directorate General of Health Services (DGHS) is a division of the ministry that provides general preventive and curative health care as well as technical help to the ministry as it launches on new programs and interventions. Family planning and reproductive health services are provided by the Directorate of Family Planning (DGFP). In collaboration with, or under the administration of, the department of Maternity, Neonatal, Child, and Adolescent Health, both directorates provide maternal, newborn, and child health services at various levels of care (MNCAH).3\n\nPrivate sector\n\nIn Bangladesh, the private healthcare industry is rapidly expanding. Particularly in metropolitan regions, the private sector offers a major percentage of healthcare services. The private sector is mainly unregulated, and it offers services through both official and non-formal, untrained providers, such as traditional birth attendants and healers.19\n\nHealth workforce\n\nCritical shortages, an insufficient skill mix, and an unequal geographic distribution of the health personnel are key roadblocks to attaining the health-related Millennium Development Goals (MDGs) in the majority of developing nations. This is also the situation in Bangladesh, where a lack of health workers makes it difficult to offer a continuum of care for women and babies.7 Bangladesh has a significant lack of public-sector health personnel, as well as a skewed distribution. There are about 0.58 employees per 1000 people, which is significantly below the WHO's cut-off of 2.28 workers per 1000 people.20 Bangladesh's health worker shortage is worse than in other South and Southeast Asian countries. Only 0.5 physicians and 0.2 nurses are available per 1000 people.21\n\nThough there are standardized training for midwives, community level workers are also getting training on “Essential Newborn Care (ENC)” still there some barriers like shortage of health professionals in low density population, difficulty in retaining trained person in hard to reach areas, higher number of vacancies in remote areas, weak recruitment transfer and posting system, weak supervision system of health worker, in appropriate ratios of health professionals in the facilities. These problems can be overcome by taking some steps like proper advocacy on filling and recruitment of vacant positions, training for NGO and government health workers etc.3 These could only be possible when our health system would be stronger. So, strengthening health systems is important to ensure proper human resource for the service delivery of newborn.22\n\nEssential newborn care\n\nBoth the mother's and the newborn's health are dependent on the initial few minutes following birth. More than three-quarters of infant fatalities occur within the first week, with up to half occurring within the first 24 hours.8 Essential Newborn Care (ENC) is a set of basic treatments provided by healthcare providers to increase the odds of a newborn's survival after birth. Birth attendants, family members, relatives, and volunteers trained in ENC techniques should give ENC interventions soon following delivery3 (Table 3).\n\nApplication of chlorohexidine in newborn care\n\nThe importance of clean umbilical cord care in the prevention of newborn infections has been demonstrated. Invasive bacteria can enter the body through freshly cut umbilical cord stumps, causing neonatal sepsis, a leading cause of death in the first week of life. Cord infection is frequently linked to cultural customs of putting various substances to the cord, as well as a lack of sanitary precautions.\n\nUntil 2013, the recommended intervention for preventing neonatal infections was dry cord care. Based on compelling new data from three studies performed in Bangladesh, Nepal, and Pakistan, the World Health Organization (WHO) has now changed its advice. These investigations, which took place between 2007 and 2013, found that cleaning the umbilical cord with chlorhexidine digluconate (CHX) decreased both newborn mortality and infection. In a pooled analysis of these three studies, CHX usage was linked to a 23% reduced risk of death when compared to dry cord care. Based on this new evidence, policymakers, national experts, and stakeholders in Bangladesh conducted a meeting to recommend that a 7.1 percent CHX solution be applied to the newborn umbilical cord stump as a single application at birth, followed by dry cord care for all newborns, whether they were born in a facility or at home.9\n\nThough necessary steps have been taken to increase use the CHX like CHX application has been incorporated in national policy, DGDA has been issued license for the production of this drug, local pharmaceutical companies are producing it at a low cost but still there are some draw backs in this intervention like it is not available countrywide, insufficient resource for the implementation, HMIS does not keep relevant information about it etc. If the following actions are taken like ensuring availability of the drug with adequate dose and application guideline in market and facilities, initiation of CHX use at all level of service delivery, including it in “National Essential Medicine List (NEML)” then it can reduce mortality.3 By strengthening our health system, we can easily take these measures to ensure CHX for newborn care.22\n\nRole of post-natal care in reducing child death\n\nBecause the majority of newborn fatalities occur during the first few days and weeks, Bangladesh's National Neonatal Health Strategy (NNHS) Guidelines highlight the significance of urgent and early postnatal check-ups for both family and providers. Family members must recognize health risk indicators early in order to receive timely assistance from qualified professionals or at appropriate institutions. Four scheduled post-natal visits are important according to the NNHS guideline: Within 24 hours following birth, on the third day, between seven and fourteen days, and within 42 days. Among these four visits, the first three are very important for the survival of neonates. In Bangladesh, as in many other low-income nations, most mothers give birth at home. Many parents encounter financial, social, and other obstacles when it comes to taking their newborns to a healthcare institution, especially in the crucial first week after birth.10 In 2009, the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF) issued a joint statement urging home visits by health experts or trained community health workers during a baby's first week of life. In high-mortality settings, where many deliveries occur outside of health facilities, research suggests that home-based newborn care treatments can avert between 30 and 60 percent of newborn fatalities. WHO and UNICEF urge that all women and their babies be examined by medical experts shortly after birth, regardless of where the birth occurs, and that mothers and newborns have a regular schedule of follow-up appointments. If their baby shows particular danger symptoms, such as a high temperature and seizures, mothers should seek medical help at once.11\n\nThough postnatal care (PNC) services are improving, desired coverage has not been achieved. Only 32% newborn achieve PNC by skilled health professionals within 48 hours of birth.\n\nSome factors are responsible for this poor coverage like high proportion of home deliveries creates obstacle to receive services, reluctance of family member regarding taking the child to facilities for health care, unpreparedness of facilities for providing PNC services, women's access to facility-based PNC and ENC is hampered by high out-of-pocket costs, health personnel have insufficient knowledge and counseling skills when it comes to basic infant care, insufficient necessary logistic supply and there is no indicator for assessing ANC PNC progress which is important for decision making. Increased resource allocation to ensure quality of service and increased awareness of good maternal and infant care practices, as well as readiness to seek appropriate care which are a part of strengthening health system could enhance the coverage PNC service.3,22\n\nManagement of birth asphyxia\n\nOnly around 5% to 10% of infants require immediate assistance to start breathing after delivery,12 and only a small percentage of them require more sophisticated treatments like heart massage, intubation, or medications. A neonatal evaluation, as well as drying and tactile stimulation for the infant, are the initial steps in treating asphyxia.13 All health personnel, particularly those who attend births, should be able to assist babies in starting to breathe, according to the NNHS. Since 2011, the National Scaling-Up of HBB (Helping Babies Breath) Programs has provided instructions and tools (a bag and mask, as well as a sucker) to almost all government and non-government medically trained delivery care workers, including Community-Based Skilled Birth Attendants (CSBAs).10\n\nHBB has been adopted in government curriculum to train CSBAs during their service training and this training was initiated. HBB training is started and managed by the Ministry of Health and Family Welfare (MOHFW) where trainers are from national level and district level. But due to some reasons this initiative could not reduce desired number of mortality. Some of the drawbacks of this initiative are lack of adequate information necessary for scaling up this intervention, the SBAs (Skilled Birth Attendant) do not include enough information on skill retention and practice, lack of resources to conduct the program and HBB monitoring is not integrated in HMIS.3 A strong health system can address these gaps and take strategic actions to save more newborn life.22\n\nUse of Antenatal Corticosteroid (ACS) for the prevention of complication related to preterm\n\nThe single most effective strategy for improving neonatal outcomes among preterm newborns is antenatal corticosteroid (ACS).16 Near-universal coverage of ACS for suspected preterm delivery might result in up to 40% fewer baby deaths due to prematurity-related complications.14 The World Health Organization supports ACS as a first-line strategy in the treatment of preterm labor to avoid respiratory distress syndrome (RDS).18 According to a Lives Saved Tool (LiST) study, ACS may save over 400,000 lives each year.15 Although the highest effect is obtained 48 hours after the initial injection, even partial or incomplete regimens can offer some benefit. ACS should be initiated as soon as the detection of preterm birth because it is difficult to predict the time of delivery.17 Although the National Newborn Health Strategy 2009 highlighted preterm as a major cause of avoidable neonatal mortality, it did not include explicit recommendations on ACS usage. Several programs, notably ACS, promoted expanding of innovative neonatal treatments in 2013. The National Technical Working Committee on Newborn Health (NTWCNH) recommended that ACS (dexamethasone) be used in premature labor both in institutions and in the community to minimize problems from preterm deliveries, particularly respiratory distress syndrome. Giving priority to this intervention national guideline and protocol have been developed. Besides local pharmaceutical companies are also producing and marketing to increase the coverage.10\n\nUnavailability of required amount of ACS, tendency of not following standard protocol in facilities, late arrival of women at the facility for delivery that decrease efficiency of ACS, lack of knowledge of health professionals regarding administration of this drug, unawareness of people about the danger signs of pregnancy are some of issues creating obstacles in this intervention. Finalization and distribution of national protocol and guideline on usage of ACS, production of ACS in a 6 mg dosage with proper indication that is listed on the National Essential Medicines List (NEML) for premature deliveries, proper training of professional on administration of ACS and proper logistic management are some of the outcomes of effective health system necessary to reduce neonatal mortality.3,22\n\nKangaroo Mother Care\n\nIn Bangladesh, Kangaroo Mother Care (KMC) is a cost-efficient, practical, and successful technique to preserve preterm and low-birthweight babies. KMC refers to the mother's (or mother substitute's) early, extended, and continuous skin-to-skin contact with her infant, both in the hospital and after discharge. It involves assistance to mothers with baby positioning, feeding (preferably exclusively breastfeeding), and the prevention and treatment of infections and respiratory problems. KMC should be given to all clinically stable infants weighing fewer than 2,000 grams, according to the evidence. It is most beneficial if it is begun within the first week of life. Understanding the value of KMC it is increasingly recognized in policy statements with proper guideline. But still there are problems like scarcity of trained nurses as they are frequently transferred from KMC, lack of proper instruction for the implementation of KMC through health system. As a part of well-organized health system identification of key champion for KMC in Bangladesh and support the Ministry of Health and Family Welfare's national level planning for scale-up and integration of KMC into its sector planning MOHFW could be the ways to increase the acceptance as well as the coverage of KMC to reduce child death.3\n\n\nDiscussion\n\nThe key findings included the National Neonatal Health Strategy was adopted in 2009 in Bangladesh for a significant step forward in the country's newborn health. With the National Technical Working Committee, the newborn health measures have been taken through a series of discussions with stakeholders, experts, and development partners. Bangladesh is in more progress in reducing neonatal deaths than most low-income countries. The neonatal mortality decline 4.0% per year in the last decade which is higher than global averages 2.1% per year; however, the decline for under 5 mortality was double this rate, 8.6% per year.23,24\n\nThe MOHFW provides the health services and formulating national policies, guidelines, and action plans within the government. The extensive changes including a National Newborn Health Strategy have occurred over the last decade in health policy related to newborn care. Along with the GoB the civil society and academics have played key roles. Due to a pluralistic health system and a diversity of policies and programmes, the local and global data and evidence have been influential, but pathways between research and action are complex.25,26\n\nThe capacity development on essential newborn care among the community health workers are still barriers with shortage of health professionals in high density population. Making sustained capacity building of government health workers within the formal health system is challenging. Another challenge include the regularly implement interventions without useful co-ordination with the government or other counterparts or without vigilant evaluation.27\n\nThere are difficulties in retaining trained person in hard to reach areas, higher number of vacancies in remote areas, weak recruitment transfer and posting system, weak supervision system of health worker, in appropriate ratios of health professionals in the facilities. For adequate health service provision qualified and motivated human resources are very much essential. In rural areas and hard to reach areas the HR shortages have reached critical levels. To address shortages of the existing workforce the strategies improving performance are essential.28\n\nThe essential newborn care is a set of basic treatments provided by HCPs to increase the odds of a newborn's survival after birth. Birth attendants, family members, relatives, and volunteers trained in ENC techniques should give ENC interventions soon following delivery. There are 16% less likely to lose their baby and 24% less likely to experience pre-term birth occurred among the women who receive midwife-led continuity of care provided by professional midwives.29\n\nThe prevention of newborn infections has been demonstrated through ensuring the care and clean umbilical cord. Invasive bacteria can enter the body through freshly cut umbilical cord stumps, causing neonatal sepsis, a leading cause of death in the first week of life. Lack of quality care at birth or skilled care and treatment immediately after birth and in the first days of life is associated with neonatal deaths within the first 28 days. Most neonatal deaths occurred due to preterm birth, birth asphyxia or lack of breathing at birth, infections and birth defects.29\n\nThe MOHFW took initiatives to ensure HBB through a comprehensive training package at national level and district level. This HBB has been adopted in government curriculum to train CSBAs during their service training. To ensure the clinical knowledge and skills of health workers HBB training is one of the significant measures. For scaling HBB program nationally, it is essential to ensure training fidelity. The maintaining high quality coordination and standardization of the course delivery by trainers and having a core team are essential.30\n\nKMC is mother's continuous skin-to-skin contact with her infant, both in hospital and in home after discharge. It involves assistance to mothers with baby positioning, exclusively breastfeeding, and the prevention and treatment of infectious diseases. For the low birth weight infants skin-to-skin care, and exclusive breastfeeding was almost universal at discharge.31\n\n\nConclusion\n\nThe neonatal mortality has been decreasing gradually in Bangladesh through taking different initiatives both at community and facility level. The national newborn strategy is one of the key documents where the protocol and procedure of proper newborn care is emphasized. The findings of this documents review need to be highlighted at policy level to take necessary initiatives for reaching the SDG target in reducing neonatal mortality on time.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nUNICEF, World Health Organization: Ending preventable newborn and stillbirths by 2030: moving faster towards high-quality universal health coverage in 2020–2025.\n\nChowdhury HR, Thompson S, Ali M, et al.: Causes of neonatal deaths in a rural subdistrict of Bangladesh: Implications for intervention. J. Health Popul. Nutr. 2010; 28(4): 375–382. PubMed Abstract\n\nMoH&FW: Bangladesh: National Newborn Health Situation Analysis Report 2014. Minist Heal Fam Welf. 2014; 112.\n\nNational Institute of Population Research and Training, Mitra and Associates, and ICF International: Bangladesh Demographic and Health Survey. Dhaka, Bangladesh, Rockville, Maryland, USA.2004; 328. Reference Source\n\nHarding EBM, Harrington LT, Lockwood CM, et al.: Perinatal and neonatal mortality. Br. Med. J. 1980; 281(6254): 1567. Publisher Full Text\n\nEnding Preventable Child Deaths before 2035_ Bangladesh Call to Action - Bangladesh _ ReliefWeb.\n\nVinten G: Book Review: Human resources for health: overcoming the crisis. J. R. Soc. Promot. Health. 2005; 125: 287–288.\n\nWorld’s mother: Saving the Lives of Mothers and Newborns. Solutions.2006. Reference Source\n\nImdad A, Mullany LC, Baqui AH, et al.: Es necesario limpiar el cordon umbilicar con antisepticos? Evidencia en pediatría [revista en Internet]. 2017 [acceso 10 de diciembre de 2018]; 13(2): 1–11. 2013;13(Suppl 3). Reference Source\n\nWelfare M of H and F: National Neonatal Health Strategy and guidelines for Bangladesh.2009. Reference Source\n\nFund WHONC: WHO/UNICEF Joint statement: Home visits for the newborn child: a strategy to improve survival. WHO Libr. 2009; 1–8.\n\nWall SN, Lee ACC, Niermeyer S, et al.: Neonatal resuscitation in low-resource settings: What, who, and how to overcome challenges to scale up? Int. J. Gynecol. Obstet. 2009; 107(SUPPL): S47–S64. Publisher Full Text\n\nJobe AH: Helping babies breathe. J. Pediatr. 2012; 160: A6. Publisher Full Text\n\nLeung C: Born too soon. Neuroendocrinol. Lett. 2004; 25(SUPPL. 1): 133–136.\n\nMiracle X, Di Renzo GC, Stark A, et al.: Guideline for the use of antenatal corticosteroids for fetal maturation. J. Perinat. Med. 2008; 36(3): 191–196. PubMed Abstract \| Publisher Full Text\n\nAmerican College of Obstetricians and Gynecologists: ACOG Practice Bulletin No. 127. Management of preterm labor. Obstet. Gynecol. 2012; 119(6): 1308–1317. Publisher Full Text\n\nAmerican College of Obstetricians and Gynecologists Committee Opinion No. 402: Antenatal corticosteroid therapy for fetal maturation. Obstet. Gynecol. 2011; 475(117): 422–424.\n\nWHO recommendations on interventions to improve preterm birth outcomes. http\n\nBangladesh Health Watch: Bangladesh Health Watch Report 2007 Health Workforce in Bangladesh: Who Constitutes the Healthcare System? Dhaka, Bangladesh: James P Grant School of Public Health; 2008.\n\nThe World Health Report 2006 - Working together for health. Geneva: World Health Organization; 2006. (accessed October 2014). Reference Source\n\nArifeen SE, Christou A, Reichenbach L, et al.: Community-based approaches and partnerships: innovations in health-service delivery in Bangladesh. Lancet. 2013; 382: 2012–2026. PubMed Abstract \| Publisher Full Text\n\nWHO: Success Factors for Women’ s and Children’ s Health Rwanda.2014; 28.\n\nChowdhury S, Banu LA, Chowdhury TA, et al.: Achieving Millennium Development Goals 4 and 5 in Bangladesh. Br. J. Obstet. Gynaecol. 2011; 118: 36–46. PubMed Abstract \| Publisher Full Text\n\nUNICEF, WHO, The World Bank, the United Nations Population Division, Levels and Trends in Child Mortality, Report 2011, 2011New York, UNICEF.\n\nWHO, UNICEF, USAID, Save the Children, WHO-UNICEF Joint Statement on home visits for the newborn child: a strategy to improve survival, 2009 Geneva World Health Organization.\n\nRubayet S, Shahidullah M, Hossain A, et al.: Newborn survival in Bangladesh: a decade of change and future implications. Health Policy Plan. 2012; 27(3): iii40–iii56. PubMed Abstract \| Publisher Full Text\n\nBangladesh Health Watch, How Healthy is Health Sector Governance? 2010 Dhaka James P Grant School of Public Health.\n\nWHO: Evidence and Information for Policy, Department of Human Resources for Health Geneva.September 2006. Reference Source\n\nWHO: Newborns: improving survival and well-being.Reference Source\n\nChinbuah MA, Taylor M, Serpa M, et al.: Scaling up Ghana's national newborn care initiative: integrating 'helping babies breathe' (HBB), 'essential care for every baby' (ECEB), and newborn 'infection prevention' (IP) trainings. BMC Health Serv. Res. 2020; 20: 739. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlmgren M: Benefits of skin-to-skin contact during the neonatal period: Governed by epigenetic mechanisms? Genes & Diseases. 5(1): 24–26. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "267924", "date": "14 May 2024", "name": "Stanley Mwita", "expertise": [ "Reviewer Expertise Pharmacy 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 study provides information on the Role of health system strengthening (HSS) in reducing neonatal mortality in Bangladesh: A document review. The topic of this manuscript fits the journal's scope. The manuscript can be indexed once the following issues are addressed:\n\nIntroduction: Rephrase the following sentence “Still there are some gaps for which we\nare lagging behind to reach our desired goal, putting emphasis on strengthening the health system is the ultimate solutions to recover these gaps.”\n\nThe methodology is not clear. It needs to be improved. It shall include: eligibility criteria, Number of documents reviewed per category, Excluded and included documents, Rationale of using document review method in this study.\n\nAdd a summary of the results presented in Table 2. Further, this table has some unnecessary study documents. It does not present a specific finding as per the study objective. I would expect key findings presented here to report on neonatal mortality and HSS, like Massawe et al.\n\nDiscuss the role of antenatal corticosteroids.\n\nAdd limitations to this 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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "267921", "date": "21 May 2024", "name": "Katherine Kalaris", "expertise": [ "Reviewer Expertise Health systems", "SRMNH", "programme implementation", "healthcare networks" ], "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 covers an important topic - health systems approaches to reduce neonatal mortality in Bangladesh. The authors provide background on the trends in child and neonatal mortality to introduce the topic. There is a brief overview of the methods, though this section needs significantly more detail for the study to be reproducible. The results provide a summary of policies and strategies for neonatal care, an overview of service delivery, and highlight specific initiatives to improve neonatal care and reduce morbidity and mortality. A list of selected documents is provided.\n\nOverall, this article needs significant work, including improvements to the language and grammar. The methods section does not provide sufficient detail - how were the documents selected, where were they searched for, were specific search terms used, were databases searched. Additionally, there is not sufficient detail on how the analysis was done. The authors say that they \"tried to figure out the success of these existing initiatives\" how was that done? How were challenges with the interventions identified? Was a framework or data charting tool used? There needs to be a link between the documents selected and the conclusion drawn. For the results - please include details on how many documents were selected (were any excluded) and summary characteristics of those documents from the table. The results covers policies and strategies, service delivery, and a summary of specific initiatives on neonatal care. if the authors want to focus on health system strengthening using the WHO health systems building blocks would be one approach to frame the results. The discussion seems to be more of only a summary of the results - what are the strengths and limitation of the research, what are areas for future research, there needs to be more consideration of the existing literature as well. Please also check coherency of statistics used throughout the document. For example, in paragraph 2 should be per 1000 live births, not percent and the NMR in the mid-2010s referenced is lower than the BDHS NMR - if this is the case than this needs to be commented on.\n\nOverall, this document review is on an important topic but it needs significant work to be approved.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-412
https://f1000research.com/articles/11-239/v1	25 Feb 22	{ "type": "Research Article", "title": "Depressive symptoms and their sociodemographic determinants among people living with HIV/AIDS in Bangladesh: a cross-sectional study", "authors": [ "Rokshana Rabeya", "Nur Alam", "Zannatul Ferdous Sonia", "Dipa Rani Mohajon", "Yasin Arafat", "Md. Kamrul Hasan", "Mohammad Delwer Hossain Hawlader", "Rokshana Rabeya", "Nur Alam", "Zannatul Ferdous Sonia", "Dipa Rani Mohajon", "Yasin Arafat", "Md. Kamrul Hasan" ], "abstract": "Background: This study aimed to determine the prevalence of depression and its associated factors among people living with HIV/AIDS in Bangladesh.\n\nMethods: This cross-sectional study, which took place in Dhaka, Bangladesh, from July to December 2020, included 338 HIV-positive people. The method used was a simple random sampling technique. The Beck Depression Inventory assessed depression in HIV-positive people (BDI).\n\nResults: More than 62 percent of the 338 people surveyed had severe depression, 30.5 percent had moderate depression, 5.6 percent had mild depression, and 1.8 percent had no depression at all. Age, being a man, being married, and having a low monthly income were all significant predictors of depression. Conclusions: This study found that depressive symptoms are highly prevalent among HIV-positive patients in Bangladesh. The authors recommend that health care providers address depressive disorders for people with HIV/ AIDS comprehensively.", "keywords": [ "HIV/AIDS", "depression", "depressive symptoms", "acquired immune deficiency syndrome (AIDS)", "Bangladesh" ], "content": "Introduction\n\nInfection with the human immunodeficiency virus (HIV) causes acquired immune deficiency syndrome (AIDS) (Krämer, Kretzschmar, & Krickeberg, 2010). Around 38 million people worldwide are infected with HIV (Global Aids Update 2020, 2020). HIV is transmitted primarily through unprotected sex, contaminated blood transfusions, hypodermic needles, and mother-to-child transmission during pregnancy, delivery, or breastfeeding (Rom & Markowitz, 2007). Sharp and Hahn (2011) first recognized it as a new disease in 1981, when an increasing number of young homosexual men died of unusual opportunistic infections and rare cancers (Sharp & Hahn, 2011). According to the World Health Organization (WHO), 36.7 million people worldwide live with HIV and AIDS, with 1.1 million people dying from the disease in 2015 (World Health Organization, 2018). In Bangladesh, the first case of HIV was discovered in 1989 (Goldberg, 2010).\n\nDepression is a mental illness marked by persistent sadness or despair that can alter a person's thoughts and feelings. It also has an impact on social behavior and physical well-being. It affects people of all ages, including children and teenagers (Deshmukh, Borkar, & Deshmukh, 2017). Depression is common among HIV-positive people. Depressed mood, loss of interest or pleasure, decreased energy, guilt or low self-worth, disturbed sleep or appetite, and poor concentration are all symptoms (World Health Organization, 2017). It obstructs daily life and lowers life quality. People living with HIV (PLHIV) had more mental health problems than people who were not infected with the virus, with those who had fewer problems being less likely to be poor and more likely to be employed, educated, and on antiretroviral therapy (ART). Being female, being in poor health, receiving poor-quality health care, and lacking material and emotional support from family and friends were all found to be more strongly linked to psychiatric morbidity (Brandt, 2009).\n\nIn PLWHA, depression is linked to increased morbidity and mortality, as well as poor adherence to antiretroviral therapy (ART), quality of life (QoL), and health-related quality of life (AQoL) (Abas, Ali, Nakimuli-Mpungu, & Chibanda, 2014). The financial cost of HIV treatment for the victim/patient is enormous, and it frequently leads to abject poverty for the sufferer and his or her family. Negative social consequences, such as stigma associated with being a PLWHA, are a problem that almost all PLWHAs face, limiting marriage and employment opportunities and possibly leading to divorce (Raguram, Weiss, Channabasavanna, & Devins, 1996). Even though depression among HIV patients is widespread in various countries, there is little evidence from Bangladesh. As a result, we conducted this research to fill a research gap that may provide evidence for future effective HIV/AIDS prevention and treatment.\n\n\nMethods\n\nFrom July to December 2020, an institution-based cross-sectional study among PLWHA in Bangladesh was conducted. Considering 67.3% population prevalence (Rai & Verma, 2015), 5% error, and 95% confidence interval, our sample size was 338. We conducted this study in all drop-in centers (DIC) of CARE Bangladesh located in Chankharpul, Swamibag, Dholpur, Hazaribagh, Noya Bajar, and Tongi of Dhaka city. We recruited adult males, females, and transgender who were advised for a routine checkup in those centers. HIV-positive patients who were not willing to participate in this study were excluded.\n\nFor this study, a purposive sampling technique was applied for selecting the HIV working organization, and after that, a simple random sampling technique was applied to recruit the study participants. A written, structured questionnaire based on the objectives and variables was used for data collection (see extended data). Only close-ended questionnaires were used to assemble data, and the interview was completed through the local language. Questionnaires were first prepared in English and then translated into the local language Bangla and again back translated into English to see the accuracy of Bangla translation.\n\nThe Statistical Package for Social Science (SPPS) version 25 was used to compile and analyze the data for this study. The questionnaire and data are available online (Rabeya et al., 2021, 2022). A chi-square test or Fisher exact was used to determine the relationship between categorical variables. The presence and strength of association between independent variables and severe depression category were determined using crude and adjusted odds ratios with a 95 percent confidence interval (CI). Variables with a “p-value” of less than 0.05 were considered significant in the bivariate logistic model.\n\nPrimeasia University's Institutional Review Board (IRB) approved the study (in Dhaka, Bangladesh. Prior to data collection, we received approval from CARE Bangladesh addition to this approval. CARE Bangladesh is a humanitarian organization to improve the socioeconomic status of women and the marginalized population in Bangladesh. The purpose of the study was explained to each respondent (HIV-positive patient). Each respondent was given the option of declining to participate in the study, and the information gathered was kept private. Before providing information, each participant was informed about the study's purpose and signed a written consent form. This study was carried out in accordance with the Helsinki Declaration at every stage.\n\nThe Institutional Review Board (IRB) of Primeasia University, Dhaka, Bangladesh, approved this study. The reference number is PAU/IEAC/22/103. Additionally, each participant was aware of the aim of the study, as well as they signed in the written informed consent form prior to providing information.\n\n\nResults\n\nTable 1 shows that a total of 338 male, female and transgender HIV positive respondents aged between 18 to more than 50 years were enrolled in the study. Demographic characteristics of the subject (n= 338) in this cross-sectional study shows that most of the participants (35.8%) belonged to age groups of 18 to 30 years, 31 to 40 years were 35.8%, 41 to 50 were 20.4%, and 50 and above were 8.0%. The mean age of the participants was 35.6 (±9.9) years. The study revealed that 297 (87.95%) were male, whereas 20 (5.95%) were female, and 21 (6.2%) were transgender. Among 338 participants, 116 (34.3%) were illiterate, 173 (51.2%) were educated up to secondary school level (10th grade), 49 (14.5%) were Higher Secondary (12th grade) and above. Occupation revealed the following participants: 14.8% were unemployed/homemakers/others, 79.6% were employed, and 5.6% were students. In terms of religion, 93.5% were Muslims, and 6.5% were Hindu. Among the respondents, 57.7% were married, 34.4% were unmarried, and 7.4% were divorced or separated. The majority (76.3%) were from nuclear families, and 23.7% were from families with multiple members (spouses/parents). Most of the respondents (68.6%) came from a family consisting of two to five family members, followed by 24.6% of respondents who were single, and 6.8% were from more than six family members. The subjects' socioeconomic status showed that 71.3% of respondents' earnings were below 10000 TK per month based on their monthly income.\n\nThe Beck Depression Inventory (BDI) scale was used to determine depression, which was divided into four categories: no depression (0–9), mild depression (10–16), moderate depression (17–29), and severe depression (30–63) (Unnikrishnan, Jagannath, Ramapuram, Achappa, & Madi, 2012). We discovered that 62.1 percent had severe depression, 30.5 percent had moderate depression, 5.6 percent had mild depression, and only 1.8 percent had no depression at all (Figure 1).\n\nTable 2 presents the results of the association between different categories of depression and various sociodemographic variables, where the significant association of depression was detected with age (p=0.013), religion (p=0.038), marital status (p<0.002), number of family members (p=040), and monthly income (p<0.001). Nevertheless, the variables like education, gender, occupation, family type did not exhibit any association with depression among HIV-positive respondents.\n\nAn adjusted multivariable model was created by forward stepwise logistic regression using the significant factors with the bivariate model. In our study, in the case of religion, Hindus were 4.9 times more prone to develop severe depression than their counterpart, Muslims (AOR=4.93; 95%CI: 1.09-22.24). Unmarried individuals had 1.9 times more chances to develop severe depression than married individuals (AOR=1.95; 95%CI: 1.00-3.80). Transgender people were more prone to develop severe depression than male and female respondents, but the association was not statistically significant in multivariable analysis. Similarly, students were more likely to develop severe depression than other occupations but did not find significant associations. Other variables such as education, family types, number of family members, and income were not significantly associated with depression of HIV patients (Table 3).\n\n1 Adjusted with age, sex, religion, marital status, and family members.\n\n* Significant p-value at p<0.05.\n\n\nDiscussion\n\nThe purpose of this study was to assess depression in PLWHA. This study included 338 HIV-positive respondents, ranging from 18 to more than 50 years old, with a mean age of 35.6 years. The average age of participants in a similar study conducted in Sub-Saharan Africa was 38.9 years, slightly higher than our study (van Coppenhagen & Duvenage, 2019). Chikezie et al. in Nigeria found that the average age of participants was 35.57 years, which is similar to our study (Chikezie, Otakpor, Kuteyi, & James, 2013).\n\nThe Beck Depression Inventory was used in this study, and it was used to categorize depression into four categories: no depression, mild depression, moderate depression, and severe depression. We discovered that 62.1 percent of people had severe depression, 30.5 percent had moderate depression, 5.6 percent had mild depression, and 1.8 percent had no depression. A similar study conducted in Brazil found that the prevalence of no depression was 46.3 percent, mild depression was 17.7 percent, moderate depression was 22.7 percent, and severe depression was 13.3 percent, which was significantly lower than our study in terms of moderate and severe depression levels (Dal-Bó et al., 2015). Another study in China found that 71.9 percent of people suffer from mild to severe depression (Su et al., 2013). In the north, west, and south of Iran, depression was found in 45 percent, 30 percent, and 56 percent of HIV patients, respectively. Furthermore, depression was prevalent in 25 percent of addicts and 58 percent of non-addicts, respectively (Doosti-Irani, Moameri, Ahmadi-Gharaei, & Holakouie-Naieni, 2017).\n\nSome of the differences in depression prevalence could be due to those countries' socio-cultural and economic contexts, such as income, political and social stability, strong familial support, and healthy social environments. This cross-sectional study found that males were suffering more from depression than females. The possible reasons could be that men are more likely to smoke, drink alcohol, eat unhealthily, and are often less aware of medical conditions and confront unemployment, economic hardship, etc. (Alkazemi, 2019). A study conducted in Kalafong Provincial Tertiary Hospital is slightly different from our study, where they found that females were more depressive than males (55.70% vs. 50.66%) (van Coppenhagen & Duvenage, 2019). In addition, several studies also reported that women had more depression, anxiety, and stress, such as Gordillo et al. (Gordillo et al., 2009) Wisniewski et al. (Wisniewski et al., 2005) Rapaport et al. (Rapaport, Clary, Fayyad, & Endicott, 2005) and Othman et al. (Othman, Fadzil, Zakaria, Jaapar, & Husain, 2015).\n\nThis study revealed that participants whose monthly household income was less were at higher risk for depression; similar findings were reported by a study conducted at three hospitals in Ethiopia, which found that income less than 200 birr's was associated with depression (Gupta et al., 2010). This could be because people in low-income countries are under pressure to rely on academics due to poverty-related factors, which leads to increased domestic work and a lack of access to health education and awareness (Al Jarad et al., 2018). Deshmukh et al. conducted a study that backs up this claim (Deshmukh et al., 2017).\n\nDorsisa et al. (2020) found that married people are more depressed than unmarried people in Ethiopia (Dorsisa, Ahimed, Anand, & Bekela, 2020), but we found that unmarried people are more likely to develop depression in our current study. Loneliness and a lack of mental support from partners to share the pain could be the cause, resulting in a variety of negative thoughts. Our research found a link between age and depressive symptoms in people aged 18 to 30, and Abebe et al. (2019) found a similar link. Understanding and conceptualizing that their HIV status increases with age and transitioning to adulthood may be fraught with developmental challenges (Abebe, Shumet, Nassir, Agidew, & Abebaw, 2019). In some studies, specific characteristics, such as age, employment status, and income level, have been linked to depression in PLWHA (Nanni, Caruso, Mitchell, Meggiolaro, & Grassi, 2014; Rabkin, 2008; Eller et al., 2014; Do et al., 2014).\n\n\nConclusions\n\nThe current study found a high prevalence of depressive symptoms among HIV-positive patients in Bangladesh. In order to improve patient care and clinical outcomes, routine screening is critical in addressing this common psychiatric condition among HIV-positive populations. Because depression is so common among HIV-positive people, policymakers should include mental health programs in routine HIV care so that depression can be detected and treated early.\n\n\nData availability statement\n\nZenodo: Availability and use of technology for e-learning in Bangladesh https://doi.org/10.5281/zenodo.5808314 (Rabeya, 2021)\n\nThis project contains the following underlying data:\n\n• Data.xls (raw data from questionnaires)\n\nZenodo: HIV/AIDS-Depression questionnaire. https://doi.org/10.5281/zenodo.5904418 (Rabeya, 2022)\n\nThis project contains the following extended data:\n\n• Data file 1. Copy of the survey administered to participants (in English).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthor contributions\n\nAll of the authors greatly aided the manuscript's development.", "appendix": "Acknowledgment\n\nThe authors would like to thank the University of Primeasia, Bangladesh, for providing technical support and the respective Bandhu Social Welfare Society (BSWS) and CARE Bangladesh authority for their kind consideration for allowing us to conduct the study.\n\n\nReferences\n\nAl Jarad A , Al Hadi A , Al Garatli A , et al.: Impact of Cognitive Dysfunction in the Middle East Depressed Patients: The ICMED Study. Clinical Practice and Epidemiology in Mental Health . 2018; 14(1): 270–283. PubMed Abstract \| Publisher Full Text\n\nAbas M, Ali G-C, Nakimuli-Mpungu E, et al.: Depression in people living with HIV in sub-Saharan Africa: time to act. 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PubMed Abstract \| Publisher Full Text\n\nDorsisa B, Ahimed G, Anand S, et al.: Prevalence and Factors Associated with Depression among HIV/AIDS-Infected Patients Attending ART Clinic at Jimma University Medical Center, Jimma, Southwest Ethiopia. Psychiatry Journal . 2020; 2020: 1–9. PubMed Abstract \| Publisher Full Text\n\nEller LS, Rivero-Mendez M, Voss J, et al.: Depressive Symptoms, Self-Esteem, HIV Symptom Management Self-Efficacy and Self-Compassion in People Living with HIV. AIDS Care . 2014; 26(7): 795–803. PubMed Abstract \| Publisher Full Text\n\nGlobal Aids Update 2020: Seizing the moment — Tackling entrenched inequalities to end epidemics. The Joint United Nations Programme on HIV and AIDS . 2020, June: 1–384. https://www.unaids.org/en/resources/documents/2020/global-aids-report\n\nGoldberg D: The detection and treatment of depression in the physicallyill. World Psychiatry . 2010; 9(1): 16–20. PubMed Abstract \| Publisher Full Text\n\nGordillo V, Fekete EM, Platteau T, et al.: Emotional support and gender in people living with HIV: effects on psychological well-being. Journal of Behavioral Medicine . 2009; 32(6): 523–531. PubMed Abstract \| Publisher Full Text\n\nGupta R, Dandu M, Packel L, et al.: Depression and HIV in Botswana: A Population-Based Study on Gender-Specific Socioeconomic and Behavioral Correlates. PLoS One . 2010; 5(12): e14252. PubMed Abstract \| Publisher Full Text\n\nKrämer A, Kretzschmar M, Krickeberg K: Modern Infectious Disease Epidemiology: Concepts, Methods, Mathematical Models, and Public Health. Springer Science & Business Media; 2010. https://www.researchgate.net/publication/321619747_Modern_Infectious_Disease_Epidemiology_Concepts_Methods_Mathematical_Models_and_Public_HealthPublisher Full Text\n\nNanni MG, Caruso R, Mitchell AJ, et al.: Depression in HIV Infected Patients: a Review. Current Psychiatry Reports . 2014; 17(1): 530–511. PubMed Abstract \| Publisher Full Text\n\nOthman Z, Fadzil NA, Zakaria R, et al.: Religiosity in Malay patients with HIV/AIDS: Correlation with emotional distress. Middle-East Journal of Scientific Research . 2015; 23(2): 170–174. Publisher Full Text\n\nRabkin J: HIV and depression: 2008 review and update. Current HIV/AIDS Reports . 2008; 5(4): 163–171. PubMed Abstract \| Publisher Full Text\n\nRabeya MR, Alam MN, Sonia ZF, et al.: HIV/AIDS-Depressive symptoms dataset . 2021. Publisher Full Text\n\nRabeya MR, Alam MN, Sonia ZF, et al.: HIV/AIDS-Depression questionnaire . 2022. Publisher Full Text\n\nRaguram R, Weiss MG, Channabasavanna SM, et al.: Stigma, depression, and somatization in South India. The American Journal of Psychiatry . 1996; 153(8): 1043–1049. PubMed Abstract \| Publisher Full Text\n\nRai P, Verma BL: A study on depression in people living with HIV/AIDS in South-West part of Uttar Pradesh, India. South East Asia Journal of Public Health . 2015; 5(1): 12–17. Publisher Full Text\n\nRapaport MH, Clary C, Fayyad R, et al.: Quality-of-life impairment in depressive and anxiety disorders. The American Journal of Psychiatry . 2005; 162(6): 1171–1178. Publisher Full Text\n\nRom WN, Markowitz SB: Environmental and occupational medicine. Lippincott Williams & Wilkins; 2007. https://books.google.com/books?hl=en&lr=&id=H4Sv9XY296oC&oi=fnd&pg=PR31&dq=Environmental+and+occupational+medicine,+Published+in+2007+in+Philadelphia+by+Wolters+Kluwer/Lippincott+Williams+%26+Wilkins&ots=xD3QkRR7JH&sig=URGuF1YZpvfhmdQ2emRSlTYqiH0\n\nSharp PM, Hahn BH: Origins of HIV and the AIDS Pandemic. Cold Spring Harbor Perspectives in Medicine . 2011; 1(1). PubMed Abstract \| Publisher Full Text\n\nSu X, Lau JTF, Mak WWS, et al.: Prevalence and associated factors of depression among people living with HIV in two cities in China. Journal of Affective Disorders . 2013; 149(1–3): 108–115. PubMed Abstract \| Publisher Full Text\n\nUnnikrishnan B, Jagannath V, Ramapuram JT, et al.: Study of Depression and Its Associated Factors among Women Living with HIV/AIDS in Coastal South India. International Scholarly Research Notices . 2012; 2012: 1–4. PubMed Abstract \| Publisher Full Text\n\nvan Coppenhagen B , Duvenage HS: Prevalence of depression in people living with HIV and AIDS at the Kalafong Provincial Tertiary Hospital Antiretroviral Clinic. The South African Journal of Psychiatry . 2019; 25. PubMed Abstract \| Publisher Full Text\n\nWisniewski AB, Apel S, Selnes OA, et al.: Depressive symptoms, quality of life, and neuropsychological performance in HIV/AIDS: the impact of gender and injection drug use. Journal of Neurovirology . 2005; 11(2): 138–143. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Depression and Other Common Mental Disorders Global Health Estimates. World Health Organization; 2017. https://apps.who.int/iris/handle/10665/254610\n\nWorld Health Organization: Fact sheets on sustainable development goals: health targets-Mental health. World Health Organization; 2018: pp. 1–8. https://www.euro.who.int/en/health-topics/health-policy/sustainable-development-goals" }	[ { "id": "145206", "date": "09 Aug 2022", "name": "Epari Venkatarao", "expertise": [ "Reviewer Expertise Depression", "Reproductive health", "Clinical trials", "NCDs", "Physical activity promotion", "Oncology. 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\nThis is an important study documenting the burden of depressive symptoms among PLHIV, which can contribute to policy formulation, both locally and globally. The study has been conducted carefully and technically the study design is appropriate to respond to the study objectives. A review of the literature has been done optimally.\nHowever, I have just two comments\nTable 1 was not needed, it can be merged with table 2 as a composite table and that would reduce the article space in the journal.\nSecondly, under the discussion section one or two articles relating to depression among people with other chronic diseases, might add value to 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?\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": "8705", "date": "08 Sep 2022", "name": "Md. Kamrul Hasan", "role": "Author Response", "response": "Comment-1: Table 1 was not needed, it can be merged with table 2 as a composite table and that would reduce the article space in the journal. Author's Response: Thanks for the suggestion. As suggested the authors merged table 1 and table 2 and add a composite table in the revised (new version) manuscript. Comment-2: Secondly, under the discussion section one or two articles relating to depression among people with other chronic diseases, might add value to the article. Author's Response: Thanks. The authors added information relating to depression among people with other chronic diseases with appropriate citations in the revised (new version) manuscript." } ] } ]	1	https://f1000research.com/articles/11-239
https://f1000research.com/articles/12-409/v1	17 Apr 23	{ "type": "Research Article", "title": "Epidemiological profile and diabetes control of Type 1 Diabetes Mellitus patients in Karbala Governorate, Iraq", "authors": [ "Abdul Razzaq Oleiwi Jasim", "Noor Abdul Razzaq", "Ali Thoulfikar A. Imeer", "Rahem Mahdi Rahem", "Abdul Amir H. Kadhum", "Ahmed A. Al-Amiery", "Abdul Razzaq Oleiwi Jasim", "Noor Abdul Razzaq", "Ali Thoulfikar A. Imeer", "Rahem Mahdi Rahem", "Ahmed A. Al-Amiery" ], "abstract": "Background: Type1 Diabetes Mellitus is a common chronic diseases among children, and associated with morbidity, mortality, and enormous healthcare expenditures.\nObjectives; to estimate the prevalence, incidence, and describe the epidemiological characteristics of Type 1 Diabetes Mellitus among children in Karbala governorate.\nMethods: A cross-sectional study was conducted among all children who attended the main DM center in Karbala Teaching Hospital for Children, Public Clinics, and Primary Health Care centers. Data was collected by structured questionnaire and biochemical and anthropometric measurements. The statistical analysis data entry was conducted using Statistical Package for Social Sciences.\nResults: Total number of Type 1 Diabetes Mellitus patients aged 0-15 years in Karbala in 2015 was 199; making 44.66/1005. Fifty-four patients were newly diagnosed patients making an incidence of 12.11/1005. There was nearly equal distribution among both sexes. Most of the cases were from urban areas and most of the patients had a Family history of Type 2 Diabetes Mellitus. Diabetic ketoacidosis was reported in 16.8% of the patients. Only 31.6% of patients had HbA1c < 7%. Half of the patients had a history of admission to the hospital for diabetes. Logistic regression analysis revealed that the only independent variables significantly correlated with poor glycemic control were lack of Self-Monitoring Blood Glucose, irregular visits to the Diabetic Center, and positive family history of diabetes.\nConclusion: Only one-third of T1DM children in Karbala city had controlled blood sugar. Lack of regular blood glucose monitoring and irregular contact with health care providers were the main determinants of uncontrolled blood sugar.", "keywords": [ "Type1 diabetes", "epidemiological characteristics", "glycemic control", "blood glucose" ], "content": "Introduction\n\nType 1 diabetes mellitus (T1DM), is one of the most frequent chronic diseases among children and represents a public health challenge for many reasons globally the incidence, which represents about 10% of the diabetes cases all over the world. It is increasing especially among children below 15 years old and is increases 3% yearly worldwide. The first presentation of 10-70% of newly diagnosed children with DM1 is Diabetic Ketoacidosis (DKA).1,2 The incidence rates differ in different populations, the lowest is observed in Venezuela and China while the highest in Sardinia and Finland. Regarding T1D incidence among Arab countries, several studies reported low incidence in Oman (2.54/100,000) and high incidence in Saudi Arabia (29/100,000). In general, the Arab countries show higher incidence and prevalence estimates in areas where DKA rates are high.3 High DKA incidence results in increased hospital admission and emergency department (ED) visits and contributes to the high costs of care for children with T1DM. In Iraq, the prevalence of T1DM increased from 7.8 in 1995 to 14.2/100000 in 2000 and to 24.7/100000 in 2014 under 15 years old children.4 Daily management of T1DM presents numerous challenges to achieving satisfactory metabolic control including multiple daily insulin injections, frequent blood glucose monitoring, frequent contact with medical professionals, and careful regulation of exercise, meal schedules, social status, and the educational level of the parents.4,5 Studies in Iraq conducted on adolescents and children with Type 1 diabetes mellitus have shown that both patient and family education was associated with a lack in several emergency room visits and hospitalizations, a reduction in overall healthcare expense. Glycemic control in adolescents and children with type 1 diabetes mellitus in post-conflict in Iraq is low. They are poor in these diagnoses in pre-school age, adolescents, and obese children (23.8% of diabetic children had glycemic control), and it is much higher reported cases in Basrah, south of Iraq (10%).5,6 While the Iraq Ministry of Health had some data on type 2 DM, there is scarce data on T1DM epidemiology. Availability of such data can help identify the burden of the problem and provide evidence that helps in the planning of health programs and allocation of financial and human resources, logistics, and treatment facilities. As the Karbala population is not different from the remaining Iraqi population, data from this study can be extrapolated and generalized to estimate corresponding figures representing Iraq. The aim of this study is to estimate the incidence and prevalence and to describe the epidemiological characteristics and the control status of T1DM among young patients in Karbala Governorate, Iraq.\n\n\nMethods\n\nSetting: Karbala Governorate is located 100 Km to the south of Baghdad, the capital of Iraq with an estimated population of 1,122,400; 445,628 of them aged ≤15 years.\n\nStudy design: A cross-sectional study to measure the prevalence and identify basic socio-demographic data and complications. Also, a longitudinal incidence design was used to measure the incidence of the disease over one year.\n\nStudy Population: This study involved all T1DM patients who lived and registered in Karbala governorate, aged ≤15 years.\n\nData of T1DM patients have been obtained from the following sources: 1) Karbala Teaching Hospital for Children where the main DM center is located, and the majority of young DM patients attended to seek medical support. 2) Public Clinics are distributed in all cities and towns throughout the country and they are the main health outlets where all patients with chronic diseases (including DM) receive their medications for a very subsidized cost. 3) Primary Health Care Centers (PH Care Cs) are distributed in all localities and provide all PHC services including school health services. Part of the activities of the school health program in these PHCs is to have a list of all students with chronic diseases (including DM) in the schools within the catchment areas of these PHCCs.\n\nA data collection form was developed to compile data on all patients with T1DM from the registries of the diabetes center in Karbala Teaching hospital for Children, all the Popular Clinics, and all the PHCs Karbala governorate. These registries were useful in developing a list of patients currently diagnosed with T1DM, and the newly registered cases during 2013. Deceased patients during this year were first looked for at the Popular Clinics by identifying defaulters from the monthly medication supply lists. Information wars were verified through telephone communication with the families and school health programs in the PHCs. Clinical and epidemiological data were collected through the direct intervention of the patients using a structured questionnaire. The questionnaire was pre-tested on a sample of 10 patients from the target study population in the Diabetes Center. The measured parameters were the following anthropometric measures: Bodyweight, using a weighting digital scale (Seca) with light clothes without shoes and the weight was approximated to the nearest 100g), Height, using a portable measure (Seca), and height was approximated nearest 1cm. Body Mass Index (BMI) is calculated using the following equation: weight (in kilogram)/squared height (in meter). Moreover, measured fasting blood glucose (using Randox kit) using the colorimetric method), and Glycosylated hemoglobin (HbA1C) (using StandBio kit, using the spectrophotometric method).\n\nWritten informed consent was obtained from the caregiver of each participant before data collection. Confidentiality of the data was maintained throughout the study. Official approval was granted from the Karbala Directorate of Health.\n\nStatistical analysis: Statistical Package for Social Sciences was used for data entry and statistical analysis. The frequency data were expressed in suitable tables, and graphs. The statistical association was tested using the chi-square test of independence and Fisher’s exact probability test when needed. Quantitative data were analyzed using an unpaired t-test. Logistic regression analysis was used to identify the significant independent determinants. A less than 0.05 of p-value of is considered significant statistically.\n\nWritten informed consent for publication was obtained. The requirement for ethical approval was waived by the Medical Ethics Review Committee of the Academic Medical Center. Patients were treated according to guidelines of the Iraq Medical Association (KNMG). Medication was administered under Karbala Teaching Hospital and Primary Health Care Centers.\n\n\nResults\n\nA total of 190 T1DM patients aged 0-15 years were identified making a prevalence of 44.66/105. Fifty-four patients were newly diagnosed patients during 2013, making an incidence of 12.11/105. The Mean age was 8.8 (±3.6) years; 55.8% were more than eight years old and 14.2% were less than five years. About 53% were females and 68% were urban residents. Breastfeeding was reported by 93.2% of the patients; 77% of them continued breastfeeding for more than six months. Positive family history of T1DM and exposure to significant stressful life events were reported in 20.5% and 21.5%, respectively. As shown in Table 1, more than one-fifth (23%) of the patients presented with diabetic ketoacidosis (DKA) as a first manifestation of the disease, and around 22% had the disease for more than six years. Only one-third of the patients (31.6%) had controlled diabetes as reflected by HBA1c; 28% had a history of severe acute complications (hypoglycemia or DKA) and 51.5% required admission either to the ER or the inpatient ward.\n\nTo identify the determinants of the control status of the disease among the young T1DM patients, we compared patients with controlled diabetes as reflected by normal HBA1c, with the group of uncontrolled disease by several sociodemographic and clinical characteristics and certain behavioral factors as demonstrated in Tables 2 and 3. The proportion of controlled disease was significantly higher among children aged ≤8 years (P=0.008), with better mothers’ and fathers’ education (P<0.001), employed fathers (P=0.017), and children with a duration of disease <6 years (P=0.034). (Table 2). Also, controlled diabetes was significantly higher among patients with regular attendance at the diabetes center (P< 0.001), blood glucose self-monitoring (P< 0.001), adherence to insulin treatment, and controlled dietary intake by the parents (P< 0.001). (Table 3).\n\nLogistic regression analysis was applied considering the poorly controlled status as the dependent variable and all the variables that were found significant in the binary analysis were included as the independent variables in the model. Three factors were found statistically significant: self-monitoring of blood glucose (OR:10.41, 95% CI:2.24-47.62) irregular attendance to the diabetes center (OR:5.62, 95%CI: 5.62-171.32), and family history with positive DM (OR:1.86; 95% CI:1.01-3.41).\n\n\nDiscussion\n\nIn the current study all the patients were 199, among them there was 54 new patients, 190 patients (95.47%) participated in this study, and the incidence rate (12.11/100,000) was higher as compared to another study in Basrah city, Southern Iraq, (7.4 per 100,000 (95% CI, 7.1-8.1).5 Japan (2.2/100,000), Iran (3.7/100,000), and lower compared to, United Kingdom (26/100,000)2 Saudi Arabia (33.5/100,000).7 While prevalence (44.65/100,000) was higher compared to study in South-Eastern Nigerian school children aged 5–17 years. (33/100,000),8 and lower than Saudi-Arabian (109.5/100,000).9\n\nMost of our patients aged more than 8 years, most of them from rural areas, Sex nearly equal distribution with only slight female predominance. A study from Al-Madinah/Saudi Arabia has reported a higher incidence for the 10–12-year age group than in younger children. Furthermore, they have reported a higher incidence of T1DM in girls than in boys and most patients from urban areas,7,10 in the current study most of the mothers were housewives, with low educational levels, in constituent with another study in Egypt in which 85% of the patients with not work mothers and about two-thirds of them had low educational level.11\n\nAchieving the ideal blood glucose level is very difficult for many patients with diabetes, therefore, in the current study, the rate of optimal glycemic control (HbA1C <7%) represented around 31.6% of the patients comprised in other studies in Kenya (28%),12 Saudi Arabia (31.2%), Egypt (60%) and two-thirds of Italian children with T1DM have HbA1c>8% despite a regionalized centers, free access to appropriate diabetes care, multidisciplinary team approach, frequent blood glucose monitoring, education and multiple insulin injections.13\n\nGood glycemic control is associated with age < 8 years, urban residence, not affected by gender, reflecting the same complex the social and economic environment in which both genders are living, another study in Saudi Arabia confirmed the same result.13 other studies found better control associated with older age14 and a few studies shows that the mean of HbA1C in girls was significantly higher than in boys.15 another study from the UK showed a linear association between residence and glycemic control in type 1 diabetes16 and Our results reflected a very low incidence of divorce in the study group, so there was no significant correlation between glycemic state and marital status of the parents.\n\nIn the current study, good blood glucose monitoring (BGM), adherence, and regularity of clinic attendance were significantly associated with better HbA1c, in agreement with several studies.14,17 The clinic visits frequently is recommended to allow for better frequent adjustment of insulin regimens, and an increases number of opportunity for motivation and education.\n\nMore knowledgeable parents on diabetes with better education are able to cope more effectively and maintain better glycemic control of their diabetic children, results in other studies showed a significant relationship between low education of parents and poor glycemic control,11,18 majority of our patients had housewife mothers and most of their fathers were self-employed, such type of life does not permit optimal looking after the diabetic Childs, then consequently it turns to uncontrolled status of HbA1C levels, we found that positive family history of T2DM was more frequent than type 1 or both, other study found that only 10–15% of the patients have a first- or second-degree relative with T1DM,1 while family history of type 1 diabetes mellitus is highly prevalent among other studied patients.19\n\nOur study is in agreement with many other studies, which concluded a significant correlation between the level of HbA1c and diabetes duration (poor glycemic control in patients with a duration of 6 years and more), adherence to dietary management, and insulin.13–14,20\n\n16.8% of the patients had DKA, 8.9% had hypoglycemia, in 2013, and by the ranking of countries according to the frequency of DKA, the current study demonstrated that the result was lower than the frequencies were observed in Saudi Arabia (44.9%), the United Arab Emirates (80%), Hungary (23%) and Finland (22%). The lowest frequencies for DKA presentation of T1DM is reported in Sweden (14%).9 Initially, a classical type of presentation was found in about two-thirds of our patients, In comparison to a study conducted in Brazil, approximately 20% of patients with previously undiagnosed DM1 initially presented with DKA 21 while another study found 68% of children had DKA.22 About half of diabetic patients are admitted to the hospital, in Sweden children newly diagnosed with type 1 diabetes mellitus are admitted to the hospital for metabolic stabilization and training, even if they are not acutely ill, diabetic ketoacidosis and hypoglycemia often lead to an emergency department (ED) visit and hospital admission. In another study, in Sudan, most of the children (81%) had a history of hospital admission with DKA.23 Mortality in patients with childhood-onset type 1 diabetes in the current study was zero, during the time of the study. No chronic complication was found in all patients. Despite the developments in clinical care in recent years, the mortality risk for people developing type 1 diabetes in childhood remains high in young adult life before the onset of chronic complications.24,25\n\n\nConclusion\n\nPoor glycemic control was in around two-thirds of the patients and about 25% of them had acute complications and problems in school achievement. About half of the patients were admitted to the hospital. The most important co-varieties of the uncontrolled glycemic state of the patients were older age children, low educational level of the parents, and rural residence factors. SMBG, irregular visits to the Diabetic Center, and family history of diabetes were significant predictors of poor rate control.\n\nThe result of this study suggests that the pediatrician and endocrinologists must be critically assess the care required to these group with considering any new approaches to improve controlling their glycemic level. Special attention should be taken to develop a public health intervention strategy to educate the population and increases their awareness about the risk factors of diabetes complications. Stress on the practice of regular visit to the care Center for the proper monitoring of the disease and preventing any complication. Our finding is confirmed the need to develop a national registry for T1DM and the need for further multicenter epidemiological research studies covering the entire country to define the nationwide T1DM incidence for the related health data in Iraq.\n\n\nAuthor contributions\n\nA.R.O.J., N.A.R., A.T.A.I., R.M.R., and A.A.H.K., were responsible for the design of the treatment protocol and done all the tests. A.A.A-A. draft the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.", "appendix": "Data availability\n\nZenodo. Epidemiological profile and diabetes control of Type 1 Diabetes Mellitus patients in Karbala Governorate, Iraq. DOI: 10.5281/zenodo.7380413. 26\n\nFigshare. dx.doi.org/10.6084/m9.figshare.6025748. DOI: https://doi.org/10.6084/m9.figshare.6025748.v1. 27\n\nThese projects contain the following data:\n\n- Source data for the figures of the article\n\n- Statistical results\n\n- Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nAuthors thank Karbala Health Directorate for facilitating this work.\n\n\nReferences\n\nPaschou SA, Papadopoulou-Marketou N, Chrousos GP, et al.: On type 1 diabetes mellitus pathogenesis. Endocr. Connect. 2018; 7(1): R38–R46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMirsadraee R, Khajedaluee M, Vakili R, et al.: Factors Associated with Newly Diagnosed Children with Diabetic Ketoacidosis. J. Pediatr. Rev. 2017; 5(1): e7462. 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Metabol. 2011; 55: 194–202. PubMed Abstract \| Publisher Full Text\n\nSaif A-Y, Ullah I, Sharef W: Demographic and clinical characteristics of type 1 diabetes mellitus in Omani children - single center experience. Oman Med. J. 2014; 29(2): 119–122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu L, Yang D, Zhang Y: Glycaemic control and its associated factors in Chinese adults with type 1 diabetes mellitus. Diabetes Metab. Res. Rev. 2015; 31: 803–810. PubMed Abstract \| Publisher Full Text\n\nLopes CLS, Pinheiro PP, Barberena LS: Diabetic ketoacidosis in a pediatric intensive care. J. Pediatr. 2017; 93(2): 179–184. PubMed Abstract \| Publisher Full Text\n\nYashwanth K, Chary R: Children with type 1 diabetes presenting with ketoacidosis: frequency, severity and clinical profile. J. Dent. Med. Sci. 2017; 16(11): 52–55. Publisher Full Text\n\nTaha Z, Eltoum Z, Washi S: Predictors of Glucose Control in Children and Adolescents with Type 1 Diabetes: Results of a Cross-Sectional Study in Khartoum, Sudan. Maced J. Med. Sci. 2018; 6(11): 2035–2039. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWasag DR, Gregory JW, Dayan C: Excess all-cause mortality before age 30 in childhood onset type 1 diabetes: data from the Brecon Group Cohort in Wales. Arch. Dis. Child. 2018; 103(1): 44–48. PubMed Abstract \| Publisher Full Text\n\nDonzeau A, Piffaretti C, Jossens A, et al.: Time trend in excess mortality in children with type 1 diabetes from 1987 to 2016 in mainland France. Epidemiology. 2018; 23(11): 38–44. PubMed Abstract \| Publisher Full Text\n\nJasim ARO: Epidemiological profile and diabetes control of Type 1 Diabetes Mellitus patients in Karbala Governorate, Iraq.2022. Publisher Full Text\n\nJasim ARO: Epidemiological profile and diabetes control of Type 1 Diabetes Mellitus patients in Karbala Governorate, Iraq.2022. Publisher Full Text" }	[ { "id": "180900", "date": "30 Jun 2023", "name": "Kalyani Pathak", "expertise": [ "Reviewer Expertise Diabetes", "Traditional medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper provides valuable insights into the prevalence and epidemiological characteristics of Type 1 Diabetes Mellitus in children in Karbala. However, it would benefit from more clarity regarding the methodology. The use of a structured questionnaire, biochemical, and anthropometric measurements are mentioned, but specific details are missing. For example, it would be helpful to know the exact questions asked in the questionnaire and the nature of the biochemical measurements. Also, it would be interesting to understand how the authors controlled for potential confounding factors.\nThe manuscript is well-structured and provides important information on Type 1 Diabetes Mellitus in children. However, it would be beneficial to provide more context on the implications of the findings. The authors mention that only 31.6% of patients had HbA1c < 7%, but the significance of this finding for the patient population or the health care system is not discussed. It would be useful to elaborate on what these results mean for future research, public health policies, or clinical practices in Karbala.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "311391", "date": "28 Aug 2024", "name": "Kamarudin Ahmad", "expertise": [ "Reviewer Expertise My research is dedicated to advancing the safety and effectiveness of medication management within community and clinical settings. It emphasizes reducing prescribing errors through pharmacist-led interventions and other systematic approaches. A significant aspect of this research involves exploring the side effects of widely used medications", "such as metformin in Type 2 Diabetes Mellitus", "by conducting comprehensive reviews of existing studies.Collaborating with healthcare professionals aims to translate research findings into practical applications that improve patient care. Research interests extend to international collaborations", "particularly between Malaysia and Thailand", "focusing on drug security", "medicines policy", "and primary care pharmacy. The ultimate goal is to enhance patient outcomes by ensuring safer and more effective use of medications." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract The abstract of the manuscript provides a concise overview of the study. However, it could benefit from a stronger emphasis on the methods section. Detailing the study design, sampling strategy, and key statistical approaches in the abstract would offer readers a clearer understanding of how the research was conducted. This would also enhance the transparency and reproducibility of the study. Suggestions:\nInclude Methodological Details: Briefly mention the logistic regression analysis and any specific inclusion/exclusion criteria used to select participants. Highlight Key Findings: Summarize the most significant results to give a snapshot of the study’s outcomes.\nIntroduction The introduction currently lacks a smooth and directive flow. It would be more effective to explicitly discuss the problem being addressed by the study, using a funnel approach that starts broad and narrows down to the specific research aims. The introduction should also incorporate a detailed explanation of the current situation, supported by relevant literature, and link these to the study's objectives. Additionally, the introduction should explain the factors influencing the variables included in the logistic regression analysis, supported by evidence from the literature. This will help set the stage for the analysis and interpretation of results. Suggestions:\nImprove Structural Flow: Revise the introduction to ensure each section logically leads to the next, connecting background information to research questions and objectives. Contextualize Variables: Explain the factors influencing the variables in the logistic regression model with supporting literature. Consistency in Terminology: To avoid confusion, use terms like \"glycemic control,\" \"blood sugar control,\" and \"HbA1c levels\" consistently throughout the introduction.\nMethods The methods section requires reordering, with the statistical analysis discussed before the ethical approval. Additionally, not all variables included in the logistic regression analysis are explained in detail. A thorough explanation of each variable, including how they were measured and why they were included in the regression model, is crucial for the reader's understanding. Suggestions:\nReorder Sections: Present the statistical analysis methods before discussing ethical approval to follow a logical sequence. Detailed Variable Explanation: Provide a clear rationale for including each variable in the logistic regression model and describe how each was measured. Clarity in Ethical Considerations: Clearly outline how ethical considerations influenced the study design, particularly in a sensitive context like pediatric diabetes. This highlights the researchers' commitment to ethical rigour.\nResults There is evidence of selective outcome reporting in the results section. Specifically, not all results from the logistic regression analysis are displayed. This selective reporting could undermine the study's credibility and the robustness of its conclusions. Suggestions:\nComprehensive Reporting: Ensure that all results from the logistic regression analysis are presented, including both significant and non-significant findings. This transparency strengthens the study's validity. Data Visualization: Incorporate more visual elements such as graphs or tables to summarize key findings, particularly from the logistic regression analysis. Visual aids help readers quickly grasp complex data and understand the relationships between variables.\nDiscussion The discussion section inappropriately includes results that should be confined to the results section. The discussion should focus on interpreting the findings, explaining the implications of the results, and discussing them in the context of existing literature. Moreover, there is a need to explain why certain variables were significant in the logistic regression analysis, supported by relevant evidence. Suggestions:\nRestructure Content: Avoid presenting new results in the discussion. Focus on interpreting the results already presented and discussing their implications. Justify Findings: Explain why certain variables were significant in the logistic regression analysis, supported by relevant literature. Highlight Study’s Limitations: Include a detailed discussion of the study's limitations, such as potential biases (e.g., selection bias or confounding factors), and how these were addressed or could be addressed in future research. This transparency strengthens the study’s credibility. Future Research Directions: Expand on the implications of the findings for future research. For example, suggest exploring significant variables further in longitudinal studies or different populations to validate the findings. Impact on Clinical Practice and Policy: Discuss how the findings could influence clinical practice or public health policy, especially in the context of pediatric diabetes management. Explore how regular monitoring and family education could be better integrated into public health initiatives.\nConclusion The conclusion currently makes statements not fully supported by the results presented. The conclusion must be directly backed by the study's findings, without overgeneralizing or making unsupported claims. Suggestions:\nAlign with Results: Ensure the conclusion accurately reflects the study's results. Avoid introducing new information or making extrapolations beyond the data presented. Clear and Concise: Summarize the key findings and their implications succinctly, reinforcing the study’s contributions without overstating them.\nAdditional Suggestions\nConsistency in Terminology:\nEnsure that the terminology is consistent throughout the manuscript. To avoid confusion, terms like \"glycemic control,\" \"blood sugar control,\" and \"HbA1c levels\" should be used uniformly.\n\nHighlighting the Study’s Limitations:\nAcknowledge potential biases, such as selection bias or confounding factors, and discuss how these were addressed or could be mitigated in future research. This enhances the study’s credibility.\n\nFuture Research Directions:\nEncourage the authors to outline specific areas for future research based on their findings, such as conducting longitudinal studies or expanding the research to different populations to validate and extend the results.\n\nImpact on Clinical Practice and Policy:\nSuggest ways the findings could inform clinical guidelines or public health policies, particularly in improving pediatric diabetes management through regular monitoring and education initiatives.\n\nData Visualization:\nI recommend using additional visual elements like graphs, charts, or tables to effectively present complex data and highlight key relationships identified in the logistic regression analysis.\n\nClarity and Consistency:\nEnsure that all manuscript sections use consistent terminology and maintain a clear, logical flow to enhance readability and comprehension.\n\nOverall Structure and Presentation\nConsistency and Clarity: Maintain consistent terminology and ensure each section transitions smoothly to the next. Detailed Reporting: Provide sufficient detail in the methods and results sections to allow replication and full understanding by readers. Transparent Reporting: To enhance the study's transparency and reliability, present all findings comprehensively, including non-significant results. Comprehensive Discussion: Focus on interpreting results, addressing limitations, and outlining future research directions without introducing new data.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "311405", "date": "12 Sep 2024", "name": "Charlotte Verroken", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well-structured. The most important concern is that the methodology needs to be more clearly described. How and in which time frame were participants recruited (were all T1DM patients invited or those that visited the clinics within a certain timeframe)? How high was the participation rate? Who performed the anthropometrics (the research team or was this information retrieved from the patient files)? Were lab analyses specifically performed for the study or also retrieved from patient files? In addition, the article might benefit from language editing.\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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-409
https://f1000research.com/articles/11-1566/v1	22 Dec 22	{ "type": "Brief Report", "title": "Functional tic-like presentations differ strikingly from Provisional Tic Disorder", "authors": [ "Amanda L. Arbuckle", "Emily C. Bihun", "Bradley L. Schlaggar", "Kevin J. Black", "Amanda L. Arbuckle", "Emily C. Bihun", "Bradley L. Schlaggar" ], "abstract": "Background: Recent years have seen a dramatic increase in new “tic” cases in teens and young adults. These individuals often present with fulminant onset of symptoms not commonly seen in Tourette syndrome (TS) and are often diagnosed with Functional Neurological Symptom Disorder (FND-tic). However, some authors have questioned whether this illness truly differs from typical Provisional Tic Disorder (PTD) and TS. Previous studies have compared FND-tic, usually a few months after symptom onset, to patients with TS, usually years after symptom onset. We sought to test whether the presenting symptoms of FND-tic differ substantially from those in patients at a similar duration of symptoms who are later diagnosed with TS. Methods: This comparative study examines clinical features summarized from published reports of FND-tic with novel data from a longitudinal study of PTD. This study came from a referral center for TS and tic disorders and included 89 children with tics whose first tic occurred a median of 3.6 months earlier, nearly all of whom were diagnosed with a chronic tic disorder at follow-up. Specifically, we examine clinical features identified in a recent literature review as supporting a diagnosis of FND-tic, including symptom characteristics, course, severity and comorbidity. Results: Several clinical features dramatically distinguish the patients diagnosed with FND-tic from those diagnosed with typical PTD. For example, coprophenomena are reported at or shortly after symptom onset in over half of FND-tic patients, whereas even several months after onset, coprophenomena had occurred in only 1 of 89 children with PTD. Six clinical features each have a positive predictive value over 90% for FND-tic diagnosis if prior probability is 50%. Conclusions: These new data provide strong evidence supporting the diagnostic validity of FND-tic as distinct from TS.", "keywords": [ "Tic Disorders/classification", "Provisional Tic Disorder", "Functional Neurological Symptom Disorder", "Conversion Disorder", "Diagnosis", "Differential", "Tourette Syndrome" ], "content": "Introduction\n\nRecent years have seen a dramatic increase in new “tic” cases in teens and young adults.1,2 These individuals often present with fulminant onset of symptoms not commonly seen in Tourette syndrome (TS), but often similar to those found in videos tagged as “Tourette” on social media.3 For instance, echopraxia and coprolalia occur in more than half of these individuals at symptom onset.3 The nature and characteristics of these symptoms, and the onset age and course of illness have led experienced clinicians to differentiate these cases from TS and to diagnose instead Functional Neurological Symptom Disorder (FND). FND with tic-like symptoms (hereinafter “FND-tic”) has been reported previously, but prior to 2019 was considered rare, occurring in < 2% of tic or tic-like cases at five major referral centers from three continents.4\n\nSome experts have expressed skepticism as to whether a new diagnosis (FND) is needed for these patients, positing that the previous understanding of TS may have been too narrow.5 Perhaps, for instance, echopraxia is present early in the course of TS much more often than has been recognized. We concur with these authors that clearly differentiating a new diagnosis from existing diagnoses is a key component of diagnostic validity.6 Substantial published data describe typical clinical features of chronic tic disorders, but almost no prospective data have been published on symptoms in the first year after tic onset.7,8 This evidence gap is crucial because most FND-tic patients at clinical centers have had symptoms for only a few months (mean 0.4 years in one study4). Here, we directly address this concern by providing for the first time substantial data on relevant clinical features during the first few months after symptom onset in children ultimately diagnosed with TS.\n\n\nMethods\n\nThe New Tics study is a prospective, longitudinal study that enrolled 89 children ages 5–10 years whose first tic occurred in the past 9 months (median 3.6 months).9 Children are assessed using multiple informants (child, parent, trained interviewer, and observation by an experienced clinician for more than an hour, including by video while the child is alone). The diagnosis in this situation is Provisional Tic Disorder (PTD), and nearly all these children (77 of 79) were diagnosed with TS (70) or a persistent tic disorder (7) when they returned at the one-year anniversary of their first tic.10 Here we report the prevalence and timing from the New Tics sample of various features that occur commonly in FND-tic patients.\n\nThe feature list was drawn from a recent review of FND-tic, a narrative review that included all primary data publications on FND-tic known to its authors as of August, 2022.3 The comparison data for FND-tic patients comes from 26 published reports, with pertinent data in 17 reports4,11–26 describing a total of 336 patients (data file available as Underlying data27). For quantitative variables, the weighted mean is provided (weighted by N in each report), along with the median and range of the summary values reported in each relevant publication.\n\nMost of these features thought to suggest FND-tic were recorded prospectively in the New Tics study, including age, sex, premonitory urges, tic suppression, coprophenomena, and family history. However, since the New Tics sample was enrolled almost entirely before the recent FND-tic upsurge, some of these clinical features were recorded indirectly. For instance, to match “severe symptoms at onset,” defined differently in various reports on FND-tic, we conservatively chose from the New Tics sample all patients with emergency department visits or disability prior to the screening visit, or a high score on the Yale Global Tic Severity Scale’s28 impairment item at screening. Details on other such choices are given in footnotes to Table 1.\n\nFND, Functional Neurological Symptom Disorder; FND-tic, Functional Neurological Symptom Disorder with tic-like symptoms; PTD, Provisional Tic Disorder; NP, number of publications; OCD, obsessive compulsive disorder; ADHD, attention deficit hyperactivity disorder; YGTSS, Yale Global Tic Severity Scale.\n\na Specified clinical features in patients with tic-like symptoms (“FND”) from the articles reviewed in Malaty et al. (2022),3 compared to participants with typical Provisional Tic Disorder from the New Tics study (“New Tics”).\n\nb NP = number of publications from the table in Malaty et al. (2022)3 that contributed data to the statistics in this row.\n\nc N = 89 except where numerator and denominator are provided.\n\nd N = 125, the number of participants who came for a screening visit after reporting recent onset of tics during initial telephone contact. Some were found during careful screening to have a prior episode of transient tics.\n\ne Excludes the two reports that defined the sample by sudden onset.4,21\n\nf The investigator assigned the most likely symptom onset date within a date range (possible onset dates) of less than 7 days.\n\ng From parents’ answers as to whether the onset of the first tic was sudden or gradual.\n\nh Investigator noted tics during the history and exam (approximately 45 minutes), but no tics when observing the child alone in the room via video camera (approximately 40 minutes).\n\ni Yale Global Tic Severity Scale.\n\nFisher’s exact test was used to find the probability of differences in frequency of features between the two populations (fisher_exact from SciPy (RRID:SCR_008058) 1.9.1).29\n\nThe New Tics study was approved by the Washington University Human Research Protection Office (IRB, protocol numbers 201109157 and 201707059), all participants assented to participation, and a parent or other legal guardian provided written documentation of informed consent.\n\n\nResults\n\nStark differences in presentation distinguish the FND-tic patients from typical PTD (Table 1). For example, coprophenomena are reported at or shortly after symptom onset in 59% of FND-tic patients. By contrast, coprophenomena had occurred in only 1 of 89 children with PTD at an average of 3.6 months after tic onset. Similarly, the TS International Database Consortium found that only 2% of TS patients in tertiary centers retrospectively reported coprophenomena at symptom onset, and only 20% ever manifested coprophenomena by an average of 5 years after tic onset.30 Movements or vocalizations that were dramatically worse in the presence of others versus when alone occurred in 47% of FND-tic patients, but in none of the New Tics PTD sample. Symptoms dramatically and persistently disrupted intended actions in 65% of FND-tic patients, but in only 2% of PTD. The prevalence of prolonged tic attacks was 65% in FND-tic, but 0–2% in PTD. Other features that differed substantially include lack of premonitory urges (53% vs. 4%) and severe symptoms at onset (48% vs. 3–6%). Table 1 and Table 2 provide details on these comparisons and include statistics on a dozen more clinical features of FND-tic that differ from the New Tics PTD sample.\n\nIn a patient with recent onset of tics, the presence of any one of the features named above raises the probability of a non-TS diagnosis from 50% (as when the clinician is ambivalent about the diagnosis prior to considering this feature) to over 90% (Table 2). Other features differ significantly but are less useful diagnostically. For instance, obsessive compulsive disorder (OCD) is more than three times less common in FND-tic than in PTD (p<.0001), but its absence only raises the probability of FND-tic from 50% to 57%.\n\nPPV, positive predictive value; NPV, negative predictive value; OCD, obsessive compulsive disorder; ADHD, attention deficit hyperactivity disorder; TS, Tourette syndrome; FND, Functional Neurological Symptom Disorder.\n\na PPV of a non-TS diagnosis for the binary features in Table 1, assuming a prior probability for FND of 2% (typical pre-pandemic prevalence at a referral center4) or 50% (representing clinical equipoise about a given patient’s diagnosis before considering this feature). NPV is shown for features more common in typical TS, equivalent to PPV for the absence of the given feature.\n\nb Fisher’s exact test; “.000” means < .0005; p values bolded if p < .05/20 = .0025.\n\nNote: Values for PPV and NPV are bolded if ≥67%.\n\n\nDiscussion\n\nWe demonstrate conclusively that patients with functional tic-like symptoms differ notably from typical tic patients at the same stage of the disorder, namely in the first few months after symptom onset. Previous reports have compared FND-tic to TS,3,31 but not to a large PTD sample. The older age of the FND-tic group could be seen as a limitation of our study, since one might posit different symptoms at different ages of tic onset might be part of the natural history of tic disorder. However, this theoretical concern does not in fact limit our conclusions, because retrospective studies of TS and a prospective study of PTD in siblings of TS probands all found peak tic onset before age 10 years old in both sexes; adult onset of tics is uncommon.7,8,32\n\nThe data presented here do not prove the etiology of the tic-like symptoms diagnosed in the cited reports; hence the limited claim that these symptoms represent a different illness than PTD/TS. However, the marked difference in presentation these data demonstrate is an important argument adduced in the cited reports to support the diagnosis of functional neurological symptom disorder. Diagnosing FND-tic is important, since to the extent of our current knowledge, its prognosis and optimal treatment differ from those of TS.3\n\nIn conclusion, these new clinical data about the first few months after tic onset prior to diagnosis of TS provide strong evidence supporting the diagnostic validity of functional tic-like symptoms as distinct from PTD and TS.", "appendix": "Data availability\n\nInformation on ‘The New Tics Study: A Novel Approach to Pathophysiology and Cause of Tic Disorders’ can be found at NIMH Data Archive and NIH RePORTER (Project Number 1R01MH104030-01A1).\n\nOpen Science Framework: Supplemental materials for publication: Functional tic-like presentations differ strikingly from Provisional Tic Disorder. https://doi.org/10.17605/OSF.IO/RSFXN 27 .\n\nThis project contains the following underlying data:\n\n- FNS_vs_TS.csv (summary table from the FND-tic publications cited by Malaty et al. 3 )\n\n- FND-tic_not_PTD.py (python script used to summarize data for Tables 1 and 2)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nAn earlier version of this article can be found on Open Science Framework (doi: https://doi.org/10.31219/osf.io/3u6bk).\n\n\nReferences\n\nAnderson KN, Radhakrishnan L, Lane RI, et al.: Changes and inequities in adult mental health-related emergency department visits during the COVID-19 pandemic in the US. 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PubMed Abstract \| Publisher Full Text\n\nHan VX, Kozlowska K, Kothur K, et al.: Rapid onset functional tic-like behaviours in children and adolescents during COVID-19: Clinical features, assessment and biopsychosocial treatment approach. J. Paediatr. Child Health. 2022 Jul; 58(7): 1181–1187. Epub 20220305. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJanik P, Milanowski Ł, Szejko N: Psychogenic tics: clinical characteristics and prevalence. Psychiatr. Pol. 2014 Jul-Aug; 48(4): 835–845. Tiki psychogenne: charakterystyka kliniczna i czestość wystepowania. PubMed Abstract\n\nOlvera C, Stebbins GT, Goetz CG, et al.: TikTok tics: A pandemic within a pandemic. Mov. Disord. Clin. Pract. 2021; 8(8): 1200–1205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaulus T, Bäumer T, Verrel J, et al.: Pandemic tic-like behaviors following social media consumption. Mov. Disord. 2021 Dec; 36(12): 2932–2935. Epub 20210924. PubMed Abstract \| Publisher Full Text\n\nBlack KJ, Arbuckle A:Supplemental Materials for Publication: Functional Tic-like Presentations Differ Strikingly from Provisional Tic Disorder. [Dataset]. OSF. Dec 2022. Publisher Full Text\n\nLeckman JF, Riddle MA, Hardin MT, et al.: The Yale Global Tic Severity Scale: Initial testing of a clinical-rated scale of tic severity. J. Am. Acad. Child Adolesc. Psychiatry. 1989; 28(4): 566–573. PubMed Abstract \| Publisher Full Text\n\nVirtanen P, Gommers R, Oliphant TE, et al.: SciPy 1.0: Fundamental algorithms for scientific computing in Python. Nat. Methods. 2020 Mar; 17(3): 261–272. Epub 20200203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreeman RD, Zinner SH, Müller-Vahl KR, et al.: Coprophenomena in Tourette syndrome. Dev. Med. Child Neurol. 2009 Mar; 51(3): 218–227. Epub 20081204. PubMed Abstract \| Publisher Full Text\n\nNilles C, Martino D, Berg L, et al.: Can the phenomenology of functional tic-like behaviours be sufficient to make the diagnosis? AACAP/CACAP annual meeting; 19 Oct 2022; Toronto, Ontario, Canada.2022.\n\nBlack KJ, Kim S, Yang NY, et al.: Course of tic disorders over the lifespan. Curr. Dev. Disord. Rep. 2021 Jun; 8(2): 121–132. Epub 2021/06/29. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "158573", "date": "25 Jan 2023", "name": "Michael Bloch", "expertise": [ "Reviewer Expertise Meta-analysis", "psychopharmacology", "tic disorders", "OCD" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 compares the clinical characteristics of the sample of patients with Functional Neurological Disorder (FND) described in the literature, with a well-characterized sample of Provisional Tic Disorder. Strengths of the manuscript include the clinical importance and novelty of the data as well as the expertise of the authors and the well-written manuscript. Overall, I think this is a manuscript worthy of indexing, although I have several critiques that could potentially improve the manuscript with revision.\nI do not believe the primary limitation of the manuscript is really acknowledged in the current draft. Mainly that the FND cases are drawn from the existing literature (where there is likely a large degree of reporting bias) versus a Provisional Tic Disorder sample that is much more general. Basically, there is quite a strong possibility that there are other characteristics associated with group difference other than diagnosis.\n\nPlease consider providing statistical tests in table 1 as well as sensitivity, specificity in table 2.\n\nI think in the conclusion the authors should discuss the importance of testing the potentially meaningful differentiators in an independent dataset.\n\nIt would also be very worthwhile in further studies to use individual participant data to determine the PPV of multiple predictors to engage in a diagnostic distinction.\n\nIf possible, a histogram exploring age and gender differences between the two groups would be helpful.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9549", "date": "17 Apr 2023", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank Dr. Bloch for his thoughtful and expert critique. Below we respond to each comment in turn. 1. I do not believe the primary limitation of the manuscript is really acknowledged in the current draft. Mainly that the FND cases are drawn from the existing literature (where there is likely a large degree of reporting bias) versus a Provisional Tic Disorder sample that is much more general. Basically, there is quite a strong possibility that there are other characteristics associated with group difference other than diagnosis. We agree and have edited the ms. to reflect this point. However, the disparity is smaller than one might think. Here is our new text in the discussion: \"The primary concern with the validity of these conclusions arises from the different potential sources of ascertainment bias in the two groups. Fortunately, these differences may not be as problematic as one might suppose. The FND-tic group is older, and if one’s information were limited to this study alone, one might posit that the natural history of tic disorder included different early symptoms at different ages of tic onset. However, the literature includes decades of previous clinical information on typical tic disorders. Retrospective studies of TS and a prospective study of PTD in siblings of TS probands all found peak tic onset before age 10 years old; adult onset of tics is quite uncommon. 7 , 8 , 33 The PTD cases were volunteers willing to participate in a rather intensive research study, and were ascertained by a variety of methods (advertising and referrals from clinicians being the most common) rather than by clinical care-seeking. However, at the screening visit for the study, over 60% of them had sought clinical care for the tics or were planning to. The two FND-tic reports from Calgary, Alberta (child and young adult) were from a prospective registry of all patients seen for tics at their center, which is the only specialty center in their region. FND-tic cases were defined by “rapid onset of complex tic-like behaviors, with escalation to peak severity within hours to days.” In other words, these reports are selected only for being seen at a specialty center for tic-like phenomena. Yet their clinical features are similar to those of the FND-tic literature overall (or even more different from the PTD group). For instance, 95% of the Calgary FND-tic child patients were of female sex, only 25% had ADHD, and only 5% had OCD. We can conclude that potential bias from selecting more interesting, severe or classic cases for published case series does not substantially alter our results. Perhaps most importantly, there are no larger published sources for data about FND-tic nor about PTD. Nevertheless, ideally the results presented here should be confirmed in a new, independent sample.\" 2. Please consider providing statistical tests in table 1 as well as sensitivity, specificity in table 2. Done. 3. I think in the conclusion the authors should discuss the importance of testing the potentially meaningful differentiators in an independent dataset. We agree. The discussion now includes: \"ideally the results presented here should be confirmed in a new, independent sample.\" 4. It would also be very worthwhile in further studies to use individual participant data to determine the PPV of multiple predictors to engage in a diagnostic distinction. That's an excellent idea. We have supplied individual participant data for the New Tics group as a supplementary file. 5. If possible, a histogram exploring age and gender differences between the two groups would be helpful. We do not have individual participant data for the FND-tic group (which came from 17 different publications), so we can't do this." } ] }, { "id": "158571", "date": "31 Mar 2023", "name": "Tammy Hedderly", "expertise": [ "Reviewer Expertise Paediatric Neurology", "Movement Disorders and Tourettes" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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, all highly experienced 'Tourettologists', have approached this topic in an interesting and novel manner to try and provide some evidence for the ability to differentiate functional tic like behaviours from those seen in provisional tic disorders, usually related to Tourettes at follow up. This is a complex area, with some researchers proposing some shared mechanisms and overlapping neurobiology in the presentations. This paper uses some usefully collected detailed (although some retrospective) clinical data of children with PTD within a few months of presentation to compare characteristics demonstrating some useful distinguishing features. The presentation of the statistics could benefit from a little more expansion and explanation, especially for the non-expert, as it seems an important aspect of this paper. For example, table 2 is not that clearly explained and could do with more detail.\nThere are some limitations, but these have been clearly highlighted by the authors, such as the difference in ages of the two groups. I therefore would support this paper as it represents a useful contribution to the ongoing exploration of the functional tic-like phenomenon. I do wonder whether the dichotomy of subjects is a problem and would welcome wider discussions by the authors, on the situations seen commonly in the clinic in which young people, demonstrate both Tourette-related tics and functional tic-like behaviours together. This paper raised a question in my mind, as to whether the authors could have included distinguishing factors for the concept of provisional or transient functional disorder, especially when appearing in the context of exposure to social media or influencers.\nThank you for an interesting contribution.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9550", "date": "17 Apr 2023", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank Dr. Hedderly for her thoughtful and expert critique. Below we respond to each comment in turn. 1. The presentation of the statistics could benefit from a little more expansion and explanation, especially for the non-expert, as it seems an important aspect of this paper. For example, table 2 is not that clearly explained and could do with more detail. Thank you. We agree. The new text in 'Results' reads as follows: \"Table 2 illustrates the clinical value of these comparisons, viewing each clinical feature as a “test” for FND-tic, with its associated sensitivity and specificity. We also provide the positive predictive value (PPV) for each feature thought to indicate FND-tic. The PPV represents how confident one can be of a FND-tic diagnosis given the presence of the listed feature. The PPV depends on the prior probability of the FND-tic diagnosis, or its likelihood before one knows whether a given patient has that feature. We provide PPV for each feature based on two prior probabilities, 2% and 50%. The first, 2%, represents the approximate rate before the pandemic of functional tic-like symptoms at clinical referral centers. 4 Clinical equipoise about a given participant’s diagnosis is represented by a prior probability of 50%, for instance if one knows that a referring clinician is ambivalent about whether the patient has PTD or FND-tic, but one has not yet read the chart nor seen the patient. Features thought to be less common in FND-tic are listed with a negative predictive value (NPV), equivalent to PPV for the absence of the given feature.\" 2. I do wonder whether the dichotomy of subjects is a problem and would welcome wider discussions by the authors, on the situations seen commonly in the clinic in which young people, demonstrate both Tourette-related tics and functional tic-like behaviours together. We agree the discussion did not address this issue previously. The discussion now includes this additional paragraph: \"We note that 22% of the patients reported in the FND-tic papers in fact had a prior history of typical tics, and many of them exhibited both tics and functional tic-like symptoms at the time they presented due to the latter. We have structured our results around whether FND-tic can be diagnosed, not whether PTD can also be diagnosed. In other words, when a patient presents with a typical history for TS but also new symptoms, the features above would allow more confidence in diagnosing FND-tic in addition to TS.\" 3. This paper raised a question in my mind, as to whether the authors could have included distinguishing factors for the concept of provisional or transient functional disorder, especially when appearing in the context of exposure to social media or influencers. The reviewer raises a very interesting question that appears to include two concepts: diagnostic utility of exposure to social media and nosology. a) The exposure to social media or other examples that the new symptoms appear to recapitulate is an important diagnostic clue. We and others have discussed that issue elsewhere (e.g. Frey et al. (2022) and Malaty et al. (2022)). Unfortunately, we have no prospectively collected data on this question from the PTD group. Anecdotally, I can report that although over a third of our New Tics study participants had a parent or sibling with tics, only once or twice was there any suggestion that the child's first tic may have appeared to be prompted by the family member's similar tic. We have added to the discussion the following paragraph: \"Other features in addition to those studied here may also be important for diagnosis. For instance, exposure to tic-like symptoms on social media was a common feature discussed in the papers discussing FND-tic in the past few years. 3 Unfortunately, we have no prospectively collected data from the PTD group on exposure to others with similar symptoms.\" b) Traditionally, functional neurological symptoms have not been diagnosed differently based on duration of the defining symptoms. The closest analogy I can think of is Briquet's syndrome (and its dramatically modified descendants such as DSM-IV somatization disorder), which required not only atypical, medically unexplained symptoms starting before midlife but also numerous symptoms over time, affecting a variety of organ systems. Personally I would not favor creating a new diagnosis to represent \"presumed functional symptoms in the first year since onset\"." } ] } ]	1	https://f1000research.com/articles/11-1566
https://f1000research.com/articles/12-408/v1	17 Apr 23	{ "type": "Method Article", "title": "Pulling wire to avoid misinsertion of filament and vascular damage improves ischemic stroke models' success rate and repeatability", "authors": [ "Fei Yang", "ZhiYun Zhang", "WeiPing Wan", "Zhen Wang", "LanQing Li", "ShuMin Li", "Hao Fu", "Wenzhi Yang", "Rong Chen", "Rongping Zhang", "XiNan Shi", "Jian Wang", "Fei Yang", "ZhiYun Zhang", "WeiPing Wan", "Zhen Wang", "LanQing Li", "ShuMin Li", "Hao Fu", "Wenzhi Yang", "Rong Chen", "Rongping Zhang" ], "abstract": "Background: Middle cerebral artery occlusion (MCAO) ischemia-reperfusion in rats is a commonly used in vivo model in stroke research, but the problems of long time-consuming, unstable neurological injury and high mortality are not conducive to an experimental animal study. In this study, the success rate of the MCAO model in rats was improved by changing the surgical approach and giving preoperative as well as postoperative care support.\nMethods: Place A-wire below the common carotid artery (CCA) to ligate the proximal end of the CCA, B-wire to fix the filament, and C-wire to pull the filament through the bifurcation of the internal carotid artery (ICA) and the external carotid artery (ECA). After temporarily clamping the ICA, insert the filament into the CCA incision, then drag the C-wire to reduce the angle between the CCA and the pterygoid artery (PPA) to avoid filament insertion into the PPA. Push forward the head of the filament to the middle cerebral artery (MCA), ventral thalamic artery (VTA), and anterior choroidal segment to block the blood supply. Neurological function scoring, triphenyltetrazolium chloride (TTC) staining and Nissl staining were applied to evaluate the model success or failure, the infarct area and neuro damage. Results: Compared with the traditional Zea longa mapping method, our method significantly reduced modeling time and surgical trauma and improved postoperative survival rate and success rate. Furthermore, our method is more consistent in terms of infarct area, and there was a significant reduction in Nissl bodies. Conclusions: The wire-lifting method pulls the vessel, reduces the angle between the CCA and the PPA, facilitates the insertion of the filament into the MCA to avoid the accidental insertion of the filament into the PPA, reduces the surgical trauma, and saves the model preparation time, which can effectively improve the survival rate and modeling success rate.", "keywords": [ "ischemic stroke", "middle cerebral artery occlusion model", "filament method", "intravascular modeling method" ], "content": "Introduction\n\nThe epidemiological study has shown that about 70% of all stroke patients worldwide suffer cerebral ischemic stroke (CIS),1 while ischemic stroke accounts for about 85% of all stroke cases in China.2 The study of reperfusion injury has become one of the hottest health topics in the world. Because the Sprague-Dawley (SD) rat vascular wall is very similar to that of humans in terms of structure and morphological changes during cerebrovascular disease,3,4 the SD rat middle cerebral artery occlusion (MCAO) model is the most commonly used animal model of stroke and can well simulate the process of ischemic stroke in humans.5 Zea Longa et al. reported a method for simulating ischemic stroke by wire embolization,6 which solves the issues of difficult surgery, long modeling time, and high mortality related to open brain simulations of ischemic stroke.7 As a result, the method is generally used in the establishment of ischemic stroke models. How to avoid thrombosis and vascular injury caused by misinsertion of the filament head into the pterygoid artery (PPA) and multiple embolus insertion is a difficult task in the preparation of the model. Zea Longa et al.6 proposed that ligation of the PPA could effectively avoid inadvertent insertion of the PPA. However, it is practically severe and detrimental to their postoperative survival because the location of the PPA closer to the posterior basal segment of the brain would make it more difficult to separate the vessels, and cause damage to the rats after separation.\n\nTo solve this problem, Kutluay Uluc et al. proposed bending the filament in a natural curve, so that they would be closer to the medial side during the intravascular alignment.8 This can partially avoid insertion into the PPA, but it is still not widely used because this method requires much experience and poor stability for the experimental operator and repeated practice to master. Although many researchers have subsequently improved this method in various ways, such as surgical technique, anesthesia, filament type, MCAO occlusion filament insertion depth, and occlusion time,9–13 there are still limitations such as long modeling time, high model mortality, and unstable model success rate.\n\nIn this study, we proposed to change the angle between the common carotid artery (CCA) and the PPA by pulling the vessel to avoid the inadvertent insertion of the filament into the PPA, and then advance the head of the filament to the middle cerebral artery, the ventral thalamic artery and the anterior choroidal artery. The MCAO is achieved by blocking the blood supply in the choroidal artery segment, avoiding damage to the rats caused by ligating the PPA and reducing operation time. At the same time, it avoids the long preparation time of the model caused by multiple filament insertion because it is more repetitive and stable compared to pulling the vessel to change the direction of the filament. By assessing the neurological function score and the stability of the cerebral infarct area, this method can significantly improve the model stability and modeling success rate.\n\n\nMethods\n\nThe animals in this experiment were sacrificed according to the 2006 Guideline of the Chinese Ministry of Science and for the Care and Use of Laboratory Animals. Our study passed the ethical review requirements of Yunnan University of Traditional Chinese Medicine, ethical review no: R-062021002.\n\nSeventy-five specific pathogen free (SPF) male SD rats, 8–10 weeks old, weighing 250–280g, were provided by Hunan Slack Jingda Biological Company. The breeding and modeling of experimental animals in this study were carried out in the SPF level experimental operation room of the university [License number: SYXK (Yunnan) K2017-0005]. The use of experimental animals followed the 3R principle and all experimental protocols were approved by the Experimental Animal Welfare Ethics Committee of Yunnan University of Traditional Chinese Medicine. Before the operation, four to five animals were housed in each cage, the rats were maintained under a 12h/12h light/dark cycle and could obtain food and water without restriction. The rats were randomly divided into1 no-surgery group (n=15),2 sham group (n=15),3 Zea longa group (n=15), and4 optimal group (n=15). At the end of the experiment, the animals were sacrificed by spinal cord dislocation after an intraperitoneal injection of sodium pentobarbital. All efforts were made to ameliorate any suffering of the animals.\n\n\n\n1. MCAO bolt line purchased from Beijing Sinon Technology Co.\n\n2. TTC (2%) staining reagents were purchased from Solebro Biotechnology Co.\n\n3. Phosphate buffer solution was purchased from VivaCell.\n\n4. Sodium pentobarbital (1%) was provided by the animal experimental center of Yunnan University of Chinese medicine.\n\n5. 4% paraformaldehyde universal tissue fixative purchased from White Shark Biotechnology Co.\n\n6. Xylene and anhydrous ethanol (75%) were purchased from Tianjin Zhiyuan Chemical Reagent Co.\n\n7. Toluidine blue staining solution (1%) and glacial acetic acid were purchased from Wuhan Google Biotechnology Co.\n\n8. Neutral gum was purchased from Sinopharm Chemical Reagent Co.\n\nAfter seven days of adaptive feeding, the rats were diet fasted for six hours before surgery. Each rat was kept in a separate cage after surgery and the wound was disinfected with iodophor twice a day to prevent infection. The food was softened with cow's milk before the rats were fed.\n\nThe operation was performed under general anesthesia (2% isoflurane in 30% O2 and 70% N2O). Heating pads and rectal thermometers were used to detect and maintain the body temperature of the rats between 36.5–37.0°C. The MCAO standard procedure Zea longa approach for the surgery was as follows: After 75% ethanol skin disinfection, a 2 cm long median incision was made in the neck, and the right side was dissected CCA, external carotid artery (ECA), internal carotid artery (ICA), occipital artery (OA), superior thyroid artery (STA), and PPA. The distal ends of OA, STA, PPA, and ECA were ligated, and CCA and ICA were temporarily clamped. Insert the filament from the ECA into the CCA and finally insert the ICA to block the main MCA and ICA. The filament was pulled out after one hour and the muscle skin was sutured. The wire lifting method of this study is specifically as follows: After skin disinfection with 75% ethanol, a 2 cm long median incision was made in the neck, and the right CCA, ECA, ICA, and carotid artery are dissected. The proximal end of the common artery was ligated to block the blood supply, the ICA was temporarily clamped. In addition, pulling thread a (Figures 1 and 2) was tied around the ECA above 2–3 mm of the bifurcation of the ICA and ECA and a 45° incision was made on the CCA below 5–6 mm of the bifurcation of the ICA and ECA. The filament was placed into the ICA through the incision access. Then the ECA arteriole clamp was loosened, the pulling thread b was tied on the CCA, and the pulling thread a, b was lifted left with the index finger and middle finger (raise the CCA by about angle 20°–30° between the ICA and PPA to avoid the filament passing into the PPA). The filament was moved slowly forward in the ICA till a hindrance was felt indicating that the filament had approached the front end of the MCA. After ischemia of one hour, the filament was removed then the muscle skin was sutured, and the wound was disinfected. The filament was not inserted in the sham group, but the other operations were the same as those in the Zea longa group. The no-surgery group did not do have any intervention. Please see underlying data (Video file).14\n\nA. In the Zea longa method. B. The optimization method of this study.\n\nA. The anatomy of the rat neck vessels: common carotid artery (CCA), external carotid artery (ECA), internal carotid artery (ICA) and pterygoid artery (PPA). D and E are the threaded paths, in which the angle between CCA and PPA is changed by pulling (a and b) two threads to the left, and CCA and ICA tend to form a straight line at the same level, and the head of the thread is easily inserted into ICA within a short period of time (a is the pulling thread, b is the pulling thread, c is the fixed thread, and d is the head of the thread).\n\nAfter the rats had been awake for 24 hours, the behavior of the rats was scored according to the Zea longa 5-level 4-point neurological scoring principle. The Zea longa 5-level 4-point system scoring principles: 1. 0 points, indicates no signs of neurological abnormalities. 2. 1 point indicates unable to fully extend the upper limb on the contralateral side of the lesion. 3. 2 points, indicates the rat rotates to the opposite side while walking. 4. 3 points, indicates a fall to the opposite side while walking. 5. 4 points, indicates no spontaneous activity with reduced consciousness. A score of 1–4 points is a successful model; rats that died accidentally during the operation, death within 24 hours of reperfusion, subarachnoid hemorrhage, and 0 points were excluded. Please see underlying data (Data file 2).14\n\nThe first day after the successful model, the rats were sacrificed after spinal cord dislocation under pentobarbital sodium anesthesia to remove the brain. The brain was washed twice with normal saline, and frozen in a refrigerator at -20°C for 10 min. After removal, the brain was placed in the brain groove and cut into five pieces at intervals of 2 mm with a coronal plane slice. The first brain cut was between the optic chiasm and the anterior pole of the brain, the second cut was on the optic chiasm, the third cut was on the pituitary gland, and the fourth place was between the pituitary gland and the posterior pole of the brain. The pieces of brain were incubated in a 0.08% TTC-0.01 mol/L pH 7.26 phosphate buffer solution stored in the dark at 37 °C for 30 min, turning once every 5 min. The brain slices were washed three times with 0.08% TTC-0.01 mol/L pH 7.26 phosphate buffer solution and the solution was then discarded. Then they were put into 40 g/L paraformaldehyde solution for 24 hours. Finally, these brain slice images were captured by a Nikon D3100 camera and ImageJ v1.8.0 analysis software was used to calculate the cerebral infarction volume of the brain slice images (Cerebral infarction volume (%) = total cerebral infarction area/total brain slice area × 100%). Please see underlying data (Data file 1).14\n\nOn the first day after the successful modeling, and after the rats were sacrificed, the heart was perfused with physiological saline and then perfused with 4% paraformaldehyde 0.1 mol/L phosphate-buffered saline (PBS, pH = 7.4). Paraffin embedding took place after brain extraction. For the paraffin section, the thickness of the clean, glass slide was about 4 μm, and the paraffin section was deparaffinized with water. The sections were placed sequentially into xylene I for 20 min; xylene II for 20 min; absolute ethanol I for 5 min; absolute ethanol II for 5 min; 75% alcohol 5 min, then washed with distilled water. For the Toluidine blue staining: the tissue sections were placed in the dye solution for 5 min, washed with water, and slightly differentiated with 1% glacial acetic acid. The reaction was terminated by washing with distilled water. The degree of differentiation was controlled under a microscope (Olympus BX43, 10X). After washing with distilled water, the sections were placed in an oven (DHG Series, Shanghai Yiheng Biological Company) and dried. Transparent mounting was performed by slicing into clean xylene for 5 min and sealing with neutral gum sealer. The pathological changes of Nissl-stained tissues in the cerebral cortex and hippocampus were observed under a mounting microscope (Olympus BX43, 10X, 40X) Please see underlying data (Data file 3).14\n\nAfter the completion of the operation, the survival time and the number and weight of the rats in each group were recorded each day.\n\nThe experimental data include at least three independent and repeated experimental values, and the values are described by the mean + standard deviation. Student’s-t Test was performed in a two-group comparison, <0.05 was considered statistically significant (SPSS 22.0, SPSS Inc, Chicago, IL, USA)\n\n\nResults\n\nIt is well known that the MCAO animal model is the main method of studying cerebral stroke in human beings. In recent years, many researchers have improved the Zea Longa MCAO method in various ways9–13 since Zea Longa first performed it.5–6 But there are still some limitations such as long modeling time, high model mortality and unstable model success rate. It is urgent to find an MCAO model that can be time saving, cause less mortality and provide a more stable model success rate than before. Efficient MCAO would facilitate the research of cerebral stroke in humans and massively contribute to the field that causes one of highest morbidity and mortality rates in cardiovascular disease. In our study, the MCAO method had a significant time saving, less mortality and a stable model success rate compared to other methods by changing the angle between the CCA and the PPA by lifting the vessel left of the CCA and ECA to avoid the inadvertent insertion of the filament into the PPA.\n\nTo compare the success rate of each group, neurological function score was adopted to evaluate the function damage of the rats. According to the model success criteria, our result indicated there was no significant difference in the neurological function scores in the sham-operated group and no-surgery group. A total of 45 rats were used in the Zea longa group and our optimal group. Ten models failed and 35 succeeded in the Zea longa method group, with a success rate of 77.77% (Figure 3A); five models failed and 40 succeeded in the optimal group, with a success rate of 88.88% (Figure 3B) and the difference was statistically significant (Figure 3C).\n\n(score >1 means success). A and B. The neurological function scores of Zea longa group and Optimization group; C. The statistics of the number of successful modeling rats by different methods, and the differences are statistically significant (* indicates p <0.05).\n\nTTC staining was performed on the brain tissue of rats in each group, and the results showed that the right and left cerebral hemispheres of the sham-operated group were evenly and symmetrically stained in coronal section, with red, gray matter and light red or porcelain white matter, and no pale infarcted areas. The ischemic side of the brain tissue of the Zea longa group and the wire group showed pale white infarcted areas in the cortex and subcortex, including the lateral basal nucleus, parietal and temporal cortex, hippocampus, striatum and other areas. The infarct volume was 18.36 ± 4.93% in the Zea longa group and 23.75 ± 2.96% in the optimization group, and the difference was statistically significant and apparently smaller within-group, indicating that the success rate and stability of the method were better than those of the traditional method (Figure 4).\n\nA. The results of TTC staining of brain tissues in each group, light red is normal brain tissue, white is infarcted tissue, and the infarcted area was measured to compare the statistical differences between groups. B. shows the comparison of the percentage of infarcted volume in each group for statistical difference comparison. ** indicates p <0.0001.\n\nThe cells in the hippocampal area of the blank group and sham-operated group were arranged regularly and neatly under light microscopy. The nucleus and nucleolus were clearly visible, with purple-blue granules or block-like structures in the cytoplasm of the nictitating bodies and naturally arranged neural protrusions. In the Zea longa group and the right hippocampal region of the optimization group, the cell arrangement was disorganized: some of the neuronal cells disappeared, the cytosol of the remaining neuronal cells was wrinkled and invaginated, the nerve fibers were abnormally curved, the intracytoplasmic Nissl disappeared and was blurred, the nucleus was solidified and homogenized, and the nucleolus was translocated (Figure 5).\n\nA. Microscopic observation of morphology of hippocampal neurons for Nissl staining, the microscopic magnification of the upper and lower parts was 100x and 400x, respectively); B. The number of Nissl vesicles in hippocampal region of different subgroups were counted to compare the statistical differences. ( indicates p <0.0001).\n\nWe recorded the model preparation time of the two different methods and found that the mean modeling time of the Zea longa group was 21.07 ± 6.11 min, while the mean modeling time of the optimization group was 17.22 ± 2.26 min. The mean modeling time of the optimization group was less than that of the Zea longa group, and the modeling time was shorter, and more importantly, the modeling time of the optimization group was more consistent and repeatable. The difference was statistically significant (Figure 6). Please see underlying data (Data file 5).14\n\nCompared to the Zea longa group, the Optimization group showed a statistically significant reduction in the modeling time. (* indicates p <0.001).\n\nThe rats fasted for six hours before surgery and after surgery each rat was kept in a separate cage and the wound was disinfected with iodine twice a day to prevent infection. The rats’ food was softened with cow's milk and placed in the cage to facilitate feeding. The animals in the blank and sham-operated groups did not die during the experiment. The bodyweight of the animals in both groups increased gradually over time, but the sham-operated group showed a decrease in body weight on the first postoperative day, which was considered to be due to the physical injury to the rats due to the surgical trauma. The Zea longa group showed a significant weight loss in the first five days after surgery and a gradual increase in weight on the seventh day, while the Zea longa group showed a weight loss in the first three days after surgery and a gradual increase in weight on the fifth day. We speculate that the main reason for this trend is that the different modeling methods caused different damage to the rats. The number of survivors in both the Zea longa group and the optimization group decreased, and the number of survivors in the optimization group decreased less than that in the Zea longa group, and the difference was statistically significant (Figure 7). Please see underlying data (Data file 4 and 6).14\n\nA. The statistics of the number of surviving rats at 10 days after surgery, comparing the differences in the number of surviving rats at different time points; B. The statistics of the body weight of rats at 10 days after surgery, comparing the changes in body weight of rats at different time points. (* indicates p <0.05, indicates p <0.01, ** indicates p <0.0001).\n\n\nDiscussion\n\nMCAO is the most commonly used experimental stroke model, which can simulate the process of ischemic stroke, but limitations such as time-consumption, high mortality, and unstable neurological deficit scores have not been addressed. The commonly used methods for the preparation of MCAO models include the wire embolization method15 and the thrombus method.16 The filament method has been continuously improved to establish a stable and reproducible MCAO model and is the closest animal MCAO model to human stroke, so most MCAO models are prepared by the filament method. However, the classical method of MCAO modeling of transient Zea longa can have a mortality rate of up to 50%,17,18 which becomes a key factor limiting the study of animal models of MCAO. The long preparation time of the model, the trauma caused by the surgery itself, the different animal strains, the way the model was prepared, and the ischemia time all contribute to the high mortality rate of the model. Some groups have reported mortality rates as high as 50% in C57BL6 mice17 and 71% in Balb/C mice,19 which may be the result of cerebral vascular variability.20 Therefore, adult SD male rats were used in this study because the cerebral arteries of SD rats are very similar to human cerebral arteries3,4,21 whereas SD rats have less intraoperative hemorrhage and male SD rats may avoid the neuroprotective effect of estrogens.22–24\n\nTransient ischemia of the brain can be achieved by inserting a filament into the MCA either via the ECA or CCA,25,26 but the traditional method of preparing the Zea longa model is prone to accidental insertion of the filament into the PPA either way due to the physiological curvature and alignment of the vessel,3 which is the main difficulty in the preparation of this model. Some investigators have suggested that this problem can be avoided by ligating the proximal end of the PPA or bending the wire peg to bring it closer to the medial alignment of the vessel.8 However, the location of the PPA is closer to the base of the brain and ligating the PPA would increase the difficulty of the procedure while causing more damage to the muscles and vessels and altering the original blood supply to the rats, which is not conducive to their survival. Although ligature of the PPA can be avoided by bending the pegs more medially, its stability and reproducibility are low, which is not conducive to the preparation of the model. We used the method of pulling the blood vessel to change the alignment of the filament by pulling the a and b wires to the upper left to change the angle between CCA and PPA, so that the CCA and ICA tend to form a straight line at the same level, and the head of the wire pins can be inserted into the ICA more easily in a short time (Figure 2). This avoids ligating the ECA and PPA and can significantly shorten the modeling time and improve the modeling efficiency. At the same time, the reduced deep muscle damage in the rat’s neck is also beneficial to postoperative recovery and improved survival rate.\n\nDuring our experiments, we found that the size of the filament’s head was crucial to the success of the MCAO model, and the size and length of the bolus head should be adjusted according to the body weight of the rat.27 Some researchers compared the effect of different sizes of filament with the success rate of MCAO models, and the results showed that filaments with silicone coated heads had a higher success rate and the infarct volume was more consistent. This may be because silicone coated plugs can fully block the MCA and block blood circulation.28 The depth of the plug also affects the success rate of the model, and in our experience, a depth of 18–20 mm is appropriate for the insertion of the plug, which is just sufficient to occlude the MCA.29 Therefore, a silicone coated filament with a 2 cm marker point in the middle section (3400A Western Thickening) was used to easily determine the depth of filament insertion and improve the success rate of modeling. It is worth noting that the time of ischemia-reperfusion also plays an important role in the success of the MCAO model, which is usually 60, 90, or 120 minutes.4 However, some researchers30 have pointed out that when the blockage time is greater than 90 minutes it triggers ischemic injury in the hypothalamus leading to spontaneous hyperthermia in the MCAO model, and the increase in body temperature leads to a larger infarct volume and increased mortality.31–33 We chose 1 hr of ischemia, which can reduce mortality but obtain good infarct results. Meanwhile, a thermostatic heating pad was used to maintain the body temperature of rats at 36.5–37.0 °C during the modeling procedure, and a rectal thermometer was used to detect the body temperature, which avoided the brain infarct volume enlargement caused by high body temperature in rats.\n\nIn addition, increased pre- and post-operative care of MCAO had a positive effect on the survival rate of the rats. The high blood glucose levels exacerbate ischemic injury in model rats,34,35 so to avoid physiological postprandial blood glucose elevation, rats were fasted six hours before surgery to maintain blood glucose in the normal range. At the same time, preoperative fasting can avoid the obstruction of the esophagus by food residues after anesthesia and avoid accidental death during the preparation of the model. Routine postoperative wound disinfection and anti-infection facilitated wound healing. At the same time, because of the temporary damage to the masticatory muscles due to surgical trauma,36 softening the food with milk reduces the difficulty of free feeding in rats and ensures the nutrition required by the organism. These improvements are conducive to improving the postoperative survival rate (Figure 7). Also, the longer-term model weight changes and survival rates were not reported by other investigators and are important for studying the cycle of pharmacological intervention after MCAO modeling.\n\n\nConclusions\n\nIn conclusion, this study proposes that by pulling the vessel to change the course of the thread embolus optimizes the long preparation time and high mortality rate of the traditional Zea longa modeling method. This makes the preparation of the MCAO model easier, it is time saving, there is a lower mortality rate, a higher successful modeling rate and better repeatability.", "appendix": "Data availability\n\nHarvard Dataverse: Underlying data for ‘Pulling wire to avoid misinsertion of filament and vascular damage and improve ischemic stroke models' success rate and reproducibility’. https://doi.org/10.7910/DVN/6PHB1D. 14\n\nThis project contains the following underlying data:\n\n• Data file 1: Infarct volume.xlsx\n\n• Data file 2: Neurological function score.xlsx\n\n• Data file 3: Nissl data.xlsx\n\n• Data file 4: Numbers.xlsx\n\n• Data file 5: Operation time.xlsx\n\n• Data file 6: Time and weight.xlsx\n\n• Video file: Operation.mp4\n\n• Figure 1: Schematic diagram of the main arteries of the head and neck of the rat during the modelling of the different groups middle cerebral artery occlusion (MCAO) and the position of the filament.\n\n• Figure 2. Schematic diagrams of the embolization paths of different methods.\n\n• Figure 3. Neurological function scores and success rates of rats in different groups.\n\n• Figure 4. Results of the volume and morphological changes of brain infarcts in rats of different groups (MCAO).\n\n• Figure 5a. Nissl staining in hippocampal region of rats in different subgroups (MCAO).\n\n• Figure 5b. Nissl staining in hippocampal region of rats in different subgroups (MCAO).\n\n• Figure 6. Modeling time of different methods.\n\n• Figure 7. The number of surviving rats and body weight changes after surgery.\n\nHarvard Dataverse: ARRIVE checklist for ‘Pulling wire to avoid misinsertion of filament and vascular damage and improve ischemic stroke models' success rate and reproducibility’. https://doi.org/10.7910/DVN/6PHB1D. 14\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\nHoward VJ, Kleindorfer DO, Judd SE, et al.: Disparities in stroke incidence contributing to disparities in stroke mortality. Ann. Neurol. 2011; 69(4): 619–627. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu S, Cui B, Mlynash M, et al.: Stroke epidemiology and stroke policies in China from 1980 to 2017: A systematic review and meta-analysis. Int. J. Stroke. 2020; 15(1): 18–28. 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PubMed Abstract \| Publisher Full Text\n\nLee R: Morphology of cerebral arteries. Pharmacol. Ther. 1995; 66(1): 149–173. Publisher Full Text\n\nBrann DW, Dhandapani K, Wakade C, et al.: Neurotrophic and neuroprotective actions of estrogen: basic mechanisms and clinical implications. Steroids. 2007; 72(5): 381–405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu R, Yang SH: Window of opportunity: estrogen as a treatment for ischemic stroke. Brain Res. 2013; 1514: 83–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInagaki T, Etgen AM: Neuroprotective action of acute estrogens: animal models of brain ischemia and clinical implications. Steroids. 2013; 78(6): 597–606. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShvedova M, Islam MR, Armoundas AA, et al.: Modified middle cerebral artery occlusion model provides detailed intraoperative cerebral blood flow registration and improves neurobehavioral evaluation. J. Neurosci. Methods. 2021; 358: 109179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrotman-Lucas M, Kelly ME, Janus J, et al.: Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice. J. Vis. Exp. 2019; 143.\n\nKuge Y, Minematsu K, Yamaguchi T, et al.: Nylon monofilament for intraluminal middle cerebral artery occlusion in rats. Stroke. 1995; 26(9): 1655–1658. discussion 8. PubMed Abstract \| Publisher Full Text\n\nShimamura N, Matchett G, Tsubokawa T, et al.: Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model. J. Neurosci. Methods. 2006; 156(1-2): 161–165. PubMed Abstract \| Publisher Full Text\n\nTang Q, Han R, Xiao H, et al.: Role of suture diameter and vessel insertion position in the establishment of the middle cerebral artery occlusion rat model. Exp. Ther. Med. 2013; 5(6): 1603–1608. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi F, Omae T, Fisher M: Spontaneous hyperthermia and its mechanism in the intraluminal suture middle cerebral artery occlusion model of rats. Stroke. 1999; 30(11): 2464–2471. discussion 70-1. PubMed Abstract \| Publisher Full Text\n\nKuluz J, Gregory G, Han Y, et al.: Fructose-1,6-bisphosphate reduces infarct volume after reversible middle cerebral artery occlusion in rats. Stroke. 1993; 24(10): 1576–1583. PubMed Abstract \| Publisher Full Text\n\nChen H, Chopp M, Welch K: Effect of mild hyperthermia on the ischemic infarct volume after middle cerebral artery occlusion in the rat. Neurology. 1991; 41(7): 1133–1135. PubMed Abstract \| Publisher Full Text\n\nMiyazawa T, Tamura A, Fukui S, et al.: Effect of mild hypothermia on focal cerebral ischemia. Review of experimental studies. Neurol. Res. 2003; 25(5): 457–464. PubMed Abstract \| Publisher Full Text\n\nKoh PO: Hyperglycemia decreases preoxiredoxin-2 expression in a middle cerebral artery occlusion model. Lab Anim Res. 2017; 33(2): 98–104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang SS, Lu YJ, Huang JP, et al.: The essential role of endothelial nitric oxide synthase activation in insulin-mediated neuroprotection against ischemic stroke in diabetes. J. Vasc. Surg. 2014; 59(2): 483–491. PubMed Abstract \| Publisher Full Text\n\nDittmar M, Vatankhah B, Fehm N, et al.: The role of ECA transection in the development of masticatory lesions in the MCAO filament model. Exp. Neurol. 2005; 195(2): 372–378. PubMed Abstract \| Publisher Full Text" }	[ { "id": "169934", "date": "03 May 2023", "name": "Claire L. Gibson", "expertise": [ "Reviewer Expertise Cerebral ischemic stroke" ], "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 refer to their specific aims (of this surgical approach) to address the issues of modelling time, high model mortality and a stable success rate. The main issue is that the results presented don't really relate then to these characteristics so the aims and validity of the study are unclear.\nIn vivo experiments are not described in accordance with ARRIVE guidelines. I cannot find information in the manuscript text that relates to the following points on the ARRIVE guidelines. Although the authors have stated on the checklist where this information is available in the particular section of the manuscript the information as currently presented does not fully match to the criteria. Comments below;\nNo justification of animal numbers (i.e. group sizes) is given. Explain how the sample size was decided. Provide details of any a priori sample size calculation, if done. – I cannot find information on sample size calculation.\n\nDescribe any criteria used for including and excluding animals (or experimental units) during the experiment, and data points during the analysis. Specify if these criteria were established a priori. If no criteria were set, state this explicitly. – I cannot find information on exclusion and inclusion criteria.\n\nFor each experimental group, report any animals, experimental units or data points not included in the analysis and explain why. If there were no exclusions, state so. – I cannot find this information.\n\nFor each analysis, report the exact value of n in each experimental group. – the total number of animals per treatment group is stated not the number per analysis.\n\nState whether randomisation was used to allocate experimental units to control and treatment groups. If done, provide the method used to generate the randomisation sequence. – manuscript states that animals were randomly allocated (doesn’t state how).\nAlso the group comparisons aren't valid - why is a non-surgical group included? The sham group included are only shams for the Zea Longa method and not the new approach the authors are proposing. A better design would have been to have a sham group for each of the surgical models being used. The number of animals per outcome measure (e.g. histology, TTC etc) is not stated.\nI'm not convinced that a 5-point neurological score is a true indicator of model success or not - this needs further justification why this scoring determines if the model was successful - surely a better measure would have been to record cerebral blood flow during surgeries and record whether a successful reduction in blood flow was obtained or not. The neurological score used here simply reports whether animals present with neurological dysfunction as indicated by the score - there will be variability in this between animals that doesn't necessarily reflect whether a successful occlusion occurred or not.\nFigure 4 - please present all TTC slices in the same orientation. What does 4**** mean? Typically significant differences are indicated by 1, 2 or 3 asterisks. The interpretation of Figure 4 is unclear - the data shows that the lesion area (%) was larger in the optimization group compared to the Zea Longa group. What is meant by the authors stated that the difference was 'apparently smaller within-group' indicating that the success rate and stability of the method were better. Do the authors mean the level of variability within each surgical group - that can't be assessed by how the data are presented and analysed here. Would be better to show lesion area as individual plots on graph.\nFigure 5 - unclear what axis on graph is referring to. Total counts are inappropriate but should be some measure of cell count per defined area.\nFigure 6 - the authors refer to this as showing that the modelling time (of the optimization group) was more consistent and repeatable - again, the authors are indicating that the optimization group has less variability within but the data as presented and analysed here don't allow for that interpretation.\n'Caused less hurt' is a non scientific interpretation. Need to refer specifically to indicators of health and wellbeing.\nFigure 7 - typically body weight is expressed as % of pre-surgical weight to take into account any individual animal differences at the time of surgery.\nIt is unclear why animals were fasted for 6h prior to surgery - this is not standard.\nIn terms of time taken to complete model - this probably varies a lot between research groups too - would be useful to set in context with others.\nSome recent publications aimed at improving validity and reducing variability of the MCAO model are not cited here - for example see the IMPROVE guidelines (JCBFM; 2017)1.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "265888", "date": "09 May 2024", "name": "Huaxin Sheng", "expertise": [ "Reviewer Expertise Ischemic stroke" ], "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 filament middle cerebral artery occlusion (MCAO) model is performed in two methods: 1) the filament is introduced into the internal carotid artery through the incision made on the external carotid artery (ECA). It is called the Longa method. 2) the filament is inserted into the internal carotid artery through the incision made on the common carotid artery (CCA). It is called the Koizumi method. This manuscript was to modify the procedure of the Koizumi, and it should be compared with the other studies performed using the Koizumi method to show the advantages of their procedure. However, they compared it to the Longa method.\n\nThe critical part is that most researchers use the Longa method for making MCAO in animals because Koizumi's method destroys the common carotid artery, which reduces cerebral blood flow by almost 50-60% and does not allow the brain to have good reperfusion after stroke. Thus, the model they modified is not commonly used in stroke research and is not a clinically relevant model.\nIn addition, they misunderstood the Longa method. It is not a long surgical procedure with high mortality.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No", "responses": [] } ]	1	https://f1000research.com/articles/12-408
https://f1000research.com/articles/11-1231/v1	31 Oct 22	{ "type": "Research Article", "title": "Evaluation of clinical efficacy of Ginkgo biloba extract in the treatment of knee osteoarthritis: a randomized clinical trial", "authors": [ "Maryam Al-Haddad", "Ahsan Bairam", "Haider Hussain", "Suhad Majeed", "Ahmad Al-Jalehawi", "Ahsan Bairam", "Haider Hussain", "Suhad Majeed", "Ahmad Al-Jalehawi" ], "abstract": "Background: Osteoarthritis (OA) is a degenerative joint disease characterized by deteriorating articular cartilage. Most patients may not get enough control of symptoms in spite of the availability of various treatment options. Because of the synergistic impact of flavonoids and ginkgolides, Ginkgo biloba (GB) has been shown to possess antioxidant and anti-inflammatory properties besides its generally safe profile. This study aims to assess the efficacy of the Ginkgo biloba extract (GBE) in patient with Knee OA.\nMethods: This is a randomized double blinded clinical trial conducted in a private orthopaedic clinic in Al-Najaf Government-Iraq. Patients were randomized into two groups; Group A administered the standard treatment (diclofenac capsule 100 milligram sustained released with paracetamol 1 gram twice daily) with GBE 120 milligram twice daily, while Group B administered the standard treatment with placebo (starch) capsule only. The Knee injury and Osteoarthritis Outcome Score (KOOS) was performed to assess the efficacy of the GBE in patient with Knee OA for eight weeks. Results: 60 patients successfully completed the eight weeks follow up. Pain, symptoms, sport, activity of daily living (ADL) scores showed significant improvement in Group A from the first two weeks after treatment with GB in comparison with group B. however, scores of quality of life (QOL) were not significantly improved in two group comparison at each assessment week. Only 10 patients reported gastrointestinal adverse effects during the study which were all mild. Conclusions: GB may provide additional beneficial health effects to patients with knee OA based on KOOS scores when add to the standard treatment regime. Registration: ClincialTrials.gov (NCT05398874, https://clinicaltrials.gov/ct2/show/NCT05398874).", "keywords": [ "Ginkgo Biloba", "KOOS", "Knee osteoarthritis" ], "content": "Abbreviations\n\nADL: Activities of daily living\n\nEGb-761: standardized Ginkgo biloba extract\n\nGB: Ginkgo biloba\n\nGBE: Ginkgo biloba extract\n\nKOOS: Knee injury and Osteoarthritis Outcome Score\n\nOA: osteoarthritis\n\nQOL: quality of life\n\n\nIntroduction\n\nOsteoarthritis (OA) is a degenerative joint disease characterized by deteriorating articular cartilage. Since the knees and hips are considered weight-bearing joints, osteoarthritis is widely spread in these locations.1 OA-related symptoms such as swelling, discomfort, and function loss are often primary reasons to visit a specialist.2,3 Risk factors for OA include aging, female gender, genetic susceptibility, and nutritional status. The prevalence of knee osteoarthritis is around 16.0% in patients aged 15 years and older and 22.9% in those aged 40 years and older.4 85% of people older than 75 years old were found to exhibit some symptoms of OA.5 Regarding gender variations, females display greater incidence of knee osteoarthritis than in males.4 In addition, obesity was reported to increase the load-bearing tension on the hip and knee joints. In fact, the risk of OA increases by 10% for every kilogram over the ideal weight.6 Painkillers are the most effective treatment available for patients with OA However, the long-term use of these compounds can impose toxic effects on the digestive7 and cardiovascular systems.8 An estimated 16,500 Americans with rheumatoid or osteoarthritis die each year from using non-steroidal anti-inflammatory drugs (NSAIDs). If the effects of NSAIDs on the gastro-intestinal tract were counted separately, they would be the 15th leading cause of death in the United State (US).9 At equipotent doses, the efficacy of various NSAIDs is similar, but there is clear individual diversity in the therapeutic responses to these medications. Some people may respond better to one treatment than other10 Dietary supplements were also considered, including glucosamine, chondroitin, vitamin D, and fish oil. At two years follow-up, high-dose fish oil showed a higher improvement in WOMAC score than vitamin D, but the other supplements showed no significant changes.11 Traditional Chinese medicine has been using the ginkgo tree for a long time. It is one of the oldest living tree species in the world.12 Standardized preparations (EGb-761) have 24% ginkgo flavonoid glycosides, 6% terpene lactones, and less than 5 parts per million ginkgolic acids.13 GBE is popular in Europe and the United States. In clinical research, 120-240 mg doses of GB demonstrated neuroprotective characteristics and other beneficial circulatory effects in elderly people, including cerebral insufficiency and cognitive consequences and peripheral circulatory impairment, particularly intermittent claudication, vertigo, and tinnitus.14 Due to the synergistic impact of flavonoids and ginkgolides, GB has been shown to possess strong antioxidant and anti-inflammatory properties.15,16 Oedema was significantly reduced with GB in animal trials; however, a greater reduction in oedema was seen when GB is combined with the NSAIDs like indomethacin, rofecoxib, and celecoxib, than with NSAIDs alone.17 Ho et al. study revealed that the cartilage was collected from OA patients who undergo total knee or total hip joint replacement surgery and prepare chondrocyte from it. Results indicated that EGb-761 displayed a dose-dependent inhibition of interleukin-1-induced Nitric Oxide production. The same study concluded that the phosphorylated types of c-Jun N-terminal kinases (JNK) were efficiently decreased by treatment with EGb-76.7 EGb-761 could also effectively reverse the chondrocyte/cartilage damage in an OA rat model.18 It is highly suggested to include non-drug strategies in the treatment of OA to prevent or reduce disease progression.19 Some patients may not get enough control of symptom despite the availability of various treatment options, while others will experience harmful consequences from the available therapeutic interventions20 and long-term use of NSAIDs. Significantly, non-specific medicines accelerate OA structural progression21 by increasing knee joint load after reducing the pain.22 Based on the above, this study aims to assess the efficacy and safety of the Ginkgo biloba extract in patient with knee OA.\n\n\nMethods\n\nThis study is a part of a Master’s degree thesis. The study was registered in and approved from the Medical Ethics Committee in the University of Kufa, college of medicine with reference: (MEC_13). The study protocol was registered in ClincialTrials.gov (identifier: NCT05398874, 1st June 2022). Trial registration to ClinicalTrials.gov took place after the trial was completed due to a logistics issue in contacting the authorized personnel.\n\nParticipants were asked to sign a printed informed consent that explained the aim of study.\n\nThis was a randomized double blinded clinical trial conducted in a private orthopaedic clinic in Al-Najaf Government in Iraq from November 1st 2021 to June 1st 2022. The doctor of clinic defined the patients that can be enrolled in the study and informed them about aim of study and take oral and written consent from them. Then the researcher randomized patients into two groups using simple randomization (Block size 4 (2A, 2B), allocation:1:1). The doctor and patients did not know in which group they were categorized. Group A received EGb-761 120mg twice daily while group B received placebo (starch) capsule. Patients in both groups administered their standard therapy of paracetamol tablet 1 gram twice daily plus diclofenac sustained release capsule 100 milligrams once daily. The treatment continued for eight weeks as demonstrated by the CONSORT flow diagram in Figure 1.\n\nOut of 145 patients assessed for their eligibility, 103 patients were selected to be enrolled in the study. However, 65 patients only (35 group A, 30 group B) completed four weeks of treatment, while 60 patients (33 patients in group A and 27 patients in group B) completed eight weeks of treatment. In each visit, patients were asked to mention any side effects that may be related to GB.\n\nPatients included in this study were diagnosed with grade 2 or grade 3 Knee OA according to Kellgren and Lawrence (K&L) classification system23 and they were aged between 38-75 years old. The exclusion criteria were patients with an allergy or contraindication to NSAIDs or GB or paracetamol, those with renal or hepatic problem, pregnancy or lactation and those who have any cardiovascular or neurological diseases.\n\nClinical assessment was done before starting treatment, after two weeks, four weeks, and eight weeks by KOOS questionnaire. KOOS includes 42 items divided into 5 subscales. These are pain, symptoms, ADL, sport and recreational activity, and finally QOL associated with knee activity.24\n\nDescriptive statistics are presented in the form of numbers and percentages for the categorical variables. Means and standard deviations are reported for numerical variables. Mixed ANOVA method was used to study the effect of time on the two groups (Group A and group B).\n\nIBM SPSS 28 for windows software was used in the analysis, and a P-value < 0.05 is considered as statistically significant.\n\n\nResults\n\nA total of 60 patients, groups A and B were 33 and 27 respectively, completed the eight weeks follow up. Females were 46 patients of the total participant while males were 14 males. The mean ages of patients in groups A and B were 54.2 and 58.0 years, respectively. All the adverse effects occurred during the study period were recorded (Table 1).\n\nThe mean KOOS score for each of the five subscales at different follow up periods as well as the main effects for the time and groups are shown in Table 2.\n\n* Refers to significant p-value.\n\nTable 2 demonstrated that there are several significant differences among different times of treatment, between groups (Group A vs B) and interaction between time and groups. Within the same group, indeed, the pain scores in group A (53.03±16.37, 55.76±16.67 and 57.94±15.16) have significantly increased at all follow up weeks two, four and eight, respectively, when compared with the baseline (38.52±13.36). In a different manner, the pain score in group B (46.74±14.51) has only increased significantly after eight weeks of treatment. As shown in Figure 2, significantly higher pain scores were observed in group A in comparison to that in group B at weeks two, four and eight of treatment with GB.\n\nA refers to group A, patients administered standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus GB.\n\nB refers to group B, patients administered standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus placebo.\n\nwk refers to week;\n\n* refers to p=0.007;\n\n# refers to p=0.003;\n\n¥ refers to p=0.005.\n\nData listed in Table 2 further showed important differences within different times, between groups and interaction between time and groups. Within group A, symptoms scores (68.00±16.33, 71.48±15.73 and 73.61±16.84) recorded at weeks two, four, and eight of treatment with GB, respectively, were all significantly higher than that at the baseline score (54.39±16.55). Within group B, the symptoms scores (58.81±17.34, 58.78±17.23 and 58.89±19.00) at weeks two, four and eight of treatment with GB, respectively, were also significantly higher than that at the baseline time (49.04±13.08). Comparison between groups (A and B) has been clearly demonstrated in Figure 3 which revealed a statistically significant improvement of symptoms scores in group A versus that in group B after two, four and eight weeks of treatment with GB.\n\nA refers to group A, patients administered standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus GB.\n\nB refers to group B, patients administered standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus placebo.\n\nwk refers to week;\n\n* refers to p=0.039;\n\n# refers to p=0.004;\n\n¥ refers to p=0.002.\n\nAs listed in Table 2, there are significant differences within different times, between groups and interaction between time and groups. ADL score in group A has dramatically increased at weeks two, four, and eight (52.06±13.12, 58.15±14.95 and 59.64±13.46) when compared with baseline score (40.36±12.15). While in group B, a significant increase in ADL score has recorded after 8 weeks of treatment with GB (46.74±12.57) in comparison with the baseline score (37.96±13.01). However, no significant differences were found after two and four weeks of treatment. Moreover, Figure 4 displayed that ADL score of group A were significantly higher than group B at all times of follow up (two, four, and eight weeks).\n\nA refers to group A, patients administered standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus GB.\n\nB refers to group B, patients administered standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus placebo.\n\nwk refers to week;\n\n* refers to p<0.001;\n\n# refers to p<0.001;\n\n¥ refers to p<0.001.\n\nAs noted in Table 2, there was significant elevation in sport scores of group A (35.30±11.92, 39.09±14.33 and 39.70±16.63) at all times of follow up, weeks two, four and eight, respectively, when compared with week 0 (21.36±10.99). On the other hand, group B showed significant improvement only at weeks two and eight (26.11±12.04 and 27.59±13.82). Intergroups comparison (group A versus B) was demonstrated in Figure 5, which showed that sport score in group A was significantly higher than group B at all times of follow up.\n\nA refers to group A, patients administered standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus GB.\n\nB refers to group B, patients administered standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus placebo.\n\nwk refers to week;\n\n* refers to p=0.004;\n\n# refers to p<0.001;\n\n¥ refers to p=0.004.\n\nData listed in Table 2 showed that QOL scores were improved significantly in group A after weeks four and eight (39.82±13.86 and 43.61±15.37, respectively) of treatment with GB as compared with the baseline score. In group B, QOL scores at weeks two, four and eight (31.30±10.90, 32.85±12.88 and 39.07±13.59) were significant higher in comparison with the baseline score (24.85±8.38). Notably in Figure 6, it was found that although QOL scores in group A were higher than that in group B, these data were statistically not significant at weeks two, four and eight of treatment with GB.\n\nA refers to group A, patients administer standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus GB.\n\nB refers to group B, patients administer standard treatment (diclofenac 100 mg sustained released and paracetamol 1 g twice daily) plus placebo.\n\nwk refers to week.\n\nTen patients experienced adverse effects during this clinical trial as listed in Table 1: eight patients in group A and two in group B. All of the recorded effects were related to the digestive system. In detail, nine patients reported gastrointestinal upset and one had constipation. All these effects were mild and tolerable, and no patient discontinued the trial due to these mild effects.\n\n\nDiscussion\n\nThis study aims to assess the efficacy of the GBE in patient with Knee OA based on KOOS. The KOOS is a knee-specific tool designed to evaluate patients' perceptions of their knee and related issues.25 The most obvious symptom of osteoarthritis is pain.26 KOOS evaluates the pain intensity through questioning the different daily activities in the last weeks from the visit. According to the results, the pain score in patients administered GB was improved more quickly, after two weeks of treatment with 240 mg/day GB, than those patients in the second group (group B) who administered the conventional therapy with placebo. These findings were in line with other similar previous studies. Clinical research conducted by Al-Rekabi in 2014 reported that twice-daily Glucosamine/Ginkgo biloba (500/50 mg) administration improved all scores of KOOS components significantly when compared with Glucosamine/Chondroitin (500/400 mg).27 GB was shown to block the enzymes cyclooxygenase-2 (COX-2) and 5-lipoxygenase, which are responsible for the conversion of arachidonic acid to prostaglandins (PGs) and leukotrienes, respectively.16,28 Since diclofenac was given orally to both groups in this study, patients in both groups reported improvement in symptoms such as morning stiffness and knee swelling. The KOOS symptom score was determined by asking the patient series of questions regarding experiencing swelling, clicking, or restriction in their ability to bend or straighten their knee. In spite of the fact that there was clear evidence of improvement in both groups, the ginkgo group showed significant difference throughout the course of the study period. This may be related to the analgesic and anti-inflammatory activities of NSAIDs which are typically obtained within weeks.29 Paracetamol may not be as effective as NSAIDs in individuals with pain and inflammation who are experiencing symptoms.30 NSAIDs are known for their anti-inflammatory impact through various mechanisms, including the COX enzyme, interleukins, chemokines, and others.31 When given orally, EGb-761 reduced thermal hyperalgesia and showed dose-dependent efficacy comparable to or greater than diclofenac in rat model of pain. Experimental results showed that the antihyperalgesic effect of GB can effectively alleviate the inflammatory pain associated with acute injury.32 It is well recognized that persons with knee OA may have difficulty to participate in physical activities due to knee pain.33 Limitations in walking, stair climbing, and squatting are common patient complaints that greatly interfere with activities of daily living and recreation22 which were improved significantly in group A of this study.\n\nInflammation affects central and peripheral nociceptive input, and it increases receptor sensitivity to nociceptive input by increasing prostaglandins and inflammatory cytokines.26 In a two-month follow-up study of 80 patients with knee OA, the combination of GB with celecoxib was associated with a higher decrease in serum malondialdehyde (MDA), matrix metalloproteinase (MMP)-1 concentration, erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) levels. Additionally, this combination resulted in a very significant increase in total antioxidant status (TAS) when compared to celecoxib alone.34\n\nThe failure of nonsurgical therapies to modify health related QOL in knee OA patients is not surprising given their inability to alleviate physical manifestations of OA.22 Individuals with osteoarthritis have a low perception of their quality of life in functional capacity, functional limitation and pain.35 During the study period of this study, QOL improved with time with but with no significant differences between study groups. Furthermore, coexisting disorders, altered nociceptive sensitivity, and psychosocial factors have been demonstrated to affect the clinical presentation and treatment response of patients.36\n\nFinally, no patient in this study reported serious adverse effects, and all the recorded adverse effects were related to the gastrointestinal system. Similarly, these records were in consistent with that reported by a previous meta-analysis study.37\n\nPatients recruited in limited number from one center may not reflect the effect on the general population. Moreover, the effects of ginkgo biloba cannot be predicted for long-term use because the trial lasted only eight weeks. Limited financial resource is also a barrier to involve more patients from different settings.\n\n\nConclusions\n\nStandard extraction of Ginkgo biloba improved the KOOS in term of pain, symptoms, sport, ADL, and QOL in patients with knee OA significantly when add to the standard treatment along the study period. In contrast to the other scores, only QOL showed no significant difference between group A and B.\n\n\nData availability\n\nFigshare: Evaluation of clinical efficacy of Ginkgo biloba extract in the treatment of knee osteoarthritis: a randomized clinical trial, https://doi.org/10.6084/m9.figshare.21259335.v2.38\n\nFigshare: Protocol for ‘Evaluation of clinical efficacy of Ginkgo biloba extract in the treatment of knee osteoarthritis: a randomized clinical trial’, https://doi.org/10.6084/m9.figshare.21259335.v2.38\n\nFigshare: CONSORT checklist for ‘Evaluation of clinical efficacy of Ginkgo biloba extract in the treatment of knee osteoarthritis: a randomized clinical trial’, https://doi.org/10.6084/m9.figshare.21259335.v2.38\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nMabey T, Honsawek S: Cytokines as biochemical markers for knee osteoarthritis. World J. Orthop. 2015 [cited 2021 Dec 24]; 6: 95–105. PubMed Abstract \| Publisher Full Text\n\nSowers MF, Karvonen-Gutierrez CA, Jacobson JA, et al.: Associations of Anatomical Measures from MRI with Radiographically Defined Knee Osteoarthritis Score, Pain, and Physical Functioning. J. Bone Joint Surg. Am. 2011 Feb 2 [cited 2021 Dec 10]; 93(3): 241–251. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamuel AJ, Kanimozhi D: Outcome measures used in patient with knee osteoarthritis: With special importance on functional outcome measures. Int. J. Health Sci. 2019; 13(1): 52–60. PubMed Abstract\n\nCui A, Li H, Wang D, et al.: EClinicalMedicine Global, regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies. EClinicalMedicine. 2020; 29-30: 100587. PubMed Abstract \| Publisher Full Text\n\nStevermer CA: Functional movement assessment for individuals with knee osteoarthritis. Iowa:Iowa State University;2010.\n\nChisholm MA, Schwinghammer TL, Malone PM, et al.: Pharmacotherapy Principles & Practice. 5th ed.McGraw-Hill Educaion;2019; 903.\n\nHo LJ, Hung LF, Liu FC, et al.: Ginkgo biloba Extract Individually Inhibits JNK Activation and Induces c-Jun Degradation in Human Chondrocytes: Potential Therapeutics for Osteoarthritis. PLoS One. 2013 Dec; 8(12): e82033. PubMed Abstract \| Publisher Full Text\n\nMcGettigan P, Henry D: Cardiovascular risk and inhibition of cyclooxygenase: a systematic review of the observational studies of selective and nonselective inhibitors of cyclooxygenase 2. JAMA. 2006 Oct 4; 296(13): 1633–1644. Publisher Full Text\n\nWolfe MM, Lichtenstein DR, Singh G: Gastrointestinal Toxicity of Nonsteroidal Antiinflammatory Drugs.1999 Jun 17. Publisher Full Text\n\nSolomon DH: NSAIDs: Therapeutic use and variability of response in adults - UpToDate.2022. [cited 2022 Aug 7].Reference Source\n\nReston J, Fontanarosa J, Giradi G, et al.: Osteoarthritis – Evidence Report Prepared for: American College of Rheumatology.2019; 1–410.\n\nGinkgo\|NCCIH:[cited 2022 Aug 7].Reference Source\n\nEMA: Assessment report on Ginkgo biloba L., folium. European Medicines Agency;2015.\n\nGinkgo Biloba leaf extract - American Botanical Council:[cited 2022 Aug 7].Reference Source\n\nChuen CP, Xia Q, Fu PP: Ginkgo Biloba Leave Extract: Biological, Medicinal, and Toxicological Effects. Journal of Environmental Science and Health Part C. 2007; 25: 211–244. PubMed Abstract \| Publisher Full Text\n\nDe Souza GA , De Marqui SV , Matias JN, et al.: Effects of Ginkgo biloba on Diseases Related to Oxidative Stress. Planta Med. 2020; 86(06): 376–386. Publisher Full Text\n\nAbdel-Salam OME, Baiuomy AR, El-batran S, et al.: Evaluation of the anti-inflammatory, anti-nociceptive and gastric effects of Ginkgo biloba in the rat. Pharmacol. Res. 2004; 49(2): 133–142. PubMed Abstract \| Publisher Full Text\n\nChen YJ, Tsai KS, Chiu CY, et al.: EGb761 inhibits inflammatory responses in human chondrocytes and shows chondroprotection in osteoarthritic rat knee. J. Orthop. Res. 2013; 31(7): 1032–1038. PubMed Abstract \| Publisher Full Text\n\nYu SP, Hunter DJ: Managing osteoarthritis. Aust. Prescr. 2015 Aug 1; 38(4): 115–119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKolasinski SL, Neogi T, Hochberg MC, et al.: 2019 American College of Rheumatology/Arthritis Foundation Guideline for the Management of Osteoarthritis of the Hand, Hip, and Knee. Arthritis Rheum. 2020; 72(2): 220–233. PubMed Abstract \| Publisher Full Text\n\nSimic M, Harmer A, van der Esch M , et al.: Do non-steroidal anti-inflammatory drugs cause osteoarthritis progression, a systematic review and meta analysis. Osteoarthr. Cartil. 2019 Apr 1; 27: S280–S282. Publisher Full Text\n\nFarr J II, Miller LE, Block JE: Quality of Life in Patients with Knee Osteoarthritis: A Commentary on Nonsurgical and Surgical Treatments. Open Orthop. J. 2013 Nov 26; 7(1): 619–623. PubMed Abstract \| Publisher Full Text\n\nKellgren JH, Lawrence JS: RADIOLOGICAL ASSESSMENT OF OSTEO-ARTHROSIS. Ann. Rheum. Dis. 1957; 16(4): 494–502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCollins NJ, Misra D, Felson D, et al.: Measures of Knee Function. Arthritis Care Res. 2011; 63(November): S208–S228. Publisher Full Text\n\nRoos EM, Lohmander LS; The Knee injury and Osteoarthritis Outcome Score (KOOS): from joint injury to osteoarthritis Pilot study.2003; 8: 1–8.\n\nFelson DT: The sources of pain in knee osteoarthritis: Editorial review. Curr. Opin. Rheumatol. 2005; 17(5): 624–628. Publisher Full Text\n\nAl-Rekabi M: Comparative Study Between The Clinical Effects of Glucosamine /Gingko Biloba & Glucosamine/Chondroitin in Treatment of Knee Osteoarthritis. kufa Journal for Nursing sciences. 2014; 4(2): 167–174.\n\nSon JK, Son MJ, Lee E, et al.: Ginkgetin, a biflavone from Ginko biloba leaves, inhibits cyclooxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells. Biol. Pharm. Bull. 2005; 28(12): 2181–2184. PubMed Abstract \| Publisher Full Text\n\nJoint Formulary Committee: British national formulary 83. London:BMJ Publishing and the Royal Pharmaceutical Society;2022.\n\nFendrick AM, Greenberg BP: A review of the benefits and risks of nonsteroidal anti-inflammatory drugs in the management of mild-to-moderate osteoarthritis. Osteopathic Medicine and Primary Care. 2009 Jan [cited 2022 Aug 21]; 3(1). PubMed Abstract \| Publisher Full Text Reference Source\n\nGunaydin C, Bilge SS: Effects of nonsteroidal anti-inflammatory drugs at the molecular level. Eur. J. Med. 2018; 50. : 116–121. AVES İbrahim KARA.\n\nBiddlestone L, Corbett AD, Dolan S: Oral administration of Ginkgo biloba extract, EGb-761 inhibits thermal hyperalgesia in rodent models of inflammatory and post-surgical pain. Br. J. Pharmacol. 2007; 151: 285–291.\n\nFukutani N, Iijima H, Aoyama T, et al.: Knee pain during activities of daily living and its relationship with physical activity in patients with early and severe knee osteoarthritis. Clin. Rheumatol. 2016; 35: 2307–2316. PubMed Abstract \| Publisher Full Text\n\nIsmael IK, Othman SH: Effects Of Ginkgo biloba Versus Celecoxib On The Levels Of Oxidative Stress And Matrix Metalloproteinase-1 In Patients With Knee Osteoarthritis. Tikrit J. Pharm. Sci. 2013; 9(1): 43–53.\n\nKawano MM, Araújo ILA, Castro MC, et al.: Assessment of quality of life in patients with knee osteoarthritis. Acta Ortopedica Brasileira. 2015; 23(6): 307–310. PubMed Abstract \| Publisher Full Text\n\nMills K, Hübscher M, O’Leary H, et al.: Current concepts in joint pain in knee osteoarthritis. Schmerz. 2019; 33: 22–29. PubMed Abstract \| Publisher Full Text\n\nHashiguchi M, Ohta Y, Shimizu M, et al.: Meta-analysis of the efficacy and safety of Ginkgo biloba extract for the treatment of dementia. J. Pharm. Health Care Sci. 2015 Apr 10 [cited 2022 Aug 11]; 1(1): 12–14. PubMed Abstract \| Publisher Full Text\n\nAl-Haddad M: data.xlsx. Figshare. [Dataset].2022. Publisher Full Text" }	[ { "id": "154848", "date": "02 Dec 2022", "name": "Ali Azeez Al-Jumaili", "expertise": [ "Reviewer Expertise Pharmacy Practice and Clinical Pharmacy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEvaluation of clinical efficacy of Ginkgo biloba extract in the treatment of knee osteoarthritis: a randomized clinical trial\nThank you for inviting me to review this manuscript. I invite authors to resubmit your manuscript after addressing all comments. Please carefully consider all issues I mentioned in a file including the authors’ responses to reviewer comments, and outline every change made point by point on the manuscript using track changes. Thanks\nAbstract\nMethod: Date and duration of the study are required here. Was GBE was given orally? As capsule?\nResults: Please add the mean age and gender of the participants.\n“Only 10 patients reported gastrointestinal adverse effects during the study which were all mild”. Please clarify from which group those 10 patients with GIT adverse effects?\nKeywords: add clinical trial and Iraq to the keywords\nMain Introduction\nPlease be consistent in the study objective between the abstract and main introduction section.\n“Based on the above, this study aims to assess the efficacy and safety of the Ginkgo biloba extract in patient with knee OA”. In the abstract, the authors only mentioned the assessment of the efficacy while in the introduction, they also mention the safety? Please be consistent in the two places with the title as well.\nMethods\nParameters measured:\nPlease clarify whether the authors used the Arabic or English version of KOOS tool?\nIf Arabic, who did the translation and validation?\nWas the KOOS self-administered, or researcher administered?\nMore information about the researchers who did the scoring (by KOOS) are needed. More information about the KOOS domains, and grading/scoring are needed.\nMore details about the recruitment and the clinic settings are needed in the method section.\nWas there incentive to the participants?\nWere their secondary outcome measures? Please specify\nPlease specify the company of the Ginkgo biloba and whether the patients purchased it or brought by the researcher?\nStatistics\nDid you mean Two-Way ANOVA or Repeated measure ANOVA by “Mixed ANOVA method”?\nDid you use ANOVA to measure the difference in the outcome within the same group across the 3 time points? Please clarify in this section.\nPlease clarify why the authors used ANOVA instead of Independent T-Test given they were 2 measured for 2 groups?\nResults\nI am wondering whether there was significant difference in the severity of the disease (OA grade) between the 2 groups at the baseline?\nTable 2: It is unclear whether these P-values are between the 2 different groups or within the same group.\nThus, I recommend having 2 separated tables: One for between the 2 groups and 2nd among the measures within the same group.\nAdverse events reported\nIf the safety was one of the study objectives, statistical analysis should be performed to compare the ADRs between the 2 groups. I also expect to have at least frequency of the ADRs for the 2 groups.\nLimitations\nPlease add the study did not control (adjust) the other confounders such as age, gender, and severity of the disease.\nConclusion\nIf you have numbers of ADRs, please add them in the conclusion section.\nAre there any clinical recommendations to rheumatologists? Please add them after 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? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9525", "date": "28 Apr 2023", "name": "Maryam Al-Haddad", "role": "Author Response", "response": "Dear reviewer Thank you for your valuable comments which indeed increase the quality of the research paper. the corrections and responses are as followed: Abstract: The corrections are made in New Version as track change. GBE was given orally as capsule dosage form. Main introduction: The corrections are reported in New Version. Method: Arabic version was used which was validated and translated originally and could be found in KOOS website. No incentive was given to the patients. There was secondary outcome, which is prepared to be published in separated article regarding measuring inflammatory biomarker. Other comments were add in the New Version. Statistics: The mixed ANOVA was used in order to measure the differences of the two arms of the study across different times. Mixed ANOVA aimed to measure: 1- Difference between groups regardless of time 2- Difference between times regardless of groups 3- Interaction between time and group that show the specific point of significant Independent T-Test measure the difference between two times only , if used that leads to inflated results. So the mixed ANOVA is the best method used in this case. Results: The base line differences was add in the new version which show no significant difference between the two groups Table 2 shows the differences between times in same group Adverse events reported: The statistics was added to new version which was insignificant between two groups. Limitation : Done as required Conclusion: Done as required Thank you for your time, and we are ready to address any further comments. Best regards." } ] } ]	1	https://f1000research.com/articles/11-1231
https://f1000research.com/articles/10-1088/v1	26 Oct 21	{ "type": "Research Article", "title": "P2P lending platforms in Malaysia: What do we know?", "authors": [ "Lan Thi Phuong Nguyen", "Wisdom Kalabeke", "Saravanan Muthaiyah", "Ming Yu Cheng", "Kwan Jing Hui", "Hazik Mohamed", "Wisdom Kalabeke", "Saravanan Muthaiyah", "Ming Yu Cheng", "Kwan Jing Hui", "Hazik Mohamed" ], "abstract": "Background - With the recent evolution of Financial Technology (FinTech), 11 peers to peer (P2P) lending platforms have been regulated by the Securities Commission in Malaysia since 2016. P2P lending platforms offer new investment opportunities to individual investors to earn higher rates on return than what traditional lenders usually provide. However, individual investors may face higher potential risks of default from their borrowers. Therefore, individual investors need to understand the potential exposure to such P2P lending platforms to make an effective investment decision. This study aims to explore the potential risk exposures that individual investors may experience at Malaysia's licensed P2P lending platforms.\n\nMethods - Based on data collected manually from nine P2P lending platforms over five months, relationships between interest rates and various risk classifying factors such as credit rating, industry, business stage, loan purpose, and loan duration are examined.\n\nResults- This study shows that loans with a similar credit rating and with or without similar loan purpose; and a business stage may offer investors significantly different interest rates. In addition, loans with shorter durations may provide investors with higher interest rates than those with longer durations. Finally, loans issued by companies from the same industry appeared to be charged with similar interest. These findings are valuable to investors to prepare themselves before making their investments at the P2P lending platforms.\n\nConclusion- With first hand-collected data, this study provides an original insight into Malaysia's current P2P lending platforms. Findings obtained for relationships between interest rates and risk classifying factors such as credit rating, industry, business stage, loan purpose and loan duration are valuable to investors of Malaysian P2P lending platforms.", "keywords": [ "P2P lending platforms", "default risk", "SMEs", "individual investors", "credit rating", "FinTech platforms" ], "content": "Introduction\n\nAs the most prominent sector in Financial Technology (FinTech), peer-to-peer (P2P) lending platforms use digital communication technology to connect lenders and borrowers online. FinTech platforms like P2P lending are easily accessed by smartphones and the internet for getting unconventional loans. Unlike traditional banks, these lending platforms do not require collateral, the ownership of a bank account, and a business plan. Borrowers are rated and categorized based on their risk profile determined by their provided personal information, including information from their social media sites.\n\nSince the first two P2P lending platforms, named “Zopa” and “Prosper”, were launched in the U.K. and U.S. in 2005 and 2006, respectively, the P2P lending industry has grown and reached Asia. The credit crunch experienced by many traditional banks in the U.S. because of the 2008 Global Financial Crisis, forced many borrowers to turn to P2P lending platforms for their short-term financing needs. Business Wire (2020) reported that the annual compounded growth rate of the global peer to peer (P2P) lending market was around 25% between 2014 and 2019. Although there has been a slowdown in FinTech investments across America and Europe since 2016, the number of P2P lending platforms continues to increase in many emerging markets in the past few years (Stern, Makinen, and Qian, 2017).\n\nSince 2016, 11 peer-to-peer (P2P) lending platforms have been granted licenses by the Securities Commission in Malaysia. Since then, the industry has experienced healthy growth in its first two and a half years of operation, as reported by Zhe (2019). Within the first nine months of operation, RM521.7 million were raised by the first six licensed P2P lending platforms to fund 1,560 small and medium enterprises (SMEs). By the end of 2019, the numbers of registered investors at the three most active platforms, Funding Societies Malaysia, Fundaztic and B2B FinPal, were 3,500, 1,700, and 1,500, respectively.\n\nKok (2021) reposted that many P2P lending platforms, i.e., Funding Societies Malaysia and Fundaztic, experienced steady growth in 2020 despite the Covid-19 pandemic. Their default rates were reported to be reasonably low at 3% and 2.5% per annum by Funding Societies and Fundaztic, respectively. There are two main reasons for this strong growth of P2P lending platforms: the cut in interest rates by Bank Negara Malaysia from 3% to 1.75% between December 2019 and July 2020, and the growth in the use of digital space during the pandemic. Thus, P2P lending platforms appear to be attractive alternative investment platforms as they offer much higher interest rates, ranging from 6% to 21% per annum across the eleven platforms.\n\nUnlike P2P lending platforms in the West, P2P lending platforms in Malaysia are designed to give out loans only to businesses and not individual borrowers. For SMEs that cannot provide sound evidence on their business performance and enough collateral, P2P lending platforms are their best financing alternatives. However, this could imply a possibly higher rate of default faced by the P2P lending industry in Malaysia than traditional lending institutions. Therefore, investors are often advised to diversify their funds across different investment notes while investing at each platform. However, having an effective diversification strategy depends heavily on two critical elements: (1) whether investors have basic financial literacy and (2) how much information of each investment note is made known to investors by each P2P platform.\n\nWhen it comes to evaluating the default risk of a borrower, information that can help arrive at one's estimated credit score is needed. For P2P lending platforms, the borrowers' credit score is based solely on information supplied by the borrower (Li, 2018). Since the information provided by P2P lending platforms are limited and not guaranteed, it would be even more challenging for investors with good financial literacy to reach a sound investment decision. Therefore, individual investors need to understand the potential exposure to such P2P lending platforms to be able to make an effective investment decision. This study explores potential exposures that individual investors may experience at licensed P2P lending platforms in Malaysia.\n\n\nMethods\n\nIndividual investors would mainly like to subscribe to platforms with affordable subscription fees. Of 11 licensed P2P lending platforms in Malaysia, only 10 platforms (Funding Societies, B2B FinPal, Fundaztic, QuicKash, AlixCo, Nusa Kapital, CapSphere, MicroLEAP, Cofundr, and Money Save) require affordable initial deposits, ranging from RM5 to RM1,000, from individual investors. CapBay was excluded from this study as it requires the highest amount of initial deposit (RM10,000) from its investors. Additionally, Funding Societies was also not considered due to the complexity of its subscription. Therefore, the final sample size was nine P2P lending platforms (B2B, Fundaztic, QuicKash, AlixCo, Nusa Kapital, CapSphere, MicroLEAP, Cofundr, and Money Save).\n\nAll available information was obtained from the nine platforms for comparative analysis (Underlying data) (Nguyen, 2021). The data collection period was from January to May 2021. Data collection was done manually as a loan campaign is only announced to investors for a maximum of seven days at each platform. Once a campaign is bided on successfully, the issue note will be taken down, and its record will no longer be visible to investors who did not invest in that note.\n\nThe lender decides whom to grant the loan primarily based on the interest rate. The percentage of the interest rate is mainly dependent on the borrower’s credit score, which is based on various loan characteristics such as the type of business, business cycle, loan purpose, loan duration, and loan amount. Therefore, understanding the relationship between the prime factor - interest rate and other secondary factors such as credit score, loan term, loan duration, loan purpose, business cycle and industry will help investors to be more aware of what to expect when making their investment decisions at those platforms.\n\nSince there is no data publicly available for individual P2P lending platforms in Malaysia, this study employs first hand-collected information for the analysis. Descriptive and cross-tabulation statistics were produced by Microsoft Excel 2010 and IBM SPSS Statistics 26, using a five-month data set to examine the potential exposure that individual investors may face at the nine selected P2P lending platforms. In addition, other tests for normality, coefficients, and multiple regression tests were also conducted. However, only descriptive statistics are reported in this paper.\n\n\nResults/discussion\n\nAfter the collapse of hundreds of P2P lending platforms in China since 2013 (Bloomberg Businessweek, October 3, 2018) due to fraudulent activities, P2P lending in Malaysia was somewhat restrictive until 2016. Malaysia is the first country in Southeast Asia to regulate its first six P2P market operators in 2016 (The Securities Commission, 2017). The Securities Commission of Malaysia (SC) requires that P2P lending platforms operating in Malaysia have a minimum paid capital of RM5million, assess and monitor the risk level of each prospective borrower, set limits and obligations, and ensure both issuers and lenders comply with relevant guidelines. SC further established that issuers must be sole proprietorship, partnership, limited liability companies, private unlimited and unlisted companies incorporated in Malaysia. Investors could be any individual or institution outside or within Malaysia, however, depending on rules set by an operator.\n\nAs shown in Table 1, information required from potential investors at the nine P2P lending platforms were varied. At each P2P lending platform, potential investors must have initial deposit amounts ranging from RM5 to RM1,000, kept at a specified third-party's account at each platform. Each platform requires each investor to provide basic personal information such as identification card (ID)/passport number, most recent bank account statement, details of sources of income and proof of current residential address. Some P2P lending platforms, such as QuicKash, Money Save, and AlixCo, require additional documents: a selfie photo or a video clip that clearly shows the face of the potential investor while holding his/her passport/ID with the first page open. Most of the platforms allow only Malaysian investors, except QuicKash, MicroLEAP, CapSphere, and AlixCo. Funding Societies, in particular, welcomes investors from four countries, namely Thailand, Indonesia, Singapore, and Malaysia for investment opportunities. Nusa Kapital specifically welcomes Malaysian Muslim investors to participate. Money Save is the only platform that assesses potential investors' financial literacy to categorize them into three groups of investors: (1) with basic investment knowledge, (2) with basic investment knowledge and experience, and (3) with sophisticated investment knowledge and experience.\n\nCredit rating is one of the most important criteria for most lending institutions, including P2P lending platforms, when deciding on the interest rate that should be charged to a borrower. A credit rating is given to a borrower based on evaluating the potential default incurred by that borrower. In Malaysia, credit ratings can be obtained from Credit Bureau Malaysia. Upon request from a lending institution, Credit Bureau Malaysia provides information such as personal identifying data, personal credit histories reported by various lenders previously, information showing the honesty and stability of a borrower, and the number of requests made by lenders and legal authorities on the borrower's credit status. Credit scores are determined based on factors that differentiate between a good and a bad borrower. A borrower with a good credit rating score can be offered lower interest rates.\n\nHowever, it is found that some P2P lending platforms in Malaysia use the scores provided by Credit Bureau Malaysia, while others use their own formula to determine the scores for their borrowers. Additionally, the credit ratings are not shared between P2P lending platforms, which leads to varying credit scores across all platforms. It is unknown to investors how these scores are decided, as a result, a borrower may get a low credit score from one P2P lending platform and a higher from another.\n\nAs shown in Figure 1, the principle that loans issued by SMEs with higher credit ratings should be charged with lower interest rates does not hold for most P2P lending platforms, except for MicroLEAP.\n\nThe results show that some loans issued by SMEs with lower credit ratings were charged with lower interest rates. For instance, at B2B a loan issued by an SME with a lower credit rating (C), was charged with an interest rate of 12% per annum. In comparison, A was charged with a higher interest rate of 12.5% per annum for a loan issued by an SME with a higher credit rating (Figure 1). Similarly, at AlixCo platform, a loan issued by an SME with a credit rating of CAA2 (Very high credit risk) (see rating classification at https:/www.alixco.com/fag/statistics) was charged with an interest rate of 14.28% per annum, which is lower than what was (15% per annum) charged for loans issued by SMEs with CAA and CAA3. At Money Save platform, loans issued by SMEs with credit rating A were charged with interest rates ranging from 12% to 14.5% per annum, while loans issued by SMEs with credit rating B were charged with a much lower range of interest rates, i.e., 9.75% - 10.75% per annum.\n\nSimilarly, at QuicKash platform, an interest rate of 12% per annum was charged to SMEs with credit ratings ranging from 7-Good to 9-Strong. For Cofundr platform, a loan issued by an SME with a credit rating of CR3-Good was charged with a higher interest rate (9.75% per annum) as compared to that (9.6% per annum) charged to a loan issued by an SME with a lower credit rating (CR4-Satisfactory). Similarly, at CapSphere, loans issued by SMEs with a credit rating D were charged with lower interest rates (7.1%, 8% and 8.8%) as compared to those (9% and 11% per annum) charged to loans issued by SMEs with a credit rating C. Also, at Nusa Kapital platform, an interest rate of 15% per annum was charged to loans issued by SMEs with different credit ratings.\n\nThe above findings show that loans with similar credit ratings may offer investors very different interest rates. This result implies that a loan issued by a borrower with a higher credit rating can offer the same or higher interest rate charged to a loan issued by a borrower with a lower credit rating.\n\nLoan purpose is the main reason for a borrower to request a loan. Lending institutions often assess a borrower's loan purpose to see the level of default risk associated with the requested loan. Regularly stated loan purposes declared at the nine P2P lending platforms are invoice financing, working capital, Covid-19 relief financing, trading, expansion, insurance premium, and equipment maintenance (Figure 2). As shown in Figure 2 there appears to be no specific interest rate applied to a particular loan purpose at the nine individual platforms. This result may suggest that loan purpose may not be one of the main criteria for a P2P lending platform for determining an interest rate for a borrower. This finding could be due to the similar risk level associated with the previously mentioned loan purposes.\n\nThese results show that loans with a similar loan purpose may offer investors significantly different interest rates. In addition, loans with different borrowing purposes can be charged with the same interest rate, suggesting that loan purposes may not indicate much difference in potential risks associated with them at the nine P2P lending platforms.\n\nDuration measures the sensitivity of the value of a debt instrument when the interest rate changes. According to the term structure of interest rates, loans with a longer duration should be charged with a higher interest rate due to the high possibility that interest rates may change during that long period.\n\nHowever, this does not seem to be the case for the nine P2P lending platforms. As shown in Figure 3, a similar interest rate was applied to loans with different maturities at the nine P2P lending platforms. At B2B FinPal, loans with short durations, i.e., 37 days, had a higher interest rate than those charged for loans with a 98-day duration. Similarly, at Fundaztic, loans with a three-month duration are charged an interest rate of 15%, while a loan with a 36-month duration is charged with a lower interest rate, i.e., 12.23%. At AlixCo, the highest interest rate (15.36%) is charged to a three-month loan, while other loans with four- and six-month durations are only charged at 15%. At MicroLEAP, a 12-month loan is charged with a higher interest rate (14%) than those (12%, 11.5% and 11%) charged to 24-, 30- and 36-month loans. At Money Save, six 1-month loans offer investors interest rates of either 14.75% or 12%, while a six-month loan offers investors a 10.75% interest rate.\n\nAt Cofundr, a one-month loan is charged with the highest interest rate of 17.5%. At CapSphere, some 12-month loans are charged with lower interest rates (8.8% per annum) than those (10% per annum) charged to six-month loans. At NursaKapital, a three-month loan is also charged with a higher interest rate (15%) than that (14%) charged to a six-month loan.\n\nThese results show that loans with a shorter duration may offer investors higher interest rates than loans with longer durations. This suggests that individual investors can look for loans with a duration of one month and still earn an interest rate that is much higher than the one offered by a 12-month loan at the nine P2P lending platforms.\n\nMost companies asking for loans at the nine P2P lending platforms are in one of the three main stages: growth, extension, and maturity. A stage that a business is going through may indicate the uncertainties that it may face. In a typical business cycle, a company goes through seven stages: seed, startup, growth, established, extension, decline, and exit. There are plenty of opportunities for growing companies; however, they may be exposed to high competition, unstable economy, and market demand, which may hamper their business. Thus, it is also a concern for lenders when granting loans to such businesses. They aim to expand the market share and achieve a new profit level for companies in their extension stages. However, if the expansion to a new product line adds considerable risk to a firm, lenders may need to evaluate loan purposes for such businesses. At a mature/decline stage, companies can experience a drop in their revenues and profits, and therefore might want to close their business. It is due to this reason that lenders find granting loans to these businesses a high risk.\n\nAs shown in Figure 4, most loans given by the nine P2P lending platforms are for companies in their growth and expansion stages. However, mixed interest rates are charged to both growing and expanding companies at most platforms, except Nusa Kapital. This finding may be mainly due to the limited data available on Nusa Kapital. The stage of a business does not reflect its entire potential risk. A similar interest rate may be charged to both growing and expanding companies on the same platform. In eight out of nine P2P lending platforms, fewer loans are given to mature businesses. There are two possible reasons for this: (1) mature businesses can get loans at traditional lending institutions at lower rates, (2) P2P lending platforms may view small mature businesses much riskier than growing and expanding businesses due to their potential closure.\n\nIn short, loans issued by companies are primarily in their growth and expansion stages. It is shown that similar interest rates may be charged to loans issued by companies at different business stages. Moreover, there is no specific range of interest rates applied to loans issued by companies at the same business stage, from the same platform. In other words, the business stage may not be an essential factor in evaluating risks by the nine P2P lending platforms.\n\nIf an industry is less sensitive to any economic downturns, companies from that industry are often expected to have stable businesses for a long time. Thus, lending institutions may prefer to provide loans for such businesses.\n\nThe ununiformed classification of industries in the nine P2P lending platforms, as shown in Figure 5, may indicate potential different risk assessments done by each platform for businesses applying for loans. The results show that at B2B the industries with two or more loans charged with a similar interest rate are: advertising (2), consumer goods (3), design (2), import and export (3), and E-commerce (2). In contrast, loans from the same industry, i.e., construction, food and beverages, packaging and distribution, and trading, can be charged with different interest rates. For AlixCo, loans that come from two classified industries: Retail/Trade (FMCG) (4) and Retail/Trade (Smartphone) (18), are charged at the same interest rates, 14.28% and 15.2%, respectively. However, loans from the E-commerce industry are charged with different interest rates, ranging from 13.8% to 15.1%. For MicroLEAP, two loans from the automobile industry are charged at the same interest rate (12%), while loans from the E-commerce industry are charged with different interest rates, ranging from 11.5% to 14%. For Money Save, it appears that loans from the same industries are charged with different interest rates. For QuicKash, only loans from E-commerce are charged at the same rate of 8% per annum. However, it cannot be determined if all future loans from E-commerce will be charged at the rate of 8% since only two loans were reported for this industry at the time of data collection. For Cofundr, loans from Automobile are charged with the same interest rate of 15%. However, there were only two loans reported for this industry.\n\nFor CapSphere, three different loans from Freight and Logistics are charged at the same interest rate of 7.1%, while loans from other industries are charged at various interest rates. Due to limited data on Fundaztic and Nursa Kapital, a conclusion cannot be reached for these two P2P lending platforms.\n\nIn short, there seems to be a pattern of having loans issued by companies from the same industry being charged with similar interest rates across the nine P2P lending platforms. Thus, investors may select loans issued by companies from industries that often offer higher interest rates to improve their income streams.\n\n\nStudy limitation\n\nThis study provides a first and original insight into the current P2P lending platforms in Malaysia. However, the analysis was based on a small sample size as it only considered nine P2P lending platforms in this country.\n\n\nConclusion\n\nThis study examined nine P2P lending platforms in Malaysia to determine the potential exposures faced by investors through various relationships between interest rates and risk classifying factors such as credit rating, industry, business stage, loan purpose and loan duration. The results of this study showed:\n\n• Loans with similar credit ratings may offer investors significantly different interest rates.\n\n• Loans with similar purposes may offer investors different interest rates.\n\n• Loans with shorter durations may offer investors higher interest rates as compared to those with longer durations.\n\n• No specific range of interest rates was applied to loans issued by companies at the same business cycle.\n\n• Loans issued by companies from the same industry appeared to be charged with similar interest.\n\nThe five-month collected data in this study provides a first and original insight into Malaysia's current P2P lending platforms, which will be valuable to potential investors to prepare themselves before making their investments at those platforms.\n\n\nData availability\n\nFigshare: P2P Lending Platforms in Malaysia: What Do\n\nWe Know?\n\nDoi: 10.6084/m9.figshare.14880369 (Nguyen, 2021).\n\nThis project contains the following underlying data:\n\nData file 1. Dataset_P2P lending in Malaysia What do we know_TIM21109.xlsx\n\nData are available under the terms of the Creative Commons International “No rights reserved” data waiver (CC BY 4.0).\n\n\nEthical approval\n\nThe Research Ethics Committee of Multimedia University approved this research to be conducted. The reference number of this approval is: TTO/REC/EA/123/2021.\n\n\nAuthor contributions\n\nAll authors contribute to the data collection. The literature review, research framework and data analysis have been conducted by Nguyen Thi Phuong Lan, Wisdom Kalabeki, and Saravanan Muthaiyah. Cheng-Ming Yu evaluated the findings and discussion of the analysis. Hazik Mohamed gave comments on the practical contribution of this project. Kwan Jing Hui assisted with the data subscription.", "appendix": "Acknowledgement\n\nWe acknowledge the valuable feedback received from the Managing Director of Stella Consulting Group through our project. We also acknowledge the collaborations between researchers from MMU, UPM and UTAR, that allowed the funding application for this project to be successfully approved by the Ministry of Education of Malaysia. We also acknowledge the work contributed by all researchers in this project.\n\n\nReferences\n\nBloomberg News: How China’s Peer-to-Peer Lending Crash Is Destroying Lives.2018.Reference Source\n\nBusiness Wire: Global Peer to Peer (P2P) Lending Market Trends, Growth, Opportunity Report 2020-2025 - ResearchAndMarkets.com.2020 December 15, 2020.\n\nChen X, Chong Z, Giudici P, et al.: Networking with Peers: Evidence from a P2P Lending Platform. Asian Development Bank Institute, Working Paper No.1080, February 2020.2020.\n\nGibilara L, Mattarocci G: Peer-to-Peer lending and real estate mortgages: Evidence from the United Kingdom. J. Euro. Real Estate Res. 2018; 11(3): 319–334. Publisher Full Text\n\nGonsalez L: Blockchain, herding and trust in peer-to-peer lending. Manag. Finance 2019; 46: 815–831. Publisher Full Text\n\nHe Q, Li X: The Failure of Chinese Peer-to-Peer Lending Platforms: Finance and Politics. J. Corp. Finan. 2021; 66: 101852. Publisher Full Text\n\nHidajat T: Unethical practices peer-to-peer lending in Indonesia. J. Finan. Crime. 2020; 27(1): 274–282. Publisher Full Text\n\nKim D: Bounded rationality in a P2P lending market. Review of Behavioral Finance. 2020; 13: 184–201. Publisher Full Text\n\nKok C: Asset Quality of P2P Players Remain inact. Star. 2021.May 22 2021.Reference Source\n\nLi Q: the Mechanism and Effectiveness of Credit Scoring of P2P Lending Platform: Evidence from Renrendai.com. China Finance Review International. 2018; 8(3): 256–274. Publisher Full Text\n\nLusardi A, Mitchell OS: Planning and Financial Literacy: How Do Women Fare?. Am. Econ. Rev. 2008; 98: 413–417. Publisher Full Text\n\nNemoto N, Storey D, Huang B: Optimal Regulation of P2P Lending for Small and Medium-Sized Enterprises. Asian Development Bank Institute, Working Paper No. 912.2019.\n\nNguyen TPL: Data Source_P2P Lending Platforms in Malaysia: What Do We Know? figshare. Dataset. 2021. Publisher Full Text\n\nRosavina M, Rahadi RA, Kitri ML, et al.: P2P lending adoption by SMEs in Indonesia. Qualitative Research in Financial Markets. 2019; 11(2): 260–279. Publisher Full Text\n\nSan-Jose L, Retolaza JL: Crowdlending as a socially innovative corporate financial instrument. Int. Perspectives on Crowdfunding. 2017; 129–149.\n\nShi X, Wu J, Hollingsworth J: How does P2P lending platform reputation affect lenders ‘decision in China?. Int. J. Bank Mark. 2019; 37(7): 1566–1589. Publisher Full Text\n\nStern C, Makinen M, Qian Z: FinTechs in China – with a special focus on peer-to-peer lending. J. Chinese Economic and Foreign Trade Studies. 2017; 10(3): 215–228. Publisher Full Text\n\nTrönnberg CC, Hemlin S: Lending decision making in banks: A critical incident study of loan officers. Eur. Manag. J. 2014; 32(2): 362–372. Publisher Full Text\n\nThe World Bank Group: The little data book on Financial Inclusion. The Global Findex database.2017.Reference Source\n\nZhe KSK: P2P Financing Industry Growing. the Edge Malaysia. 2019.December 26, 2019.Reference Source" }	[ { "id": "98013", "date": "10 Nov 2021", "name": "Dongwoo Kim", "expertise": [ "Reviewer Expertise P2P lending" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 examined nine P2P lending platforms in Malaysia to determine the potential exposures faced by investors through various relationships between interest rates and risk classifying factors such as credit rating, industry, business stage, loan purpose and loan duration. This paper is meaningful in clarifying the relationship between interest rates and other information on multiple P2P lending platforms based in Malaysia for the first time. However, there are some issues that the author should address:\n1. Citation: some of the references are not displayed in the body text.\n2. Samples: it is necessary to clarify the number of samples for each platform to be analyzed.\n3. Methods: it is necessary to clarify the reason for omitting the results of regression analysis and normality analysis as well as containing only the contents of descriptive statistics.\n4. P2P lending is a field that has recently received a lot of attention academically and practically. Therefore, more and more related studies are being published. Among them, there are several studies that have revealed the relationship between various types of borrower information and loan interest rates, which are of interest to the authors, and should be investigated by them.\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": "8006", "date": "04 Apr 2022", "name": "Thi Phuong Lan Nguyen", "role": "Author Response", "response": "Dear Prof. Dr. Dongwoo Kim, First of all, we would like to express our sincere thanks and gratitude for being one of the reviewers for our research paper. Upon receiving all your constructed comments, we have revised and made corrections accordingly. The following are our responses to your given comments: 1. For the comment #1: Citation: some of the references are not displayed in the body text. We have added more references and corrected citations as suggested. 2. For the comment #2: Samples: it is necessary to clarify the number of samples for each platform to be analyzed. We have added details of the sample of loan campaigns recorded from the nine P2P lending platforms (page 8). The following table is added: Table 1: Number of Loan Campaigns Recorded from January to May 2021 at the Nine Malaysian P2P Lending Platforms (Page 9) 3. For the comment #3: Methods: it is necessary to clarify the reason for omitting the results of regression analysis and normality analysis as well as containing only the contents of descriptive statistics. We have also explained the analysis method used for the study (Page 8). 4. For the comment #4: P2P lending is a field that has recently received a lot of attention academically and practically. Therefore, more and more related studies are being published. Among them, there are several studies that have revealed the relationship between various types of borrower information and loan interest rates, which are of interest to the authors, and should be investigated by them. We have also added a section of literature review to show the related works and the gap in literature the study aims to fill in (see Page 3). We are looking forward to having further feedback and approval. Many thanks, On behalf of the team, Lan Nguyen (Ph.D.)" } ] }, { "id": "126134", "date": "04 Mar 2022", "name": "Ryan Randy Suryono", "expertise": [ "Reviewer Expertise Fintech", "P2P Lending" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 potential exposures that individual investors may experience at licensed P2P lending platforms in Malaysia.\n1. In the introduction, it is necessary to explain the reason for the the research. Why was this research was conducted? Why Malaysia? This section has very few references.\n2. The methodology section should be explained step by step. What is the process for determining the feasibility of a case study? What is the process of data collection and analysis until it becomes the research result?\n3. The results section should produce an in-depth discussion. How are lessons learned from the findings? Do the results support other research? What are the theoretical and practical implications? Can the author also compare P2P Lending practices in other countries? It might be an interesting review if there are some benchmarks.\n4. Overall, this article needs a lot of improvement in substance by enriching the study literature. Please correct the citation and proofread.\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": "8007", "date": "04 Apr 2022", "name": "Thi Phuong Lan Nguyen", "role": "Author Response", "response": "Dear Prof. Dr. Ryan Randy Suryono, First of all, we would like to express our sincere thanks and gratitude for being one of the reviewers for our research paper. Upon receiving all your constructed comments, we have revised and made corrections accordingly. The following are our responses to your given comments: 1. For the comment #1: In the introduction, it is necessary to explain the reason for the research. Why was this research was conducted? Why Malaysia? This section has very few references. We have added the justification for the study as suggested (see Page 3). We have also added a section of literature review to show the related works and the gap in literature the study aims to fill in (see Page 3). 2. For the comment #2: The methodology section should be explained step by step. What is the process for determining the feasibility of a case study? What is the process of data collection and analysis until it becomes the research result? We have added details of the sample of loan campaigns recorded from the nine P2P lending platforms (page 8). The following table is added: Table 1: Number of Loan Campaigns Recorded from January to May 2021 at the Nine Malaysian P2P Lending Platforms (Page 9) We have also explained the analysis method used for the study (Page 8). 3. For the comment #3: The results section should produce an in-depth discussion. How are lessons learned from the findings? Do the results support other research? What are the theoretical and practical implications? Can the author also compare P2P Lending practices in other countries? It might be an interesting review if there are some benchmarks. We have added more interpretation of the results, linked them to past studies and practices, and provided implications and suggestions to each finding section. (see Page 9 - Page 14) 4. For the comment #4: Overall, this article needs a lot of improvement in substance by enriching the study literature. Please correct the citation and proofread. We have also added a section of literature review to show the related works and the gap in literature the study aims to fill in (see Page 3). We have added more references and corrected citations as suggested. The article has gone through a few rounds of proofreading. (see page 15 – 16) We are looking forward to having further feedback and approval. Many thanks, On behalf of the team, Lan Nguyen (Ph.D.)" }, { "c_id": "8051", "date": "11 Apr 2022", "name": "Ryan Randy Suryono", "role": "Reviewer Response", "response": "All suggestions for revision have been done well. Authors have added more interpretations of the results, linked them to previous studies and practice, and provided implications and suggestions for each section of the findings listed on Page 9 - Page 14. Suggestions for further research, conducting a qualitative approach/empirical study, can also conduct Focus Group Discussions with the P2P Lending platform in Malaysia and capture the P2P Lending landscape. Propose ideas for the industry. Good Luck!" } ] } ]	1	https://f1000research.com/articles/10-1088
https://f1000research.com/articles/12-198/v1	20 Feb 23	{ "type": "Research Article", "title": "Disease association and comparative genomics of compositional bias in human proteins", "authors": [ "Christos E. Kouros", "Vasiliki Makri", "Christos A. Ouzounis", "Anastasia Chasapi", "Christos E. Kouros", "Vasiliki Makri", "Christos A. Ouzounis" ], "abstract": "Background: The evolutionary rate of disordered proteins varies greatly due to the lack of structural constraints. So far, few studies have investigated the presence/absence patterns of intrinsically disordered regions (IDRs) across phylogenies in conjunction with human disease. In this study, we report a genome-wide analysis of compositional bias association with disease in human proteins and their taxonomic distribution. Methods: The human genome protein set provided by the Ensembl database was annotated and analysed with respect to both disease associations and the detection of compositional bias. The Uniprot Reference Proteome dataset, containing 11297 proteomes was used as target dataset for the comparative genomics of a well-defined subset of the Human Genome, including 100 characteristic, compositionally biased proteins, some linked to disease. Results: Cross-evaluation of compositional bias and disease-association in the human genome reveals a significant bias towards low complexity regions in disease-associated genes, with charged, hydrophilic amino acids appearing as over-represented. The phylogenetic profiling of 17 disease-associated, low complexity proteins across 11297 proteomes captures characteristic taxonomic distribution patterns. Conclusions: This is the first time that a combined genome-wide analysis of low complexity, disease-association and taxonomic distribution of human proteins is reported, covering structural, functional, and evolutionary properties. The reported framework can form the basis for large-scale, follow-up projects, encompassing the entire human genome and all known gene-disease associations.", "keywords": [ "disease-associated gene", "low complexity", "compositional bias", "intrinsically disordered protein (IDP)", "intrinsically disordered region (IDR)", "phylogenetic profile", "human genome", "human disease" ], "content": "Introduction\n\nThe classical view that protein function requires a defined three-dimensional (3D) structure has been challenged by recent developments where many proteins and protein regions are shown to perform distinct biological functions, despite their propensity for disordered conformations. These intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDRs) are defined as lacking a precise 3D folding pattern. The difference between ordered and disordered proteins is already reflected at the primary structure level, with IDPs being characterized by regions typically enriched in specific amino acids, resulting in an overall low sequence complexity. Specifically, IDPs and IDRs contain substantially fewer residues that promote order (typically C, F, I, L, N, V, W and Y) and are substantially enriched in residues that promote disorder (typically A, E, G, K, P, Q, R, and S) (Williams et al., 2000; Dunker et al., 2001; Harbi et al., 2011).\n\nWith the increasing number of predicted and experimentally validated IDPs and proteins containing IDRs, disordered proteins and regions are no longer considered as exceptions, but rather the object of extensive study with regard to their structure and function. A wide range of disorder predictors has been successfully developed over the past years, adopting different approaches such as Compositional Bias Detection (Harrison, 2021; Promponas et al., 2000; Wootton & Federhen, 1993), residual energy-based disorder prediction (Dosztanyi et al., 2009, 2005) and others (Linding et al., 2003; Tang et al., 2021; Walsh et al., 2012; Wang et al., 2016; Zhang et al., 2012). Integrative tools have made their appearance, such as MobiDB-lite (Necci et al., 2017), a data fusion tool making use of eight distinct predictors. The prediction accuracy of such tools varies greatly, with deep learning-based methods typically outperforming methods based on physicochemical characteristics (CAID Predictors et al., 2021). DisProt, a manually curated, dedicated database for IDPs (Sickmeier et al., 2007) has developed into the main resource for IDP/IDR information (Hatos et al., 2019; Quaglia et al., 2022).\n\nMultiple computational and experimental analyses of a wide range of species at the genome level have established widespread presence of intrinsic disorder across the tree of life (Hatos et al., 2019; Ntountoumi et al., 2019; Peng et al., 2015; Ward et al., 2004). In fact, proteins at all taxonomic levels, including viruses, exhibit noticeable intrinsic disorder that apparently increases with organism complexity. Disorder presence is particularly prominent in eukaryotes, in which at least half of their genome-encoded proteins possess long IDRs (Ahrens et al., 2017; Basile et al., 2019; Peng et al., 2015; Ward et al., 2004; Xue et al., 2012). This high prevalence of IDPs and IDRs in eukaryotes indicates that key functions, such as cell signalling and regulation, are transiently associated with intrinsic disorder in nucleated cells (Bürgi et al., 2016; Tantos et al., 2012).\n\nThe same trend holds for an ever-increasing emergence of disease-associated genes in more recent speciation events (Dickerson & Robertson, 2012; Lopez-Bigas & Ouzounis, 2004), raising the question whether specific residues can be directly implicated in particular diseases. A correlation between intrinsic disorder and various human diseases such as cancer, diabetes, amyloidosis, and neurodegenerative diseases has already been established in specific cases (Choudhary et al., 2022; Monti et al., 2021, 2022), and is emerging as a significant biomedical research endeavour.\n\nDue to a lack of structural constraints, the evolutionary rate of disordered proteins varies, with some IDPs/IDRs being highly conserved while others appearing particularly diversified (Brown et al., 2011; Khan et al., 2015; Xue et al., 2013). So far, few studies have investigated the phylogenetic profiling of IDRs in conjunction with human disease (Pajkos et al., 2020). To assess this hypothesis, we use a curated list of 100 annotated proteins from the human genome with well-characterised compositionally biased regions (CBRs) (Mier et al., 2020), as a first step for the comparative genomics of compositionally biased genes, some of which are in fact disease associated. We identify those instances known to be linked with human disease and assess their phylogenetic depth. This framework, with human queries against multiple species, forms the basis for follow-up, large-scale studies that would encompass the entire human genome and all known gene-disease associations.\n\n\nMethods\n\nThe Human Genome protein set recorded in the Ensembl database (GRCh38.p13) was retrieved, containing ~119K gene transcripts to be used as reference (Yates et al., 2016).\n\nFor the disease mapping on gene transcripts, the DISEASES database was chosen, which integrates disease-gene associations derived from text mining, as well as manually curated disease–gene associations, cancer mutation data, and genome-wide association studies from existing databases (Pletscher-Frankild et al., 2015). Specifically, the “Knowledge channel” was selected, containing manually curated associations from GHR (Koos & Bassett, 2018) and UniProtKB (The UniProt Consortium et al., 2022), a total of 7269 disease-gene, high-confidence associations.\n\nDisease associations are provided with the use of Disease Ontology identifiers (DOID) (Schriml et al., 2019). For each entry of the Ensembl dataset, DOIDs were mapped from the DISEASES knowledge channel dataset and added to the header description of the corresponding gene transcript.\n\nFor phylogenetic analysis, the Uniprot Reference Proteome (URP) dataset was selected, containing a total of 11297 proteomes, excluding viruses. The URP set has been selected manually and algorithmically among all proteomes, to provide broad coverage of the tree of life, representing the taxonomic diversity found within UniProtKB and including the proteomes of well-studied model organisms and other proteomes of interest for biomedical research (Chen et al., 2011). Specifically, the URP (version: Reference_Proteomes_2022_04) contains 349 Archaeal, 8763 Bacterial and 2185 Eukaryotic proteomes.\n\nThe low complexity query set investigated both for its disease association and phylogenetic depth was previously recorded (Mier et al., 2020) and contains 100 human proteins with characteristic compositional bias.\n\nThe computational pipeline cogent_utils, part of CGG toolkit v1.0.1. (Vasileiou et al, submitted), was used to create a CoGenT-style sequence collection (Janssen et al., 2003) from Ensembl GRCh38.p13 as well as the URP, selected as a robust and convenient identifier encoding scheme both for human interpretation and programming convenience. Specifically, cogent_utils enables header modification for all entries of FASTA sequence files, based on user-defined criteria. Below we present the example of the oleosine protein of Camellia sinensis, as it appears originally in URP and after cogent_utils transformation:\n\nURP original header\n\n>tr\|A0A7J7IAQ7\|A0A7J7IAQ7_CAMSI Oleosin OS=Camellia sinensis OX=4442 GN=HYC85_002860 PE=3 SV=1\n\nModified header\n\n>UP000593564-00004442-Came_sine-22-000001-E-000699 tr\|A0A7J7IAQ7\|A0A7J7IAQ7_CAMSI Oleosin OS=Camellia sinensis OX=4442 GN=HYC85_002860 PE=3 SV=1\n\nThe first part of the header has been added, and corresponds to the following format: [URP identifier]-[NCBI Taxonomy ID]-[organism name]-[URP year release]-[proteome counter]-[taxonomic domain]-[protein counter].\n\nMagicMatch v1.0.1 (Smith et al., 2005) was used for sequence matching across databases to verify the identity of the reference proteome collection against the modified identifier space.\n\nFor the detection of compositional bias as a proxy for low-complexity sequence tracts, we deployed CAST v1.0.1 (Promponas et al., 2000), for all protein sequences of the human genome. The CAST algorithm was applied on the DOID annotated Ensembl FASTA format dataset using default parameters, i.e. threshold score 40 for reported regions. The outcome of the analysis were 2 files dividing the original dataset; one containing all entries where low complexity regions were detected and one containing all remaining entries.\n\nSearching with query datasets against Proteomes for the creation of phylogenetic profile patterns was performed with DIAMOND blastp (Buchfink et al., 2021), using the URP dataset as target database and adjusting the alignment algorithm to enable compositional bias statistics (option: --comp-based-stats 3), conditioned on sequence properties (Yu & Altschul, 2005). All hits considered as significant recorded an E-value<0.001 and exhibit sequence similarities of 21% and above.\n\nFor the calculation of amino acid frequencies across the Ensembl protein set, the BioPython Bio.SeqUtils.ProtParam module (Cock et al., 2009) was used, which takes input files of sequences (typically FASTA or FASTQ), counts all the letters in each sequence, and returns a summary table of their counts and percentages. The output was used for data normalisation as explained in Figure 1.\n\nSum of CAST scores for all compositionally biased occurrences by amino acid, normalised with respect to general amino acid frequency in the human genome (i.e. total score/frequency). The column colour corresponds to an amino acid classification according to the chemical nature of their side chains (Katchalski-Katzir et al., 2006).\n\nThe phylogenetic profile heatmap (Figure 4) was produced using the heatmap3 R library (Zhao et al., 2014) with default dissimilarity matrix calculation parameters.\n\nThe 2×2 chi-square test, comparing low complexity presence in protein transcripts and disease-association (Table 1) was performed with 0.01 significance threshold and no Yates continuity correction.\n\nLifemap, an interactive cartography-type tool to explore the NCBI taxonomy was chosen for the visualisation of the taxonomic distribution of data subsets (de Vienne, 2016). For each visualisation, a list of the NCBI IDs of interest were used as input for the tool, which were retrieved, in each case, from the phylogenetic profiling hit list. NCBI taxonomy ID visualisations are provided for all UPR hits of ALG13, SIX3 and RP9 (Figure 5).\n\n\nResults\n\nThe dataset upon which all transformations and analyses were performed was the Ensembl Human Genome export (GRCh38.p13), containing 119068 gene transcripts. The dataset was annotated with regard to disease association, using curated associations from GHR and UniProtKB, which are indexed in the DISEASES database (Pletscher-Frankild et al., 2015). Of these, 3625 transcripts are confidently associated with disease, whereas the remaining 115443 are not verified for any strong disease association in the “knowledge channel” of DISEASES.\n\nTo remove noise, e.g. putative or alternative mini-transcripts (some with multiple stop codons), the Ensembl dataset was filtered and all transcripts with length <80 amino acid residues were removed, with the exception of short transcripts with at least one disease (i.e. DOID) association. The filtered set contains 102702 transcripts, which include all 3625 instances associated with disease (Table 1).\n\nFor the evaluation of low complexity presence in the transcripts of the human genome we performed compositional bias detection using CAST (Promponas et al., 2000). Out of the 102702 transcripts of the filtered Ensembl human genome dataset, compositional bias was detected in 38095 instances, with at least one compositionally biased sequence tract. Cross-evaluation of compositional bias and disease-association presence in the dataset using chi-square test of independence, revealed a significant bias towards low complexity regions in disease-associated, X2 (1, N = 102702) = 306.8467, p-value < 0.00001 (Table 1). This significant pattern alone provides a strong indication for the involvement of low complexity in human disease on genome scale, seen here for the first time, complementing previous, well-established classifications of protein structure and function (Ouzounis et al., 2003).\n\nExamination of the low complexity gene dataset features highlighted the significant divergence among amino acid-related, low complexity frequencies. Figure 1 shows the amino acid-specific rich regions, expressed by the sum of CAST scores for each compositionally biased region and normalised with respect to general amino acid frequency in the human genome as calculated from the filtered Ensembl dataset using Biopython protein analysis modules (Cock et al., 2009). Charged, hydrophilic residues appear over-represented, while hydrophobic, order-promoting amino acids are less frequent, in agreement with what is known about IDP/IDR composition (Williams et al., 2000; Dunker et al., 2001; Harbi et al., 2011). The striking over-representation of serine/threonine (S/T) tracts, along with glutamate/glutamine (E/Q) and proline (P) followed by lysine (K) is indicative of the main residue types that might affect functional properties of human proteins, including their potential association with known phenotypes, such as polyglutamine tracts with neurodegenerative diseases (Bunting et al., 2022).\n\nFor the assessment of the relationship among disease association and compositional bias across the human proteome, the associated DOID vector for each amino acid enriched region was used as a multidimensional clustering parameter for Principal Component Analysis (PCA) (Figure 2). Consistent with the above, the presence of amino acid types in low complexity regions (e.g. S, E, P, Q) exhibit the highest contribution to the main principal components with regard to disease association, thus amplifying the link between low complexity and disease and establishing a direction for further study.\n\nThe colour coding of each amino acid is the same as Figure 1 and reflects the chemical nature of their side chains. DOID=Disease Ontology identifier.\n\nTo further our investigation into the phylogenetic depth of low complexity proteins with or without known disease associations, we selected a published list of 100 human proteins with well-characterised compositionally biased regions (Mier et al., 2020). The proteins were mapped to the enriched human genome datasets derived from Ensembl. Out of the 100 proteins in this curated dataset, 17 are confidently associated with disease, with one or more associated DOIDs, covering a wide range of disorders from metabolic and cardiovascular diseases to autoimmune conditions and cancer (Figure 3).\n\nThe first column corresponds to the amino acid that appears to have enriched presence in each case. The last column’s size is proportional to the sum of CAST scores for amino acid rich regions in each gene. Although specific genes have originally been listed as exemplary for one compositional bias type, in this analysis they can be observed more than once along their DOID associations, as the result of CAST analysis for the derivation of total scores. DOID=Disease Ontology identifier.\n\nTo examine in more detail the emergence of compositional bias for the curated dataset of 100 human proteins as an exemplary case, protein sequence alignment was performed against the URP dataset. Homologues were detected in >11000 species, with just 269 cases not containing any of these regions, largely corresponding to Archaeal and Bacterial taxa. This preliminary, targeted comparative analysis using a limited query of 100 human proteins is a first glimpse into the dynamics of compositional bias across phylogenies. Our ongoing effort to investigate the presence of compositional bias and the connection to human disease will assess these discovered phylogenetic patterns across the entire human genome in the near future. The complete phylogenetic profiling matrix is provided as Extended data (Chasapi, 2022).\n\nFocusing on the 100-gene subset with confident disease associations, most disease-associated genes had detectable homologues across Eukaryotic organisms, with only a few, scarce Bacterial hits (Figure 4). An exception is the DnaJ heat shock protein family (Hsp40) member C5 (DNAJC5) which exhibits an extended phylogenetic depth, covering 86% of the URP (i.e. 9751 proteomes), verifying the observation as a well-known, abundant domain (Stetler et al., 2010; Qiu et al., 2006).\n\nThe heatmap range reflects the dissimilarity matrix of the plotted values. The row side colours correspond to the log of the sum of CAST scores for all detected compositionally biased regions for each protein (darker colour indicates higher sum of CAST scores). The column side colours indicate the taxonomic level of each target proteome (orange = eukaryotes, purple = bacteria). DNAJC5 is not displayed.\n\nThe remaining, 16 disease-associated genes were detected in 1350 proteomes with one or several hits. Figure 4 shows the phylogenetic profile map of these genes across the URP target proteome set. Most genes display homologues in higher eukaryotic organisms, whereas, with the exception of the E3 ubiquitin-protein ligase RLIM, almost no homologous genes are detected in plant genomes. Similarly, the subunit of the rod cyclic GMP-gated cation channel (CNGA1) is the only query gene with ion channel homologues in ciliates and fungi, with the exception of the Ascomycota. In the case of genes with an overall high CAST score, there seem to be more sequence hits, both in number and in taxonomic distribution. This can be, in part, due to the sequence alignment analysis which was tailored to compositionally biased sequences, thus increasing hit sensitivity.\n\nThis comparative genomics framework is a useful tool both for the investigation of tendencies among gene sets confidently associated with diseases, containing compositionally biased regions, as well as for the identification of specific taxonomic signatures for each gene. A selected number of specific cases are reviewed below.\n\nThe protein encoded by ALG13 is a subunit of a bipartite UDP-N-acetylglucosamine transferase, which heterodimerizes with asparagine-linked glycosylation 14 homolog to form a functional UDP-GlcNAc glycosyltransferase that catalyses the second sugar addition of the highly conserved oligosaccharide precursor in endoplasmic reticulum N-linked glycosylation. ALG13 has been associated with several disease conditions including developmental and epileptic encephalopathy as well as genetic intellectual disability (Epi4K Consortium & Epilepsy Phenome/Genome Project, 2013; Bissar-Tadmouri et al., 2014; Ng et al., 2020). ALG13 homologs are detected in 248 proteomes. Moreover, all hits correspond to higher Eukaryotes, specifically to the infraphylum Gnathostomata, including mostly Euteleostomi representatives. Figure 5A shows a general view of the tree of life, highlighted for species where ALG13 homologue hits were retrieved, whereas Figure 5B provides a closer look of the same result. The restricted phylogenetic depth of ALG13 may indicate that the interaction pathways including ALG13 are restricted to functions specific to bony vertebrates, a hypothesis that can be assessed by jointly analysing all participating proteins for their evolutionary emergence.\n\nA) a broad view of the tree of life, visualised be Lifemap (de Vienne, 2016). ALG13 hits are highlighted in blue. B) A zoomed in view of all ALG13 hits. C) SIX3 hits that belong to the Ascomycota phylum and are not found for any other gene of the dataset. D) RP9 hits that correspond to the Dictyostelia clade and are not found for any other gene of the dataset.\n\nSIX Homeobox 3 (SIX3) encodes a member of the sine oculis homeobox transcription factor family. The expressed protein plays a role in brain and eye development, and its mutations are associated with Holoprosencephaly and Schizencephaly abnormalities (Wallis et al., 1999, 3; Hehr et al., 2010, 3). SIX3 homologues were detected in 869 reference proteomes, including filamentous ascomycetes proteome sequences in which SIX3 is the only disease-associated gene with significant hits (Figure 5C). A follow-up study could further investigate this distinct conservation pattern.\n\nRetinitis Pigmentosa 9 (RP9 or PAP1) is thought to be a target protein for the PIM1 serine/threonine protein kinase. The protein localises in nuclear speckles and has a role in pre-mRNA splicing. Mutations in the RP9 gene result in autosomal dominant retinitis pigmentosa (Maita et al., 2004; Keen et al., 2002). The comparative genomics analysis of RP9 presence detects homologues in 507 species, including all representatives of the Dictyostelia clade, that were uniquely matched to RP9 among all disease genes (Figure 5D). Dictyostelium discoideum, the most studied representative of Dictyostelia (i.e. dictyostelid cellular slime molds), has been used extensively as model organism for cell communication, differentiation, and programmed cell death studies (Kawabe et al., 2019; Strassmann et al., 2000). The specific presence of RP9 homologues in Dictyostelia including D. discoideum, raises questions about their specific roles in this taxon and the possibility that functional analysis can shed further light into the human disease.\n\n\nDiscussion\n\nA major research objective for biomedical research is the detection of genetic factors involved in human disease at multiple levels including variation, gene expression and cellular roles. The evolutionary perspective of human disease is less appreciated, compared to the functional genomics of human genes and proteins, by either computational or experimental means. Combining evolutionary characters to structural features such as IDR presence which has yet to be systematically studied in conjunction with specific disease classes, can provide a novel analysis framework of the human genome with respect to disease.\n\nIn this study, we report a genome-wide analysis of the compositional bias association with disease in human proteins and their taxonomic distribution. It is the first time that a combined genome-wide analysis of these aspects is reported, from various structural, functional and evolutionary angles. Our analysis includes novel views on the relation between compositional bias and disease-association, demonstrating a strong correlation between the two features. Delving deeper into the contribution of specific amino acids to compositionally biased regions of disease-associated genes across the human genome, we demonstrate that charged, hydrophilic residues are over-represented in genes with confident disease associations.\n\nWe adopt a comparative genomics perspective for the evaluation of disease association of compositional bias in human proteins, using a curated list of 100 human proteins, as a first step towards this direction in a controlled manner. We delineate conservation patterns of the annotated gene set across taxonomic categories, taking advantage of the great plethora of sequenced genomes across the tree of life, using a total of 11297 representative proteomes.\n\nThe described framework of structurally and functionally annotated gene queries against multiple species has been developed with the view of future directions, encompassing the entire human genome and all known gene-disease associations. This will potentially allow us to elucidate specific evolutionary patterns of groups of genes involved in the same disease, serving as a tool to better understand the underlying mechanisms and identify appropriate model organisms for experimental investigation.", "appendix": "Data availability\n\nAll data underlying the analyses are available as part of the article or as referenced external data sources and no additional source data are required.\n\nZenodo: Phylogenetic profile of 100 annotated low complexity proteins against the Uniprot Reference Proteome dataset. https://doi.org/10.5281/zenodo.7486339 (Chasapi, 2022).\n\nThis project contains the following extended data:\n\n- cb100-query-20221223.map (The phylogenetic profile of the 100 selected annotated low complexity proteins against the Uniprot Reference Proteome dataset)\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 the IDP implementation study community participating in the Elixir Commissioned Service entitled “Standardising Intrinsically Disordered Proteins (IDPs) Data” for the useful knowledge exchange and excellent collaboration on the topic of IDP standardisation.\n\n\nReferences\n\nAhrens JB, Nunez-Castilla J, Siltberg-Liberles J: Evolution of intrinsic disorder in eukaryotic proteins. 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Publisher Full Text" }	[ { "id": "164957", "date": "03 Mar 2023", "name": "Stella Tamana", "expertise": [ "Reviewer Expertise Bioinformatics", "Compositionally Biased Regions", "Comparative genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe presented manuscript “Disease association and comparative genomics of compositional bias in human proteins” focuses on the very interesting, but challenging to analyze and interpret, study of the structural, functional and evolutionary properties of compositional bias in human proteins. The authors report novel findings and a strong correlation between compositional bias and disease association. Studying compositional bias and disease association, both computationally and experimentally, has been quite challenging over the years, mainly because proteins with compositional bias tend to conform into non-globular structures or be in a disordered state and thus, requiring special treatment in fundamental steps of comparative genomic analyses and experimental procedures to determine their three-dimensional (3D) structures. This is the first time that, successfully, a computational framework, combined genome-wide disease-association analysis of compositional bias with regards to their functional, structural, and evolutionary properties. The presentation is very clear, well-structured, and easy to comprehend. The reported computational framework and methodology take advantage of up-to-date computational tools and statistical packages. This paper is of interest to scientists within the field of human diseases and variant interpretation as elucidating specific functional and structural patterns of groups of genes (especially those presenting extreme compositional bias) involved in the same disease will ultimately help discover the underlying mechanisms and develop new experimental procedures.\n\nMinor comments:\nIntroduction – Disordered proteins exhibit specific patterns at the sequence level\nThis section will be greatly enhanced if the authors consider adding a paragraph (or 2-3 lines) of the structural characteristics and underlying mechanisms of IDPs/IDRs and thus, giving the reader the opportunity right from the start of the paper to understand the correlation between compositional bias and disease-association. For example, providing in a bit more detail that order-promoting residue types are commonly found within the hydrophobic cores of foldable proteins as opposed to disorder-promoting residues typically located at the surface of foldable proteins (for references see Theillet et al., 20131; Uversky, 20132). Also, another example could be that hydrophobic enriched regions are prone to induce either self-aggregation or/and intermolecular interactions with surrounding proteins when exposed and thus, trigger aggregation (for reference see Grignaschi et al., 20183).\n\nLine 1-3: please consider adding references.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9533", "date": "21 Apr 2023", "name": "Anastasia Chasapi", "role": "Author Response", "response": "\"Introduction – Disordered proteins exhibit specific patterns at the sequence level This section will be greatly enhanced if the authors consider adding a paragraph (or 2-3 lines) of the structural characteristics and underlying mechanisms of IDPs/IDRs and thus, giving the reader the opportunity right from the start of the paper to understand the correlation between compositional bias and disease-association. For example, providing in a bit more detail that order-promoting residue types are commonly found within the hydrophobic cores of foldable proteins as opposed to disorder-promoting residues typically located at the surface of foldable proteins (for references see Theillet et al., 20131; Uversky, 20132). Also, another example could be that hydrophobic enriched regions are prone to induce either self-aggregation or/and intermolecular interactions with surrounding proteins when exposed and thus, trigger aggregation (for reference see Grignaschi et al., 20183). \" We thank the reviewer for the suggestion. The text has been enriched accordingly. \"Line 1-3: please consider adding references.\" Done" } ] }, { "id": "164958", "date": "13 Mar 2023", "name": "Gregory Amoutzias", "expertise": [ "Reviewer Expertise Bioinformatics", "evolution", "sequence 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 authors identified disordered regions in human proteins, they investigated their annotation and their association with diseases and then investigated the presence/absence pattern of these disordered proteins in homologs from various taxa. Interestingly, the authors found that disease-related proteins tend to have LCRs that are enriched in charged, hydrophilic amino acids. In addition, they investigated the phylogenetic distribution of these proteins. The authors discuss in detail some very interesting disease-associated genes with LCRs and their distinct phylogenetic depth. This is an interesting and very useful study that sheds more light (also from an evolutionary point of view) on the intriguing link between LCRs and disease, and importantly, it functions as a pilot study for a larger-scale planned analysis by the authors.\nComments/suggestions:\nIn introduction, I would also add a paragraph and explain in more detail the terms 'compositional bias' and 'low complexity' and explain that they are strongly linked to IDRs. Concerning the LCRs part, I would include the references of Wooton et al. (19941), of Karlin et al. (20022), of Schaper et al. (20143) and explain that they were originally thought of as junk or linker regions, but not anymore (Haerty et al., 20104).\nIn Methods: “Searching with query datasets against Proteomes for the creation of phylogenetic profile patterns was performed with DIAMOND blastp”. I understand that the authors search for homologs and not only orthologs, is that correct? Also, concerning the cutoff of 21% sequence similarity, is it for any local alignment that DIAMOND reports, or is it for a certain query coverage too?\nMaybe Figure 1 and Table 1 could be moved to Results.\nIn Results, third paragraph. The authors clearly identify a strong compositional bias for disease-associated transcripts. It would be very informative to mention the enrichment fold of 1.8 ((1845/1780) / (36250/62827)). Also, better mention the p-value as 1e-5. I wonder if disease-associated genes tend to have more transcripts on average than non-disease genes? If they do, are the transcripts-isoforms more frequently retaining the compositionally biased region? Maybe the authors could repeat this chi-square test, where they use the longest transcript per gene.\nSome thoughts for the future, when the authors perform their planned large-scale analysis: Do disease-associated genes and transcripts with compositional bias have more wide or more restricted gene expression profiles?\nResults: “The striking over-representation of serine/threonine (S/T) tracts, along with glutamate/glutamine (E/Q) and proline (P) followed by lysine (K) is indicative of the main residue types that might affect functional properties of human proteins, including their potential association with known phenotypes, such as polyglutamine tracts with neurodegenerative diseases (Bunting et al., 2022)” It would also be informative to add that tandem repeats of short oligopeptides that are rich in glycine, proline, serine or threonine are capable of forming flexible structures that bind ligands under certain pH and temperature conditions (Matsushima et al., 20085). Maybe also add Williamson et al. (19946), concerning proline rich regions.\nI would recommend that the authors explain somewhere in detail what the CAST score means.\nConcerning the section of phylogenetic profiling, first paragraph: It would be informative to show what is the enrichment of disease in the well characterized CB proteins, compared to the background, and the statistical significance (Hypergeometric test probably). For example, the background is X disease-proteins in Y total proteins (or genes) of the genome. Enrichment: (17/100)/(X/Y). Same section, second paragraph: I guess its pairwise local sequence alignment with DIAMOND? Same section, third paragraph: “Focusing on the 100-gene subset with confident disease associations”. I guess these are the 17 proteins of the 100 gene subset with known IDs?\nWithin the abstract, the authors use all three related terms, intrinsically disordered regions (IDRs), compositional bias, low complexity regions (LCRs). Maybe they could use only one (in the abstract). Also, they should explain in the Introduction their inter-changeability.\nAbstract: “The evolutionary rate of disordered proteins”. I would rephrase that as disordered protein regions.\nAbstract: Low complexity proteins or proteins with LCRs?\nIn keywords, I would rephrase towards: low complexity region (LCR).\nIn Introduction, please correct the CAID Predictors et al reference.\nIn Introduction: “are transiently associated with intrinsic disorder in nucleated cells”. Could the authors please explain in more detail what they mean with the term “transiently”?\nIntroduction: the paragraph that discusses the link between intrinsic disorder and disease: It would be nice to briefly mention one specific example of how intrinsic disorder is associated with human disease.\nIntroduction: “while others appearing particularly diversified”. Maybe the term “rapidly evolving” would be better.\nIn Methods, when first mentioning GHR, I would include the entire name “Genetics Home Reference”.\nIn Methods: “a total of 7269 disease-gene, high-confidence associations”. It would be even more informative if the number of unique genes and the number of unique diseases in these associations were included too.\nIn Methods, could the authors please elaborate more on what MagicMatch does? I did not exactly understand this part: “to verify the identity of the reference proteome collection against the modified identifier space”.\nConcerning the calculation of amino acid frequencies across the Ensembl protein set, was that done for all protein isoforms of a certain gene, or only for one representative isoform (i.e. the longest one) from each gene?\nIn table 1, the numbers are for human genes or transcripts/protein isoforms?\nIn Methods: “For each visualisation, a list of the NCBI IDs”. Which types of IDs?\nResults: “(GRCh38.p13), containing 119068 gene transcripts”. Could the authors also mention the number of genes?\nResults: “Examination of the low complexity gene dataset features highlighted the significant divergence among amino acid related, low complexity frequencies.” Maybe better use the term difference because divergence relates to conservation.\nResults: “Charged, hydrophilic residues appear over-represented”. I would also mention them in parenthesis.\nIn figure 3, this must be the subset of 17 disease-associated genes from the set of 100 human proteins with well-characterised compositionally biased regions? . The title of Figure 3 should be changed accordingly. Is it possible to also include a key of DOIDS-disease next to the figure, or is it too many of them?\nFigure 4 legend: could the authors explain this more?: “The heatmap range reflects the dissimilarity matrix of the plotted values.”. I am not sure I understood figure 4. Does the figure only show if a homologue is present in a certain species, or does it show as well if the homologue also contains an LCR or CB region as well? Does this correspond to the red/blue colour of the matrix? Concerning figure 4, as a thought/suggestion for future studies, when the authors move to larger-scale analyses, maybe they could also include an analogous analysis, where they show the presence of orthologs, not homologs. For that, they would have to use best reciprocal blast and one representative protein (the longest) from each gene of a genome. Or, maybe the authors could do that using the orthology presence from the OMA database (https://omabrowser.org/oma/home/).\nFigure 5: visualized by Lifemap.\nConcerning supplementary .map file, I would simply convert it to a csv file for import in excel. I would also add two columns, one with the species name of the proteome and another one with the wider taxonomic group that the species belongs to. Also, could the authors explain what the numbers in the cells correspond to, I guess it's the number of homologs in that species?\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": "9534", "date": "21 Apr 2023", "name": "Anastasia Chasapi", "role": "Author Response", "response": "“In introduction, I would also add a paragraph and explain in more detail the terms 'compositional bias' and 'low complexity' and explain that they are strongly linked to IDRs. Concerning the LCRs part, I would include the references of Wooton et al. (19941), of Karlin et al. (20022), of Schaper et al. (20143) and explain that they were originally thought of as junk or linker regions, but not anymore (Haerty et al., 20104).” The introduction has been enriched to accommodate most points raised by both reviewers. “In Methods: “Searching with query datasets against Proteomes for the creation of phylogenetic profile patterns was performed with DIAMOND blastp”. I understand that the authors search for homologs and not only orthologs, is that correct? Also, concerning the cutoff of 21% sequence similarity, is it for any local alignment that DIAMOND reports, or is it for a certain query coverage too?” We thank the reviewer for the comment. For the phylogenetic profiling analysis, only sequence similarity, not alignment geometry, was taken into consideration. We have added an explanatory phrase in the Methods section. “Maybe Figure 1 and Table 1 could be moved to Results.” Table 1 and Figure 1 have been moved. “In Results, third paragraph. The authors clearly identify a strong compositional bias for disease-associated transcripts. It would be very informative to mention the enrichment fold of 1.8 ((1845/1780) / (36250/62827)). Also, better mention the p-value as 1e-5. I wonder if disease-associated genes tend to have more transcripts on average than non-disease genes? If they do, are the transcripts-isoforms more frequently retaining the compositionally biased region? Maybe the authors could repeat this chi-square test, where they use the longest transcript per gene.” Enrichment fold and p-value modification have been added. No reference to transcript abundance has been attempted, an interesting question for further research in the future. “Some thoughts for the future, when the authors perform their planned large-scale analysis: Do disease-associated genes and transcripts with compositional bias have more wide or more restricted gene expression profiles?” We thank the reviewer for the very interesting thought. We have not looked into gene expression for the time being, but it would be a very informative functional parameter for follow up study. “Results: “The striking over-representation of serine/threonine (S/T) tracts, along with glutamate/glutamine (E/Q) and proline (P) followed by lysine (K) is indicative of the main residue types that might affect functional properties of human proteins, including their potential association with known phenotypes, such as polyglutamine tracts with neurodegenerative diseases (Bunting et al., 2022)”. It would also be informative to add that tandem repeats of short oligopeptides that are rich in glycine, proline, serine or threonine are capable of forming flexible structures that bind ligands under certain pH and temperature conditions (Matsushima et al., 20085). Maybe also add Williamson et al. (19946), concerning proline rich regions.” We thank the reviewer for the suggestion. The text has been enriched accordingly. “I would recommend that the authors explain somewhere in detail what the CAST score means.” Done (figure 1 legend) “Concerning the section of phylogenetic profiling, first paragraph: It would be informative to show what is the enrichment of disease in the well characterized CB proteins, compared to the background, and the statistical significance (Hypergeometric test probably). For example, the background is X disease-proteins in Y total proteins (or genes) of the genome. Enrichment: (17/100)/(X/Y).” We thank the reviewer for the suggestion. We have chosen a 100-protein low complexity query set, with characteristic CB for further exploration regarding disease association and phylogenetic depth. However, this query set is not necessarily representative of the whole genome, and we therefore chose not to make any extrapolation. “Same section, second paragraph: I guess its pairwise local sequence alignment with DIAMOND?” Indeed, as described in the methods section. Reference to methods has been added. “Same section, third paragraph: “Focusing on the 100-gene subset with confident disease associations”. I guess these are the 17 proteins of the 100 gene subset with known IDs?” Indeed. A small clarification has been added. “Within the abstract, the authors use all three related terms, intrinsically disordered regions (IDRs), compositional bias, low complexity regions (LCRs). Maybe they could use only one (in the abstract). Also, they should explain in the Introduction their inter-changeability.” Done “Abstract: “The evolutionary rate of disordered proteins”. I would rephrase that as disordered protein regions.” Done “Abstract: Low complexity proteins or proteins with LCRs?” Thank you, it is indeed more accurate and has been changed. “In keywords, I would rephrase towards: low complexity region (LCR).” Done “In Introduction, please correct the CAID Predictors et al reference.” Thank you for your comment, it has been corrected. “In Introduction: “are transiently associated with intrinsic disorder in nucleated cells”. Could the authors please explain in more detail what they mean with the term “transiently”?” “Transiently” was replaced with “dynamically” for better clarity. “Introduction: the paragraph that discusses the link between intrinsic disorder and disease: It would be nice to briefly mention one specific example of how intrinsic disorder is associated with human disease.” A paragraph explaining the role of alpha-synuclein and relation in PD onset has been added. “Introduction: “while others appearing particularly diversified”. Maybe the term “rapidly evolving” would be better.” Done “In Methods, when first mentioning GHR, I would include the entire name “Genetics Home Reference”.” Done “In Methods: “a total of 7269 disease-gene, high-confidence associations”. It would be even more informative if the number of unique genes and the number of unique diseases in these associations were included too.” Thank you for the comment, we have added the values. “In Methods, could the authors please elaborate more on what MagicMatch does? I did not exactly understand this part: “to verify the identity of the reference proteome collection against the modified identifier space”.” We thank the reviewer for the comment. MagicMatch is used so that a sequence-identity check at 100% is performed based on sequence only, no comparison of identifiers. A clarification has been added in the Methods section. “Concerning the calculation of amino acid frequencies across the Ensembl protein set, was that done for all protein isoforms of a certain gene, or only for one representative isoform (i.e. the longest one) from each gene?” The calculation concerns the whole Ensembl protein dataset, therefore all reported isoforms for each gene. “In table 1, the numbers are for human genes or transcripts/protein isoforms?” We thank the reviewer for the observation. The numbers correspond to gene transcripts, we have changed the Table legend. “In Methods: “For each visualisation, a list of the NCBI IDs”. Which types of IDs?” It has been clarified in the text. “Results: “(GRCh38.p13), containing 119068 gene transcripts”. Could the authors also mention the number of genes?” Done “Results: “Examination of the low complexity gene dataset features highlighted the significant divergence among amino acid related, low complexity frequencies.” Maybe better use the term difference because divergence relates to conservation.” Done “Results: “Charged, hydrophilic residues appear over-represented”. I would also mention them in parenthesis.” Done “In figure 3, this must be the subset of 17 disease-associated genes from the set of 100 human proteins with well-characterised compositionally biased regions? . The title of Figure 3 should be changed accordingly. Is it possible to also include a key of DOIDS-disease next to the figure, or is it too many of them?” The title has been changed accordingly. Regarding the DOIDs, we have chosen to include only the IDs as some of them are quite descriptive and would result in a “noisy” figure “Figure 4 legend: could the authors explain this more?: “The heatmap range reflects the dissimilarity matrix of the plotted values.”. I am not sure I understood figure 4. Does the figure only show if a homologue is present in a certain species, or does it show as well if the homologue also contains an LCR or CB region as well? Does this correspond to the red/blue colour of the matrix? Concerning figure 4, as a thought/suggestion for future studies, when the authors move to larger-scale analyses, maybe they could also include an analogous analysis, where they show the presence of orthologs, not homologs. For that, they would have to use best reciprocal blast and one representative protein (the longest) from each gene of a genome. Or, maybe the authors could do that using the orthology presence from the OMA database (https://omabrowser.org/oma/home/).” We thank the author for the comment. The heatmap represents the number of homologues found across species, without specifying whether these homologues contain LCR/CB regions. The colour range is indicative of this matrix of values, with normalization applied across rows (each row is scaled to have a mean of 0 and standard deviation of 1). We have clarified these points in the figure legend and Methods. Although in this study all analyses concern homologue detection, we welcome the reviewer’s comment for performing similar comparisons for orthologue presence, potentially annotating LCR presence in parallel “Concerning supplementary .map file, I would simply convert it to a csv file for import in excel. I would also add two columns, one with the species name of the proteome and another one with the wider taxonomic group that the species belongs to. Also, could the authors explain what the numbers in the cells correspond to, I guess it's the number of homologs in that species?” We thank the reviewer for the suggestions. The file has been converted to .csv and the MagicMatch identifier has been separated in respective columns, for better readability. These include reference proteome identifier, NCBI taxonomy ID, species code, taxonomic domain (eukaryota/bacteria/archaea). Moreover, a column with the full species name has been added. Regarding the numbers, they indeed correspond to homologs, an information that has been added to the Zenodo file description along the description of the rest of the columns." } ] } ]	1	https://f1000research.com/articles/12-198
https://f1000research.com/articles/11-1457/v1	08 Dec 22	{ "type": "Research Article", "title": "Carotenoids from female Grapsus albolineatus as potential anti-ageing compounds", "authors": [ "Darus Saadah Johanis Paransa", "Kurniati Kemer", "Desy Maria Helena Mantiri", "Rene Charles Kepel", "Dininurilmi Putri Suleman", "Ara Deani Soemantri", "Dikdik Kurnia", "Kurniati Kemer", "Desy Maria Helena Mantiri", "Rene Charles Kepel", "Dininurilmi Putri Suleman", "Ara Deani Soemantri", "Dikdik Kurnia" ], "abstract": "Introduction: Grapsus albolineatus is thought to have a high concentration of carotenoid pigments. Although studies on male G. albolineatus have been conducted, no studies on pigment extraction from female G. albolineatus have been carried out. Carotenoids have a high ability to fight free radicals. Previous research has shown that carotenoids can fight free radicals that cause premature skin-aging. The purpose of this research was to find out what kinds of carotenoids are found in G. albolineatus and whether they can act as natural inhibitors of proteins that cause premature aging, such as glucogenase, elastase, and hyaluronidase enzymes. Methods: Carotenoids were extracted from G. albolineatus using column chromatography and high-performance liquid chromatography (HPLC); molecular docking and visualization were done with Autodock 4.2 and Discovery Studio/Biovia, respectively.\n\nResults: According to HPLC data, there are carotenoid pigments such as didehydroastaxanthin, tetrahydroastaxanthin, dihydroastaxanthin, diatoxanthin, astaxanthin, and adonixanthin. According to molecular docking experiments, pigment carotenoids from G. albolineatus are efficient inhibitors of protein elastase and hyaluronidase. Conclusions: G. albolineatus carotenoids have the potential to be anti-aging since they are more effective as protein elastase and hyaluronidase inhibitors than their native inhibitors.", "keywords": [ "Grapsus albolineatus", "glucogenase", "elastase", "hyaluronidase", "carotenoids." ], "content": "Introduction\n\nSkin is one of the largest organs in the human body and covers almost its entire.1,2 Skin has various functions such as protecting muscles, bones, and internal organs.3 Skin can experience aging that in unavoidable due to intrinsic factors, influenced by age, genetics, hormones, and blood sugar levels.4 While the majority of extrinsic variables are caused by continuous exposure to ultraviolet (UV) rays, pollution, diet, and smoking.5,6\n\nUV radiation is associated with an increase in oxygen radical species on the skin. Excessive reactive radical species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) can worsen pigmentation and aging of the skin, caused pigmentation disorders, rough skin and skin wrinkling.7 In addition to oxidative damage to the skin, ROS are known to activate hyaluronidase, glucogenase, and elastase enzymes.8 Increased activity of this enzyme can reduce hyaluronic acid, elastasin, and collagen. These components are key elements of the skin that act on elasticity, flexibility, and moisture provision, allowing the skin to seem young and healthy.9,10\n\nTherefore, to prevent premature aging, the skin needs an antioxidant as well as an enzyme inhibitor which acts as a natural inhibitor of enzymes that are activated due to UV radiation. Carotenoids are a class of compounds that have excellent antioxidant activity.11,12 Vegetables and fruits are considered as the most important sources of carotenoids in the human diet; animal products also contain large amounts of carotenoids.13,14\n\nThe lightfoot crab from North Sulawesi, G. albolineatus, is known to contain high levels of carotenoids. G. albolineatus is a large crab with a large population. Grapsus albolineatus Latreille has a convex carapace with low tubercles; lateral, rounded margins, with one anterior tooth; the length of the front is equal to the length of the posterior margin of the carapace. The carapace is greenish-blue with paler areas; lateral margins and tubercles white; legs mottled in brown with one orange spot at the tip of meri.15 This species has markedly arched carapace lateral margins; outer infraorbital angle with acute tooth; third maxillipeds with long merus, slightly shorter than the ischium; the male abdomen has five somites about 2.3 times as broad as long; G1 is slightly curved. G. albolineatus is a large-sized species. Its world distribution includes the Indo-West Pacific: East Africa, Mauritius, Somalia, Socotra, Red Sea, Gulf of Aden, Southern Oman, Persian Gulf, Gulf of Oman, Pakistan, India, Sri Lanka, Bay of Bengal, Nicobar Islands, Andaman Sea, Mergui Archipelago, China, Japan, Indonesia, Singapore, Cocos (Keeling) Islands, Australia, and Hawaii. Its habitat is rocky, intertidal substrate.16\n\nIt is widely distributed in the seas of the SouthWest Pacific. Male G. albolineatus have been recognized as having varying quantities of carotenoid pigments, while female G. albolineatus have not been studied for the kinds of carotenoid pigments they contain and their separation.17\n\nTherefore, this study aimed to explore the pigment and separation of carotenoids in female G. albolineatus as well as to test carotenoids as anti-aging and natural inhibitors of hyaluronidase, glucogenase, and elastase enzymes using a molecular docking approach.\n\n\nMethods\n\nThe researcher submitted a research permit (ethical clearance) to the UB (Universitas Brawijaya) Ethics committee team. Part of this research was conducted at the university.\n\nEthical clearance was obtained by including a research proposal, in which the method had a 3R stage, namely: 1) Reduction: using 3 animals, 2) Replacement: using 1 gram of the carapace organ, 3) Refinement: To avoid distress in animals, the researcher used 95% alcohol which functions as an analgesic.\n\nThis research was funded by the Institution of Research and Community Service Sam Ratulangi University (UNSRAT) with assignment letter Number 673/UN12.13/LT/2019. which was signed by the chairman of Institution of Research and community service UNSRAT, after being passing evaluation by the university reviewer.\n\n\n\nSampling was carried out by exploring the intertidal area at the lowest low tide at night. The sample was put into a container filled with a little seawater, to ensure the crabs did not die. The dorsal carapace of the 3.7 cm crab in that section was smeared with 95% alcohol, when the crab fainted, surgery was carried out and then extraction was carried out.\n\nCrab samples were first injected to obtain blood from G. albolineatus using a five-mile injection and then dissected in order to separate the hepatopancreas and gonads. The next step was to separate the epidermis layer, which is a thin membrane connected to the interior of the carapace, and the black epidermis layer with tweezers and a laboratory knife. The organ carapace was crushed and immersed for five minutes in 2N hydrodrochloric acid. Then we strained it through filter paper into a separator flask,added petroleum ether and distilled water to make two layers. The absorption peak was then calculated using a UV-Vis spectrophotometer with a wavelength range of 380-550 nm.\n\nThe absorbance of the carapace pigment extract was measured using a UV-Vis spectrophotometer at 380-550 nm wavelength. The spectrogram’s shape is a curve with the X axis representing wavelength, and the Y axis representing absorbance, and the highest absorption peak at a certain wavelength can be read. Each spectrogram produced can be used to calculate the amount and concentration of G. albolineatus pigments utilized in this investigation.18,19\n\nAfter that, the entire pigment extract was separated using column chromatography. The n-hexane-acetone was the solvent employed in the mobile phase (70:30). The X fraction was collected, which was then accommodated and examined for absorption with a UV-Vis spectrophotometer at a wavelength of 380-550 nm, yielding the spectrophotometer’s highest absorption peak.18,19\n\nHigh performance liquid chromatography (HPLC) type LC.20ADVP with photodiode array detector (PDA; SPD-M20A-Shimadzu). The HPLC column used was C30 (YMC carotenoid, 150 × 4.6 mm I.D) with a column temperature of 30°C and a flow rate of 1 mL/min with a gradient system of H2O, methanol and methyl tertiary butyl ether. The sample used was 20 μL. Carotenoids were separated in a gradient form for 50 min using a mixed solution of methanol, methyl tertiary butyl ether, and water (80:5:15) at a flow rate of 1 mL/min. After 10min, the water content was lowered linearly, and the acetone was added to get the acetone proportion up to 20 percent at 50 min after injection. The final composition of the gradient mixture was methanol: methyl tertiary butyl ether (80:20).\n\nThe binding mechanism to the active site of three enzymes was investigated using molecular docking: fibroblast collagenase (1CGL), porcine pancreatic elastase (4YM9), and hyaluronidase (1FCV).20–22 These three receptors are available on the Protein DataBank (PDB) website and may be saved as a PDB file.23\n\nEnzyme preparation was accomplished by eliminating water and cofactors from each enzyme. The protein was first optimized by adding polar hydrogen, followed by the addition of atomic charge with Kolman charge and nonpolar hydrogen. The receptor was then stiffened and stored in PDBQT (Protein DataBank, partial charge (Q), and atom type) format.9\n\nThe Canonical SmilesY of each ligand utilized in ligand preparation was acquired from Pubchem.24 Subsequently, Canonical Smiles was converted to Chemdraw 2D 16.0 to produce a 2D compound, which was then modified into 3D using Chemdraw 3D and a reduced calculation was done. The file was stored as a PDB file. The ligand’s PDB file was then mixed with polar hydrogen and given the gasteiger atomic charge and torque on each ligand, after which the ligand was stored in PDBQT format.\n\nThe AutoDock 4.2.6 software was used to simulate docking.25 At each active site, molecular docking was performed. The active site is determined by verifying the docking of each inhibitor and the corresponding receptor. A grid box was created in the middle of the ligand location to determine the coordinates of the receptor’s active side. Then, for 1CGL and 4YM9 receptors, dimensions of 50×50×50 points were utilized, while for 1FCV, box dimensions of 60×60×60 points were chosen, with a grid point spacing of 0.375 Å. The Lamarckian Genetic Algorithm was the calculation employed in this docking approach. The optimal conformation was chosen based on the lowest bonding energy among the most conformational populations.\n\nTo observe the interactions that occur between the ligand and the receptor, docking visualization was performed using Biovia Discovery Studio 2020.26 Interactions are depicted in three and two dimensions, respectively. Hydrogen bonding and hydrophobicity were utilized to analyze intermolecular interactions.\n\n\nResults\n\nThe highest wavelength of the content and concentration of total pigment extract in G. albolineatus crabs, particularly in the carapace, was 474 nm. As a result, the overall pigment extract content and concentration value was 4.33 μg/g, whereas the pigment content in the carapace organ was 4.46 μg/g.\n\nThe column chromatography separation was repeated twice with an n-hexane-acetone mobile phase (70:30). A total pigment extract divided into four fractions: F1, F2, F3, and F4. A UV-Vis Spectrophotometer with a wavelength range of 380-550 nm was used to examine the four fractions. The presence of x-carotene pigments was indicated by wavelengths of 426, 448, and 475 nm, whereas the presence of zeaxanthin pigments was indicated by wavelengths of 426, 450, and 475 nm in the second fraction; the pigment types in F3 and F4 were unidentified.27\n\nF1 was not processed through the second round. However, F2, F3, and F4 were separated again using column chromatography: F2.1 was characterized as echinenone and F2.2 as astase. Furthermore, the pigment was not identified in F3.1 and F4.1. Krocoxanthin was found in F3.2, alloxanthin was identified in F3.3, and pyrhoxanthin was identified in F4.2.\n\nTo generate 61 maximum absorption peaks, the findings of one column chromatography fraction were separated using HPLC with a propagation time of 50 minutes. The absorption spectra of each peak from the HPLC chromatogram were identified at 463 nm, and those with an area per-centage greater than 3% were investigated by examining the spectral pattern using an HPLC UV-Vis absorption spectrophotometer\n\nThe HPLC fraction one findings showed eleven absorption peaks. Table 1 shows the absorption generated at the highest peak. The greatest absorption peak at 473 nm was determined to be a kind of didehydroastaxanthin pigment, while the maximum absorption peak at 476 nm was determined to be a type of tetrahydroastaxanthin. (Tables 1-3). The HPLC separation was carried out at a propagation time of 50 minutes and generated 65 absorption peaks in the findings of the second fraction column chromatography.\n\nAt a wavelength of 436 nm, the absorption spectrum region with a larger percentage results in the formation of a maximum absorption peak. The highest absorption in Table 2 helps determine the kind of carotenoid pigment.28\n\nSix spectrum peaks were identified from the separation of nine absorption peaks by HPLC. Only five types of pigments could be identified using the six peak spectral pattern. The HPLC separation resulted in the formation of an absorption peak with a peak area of 3%in Table 3. The greatest absorption peaks were discovered by the absorption of HPLC UV-Vis spectrophotometer, namely the 8th, 14th, 15th, 16th, 17th, and 34th absorption peaks, allowing the kind of pigment to be determined. Figure 1 presents the visualization of the compound structure of the pigment types produced by the HPLC analysis.\n\nThe value of the bond energy is generated from the total final intermolecular energy, namely from van der Waals bonds, hydrogen bonds, de-solvation energy, and electrostatic energy, then from the final total internal energy, tor sional energy and unbound system’s energy. This summa tion of energy is usually known as the estimated free energy of binding (EFEB). The inhibition constant was also estimated by using Autodock 4.2.6 at 298 K. The low value of the inhibition constant was related to the biological activity.29\n\nThe carotenoids discovered by HPLC (Figure 1) were used as test ligands for binding proteins that contribute to skin aging. According to Table 4, after compared to other carotenoid compounds, adonixanthin had the lowest binding energy with -6.61 kcal/mol and the highest inhibition constant with 14.23 mM. The binding energy of these carotenoids, however, is not better than the native ligand (-7.83 kcal/mol with the lowest inhibition constant of 1.81 mM). The visualization of molecular docking results is shown in Figures 2-4.\n\nAccording to the results of the interaction between carotenoids and the protein elastase, carotenoid pigments have a higher binding energy than natural ligands, which only have a binding energy of -5.55 kcal/mol and an inhibition constant of 85.31 mM (Table 5). Adonixanthin and astaxanthin have the highest binding energies, with binding energies of -9.03 kcal/mol and -9.01 kcal/mol, respectively.\n\nThe carotenoid compounds from G. albolineatus had a lower binding energy than the natural hyaluronidase ligand, which had a binding value of -4.17 kcal/mol. Astaxanthin had a binding energy of -2.27 kcal/mol and is only bound to four amino acid residues, whereas didehydroastaxanthin had a binding energy of -7.71 kcal/mol (Table 6).\n\n\nDiscussion\n\nG. albolineatus is a large crab with a large population along the rocky intertidal zone.30 G. albolineatus can be found off the coast of Kalasey, Manado Bay, North Su-lawesi, in Indonesia. Carotenoid pigments, such as carotene, echinonen, and cantaxanthin, are found in hemocyanin, hepatopancreas, epidermal layer, and outside the carapace in male G. albolineatus from Indonesia.17\n\nBased on HPLC results from the carapace of the female G. albolineatus, several types of carotenoids were found, including didehydrodiastaxanthin, tetrahydroastaxanthin, dihydroastaxanthin, diatoxanthin, astaxanthin, and adonixanthin. Identification of carotenoids from female G. albolineatus is a novelty.\n\nCarotenoids provide effective antioxidant protection against peroxyl radicals during the photo-oxidation process. Carotenoids have a maximum wavelength of 450 nm due to their compound structure.11,31 Several studies have shown that people with high levels of carotenoids in their skin have skin that looks younger than their age, while those with low levels of carotenoids have skin that looks older.32 This is presumably because skin aging is linked to UV radiation, and carotenoids are thought to be effective radical scavengers.33\n\nSkin exposition to high levels of free radicals can activate collagenase, elastase, and hyaluronidase enzymes which cause a decrease in collagen, elastin, and hyaluronic acid, causing aging of the skin. Molecular docking studies have been used to figure out how carotenoids suppress the enzymes collagenase, elastase, and hyaluronidase, and Table 5 show the binding energy of each component. Figures 1-3 show an image representation of the bonding that occurs. Native inhibitors had a higher binding energy in collagenase protein (1cgl) than carotenoids derived from G. albolineatus. This is because the native nhibitors interact with the amino acid residues His222, His218, and His228 via hydrogen interactions.29,34\n\nMeanwhile, the interaction between these residues in the interaction between carotenoid compounds and the collagenase receptor is only found in hydrophobic interactions. Torsion energy is another factor that influences binding energy. A positive value for the torsional energy between carotenoids’ interactions that is too large is thought to have an effect on the small binding energy produced. However, adonixanthin (Table 4) has a relatively high binding energy and an inhibitory value of 14.23 mM, which determines the bioavailability of this component for use as a drug29 (Figure 2 and 3).\n\nAccording to the literature, elastases are serine proteases whose main function is the cleavage of peptide bonds of many proteins, including elastin, which is responsible for the elasticity of connective tissues and is primarily found in the lungs, arteries, and ligaments.35,36 It has been identified that binding to Tyr35, His40, and Val216 has a significant influence on the strength of the binding energy between the ligand and the target receptor in the molecular docking interaction between carotenoids and elastase. The docking results revealed that astaxanthin and adonixanthin had the highest binding energies, i.e. -9.01 and -9.03 kcal/mol, respectively. Based on binding energy results, the carotenoid compounds from G. albolineatus in Table 6 can be used as elastase inhibitors when compared to native inhibitors (Figure 3).\n\nIn the interaction that occurs between the complex ligand and protein hyaluronidase, from the results when compared with native inhibitors, residues Tyr227, Phe46, and Pro18 are thought to have an effect on increasing the binding energy of the complex formed, and hydrophobic bonds are preferred. The absence of interaction between native and Pro18 reinforces that the interaction that occurs has a major influence on the resulting bond energy. In addition, astaxanthin only produced a binding energy of -2.27 kcal/mol. As a result of this discovery, it is possible to conclude that astaxanthin has an excessively large inhibition constant, resulting in a low bioavailability value.9 Meanwhile, other carotenoids with high binding energy, such as didehydroastaxanthin, tetrahydroastaxanthin, diatoxanthin, and adonixanthin, have the potential to be used as natural inhibitors of hyaluronidase protein. Because the test ligands’ interactions are anchored in the same location as the active site, these compounds can be used as competitive inhibitors of collagenase, elastase, and hyaluronidase proteins.\n\n\nConclusions\n\nThe pigment content of the carapace was 4.46 mg based on quantitative measurement of total pigment extraction in female G. albolineatus. Qualitatively, the carapace was recognized as containing carotenoid pigments such as didehydroastaxanthin, tetrahydroastaxanthin, dihydroastaxanthin, diatoxanthin, and astaxanthin based on HPLC. Carotenoids are naturally occurring antioxidants that play a vital role in skin regeneration. According to molecular docking results, carotenoid compounds from G. albolineatus are stronger inhibitors than native inhibitors, which competitively bind to the same catalytic residues on elastase and hyaluronidase protein. However, in vivo and in vitro studies are required to confirm this approach so that this data may be utilized as a contender for medication prospects, particularly in the cosmetic area.", "appendix": "Data availability\n\nFigshare: Potential Carotenoids as Anti-aging from Female Grapsus albolineatus, https://doi.org/10.6084/m9.figshare.19947758 37\n\nThis project contains the following underlying data:\n\n- hasil docking-Dr. darus.xlsx\n\n- dock1cgl-native.dlg\n\n- dock4ym9-native.dlg\n\n- dockasta1cgl.dlg\n\n- dockasta4ym9.dlg\n\n- dockbeta1cgl.dlg\n\n- dockbeta4ym9.dlg\n\n- dockdehydro1cgl.dlg\n\n- dockdehydro4ym9.dlg\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 express our deepest gratitude for the financial support of Sam Ratulangi University, Manado, Indonesia, through the Excellent University Fundamental Research (RDUU) Scheme, Fiscal Year 2019\n\n\nReferences\n\nWang M, Luo Y, Wang T, et al.: Artificial skin perception. 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Publisher Full Text" }	[ { "id": "159375", "date": "02 Feb 2023", "name": "Mosad A Ghareeb", "expertise": [ "Reviewer Expertise Chemistry of natural products and their biological applications" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current study includes the chemical characterization of female Grapsus albolineatus for the presence of bioactive carotenoids including didehydroastaxanthin, tetrahydroastaxanthin, dihydroastaxanthin, diatoxanthin, astaxanthin, and adonixanthin. The study also deals with evaluation of these carotenoids as natural inhibitors of proteins that cause premature aging, such as glucogenase, elastase, and hyaluronidase enzymes via molecular modeling like docking.\nThe article is well organized, the introduction is sufficient, the methods and tools used are sufficient, and the references are recent and appropriate to the subject of the study.\nMoreover, the study is considered a new addition to the field of drug discovery from natural sources as a safe alternative to synthetic drugs.\nWe recommend in the future that these compounds should be re-evaluated in vivo using experimental animal models and conducting further toxicological investigations.\n\nAbstract:\nThe section ''The purpose of this research was to find out what kinds of carotenoids are found in G. albolineatus and whether they can act as natural inhibitors of proteins that cause premature aging, such as glucogenase, elastase, and hyaluronidase enzymes'' should be located under the sub-title ''Aim''.\n\nBinding energy range should be mentioned in the docking results.\nMethods:\nTotal pigment extraction\nThis section should be reinforced by a relevant citation.\nHigh performance liquid chromatography analysis\nThis section should be reinforced by a relevant citation.\nResults:\nTable 1: The column title ''pigment type'' should be written as ''Pigment type''.\n\nConclusion:\nThe conclusion should be supported by the results.\nAbbreviations:\nList of abbreviations should be inserted by the end of the manuscript before references.\nReferences:\nAll scientific names species should be written in italic fonts:-\nRef. 10: euphorbia retusa. Ref. 17: grapsus albolineatus. Ref. 30: grapsus albolineatus.\n\nThe first letter of genus name should be written in uppercase letter:-\nRef. 10: euphorbia retusa. Ref. 17: grapsus albolineatus. Ref. 30: grapsus albolineatus.\n\nThe word ''in vitro'' should be written in italic font.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "9311", "date": "06 Feb 2023", "name": "Darus Paransa", "role": "Author Response", "response": "Thank you very much reviewing the manuscript. We really appreciate your time and assessment, which will help us to maintain quality of our contents." } ] } ]	1	https://f1000research.com/articles/11-1457
https://f1000research.com/articles/12-406/v1	14 Apr 23	{ "type": "Research Article", "title": "Cell-type classification of cancer single-cell RNA-seq data using the Subsemble ensemble-based machine learning classifier", "authors": [ "David Chen", "Parisa Shooshtari", "David Chen" ], "abstract": "Background The advent of single-cell RNA sequencing (scRNA-seq) has provided a high-resolution overview of the cellular heterogeneity of different tissue types. Manual cell type annotation of gene expression datasets remains a useful but time-intensive task. Ensemble machine learning methods leverage the predictive power of multiple classifiers and can be applied to classify high-dimensional gene expression data. Here, we present a novel application of the Subsemble supervised ensemble machine learning classifier used to classify novel cells with known cell type labels using gene expression data. Methods First, we tested the classification performance of different pre-processing steps used to normalize and upsample the training dataset for the Subsemble using a colorectal cancer dataset. Second, we conducted a cross-validated performance benchmark of the Subsemble classifier compared to nine other cell type classification methods across five metrics tested, using an acute myeloid leukemia dataset. Third, we conducted a comparative performance benchmark of the Subsemble classifier using a patient-based leave-one-out cross-validation scheme. Rank normalized scores were calculated for each classifier to aggregate performance across multiple metrics. Results The Subsemble classifier performed best when trained on a dataset that was log-transformed then upsampled to generate balanced class distributions. The Subsemble classifier was consistently the top-ranked classifier across five classification performance metrics compared to the nine other baseline classifiers and showed an improvement in performance as the training dataset increased. When tested using the patient-based leave-one-out cross-validation scheme, the Subsemble was the top-ranked classifier based on rank normalized scores. Conclusions Our proof-of-concept study showed that the Subsemble classifier can be used to accurately predict known cell type labels from single-cell gene expression data. The top-ranked classification performance of the Subsemble across two validation datasets, two cross-validation schemes, and five performance metrics motivates future development of accurate ensemble classifiers of scRNA-seq datasets.", "keywords": [ "Subsemble", "scRNA-seq", "machine learning", "deep learning", "supervised", "classification", "ensemble", "cell-type annotation" ], "content": "Introduction\n\nTranscriptomic analyses are an increasingly important method to characterize biological processes, cell lineages, and the genetic profiles of cell states by quantifying all of the RNA in a cell population (Wirka et al., 2018). The advent of single-cell RNA sequencing (scRNA-seq) has increased the resolution to which analyses of cell type heterogeneity can be used to identify rare cell populations, reveal gene regulation networks, as well as trace cell lineages and map their fates (Hwang et al., 2018; VanHorn & Morris, 2021). Moreover, the growth in the number of scRNA-seq datasets and the resulting need to annotate cell type labels for each cell in these datasets remains a time and labor-intensive task. Automation of cell type annotation has previously been addressed using supervised (e.g. scPred (Alquicira-Hernandez et al., 2019), ACTINN (Ma & Pellegrini, 2019), LAmbDA (Johnson et al., 2019)), semi-supervised (e.g. CALLR (Wei & Zhang, 2021), semiRNet (Dong et al., 2022), scNym (Kimmel & Kelley, 2021)) and unsupervised (e.g. ScType (Ianevski et al., 2022), UNIFAN (Li et al., 2022), cellVGAE (Buterez et al., 2022)) machine learning methods.\n\nStandalone machine learning classifiers and classical statistical methods used to annotate cell types may not be able to de-noise and fully leverage the complex parameter space from high-dimensional scRNA-seq datasets in order to make accurate, robust predictions (Asada et al., 2021). In comparison, deep learning classifiers are prone to overfit on training data, especially in cases where imbalanced classes lead to increased classification performance when tested on majority classes while trading for reduced classification performance when tested on minority classes (Oller-Moreno et al., 2021). Data augmentation using statistical techniques such as Synthetic Minority Oversampling Technique (SMOTE) (Chawla et al., 2002) and more complex generative adversarial network models (Marouf et al., 2020) can be used to generate realistic observations needed to correct for class imbalance that may lead to suboptimal machine learning classification performance (Krawczyk, 2016).\n\nEnsemble learning involves aggregating predictions from multiple individual machine learning classifiers to improve overall classification performance (Dietterich, 2000). Moreover, ensemble classifiers can be optimized by weighting the contribution of individual classifiers to the final prediction based on their historical performance (Akhter et al., 2021). For instance, previous studies have shown that ensemble machine learning models outperform individual machine learning classifiers when tasked with detecting doublets (Xiong et al., 2022), predicting RNA velocity (Wang & Zheng, 2021), clustering cells (Geddes et al., 2019), and impute read counts of dropout events (Lu et al., 2021) using scRNA-seq data as input.\n\nThe Subsemble is a subset ensemble prediction method that has been proposed to be scalable to large datasets by training individual classifiers on data subsets in parallel, employs a multi-layer model architecture that re-weights the contribution of individual classifiers to optimize the final prediction, and has been shown to consistently outperform individual classifiers across multiple test datasets (Sapp et al., 2014). The Subsemble improves model fit to the training data by combining subset-specific fits from multiple individual classifiers into a single meta prediction (Sapp et al., 2014).\n\nHere, we introduce a novel proof of concept of the Subsemble supervised classifier coupled with a feature normalization and upsampling pipeline to accurately predict cell type labels from single-cell gene expression data (RRID: SCR_022784). First, we identified a novel feature normalization and upsampling pre-processing pipeline that optimized classification performance of a baseline support vector classifier. Second, we compared the supervised classification performance of the proposed pre-processing pipeline and Subsemble classifier to predict cell type labels from gene expression data against nine other machine learning classifiers, deep learning classifiers, and an existing cell type annotation method using a 10-fold stratified cross-validation scheme and five classification performance metrics. Third, we tested if information loss from the training data when using five-fold, 10-fold and 20-fold stratified cross-validation schemes affected classifier performance. Fourth, we benchmarked classifier performance when trained on gene expression data associated with one subset of patients and trained on a second subset of patients using a leave-one-out cross-validation scheme within the same dataset.\n\n\nMethods\n\nTwo scRNA-seq datasets with cell type labels were used to evaluate the classification performance of the proposed Subsemble classifier compared to existing machine learning-based classifiers. Each dataset was formatted as one cell by gene expression matrix and one table that mapped each cell to a known cell type label. The two datasets were downloaded using the TMExplorer tool (Christensen et al., 2022).\n\nThe first scRNA-seq dataset from Li et al. (2017) was sourced from 11 primary colorectal tumors. The dataset consisted of the read count matrix associated with 359 cells and 57,241 genes as well as the cell type label of each cell. Cells were labelled with one out of five possible cell type labels. This dataset was used to test if different data pre-processing steps of the model training dataset, such as log-transformation and Synthetic Minority Oversampling Technique (SMOTE) upsampling, would increase Subsemble classification performance compared to training the same model with the raw read counts.\n\nThe second dataset from van Galen et al. (2019) was sourced from 40 bone marrow aspirates of 16 acute myeloid leukemia patients. The dataset consisted of the read count matrix associated with 22,284 cells and 27,899 genes as well as the cell type label of each cell. Cells were labelled with one out of six possible cell type labels. First, this dataset was used to evaluate the 10-fold, stratified, cross-validated performance of the proposed Subsemble classifier across five different performance metrics compared to nine different machine learning and deep learning-based classifiers. Second, this dataset was also used to evaluate the leave-one-out cross-validated performance of the proposed Subsemble classifier when trained on a dataset subset associated with all but one patient and tested on a dataset associated with the hold-out patient.\n\nEach of the two scRNA-seq expression datasets was reduced to 100 principal components using principal component analysis, log-transformed, and upsampled using SMOTE to generate balanced classes based on cell type labels.\n\nThe proposed Subsemble model is a supervised ensemble-based classifier that partitions the training scRNA-seq expression dataset into 10 subsets, outputs the probability that each cell belongs to a known cell type class assigned by one base layer of multiple different machine learning and deep learning classifiers on each subset, and fits one meta layer consisting of one machine learning classifier on the cell type class probabilities (Figure 1) (Chen & Shoostari 2022a). The base layer consists of one XGBoost classifier, one Random Forest classifier, one Multi-layer Perceptron classifier, and three Support Vector classifiers initialized with three different kernels (linear, third-degree polynomial, and radial base function). The meta layer consists of one Support Vector classifier initialized with the radial base function kernel. The trained Subsemble model can then be used to predict the cell type of an unknown cell from expression data (see Figure 1 for model architecture schematic). The Subsemble model does not predict class probabilities associated with each cell.\n\nA single cell gene expression matrix with known cell type labels is used to train the Subsemble classifier. First, the training dataset is randomly partitioned into 10 random subsets. Second, the Subsemble classifier is trained individually on each subset and the predictions from each subset are pooled together to make a final cell type prediction when used to predict cell type from the testing dataset. Four types of base learners, including XGBoost (XGB), Random Forest (RF), Multi-layer Perceptron (MLP), and Support Vector Classifier (SVC), are trained on each subset of the training dataset. The output of each base machine learning classifier is the class probability that the cell belongs to each of the known cell type labels. Next, the meta learner Support Vector Classifier (SVC) is trained on the output class probabilities from the base learners for each cell across the 10 random subsets of the training dataset. Third, a single cell gene expression matrix without known cell type labels is used to test the Subsemble classifier. The Subsemble classifier will make a single prediction of a known cell type label observed in the training dataset.\n\nFive performance metrics were used to evaluate the classification performance of the proposed Subsemble classifier compared to known machine learning classifiers such as Naïve Bayes (NB), AdaBoost (ADA), Decision Tree (DT), K-nearest Neighbors (KNN), Random Forest (RF), and Support Vector Classifier (SVC), an extreme gradient boosting classifier (XGBoost), a deep learning classifier (Multi-layer Perceptron), and a deep learning-based annotation method (ACTINN). Classification performance metrics associated with classifier were generated for each fold in the dataset cross-validation scheme and repeated five times. The same random seed was used to generate the same folds in each cross-validation scheme. The same dataset pre-processing steps were used to process the input data used to benchmark each classifier and ensure that the difference in classification performance can be attributed to the classifier used. Classification performance metrics are reported as the median value across all repeats and folds of the cross-validation scheme used.\n\n‘Accuracy’ measured the number of correct predictions made by the classifier divided by the total number of predictions made. ‘Precision’ measured the number of true positives divided by the number of true positives and the number of false positives. ‘Recall’ measured the number of true positives divided by the number of true positives and the number of false negatives. ‘F1-score’ measured the product of precision and recall multiplied by two, then divided by the sum of precision and recall. Matthew’s Correlation Coefficient was used to measure classifier performance (Matthews, 1975) and is known to fairly represent classifier performance tested on imbalanced datasets (Chicco et al., 2021).\n\nTo rank the classification performance of the nine baseline classifiers and the proposed Subsemble classifier across the five classification performance metrics, we employed a rank normalized scoring approach previously used to benchmark the performance of machine learning classifiers when trained and tested on drug discovery (Korotcov et al., 2017) and raman spectroscopy datasets (Chen, 2021). Rank normalized scores for each classifier were calculated by first ranking each classifier by their performance in each metric, where a classifier with a lower rank represents relatively increased performance compared to a classifier with a higher rank. Next, the ranks for each metric were summed together for each classifier and ordered from lowest to highest based on the rank sum, where a classifier with a lower rank sum represents relatively increased performance across multiple performance metrics compared to a classifier with a higher rank sum.\n\n\nResults\n\nTo optimize the classification performance of the pre-processing steps used, we tested the effect of using raw read counts, upsampled read counts using SMOTE, to create balanced class distributions, log2-transformed read counts, and both log2-transformed and upsampled read counts from the Li et al. colorectal cancer dataset on five metrics used to measure the performance of the Subsemble. Using five repetitions of a 10-fold stratified cross-validation technique to train and test the Subsemble, we found that the median accuracy of 94.44% across all folds and repetitions was highest when training and testing the Subsemble using log-transformed and upsampled read counts (Figure 2). In comparison, the median accuracy when using log-transformed read counts was 93.44%, followed by a drop-off in accuracy when using upsampled read counts (88.89%) and raw read counts (88.57%). Likewise, the Subsemble performed best in precision (94.61%), recall (94.44%), F1 score (93.62%), and Matthew’s Correlation Coefficient (86.41%) when trained and tested using log-transformed and upsampled read counts compared to other read count pre-processing methods. Moreover, log-transformation and SMOTE upsampling of read counts generally resulted in decreased variance in classification performance across each of the five performance metrics compared to log-transformation alone or when using raw read counts. Rank normalized scores of Subsemble classification performance measured across five metrics and tested using four different pre-processing pipelines showed that the log2-transformation and SMOTE upsampling of raw read counts consistently ranked the highest among other pre-processing pipelines (Supplementary Table 1) (Chen & Shoostari, 2022b).\n\nThe Subsemble classifier shows higher classification performance when trained and tested on single cell expression data that has been pre-processed using log2-transformation for data normalization and SMOTE upsampling to generate balanced class proportions compared to pre-processing with each step alone and to no pre-processing conducted (raw read counts).\n\nFirst, we benchmarked the classification performance of the proposed Subsemble classifier compared to nine machine learning classifiers, deep learning classifiers, and an existing cell type annotation method, using the van Galen et al. acute myeloid leukemia dataset and a ten-fold stratified cross-validation scheme for classifier training and testing. We used the log2- transformation for data normalization and SMOTE upsampling to generate balanced class distributions for the training datasets used to train each classifier in the cross-validation benchmarks using the van Galen et al. acute myeloid leukemia dataset.\n\nThe Subsemble classifier consistently performed with the highest performance across all metrics, including median accuracy (88.65%), median precision (88.9%), median recall (88.65%), median F1 score (88.64%), and median Matthew’s Correlation Coefficient (85.81%) (Figure 3A). The ACTINN deep learning cell type annotation method was the second highest ranked classifier based on median performance across all five metrics, followed by the XGBoost classifier and Support Vector classifier. The variance of each metric measured from five repetitions of the cross-validation scheme and ten folds in each scheme was comparable between the Subsemble and other classifiers, with the exception of the AdaBoost classifier which showed a marked increase in performance variance across different folds.\n\nA) Classification performance measured using five metrics across all folds of the 10-fold cross-validation scheme. B) Median accuracy of cell type-specific predictions across all folds in the cross-validation scheme. C) Median F1 score of cell type-specific predictions across all folds in the cross-validation scheme.\n\nRank normalized scores ranked the Subsemble as the top performing cell type classifier, followed by the ACTINN deep learning annotation method, XGBoost classifier, and Support Vector Classifier (Supplementary Table 2) (Chen & Shoostari, 2022b). To test whether different classifiers showed biases in class-specific classification performance, we calculated the accuracy and F1 score metrics associated with each of the six cell type labels included in the van Galen et al. acute myeloid leukemia dataset (Figure 3B and C, respectively). We observed that the Granulocyte Monocyte Progenitor (GMP) cell type was a minority class and consistently showed relatively decreased median accuracy and median F1 score across all classifiers. Moreover, we noted that the Subsemble generally performed comparably or out-performed other classifiers when tasked with classification of all cell types except for the GMP cell type.\n\nSecond, we tested if the number of folds used for the stratified cross-validation scheme including five folds, 10 folds, and 20 folds, affected the median accuracy and F1 score performance of the proposed Subsemble classifier and nine other classifiers and annotation methods (Figure 4A and B, respectively). We expected that as the number of folds increased and a larger proportion of the entire dataset was used for training the classifiers, there would be an increase in the classification performance when tested on the hold-out fold of the dataset. We observed that as the number of folds in the stratified cross validation scheme increased from five-fold to 20-fold, there was a marked increase in the median accuracy and median F1 score for the Subsemble (+3.67% accuracy, +3.97% F1 Score), K-nearest neighbors (+5.97% accuracy, +5.21% F1 score), Naïve Bayes (+2.40% accuracy, +2.54% F1 Score) classifiers and AdaBoost (+2.57% accuracy, +2.19% F1 score) classifiers.\n\nA) Median accuracy performance across all folds of the five-fold, 10-fold, and 20-fold cross-validation scheme. B) Median F1- core performance across all folds of the five-fold, 10-fold, and 20-fold cross-validation scheme.\n\nRank normalized scores of median accuracy, aggregated across accuracy benchmarks using a five-fold, 10-fold, and 20-fold stratified cross-validation scheme, showed that the Subsemble was the top performing cell type classifier when trained and tested using five, 10, or 20 folds, followed by ACTINN, XGBoost, and SVC (Supplementary Table 3) (Chen & Shoostari, 2022b). We noted that ACTINN was the top performing cell type classifier based on median F1 score when tested using a five-fold stratified cross-validation scheme, while the Subsemble was the top performing cell type classifier based on median F1 score when tested using a 10-fold and 20-fold stratified cross-validation scheme. This shows that the ACTINN neural network based classifier may perform well in scenarios where large training datasets are not available, but the Subsemble continues to outperform individual classifiers as the training dataset size increases. Rank normalized scores of median F1 score showed that the Subsemble was the top performing cell type classifier, followed by ACTINN, XGBoost, and Support Vector Classifier (Supplementary Table 4) (Chen & Shoostari, 2022b).\n\nThird, we compared the classification performance of the Subsemble classifier to the nine other classifiers and annotation methods when trained on the van Galen et al. acute myeloid leukemia data associated with all but one patient and tested on data associated with the hold-out patient using a leave-one-out cross-validation scheme based on patient. The Subsemble classifier reported the highest median accuracy (87.00%), median precision (94.27%), median recall (85.72%), median F1 score (87.79%), and median Matthew’s Correlation Coefficient (59.97%) (Figure 5). We noted that Subsemble generally showed the least variance in each performance metric across different folds as well as the smallest range compared to the nine other classifiers and annotation methods. Rank normalized scores aggregated across all five performance metrics for each classifier showed that the Subsemble was the top-ranked cell type classifier, followed by Support Vector Classifier, XGBoost, and ACTINN (Supplementary Table 5) (Chen & Shoostari, 2022b).\n\n\nDiscussion\n\nSingle cell RNA sequencing is a powerful method to analyze the transcriptome of a tumor sample at single cell resolution, providing an overview of cellular heterogeneity based on cell type and cell state. To automate the time and labor-intensive task of manual cell type annotation, we employed the Subsemble ensemble classifier trained on known cell types to accurately classify cells based on single cell gene expression data as a novel application of the Subsemble. In our proof-of-concept study, we showed that the log-transformation and SMOTE-upsampling pre-processing pipeline coupled with the Subsemble classifier consistently performed as the top-ranked classifier across five classification performance metrics, two different validation datasets, and two different cross-validation schemes.\n\nData normalization using log-transformation (Lytal et al., 2020) and upsampling of RNA sequencing data using SMOTE (Yap et al., 2021) have previously been reported to individually improve performance in classification tasks. This study uniquely applied both pre-processing steps to transform gene expression data from single cell RNA sequencing and showed that using both pre-processing steps increased Subsemble classification performance compared to each standalone step or using raw read counts as the training dataset input. The improvement of each of the five classification performance metrics when using both pre-processing steps to transform the Li et al. colorectal cancer dataset suggests that log-transformation and SMOTE upsampling may be useful for scaling and generating balanced class proportions recommended for training machine learning classifiers (Johnson & Khoshgoftaar, 2019).\n\nPrevious studies have shown that ensemble methods were superior in overall classification performance and tend to perform at least comparably if not better than individual machine learning classifiers (Dietterich, 2000; Zhao et al., 2020). The higher overall classification performance and lower variance of each performance metric suggest that the Subsemble is more consistent in its predictions across the entire test dataset. Moreover, as we increased the number of folds in the stratified cross-validation scheme, we observed that the Subsemble increased in median accuracy and F1 score which suggests that Subsemble performance can be further improved with larger training datasets. Finally, the accurate cell type predictions made by the Subsemble when trained on gene expression data and cell type labels associated with all patients but one and tested on the hold-out patient suggest that the Subsemble can be applied to cell type prediction of unlabelled gene expression data when trained on data generated from the same experiment and technical conditions.\n\nAcross the different performance benchmark tests, we noted that the individual Support Vector machine learning classifier and XGBoost boosting classifier were generally top-performing cell type classifiers across each of the performance metrics. The Support Vector machine learning classifier is computationally efficient and generalizable to a variety of classification tasks, including high-dimensional single cell RNA sequencing datasets (Karamizadeh et al., 2014). The XGBoost classifier is an ensemble gradient boosting model that is faster in training time than other implementations of gradient boosting and uses an iterative approach to continuously refine classification predictions (Santhanam et al., 2017). By incorporating both the Support Vector and XGBoost classifier as base learners in the Subsemble, our proposed model takes advantage of both classifiers’ strengths to optimize the final cell type prediction.\n\nThe performance of supervised machine learning-based cell type classifiers depends on the number of cell type observations and diversity of cell types within the training dataset. The poor performance of the Subsemble in the classification of the minority GMP cell type in the van Galen et al. acute myeloid leukemia dataset, alike to the other classifiers, requires a larger number of cell type observations with balanced class proportions in the training dataset. Thus, future work will aim to develop a Subsemble classifier pre-trained on an integrated gene expression dataset of multiple different reference cell types, including rare cell types, and sequenced using different technologies (Korsunsky et al., 2019), different tissue subtypes (Travaglini et al., 2020), and different patient clinical conditions to improve classification sensitivity (Mereu et al., 2020). Moreover, further classification performance benchmark studies of SMOTE upsampling compared to random undersampling when applied to high-dimensional data such as scRNA-seq gene expression datasets should be conducted to validate the pre-processing steps used to transform the training datasets for cell type machine learning classifiers (Blagus & Lusa, 2013).\n\nEnsemble methods require multiple individual machine learning classifiers to be trained independently which increases the computational resources and time required to train the ensemble model compared to training standalone classifiers or using statistical methods for cell type classification. We plan to improve the Subsemble classifier by training individual classifiers using multiple different training subsets in parallel and conducting a hyperparameter grid search to optimize both classification performance and training time. Nevertheless, our proof-of-concept study showed that the Subsemble classifier trained on two validation single cell gene expression datasets is an accurate classifier of cell type labels compared to individual machine learning classifiers, deep learning classifiers, and ACTINN. Further benchmark tests comparing the Subsemble classifier to other supervised and unsupervised cell type annotation methods will be conducted.\n\n\nConclusions\n\nIn conclusion, our proof-of-concept study is a novel application of the Subsemble ensemble classifier to supervised classification of scRNA-seq gene expression data. Data normalization and upsampling coupled with the Subsemble classifier showed overall improved classification performance compared to nine other cell type classification methods when tested using five-fold, 10-fold, and 20-fold stratified cross-validation schemes. The Subsemble classifier also showed superior performance when tested using a patient-based leave-one-out cross-validation scheme. The superior classification performance of the Subsemble classifier across two different scRNA-seq gene expression datasets, two cross-validation schemes, and five performance metrics motivates future development of ultra-fast and accurate ensemble cell type classifiers and larger-scale systematic benchmark tests compared to other cell type annotation methods.", "appendix": "Data availability\n\nThe Li et al. and van Galen et al. single cell RNA sequencing gene expression datasets and respective cell type labels used to train and test the Subsemble cell type supervised classifier can be accessed on Gene Expression Omnibus at accessions GSE81861 and GSE116256 respectively.\n\nFigshare: Figure Data. https://doi.org/10.6084/m9.figshare.20484153.v1 (Chen & Shoostari, 2022a).\n\nThis project contains the following underlying data:\n\n- Figure_2 (Folder containing all benchmark metrics calculated after testing the performance of the Subsemble Cell type for figure 2)\n\n- Figure_3 (Folder containing all benchmark metrics calculated after testing the performance of the Subsemble Cell type for figure 3)\n\n- Figure_4AB (Folder containing all benchmark metrics calculated after testing the performance of the Subsemble Cell type for figure 4a and 4b)\n\n- Figure_4C (Folder containing all benchmark metrics calculated after testing the performance of the Subsemble Cell type for figure 4c)\n\nFigshare: Supplementary Table 1-5. https://doi.org/10.6084/m9.figshare.20484144.v1 (Chen & Shoostari, 2022b).\n\nThis project contains the following extended data:\n\n- ST1. Pre-processing_Pipeline_Rank_Normalized_Scores.csv\n\n- ST2. Ten-Fold_CV_Rank_Normalized_Scores.csv\n\n- ST3. CV_Fold_Accuracy_Rank_Normalized_Scores.csv\n\n- ST4. CV_Fold_F1_Score_Rank_Normalized_Scores.csv\n\n- ST5. Patient_LOOCV_Rank_Normalized_Scores.csv\n\nData are available under the terms of the Creative Commons Attribution 4.0 International Public License (CC-By 4.0).\n\n\nReferences\n\nAkhter MP, Zheng J, Afzal F, et al.: Supervised ensemble learning methods towards automatically filtering Urdu fake news within social media. PeerJ. Comput. Sci. 2021; 7: e425. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlquicira-Hernandez J, Sathe A, Ji HP, et al.: Scpred: Accurate supervised method for cell-type classification from single-cell RNA-seq data. Genome Biol. 2019; 20(1): 264. 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PubMed Abstract \| Publisher Full Text\n\nMereu E, Lafzi A, Moutinho C, et al.: Benchmarking single-cell RNA-sequencing protocols for cell atlas projects. Nat. Biotechnol. 2020; 38(6): 747–755. PubMed Abstract \| Publisher Full Text\n\nOller-Moreno S, Kloiber K, Machart P, et al.: Algorithmic advances in machine learning for single-cell expression analysis. Curr. Opin. Syst. Biol. 2021; 25: 27–33. Publisher Full Text\n\nSanthanam R, Nishant U, Raman S, et al.: Experimenting XGBoost Algorithm for Prediction and Classification of Different Datasets. Int. J. Control. Theory Appl. 2017; 9.\n\nSapp S, van der Laan MJ , Canny J: Subsemble: An ensemble method for combining subset-specific algorithm fits. J. Appl. Stat. 2014; 41(6): 1247–1259. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTravaglini KJ, Nabhan AN, Penland L, et al.: A molecular cell atlas of the human lung from single-cell RNA sequencing. Nature. 2020; 587(7835): 619–625. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Galen P , Hovestadt V, Wadsworth MH, et al.: Single-cell RNA-seq reveals aml hierarchies relevant to disease progression and immunity. Cell. 2019; 176(6): 1265–1281.e24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVanHorn S, Morris SA: Next-generation lineage tracing and fate mapping to interrogate development. Dev. Cell. 2021; 56(1): 7–21. PubMed Abstract \| Publisher Full Text\n\nWang X, Zheng J: Velo-Predictor: An ensemble learning pipeline for RNA velocity prediction. BMC Bioinformatics. 2021; 22(10): 419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWei Z, Zhang S: CALLR: A semi-supervised cell-type annotation method for single-cell RNA sequencing data. Bioinformatics. 2021; 37(Supplement_1): i51–i58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWirka RC, Pjanic M, Quertermous T: Advances in transcriptomics. Circ. Res. 2018; 122(9): 1200–1220. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "169736", "date": "25 Apr 2023", "name": "Saskia Freytag", "expertise": [ "Reviewer Expertise Single cell and spatial transcriptomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nChen and Shooshtari present a benchmarking study of automatic cell classifiers. The benchmarking study is technically sound but could be improved in a number of places to increase the study's value to end-users:\nI think for this type of study it would be important to compare the computational requirements of each of the methods. This would allow users to get a feeling for how much extra compute is required to get potentially modest gains in performance.\n\nOne common way of using automatic classifiers is to annotate cells with the most common label assigned by the classifier for other cells belonging to the same cluster. It would be interesting to see how this approach changes the performance.\n\nIt was unclear on how historical performance was used to weight the different methods used in the Subsemble classifier. Where did these come from?\n\nI really liked the last investigation of changing the reference dataset, as this is more closely related to the situation we face in reality. In fact, I would recommend an expansion of this type of investigation and also use reference datasets that are unrelated to dataset of interest (i.e. different study investigating the same disease). This would be much closer to the situation that practitioners face in real life. The drop observed in performance in the test with the hold-out patient points to the effect of the reference dataset on performance and should therefore be discussed and further investigated.\n\nIn general, all datasets used in this benchmarking study are on the small side and do no longer reflect the standard in the field.\n\nIt should be clarified that the original labels in the study where taken as the ground truth and the limitations of this assumption should be discussed.\n\nIt was unclear how exactly the data was processed prior to PCA (i.e. log-transformation of the counts or PCA). I would therefore recommend adding additional 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? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "233659", "date": "24 Jan 2024", "name": "Yue Cao", "expertise": [ "Reviewer Expertise single-cell", "spatial omics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Chen et al. demonstrated a proof-of-concept using the Subsemble model to perform cell type classification on scRNA-seq data. It is shown to outperform nine other common classifiers such as random forest. The authors further showed that upsampling using SMOTE achieved good performance. While the concept is interesting, the study is currently limited to two relatively small and simple datasets and does not have sufficient evidence to robustly support the performance of Subsemble and SMOTE. It is suggested that the authors incorporate a greater number of datasets, more complicated datasets, as well as more imbalance cases to illustrate the power of Subsemble and SMOTE.\n\nSMOTE is a popular technique that deals with data imbalance by upsampling minority classes. In this study, the motivation behind using SMOTE is not clearly articulated. Did the two datasets have clear data imbalance or rare cell types? If so, this needs to be stated in the Methods and Results section.\n\nThe authors stated that “The Subsemble is a subset ensemble prediction method that has been proposed to be scalable to large datasets”. However, the sizes of the two datasets, one with 359 cells and one with 22, 284 cells, are not very large by current standards. Nowadays it is common to see scRNA-seq datasets with millions of cells. Data with a few hundred cells are rarely generated or analysed these days. The authors should consider testing on larger datasets, for example, the Tabula Muris dataset with 1 million cells.\n\nSimilarly, the number of cell types in the two datasets is relatively small, with only 5 and 6 cell types. The author should consider testing on more complicated datasets with a greater number of cell types.\n\nThe advantage of using SMOTE is not clear. In Figure 2, there is no visible difference between the log-transformed count and the log-transformed up sampled count across all five evaluation metrics. One potential reason could be the characteristic of datasets, that they are not imbalanced enough to benefit from up sampling. The authors should consider using alternative cases to better illustrate the advantage of SMOTE. For example, subsampling to create increasingly imbalanced datasets (eg, two cell types at the ratio of 1:10, 2:10, 3:10 etc.) and investigate at what data imbalance ratio does SMOTE significantly increase model performance.\n\nThe authors mentioned the datasets were “reduced to 100 principal components using principal component analysis, log-transformed, and upsampled using SMOTE”. Could the authors explain the rationale for using PCs over using the count matrix? What is the rationale for selecting 100 PCs? Also, why is log transformation written after PCA? The log normalisation should be performed before PCA.\n\nApart from model performance, the cost of running a model is also a key consideration in practice. Would an ensemble model significantly increase the computational resource requirement? The authors should report the running time and memory usage of Subsemble with other models.\n\nIs the work 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": [] }, { "id": "233658", "date": "24 Jan 2024", "name": "Zhixiang Ren", "expertise": [ "Reviewer Expertise deep learning", "bioinformatics", "single cell RNA sequencing data analysis", "drug discovery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this is an interesting work that presents a compelling approach utilizing a Subsemble-based ensemble learning method for cell annotation in scRNA seq data, complemented by SMOTE up-sampling to balance data categories. The manuscript establishes a reliable benchmark to assess the performance differences between the proposed ensemble learning approach and traditional machine learning classification models, convincingly demonstrating the feasibility of your method. However, there are still some aspects that can be improved:\n\nThe manuscript needs to provide more details in the Method section. For the data preprocessing, it would be beneficial to provide parameters and necessary formulas for each step to enhance understanding. The current statement is somewhat brief. The manuscript should also clarify whether the data has been processed with quality control, normalization before up-sampling. Moreover, batch effect is a significant concern in multi-sample analysis, and an evaluation should be included to demonstrate the impact of batch effects on the data.\nFor the model architecture, more detailed algorithmic information is required, including essential formulas and details on hyper-parameter tuning. I also suggest adding ablation experiments to validate that the base learners in the current setup offer optimal performance. Testing the performance impact of removing or replacing models in the base learner could further strengthen your findings.\nComparisons with state-of-the-art methods in cell annotation, such as SingleR and scBERT, are currently missing. It is recommended to include performance comparisons with these methods to highlight the novelty and practical value of your approach. The figure resolution requires enhancement for better clarity and understanding.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-406
https://f1000research.com/articles/12-405/v1	14 Apr 23	{ "type": "Data Note", "title": "Dataset of companies’ profitability, government debt, financial statements' key indicators and earnings in an emerging market: Developing a panel and time series database of value-added tax rate increase impacts", "authors": [ "Mahfoudh Hussein Mgammal", "Ebrahim Al-Matari", "Ebrahim Al-Matari" ], "abstract": "Company profitability is a crucial indicator that can be used for developing and sustaining trust in accounting information and, thus, inefficient capital markets. Companies with good financial statements' key indicators have a more extensive customer base and can diversify their revenue streams, making them more resilient to economic downturns. Assembling and managing taxes is a critical underpinning to protecting a country's financial intensity and developing a country's tax-system. VAT is a primary source of financial gain in developing nations, which differs from economic income in developed countries, where economic income is primarily derived from tax income. In emerging economies, the existing practice requires firms to effectively and efficiently publish annual-reports and indicators on market-websites, as users rely heavily on timely-information and need it to make decisions. However, these practices fell short of expectations, requiring more research. These variables are crucial for most accounting/economics/taxation research models and the lack of easily attainable data in well-known databases (e.g., ARGAAM; DataStream). This article is primarily a dataset for analysing taxation, performance variables, and key financial-statement indicators. The data describes the raw, combined, and filtered information at the company level, such as company profit and government debt in Saudi Arabia. It combines a firm-level panel dataset sample of company profit that its measures scaled by total assets and include: earnings before interest, taxes, decrease and amortisation, earnings before interest and taxes, earnings after taxes and earnings before taxes—moreover, the time series dataset sample of 11 financial statements' key indicators. The dataset results from 494 company-year observations (226-panel data sample and 268-time series data sample) from 2019 to 2020. Data has been collected from taxation reports, corporate annual reports, ARGAAM database, FinBox database, the Trading Economics database and the Tadawul-market website in Saudi Arabia.", "keywords": [ "Earnings", "Government Debt", "Performance", "Taxation", "Shareholders' Equity", "Profitability" ], "content": "Introduction\n\nThe reasoning and setting behind the creation of this dataset are to help the researcher to perform a comparative analysis across different disciplines to study the role of these variables in a specific phenomenon or the factors enlightening value-added tax (VAT) usefulness and effectiveness. For example, estimate the consequences of a VAT rate increase on profitability, government debt etc. This dataset helps complete two of the few studies investigating the VAT effect in the Kingdom of Saudi Arabia (KSA). There was an encouragement to observe the effect of imposing the new 15% VAT on the profitability of nonfinancial Saudi-listed companies. This dataset is in two types: a firm-level panel dataset sample and a time series sample (Mgammal, 2021; Mgammal, Al-Matari, and Alruwaili, 2023).\n\n\nMethods\n\nThis dataset article contains proxies to measure the economic impacts of VAT as used by several prior researchers (Mgammal, 2021). The representatives for measuring the economic effects of VAT were manually collected and built using secondary data obtained from publicly available data from 2019 to 2020. In Table 1, ProFtEBITDA means company profit measured by earnings before interest, taxes, devaluation, and amortisation (EBITDA) and scaled by total assets and data collected from FinBox database tools. Data were hand collected from companies’ tax reports. We specify companies and identify them with inclusion/exclusion criteria, as mentioned in Table 3. The inclusion/exclusion criteria of all data in this article are as follows: we included nonfinancial companies and excluded finance firms. Then we filtered the sample by excluding companies with annual reports unavailable for two years. The fiscal year-end date is not 31/12/of each year, and the accounting period is over 12 months. Consequently, the final dataset is 494 company-year observations (226-panel data sample and 268-time series data sample) from 2019 to 2020. This final sample is the foundation that can be used for analysis in future research. SIZE2020 and SIZE2019 are the mean company size in 2020 and 2019, measured by the natural logarithm of total assets and data gathered from companies’ annual reports. EAT2020 and EAT2019 are the mean company earnings after tax in 2020 and 2019 and were measured by deducting all expenditures and revenue taxes from the business’s revenues. Following prior research, two variables were added, GvD2020 and GvD2019, which meant government debt in 2020 and 2019 and was measured as government debt over the gross domestic product (GDP). Data for this were extracted from the Trading Economics (https://tradingeconomics.com/) database. Regarding the process for accessing the data, the data was hand collated directly from the Trading Economics website using many Saudi Arabia Indicators reports, especially GDP Indicators reports. In this context, the data collected from the Trading dataset are the same inclusion/exclusion criteria as mentioned above, and we started data collection on 23/01/2021 to 30/03/2021.\n\nAs in Table 2 below, an index containing 11 items was included: BALANCE SHEET: total assets, total equity, and liabilities-equity. INCOME STATEMENT: total income, total revenues, total expenses, and net income. CASH FLOW: changes in operation. Activity, changes in investing act, changes in the financing act, and cash at the end of the period. These factors help control for potential impacts when analysing how a VAT increase will affect a firm. We take each into account as each has a component of the likely effect of a VAT increase. Collectively, these classifications were based on data available on the Tadawul market website as we clarify in inclusion/exclusion criteria above. It is a periodic data set of most Saudi registered companies from before the introduction of the new VAT rate of 15% in 2019 to after the introduction of the new VAT rate in 2020. The dataset framework was chosen when the new 15% VAT was introduced due to public access to VAT information for nonfinancial companies. Tadawul requires all public companies to publish their financial statements on the Tadawul website quarterly and annually (https://m5.gs/OXFqam) (Tadawul, 2019-2020). These data in the file can help build additional variables, such as measuring the effect of VAT before and after increasing its rate using unique techniques such as the difference-in-difference (DID) approach and the autoregressive integrated moving average (ARIMA) modelling approach. Nevertheless, since the literature does not provide common definitions or metrics, we leave the creation of these additional variables at the discretion of potential users. Thus, users can use this dataset to create these measurements from their perspective (Baatwah and Aljaaidi, 2021; Mgammal, 2021).\n\nThe dataset included in this article contains three files describing and defining the sample and variables. Excel file 1 consists of all raw and filtered data for the variables for the panel data sample. Excel file 2 depicts time-series and cross-sectional data for nonfinancial firms listed on the Saudi market for the second and third quarters of 2019 and the third and fourth quarters of 2020. Excel file 3 presents the raw material of variables used in measuring the company’s profitability of the panel data sample. The period of this data is selected from an extensive section of registered companies in Saudi Arabia from 2019 before imposing the new VAT rate, 15%, to 2020 after setting the new VAT rate. The major segments are consumer discretionary, information technology, energy, consumer staples, materials, health care, industrials, communication services, real estate, utilities, and financials. The sample framework was chosen due to the time of implementation new 15% VAT rate and the public access to information about VAT if nonfinancial companies, where Tadawul forces all listed companies to publish their financial statements publicly quarterly and annually on the Tadawul website. Financial companies were excluded from the sample framework as they have unique treatments, and some previous studies investigated the effects of VAT on KSA Banks. The final panel data sample framework is 131 listed companies and 268 observations for the time sires’ sample, as depicted in Table 3 below.\n\n* In the Kingdom of Saudi Arabia (KSA), value-added tax (VAT) was first introduced in all industries as a 5% VAT on goods and services as of Jan. 1, 2018, and, because of COVID-19, the Kingdom of Saudi Arabia (KSA) increased the VAT from 5% to 15% on July 1, 2020.\n\nWe utilise balanced panel data as it is a more sensitive measurement of the modifications that could occur between points in time (Cavana, Delahaye, & Sekeran, 2001). Additionally, the outcomes created are more robust, consistent, and stable, enabling a generalisation of the population to be more meaningful and representative. Therefore, the final dataset sample is 226 observations were specified to be eligible for implication in the analyses. It is the basis for the research, i.e. multivariate, bivariate, additional tests and descriptive.\n\nTo recognise and determine the situation of every concept, descriptive statistics were utilised to clarify. Table 4 displays the statistics of descriptive (standard deviation, median, mean, minimum, maximum values and degrees of freedom) for 113*2 = 226 observations of all variables of the panel data sample and 268 observations for the time series sample.\n\n\n\n• This dataset is essential because it covers data on variables rarely overlooked in accounting, taxation and business performance research models, collectively or individually, but appeal to a wide range of stakeholders. For example, it enables capital markets regulators, standard setters, practitioners and users of financial reporting to easily access long-term data to assess the effectiveness and efficiency of VAT in controlling the risk of tax evasion in fast-growth markets.\n\n• This dataset is valuable for interdisciplinary studies investigating the role of these variables in specific phenomena or factors enlightening VAT and company performance effectiveness.\n\n• This dataset is beneficial because it contains data on profitability collected to measure company profit using earnings before interest, taxes, depreciation, and amortisation (EBITDA) and scaled by the total assets data set. Further, the dataset has been arranged into individual and multiple measurements.\n\n• The data allows researchers to scrutinise the influence of VAT increase on various accounting matters, such as corporate governance mechanisms, the performance of companies and the quality of financial reporting. Furthermore, this dataset is valuable to related parties, e.g., investors, stakeholders, tax authorities, decision-makers, managers and market regulators in assessing and reviewing the tax system in Saudi Arabia. This assessment will give them assurance in making different decisions.\n\n• These data can be analysed and/or compared to other emerging economies and G20 countries (https://www.g20.org/en/). It can be used in discussing the tax system and VAT rate regarding which parties are responsible for adding some VAT incentives in the tax system and updating the tax system sideways with VAT implementation to advantage from the effectiveness of VAT in the KSA. Because KSA could be similar to other countries in G20 and Gulf Cooperation Council (GCC).\n\n• The data is also beneficial for studies on VAT incentives efficiency using data in the long term.", "appendix": "Data availability\n\nHarvard Dataverse: Dataset of companies’ profitability, government debt, Financial Statements’ Key Indicators and earnings in an emerging market: Developing a panel and time series database of value-added tax rate increase impacts. https://doi.org/10.7910/DVN/HEL3YG (Mgammal and Al-Matari, 2023).\n\nThe project contains the following underlying data:\n\n• Final Data - Panel dataset- DiB-2.xlsx. (Excel file 1 consists of all raw and filtered data for the variables for the panel data sample).\n\n• VAT-Final-Time seires dataset DIB-2.xlsx (Excel file 2 depicts time-series and cross-sectional data for nonfinancial firms listed on the Saudi market for the second and third quarters of 2019 and the third and fourth quarters of 2020).\n\n• Raw matiral – DiB-2.xlsx. (Excel file 3 presents the raw material of variables used in measuring the company’s profitability of the panel data sample).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nBaatwah SR, Aljaaidi KS: Dataset for audit dimensions in an emerging market: Developing a panel database of audit effectiveness and efficiency. Data Brief. 2021; 36: 107061. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCavana R, Delahaye BL, Sekeran U: Applied business research: Qualitative and quantitative methods. Australia: John Wiley & Sons; 2001.\n\nMgammal MH: Does a Value-Added Tax Rate Increase Influence Company Profitability? An Empirical Study in the Saudi Stock Market. J. Australas. Tax Teach. Assoc. 2021; 16(1): 92–128.\n\nMgammal MH, Al-Matari E: Dataset of companies’ profitability, government debt, Financial Statements’ Key Indicators and earnings in an emerging market: Developing a panel and time series database of value-added tax rate increase impacts [Developing a panel and time series database]. 2023. Publisher Full Text\n\nMgammal MH, Al-Matari EM, Alruwaili TF: Value-added-tax rate increases: a comparative study using difference-in-difference with an ARIMA modeling approach. Humanit. Soc. Sci. Commun. 2023; 10: 121. Accepted. (In press). PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "172936", "date": "24 May 2023", "name": "Saeed Rabea Baatwah", "expertise": [ "Reviewer Expertise I am interested in auditing", "financial reporting", "CSR", "taxation", "and emerging countries." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reviewing this interesting data article. I think that researchers can test interesting issues in the context of VAT and in Saudi Arabia using this dataset. I would suggest to the authors that it will be more reflective of the dataset if the title is updated to \"Dataset of value-added tax, companies’ profitability, government debt, financial statements' key indicators, and earnings in an emerging market: Developing a panel and time series dataset\". Furthermore, I would suggest the authors review the paper, as there are some minor typos. Overall. I expect greater value from this dataset and its potential to help draw economic implications.\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": "9727", "date": "08 Jun 2023", "name": "Mahfoudh Mgammal", "role": "Author Response", "response": "I would like to thank you for this great feedback and suggestions. We agree with you that updating the title to \"Dataset of value-added tax, companies’ profitability, government debt, financial statements' key indicators, and earnings in an emerging market: Developing a panel and time series dataset\" would be more reflective of the dataset. It's also good to hear that the dataset has the potential to draw economic implications. Moreover, we confirmed that we reviewed and corrected the paper for minor typos as per requested. In this regard, we looking forward to seeing the greater value that this dataset can bring to researchers studying VAT and Saudi Arabia." } ] }, { "id": "230650", "date": "08 Feb 2024", "name": "Faozi Almaqtari", "expertise": [ "Reviewer Expertise Governance", "Financial Performance", "Auditing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me chance review of this intriguing data article. I believe that by utilizing this dataset, researchers can explore compelling issues within the realm of value-added tax (VAT) in the specific context of Saudi Arabia. I would kindly suggest that the authors carefully review the paper, as there are a few minor typos that could be addressed. In summary, I anticipate significant value emanating from this dataset, foreseeing its potential to contribute valuable insights and implications for economic considerations.\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": "11036", "date": "03 Apr 2024", "name": "Mahfoudh Mgammal", "role": "Author Response", "response": "Thank you for your thorough review and insightful comments on our data article. We appreciate your attention to detail and your anticipation of the significant value that this dataset holds for researchers interested in the realm of value-added tax (VAT) in Saudi Arabia. We certainly take your suggestion to heart and carefully review the paper to address any minor typos or errors that may be present. Ensuring clarity and precision in our work is essential for facilitating understanding and replication by others in the scientific community. Your confirmation of the clarity and appropriateness of the dataset creation rationale, protocols, technical soundness, and accessibility of the presented datasets is reassuring. We strive to provide comprehensive and transparent documentation to enable replication and further exploration by interested researchers. Once again, we thank you for your valuable feedback and look forward to the potential contributions and insights that this dataset may offer to economic considerations in the context of VAT in Saudi Arabia." } ] } ]	1	https://f1000research.com/articles/12-405
https://f1000research.com/articles/12-400/v1	14 Apr 23	{ "type": "Research Article", "title": "Introducing technology of planting methods to increase rice production for sustainable farmers’ income", "authors": [ "Julian Witjaksono", "Rita Indrasti", "Bungati Bungati", "Jefny B Markus Rawung", "Siti Sehat Tan", "Abdul Gaffar", "Nandang Sunandar", "Enti Sirnawati", "Conny N Manoppo", "Muhammad Assagaf", "Wawan Sulistiono", "Donald Sihombing", "Wahyu Handayati", "Resmayati Purba", "Asmin Asmin", "Rita Indrasti", "Bungati Bungati", "Jefny B Markus Rawung", "Siti Sehat Tan", "Abdul Gaffar", "Nandang Sunandar", "Enti Sirnawati", "Conny N Manoppo", "Muhammad Assagaf", "Wawan Sulistiono", "Donald Sihombing", "Wahyu Handayati", "Resmayati Purba", "Asmin Asmin" ], "abstract": "Background: Rice self-sufficiency is still a great concern in Indonesia; in order to feed the population of 270 million people, farmers must implement new technology to boost rice production. This study aimed to provide information on the differences between direct seed planting and its conventional counterpart in terms of productivity and farmer income. Secondly, we aimed to characterize the productivity and farmer income for two legowo planting systems (legowo 4:1 and legowo 2:1). Finally, farmers’ perceptions of the legowo implementation were evaluated. Methods: This study was carried out in Konawe District at the farm level in Karandu Village, Anggotoa Sub District of Southeast Sulawesi Province, Indonesia. The research design was implemented to allow comparison between legowo 4:1 and legowo 2:1. 60 farmers who implemented legowo 2:1 and legowo 4:1 respectively, were selected. Moreover, 30 farmers who did not implement both 4:1 and 2:1 were selected as the conventional planting method. This research design was implemented in order to compare between legowo and non legowo planting systems, as mentioned above, 60 respondents will compare with 30 farmers in terms of yield and economic advantage. Lastly, in order to determine technological perceptions, a total of 150 farmers were interviewed. Results: The study case results showed that direct seed planting is much more profitable than its conventional counterpart. The introduction of legowo 4:1 was highly significant in boosting rice production compared to legowo 2:1. In terms of perception, we found that the low cost and the availability of legowo are the most important parameters for the adoption of this technology. Conclusions: In summary, we recommend the implementation of the legowo 4:1 planting system to boost rice productivity. By reducing labor costs, the direct seeding method increased farmers’ income.", "keywords": [ "legowo", "rice", "technological innovation", "farmers", "income" ], "content": "Introduction\n\nRice, the main cash crop of farmers, is a strategic and important crop in Indonesia. This staple food is consumed by more than 90% of the population comprising 270 million people with rice consumption has reached 97.40 kg/capita/year. The Indonesian government has launched a program to increase the planting area for food security and rice self-sufficiency, which aims to not only increase the planting area but also boost rice production.1\n\nSoutheast Sulawesi Province is one of the rice production areas supporting this national program. One of the potential areas producing rice in Southeast Sulawesi is Konawe District, with the largest paddy planting area. It has been stated that the rice productivity of this region is still low compared to national productivity due to the low adoption of technological innovations.2 A research study3 unveiled a few procedures that could be executed to expand rice production. One planting design broadly used for rice development in Indonesia is jajar legowo, known as Jarwo Rice Planting System.\n\nThe Jarwo switches back and forth between at least two (usually two or four) crop lines and one free space column. Hence, this planting system makes ideal use of space for development and expands the yield to excess of 160,000 harvested plants per hectare.4 Changes in plant division are one of the most significant agronomic practices for expanding crop yield. Jajar legowo has been well received because of its benefits in crop upkeep and higher number of plants per area.5 Likewise,6 detailed that jajar legowo is a proper innovation that increases rice yield and farmer income compared with the conventional strategy.\n\nThe legowo planting system has been recognized as a technology to grow rice on paddies in a rice farming system. This technology has three patterns of planting: 2:1, 4:1, and 6:1. This study was carried out to determine the resulting productivity and farmer income due to the implementation of the legowo planting system. In this study, we determined and compared the economic advantages of the legowo 4:1 and 2:1 planting systems. Furthermore, to forgo the potentially high cost of the legowo planting system due to labor, we implemented direct seed planting. Thus, as part of the system design, direct seed planting is compared with its conventional counterpart. Lastly, to assess the performance of the legowo planting system, we evaluated perceptions of it among the farmers who implemented the legowo planting system. Information on sensitive attributes was identified in this study and can be used as a consideration for stakeholders in developing strategies to maximize rice productivity.\n\nMore specifically, Konawe district is the region which has implemented legowo planting system widely across the county and adopted rapidly by farmers particularly in Karandu Sub District. The overall goal of this examination was to survey mechanical advancement of the paddy cultivating framework in Karandu County, Anggotoa Sub Region, Konawe Region of the Southeast Sulawesi Province in Indonesia. Specific goals were as follows:\n\n(1) To describe the introduction of direct seed planting technology and a transplanting method;\n\n(2) To identify the economic advantages resulting from the introduction of the legowo 4:1 and 2:1 planting systems;\n\n(3) To identify farmers’ perceptions of the legowo planting system.\n\nThis paper discusses the implementation of technological innovation in terms of transplanting as the conventional method and direct seed planting, the introduction of the 2:1 and 4:1 Legowo planting systems, and the perceptions of farmers who adopted the Legowo planting system. The study’s purpose is to prove that this technology significantly increases rice production and is highly profitable compared to its conventional counterpart.\n\n\nMethods\n\nAn action research approach was implemented to deploy the innovative legowo planting system such that it may spread across the county. This research was designed as three study models: (1) a study to compare between direct seed planting and a transplanting method as its conventional counterpart; (2) an economic study comparing the Legowo 4:1 and Legowo 2:1 planting system; and (3) observations and interviews on farmers’ perceptions of the introduction of the Legowo planting system by open questions based on the interview guide (see extended data).\n\nA purposive sampling was employed wherein 60 farmers were selected at the farm level in Karandu village as respondents who adopted legowo 4:1 and legowo 2:1 planting system, respectively. A total 120 farmers who live in Karandu village and implemented those planting system as the representative of the farmers population have been selected as the respondents. As the comparison between legowo planting system and its counterpart 30 farmers were selected randomly. All the respondents have been interviewed through the questionnaire (see extended data) as the semi structured question.\n\nThe instrument for obtaining information was a semi structured open questionnaire33 for the proficient respondents, while interview schedules were utilized to help the non-instructed or uneducated respondents. The measured variables were rice production and productivity of the farming system when implementing the direct seeding method along with a farm business analysis of the rice farming system, consisting of variable costs such as fertilizer, pesticides, herbicides, and labor costs. Finally, for the third objective, we asked the study farmers about their perceptions of the introduction of the legowo planting system in terms of the profitability, productivity, technological implementation, availability, and cost associated with its introduction.\n\nEconomic analysis of rice with different planting methods was calculated by adding the cost of all input parameters such as the cost of seeds, fertilizers, pesticides, fuel, labors, etc. and cost of operation machinery. Costs of fertilizers, seeds and pesticides were calculated on the basis of the available market price in the corresponding years. Net income (idr/ha) were worked out by subtracting the total cost of cultivation of each planting method from the gross income of respective planting systems that was calculated to ascertain economic viability of different planting method (legowo 2:1, legowo 4:1 and its conventional).\n\nFor this study we used qualitative and quantitative data. The quantitative data was tabulated using Microsoft Excel 2010 (14.0) then analyzed so that relevant inferences could be drawn. The data was analyzed using descriptive statistics. Descriptive analysis has been widely used among studies in various sciences, including economics and sociology.8,9 Moreover, profitability analysis was conducted to analyze production costs and revenues via quantitative analysis. Financial investigations were utilized to decide whether farmers would receive a greater and steady income from the use of innovation and was appraised as to whether the farmer had sufficient capital to adopt the innovations or not.10–12\n\nThis study was approved by the Ethics Board of The Provincial of Research and Development of Southeast Sulawesi (project number 13/2017) as the funding body that content its ethical statement. Written informed consent to participate was obtained before the interviewing process. This project is funded by the provincial government not by The National Research and Innovation Agency.\n\n\nResults\n\nThe direct seed planting system applied in rice paddies is a cultivation technique that does not require an initial nursery phase. This system is most widely used by Indonesian farmers in the cultivation of lowland rice.13 Direct seeding methods have several advantages over transplanting. In addition to their higher economic returns, directly cultivated crops are quicker and simpler to plant, less labor intensive, and require less water.14 In wet fields, direct cultivation should be possible through either transferring or penetrating seeds into the mud using a drum seeder. Drum seeders are utilized for quick planting, and this is broadly executed by farmers at the study site. This tool works best on a very much evened out, smooth, and wet seedbed.13 Seeders might be obstructed if the dirt is tacky or if there is inadequate planning.\n\nDirect seed planting using equipment and pulling by human force is commonly used as an innovation to accelerate the planting season, especially in areas with fewer farm workers. Based on the survey at the field farm, study findings at the farm level in Karandu Village indicated that the conventional method of transplanting is commonly used by farmers. A comparison between direct seed planting and its conventional counterpart in terms of productivity and income can be seen in Table 1.\n\nTable 1 show that the implementation of direct seeding in the farming system results in higher productivity for farmers compared to its conventional counterpart (transplanting method). Moreover, the direct seed method results in higher income in comparison to the transplanting method. The results of the farm business analysis showed that the expenditure in the paddy farming system using the conventional system (transplanting) is IDR 7,230,000 per ha, higher than that using direct seeding (IDR 6,230,000 per ha). Thus, the direct seeding system is able to reduce the cost by IDR 1,000,000 per ha. This fits with the study finding by16 that direct seeding is able to decrease the production cost and lower the labor cost,17,3 while the conventional system (transplanting) has high labor costs associated with the care of seedlings and their planting. Direct cultivation saves on work as it dispenses with the nursery raising and transplanting of seedlings. Further, the work is spread out over a more extended period in direct cultivation than in transplanting, where more work is needed at the time of transplant.3\n\nThe study findings in Table 1 show that rice productivity using direct seeding (drum seeder) is higher than that of using transplanting (6347 and 5196 kg/ha, respectively). The higher grain yield of direct cultivation when contrasted with transplanting is primarily a result of higher panicle number, higher 1000 grain weight, and lower sterility rate.18 Additionally, the greatest overall gain was found for farmers utilizing a drum seeder (IDR 18,332,400) in contrast with transplanting (IDR 14,810,650). The impact of the planting framework on the grain yield, straw yield, cost of development, overall gain, and returns per rupee on resources put into rice developed on sandy dirt topsoil soil were assessed in Bangalore.3 The authors observed that direct cultivation using a drum seeder delivered a higher overall gain of 34,953 rupee per ha and higher returns per rupee (3.12) in contrast with a total compensation of 30,420 rupee per ha and returns per rupee (2.66) recorded in the transplant framework.19 A significant justification of the advantage of direct cultivation for farmers is the increasing expense of development and diminishing benefits with customary work (transplanting). A review of 77 studies in various locations showed that different techniques for direct cultivation have reduced the expense of production by USD 9–125 per ha and required only ordinary work to produce the increased revenue of direct cultivation, as compared with transplanting.19 Farmers probably lean toward more innovative methods that give higher benefits, notwithstanding comparable or somewhat lower yield.7\n\nTo address the gap between rice production and demand, the use of innovations in farming is important as it is one of fundamental prerequisites of rural development.20 Without innovation, agrarian advancement will decline; then, individuals’ requirements for food cannot be met on the grounds that, as per Malthus’ hypothesis, the populace will expand exponentially.21\n\nIn the legowo planting framework, clusters are considered. The legowo column design alternates between at least two lines of rice planting and one clear line. The objective of legowo planting is that the plant populace per hectare can be maintained and even improved.22,23 An investigation by24 discovered that the execution of the legowo planting framework could expand rice production by 35% compared with a non-legowo planting framework. The legowo planting framework is one part of a harvest incorporated into the board framework, which is a development strategy designed by organizing divisions among clusters and between columns so that bunches of rice are compacted in lines, with greater distance between the lines.25\n\nAn analysis of the farming system (Table 2) was carried out to describe grain yield and farmer income when implementing the legowo planting systems, both 4:1 and 2:1, compared with the conventional counterpart. The study findings based on the legowo planting system showed that rice productivity under the 4:1 and 2:1 systems (6850 and 6120 kg/ha, respectively) was much higher than that under the conventional counterpart (5232 kg/ha).\n\nMore specifically, based on the second study model, the study results (Table 2) show that legowo 4:1 is significantly better than Legowo 2:1 based on the evaluated parameters. This is also supported by the study findings,5,26 due to the jajar legowo 2:1 (two lines) planting framework utilizes separation of 25 cm × 12.5 cm × 50 cm; this results in a lower plant number per hectare than that under jajar legowo 4:1 (four columns full), which has plant dispersal of 25 cm × 12.5 cm × 50 cm, resulting in a plant number of ±256.000 clusters/ha. In addition, legowo 4:1 increases the crop population by 60% compared to legowo 2:1 which, in turn, increases the population by 30% in comparison with the conventional method (25 cm × 25 cm). Both planting systems (legowo 2:1 and 4:1) affect the plant population per unit area and the number of productive tillers and could affect crop production. The reason for these changes is also linked to the effect of fringe plants that are expected to be productive, delivering better results. Moreover, in terms of economic advantages, the results show that rice was significantly more profitable for farmers who implemented legowo 4:1 instead of legowo 2:1 or the conventional counterpart (IDR 17,258,850, IDR 15,375,000 and IDR 11,265,335, respectively).\n\nIn addition, based on the financial analysis of the farming system, farmers who implemented the legowo planting system were directly able to increase their farming income and lower the cost of farming production compared with the conventional counterpart; this is in line with the results of research conducted by.23,27 Moreover, our study findings indicated that the legowo planting system was able to increase grain production and increase farmers’ income. This is in line with study results by.5 According to,22 the implementation of the legowo planting system is able to increase the productivity and income of paddy farmers, and it is recommended that it be widely implemented.28 Moreover, a legowo study by25 showed that the adoption of the legowo planting system resulted in an increase in farmer income from IDR 5,926,000 (before adoption) by IDR 912,000 (after adoption).\n\nA recent study assessed farmers’ perspectives on a given innovation regarding their perceived needs and related involvement.29 As indicated by,30 a new innovation should be altered to fit the local interest and specific considerations. Above all, the cost of the new innovation should be comparable to the costs of the currently accessible substitute advances to guarantee quick take-up and increasing use of the new farming innovation. Below, Table 3 portrays farmers’ insights into the legowo planting framework as a development innovation that has been broadly received.\n\nThe study findings (Table 3) show that the farmers’ stated main reason for implementing the legowo planting system is mostly that the technology is easy to implement (91.7%), feasible (80%), and results in high productivity (91.7%), and 100% indicated that the technology is available and cheap. In a study of the application of agricultural innovations, a measurement of farmers’ perceptions of technological innovations in jarwo super rice carried out in Gorontalo Regency indicated that most farmers (80%) agreed or strongly agreed that application of the legowo planting system could increase rice production. This was also evidenced by the average yield of rice with the application of the legowo planting system being higher than that using farmers’ existing technology28 and matches with the study findings that farmers prefer this technology (legowo planting system) due to its high grain yield. In terms of feasibility, the study findings are in line with research by31 that revealed that farmers mostly agree with the Legowo planting system technology because it is proven that this technology results in a higher income than compared with the planting system that was commonly practiced by farmers. Moreover, in terms of the legowo planting system’s ease of implementation, the results of our study are consistent with the results of research by32 Hutapea et al. (2017) and according to28 in terms of ease of application, farmers stated that the components of the legowo technology were easy to understand and easy to apply in the field.\n\n\nConclusions and recommendations\n\nThe first study model herein found that direct seeding using a drum seeder resulted in increased rice productivity compared with the transplanting model. This increases farmer income via a reduction in variable costs, particularly labor costs. In the adoption of the legowo planting system as a technological innovation, the findings were that legowo 4:1 is profitable, increasing farmer income significantly compared to legowo 2:1. Both systems increased farmers’ income and rice productivity compare to their conventional counterpart.\n\nIn the analysis of perceptions, firstly, most farmers adopted the technology due to the easy access of the technology, and it is also important that the implementation of new technology is not costly. Secondly, the technology adopted by farmers should not be complicated. Thirdly, the legowo planting system is able to increase rice production compared to its conventional counterpart.\n\n\nAuthors’ contribution\n\nJ. Witjaksono did conceptualization, funding acquisition, investigation, and writing-original draft preparation. R. Indrasti contributed conceptualization, visualization and writing-review and editing. N. Sunandar did conceptualization and visualization. S. S. Tan interpreted writing-review and editing. Jefny, B. M. Rawung, Conny, N. Manoppo, Donald. Sihombing, and Wahyu Handayati writing-review and editing. A. Gaffar, and E. Sirnawati designed methodology. M. Assagaf and W. Sulistiono conceptualization and writing-review and editing. R Purba, Bungati and Asmin writing-review and editing. All authors read and approved the final manuscript.", "appendix": "Data availability\n\nZenodo. Legowo Planting System, 10.5281/zenodo.7580482. 33\n\nThis project contains the following extended data:\n\n‐ KUISIONER PENELITIAN.doc (Research questionary of legowo planting system and its counterpart).\n\n‐ Panduan Wawancara.doc (Points of question for interview guide).\n\n‐ Data Legowo (Tabular Data)\n\n‐ Ethic Clearance (Informed Consent of Respondent)\n\n‐ Project Approval\n\nData is available under the terms of the: Creative Commons Attribution 4.0 International (CC BY 4.0)\n\n\nAcknowledgements\n\nThank you for our local government of Southeast Sulawesi Province for this research grant and also for supervisor board as the steering committee for the selection process of this project.\n\n\nReferences\n\nMulyaqin T, Yusron M, Siagian V: Energy Analysis of Rice Production in Banten Province, Indonesia. 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Publisher Full Text" }	[ { "id": "204649", "date": "20 Sep 2023", "name": "Sanusi Saheed Olakunle", "expertise": [ "Reviewer Expertise Resource and production economics", "Food Security", "agricultural programmes and policy", "sustainable development and gender issues." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 team should provide details of the analytical tools that are used to arrived at the results stated in the table particularly on productivity and economic advantages of direct seedling compared to counterpart.\nSecondly, the researchers should also endeavour to state categorically in clear terms their recommendations based on their findings. The conclusion and recommendation section in the text only summarizes the conclusion of the authors but no recommendations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "221966", "date": "23 Nov 2023", "name": "Daniel Micheal Okello", "expertise": [ "Reviewer Expertise Agricultural Economist" ], "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 need to revise the analytical approach to show significant difference between technologies/practices. Just presenting figures without any statistical test to show significance makes it difficult to accept the claim that for instance direct seed planting is much more profitable than its conventional counterpart. Yes, there could be differences in the figures, but are they significantly different?\nThe lack of statistical test makes it not possible to comment on the discussions and conclusions from this paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-400
https://f1000research.com/articles/12-396/v1	13 Apr 23	{ "type": "Research Article", "title": "Intrinsic and extrinsic motivational factors of frontline health workers in Ghana", "authors": [ "Moses Aikins", "Samuel Amon", "Samuel Agyei Agyemang", "Patricia Akweongo", "Kaspar Wyss", "Samuel Amon", "Samuel Agyei Agyemang", "Patricia Akweongo", "Kaspar Wyss" ], "abstract": "Background: Human resources are vital for delivering health services, and health systems cannot function effectively without enough skilled, motivated, and well-supported health workers. Even though several studies have addressed job satisfaction among healthcare professionals in different parts of the world, there is limited research examining intrinsic and extrinsic motivational factors involved in job satisfaction, particularly in low-and-middle-income countries. Methods: This was a facility-based quantitative cross-sectional study conducted in 34 primary health care facilities, across three out of the 26 districts in the Eastern Region of Ghana, between February and March 2018, analyzing job satisfaction and underlying factors. Randomly selected public health workers (n=287) from health facilities were included in the study. The two-factor theory was adopted as the measurement model for this study. Results: The odds of staff that had received management training in the past 12 months being intrinsically satisfied was 3.73 (95% CI:2.03,6.86) times higher than staff with no management training (p<0.001). Compared to staff working in hospitals, Community-based Health Planning and Services (CHPS) staff showed lower levels of satisfaction on factors relating to intrinsic motivation. Technical staff had higher odds of being intrinsically and extrinsically satisfied compared to non-technical staff. Conclusions: Public healthcare workers working at primary health facilities in Ghana, at higher levels of service delivery, showed higher satisfaction compared to lower-level staff, both in terms of intrinsic and extrinsic factors underlying satisfaction. Thus, to enhance healthcare services, health facilities’ administrators should take measures to improve the working conditions of staff at all levels of service delivery, since the hierarchy of service delivery in the health system makes the contributory role of all staff critical in improving the quality of primary healthcare.", "keywords": [ "Job satisfaction", "intrinsic", "extrinsic", "primary healthcare workers", "Ghana" ], "content": "Introduction\n\nJob satisfaction has been defined as “the balance between factors of working stressors and rewards”.1 Job satisfaction has been also described as: “A pleasurable or positive emotional state which is the result of someone’s working assessment or someone’s working experience. Job satisfaction results from the perception that [someone’s] job fulfills or allows the fulfillment of important values, given that these values are in accordance with individual needs”.2\n\nPrevious literature on the job satisfaction of health workers has demonstrated that access to training, ability to perform key tasks, recognition and shift schedule were associated with job satisfaction.3–5 A study on developing countries also found that job dissatisfaction results from key attributes such as adverse work environment, leadership problems, inadequate equipment and supplies, lack of recognition, stress due to heavy workloads, and limited opportunities for career development and advancement.6\n\nStudies on intrinsic and extrinsic factors of job satisfaction over the years have shown mixed results. Some studies indicate that both intrinsic and extrinsic factors can be sources of satisfaction and/or dissatisfaction.7,8 Furthermore, both intrinsic and extrinsic motivational factors in various degrees impact the work performance of nurses in Ghana.9 Additionally, Goetz et al. observed that even though both intrinsic and extrinsic issues are essential for dentists, intrinsic motivating factors have the most positive impact on their job satisfaction.10\n\nOther studies have shown that intrinsic and extrinsic factors are important when measuring job satisfaction,11 and intention to leave employment.12,13 However, studies by Djordjević et al. and Ayalew et al. also found out that health workers place a higher value on extrinsic motivation.14,15 Conversely, Tung et al. noted that a healthcare work environment that supports intrinsic motivation and improves work hours may reduce physician burnout and job dissatisfaction.16 In the education sector, private basic school teachers were more dissatisfied with their intrinsic factors of job satisfaction than their counterparts in the public sector.17\n\nIn this study, intrinsic factors are issues that relate to health worker’s need for a sense of self-actualization and self-accomplishment on the job. Intrinsic job satisfaction is derived from multiple intrinsic factors experienced in the job, such as the responsibility, commitment, self-directiveness, and skill development associated with doing the work. On the other hand, extrinsic factors define the external context and reward system within which the health worker carry out roles and responsibilities. Extrinsic factors include working conditions, supervision, support systems, company policies, work recognition, and external rewards such as allowances and salaries etc.\n\nHealth systems cannot function effectively without an adequate number of skilled and motivated health workers.4 Developing highly motivated, skilled and supported health workers is vital in advancing health in every health system and consequently, achieving national and universal health goals.18,19 Thus, work climate and motivated human resources are critical for adequate health service provision.20 The World Health Organization (WHO) estimates that shortages of about 12.9 million health care workers will be encountered by 2035 in South Eastern Asia and Africa (47% and 25%, respectively) and will represent only 1% of health care workers in the Europe region. The same report further postulates that an estimated 40% of health care workers in the world will vacate their jobs in the next decade due to lack of incentives.21\n\nDeveloping countries like Ghana face serious issues with health professionals. Recently, Ghana was included in the list of countries with severe health worker challenges by the World Health Organization (WHO). The WHO further predicted that the persistence of the challenges can significantly affect quality health service delivery.22 The undesirable features of the healthcare systems of many developing countries including Ghana coupled with poor living conditions, lack of professional development opportunities and work overload among other factors, causes health workers to migrate to developed countries,23,24 where they have better remuneration and conditions of work, easy access to technology and opportunities for intellectual growth.25 Driving factors for workforce shortages are insufficient numbers of new health cadres joining the health workforce, as well as losses due to out-of-country migration and abandonment. As a consequence, Ghana is not only losing skilled professional staff but it is also losing investments made in educating and training nurses.26 Further negative impacts of job dissatisfaction include non-conformance with medical and work procedures and policies, increases in work accidents, and organizational conflicts,27 which may increase the rate of medical errors, hence jeopardizing patient safety.28\n\nFew studies have investigated intrinsic and extrinsic job satisfaction at the primary care level in Ghana. Realizing the significance of job satisfaction for health workers’ performance, this study was conducted to assess the overall job satisfaction of healthcare workers in primary healthcare facilities in the Eastern Region of Ghana, to highlight various factors that cause job satisfaction and make recommendations that might improve the current statistics on job satisfaction.\n\n\nMethods\n\nPrior to data collection, ethical approval for the study was obtained from was the Research Ethics Committee of the Liverpool School of Tropical Medicine, United Kingdom (LSTM; ID No.: 17-046) and the Ghana Health Service Ethical Review Committee, Ghana (ID No.: GHS-ERC:009/12/17). As mentioned, written informed consent was also obtained from the study participants. The privacy, confidentiality, and anonymity rights of participants were always observed.\n\nThis was an institution-based quantitative cross-sectional study conducted in 34 primary health care facilities across three out of the 26 districts in the Eastern Region of Ghana, between February and March 2018. The region is located approximately 80 kilometers north of Accra, the capital of Ghana. These selected primary health care facilities provide services for about 343,236 inhabitants. The study is part of the baseline assessment of the PERFORM2Scale project (P2S), which sought to scale up district healthcare management, strengthening intervention.29\n\nThe main characteristics of the PERFORM2Scale project (P2S) study districts are shown in Table 1. Randomly selected public health workers from these health facilities were included in the study. This study included all categories of health staff – clinical and non-clinical, who had served for more than a year in the health facility at the time of the data collection.\n\nThe sample size calculation was based on a published sample size table by Israel (2009). With an estimated number of clinical health staff (i.e., professional groups) within the three districts at 600, a precision level of ±5%, confidence level of 95% and degree of variability of 0.5, and a potential non-response rate of 5%, the estimated sample size of health workers was 287. Apart from the district hospitals which were purposively selected, health centers and Community-based Health Planning and Services (CHPS) were randomly selected using a simple random sampling approach. The calculated sample size was proportionally allocated to the four health facilities. Therefore, the 287 study participants were selected from district hospitals (119), health centres (119), and CHPS (49).\n\nThe study participants were randomly selected. By this, full list or register of clinical and non-clinical staff was obtained from each study facility. Thus, the staff register constituted the sample frame, grouped into clinical and non-clinical staff. To anonymize participants, each eligible staff member was given a code, which was written on a piece of paper and placed in an opaque box, grouped into clinical and non-clinical staff. The required number of staff were then randomly picked for each category. Clinical and non-clinical staff who participated in the study included physicians; nurses and midwives; pharmacists and associated professionals; community health workers and managers. Casuals and auxiliary staff were excluded from the study. The research team contacted selected participants by phone to briefly inform them about the study and seek consent for their participation. To ensure participation, suitable dates for interviews were agreed for participants who consented to be part of the study and follow up confirmation phone calls were made a day before the interviews. When a selected participant refused to be part, he/she was replaced by a randomly selected participant, using the same sampling approach. Overall, three clinical staff approached refused to be part of the study.\n\nData were collected between February and March 2018. A self-administered paper-based questionnaire was used for data collection. The questionnaire was pre-tested with 15 health workers (5% of the planned sample) at a health facility outside of the study area and results were assessed by the researchers. Pre-testing was done with the aim of checking the language, comprehension of the questions, and sequence of question among the participants and over the duration of an interview. Assessment of the pre-test findings confirmed suitability of language used, understanding of questions and estimated interview duration. However, the sequence of sections was fine-tuned to make it more logically fit for purpose. By this, sections pertaining to district and facility levels management as well as specific job satisfaction (Sections III, IV, and V respectively) were moved to the later part of the questionnaire (VII, VIII, and IX respectively).\n\nPrior to data collection, the aim of the study was explained to the participants face-to-face. Written informed consent was obtained from all study participants. To ensure the internal validity of collected data, three research assistants were trained on the data collection tool. All questionnaires were completed in English. Data collected included the background and job characteristics of health workers, and job satisfaction indicators. The reliability and internal consistency of the tool was again checked using Cronbach’s alpha test and was found to be acceptable. The measure of internal consistency of job satisfaction scales found a good internal reliability for overall job satisfaction (α=0.87), intrinsic job satisfaction (α=0.70) and extrinsic job satisfaction (α=0.83). Returned questionnaires were checked for completeness and accuracy every day after field work by the field supervisor. All data were stored in a locked compartment at the University of Ghana School of Public Health, Department of Health Policy Planning and Management - and only the core research team had access to the forms. The underlying and extended data of this study are available on Figshare.52\n\nThe Herzberg two-factor theory30 was modified/adapted as the measurement model for this study (see Figure 1). This theory was chosen because it is one of the most commonly used theories in job satisfaction research.30 The model establishes two distinct sets of factors for job satisfaction. The satisfiers/motivators or the intrinsic factors such as work methods, work variations, skills application, responsibility, commitment, and client respect, result in satisfaction when adequately fulfilled. Whereas dissatisfiers or extrinsic factors (e.g., those associated with physical working conditions, work recognition, remuneration, working hours, management support, conflict resolution, job description), as in this study, cause dissatisfaction when deficient. The intrinsic factors typically are largely administered by the health worker and the extrinsic factors are under the control of the management.\n\nA five-point Likert scale with a value ranging from 1 (very dissatisfied) to 6 (very satisfied) was used to measure the intrinsic and extrinsic factors relating to satisfaction. The participants indicated their feeling for 20 items, as elicited by the questionnaire.52 These items were re-grouped into intrinsic and extrinsic job satisfaction factors afterwards. Participants answered in terms of how ‘satisfied’ or ‘dissatisfied’ they were with a few intrinsic (n=6) and extrinsic (n=14) job-related items. A summary of items covered in the questionnaire relating to intrinsic and extrinsic job satisfactions are shown in Table 2. These key influencing items were derived from the literature. In this study, to determine the general satisfaction level, respondents with a composite average score equal to or less than 3.5 (on a scale of 1–5) were classified as dissatisfied, and those with a composite average score above 3.5 were considered as satisfied.\n\n\n\n1. Freedom to choose work methods\n\n2. Variety in work\n\n3. Opportunities to use abilities\n\n4. Amount of responsibility\n\n5. Commitment to quality work\n\n6. Sense of client respect\n\n\n\n1. Physical working conditions\n\n2. Co-workers relationship\n\n3. Work recognition\n\n4. Remuneration\n\n5. Hours of work\n\n6. Acceptance of staff suggestions\n\n7. Disagreements resolution\n\n8. Colleagues’ support\n\n9. Team coordination\n\n10. Support for knowledge updates\n\n11. Management support\n\n12. Personnel problem resolution\n\n13. Conflict resolution\n\n14. Job description all-encompassing\n\nThe 20 items questionnaire data collected were entered into EPI INFO version 7.2.2.2 and all statistical analyses were performed using STATA (https://www.stata.com/) version 13.1. A freely accessible software that can also complete this analysis would be R (programming language) (https://www.r-project.org/). Descriptive analysis of continuous variables (i.e., variables that take infinite set of values) was estimated using means and deviations while that of categorical variables (nominal or ordinal variables with characteristics that cannot be quantifiable) was assessed using frequencies and proportions. Based on responses to the 5-point Likert scale factors of intrinsic and extrinsic satisfaction, composite mean scores (CMS) were calculated and classified into “satisfied” (CMS=3.5+) and “dissatisfied” (CMS<3.5). Univariate and multivariate logistic regression analysis was done to test association between outcome variables (i.e., intrinsic and extrinsic job satisfaction) and staff background factors. For all measures of association, significance levels were set at 0.1%, 1% and 5%.\n\n\nResults\n\nTable 3 shows the background characteristics of the 287 primary healthcare worker respondents in this study. The majority were men (75.3%). Most of them were aged 30-39 years (53.6%). The majority were technical staff (78.4%). More than half of the healthcare workers (58.6%) had been working for more than two years. Most of them worked in either a hospital (41.5%) or a health centre (41.5%).\n\nFigure 2 shows job satisfaction levels differentiated by technical staff, sex, work duration and facility type. The majority of both technical (physicians; nurses and midwives) and non-technical staff (pharmacists and associated professionals; community health workers and managers) were extrinsically and intrinsically satisfied. Most of the primary health care workers were more extrinsically satisfied than intrinsically satisfied. Also, most males and females were more extrinsically satisfied than intrinsically satisfied. There was no significant difference in the proportion of staff working in the hospitals who were extrinsically and intrinsically satisfied, compared to those working at other facilities. However, most staff working at CHPS facilities were more extrinsically satisfied.\n\nAs shown in Table 4, there was a statistically significant relationship between management training and intrinsic job satisfaction (<0.01), as well as extrinsic job satisfaction (<0.001). However, there was no statistically significant relationship found between all other background characteristics of primary healthcare workers and job satisfaction.\n\na Chi-square test.\n\n* p<0.001,\n\n p<0.01,\n\n* p<0.05 significant levels.\n\nTable 5 shows the staff background factors associated with intrinsic job satisfaction. A univariate logistic regression found a statistically significant association between intrinsic job satisfaction and management training (p<0.001). After controlling for observed confounding factors, multivariate logistic regression analysis found a statistically significant association between intrinsic job satisfaction and management training in the past 12 months (p<0.01) as well as the gender of primary health care worker (p<0.05). Those health workers who benefitted from management training in the past 12 months, rated intrinsic factors associated to satisfaction significantly higher at 2.8 (95% CI: 1.53,5.13) than those who had not undergone management training (p<0.001). Compared to staff working in hospitals, CHPS staff were less satisfied. Technical staff had higher odds of being satisfied compared to non-technical staff.\n\n* p<0.001,\n\n p<0.01,\n\n* p<0.05 significant levels.\n\nTable 6 shows the staff background factors associated with extrinsic job satisfaction. Similar to intrinsic satisfaction, a univariate logistic regression found a statistically significant association between extrinsic factors associated to job satisfaction and management training (p<0.001). After controlling for observed confounding factors, multivariate logistic regression analysis found a statistically significant association between extrinsic job satisfaction and the gender of the primary health care worker (p<0.05). The odds of staff that had received management training in the past 12 months being intrinsically satisfied were 3.73 (95% CI: 2.03, 6.86) times higher than staff with no management training (p<0.001). Compared to staff working in hospitals, CHPS staff were less extrinsically satisfied. Compared to non-technical staff, technical staff had higher odds of being extrinsically satisfied.\n\n* p<0.001,\n\n p<0.01,\n\n* p<0.05 significant levels.\n\n\nDiscussion\n\nIn this study, we analysed intrinsic and extrinsic factors related to job satisfaction and among primary healthcare staff in Ghana. Overall, most of the primary healthcare workers were intrinsically and extrinsically satisfied irrespective of the professional category they belong to. The perception of overall job satisfaction was higher than in a similar study in Ghana,3 as well as reports from other low-and-middle-income countries.31–36 On the other hand, it is lower than results from Nigeria.37 The potential reasons for the discordance might be due to: variations in the working context investigated; health infrastructure; administrative dynamics; organizational culture, values and setup; socio-demographic characteristics; study geographical areas, as well as the study time and tools used to measure the outcome variable.38\n\nEvidence from this study suggests that most primary healthcare workers were more satisfied extrinsically than intrinsically, which is contrary to other studies.39,40 Contextual dynamics could be the reason for the differences in the findings. According to Herzberg’s (1959)41 two-factor theory, the presence of motivator elements such as achievement, recognition, good working conditions, responsibility, advancement, and personal growth drive staff to work harder towards actualization of personal and organizational goals, without any external reward or coercion.30 Similar to findings from other studies,42 this study found a strong relationship between management training (or capacity development) and intrinsic job motivation, as well as extrinsic motivation.\n\nSimilar to findings of this study, previous reports have alluded to a directly proportional relationship between age and, intrinsic and extrinsic factors related to job satisfaction.39,43,44 Evidence from this study also corroborates that of others39,45 in which older health workers had a lower likelihood of job satisfaction compared to younger ones. This could be attributed to the sense of privilege associated with being young and employed in Ghana, given the high level of unemployment among the youth. Older primary health staff in Ghana tend to be dissatisfied mainly due to issues pertaining to compensation, working conditions, and diminished intrinsic values over time. Thus, the results of this study corroborate the discovery by Hatinoğlu and Ergün45 that age has an effect on both intrinsic and extrinsic job motivation.\n\nThe study results show that more experienced primary health care staff were intrinsically and extrinsically satisfied with their job than less experienced staff. This finding concurs with reports from other African countries46,47 as well as Asia.48 Existing literature cites helping sick patients as the main source of job satisfaction for technical staff.40 By this, a majority of health staff tend to be intrinsically satisfied due to the fulfillment that emanates from assisting patients and working for the public good, which outweighs dissatisfaction related to compensation and working conditions.40 Although not statistically significant, the majority of male staff are more intrinsically and extrinsically satisfied than their female counterparts. This finding concurs with those by Alshmemri.49 This can be ascribed to the emotional stress related to societal issues like the family and social responsibilities borne by women.39,50\n\nFurthermore, most primary healthcare staff working at lower-level facilities (i.e., CHPS) were less satisfied, both in terms of intrinsic and extrinsic factors, compared with those in higher-level facilities. Working conditions have been cited in the literature as very important for job satisfaction in public health facilities.39 Meanwhile, most staff working at lower-level facilities in Ghana have relatively poor working conditions, which may have accounted for the lower level of intrinsic and extrinsic job satisfaction among CHPS health workers compared to their counterpart at higher levels. Prestige and social recognition are typically higher for staff working at higher levels. Also, institutional efforts directed at increasing employees’ motivation differ by the demographic characteristics of employees and the organization’s structure, values and types of duties.51\n\nThe authors of this study are not aware of any other national study looking at job satisfaction among primary health workers and its associated factors. This study fills that important knowledge gap. A possible limitation relates to social desirability of a study such as ours, which could lead to artificially inflated and/or deflated responses. However, a self-administered questionnaire was used to mitigate this effect. Additionally, the use of one out of sixteen regions, and six districts out of thirty-three districts in the selected region (Eastern Region) limits the generalizability of the present findings. Also, the sample did not include private sector staff, which may have shed more light on the context. Furthermore, these results were not triangulated with qualitative data, which may have provided rich context to the quantitative findings.\n\n\nConclusions\n\nIn conclusion, public healthcare workers at primary health facilities in Ghana, at higher levels of service delivery, were intrinsically and extrinsically more satisfied compared to lower level staff. Thus, to enhance healthcare services, health facilities’ administrators should take measures to improve intrinsic and extrinsic working conditions of staff at all levels of service delivery, since the hierarchy of service delivery in the health system makes the contributory role of all staff critical in improving the quality of primary healthcare. Additionally, staff training has a significant effect on both intrinsic and extrinsic job motivation of primary healthcare workers. Hence, regular staff training must be institutionalized to contribute to the sustenance of job motivation.", "appendix": "Data availability\n\nFigshare: ‘Raw/Expanded-Data_Health Worker Job Satisfaction’. https://doi.org/10.6084/m9.figshare.21433035.v2. 52\n\nThis project contains the following underlying data:\n\n– Expanded-Data.xlsx. (Anonymised job satisfaction dataset with data processing extensions).\n\n– Raw-Data.xlsx. (Anonymised job satisfaction raw dataset).\n\n– Explanation-of-variables.xlsx. (Explanation of variable codes used in dataset).\n\nFigshare: ‘Raw/Expanded-Data_Health Worker Job Satisfaction’. https://doi.org/10.6084/m9.figshare.21433035.v2. 52\n\nThis project contains the following extended data:\n\n– Health-worker-satisfaction-survey-tool_Ghana.pdf. (Questionnaire used as data collection. tool).\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 wish to express their profound gratitude to all participants of the study at the national, regional, district, sub-district, and community levels in Ghana’s health sector.\n\n\nReferences\n\nCorey-Lisle P, Tarzian AJ, Cohen MZ, et al.: Healthcare reform: Its effects on nurses. J. Nurs. Adm. 1999; 29: 30–37. Publisher Full Text\n\nTovey EJ, Adams AE: The changing nature of nurses’ job satisfaction: An exploration of sources of satisfaction in the 1990s. J. Adv. Nurs. 1999; 30: 150–158. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFahrenkopf AM, Sectish TC, Barger LK, et al.: Rates of medication errors among depressed and burnt out residents: Prospective cohort study. BMJ. 2008; 336: 488–491. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartineau T, Raven J, Aikins M, et al.: Strengthening health district management competencies in Ghana, Tanzania and Uganda: lessons from using action research to improve health workforce performance. BMJ Glob Health. 2018; 3(2): e000619. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlshmemri M, Shahwan-Akl L, Maude P: Herzberg’s two-factor theory. Life Sci. J. 2017; 14: 12–16.\n\nBekru ET, Cherie A, Anjulo AA: Job satisfaction and determinant factors among midwives working at health facilities in Addis Ababa city, Ethiopia. PLoS One. 2017; 12: e0172397. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeleto A, Baraki N, Atomsa GE, et al.: Job satisfaction and associated factors among health care providers at public health institutions in Harari region, eastern Ethiopia: A cross-sectional study. BMC. Res. Notes. 2015; 8: 394. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaile D, Gualu T, Zeleke H, et al.: Assessment of Job Satisfaction and Associated Factors among Nurses in East Gojjam Zone Public Hospitals Northwest Ethiopia, 2016. J. Nurs. Care. 2017; 06: 06. Publisher Full Text\n\nKhadka D, Chaulagain N: Factors Influencing Job Satisfaction among Healthcare Professionals at Tilganga Eye Centre, Nepal. Int. J. Sci. Technol. Res. 2018; 1: 32–36.\n\nKumar R, Ahmed J, Shaikh BT, et al.: Job satisfaction among public health professionals working in public sector: A cross-sectional study from Pakistan. Hum. Resour. Health. 2013; 11: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSusanth BS: Human resource development HRD practices in public sector undertakings a case study of electronics corporation of India limited ECIL Hyderabad. Sri Krishnadevaraya University, Sri Krishnadevaraya Institute of Management; 2011.\n\nKolo E: Job satisfaction among healthcare workers in a tertiary center in kano, Northwestern Nigeria. Niger. J. Basic Clin. Sci. 2018; 15: 87. Publisher Full Text\n\nGeta A, Biks GA, Dellie E, et al.: Job Satisfaction and Associated Factors among Health Professionals Working at Public and Private Hospitals in Bahir Dar City, Northwest Ethiopia: A Comparative Cross-Sectional Study. Biomed. Res. Int. 2021; 2021: 1–13. Publisher Full Text\n\nAl-Haroon HI, Al-Qahtani MF: The demographic predictors of job satisfaction among the nurses of a major public hospital in KSA. J. Taibah Univ. Med. Sci. 2019; 15: 32–38. Publisher Full Text\n\nSalem OA, Baddar F, AL-Mugatti HM: The Relationship between Nurses Job Satisfaction and Organizational Commitment. J. Nurs. Health Sci. 2016; 5.\n\nHerzberg F, Mausner B, Snydermann B: The motivation to work. New York: Wiley; 1959.\n\nDaneshkohan A, Zarei E, Mansouri T, et al.: Factors affecting job motivation among health workers: A study from Iran. Global J. Health Sci. 2014; 7: 153–160. PubMed Abstract \| Publisher Full Text\n\nInceoglu I, Segers J, Bartram D: Age-related differences in work motivation. J. Occup. Organ. Psychol. 2012; 85: 300–329. Publisher Full Text\n\nStulz V: Job satisfaction of Australian nurses and midwives: A descriptive research study. J. Adv. Nurs. 2012; 29: 19–27.\n\nHatinoğlu A, Ergün D: The effects of socio-demographic characteristics and working conditions on the work motivations of employees working in health services in Turkey. Revista Argentina de ClínicaPsicológica. 2020; 29: 1848–1859.\n\nJacinta M: Improving nurse retention: Factors influencing job satisfaction of all cadres of nurses in Machinga district. University of Malawi, College of Medicine; 2008.\n\nSemachew A, Belachew T, Tesfaye T, et al.: Predictors of job satisfaction among nurses working in Ethiopian public hospitals, 2014: Institution-based cross-sectional study. Hum. Resour. Health. 2017; 15: 31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlostaz Z: Job Satisfaction among Critical Care Nurses in Al Baha, Saudi Arabia: A Cross-Sectional Study. Gulf Med. J. 2016; 5: 64–68.\n\nAlshmemri M: Job satisfaction of Saudi nurses working in Saudi Arabian public hospitals. RMIT University, Health Sciences; 2014.\n\nAlmalki M, Fitzgerald G, Clark M: Health care system in Saudi Arabia: an overview. East Mediterr. Health J. 2011; 17: 784–793. Publisher Full Text\n\nHeidarian AR, Jafari Kelarijani SE, Jamshidi R, et al.: The relationship between demographic characteristics and motivational factors in the employees of social security hospitals in Mazandaran. Caspian J. Intern. Med. 2015; 6: 170–174. PubMed Abstract\n\nAmon S: Raw/Expanded-Data_Health Worker Job Satisfaction.xlsx. Dataset. figshare. 2022. Publisher Full Text" }	[ { "id": "169560", "date": "05 Jul 2023", "name": "Matilda Aberese-Ako", "expertise": [ "Reviewer Expertise Anthropology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments\nThis is an interesting study, it is timely and well written. However, I have a few comments that can help to strengthen the quality of the paper.\nGaps in the study\nThe authors suggest that extrinsic factors are mostly caused by management issues. This implies that other contextual factors such as national activities, health care policies, labour policies and environmental factors are not in any way linked to intrinsic and extrinsic motivation?\n\nThe authors refer to motivation and job satisfaction throughout the manuscript as if they mean the same, authors need to clarify.\n\nAuthors refer to management training as an indicator, it will be interesting to know whether all health workers in Ghana undergo management training.\n\nWhy were the three districts out of the of the lot in the Eastern region of Ghana?\n\nThe use of etc in several places including in one of the figures, the use of i.e, eg, missing articles such as “a”, connectors such as “to” among others need to be worked on.\nEthical consideration\nSome information is missing from the first sentence\nStudy design and setting\nThe last sentence is a bit confusing, kindly rephrase\n\nThe information on the PERFORM2Scale project is too scanty\nSample size and sampling techniques\nWhat are the authors referring to in the following statement: “The calculated sample size was proportionally allocated to the four health facilities.”?\nMeasurements\nThe authors state the following: “The satisfiers/motivators or the intrinsic factors such as work methods, work variations, skills application, responsibility, commitment, and client respect, result in satisfaction when adequately fulfilled. Whereas dissatisfiers or extrinsic factors (e.g., those associated with physical working conditions, work recognition, remuneration, working hours, management support, conflict resolution, job description), as in this study, cause dissatisfaction when deficient. The intrinsic factors typically are largely administered by the health worker and the extrinsic factors are under the control of the management.”\nAre authors implying that satisfiers/motivators are intrinsic factors only and dissatisfiers are extrinsic only?\nPredictors associated with intrinsic and extrinsic job satisfaction\nAuthors report figures using two decimal places and others three decimals?\n\nAuthors indicate gender as a factor in the results but fail to elaborate.\nDiscussion\nIn the entire section the authors compare several studies to theirs without giving a brief on those studies\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "204612", "date": "15 Sep 2023", "name": "Cornelia Van Diepen", "expertise": [ "Reviewer Expertise Work-related health of healthcare professionals", "Person-centred care" ], "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 comment\nAlthough the premise is interesting. The statistical analyses is flawed and the conclusion do not fit. The analysis created odds ratios that do not seem to match with the descriptive table. Table 4 provides a skewed representation of the data by taking column percentages instead of row percentages.\nTable 4 should show the row percentages as for example this will reveal the division of satisfaction/dissatisfaction among women and among men. This would show that for both intrinsic and extrinsic satisfaction the prevalence as very similar. (women; intrinsically satisfied 57.7%; men: 58.8%).\nSimilarly, on the question of trained/not trained, 147 out of the total 287 participants were untrained and satisfied. That is 51.2% of the sample. Table 4 even shows that the trained participants were more dissatisfied than satisfied and the odds of 2.8 shows has trained as a reference not the other way around. These are just the results I have recalculated and I am not sure if the rest is done correctly.\nThe main results (as presented in the abstract) are therefore based on faulty calculations. This is luckily not unsalvageable and the authors can redo table 4 (with row percentages) and analyse the odds ratios again to make sure they are correct.\n\nThe redone analyses will then affect the discussion, conclusion and abstract. So I will not further comment on those.\n\nThere are a few other things, I think can be improved:\nThe title does not include 'job satisfaction' even though that is the main outcome so it should be in there.\n\nThe definitions in the first paragraph are nice but could be better incorporated in the introduction text.\n\nYou apply the Herzberg two factor theory, this needs to be explained in more detail in the introduction to help the reader understand how you are looking at job satisfaction.\n\nThe aim should be the same everywhere.\n\nThe sequence of the methods is a bit odd as you start with the ethics even though the reader doesn't know yet what you are doing (so they don't know what you need ethical review for).\n\nFigure 2 is of poor quality and does not really add anything when you have table 4.\n\nSo the study is interesting but the results need to be correct.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-396
https://f1000research.com/articles/12-395/v1	13 Apr 23	{ "type": "Research Article", "title": "Prevalence rates of mucopolysaccharidosis in Iraq: a retrospective cross-sectional observational study", "authors": [ "Furqan M. Abdulelah", "Mohammed Mahmood Mohammed", "Rabab Hassan Baaker", "Mohammed Mahmood Mohammed", "Rabab Hassan Baaker" ], "abstract": "Background: Mucopolysaccharidosis (MPS) is a rare hereditary inborn error of metabolism that represents the largest heterogeneous group of lysosomal storage diseases (LSD) and is characterized by multiorgan impairment due to glycosaminoglycans (GAGs) accumulation in various tissues and organs, resulting in severe frailty and early death. This research aimed to figure out the specific and overall birth prevalence of mucopolysaccharidosis among Iraqi children, as well as the frequencies of each type, and to compare the results with epidemiological data from other Arabian countries. Methods: Information was collected and investigated from registered patients diagnosed with MPS in five metabolic centers in Iraq between 2010 and 2020. The numbers of live births in Iraq were obtained from the Ministry of Health and Environment (Health and vital statistics department) for the period mentioned above. Birth prevalence was calculated, and Poisson distribution for confidence intervals (95%) was considered through the implementation of MedCalc statistical software. The Hardy-Weinberg equation was used to calculate carrier frequency. Results: The overall prevalence of MPS at birth is 2.97 per 100,000 live births; different forms of MPS manifest at varied frequencies. MPS VI was the most often reported form in the Iraqi population (1.32 per 100,000 live births, or 44.41% of all MPS cases), followed by MPS IVA and MPS I (0.625 and 0.593 per 100,000 live births, respectively). The higher frequency rate of MPS VI was also reported in neighboring countries, including UAE and Saudi Arabia, which were 2.51 and 8.0 per 100,000 live births, respectively. Conclusions: The health systems should highly consider data obtained from prevalence studies in all affected countries, including health care specialists, clinical genetics, and workers in laboratories involved in MPS diagnosis.", "keywords": [ "Mucopolysaccharidosis", "Inborn error", "Inherited metabolic diseases", "Iraqi children", "Carrier frequency", "the prevalence" ], "content": "Introduction\n\nMucopolysaccharidosis (MPS) is a rare inherited metabolic disorder that is the most heterogeneous group of lysosomal storage diseases (LSD). It is characterized by multiorgan impairment caused by glycosaminoglycan (GAG) buildup in various tissues and organs, eventually resulting in severe debilitation and fatality in early life.1 There are 11 types/sub-types of MPS, each caused by a specific lysosomal enzyme dysfunction or deficiency that contributes to GAG breakdown and, as a result, causes a variety of clinical manifestations such as coarsening of facial characteristics, hepatosplenomegaly, cognitive impairment, dysostosis multiplex, hernias, corneal cloudiness, kyphoscoliosis, cardiac complications, etc.2 Different types usually have mutual symptoms, particularly MPS I and II, but MPS III and VII are characterized by severe neurological features and hydrops fetalis, respectively.3 These clinical symptoms are involved in the diagnosis of MPS. However, implementing newborn screening programs and enzymatic and molecular assays for early and precise diagnosis of asymptomatic suspected individuals is imperious for improving therapeutic outcomes.4,5 Enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation are conventional therapeutic approaches for most MPS types/subtypes. However, the emergence of novel therapeutic strategies like gene therapy, pharmacological chaperone, and substrate reduction therapy is on the way to proving its effectiveness, especially for managing neurological manifestations and other intractable symptoms.6 All MPS types are inherited by autosomal recessive inheritance except MPS II, which is inherited as an X-linked pattern.7 The first case of MPS was diagnosed in 1917 by Charles Hunter; ethnicity and geographical region may affect the phenotypic characteristics of MPS.8 This is the first epidemiological survey regarding MPS in Iraq, although there were similar studies considering the epidemiology of MPS disorders conducted in the province of Arabian nations; this retrospective epidemiological study of MPS was undertaken to calculate the birth prevalence rate and frequencies of different types of MPS for a period of one-decade from 2010 to 2020 in Iraq. In 2017, the overall prevalence of MPS in Saudi Arabia (a neighbor of Iraq) was estimated and calculated by Nouriya A. Al-Sannaa et al. as 15.64 per 100,000 live births.9 In 2013, a study of MPS prevalence in the United Arab Emirates (UAE) was achieved by Fatma A. Al-Jasmi et al. and revealed an overall prevalence of 5.5 per 100,000 live births.10 Ben Turkia H et al. in 2009 surveyed MPS epidemiology with the estimation of a combined MPS majority in Tunisia, and the result revealed 2.3 per 100,000 live births.11 Despite this, the worldwide studies regarding MPS epidemiology were insufficient; the overall birth prevalence of MPS in Portugal, Brazil, the USA, Malaysia, etc., was calculated.12\n\nThe reported epidemiological surveys are expected to be underestimated; this is attributed to insufficient data regarding the diagnosis of rare diseases.13 Therefore, further epidemiological studies regarding the prevalence of different MPS types become essential to estimate both the individual and overall influence of LSDs on patients, their families and the public and decide which therapeutic approach should be commenced to improve the outcomes prior to irreversible impairment effectively ensues. Furthermore, these data will be necessary to accurately assess the cost of these illnesses to community health care organizations, and they will serve as the basis for establishing screening and therapeutic programs.14\n\nThis study was designed to calculate the individual and overall birth prevalence of mucopolysaccharidosis in Iraq and the frequencies of each type. In the discussion, we compare the outcomes with described epidemiological statistics from other Arabian countries.\n\n\nMethods\n\nOn July 26, 2022, the Ministry of Health and Environment, Al-Rasafa Health Directorate Training and Human Development Center Research Committee approved the research proposal as requested from the College of Pharmacy, University of Mustansiriyah, which defined the current study’s aims and projected data collection procedures (ethics board approval code: 386). An English version was provided on 16th October 2022.\n\nThe research also approved by Mustansiriyah University College of Pharmacy Research Ethics Committee at 28/6/2022 with approval number1 research number.13 The researcher explained the study’s goal and gave an information sheet to each responder and obtained signed permission to participate. There were no incentives given to the patients.\n\nThe present research was an observational, retrospective, cross-sectional study of patients who had already been diagnosed with MPS.\n\nTwo types of data were obtained. Firstly, the live births between 2010 and 2020 were obtained from the central registry of the Ministry of Health. The data were obtained as pdf file after direct visit to Ministry of Health, Environment of Iraq. Secondly, from July to October 2022, information was collected and investigated from populations diagnosed with MPS in five metabolic centers (there are only five metabolic centers available in Iraq), listed below, in Iraq, as seen in Figure 1.\n\n1. Child’s Central Teaching Hospital/Metabolic diseases unit/Baghdad governorate.\n\n2. Al-Kadhimiya Teaching Hospital/Metabolic diseases unit/Baghdad governorate.\n\n3. Medical City/Pediatric Teaching Hospital/Baghdad governorate.\n\n4. Faiha Specialized Diabetes, Endocrine, and Metabolism Center (FDEMC)/Basra governorate.\n\n5. Raparin Children’s Hospital/Erbil governorate.\n\nThe patients were identified from their patient files at the five centers, and asked to participate in this research during their weekly hospital visits to get ERT supplements. Written informed consent to allow their data records to be used in this research was obtained from the patients. For patients that were minors, parental consent was obtained during these visits. Approval to access the patient records was approved by each center.\n\nAll patients with confirmed MPS disorder were included in this study.\n\nStudy size\n\nSince the research is concerned with estimating prevalence, no estimation of sample size was performed; consequently, all individuals diagnosed with this condition were included in the study.\n\nData analysis and calculation of prevalence\n\nThe numbers of live births in Iraq were obtained from the Ministry of Health and Environment (Health and vital statistics department) for 2010-2020. Birth prevalence is calculated as (the number of defectively birthed cases in a definite province and time * 100,000) ÷ (the number of total live births in that province and time).13 Poisson distribution for the calculation of confidence intervals (95%) for the birth prevalence was considered through the implementation of MedCalc statistical software (version 20.218).15 In addition, the Hardy-Weinberg equation was used to calculate carrier frequency.16 The authors attempted to remove data that was inaccurate (e.g. family history of MPS was not complete), corrupted, poorly formatted, duplicated, or incomplete from a database. There is potential for data to be duplicated or mislabeled when merging different data sources from the Ministry database or hospitals. Therefore, data that did not belong in the live birth dataset was removed.\n\nWe attempted to eliminate selection bias during patient evaluation by having each individual’s diagnosis confirmed before enrollment. This was done by comparing recorded cases between the hospitals and the Ministry of Health’s central registry.\n\nWe tried to avoid geographic bias, i.e. the propensity to pick research locations for studies in a manner that excludes or decreases selection in specific geographic regions, by including all MPS specialised centers in Iraq.\n\nPoisson distribution for the calculation of confidence intervals (95%) for the birth prevalence was considered, a Poisson distribution is a discrete probability distribution. It gives the probability of an event happening a certain number of times (k) within a given interval of time or space. This was employed through the implementation of MedCalc statistical software (version 20.218).\n\n\nResults\n\nLive births documented in the Iraqi’s annual statistic report organized by the Ministry of Health from 2010 until 2020 totaled 12,319,467. In the past decade, we found 367 patients (more than half of them were males and aged older than 10 years as presented in Table 1) diagnosed with MPS I, II, III, IV, and VI, and no individuals have been diagnosed with MPS VII or IX. From our data, the overall birth prevalence of MPS is 2.97 per 100,000 live births; data on the birth prevalence rates are summarized in Table 2. Data regarding different MPS type frequencies are illustrated in Figure 2.\n\na Total number of MPS patients diagnosed between 2010-2020.\n\nb Carrier per live birth.\n\nc Two cases are female.\n\n\nDiscussion\n\nThe overall birth prevalence of MPS in the Iraqi population (2.97 per 100,000 live births), when compared with the birth prevalence of other Arabian countries, such as the UAE (5.5 per 100,000 live births) and Saudi Arabia (15.64 per 100,000 live births), was lower.9 However, it is similar to the birth prevalence in Tunisia (2.3 per 100,000 live births).17 The fact that there are only five specialized metabolic centers for diagnosis and registration of MPS cases in Iraq significantly facilitates data collection for this study; hence, we are highly confident (Poisson Confidence Interval 95%) that only a few instances might be undiagnosed. This could be attributed to several causes, including poor health awareness regarding implementation of newborn screening program, particularly for affected families, even after the first birthed child was diagnosed with MPS, unavailability of specialized centers that perform molecular analysis for diagnosis of suspected cases with an inborn error of metabolism. Therefore, several MPS cases might be undiagnosed.\n\nAdditionally, the poor financial status of most affected families may contribute to the underestimation of MPS cases, despite ERT and Diagnostic test presented as free medical services from government, most of poor families refuse to receive these services because they don’t have enough money for transportation and therefore neglect the diagnoses of other children who are recently born to MPS affected families. This fact was identified through phone communication with these families. Various types of MPS show different frequencies. In the Iraqi population, MPS VI was the most frequently reported type (1.32 per 100,000 live births, representing 44.41% of all MPS cases), followed by MPS IV A and MPS I (0.625 and 0.593 per 100,000 live births, respectively). The higher frequency rate of MPS VI among other MPS types was also reported in neighboring countries, including UAE and Saudi Arabia, which were 2.51 and 8.0 per 100,000 live births, respectively.9,10 Obviously, this could be attributed to the regional effect of similar variants or the high consanguinity rate, which increases the frequency of diseased allele transmission,18 which could be in accordance with the Iraqi situation. Typically, the correlation between consanguinity and homozygosity should be confirmed by genotyping, but molecular studies are still not available as a diagnostic tool in the Iraqi public health system. It is worth mentioning that the most prevalent type of MPS reported in almost all published studies was MPS III. Poorthuis et al. (1990) said that MPS II was more prevalent; this variation could be attributed to differences in ethnicity and geographical origin. In this study, it is imperative to mention the two children who are females affected by hunter MPS (type II), which attributed to skewed X-chromosomal inactivation and Iduronidase (IDS) de novo mutations, these two cases should receive genetic counseling with regard to their reproductive decisions.19\n\nA limitation of our study is that there are no specialized laboratories to perform genetic analysis for confirmation of MPS diagnosis; thus, some cases may be missing, particularly mild cases that are difficult to diagnose. As a result, while providing comprehensive data on the epidemiological profile of MPS in Iraq, the total numbers obtained in this study may be underestimated. In addition, access to data at the registration centers was also challenging and time-consuming. This is due to the privacy of data recorded in these centers and the distant regions where the centers are located.\n\n\nConclusion\n\nIn conclusion, data obtained in this prevalence studies should be highly considered by the health system, including health care specialists, clinical genetics, and workers in laboratories involved in MPS diagnosis. Such information is essential for decision-makers to estimate the disease’s genetic and social burden to the public. Increasing awareness among affected families for early diagnoses, such as newborn screening programs for a new birth family member, may be needed. Estimating prevalence rates is essential and required for calculating the financial burden of therapeutic approaches for stabilizing MPS disorders like ERT.", "appendix": "Data availability\n\nZenodo: Underlying data for Prevalence Rates of Mucopolysaccharidosis in Iraq: a retrospective cross-sectional observational study, https://doi.org/10.5281/zenodo.7783396. 20\n\nThis project contains the following underlying data:\n\n‐ Article data.xlsx (Prevalence Rates of MPS cases in a different healthcare center in Iraq)\n\nThe live births data can be obtained from the Iraqi Ministry of Health using contact details from their website. A paper-based request should be sent to the Department of Planning in the Statistics unit.\n\nZenodo: Patient’s Consent Form, https://doi.org/10.5281/zenodo.7652173. 21\n\nZenodo: Data sheet, https://doi.org/10.5281/zenodo.7652185. 22\n\nZenodo: Information sheet, https://doi.org/10.5281/zenodo.7714213. 23\n\nZenodo: STROBE checklist for ‘Prevalence Rates of Mucopolysaccharidosis in Iraq: a retrospective cross-sectional observational study,’ https://doi.org/10.5281/zenodo.7714215. 24\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe thank Dr. Arabia and Dr. Dhaigham for their support in data collection from Child’s Central Teaching Hospital, Metabolic diseases Unit, Baghdad and Faiha Specialized Diabetes, and the Endocrine and Metabolism Center (FDEMC), Basra, respectively.\n\n\nReferences\n\nLeadley RM, Lang S, Misso K, et al.: A systematic review of the prevalence of Morquio A syndrome: challenges for study reporting in rare diseases. Orphanet J. Rare Dis. 2014; 9(1): 1–17.\n\nKobayashi H: Recent trends in mucopolysaccharidosis research. J. Hum. Genet. 2019; 64(2): 127–137. PubMed Abstract \| Publisher Full Text\n\nSuarez-Guerrero JL, Higuera PJIG, Flórez JSA, et al.: Mucopolysaccharidosis: clinical features, diagnosis and management. Rev. Chil. Pediatr. 2015; 87(4): 295–304. PubMed Abstract \| Publisher Full Text\n\nColmenares-Bonilla D, Colin-Gonzalez C, Gonzalez-Segoviano A, et al.: Diagnosis of Mucopolysaccharidosis based on history and clinical features: Evidence from the Bajio Region of Mexico. Cureus. 2018; 10(11): 1–12. Publisher Full Text\n\nZhou J, Lin J, Leung WT, et al.: A basic understanding of mucopolysaccharidosis: Incidence, clinical features, diagnosis, and management. Intractable Rare Dis. Res. 2020; 9(1): 1–9. Publisher Full Text\n\nSawamoto K, Stapleton M, Alméciga-Díaz CJ, et al.: Therapeutic options for mucopolysaccharidoses: current and emerging treatments. Drugs. 2019; 79(10): 1103–1134. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Sannaa NA, Al-Abdulwahed HY, Al-Majed SI, et al.: The clinical and genetic spectrum of Maroteaux-Lamy syndrome (mucopolysaccharidosis VI) in the Eastern Province of Saudi Arabia. J. Community Genet. 2018; 9: 65–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilson A, Lavery C, Stewart F, et al.: Mucopolysaccharidosis and adulthood: genetics, inheritance, and reproductive options. J. Child Sci. 2018; 08(01): e138–e143. Publisher Full Text\n\nAbdulelah FM: Underlying data for Prevalence Rates of Mucopolysaccharidosis in Iraq: a retrospective cross-sectional observational study.2023. Publisher Full Text\n\nAbdulelah FM: Patient’s Consent Form. [Data set]. Zenodo. 2023. Publisher Full Text\n\nAbdulelah FM: Data sheet. [Data set]. Zenodo. 2023. Publisher Full Text\n\nAbdulelah FM: Information sheet. [Data set]. Zenodo. 2023. Publisher Full Text\n\nAbdulelah FM: STROBE checklist for ‘Prevalence Rates of Mucopolysaccharidosis in Iraq: a retrospective cross-sectional observational study’.2023. Publisher Full Text" }	[ { "id": "308852", "date": "12 Aug 2024", "name": "Laith G. Shareef", "expertise": [ "Reviewer Expertise Clinical Pharmacy and therapeutics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIncidence and prevalence data are important to back up health system decisions and are necessary to calculate the cost–benefit of new therapies and treatment.\nThis research aimed to figure out the specific and overall birth prevalence of mucopolysaccharidosis among Iraqi children, as well as the frequencies of each type, and to compare the results with epidemiological data from other Arabian countries.\nIn the introduction, in which region the authors refer in this sentence: “The reported epidemiological surveys are expected to be underestimated; this is attributed to insufficient data regarding the diagnosis of rare diseases.” the meaning here is unclear \"This study was designed to calculate the individual and overall birth prevalence of mucopolysaccharidosis in Iraq and the frequencies of each type. In the discussion, we compare the outcomes with described epidemiological statistics from other Arabian countries\" please revise the aim of the study at the end of the introduction regarding individual and overall birth prevalence, and take into account that discussing your results with others previously published works can’t be considered as an aim.\nCross-sectional studies are observational studies that analyze data from a population at a single point in time. They are often used to measure the prevalence of health outcomes, understand determinants of health, and describe features of a population. definition of cross-sectional study design means collecting data at a time but not retrospectively or prospectively. Please revised your study design at the method section\nIn the result, table 1 representing demographic data and surgical history of MPS patients, what is the importance of the surgical history information regarding the aim of this study.\nIn table 2 entitled Prevalence of MPS in the Iraqi population; the title of the first column is inappropriate, please to replace it with MPS types, and it would be better to add the sex with its frequency regarding each type.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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/12-395
https://f1000research.com/articles/11-589/v1	30 May 22	{ "type": "Software Tool Article", "title": "GRAPE: genomic relatedness detection pipeline", "authors": [ "Alexander Medvedev", "Mikhail Lebedev", "Andrew Ponomarev", "Mikhail Kosaretskiy", "Dmitriy Osipenko", "Alexander Tischenko", "Egor Kosaretskiy", "Hui Wang", "Dmitry Kolobkov", "Vitalina Chamberlain-Evans", "Ruslan Vakhitov", "Pavel Nikonorov", "Alexander Medvedev", "Andrew Ponomarev", "Mikhail Kosaretskiy", "Dmitriy Osipenko", "Alexander Tischenko", "Egor Kosaretskiy", "Hui Wang", "Dmitry Kolobkov", "Vitalina Chamberlain-Evans", "Ruslan Vakhitov", "Pavel Nikonorov" ], "abstract": "Classifying the degree of relatedness between pairs of individuals has both scientific and commercial applications. As an example, genome-wide association studies (GWAS) may suffer from high rates of false positive results due to unrecognized population structure. This problem becomes especially relevant with recent increases in large-cohort studies. Accurate relationship classification is also required for genetic linkage analysis to identify disease-associated loci. Additionally, DNA relatives matching service is one of the leading drivers for the direct-to-consumer genetic testing market. Despite the availability of scientific and research information on the methods for determining kinship and the accessibility of relevant tools, the assembly of the pipeline, that stably operates on a real-world genotypic data, requires significant research and development resources. Currently, there is no open source end-to-end solution for relatedness detection in genomic data, that is fast, reliable and accurate for both close and distant degrees of kinship, combines all the necessary processing steps to work on real data, and is ready for production integration. To address this, we developed GRAPE: Genomic RelAtedness detection PipelinE. It combines data preprocessing, identity-by-descent (IBD) segments detection, and accurate relationship estimation. The project uses software development best practices, as well as Global Alliance for Genomics and Health (GA4GH) standards and tools. Pipeline efficiency is demonstrated on both simulated and real-world datasets. GRAPE is available from: https://github.com/genxnetwork/grape.", "keywords": [ "kinship and relationship estimation", "identity-by-descent", "snakemake workflow", "bioinformatics pipeline", "phasing and imputation", "sequencing data", "genetic testing companies", "genotype" ], "content": "Introduction\n\nDistant relationship estimation has both scientific and commercial applications. Scientific applications may include the identification of monogenic (single gene) Mendelian diseases.1,2 Also, relatedness detection can be used during data quality control for genome-wide association studies (GWAS), since close relatives should be excluded to ensure that no pair of individuals is more closely related than second degree relatives.3,4 Otherwise, GWAS may suffer from high rates of false positive results. The potential commercial application of relationship estimation is utilized by direct-to-consumer genetic testing companies to find possible distant relatives for their customers.5 The applicability of existent tools and pipelines to both applications is limited. Currently, there is no open-source end-to-end solution for relatedness detection in genomic data, that: (a) is reliable and accurate for both close and distant degrees; (b) includes all necessary processing steps to work with real data; (c) is flexible enough to be adapted for various applications; (d) is user-friendly and ready for production integration. Specifically, for commercial usage, the pipeline should deal with data heterogeneity and efficiently process newly added data samples. Samples can be genotyped with different chips and may have been passed thought different quality controls. Typical databases of genetic testing companies contain a lot of data from the previously used chips, which need to be combined together during the processing. Scientific database may also contains heterogeneous data. For example, UK Biobank dataset6 contains samples which were genotyped using two different chips.\n\nDriven by the idea of open and user-led innovation, we have developed GRAPE (Genomic RelAtedness detection PipelinE), the first open-source end-to-end solution for relatedness detection that is able successfully address the above-mentioned difficulties. As a preliminary step, we comprehensively studied available approaches and software instruments for relatedness estimation from genotype data, such as identity-by-descent (IBD) segments detection tools: GERMLINE,7 IBIS,8 RaPID,9 PhasedIBD10; relationship inference tools: DRUID,11 ERSA,12,13 KING14; other tools, which may be required during data preprocessing steps, like algorithms of phasing (Eagle15) and genotype imputation (Minimac16). Then, we selected several perspective tools and joined them together into the user-friendly GRAPE pipeline.\n\nGRAPE adapts the best practices for software development, including the Snakemake17 workflow management system, Conda18 virtual environments, Docker19 containerisation, Funnel task execution service, which implements GA4GH Task Execution Schema,20 and CI/CD with automatic testing. The pipeline requires a single multi-sample VCF file as input and has a separate workflow for downloading of reference datasets and checking their consistency. IBD segments detection workflows of GRAPE can work with both phased and unphased data. As real-world datasets are often heterogeneous and inconsistent, GRAPE incorporates various data preprocessing and quality control (QC) options. GRAPE has a modular architecture that allows switching between tools and adjust tools parameters for better control of precision and recall levels. The pipeline also contains a simulation workflow with an in-depth evaluation of pipeline accuracy using simulated and reference data.\n\nGRAPE can work like a standalone version as well as from the dedicated Docker container. We recommend to use the containerized version of GRAPE since it has all the dependencies already installed. We published GRAPE image in both Docker Hub and Dockstore21 repositories to satisfy GA4GH22 standards for sharing Docker-based tools. As a results we got a robust, reliable, and easy-to-use tool. Analysis for 10k samples with 600k SNPs requires half an hour, and about 22 hours have been required to process 100k samples dataset. Precision/recall analysis for GRAPE was performed using simulated datasets. We compared GRAPE with TRIBES,23 another open-source pipeline for relatedness detection, and showed the advantages of our solution in a sense of the precision/recall metrics.\n\n\nMethods\n\nThis section describes the input data, reference datasets, and the main pipeline steps. The scheme of the pipeline is presented in Figure 1. As input, GRAPE uses a single VCF file containing the genotypes of a set of individuals. Configuration of the pipeline is managed by config.yaml file, or via the parameters of the GRAPE launcher. Reference data should be previously downloaded and stored on a hard drive. After that, the pipeline performs relationship inference accordingly to one of the three possible workflows (described in a corresponding section below). The main pipeline steps are:\n\n1. Downloading of the reference datasets.\n\n2. Quality control and data preprocessing.\n\n3. Application of the relationship inference workflow.\n\nGRAPE also includes simulation workflow to evaluate precision/recall metrics on the simulated data. To simulate artificial pedigrees, Ped-sim tool24 is used, while unrelated founders for the simulation are taken from the 1000 Genomes Project data.25\n\nThere are two main approaches for relationship estimation implemented in GRAPE. The first one is based on allele frequencies calculation (KING14). This approach is optionally used for the first three degrees of relatedness, and for calculation of the kinship coefficients. The second approach relies on searching of pairwise identical-by-descent (IBD) segments. There are two options for this purpose based on two different tools: (a) IBIS8 and (b) GERMLINE.7 IBIS is a fast tool that can operate with unphased data. IBIS performs an IBD detection using homozygous single nucleotide polymorphisms (SNPs) only. It breaks the genome into windows of fixed length and searches for homozygous SNP mismatches for each genotype window. If the number of mismatches does not exceed some predefined threshold, the window becomes a part of an IBD segment. In contrast, GERMLINE is slower and can work only with phased data, but under some circumstances it may produce higher precision results. Once the pairwise IBD segments search is completed, relationship degrees are estimated using the ERSA tool.12\n\nGRAPE requires various reference data to perform preprocessing, quality control, phasing, and imputation. Reference data is also required for the simulation workflow. In order to facilitate collection of reference data, we created a separate workflow that automates this step. It can be run by specifying reference command to the GRAPE pipeline launcher. The workflow downloads data, unpacks it, and performs the required post-processing procedures. If phasing and genotype imputation are required, one should also add the additional flags--phase and --impute to the command. It affects the amount of downloaded data. If these flags are specified, the workflow downloads additional reference dataset to make phasing and imputation possible.\n\nThere is another option to download all required reference data as a single file. This file is prepared by us and preloaded on our side in the cloud. It can be done by specifying the additional flag --use-bundle to the workflow. This way is faster, since all the post-processing procedures have already been performed. Reference files consist of three main groups.\n\n• Files for the preprocessing. These files include genetic recombination maps for mapping SNPs coordinates from base pairs (bp) to centimorgans (cM); files with the SNPs information from the 1000 Genomes Project for the SNPs quality control; reference genome of hg37 build; liftOver chain file.\n\n• Files for phasing and imputation. Phasing is required as a preliminary step for GERMLINE tool, if input data is unphased. Upon the phasing is done, genotype imputation can be additionally applied. These files are space demanding and require a considerable amount of post-processing time.\n\n• Files for simulation. These files include phased per-chromosome files from the 1000 Genomes Project; Affymetrix chip data that is used as a source of founders for the simulation; sex-specific recombination maps for better Ped-sim simulation results.24\n\nGRAPE have a versatile and configurable preprocessing workflow. One part of the preprocessing is required and must be performed before the relationship inference workflow. It is launched by the preprocess command of the GRAPE. Along with some necessary technical procedures, preprocessing includes the following steps.\n\n• [Required] SNPs quality control by minor allele frequency (MAF) and the missingness rate. We discovered that blocks of rare SNPs with low MAF value in genotype arrays may produce false positive IBD segments. To address this problem, we filter SNPs by minor allele frequency. We remove SNPs with a MAF value less than 0.02. Additionally, we remove multiallelic SNPs, insertions/deletions, and SNPs with the high missingness rate, because such SNPs are inconsistent with IBD detection tools.\n\n• [Required] Per-sample quality control, using missingness and heterozygosity. Extensive testing revealed that samples with an unusually low level of heterozygosity could produce many false relatives matches among individuals. GRAPE excludes such samples from the analysis and creates a report file with the description of the exclusion reason.\n\n• [Required] Control for strands and SNP IDs mismatches. During this step GRAPE fixes inconsistencies in strands and reference alleles.\n\n• [Optional] LiftOver from hg38 to hg37. Currently GRAPE uses hg37 build version of the human genome reference. The pipeline supports input in hg38 and hg37 builds. One should specify the genome build version by the dedicated flag --assembly of the pipeline launcher. If hg38 build is selected (--assembly hg38), then GRAPE applies the liftOver tool to the input data in order to match the hg37 reference assembly. This parameter should also be specified during the simulation workflow.\n\n• [Optional] Phasing and imputation. GRAPE supports phasing and genotype imputation. GERMLINE IBD detection tool requires phased data. So, if input data is unphased, one should include phasing (--phase flag) into the preprocessing before running the GERMLINE workflow. If input data is highly heterogeneous in a sense of available SNPs positions, we recommend to include imputation procedure as well (--impute).\n\n• [Optional] Removal of imputed SNPs. We found that if the input data is homogeneous in a sense of SNPs positions, the presence of imputed SNPs does not affect the overall IBD detection accuracy of the IBIS tool, but it significantly slows down the overall performance. For this particular case, when input data initially contains a lot of imputed SNPs, we recommend to remove them by specifying --remove-imputation flag to the GRAPE launcher. GRAPE removes all SNPs which are marked with the IMPUTED flag in the input VCF file.\n\nThere are three relationship inference workflows implemented in GRAPE. These workflows are activated by the find command of the launcher. Workflow selection is made by the --flow parameter.\n\n1. IBIS + ERSA, --flow ibis. During this workflow IBD segments detection is performed by IBIS,8 and estimation of relationship degree is carried out by means of ERSA algorithm.12 This is the fastest workflow.\n\n2. IBIS + ERSA & KING, --flow ibis-king. KING14 is a well-known method for the inference of close relationships. It’s fast and can work with unphased data. During this workflow, GRAPE uses KING tool for the first three degrees of relationships, and IBIS + ERSA approach for higher order degrees (see Figure 1). Comparison of evaluation time between IBIS + ERSA and IBIS + ERSA & KING workflows is presented in Figure 3.\n\n3. GERMLINE + ERSA & KING, --flow germline-king. The workflow uses GERMLINE for IBD segments detection. KING is used to identify relationships for the first three degrees, and ERSA algorithm is used for higher order degrees. This workflow was added to GRAPE mainly for the case when input data is already phased and accurately preprocessed.\n\nWe added a simulation workflow into GRAPE to perform a precision/recall analysis of the pipeline. It’s accessible by simulate command of the pipeline launcher and incorporates the following steps: (1) pedigree simulation with unrelated founders; here we use the Ped-sim simulation package24; (2) relatedness degrees estimation; (3) comparison between true and estimated degrees. The source dataset for the simulation is taken from CEU (Northern Europeans from Utah) population data of 1000 Genomes Project.25 As CEU data consists of trios, we picked no more than one member of each trio as a founder. We also ran GRAPE on selected individuals to remove all cryptic relationships up to the 6th degree. Then, we randomly assigned sex to each individual and used sex-specific genetic maps to take into account the differences in recombination rates between men and women.26 Results of our precision/recall analysis for the simulated datasets are presented in the corresponding Results section.\n\nDistribution of IBD segments among non-related (background) individuals within a population may be quite heterogeneous.13 There may exist genome regions with extremely high rates of overall matching, which are not inherited from the recent common ancestors.27 Instead, these regions more likely reflect other demographic factors of the population. The implication is that IBD segments detected in such regions are expected to be less useful for estimating recent relationships. Moreover, such regions are potentially prone to false-positive IBD segments.\n\nGRAPE can use two different approaches to address this issue. The first one is based on genome regions exclusion mask, wherein some genome regions are completely excluded from the consideration. This approach was proposed by authors of the ERSA algorithm, see Ref. 13. The mask was computed based on whole-genome sequencing data for European individuals. The computed mask is built-in into ERSA 1.0 algorithm and is used by GRAPE by default.\n\nThe second approach is based on the so-called IBD segments weighing. This idea reminds one proposed in the Ancestry DNA Matching White Paper28 (see description of their Timber algorithm there). The key idea is to down-weight IBD segment, i.e. reduce the IBD segment length, if the segment crosses regions with a high rate of matching. The approach can be briefly described as follows. At first, one should evaluate IBD segments Bj for some background population. After that, we break chromosomes into windows Wi of fixed length and compute total overlap length ci between found IBD segments Bj and each window Wi, ci=∑j\|Wij\|, where Wij is an overlap between the segment Bj and the window Wi. Obtained overlap lengths are transformed into weights wi which are assigned to the windows, wi=fci∈01. Here f is a weighing function that reflects the following heuristic: if the overlap length ck for a window Wk is a relative outlier among all ci, then the value of fck is close to 0, otherwise it’s close to 1. Given that weights for all windows are computed, for each IBD segment G one can compute its weighted length Gw using the formula\n\nGRAPE provides an ability to compute the weight mask from the VCF file with presumably unrelated individuals. It breaks each chromosome into 1cM windows to compute overlaps. After that, GRAPE detects outliers among overlap lengths by means of the minimum covariance determinant (MCD) algorithm,29 and then determines the outliers upper bound h. This upper bound is used to compute weights, wi=h/ci, if ci>h; and wi=1 otherwise. Computed mask can be further used as a parameter for relationship inference workflow (see the --weight-mask parameter). As an example, Figure 2 depicts the weight mask computed for the individuals of East Asian Ancestry taken from 1000 Genomes Project.25 To detect IBD segments, IBIS workflow was used with parameters --ibis-seg-len 5, --ibis-min-snp 400. High matching regions (wi≈0) are marked with the blue color. For comparison, ERSA 1.0 masked regions are depicted with the pick hatching.\n\nIBIS workflow parameters: --ibis-seg-len 5, --ibis-min-snp 400.\n\nGRAPE can be run inside a Docker container. This way is recommended. Another option is to run the pipeline from scratch with all the dependencies pre-installed. We successfully ran the pipeline on Ubuntu 18.04 Linux distribution30 with eight CPUs and 32 GB of RAM to evaluate the performance on huge datasets with up to 100k samples and millions of SNPs.\n\nGRAPE can utilize multiple cores. For that, one should specify the cores number via the --cores parameter. The default number of cores is equal to the total number of available CPUs minus 1.\n\nThe pipeline can be run using Funnel,31 a lightweight task scheduler that implements Task Execution Schema20 developed by GA4GH.22 The scheduler can work in various environments, from a regular virtual machines to Kubernetes cluster with the support of resource quotas. We provide several examples of the task specifications for Funnel. Each sample represents a JSON file with the task description. These files are available in the GRAPE GitHub repository within the corresponding funnel subfolder.\n\nTo estimate the performance of the GRAPE pipeline we used a machine with eight CPU cores, Intel(R) Xeon(R) CPU E5-2686 v4 @ 2.30 GHz, and 32 GB of RAM. The fastest IBIS + ERSA (--flow ibis) relatedness inference workflow takes about 22 hours to process 100k individuals dataset, see Figure 3. IBIS tool has a quadratic time complexity with respect to the total number of individuals. The addition of KING (--flow ibis-king) increases the total running time by roughly 50%. Performance analysis confirmed that IBIS is a simple and efficient tool. It allows the pipeline to process hundred of thousands of individuals in a reasonable amount of time.\n\nGRAPE was evaluated on a machine with 8 CPU cores, Intel(R) Xeon(R) CPU E5-2686 v4 @ 2.30GHz, and 32 GB of RAM. Both axes are in logarithmic scale.\n\n\nUse cases\n\nThis section gives examples of the GRAPE pipeline commands which have to be run to infer relationships, or to evaluate precision/recall metrics on a simulated dataset.\n\nAs the first step, reference data must be downloaded. We suppose that reference data to be stored in/media/ref directory. Here and below we specify the --real-run flag. Without this flag GRAPE performs a dry-run.\n\nListing 1. Reference downloading for the IBIS + ERSA workflow.\n\n\n\nAs the second step, preprocessing is performed. We suppose that the input file is located at/media/input.vcf.gz, and it’s in hg38 build. Input file location specified by the flag --vcf-file. GRAPE working directory is/media/data. It’s specified by the --directory flag.\n\nListing 2. Preprocessing for the IBIS + ERSA workflow.\n\n\n\nThe third step is the relationship inference. It is launched with the the find command. We use IBIS + ERSA workflow that corresponds to the ibis value of the --flow parameter (default).\n\nListing 3. Relationship inference with the IBIS + ERSA workflow.\n\n\n\nGRAPE has an ability to specify additional parameters to the ERSA and IBIS algorithms to control the sensitivity and the false positive rate. These parameters are described below. Default GRAPE parameters are quite conservative. They provide a low false positive rate and low sensitivity in high (9+) degrees.\n\n• [IBIS] --ibis-seg-len. Minimum length of the IBD segment to be found by IBIS. Higher values reduce false positive rate and give less distant matches (default = 7 cM).\n\n• [IBIS] --ibis-min-snp. Minimum number of SNPs per IBD segment to be detected (default = 500 SNPs).\n\n• [ERSA] --zero-seg-count. Mean number of shared segments for two unrelated individuals in the population. Smaller values tend to give more distant matches and increase the false positive rate (default = 0.5).\n\n• [ERSA] --zero-seg-len. Average length of IBD segment for two unrelated individuals in the population. Smaller values tend to give more distant matches and increase the false positive rate (default = 5 cM).\n\n• [ERSA] --alpha. ERSA significance level12 (default = 0.01).\n\nThe first and second steps are the same as for the previous use case. The third step is launched with specifying of the ibis-king flow parameter. For this case GRAPE performs an estimation of the first three degrees of relationship with KING. Other degrees are estimated with ERSA (see Figure 1). The resulted report of individual relationships for this case contains additional king_degree and kinship columns. KING algorithm has no additional parameters.\n\nListing 4. Relationship inference with the IBIS + ERSA & KING workflow.\n\n\n\nAt first, reference data must be downloaded. The GERMLINE tool works with phased data only. So, if input data is unphased, one should download additional reference dataset to perform phasing. For that purpose we use a reference panel from 1000 Genomes Project. This panel takes a considerable amount of disk space (∼25 GB), and requires significant time to download. To download this panel one should specify the --phase flag while using the reference command.\n\nListing 5. Reference downloading.\n\n\n\nThe second step is the data preprocessing. We use the --phase flag to apply the phasing procedure during this stage.\n\nListing 6. Preprocessing for the GERMLINE + ERSA & KING workflow.\n\n\n\nThe third step is the relationship inference. One should specify --flow germline to use the GERMLINE tool for the IBD segments detection. ERSA parameters are the same as for the IBIS + ERSA workflow. Parameters of the GERMLINE are not configurable. Currently, we use the following sets of GERMLINE parameters: -min_m 2.5, -err_hom 2, -err_het 1. See GERMLINE documentation for the parameters description.7\n\nListing 7. Relationship inference with GERMLINE + ERSA & KING workflow.\n\n\n\nTo perform simulation, at the first step one should download full reference dataset by using the reference command with the --phase and --impute flags enabled. The second step is the simulation workflow. For that, one should use the simulate command of the GRAPE launcher.\n\nListing 8. Evaluation of the IBIS + ERSA workflow on a simulated dataset.\n\n\n\nAlong with the parameters for preprocessing, ERSA and IBIS tools, simulation workflow has two additional parameters listed below.\n\n• --sim-params-file. File with parameters of simulation for the Ped-sim tool. For more information see.24 We have prepared several simulation parameters files, and stored them in the GRAPE repository on GitHub.\n\n• --sim-samples-file. File with a list of individuals from the 1000 Genomes Project which are used as founders for the simulations. One can choose ceph_unrelated_all.tsv (unrelated individuals from CEU population), or all.tsv (all individuals available in 1KGP).\n\nThe output files include a list of kinship matches found in the simulated dataset, precision/recall plots, and a confusion matrix to compare the detected degrees of relationship with the true degrees. Detailed information on the computed metrics is presented in the Results section.\n\nThe first step is to download the reference dataset (see the previous section). The second step is to run the simulate command, specifying germline-king flow and the --phase flag.\n\nListing 9. Evaluation of the GERMLINE + ERSA & KING workflow on a simulated dataset.\n\n\n\nGRAPE has a dedicated command to compute the weight mask, compute-weight-mask. It performs IBD segments detection for the input VCF file and then analyse IBD segments distribution to compute the weight mask. The resulting files consist of a weight mask file in JSON format and a visualization of the mask (see Figure 2).\n\nListing 10. Computation of the IBD segments weighing mask.\n\n\n\nFor the example above the resulting files are store in the/media/background/weight-mask/directory.\n\nTo apply weight mask during the relatedness detection (find command), one should specify the mask file with the --weight-mask parameter. When used, the ERSA 1.0 exclusion mask is disabled.\n\nListing 11. Usage of the IBD segments weighing mask.\n\n\n\n\nResults\n\nTo test the accuracy and flexibility of the pipeline, we performed extensive testing on both real and simulated datasets. As a sanity check, we took the Allen Ancient DNA Resource (AADR) dataset32 and made sure that GRAPE does not produce any kinship matches between ancient and present-day individuals.\n\nNext, we have run Khazar origins dataset33 through the GRAPE. The Khazar dataset contains 1770 samples from 106 Jewish and non-Jewish populations. The dataset contains a significant amount of data from small homogeneous populations. The KING analysis was previously applied to this dataset by the authors33 to identify close relatives up to the third degree. We applied GRAPE and found 1715 putative relationships. Most of them have a degree of 4+. This result highlights the fact that the total length of IBD segments in small homogeneous populations is several orders of magnitude higher then for the heterogeneous populations in Europe and Asia.34,35 This is an obvious obstacle for the relatedness detection. There is no method known to us to address this issue while using genotypic data obtained from SNP arrays. Whole genome sequencing has a potential to solve this problem, since rare mutations should break long IBD segments.\n\nAs for the data from genetic testing companies, GRAPE has been successfully applied to the database of 100k+ customers of Atlas Biomed,36 a company that provides direct-to-consumer genetic tests. During this test GRAPE was proven to be able to handle diverse data obtained from different chips, reference alignments, and other possible data inconsistencies.\n\nFinally, GRAPE was evaluated on simulated datasets. We performed the simulation using unrelated founders from 1KGP Affymetrix genotype chip data. This chip contains approximately 900k SNPs. The simulation was carried out with the Ped-sim package. Using this tool, we produced several pedigree structures with eight generations, and a maximum degree of relationships equal to 14. Then we joined all of the pedigrees into one dataset, and performed the precision/recall analysis of the GRAPE pipeline for the different flows. For each degree i of relationships we computed precision and recall metrics:\n\nHere TPi, FPi, FNi are the numbers of true positive, false positive, and false negative relationship matches predicted for the degree i. In our analysis we used non-exact (fuzzy) interval metrics. For the 1st degree, we require an exact match. For the 2nd, 3rd, and 4th degrees, we allow a degree interval of ±1. For example, for the 2nd true degree we consider a predicted 3rd degree as a true positive match. For the 5th+ degrees, we use the ERSA confidence intervals which are typically 3-4 degrees wide. For 10th+ degrees, these intervals are 6-7 degrees wide. We also plot a confusion matrix for the predicted vs true degrees.\n\nResults of the simulation for the IBIS + ERSA & KING workflow is presented in Figure 4. The following set of parameters was used: --ibis-seg-len 7, --ibis-min-snp 500, --zero-seg-count 0.5, --zero-seg-len 5, --alpha 0.01. Confusion matrix is presented on the right panel in Figure 4. There -1 stands for no-relationship. The pipeline shows recall above 90%+ for degrees from 1 to 5. It detects all relatives with 1-4 degrees. GRAPE found no false positive matches, i.e. it does not find any relationships among unrelated individuals, Recall−1=1. Precision is above 90% among all detected degrees. We set these parameters as default for this GRAPE workflow. These parameters are quite conservative, i.e. they provide high precision but low sensitivity.\n\nParameters of the workflow: --ibis-seg-len 7, --ibis-min-snp 500, --zero-seg-count 0.5, --zero-seg-len 5, --alpha 0.01.\n\nOne can relax GRAPE parameters to get more relatedness matches for high degrees. On the other hand, the number of false positive matches increases as well. Figure 5 shows the simulation results for the IBIS + ERSA & KING workflow for slightly relaxed parameters: --ibis-seg-len 5, --ibis-min-snp 400, --zero-seg-count 0.1, --zero-seg-len 5, --alpha 0.01. The number of detected relationships increases significantly for higher (≥8) degrees. But false positive matches arise as well, i.e. Recall−1≠1.\n\nParameters of the workflow: --ibis-seg-len 5, --ibis-min-snp 400, --zero-seg-count 0.1, --zero-seg-len 5, --alpha 0.01.\n\nSimulation results for the GERMLINE + ERSA & KING workflow is presented in Figure 6. We used the same ERSA parameters as for Figure 4: --zero-seg-count 0.5, --zero-seg-len 5, --alpha 0.01. In comparison to Figure 4, one can see that GERMLINE slightly decreases recall for 6th and 7th degrees, but improves it for higher (≥8) degrees. Precision is above 95% among all detected degrees. No false positive matches were found, i.e. Recall−1=1. Our experiments showed that, in comparison to IBIS, GERMLINE is better suited for a careful analysis of relatively small cohorts with phased data. The IBIS algorithm is less sensitive, but for segments with length above 7 cM, it produces the same results as GERMLINE, while working with unphased data.\n\nParameters of the workflow: --zero-seg-count 0.5, --zero-seg-len 5, --alpha 0.01.\n\nOur simulation experiments showed that both weighing and exclusion approaches reduce the false-positive rate and significantly improves the overall performance of the pipeline. By the way, weighing mask can be better adapted to specific ancestries, and after additional parameters tuning may slightly outperform ERSA 1.0 approach. In Figure 7 comparison between between the ERSA 1.0 exclusion mask and the GRAPE weight mask is presented for a simulated dataset with the founders of East Asian Ancestry from 1KGP. IBIS workflow was used. Weight mask was taken from Figure 2. Parameter --zero-seg-count was varied while using weight mask to achieve the same level of precision. One can see, that with approximately the same precision, the weighing approach gives several percentages higher recall.\n\nMetrics differences are marked with colors: green, if the metric has increased while using weighing; and red, if the metric has decreased. Common parameters for the both workflows: --ibis-seg-len 5, --ibis-min-snp 400 --zero-seg-len 5, --alpha 0.01. Parameter --zero-seg-count equals 0.1 while using weight mask, and 0.3 while using original ERSA 1.0 exclusion mask.\n\nIn the end, we compared GRAPE with TRIBES.23 TRIBES is an earlier open-source pipeline for relatedness detection. The pipeline combines the GERMLINE algorithm for IBD segments detection and the calculation of the genome proportion with zero alleles inferred IBD (IBD0) for each pair to detect the relatedness. If the data is not phased, TRIBES provides an ability to phase data with the EAGLE tool. This part of TRIBES is similar to one of the corresponding GRAPE workflows of IBD segments detection. In contrast to GRAPE, TRIBES estimates degrees of relationship according to expected IBD0 segments proportion ranges. GRAPE uses the ERSA algorithm, which, to our knowledge, is a more advanced approach.\n\nWe have run the TRIBES pipeline on the same simulated datasets. Since the simulated datasets contain unphased data, we also applied built-in phasing procedure from TRIBES. The result of the analysis is presented in Figure 8. TRIBES has demonstrated high detection power for distant relationships of up to the 12th degree. Given that many 13+ degree relatives do not share any IBD segments, this is near a theoretical limit. However, TRIBES produces a huge number of false positive matches, see confusion matrix in the right panel of the Figure 8. Since TRIBES lacks options that allow users to control false positives rates by varying pipeline parameters, it becomes a crucial drawback. This obstacle does not allow the TRIBES pipeline to be adapted for applications, where desired precision/recall rate may vary depending on different business or research objectives.\n\n\nConclusions\n\nIn the current paper, we introduced GRAPE: genomic relatedness detection pipeline. We performed the careful selection of tools, and combined various preprocessing steps, IBD segments detection tools, and algorithms of estimations of relationship into a single pipeline. One of the possible workflows of the pipeline is based on IBIS tools and can work with unphased data. It’s suitable for the analysis of large cohorts in a relatively short time. Using this workflow GRAPE can perform relationship estimation among 100k samples in 22 hours. Another possible workflow is based on GERMLINE IBD segments detection tool and works with phased data. Our experiments showed that GERMLINE has the most detection power, while IBIS option is the fastest, easiest to use, and has sufficient accuracy for 1-8 degrees of relationship. Finally, we compared GRAPE with TRIBES, another relatedness detection pipeline. In contrast to GRAPE, TRIBES produce a huge number of false positive matches, requires phased data, and lacks important preprocessing and evaluation options, which makes it impractical for various applications. GRAPE is proved to be a reliable and accurate tool for the analysis of close and distant degrees of kinship. It provides an ability to control a false positive rate, can work with heterogeneous data obtained from various chips, and is ready for production integration.\n\n\nData availability\n\nPublicly available datasets were used to test GRAPE. These datasets are available from:\n\n• 1000 Genomes Project25;\n\n• URL: https://www.internationalgenome.org/data-portal/data-collection/phase-3.\n\n• Khazar Origin for Ashkenazi Jews33;\n\n• URL: https://evolbio.ut.ee/khazar.\n\n• Allen Ancient DNA Resource (AADR)2;\n\n• URL: https://reich.hms.harvard.edu/allen-ancient-dna-resource-aadr-downloadable-genotypes-present-day-and-ancient-dna-data.\n\nGRAPE pipeline can be accessed from the following pubic available resources:\n\n• GitHub: https://github.com/genxnetwork/grape.\n\n• Docker Hub: https://hub.docker.com/r/genxnetwork/grape.\n\n• Dockstore: https://dockstore.org/organizations/GenX/collections/GRAPE.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.6482561.37\n\nLicense: GPLv3.", "appendix": "Acknowledgments\n\nAn earlier version of this article can be found on bioRxiv (doi: https://doi.org/10.1101/2022.03.11.483988).\n\n\nReferences\n\nPosey JE, O’Donnell-Luria AH, Chong JX, et al.: Insights into genetics, human biology and disease gleaned from family based genomic studies. 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Publisher Full Text" }	[ { "id": "139363", "date": "12 Jul 2022", "name": "Ying Zhou", "expertise": [ "Reviewer Expertise population genetics", "computational genetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMedvedev et al. presented a user-friendly pipeline for detecting both the distant and the close relatedness. The design is straightforward, and this pipeline includes different inference strategies that can deal with both phased and unphased data, and it can be run inside a Docker container, which makes it very applicable in real analysis. It is also appreciable that authors devoted lots of times in building this pipeline. I have only three comments:\nI may understand why to use the King software for close relatedness inference while using IBD based method for distant relatedness inference. It is because large IBD cutoff for example 7cM could lead to loss of IBD segments and bias the kinship estimation. However, when the target population is structured or admixed, the King software may not work as well as IBD based inference.\n\nWhat should I do if I need to run analysis on a large sample size, for example, UK biobank (n~=500k)? The largest test data in this work is 100k, while some other methods declare they can deal with kinship estimation on much larger sample sizes, see ref1.\n\nIf I want to find out all potential relatives of one target sample in a reference database, with this pipeline, I need to merge the data first and then detect all relatedness. This is not efficient. An alternative solution is to use query based IBD detection, see ref2.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "9458", "date": "13 Apr 2023", "name": "Aleksandr Medvedev", "role": "Author Response", "response": "I may understand why to use the KING software for close relatedness inference while using IBD based method for distant relatedness inference. It is because a large IBD cutoff for example 7cM could lead to a loss of IBD segments and bias the kinship estimation. However, when the target population is structured or admixed, the KING software may not work as well as IBD-based inference. [paper updated] Answer: For the close relatedness inference we use the KING ibdseg option by default, which also uses IBD segments for the degree estimation. Also, we calculate the kinship coefficient using the legacy KING kinship option and also report kinship-based degrees in our output file. We changed the description of the usage of KING in our paper. What should I do if I need to run the analysis on a large sample size, for example, UK biobank (n~=500k)? The largest test data in this work is 100k, while some other methods declare they can deal with kinship estimation on much larger sample sizes, see https://academic.oup.com/bioinformatics/article/36/16/4519/5858978. Answer: Based on Figure 3 and the fact that the IBIS and ERSA algorithms have O(n^2) complexity when n is the number of samples, we can estimate that running time on n~=500k will be ~20-25 days on the system specified in our article. This is comparable with time required for phasing a dataset of this size. We looked into IBDKin and we are willing to evaluate it in our future GRAPE version as a King substitution. However, as far as we understand, it does not infer distant degrees yet. Answer: In the new article version we added beta RaPID support and compared it with IBIS and GERMLINE. RaPID-based workflow took 45 min to run on the 100K dataset and 6 hours to run on the 500K dataset. But the dataset was already phased. If I want to find out all potential relatives of one target sample in a reference database, with this pipeline, I need to merge the data first and then detect all relatedness. This is not efficient. An alternative solution is to use query-based IBD detection, see https://academic.oup.com/bioinformatics/article/35/14/i233/5529240. Answer: We plan to implement a query-based IBD detection for IBIS in one of the future GRAPE releases. We checked the paper about PBWT queries and it appears that it works only with haplotypes and not genotypes, i.e. phased data. However, we plan to test RaPID and hapIBD on phased data as a GERMLINE replacement and possibly implement query-based IBD detection for them." } ] }, { "id": "143937", "date": "03 Aug 2022", "name": "Ardalan Naseri", "expertise": [ "Reviewer Expertise Computational Biology", "Bioinformatics", "Population Genetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMedvedev et al. have developed a user-friendly software/pipeline for relatedness inferences from genetic data. The pipeline includes data preprocessing, identity-by-descent (IBD) segment detection, and relationship inferences. The pipeline has incorporated several software inside a Docker that enables a very convenient solution for users aiming to infer relationships from genetic data.\n\nThe authors have documented the performance of the pipeline regarding the accuracy and run time using both simulated and real-world data. There are a few suggestions/comments that could improve this work:\nThe memory usage of the pipeline has not been mentioned in the article. It would be useful to provide more information about the required memory usage.\n\nCurrent biobank-scale data contain hundreds of thousands of individuals. The run time for 100k individuals was reported as 22 hours using 8 CPU cores (for IBIS + ERSA). The user may want to know if they can run the pipeline in large panels within a reasonable time. The run time of the pipeline using IBD detection (with GERMLINE) was also not included. More recent IBD detection tools (e.g. hapIBD, RaPID and iLASH) can handle very large cohorts efficiently. Incorporating other IBD detection tools might also improve the efficiency of the pipeline regarding the run time and/or memory usage.\n\nRobustness against genotyping errors, different marker densities, and different populations have not been studied well. The authors have pointed out the limitation with a small isolated population though.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "9459", "date": "13 Apr 2023", "name": "Aleksandr Medvedev", "role": "Author Response", "response": "Medvedev et al. have developed a user-friendly software/pipeline for relatedness inferences from genetic data. The pipeline includes data preprocessing, identity-by-descent (IBD) segment detection, and relationship inferences. The pipeline has incorporated several software inside a Docker that enables a very convenient solution for users aiming to infer relationships from genetic data. The authors have documented the performance of the pipeline regarding the accuracy and run time using both simulated and real-world data. There are a few suggestions/comments that could improve this work: The memory usage of the pipeline has not been mentioned in the article. It would be useful to provide more information about the required memory usage. Answer: We implemented several batch preprocessing and postprocessing routines to ensure that for the dataset of every size memory usage will not be more than 16GB RAM. Current biobank-scale data contain hundreds of thousands of individuals. The run time for 100k individuals was reported as 22 hours using 8 CPU cores (for IBIS + ERSA). The user may want to know if they can run the pipeline in large panels within a reasonable time. The run time of the pipeline using IBD detection (with GERMLINE) was also not included. More recent IBD detection tools (e.g. hapIBD, RaPID and iLASH) can handle very large cohorts efficiently. Incorporating other IBD detection tools might also improve the efficiency of the pipeline regarding the run time and/or memory usage. Answer: Based on Figure 3 and the fact that IBIS and ERSA algorithms has O(n^2) complexity, when n is the number of samples, we can estimate that running time on n~=500k will be ~20-25 days. This is comparable with time required for phasing a dataset of this size. We are planning to investigate accuracy and performance on hapIBD, RaPID and iLASH for phased datasets, but unique advantage of IBIS-based workflows is that it does not require phasing. We chose GERMLINE as the well-known method with an appropriate license. iLASH does not have an MIT or GPL-like license. Answer: In the new article version we added beta RaPID support and compared it with IBIS and GERMLINE. RaPID-based workflow took 45 min to run on 100K dataset and 6 hours to run on 500K dataset. But the dataset was already phased. Robustness against genotyping errors, different marker densities, and different populations have not been studied well. The authors have pointed out the limitation with a small isolated population though. Answer: IBIS-based workflows detect relatively big IBD segments (more than 5-7 cM). We hope that it will provide some robustness to genotyping error. We also provide a —remove imputation option in the preprocessing workflow, because we found out that some imputed regions produce many false-positive IBD matches. Answer: IBIS and RaPID-based workflows should not detect many false-positive IBD segments in the low-density regions, because they constrain both minimum number of SNPs in an IBD match and minimum cM length. However, low-density regions could produce many false-negatives." } ] } ]	1	https://f1000research.com/articles/11-589
https://f1000research.com/articles/12-392/v1	13 Apr 23	{ "type": "Research Article", "title": "Social cognition and human aesthetic niche—the evolving human cognition as a participant of human niche construction", "authors": [ "Xuanqi Zhu" ], "abstract": "Aesthetic phenomena have been intertwined with the life of humans to a significant degree that is not observed in non-human animals. The complexity and subtlety of it have long been regarded as one symbol of human’s exceptional cognitive power. This power can sometimes be misrepresented by a view that regards human aesthetic behaviours as innate/encoded and automated settings that are brought along with the human brain. In scenarios like this, the naturally selected brain takes up a role as some super explanator—by appealing to the configurations of the neural connectivity as reasons/causations for observed phenotypic traits. Therefore, this line of thinking can sometimes obscure the role played by the sociocultural background in affecting those configurations. By drawing upon the notion of niche construction, I will propose a nature-nurture coevolving framework for understanding human cognitive evolution. It will be argued that the evolutionary trajectory of human cognition is heavily defined by and is, therefore, better understood through the lens of a human cultural niche and of contextualised/context-dependent expressions of human behavioural traits. This view will be delivered by highlighting the dynamics between selective pressures and the differential expression of human phenotypic traits and acknowledging the evolutionary causal role of human cultural behaviours and practices. Finally, I argue that a major evolution of social cognition was brought about through an aesthetic tradition of the Acheulean and conclude by briefly proposing a potential subject for future study. The basic research method applied in this article is theoretical deduction. Specifically, a restricted interdisciplinary investigation that concerns academic literature from relevant fields (centring on the topic of niche construction) of archaeology, evolutionary biology and human cognition was used. Furthermore, through a process of assessing and identifying of plausible evidence, the abovementioned arguments of this study are generated.", "keywords": [ "Aesthetic niche", "cognitive evolution", "cultural evolution", "gene-culture coevolution", "human self-domestication", "social cognition", "niche construction theory" ], "content": "Introduction\n\nAmong the known animals on earth, modern humans are believed to have the most complex arrangements of cognitive abilities. Those abilities include, e.g., abstract reasoning, hierarchical language system, long-term memory, self-awareness, shared intentionality, etc. The unique potency of human cognition is sometimes represented by the knowledge of perhaps the most typical trait of humans, an unparalleled computing device, an exquisite problem-solving system, and a remarkable biological adaptation—the human brain. This human-unique equipment possesses a myriad of functions that are crucial for human survival (e.g., emotion processing, selective attention, memory retrieval, motor control, etc.). Usually, many of these functions are organised in different ways to bring about other higher-level functions involved in language using, tool making, and social cognitive tasks encompassing the use of theory of mind, recognition of group identity, social signalling and so on. Together, at the behavioural level, these functions give rise to those phenotypic traits that are conspicuously human—e.g., behaviours based on moral knowledge or local norms, and artistic behaviours such as painting or music-making. It is with this general understanding, the evolution of human cognition is extensively explored, through topics aiming at specific patterns in human behavioural diversity, by researchers from diverse fields ranging from archaeology, and cognitive neuroscience, to evolutionary biology, psychology, and philosophy.\n\nMore specifically, research topics focusing on our distinct Homo ancestors have produced rich resources for understanding the evolution of cognition in prehistoric times. For instance, the study on Neanderthal’s life mode and caring behaviours (e.g., Spikins et al., 2014, 2019; Mondanaro et al., 2020) have provided us insights into how possible cognitive adaptations might have evolved under challenging conditions within the North Temperate Zone. To push further into the distant past of the human lineage, studies based on the longest known technological tradition (i.e., the handaxe making tradition) of the Acheulean industry, have also suggested that significant episodes of cognitive evolution must have taken place throughout over 1.5 million years of development since~1.78million years ago. To put it very briefly, these studies imply that plausibly by the Late Acheulean (about 500,000 to 300,000 years ago), and before the advent of Homo sapiens, the hominin cognition had evolved with capacities by which complex behaviours, such as systematic large game hunting, efficient teaching and learning (supposedly a quasi-apprenticeship system), multi-stage tool making, or even aesthetic practices, were initiated and sustained (e.g., Stapert, 2007; Currie, 2011; Hewlett et al., 2011; Hodgson, 2011, 2015; Domínguez-Rodrigo et al., 2012; Layton et al., 2012; Hiscock, 2014; Sinclair, 2015; Dira and Hewlett, 2016; Boyette and Hewlett, 2017; Diez-Martín, et al., 2018, 2019; White and Foulds, 2018; White et al., 2019; Currie and Zhu, 2021).\n\nMeanwhile, from a more proximate perspective, with the help of experimental methodology (e.g., neuroanatomy and neuroimaging), especially the functional magnetic resonance imaging (fMRI), researchers now can probe into human cognition in a quantitatively more precise way. This is usually done through experiments relying on human participants and comparative experiments between humans and other primates (e.g., chimpanzees and bonobos). However, for our current concern about human cognitive uniqueness and its evolution, recent studies have generated intriguing implications. That is, while comparative studies have pointed to the fact that we can reasonably identify many comparable (analogous) cognitive functions in non-human primates, the differences thus become explanatorily important.\n\nFor example, it has been argued that the white matter in human brain has been expanding disproportionately (e.g., Gazzaniga, Ivry, and Mangun, 2014; Donahue et al., 2018). Compared with other primates, human prefrontal cortex shows a much higher constituent ratio in its white matter (which is mainly made up of axons) than in the grey matter (which consists of large bodies of cells). Considering the significance of the prefrontal cortex for human cognition, therefore, this disproportionate development of white matter seems to imply some human-specific evolutionary selection. In another experiment, Donahue et al. (2018) have carried out detailed measurements comparing the absolute volumes of the prefrontal cortex, grey matter, and white matter among human participants (n=60), chimpanzees (n=29) and macaques (n=19)1. The outcomes have demonstrated that the ‘white matter underlying human PFC is particularly large compared with nonhuman primates’ (Donahue et al., 2018, p.E5188). Again, since those axons’ (the white matter’s only constituent) job is to process and send nerve signals from and to other body regions, the fact that humans have disproportionately highly developed white matter favours the speculation that strong selective pressures must have come from some persistent need for a refined, well-tuned, or sophisticated synergy that better exploits and connects various other parts of the brain and body. In the next sections, it will be suggested that human cumulative cultural niche construction is a source of selective pressures on brain connectivity. Here I quote Gazzaniga, Ivry, Mangun, ‘the cognitive capabilities that are uniquely human may be more a result of how our brains are connected rather than due to an increase in the number of neurons’2.\n\nAnother related example is the FOXP2 gene, which is believed to be a key factor for the development of human language ability. Briefly speaking, the FOXP2 gene exerts its power by regulating the expressions of genes that are involved in language using. So, it has long been regarded as crucial for human cognitive uniqueness. However, studies have shown that the FOXP2 gene is not unique to modern humans (Neanderthals and other animal such as some species of birds, too, have the FOXP2 gene), and there is no recent selection for it (Krause et al., 2007; Gazzaniga, Ivry, and Mangun, 2014; Atkinson et al., 2018). According to Gazzaniga, Ivry and Mangun (2014, p.503), the FOXP2 regulates a wide array of genes that are involved with e.g., morphogenesis, intracellular signalling, neuron outgrowth, and learning, etc. Thus, just as the above example has implied, what might be the case is not some single gene that alone codes for unique human linguistic behaviours. Nevertheless, what really leads to the uniqueness here is probably a set of context-dependent factors that specifies the regulatory rules for the FOXP2 gene. In other words, it was the contingent interactions between the human niche and the expression of many other phenotypic traits (more basic ones) which eventually gave birth to the higher-level phenotype of language using.\n\nTo summaries the introductory part, firstly, based on the evidence at hand, researchers have argued that complex cognition (manifested through the material cultures such as the Acheulean industry) might well have existed in the distant past of the human lineage, way before the presence of Homo sapiens. Secondly, the above studies of neural and genetic substrates of human cognition have indicated that, in the evolutionary sense: 1. There might be certain elements in hominin’s everyday life (which are demanding in terms of neural cross reference and integration of sensory information) that motivate complex organization of behaviours; 2. These elements might have managed to get involved in the way of subsistence of different hominin societies through mechanisms like social transmission; 3. Thus, they became causally accountable in explicating the course of human cognitive evolution, e.g., by inducing effects of Baldwinisation3. That said, the significant role of culture4 has been brought to the foreground. Such an implication, by acknowledging a deep interweaving relation between cognition and culture, echoes with the recent findings of theories (e.g., niche construction theories and epigenetic theories) which advocate that cultural factors can produce stably transmitted phenotypic traits at the population level, emphasises the possibility that human cognition (and human evolution in general) is, to a considerable extent, culturally driven and flexible.\n\nThis current research will adopt an interdisciplinary perspective, with a generally deductive manner of theoretical analysing and integrating, that draws upon key works in evolutionary biology and archaeology to touch on the topic of cognitive evolution. In short, the general topic of human cognitive evolution will be addressed by proposing an inclusive evolutionary framework on human aesthetic tradition—one that acknowledges an aesthetic niche. That is, to see human aesthetic activities/practices as a mode of niche construction.\n\nThat said, before such a framework could be obtained, this research will examine:\n\n1. The notion of niche, the theory of niche construction, and the postulated relations between niche construction, organism evolution, social learning, and cultural evolution. In this regard, key works by Laland (e.g., 2014), Oldling-Smee (e.g., 2003), and others will be discussed. Such a discussion will serve as a foundation for the conception of ‘human aesthetic niche’.\n\n2. Based on 1., the deep history of human aesthetic culture/tradition, as an important aspect of human aesthetic niche that affects cognitive evolution, will be explored. Being perhaps a stone technology that had persisted the longest time span in human lineage, the Acheulean handaxe tradition will be focused upon. Specifically, evidence from key literature on the economics in handaxe manufacturing (e.g., Lycett et al., 2016; de la Torre, 2016; Pargeter, Khreisheh, and Stout, 2019), transmission of skills in palaeolithic groups (e.g., Hiscock, 2014; Diez-Martín et al., 2018), handaxe morphology (e.g., Beyene et al., 2013; Key and Lycett, 2017a; Shipton, Clarkson and Cobden, 2018; White and Foulds, 2018) will be discussed and incorporated to generate claims about the evolution of social cognition in Acheulean times.\n\nIn the following sections, I will first (in Section 2) take a step back and provide a general theoretical framework of evolution that sustains both the cultural and the biological contribution. It will be argued that the uniqueness we see currently in our cognitive prowess is likely to be an evolutionary consequence of the constant interaction between our unique reliance on the accumulating human culture and selective mechanisms. Then (in Section 3), I will offer discussions on aesthetic activities such as the appreciation of Acheulean handaxes, arguing that the cognition involved in aesthetic activities is mediated by this biocultural framework.\n\n\nMaking our conception of human evolution more inclusive\n\nThis section intends to articulate the hybrid nature of human evolution and to suggest a more inclusive framework for understanding human cognitive evolution.\n\nTo do this, I will appeal to the notion of niche construction. But before that, let us recall a more classic framework of evolution, the Modern Synthesis. Put in a few sentences, the modern synthesis is a view through which stably transmitted phenotypic traits of an organism are treated as, in essence, genetic traits (Danchin, et al., 2011; Scott-Phillips, et al., 2014; Laland et al., 2015). Or to put it another way, genetic variations and corresponding genetic inheritance should present as a prerequisite condition for any observed cross-generationally preserved phenotypic traits (with cognitive patterns being an important type).\n\nHowever, as is well-known, at the ontogenetic level, phenotypic traits can be derivable. Through mechanisms such as social learning and through molecular level mechanisms such as epigenetic methylation, the brain (neural connectivity) exhibits outstanding cognitive flexibility. That is, neurological changes could occur when individuals are exposed to major changes in their selective environments (e.g., individuals who experience long-term exposure to alcohol can develop a dependence which could be identified cognitively and behaviourally, but clearly in this case, the sociocultural background could play some part, in addition to one’s predispositions to alcohol5). A question can be asked, could traits like this (and other phenotypic traits that are socially learned or culturally triggered) become inheritable? The answer is possibly a ‘yes’. Recent studies have identified cases of trans-generation inheritance of derived traits—e.g., the specific way of tool using in human-raised crows (Danchin et al., 2011); an inherited aversion of specific olfactory stimuli among experimental rats (Dias and Ressler, 2014); similarly, acquired taste aversion is identified in fruit flies (Heyes, Chater and Dwyer, 2020); high level expression of lactose tolerance in human populations with a dairy tradition, and the increased copies of the salivary amylase genes in agricultural societies (Laland and Sterelny, 2006, Laland et al., 2014, 2016; Menary, 2014; Papineau, 2005b; Perry George, et al., 2007; Scott-Phillips et al., 2014). These findings have called the Modern Synthesis into question. It is important to see not just the underlying configurations of the genetic inheritance, but also the dynamics in the sociocultural background which contributes to these changes, if we are to gain a fuller explanation for human cognitive and other phenotypic traits (e.g., an alcohol dependence and related behaviours).\n\nThe worries for the Modern Synthesis have stimulated the development of alternative theories in the field of evolutionary biology. As evidence grows, the niche construction theory has been gaining traction recently (Laland, 2008, 2013; Laland et al., 2014, 2015, 2016). ‘Niche construction’, refers to the phenomena where organisms actively modify their own and others’ niches (Day, Laland, Odling-Smee, 2003; Odling-Smee, Laland and Feldman, 2003; Scott-Phillips et al., 2014; Laland, Matthews, Feldman, 2016). Moreover, since such modifications usually have the potential to influence selective pressures in the environments, novel episodes of evolution could incur due to significant changes in selective pressures, i.e., niche construction induced evolution. In this sense, the idea of niche construction, by taking the fact (that is, through their environment-shaping activities, organisms can actively affect the evolutionary trajectories of their own) into consideration, brings to the foreground this more inclusive nature of evolution. As a result, for many who advocate a fundamental role of niche construction for organism evolution, this perspective takes up the name ‘niche construction theory’ or ‘NCT’ (Laland and Sterelny, 2006; Kendal et al., 2011; Laland and O’Brien, 2011; Odling-Smee et al., 2013; Laland et al., 2014, 2015; Laland, Matthews, Feldman, 2016; Laland, Odling-Smee, and Endler, 2017).\n\nOther than the different senses in which the term ‘niche’ is previously used (Grinnell, 1917; Elton, 1927; Hutchinson, 1944, 1957; Holt, 2009), when used in NCT, the notion ‘niche’ is defined as, ‘the sum of all the natural selection pressures to which the population is exposed.’6 This is a broad and inclusive definition, which also captures the evolving nature of a niche. It allows for the sum total of the conditions where a species in question, through constant interactions with the natural context, maintains the development and persistence of its own7. By taking this perspective of regarding species as subjects engaging in different niche constructing processes, the following points become important:\n\n1. Evolution to a considerable extent is subject to a relationship, which is dynamic and mutual, between organisms and their surroundings—a situation in which organisms are always constrained by selective factors in the environments, and in respond to those factors, alterations to such environments are always made by those organisms as well.\n\n2. The constraining/selective environmental factors and the organism’s acts that elicit novel constraints play the crucial roles that usually produce evolutionary outcomes.\n\n3. Selective pressure is the agent through which these two parties form an evolving equilibrium.\n\nExamples of niche construction in the organismal world are numerous. For current purpose, I will leave some of those much-discussed cases—e.g., of yeast, earth worms, hermit crabs, and beavers (Laland et al., 2016; Bateson, 2017)—aside and focus on the human niche, especially, the cultural niche construction8 in human populations.\n\nAs a matter of fact, primates do seem to have employed various culturally derived ways of niche construction. For instance, in wild chimpanzee groups, the behaviour of making a primitive form of ‘sponge’ from leaves is regularly found. This behaviour buffers the need of water consumption and is believed to be socially learned (Whiten et al., 1999; Hobaiter et al., 2014; Lamon et al., 2018). Meanwhile, the presence of Japanese macaques’ food-washing activity is suggested to be another example of a behavioural pattern which get transmitted cross-generationally through a cultural manner (Kawamura, 1959; Kawai, 1965; Scheurer and Thierry, 1985). Nevertheless, human populations, still, exhibit an unparalleled level of dependence and exploitation on the wealth of cultural resources in respect of their niche constructing activities.\n\nWhenever we think of ourselves (modern humans), it occurs intuitively and obviously that modern humans are probably the only species that not only modifies its environments in a notably comprehensive scale, but also relies so tightly and heavily on cultural practices in achieving those modifications. With the help of technology, modern humans have been carrying out planet-level cultural niche construction. The earth’s landscape has been profoundly transformed into cities, farmlands, or industrial lands. Natural resources are being transported and re-distributed worldwide through oil tankers, airliners, passenger trains etc. The development in telecommunication (e.g., internet, smartphones, personal computers, etc.) largely enables to a much more efficient exchange of human knowledge across continents, regions, and nations. Furthermore, by sending satellites and research stations, the human culture is accelerating its pace into the outer space.\n\nApart from the above overview of the magnitude of human cultural niche construction is and how it could have permeated nearly every aspect of human life, there is well-tested evidence showing human-led cultural influences on human genetics. Some famous examples include the spread of phenotypic traits of lactose tolerance, malaria resistance, and starch digestion in human populations (Perry et al., 2007; Rendell et al., 2011; Laland and O’Brien, 2011; Laland, Matthews, and Feldman, 2016). These cases have pointed to a possible fact: human culturally acquired behaviours can lead to non-random evolutionary consequences through a two-way mechanism that acts bilaterally and simultaneously—the buffering of specific selective pressures on one hand (e.g., the pressures for stable food sources that buffered by regular intake of dairy products or starchy crops), the emerging of novel pressures brought about on the other hand (e.g., selection for increased lactose tolerance or amylolytic capacity).\n\nWhat has been said above has focused on just one type of the outcomes of cultural niche construction: that is, for a cultural practice, the process of cultural niche construction can lead to its refinement and stabilization over time. But when a cultural practice is put into a network of cultural practices like in the real-world condition, another type of outcome that bears equal evolutionary potency is the impact this niche constructing practice may have upon other related practices.\n\nBehavioural patterns that are culturally acquired and transmitted usually cause changes in evolutionary fitness of other cultural traits (by indirectly mediating the course of transmission and the expression of relevant genes). On the analogy of the dam-building beavers9, human behavioural traits, when they are organised and expressed through habitual ‘practices’ or ‘traditions’, often exert selective effects on other phenotypic traits that are not so immediately related to the practice itself. Socially learned patterns like, for instance, local norms related to mate choice, religious or superstitious activities, specific technological practices, or even preferences in food choice, could produce far-reaching (but consequential) influence on many other behavioural patterns that coexisting in the same community. Particularly, when these culturally led traits are learned rapidly among industrialized human societies (with the support of technologies and education systems), and when paired with mechanisms such as sexual selection, or assortative mating, they produce strong culture-grounded selections in human population. From this aspect, we can see two notions are relevant: cultural selection and cultural evolution.\n\nCultural selection emerges when: 1. Some adaptive phenotypic trait is socially learned and spread among individuals of a population; 2. Major sources of selective pressures to which this population exposed stay stable; 3. This trait can be traditionalised, and the cumulative cultural inheritance could then lead to scaffolded learning; 4. Cultural selection for this trait appears.\n\nCultural evolution, accordingly, is the process in which diverse kinds of phenotypic traits, that are subject to persistent cultural selections, are manifested through behaviours, practices, and traditions of cultural niche construction of a population. What is so important here is that, considering evidence of niche construction we have so far (e.g., the spread of human alleles due to human farming, the dam-building regulated beaver physiology and morphology), it is reasonable to expect that, in the long term, cultural evolution could exert selective pressures on a wider spectrum of genes. If a cultural practice somehow is expressed (or inhibited) in a stable manner, certain genes (partially underlying that practice) can incur either an enhanced or a suppressed expression: think of the differential expressions of amylase genes that contribute to human starch digestion that depend on different levels of the cultural practice of starchy crops farming. At the same time, regardless of whether it is an over-expression or a suppression, the same practice can still affect the expression and the adaptive value of other genes. As a matter of fact, the current human life is inevitably filled with numerous products of the Digital Age. Through a systematic integration with individual’s life, this process of digitalisation and the novel selective pressures it induces might have left its mark in our species biologically. Indeed, there are recent studies which have suggested a correlation between increased risk for suffering an intervened early-stage brain development, ADHD, or depression and habits that contribute to prolonged screen time on digital devices (Madhav, et al., 2017; Hutton et al., 2020). However, to determine whether this correlation is a causal one or whether such culturally acquired traits have produced inheritable cognitive changes, further research is needed.\n\n\nHuman aesthetic niche and the deep history of cultural intervention in cognitive evolution\n\nIn line with what discussed previously, for species that depend heavily on their cultural niche, the cultural intervention is expected to play a leading role in driving the gene-culture coevolution process. Therefore, it is certainly not a bold guess to assume that modern human’s evolution has probably been (and will continue to be) mediated by its cultural activities. This, as I will suggest, had happened in the deep past of our hominin ancestors’ societies dating back to perhaps the Lower and the Middle Palaeolithic.\n\nBy relying on the technological practice of handaxe making starting from ~1.78 million years ago, this cultural behaviour gradually obtained a new role in social signalling through a joint force with existing aesthetic niche and a process of Baldwinisation, and eventually (throughout a over 1 million years’ time span of development), had led to presumably the earliest presence of a complex form of human cognition, i.e., a culturally grounded aesthetic social cognition. To have a better grasp of this picture, a clarification on several key notion is needed.\n\nThe notion ‘human aesthetic niche’ (used in a general sense, including that of our Homo ancestors) is not to be thought as a subset of the human cultural niche. In line with our discussion of the notion ‘niche’ and ‘niche construction’, the notions of ‘aesthetic niche’ and ‘aesthetic niche construction’ can be defined accordingly as: the total of the selective pressures to which a human population is exposed during the process of aesthetic niche construction, and the specific way of niche construction that relies on human aesthetic resources. Although it is usually the case for modern humans that an example of aesthetic niche construction constitutes an example of cultural niche construction10. However, we should only see the human aesthetic niche as in effect a sub set of the total ecological niche of human, which overlaps (though largely) with the human cultural niche.\n\nThis is supported by the studies of those who advocate an evolutionary psychological theory for human aesthetic mind. In other words, the fact that humans inherit a repertoire of aesthetic sensibilities and preferences for characteristics of the natural world and of the human body. Related topics that are well-tested include: e.g., preference for savanna-like landscapes, above-average body height, a ~0.8 waist to hip ratio, or the averageness of faces, etc. (there is a large body of research on such issues, see e.g., Miller, 2001; Fink and Penton-Voak, 2002; Rhodes, 2006; Buss, 2008, 2016; Dutton, 2009, 2010; Miller and Maner, 2010; Rusch and Voland, 2013). In this regard, we see that aesthetic niche construction could happen outside (i.e., with no reference to cultural resources) the overlapping area with the cultural niche. Besides, as implied by the general dynamics of niche construction, we should not exclude the possibility for fluidity/flexibility since there is no hard line between them.\n\nRoughly, the Baldwin effect is often used to denote the process during which behavioural traits that were initially derived gradually become internalised among individuals of a population (Sterelny, 2004; Papineau, 2005a; Crispo, 2007). An episode of Baldwinisation could happen, theoretically, on following conditions: 1. Some change in selective pressures is caused, making a specific behavioural pattern (let us call it X) adaptive. 2. Such change takes place through a non-radical way, and it persists long enough so that a small portion of the population could acquire X through individual trial and error. 3. Recognition of those ‘fitter’ individuals thus promote social learning of X, which stabilise X as a species-specific mode of behaviour. 4. A stabilised state of performing trait X induces positive selection for elements (at both the phenotypic level and the genotypic level) that facilitate the acquisition of X, thus, gradually makes X innate. As Sterelny describes, this effect is ‘a transition from development contingent on rich and specific environmental signals to development which is insensitive to environmental variation.’11 Indeed, there can be an evolutionary trade-off happening between the cost of learning and the benefits of making the object of learning encoded. When benefits outweigh the cost, this transition could ensue. In spite of this, the Baldwin effect is more like a possible outcome of niche construction. Niche construction provides the major source of adaptive traits, meanwhile the process of Baldwinisation relies on stable external inputs—i.e., persistent selective pressures—to allow the internalisation of the target traits. For sure, niche construction results in modified pressures which usually contribute to the retention of some derived behavioural pattern, but it does not necessarily make it encoded.\n\nThe Acheulean industry is a Palaeolithic technological tradition that can be characterised by its continued reliance on the production of a distinctive type of stone tool, the ‘handaxe’. With the development of this tradition, especially from the middle to the late Acheulean, sophisticated handaxes were produced in large quantities. Among them, characteristics of obvious aesthetic concern—e.g., high level of symmetry, well-arranged scars, finely processed edge line, even design features such as hollowing—are found.\n\nBased on the relation between the two classes of niche (aesthetic and cultural) we have just mentioned. The reason why this handaxe making tradition is so important is that: through what we have restored about the development of this material culture, the generations of making and refining (presumably from ~1.78 million years ago to ~500,000 to 300,000 years ago) of this practice had led to an emergence of a profound social learning system, which then gave birth to a culturally grounded aesthetic cognition—a mode of cognition, I suggest, that might be the ancestor of the kind we have when we appreciate arts.\n\nFor sake of a more focused and concise argument here, I will leave aside specifics such as technological issues in specimen evaluation and provide key milestones that would sustain this proposed scenario of cognitive evolution.\n\nAt its very beginning, there must be a transition from the previously dominant form of stone tool—the Oldowan choppers and flakes—to Acheulean handaxes. That is, there must be selective pressures that were buffered by the use of handaxes which enabled the handaxe to outperform Oldowan tools in the first place. According to studies on handaxe practicality (Galán and Domínguez-Rodrigo, 2014; Key and Lycett, 2017b), handaxes are considerably more efficient when dealing with heavy-duty tasks such as butchering (in which a handaxe could be used as a multi-purpose tool for de-fleshing, cutting, or smashing). Meanwhile, analyses based on animal fossils (Layton et al., 2012) and the identification of the earliest known case of porotic hyperostosis12, a likely marker of habitual meat intake (Domínguez-Rodrigo et al., 2012) both point to the possibility that systematic group hunting could have existed in early Acheulean, thus heavy-duty work might reasonably constitute a significant part of the daily routine of Acheulean individuals.\n\nSecondly, as the handaxe became prevalent, a result of this widely employed mode of niche constructing practice would be a set of strong selective pressures on factors which facilitate the practice of handaxe making. Considering those facts about the costly nature (at both individual and group levels) of maintaining a handaxe making tradition:\n\n1. Making a functioning handaxe is cognitive demanding, even for present learners, it takes years to become an expert (Pargeter, Khreisheh, and Stout, 2019), the maker would need a mental template during the processing, and a set of technical skills to organise, monitor and adjust a long and subtle sequence of actions (e.g., to make centripetal blows on the edge; to keep the striking angle less than 90 degrees to ensure a conchoidal fracture). All these would need to be carried out well to overcome copying errors (Lycett et al., 2016; de la Torre, 2016).\n\n2. On the hand, for handaxe making practice to become stabilised as a tradition, the makers and their societies would also need a substantial volume of other knowledge that is not directly related to the manufacturing process (Hiscock, 2014; Diez-Martín, et al., 2018). This would include: a. a basic understanding of practical physics which helps with determining suitable raw materials; b. the knowledge of geological mapping of various kinds of resources (e.g., locations of raw materials and source of water); c. the knowledge of dealing with challenging situations—e.g., the regular need of searching and transporting of raw materials would mean a higher frequency of predator encountering; d. the knowledge of avoiding or coping with physical risks during the knapping—e.g., a wound in hand might lead to severe infection or death, which could eventually affect the sustainability of the group.\n\n3. Apart from 1 and 2, the nature of lithic processing makes it, to some extent unforgiving. The physics of stone leaves little room for mis-conduction especially at the finishing stage where the material is thin. A mistake could destroy the whole time-consuming, strike-by-strike, and risky process of making.\n\nTherefore, factors that buffering these costs (i.e., selective pressures) would be under selection.\n\nIt is at this stage, I suggest, that the Acheulean handaxe tradition had promoted the social learning in Palaeolithic populations. For the societies that integrated so tightly with this stone technology of handaxe production, it would be crucial for individuals to become a good handaxe maker, and for the group to preserve and share the valuable knowledge required in handaxe making. Considering the costly nature and the complexity involved in this technology, during the middle to the late Acheulean, a developed social learning system might be in place. Within this system, a behavioural pattern that bonds experienced individuals with other less experienced ones could take place. That is, through a possible teaching-learning relationship, the cost of high-fidelity transmissions of valuable skills and knowledge between individuals and over generations would be decreased. In this manner, a concept/idea of someone being a good teacher/maker could emerge and be recognised in an Acheulean society. The preference for a good ‘teacher’ and the acknowledgement of expertise, would probably lead to a competitive social context: only those who have successfully shown their skills would be treated as the one others could learn from. Eventually, this seemed to have laid the ground for the aesthetic culture we see in those well-made handaxes. In this sense, the exaggerations (e.g., being large in size, having high degree of symmetry) in handaxe morphological traits are to be understood as honest signals.\n\nFurthermore, it is possibly due to the perceptual fluency13 and perceptual bias at the beginning (Hodgson, 2011, 2015, 2019; Flavell et al., 2020) and a level of Baldwinisation (as an outcome of Acheulean aesthetic niche construction) at later time that symmetry became a type-feature (in the vein, an effective honest signal too) of handaxe. According to the studies of this core feature of handaxe—e.g., in general, a chronological increase in handaxe symmetry can be seen based on specimens from sites around the world; handaxe symmetry plays little or no role in terms of handaxe practicality; high level of symmetry in handaxe is more like a deliberate design rather than a by-product of the making process (Saragusti et al., 1998; Machin, Hosfield and Mithen, 2007; Beyene et al., 2013; Hodgson and Verpooten, 2014; Iovita et al., 2017; Key and Lycett, 2017a; Shipton, Clarkson, and Cobden 2018; White and Foulds, 2018)—we can reasonably regard handaxe symmetry as an aesthetic feature.\n\nWith evidence at hand, I claim that it could be the case that because of the persistent practice of handaxe making, at least by the middle to late Acheulean, hominins’ societies had become in essence aesthetically and culturally intertwined. It is in those societies, an aesthetic culture and correspondingly, a culturally grounded aesthetic cognition emerged.\n\n\nSelf-domestication in Palaeolithic populations?\n\nIn the case of human niche construction, one important consequence of human cultural niche construction is the self-induced cultural selection on human population itself. Therefore, the human self-domestication (HSD) hypothesis is consistent with the general framework suggested above.\n\nThis hypothesis is about the idea that, like the process of animal domestication, modern humans (by way of conducting purposeful activities that select for prosociality) are domesticating themselves, leading to biological adaptations in the population. Studies have suggested that the presence of a set of morphological, physiological, and cognitive traits in human (e.g., reduction in brow ridge, decrease in facial length, prolonged development period, reduced reactive aggression and cooperative communication, etc.) is caused by this selection (Hare, 2017; Chirchir, 2020; Gleeson, 2020). Based on such evidence, many have suggested that HSD is important in terms of understanding human phenomena such as language (Benítez-Burraco and Progovac, 2020), others have suggested possible causes of HSD appealing to selection against aggression (Hare, 2012, 2017), proactive aggression (Wrangham, 2018, 2021), and neural crest cell development (Gleeson, 2020).\n\nAnalogous to what has been discussed in Section 3, by focusing on a specific cultural practice, the feedback loop between pressure-buffering activities and novel selective pressures could significantly modify the social dynamics of a hominin’s society (e.g., a late Acheulean society which exhibits a complex social learning system), favouring a high level of prosociality as well. However, if self-domestication did occur in this case, it would have a cultural root.\n\n\nConcluding remarks\n\nBy drawing on the notion of niche and theories of niche construction, and by emphasising the evolutionary significance of human cultural (and aesthetic) niche construction, I suggest that the bio-cultural nature of the evolution of human cognition can unfold in a systematic and unified manner. Cognitive evolution is subject to a reciprocal mechanism in which human phenotypic traits are mediating and at the same time mediated by the human niche. More specifically, it is through the concerted reciprocity between a cultural practice relying on stone processing and the mediated environments, a cultural cognition that is also aesthetic appeared for the first time in the human lineage.", "appendix": "Data availability statement\n\nNo data are associated with this article.\n\n\nAcknowledgement\n\nI would like to thank Penny Spikins for her helpful discussions on relevant topics.\n\n\nReferences\n\nAtkinson EG, et al.: No evidence for recent selection at FOXP2 among diverse human populations. Cell. 2018; 174(6): 1424–1435.e15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBateson P: Behaviour, Development and Evolution. Cambridge, UK: Open Book Publishers; 2017.\n\nBeckwith C: Niger's Wodaabe: People of the Taboo. National Geographic Washington, D. C. 1983; 164(4): 483–509.\n\nBenítez-Burraco A, Progovac L: A four-stage model for language evolution under the effects of human self-domestication. Lang. 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Hum. Sci. 2021; 3. Publisher Full Text\n\n\nFootnotes\n\n1 As the data shows: the proportion of prefrontal cortex gray matter for human can be ~1.6 to 1.9-fold greater than for macaques and ~1.2-fold fold greater compared with chimpanzees. However, regarding the fractions of prefrontal cortex white matter-to-total white matter volume, ratios of ~12%, ~7%, and ~5% are obtained respectively for human, chimpanzee, and macaque. Thus, the disparity becomes salient, a 2.4-fold difference between human and macaque, and a 1.7-fold between human and chimpanzee (Donahue, et al. “Quantitative assessment of prefrontal cortex in humans relative to nonhuman primates.” Proceedings of the National Academy of Sciences 115.22 (2018): E5183-E5192).\n\n2 Gazzaniga, Michael S.; Ivry, Richard B.; Mangun, George R. Cognitive Neuroscience: The Biology of the Mind, 4th Edition, 2014, p. 509.\n\n3 In a nutshell, the Baldwin effect refers to the phenomena in which some derived characteristic in some organismal population becomes more innate and less sensitive to environmental conditions (further discussion will be given in Section 3).\n\n4 By ‘culture’, I intend the whole chain of evolutionary dynamics that it entails—e.g., cultural innovation, social learning, cultural inheritance, and cultural evolution, etc.\n\n5 Early developmental stage alcohol use is believed to be another possible cause as well. Other than this, scholars have suggested that cultural should be hold responsible for many addictive disorders. Also, it is still an unsettled question that whether we should regard addictive disorders as diagnosable brain diseases or not. (For further discussions on this issue, see for example Leshner, 1997; Foddy and Savulescu, 2006; Levy, 2013; Pickard and Pearce, 2013; Sinnott-Armstrong and Pickard, 2013; Cuthbert, 2014).\n\n6 Odling-Smee, F. J., Laland, Kevin N., and Marcus W. Feldman. Niche construction: The neglected process in evolution. Princeton University Press, 2003, p. 40.\n\n7 Let us say, for some species ‘X’, the ‘X’ niche therefore incorporates the sum of interactive possibilities or potentials existing in the accessible ecosystem that could affect ‘X’ species’ fitness over time.\n\n8 For clarification, the term ‘cultural niche construction’ used here refers to the aspect of human niche construction that depends on human cultural resources.\n\n9 The behavioural mode of building dams has brought about drastic changes to beaver’s niche by placing a semi-aquatic environment, which is an outcome of an ecosystem full of dams, at the centre of the species. Not only have such surroundings (directly) modified selective pressures on dam-building relevant genes like those expressed by the evolved trait of beaver’s webbed feet, but also (indirectly) affected, as I quote Laland (2004, p. 317), ‘genes that are expressed in quite different traits, such as beaver teeth, tails, feeding behaviour, susceptibility to predation, diseases, and life history.’\n\n10 Modern humans inhabit a highly aestheticized world in which aesthetic niche construction is deeply intertwined with cultural niche construction (e.g., Currie, 2012). Think of artefacts such as body decorations (e.g, tattoos, clothes, hair dress), architecture (e.g., museums, galleries, stadiums), many other daily objects (e.g., tools, gifts, commercial ads, food packages) and the fine arts. This is also true in non-industrialised societies of present day: e.g., the ‘Potlatch’ tradition among indigenous societies of the North America (Boas and Hunt, 1897; Piddocke, 1965; Ringel, 1979; Harkin, 2001), the Gerewol festival celebrated by the Wodaabe people of Niger (Beckwith, 1983), or the ‘Sing-Sing’ gathering and the ‘Moka’ exchange of Papua New Guinea highlanders (Strathern and Strathern, 1971; Feil, 1987; Strathern, 2007).\n\n11 Sterelny, K. (2004), A review of Evolution and learning: the Baldwin effect reconsidered edited by Bruce Weber and David Depew. Evolution & Development, 6, p299.\n\n12 A pathological condition in which one’s bones and related tissues become spongy and porous. The presence of this condition is believed to be a result of anaemia.\n\n13 The phenomenon in which the processing of certain kinds of stimulus generates a subjective feeling of ease. A fluent processing could lead to positive assessment of the stimulus perceived." }	[ { "id": "191097", "date": "15 Aug 2023", "name": "Andreas De Block", "expertise": [ "Reviewer Expertise Philosophy of science", "cultural evolution" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper studies how niche construction theory can help to make evolutionary sense of human aesthetics. Its central conjecture is that human aesthetic skills, and maybe also aesthetic preferences have been shaped through a positive feedback mechanism that was generated through the persistent practice of hand axe making. I am largely sympathetic to the conclusion of the paper, and I like many of the ideas that the author touches upon. However, I cannot recommend indexing the paper it in its current form because it does not mention, let alone discuss much of the relevant literature. Furthermore, there are many elements that should be better explained and/or argued for. Also, some parts/sections do not really contribute to the central argument.\nI will now provide more detail on each of these critiques:\nIncomplete literature:\nthe paper applies niche construction theory to human aesthetics. Yet, niche construction theory is a general framework, and a framework that might not always have the added value that Laland and others think it has. If you are arguing that it has added value, you have to look into how other frameworks/theories deal with this issue. In that sense, I was surprised that the author ignores most of the gene-culture co-evolution theories and cultural evolutionary theories (Dual inheritance theory, Heyes’ cultural gadgets theory, Sperber’s epidemiology of representations), given that most of the empirical literature on the cultural evolution of human cognition starts from these frameworks (and not from NCT). In general, it remains unclear why NCT is needed to make sense of gene-culture co-evolution.\n\nThe paper mentions the evolution of ‘expertise’ and pedagogy but neglects the rather extensive literature on ‘natural pedagogy’ (see, e.g., Gergely, György, Katalin Egyed, and Ildikó Király, 2007, “On Pedagogy”, Developmental Science, 10(1): 139–146, but also the work of Henrich and Heyes on this issue).\n\nThere is quite some literature on signaling and art. The author suggests the relevance of signaling for understanding aesthetics on two occasions, but does not really explain this idea, nor refers to the relevant literature (for a recent overview/review of this literature, see Verpooten, J. (2023). Signaling and the cultural evolution of art. In: Tehrani, J., Kendal, J., Kendal, R. (eds) The Oxford Handbook of Cultural Evolution, Oxford University Press).\n\nIt is sometimes unclear what exactly the author is arguing for:\nDefinitions/explanations are sometimes circular. For instance, it is said that “[c]ultural selection emerges when […] cultural selection for this trait appears.” My impression is even the definitions of ‘aesthetic niche’ and ‘aesthetic niche construction’ are equally circular.\n\nMany of the terms are insufficiently explained or defined. I already mentioned how the author refers to (honest) signaling, without explaining it. Likewise, the author uses ‘scaffolded learning’ as a technical term, but it cannot be assumed that the readers of this article know what this term means. Also, what does it mean that a trait can be ‘traditionalized’?\n\nMost importantly, the author does not provide much evidence on how hand axe making has shaped our cognitive aesthetic psychology (TBC: I think it probably has). Also, it remains unclear what aspects of our psychology/behavior have been shaped by this practice, according to the author. They mention the transmission of skills, but are quite vague what these skills are, and whether they also think that it has shaped particular aesthetic preferences. One of the more interesting ideas of the paper is that “ By relying on the technological practice of handaxe making starting from ~1.78 million years ago, this cultural behaviour gradually obtained a new role in social signaling through a joint force with existing aesthetic niche and a process of Baldwinisation”. The author argues that buffering the costs of hand axe making can be assumed to be under selection. They might be correct in thinking that. However, they also write that “even for present-day learners, it takes years to become an expert”. This is strange, as the Baldwinisation of hand axe making seems to entail that we would all have a knack for hand axe making (which obviously isn’t the case).\n\nThis is only a minor issue, but the author says they rely on ‘theoretical deduction’, whereas the method actually seems closer to abduction.\n\nLargely Irrelevant information/claims:\nThe self-domestication section is only vaguely related to the rest of the paper. I guess many hypotheses are consistent with the general framework that this paper is suggesting. Is the author arguing that the framework of this paper strengthens the self-domestication hypothesis, or is it the other way round?\n\nThere have been many reports of the death of the modern synthesis, but – to paraphrase Mark Twain – we can assume that these reports are greatly exaggerated. I think that claims that niche construction theory (of cultural evolution, or ..) poses great problems for the Modern Synthesis are needlessly controversial. They distract from the central contributions of this paper.\n\nThere is a long footnote on alcohol dependence that is redundant.\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 source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "191090", "date": "01 Sep 2023", "name": "Larissa Mendoza Straffon", "expertise": [ "Reviewer Expertise Cognitive archaeology", "visual art", "human evolution", "cognitive evolution", "origins of art & aesthetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 text touches upon a crucial theme to understanding the origins of the modern human mind, which is the evolution of aesthetic cognition. In that sense, it is an interesting and topical paper. I do have several recommendations to improve the content and overall coherence of the manuscript.\n\n1. The main hypothesis should be laid out explicitly.\n\nIn my reading of the paper, the main hypothesis is that hominins’ cultural (socially learned) preferences shaped and were shaped by handaxe production and the visual features of these artefacts, particularly symmetry. These preferences eventually became innate through the Baldwin effect, bringing about aesthetic cognition by the Late Acheulean. This proposal (if it indeed is what the author intended) only becomes clear in the latter part of the text, due to which the first sections of the paper sometimes feel disconnected. It would help if the main hypothesis were laid out explicitly and earlier in the text.\n2. Some key concepts need to be either defined, specified, or elaborated.\n\nHandaxe. The paper in fact does not include any detailed definition of what an Acheulean handaxe is (a bifacial stone tool) or how its production differs from that of previous (Oldowan) or later (Levallois) stone tools. Readers with no background in archaeology will find the concept confusing and the argument difficult to follow without such a definition.\n\nCultural evolution. Section 2.3 is missing references to basic literature on cultural evolution (e.g., Heyes, 2019; Mesoudi et al., 2006; Micheletti et al., 2022) and instead offers idiosyncratic definitions of cultural selection and cultural evolution. I suggest adhering to the existing definitions so that the paper is compatible with current debates in the field of cultural evolution.\n\nAesthetic cognition. The paper lacks a proper definition of aesthetic cognition. Conventionally, this is understood as the ability to appreciate and appraise ‘beauty’, but whether the author adopts this definition is uncertain. Section 3.1 hints indeed at ‘preferences’, but given that the concepts of aesthetics, beauty, and preference are much debated, a minimal definition should be provided. Furthermore, it is not explained how or why ‘preference’ should be interpreted as either a cultural or cognitive process, particularly since non-human animals also exert preferences in several domains (Maia, 2016).\n\nAesthetic niche construction. The definition of aesthetic niche construction offered on page 7 is completely circular. It reads as ‘aesthetic niche construction is the construction of an aesthetic niche’.\n\nSymmetry. The discussion on symmetry in Section 3.2 is shallow and should be fleshed out. Symmetry has traditionally been the most important feature by which it is argued that handaxes are intentionally aesthetic objects (Wynn & Berlant, 2019), so it should be expanded.\n\nHominin populations. Given that handaxes were produced by several human species (H. erectus, H. heidelbergensis, Neanderthals, and early H. sapiens) over a long span of space and time, if the hypothesis is to carry any weight, it needs to be clarified to which populations the proposal presented by the paper applies. This does not mean that there should be an extensive description of lineages and fossils, but some species specificity is crucial to the argument since the circumstances of any cultural or cognitive change unlikely apply equally to all these different hominins.\n\n3. Some key arguments also need further elaboration, and the discussion should be expanded to include some essential literature on handaxe production, function, and distribution.\n\na) Handaxes were aesthetic objects. The suggestion that handaxes were the first aesthetic objects has been proposed in the past by various authors. Some of this essential literature is currently missing and should be at least mentioned, if not discussed (e.g., Kohn & Mithen, 1999; Mithen, 2003; Wynn & Berlant, 2019; Wynn & Gowlett, 2018). The originality of the present proposal is the introduction of niche construction as a theoretical framework. Unfortunately, the argument is underdeveloped and the causal links between the proposed ‘evolutionary stages’ are not clearly expressed.\n\nb) A transition from Oldowan choppers to Acheulean handaxes was driven by cultural selection. Handaxes first emerged in East Africa some 1.7 million years ago and eventually spread to most of the Old World, but this process was neither fast nor uniform. For example, handaxes have a higher recurrence in Africa and Europe than in Asia (Dennell, 2016), and in many regions handaxes coexist with previous Oldowan technologies (Dominguez-Solera et al., 2022). How such a gradual, mosaic-like development involving different hominin species should be interpreted as a true ‘transition’ from one cultural or cognitive ‘stage’ to another is not explained. On this point the paper would benefit from including some specific examples of archaeological sites rather than mere generalizations.\n\nc) Handaxe-making involved higher costs than Oldowan tools. This is overall accepted in archaeology, but there should be some detailed description of the key differences between the Oldowan and the Acheulean in terms of raw material procurement and stone knapping strategies. It might help, for example, explaining the manufacturing steps involved in making a chopper vs. a handaxe.\n\nd) Emergence of competitive social learning was triggered by the aesthetic judgment of handaxes. Whether any form of social learning can be inferred from the manufacture of Acheulean stone tools remains an open debate among cognitive archeologists. Some authors caution that there is little evidence to support social learning (e.g., Lycett, 2019), while others favour the emergence of proper teaching in the Acheulean (e.g., Shipton, 2019). The paper should acknowledge such discussions. In addition, the conclusion that social learning would become competitive by the introduction of aesthetic appraisal is not well supported. It is likely that social transmission of knowledge in the Lower Palaeolithic occurred mostly between kin, in which case social learning would be cooperative rather than competitive. Moreover, some archaeological assemblages include only a small percentage of highly symmetrical (‘beautiful’) handaxes, while in others (e.g., Boxgrove) all the tools are uniformly ‘attractive’ (Wynn & Berlant, 2019). This makes it difficult to conclude whether handaxe form was determined by cultural or functional factors or whether social competition played any role. Finally, this particular argument seems to move toward the notion of handaxes as sexual or social displays (Kohn & Mithen, 1999). If that is the case, the corresponding literature should be included and discussed.\n\nReferences: Dennell, R. (2016). Life without the Movius line: The structure of the east and Southeast Asian Early Palaeolithic. Quaternary International, 400, 14-22. Domínguez-Solera, S. D., Martín-Lerma, I., Moreno, D., & Pérez-Garrido, C. (2022). Lower paleolithic butchery knives and carpentry tools: MODE 1 industry of “El Pino”(Campos del Paraíso, Cuenca, Spain). Journal of Archaeological Science: Reports, 42, 103377. Heyes, C. (2019). Précis of cognitive gadgets: The cultural evolution of thinking. Behavioral and Brain Sciences, 42, e169. Kohn, M., & Mithen, S. (1999). Handaxes: products of sexual selection?. Antiquity, 73(281), 518-526. Lycett, S.J. (2019) Cultural Transmission from the Last Common Ancestor to the Levallois Reducers. What can we infer? In K. Overmann & F. Coolidge (eds.), Squeezing minds from stones: Cognitive archaeology and the evolution of the human mind, 251-277. Maia, C. M., & Volpato, G. L. (2016). A history-based method to estimate animal preference. Scientific Reports, 6(1), 28328. Mesoudi, A., Whiten, A., & Laland, K. N. (2006). Towards a unified science of cultural evolution. Behavioral and brain sciences, 29(4), 329-347. Micheletti, A. J., Brandl, E., & Mace, R. (2022). What is cultural evolution anyway?. Behavioral Ecology, 33(4), 667-669. Mithen, S. (2003). Handaxes: the first aesthetic artefacts. In Evolutionary aesthetics (pp. 261-275). Berlin, Heidelberg: Springer Berlin Heidelberg. Shipton, C. (2019). The evolution of social transmission in the Acheulean. In K. Overmann & F. Coolidge (eds.), Squeezing minds from stones: Cognitive archaeology and the evolution of the human mind, 332-354. Wynn, T., Berlant, T. (2019). The handaxe aesthetic. In K. Overmann & F. Coolidge (eds.), Squeezing minds from stones: Cognitive archaeology and the evolution of the human mind, 278-303. Wynn, T., & Gowlett, J. (2018). The handaxe reconsidered. Evolutionary Anthropology: Issues, News, and Reviews, 27(1), 21-29.\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 source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-392
https://f1000research.com/articles/12-390/v1	13 Apr 23	{ "type": "Systematic Review", "title": "Microplastic content of over-the-counter toothpastes - a systematic review", "authors": [ "Kavery Chengappa S", "Ashwini Rao", "Aparna K S", "Praveen S Jodalli", "Ramya Shenoy Kudpi", "Kavery Chengappa S", "Aparna K S", "Praveen S Jodalli", "Ramya Shenoy Kudpi" ], "abstract": "Background: Microplastic particles are used as ingredients in personal care products such as face washes, shower gels and toothpastes and form one of the main sources of microplastic pollution, especially in the marine environment. In addition to being a potential pollutant to the environment, the transfer of microplastics to humans can become a severe threat to public health. This systematic review was conceptualized to identify evidence for the presence of and characteristics of microplastics in toothpaste formulations. Methods: The PICOS Criteria was used for including studies for the review. Electronic databases of Scopus, Embase, Springer Link, PubMed, Web of Science and Google Scholar were searched, as well as hand and reference searching of the articles was carried out. The articles were screened using the software application, Covidence® and data was extracted. Results: This systematic review showed that toothpastes from China, Vietnam, Myanmar and the UAE, reported no evidence of microplastics and those from Malaysia, Turkey and India reported the presence of microplastics. The shape of the microplastics present in these toothpastes were found to be granular, irregular with opaque appearance and also in the form of fragments and fibers and the percentage weight in grams ranged from 0.2 to 7.24%. Malaysia releases 0.199 trillion microbeads annually from personal care products into the environment and toothpastes in Turkey release an average of 871 million grams of microplastics annually. Similarly, in India, it has been reported that 1.4 billion grams of microplastic particles are emitted annually from toothpaste. Conclusions: The findings of this systematic review provide evidence that toothpastes, at least in some parts of the world, do contain microplastics and that there is a great risk of increase in the addition of microplastics to the environment by the use of toothpaste.", "keywords": [ "in vitro study", "microbeads", "microplastics", "systematic review", "toothpaste" ], "content": "Introduction\n\nA future without plastics is becoming more and more difficult for the younger generation to imagine. Because of their durability and lack of biodegradability, plastic materials are both more appealing to humans and harmful to the environment. Environmental experts today are extremely concerned about microplastics. Microplastics are defined by the United Nations Environment Programme as, solid phase materials, less than 5mm in size, water insoluble, nondegradable and made of plastic.1 They are divided into primary and secondary microplastics based on the manner of development. While secondary microplastics are produced when large plastics are broken up into tiny detritus, primary microplastics are plastics designed to have a microscopic size.2 There are various types of primary microplastics such as polypropylene, polymethyl methacrylate, polyethylene terephthalate, polymethyl methacrylate, nylon, and polyethylene.3\n\nMicroplastic particles that are manufactured for their use as ingredients in personal care products are called microbeads or cosmetic microplastics and although they are characterized by a size less than 0.8 mm, most of the microbeads are less than 0.1 mm.4 As a result, since traditional wastewater treatment systems are not built to remove microplastics, they bypass these treatment facilities and are subsequently transferred into river and sea waters, where they persist forever because of their non-biodegradable nature.5 Presence of microplastics in sea water, freshwater, fruits and bottled water have been found across countries.6–9\n\nWhere do these microplastics in the environment come from? It was in the 1990’s that it was first recognized that personal care products such as face washes/scrubs, shower gels and toothpastes formed one of the main sources of microplastic pollution, especially in the marine environment.10 These microplastics when subjected to ultraviolet radiation, gets degraded and absorb persistent organic pollutants (POPs) like polychlorinated biphenyls (PCBs), becoming more toxic in the long-term.11 Once these microplastics make their way into the marine environment, they are ingested by marine organisms at the base of the food chain.2 Studies have reported toxic effects, such as structural changes to the gills, necrosis in other tissues as well as a heightened immune response in mussels that were exposed to microplastics in water bodies.12,13\n\nIn addition to being a potential pollutant to the environment,3,14 the transfer of microplastics to humans via their engulfment by aquatic animals can become a severe threat to public health.15 Studies have not only identified microplastics in human placenta in utero16 but have also shown evidence of interactions between microplastics and gut epithelium leading to oxidative impairments and disturbance in the inflammatory intestinal balance, impacting the epithelial permeability of the gut and toxicity of immune cells.17 It has been reported18 that prolonged use of toothpaste containing microbeads aggravates the inflammatory process of the gingiva and that the risk of these being unintentionally ingested on a day-to- day basis could also be potentially dangerous to health.3\n\nAlthough many studies have confirmed the presence of microbeads in face washes/scrubs in significant quantities,11,19–21 very few evidence exists22–24 with respect to the presence of microbeads and their characteristics in toothpaste.\n\nLebreton and Andrady25 have reported that 60–99 million metric tonnes of mismanaged plastic waste were produced worldwide in 2015 and have predicted that the world’s microplastic emission would triple by 2060 to reach up to 170–270 million metric tons/year, thus portraying a bleak picture. Considering these glaring evidence, the United Nations Environment Program has recommended an eventual phase-out and ban of microplastics in personal care products and cosmetics.1\n\nHowever, is it even achievable? Microplastics were originally used in personal care, cosmetics, and cleaning products (PCCPs) as abrasives for their scrubbing effects. But now they are believed to carry out several other crucial functions, including acting as binders, bulking agents, emulsifiers, exfoliants, film formers, viscosity regulators, opacifying agents, glitters, skin conditioners, tooth polishing agents, moisturisers, and stabilizers.26\n\nThis systematic review was conceptualized to identify evidence for the presence of microplastics in toothpaste formulations, with the hope that this evidence might help in understanding where we stand in our goal of phasing out and eliminating microplastics from toothpaste formulations all over the world. The findings would have implications for research as well as policy implications too.\n\nThere is evidence to show that microplastics are present in personal care products like face washes and toothpastes.11,19–24 The constituents in these products constitute micropollutants, due to their capacity to cause adverse effects on health and on the environment. These products, especially toothpastes, could be potentially dangerous3 since they could not only be unintentionally ingested on a day-to-day basis but are also a source of environmental pollution as they are carried in the water.\n\nTo identify relevant literature that assesses the presence and characteristics of microplastics in toothpastes and to analyse and integrate the evidence in a systematic manner.\n\nThis review tries to answer the following questions:\n\n• Do toothpastes contain microplastics?\n\n• If present, what are the characteristics of these microplastics?\n\n\nMethods\n\nInclusion criteria\n\nThe PICOS Criteria was used for including studies for the review:\n\ni. Population/participants and conditions of interest: Toothpaste samples\n\nii. Interventions/exposures: None\n\niii. Comparisons or control groups: Any or no comparison\n\niv. Outcomes of interest: Presence and characteristics of microplastics\n\nv. Study designs: In-vitro studies\n\nExclusion criteria\n\nThe review excluded studies that were published in languages other than English and those studies whose abstracts or full text were unavailable.\n\nThe electronic databases of Scopus, Embase, Springer Link, PubMed, Web of Science and Google Scholar were searched. Hand and reference searching of the articles was also carried out.\n\nThe search strategy involved use of the following key terms including the Boolean ‘OR’ and ‘AND’ operators: “microbeads” OR “microplastics” AND “toothpastes” sort by: relevance, Filters: English and the search was carried out between June and December 2022.\n\nThe articles were selected and compiled by two reviewers (KC and AR), from these databases and assessed based on the inclusion criteria. The software application, Covidence® (Covidence, RRID:SCR_016484) URL: https://www.covidence.org/home) was utilised for the selection process.\n\nTwo reviewers, reviewer number 1 (KC) and reviewer number 2(AR), independently screened the articles. Disagreements were resolved with the help of a third reviewer (AKS). The software application, Covidence® (Covidence, RRID: SCR_016484) URL: https://www.covidence.org/home) was utilised for the process of screening and extraction of data.\n\nData was sought for the outcomes namely, content and characteristics of microplastics in toothpastes. The data extraction template by Covidence was customized for this review and the review was reported according to the standards of Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020.27\n\nUsing the ‘Quality Assessment Tool for In vitro Studies’ (QUIN),28 the chosen papers’ quality was evaluated. This tool has 12 criteria that must be assessed and gives a point value: adequately specified = 2, inadequately specified = 1, and not specified = 0. The criteria that did not apply were excluded from the calculation of scores. The first reviewer (KC) and second reviewer (AR) each independently evaluated the papers’ quality. Discussions were held to reach an agreement if there were any disagreements.\n\nUsing the algorithm, “Final score = (Total score×100)/(2×number of criteria applicable)”, the scores were then combined to get a final score based on which the studies were graded. A score of >70% indicated a low risk of bias in the article, a score of 50% to 70% indicated a medium risk of bias, and a score of <50% indicated a high risk of bias.\n\n\nResults\n\nA total of 905 articles were obtained, of which, 695 were from Google Scholar, 128 from Springer Link, 23 from Embase, 22 from PubMed, 20 from Scopus and 17 from Web of Science.\n\nIdentification of duplicates was done, resulting in the removal of 46 articles and 859 articles were selected for level 1 title screening. After title screening, 808 articles were excluded since they did not confirm with our inclusion criteria and 16 articles were found to be eligible for the next process of full text review. When the full text review was carried out, ten articles were excluded and finally six studies were included for systematic review (Figure 1).\n\nThe studies included and their characteristics are summarized in Table 1. Although no filter of ‘Results by year’ was applied, keyword search showed that evidence was available only since 2017.\n\nThe number of toothpastes samples included, varied from a minimum of three in the study by Mon and Nakata29 to a maximum of 135 samples of toothpastes belonging to 23 brands, in the study by Lei et al.3 These studies reported the type of microplastic present along with the particle size, shape, color and particle weight. While most studies used Fourier Transform Infrared Spectrometer (FTIR), some studies further used ATR (Attenuated total reflection) and microscopic analysis11 to identify the type and characteristics of the microplastic particles in toothpastes.\n\nOut of the 6 studies selected for the final review, three studies, with toothpastes from China,3,29 Vietnam,29 Myanmar29 and the UAE,30 reported no evidence of microplastics in the toothpaste samples that were analyzed. Among the remaining three studies, two studies, one from Malaysia22 and another from Turkey23 reported the presence of microplastics with composition of polyethylene whereas the study from India24 reported the presence of microplastics with composition of cellophane, polypropylene, polyvinyl chloride and polyamide.\n\nAlthough microplastics are defined as water insoluble, nondegradable plastic less than 5mm in size, the particle sizes of microplastics were found to range from 3.5 μm in the study by Madhumitha et al.24 from India to 4–20 μm in the study by Ustabasi and Baysal23 in Turkey and 3–145 μm in the study by Praveena et al.22 in Malaysia.\n\nRegarding the shape of the microplastics present in toothpastes, it was reported to be granular,22 irregular with opaque appearance23 and as colorless fragments and fibers.24\n\nThe weight of microplastics in the product was reported as 7.24 % by Praveena et al.,22 whereas Ustabasi and Baysal23 and Madhumitha et al.24 reported a range of 0.4–1% and 0.2–0.9% respectively.\n\nThree studies22–24 also did the environmental risk assessment for microplastics released through the toothpaste. Praveena et al.22 reported that five facial cleansers and five toothpastes released 0.199 trillion microbeads per year in Malaysia. Ustabasi and Baysal23 reported an estimated average of 871 million grams of microplastics released every year from toothpastes in Turkey. Madhumitha et al.24 have reported an average yearly emission rate of 1.4 billion grams of microplastic particles from toothpaste in India.\n\nUsing the ‘Quality Assessment Tool for In vitro Studies’ (QUIN), the selected articles were assessed and graded to obtain the risk of bias. Out of the 12 criteria given by QUIN, three were excluded since these criteria did not apply to this review and so the studies were graded based on 9 criteria. All the studies showed a medium risk of bias since the final scores ranged from 50–70%. Most of the articles had adequately specified the aims/objectives, given detailed explanation of methodology, method of measurement of outcome, statistical analysis and presented the results adequately. The most common criteria which were not specified included, detailed explanation of sampling size calculation and the sampling technique. The criteria of outcome assessor details and blinding was also not specified in all the six selected articles which led to all studies being categorized as showing medium risk of bias (Table 2).\n\n\nDiscussion\n\nMicroplastics in water are susceptible to physical forces, fluctuating temperatures, ultraviolet radiation, oxidation, and salinity. They also become coated with bacterial biofilm. Since they settle to the bottom and have altered surface morphologies and higher densities, these weathered or conditioned microplastics are more readily available to a wide range of marine organisms.31 However, polyethylene is a type of microplastic that floats on the water’s surface due to its specific density of <1,32 and therefore it is available to a range of planktonic species as well as fish and seabirds that eat at the water’s surface.\n\nAlthough evidence shows that microplastics are contaminating our environment in great proportion and constitute an environmental hazard leading to health impacts, they are still being incorporated in personal care products including toothpastes. Our focus was on microplastics in toothpastes, and we found a relative dearth of studies in this area. Only six studies fulfilled our inclusion criteria and were included in this systematic review, showing evidence only from a few countries like China,3,29 Malaysia,22 Turkey,23 Vietnam,29 Myanmar,29 UAE30 and India,24 out of which 3 studies22–24 showed evidence of their presence in toothpaste.\n\nThe existence of microplastics was reported in three investigations, two22,23 of which identified polyethylene as the microplastic while the third, an Indian study,24 identified cellophane, polypropylene, polyvinyl chloride, and polyamide. Interestingly, the samples from India did not include polyethylene, a common polymer found in toothpastes in other studies.\n\nThe size of the microplastic particles found in the selected studies ranged from 3.5 μm to 145 μm. Because of their small size, microplastics flow through wastewater systems and are probably not detected by wastewater treatment facilities. Microplastics are available to microscopic species at the base of the food chain because of their limited size range and resistance to degradation in the environment. As a result, they enter the marine environment where they are consumed by marine life and then they enter the human body.11\n\nRegarding the shape of the microplastics present in toothpastes, studies reported them to be granular,22 irregular in appearance23 and as fragments and fibers.24 Evidence33 shows that these microplastics are not always spherical but have a variety of irregular shapes. Despite the fact that the word “bead” refers to spherical particles and despite the widespread misconception that microbeads are the coloured spherical microparticles found in toothpastes,11 microbeads are typically of irregular form to improve abrasion.4,34 In reality, PCCPs may contain multiple types of bead shapes, and irregular shapes contribute to larger specific surface areas and therefore more friction.35\n\nRegarding the color of the microplastics present in toothpastes, one study23 reported opaque appearance and another24 as colorless. Microbeads have been shown to have different colors such as white, transparent, opaque, blue, red and orange.11\n\nThree studies22–24 also did the environmental risk assessment for microplastics released through the toothpaste. Praveena et al.22 reported that five facial cleansers and five toothpastes released 0.199 trillion microbeads per year in Malaysia. Ustabasi and Baysal23 reported that an estimated average of 871 million grams of microplastics are released every year from toothpastes in Turkey. According to Madhumitha et al.,24 in India, 1.4 billion grams of microplastic particles are emitted from toothpaste each year on an average. Using toothpaste might carry a significant danger of increasing the amount of microplastics that are added to the environment.36\n\nThe quality assessment of the selected articles showed that all the studies were classified under medium risk of bias. None of the articles gave any detailed explanation of the sample size calculation and the sampling technique. The criteria of outcome assessor details and blinding was also not specified in all the six selected articles. This highlights the need for maintaining scientific rigor in every research irrespective of where they stand in the hierarchy of evidence.\n\nThere is limited evidence available with respect to the presence of and characteristics of microplastics in toothpastes. Out of the 195 countries in the world, data is available only from a few countries like China, Vietnam, Myanmar, UAE, Malaysia, Turkey and India, and here too, data is not available for all the toothpastes available. More evidence of good quality is needed from all parts of the world to find out the role of toothpastes in adding to the microplastic burden of the environment.\n\n\nConclusions\n\nThe result of this systematic review shows that toothpastes, at least in some regions of the world, do contain microplastics and there is a significant danger that using these toothpastes will increase the amount of microplastics added to the environment. Even if it is begun to take steps now, to fully outlaw microbeads internationally, the environment would continue to contain microplastics for a very long time. It is therefore high time to initiate urgent action to curb the menace of microplastics in the environment by eliminating its presence in personal care, cosmetics and cleaning products globally.\n\nEvidence from this systematic review shows that very few studies have been done to identify the presence of microplastics in toothpastes. Further research with lower risk of bias needs to be carried out in all parts of the world for conclusively determining the role of microplastics present in toothpaste in contributing to the microplastic burden of the environment, and to determine the possible long-term toxicity of these compounds on the human body.\n\n\n\n• Source management is a solid strategy to reduce the growing ecological risk posed by microplastics, and since PCCPs are the principal source of primary microplastics, their inclusion should be phased out in favour of more ecologically friendly additives.\n\n• Even though a few nations have passed laws restricting the use of microplastics in healthcare items, many do not enforce these laws strictly. To combat the growing environmental hazard posed by microplastics, a regulatory approach, stricter testing of toothpaste samples before they are let into the market, and stricter enforcement of relevant regulations are urgently required.\n\nSince this was a systematic review of in vitro studies, it could not be registered in PROSPERO. However, the review protocol can be found in the Extended data.37", "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: Microplastic content of over-the-counter toothpastes – A systematic review, DOI: https://doi.org/10.6084/m9.figshare.22179772.v1. 38\n\nThe project contains the following extended data:\n\n‐ Protocol.docx\n\nFigshare. PRISMA checklist. Microplastic content of over-the-counter toothpastes – A systematic review, DOI: https://doi.org/10.6084/m9.figshare.22179772.v1. 38\n\nThe project contains the following reporting guidelines:\n\n‐ PRISMA checklist.docx\n\n‐ PRISMA flow chart.docx\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\nUnited Nations Environment Programme: Plastic in cosmetics: are we polluting the environment through our personal care?2015. [accessed on: 20 January 2023]. Reference Source\n\nLi WC, Tse HF, Fok L: Plastic waste in the marine environment: A review of sources, occurrence and effects. Sci. Total Environ. 2016 Oct 1; 566-567: 333–349. PubMed Abstract \| Publisher Full Text\n\nLei K, Qiao F, Liu Q, et al.: Microplastics releasing from personal care and cosmetic products in China. Mar. Pollut. Bull. 2017 Oct 15; 123(1-2): 122–126. 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PubMed Abstract \| Publisher Full Text\n\nMadhumitha CT, Karmegam N, Biruntha M, et al.: Extraction, identification, and environmental risk assessment of microplastics in commercial toothpaste. Chemosphere. 2022 Jun; 296: 133976. Epub 2022 Feb 14. PubMed Abstract \| Publisher Full Text\n\nLebreton L, Andrady A: Future scenarios of global plastic waste generation and disposal. Palgrave Commun. 2019 Jan 29; 5: 6. Publisher Full Text\n\nZhou Y, Veeramuthu A, Amobonye A, et al.: Current research trends on cosmetic microplastic pollution and its impacts on the ecosystem: A review. Environ. Pollut. 2023 Mar 1; 320: 121106. Epub 2023 Jan 18. PubMed Abstract \| Publisher Full Text\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021 Mar 29; 372: n71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSheth VH, Shah NP, Jain R, et al.: Development and validation of a risk-of-bias tool for assessing in vitro studies conducted in dentistry: The QUIN. J. Prosthet. Dent. 2022 Jun 22; S0022-3913(22): 00345–00346. PubMed Abstract \| Publisher Full Text\n\nMon EE, Nakata H: Occurrence of microplastics in cosmetic products collected from Myanmar. IOP Conf. Ser.: Earth Environ. Sci. 2020; 496: 012011. Publisher Full Text\n\nElkashlan M, Poulose V, Habib R, et al.: Analysis of the Solid Contents of Toothpastes Available in UAE (United Arab Emirates) Markets. J. Environ. Prot. 2022 Jul 13; 13(7): 539–556. Publisher Full Text\n\nVan Landuyt KL, Nawrot T, Geebelen B, et al.: How much do resin-based dental materials release? A meta-analytical approach. Dent. Mater. 2011 Aug; 27(8): 723–747. Erratum in: Dent Mater. 2013 Aug; 29(8): 919. PubMed Abstract \| Publisher Full Text\n\nEriksson C, Burton H: Origins and biological accumulation of small plastic particles in fur seals from Macquarie Island. Ambio. 2003 Sep; 32(6): 380–384. PubMed Abstract \| Publisher Full Text\n\nIsobe A: Percentage of microbeads in pelagic microplastics within Japanese coastal waters. Mar. Pollut. Bull. 2016 Sep 15; 110(1): 432–437. PubMed Abstract \| Publisher Full Text\n\nHabib RZ, Salim Abdoon MM, Al Meqbaali RM, et al.: Analysis of microbeads in cosmetic products in the United Arab Emirates. Environ. Pollut. 2020 Mar; 258: 113831. Epub 2019 Dec 17. PubMed Abstract \| Publisher Full Text\n\nSun Q, Ren SY, Ni HG: Incidence of microplastics in personal care products: An appreciable part of plastic pollution. Sci. Total Environ. 2020 Nov 10; 742: 140218. Epub 2020 Jun 22. PubMed Abstract \| Publisher Full Text\n\nChandran T, Vishnu U, Warrier AK: Microplastics in Dentistry—A Review.Muthu SS, editor. Microplastic Pollution. Singapore: Springer Nature; 2021; pp. 157–174. e-ISBN 978-981-16-0297-9. Publisher Full Text\n\nChengappa KS, Rao A, Aparna KS, et al.: Microplastic content of over-the-counter toothpastes – A systematic review. figshare. Online Resource.2023.\n\nChengappa KS, Rao A, Aparna KS, et al.: Microplastic content of over-the-counter toothpastes – A systematic review. figshare. Online resource.2023. Publisher Full Text" }	[ { "id": "169445", "date": "26 May 2023", "name": "Arun Simon", "expertise": [ "Reviewer Expertise Dental Public Health and Sociology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 looks interesting and pertinent in the current times of heightened environmental pollution concerns. The authors have correctly established the need for this study highlighting the importance of this topic in the current scenario. This review article is a step in the right direction to help build evidence in this topic. The authors have also touched upon on the major databases to select the articles and use of Covidence software gives confidence in their selection process. Overall the conclusions can be believed and looks appropriate for the sated objectives of the study. However the authors can make clear some of the following points\nDuring literature search, did they find any article in language other than English?\n\nWhat were the efforts taken to minimize errors in data extraction?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "9713", "date": "29 Nov 2023", "name": "Ashwini Rao", "role": "Author Response", "response": "Dear sir, Thank you for the review. We would like to clarify on the two points mentioned, We have mentioned that articles in languages other than English were excluded from this systematic review. To minimize errors in data extraction, two reviewers, reviewer number 1 (KC) and reviewer number 2(AR), independently extracted the data and any disagreements were resolved with the help of a third reviewer (AKS)." } ] }, { "id": "189319", "date": "26 Jul 2023", "name": "Harsh Priya", "expertise": [ "Reviewer Expertise Dental Public Health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Authors\nThank you for this manuscript. It is very pertinent for the current times. It is a futuristic clarion call for the much talked about CLIMATE CHANGE with dental fraternity as one of the stakeholders.\nI would suggest following points.\nAbstract:\nMethods section of Abstract - Line \"The articles were screened using the software application, Covidence® and data was extracted.\" can be better framed.\n\nResults section of Abstract - \"0.199 trillion microbeads\" - Is the grams / unit missing? Kindly recheck! Else can write (\"Microbeads emission in number\"/ \"Total microbeads emission in number\")\n\nIntroduction:\nLine: \"The findings would have implications for research as well as policy implications too.\" this sentence can be improved.\n\nSearch Strategy:\nAny of the terms used were MeSH terms? Can include MeSH terms!\n\nRegarding the Identification and Visualisation of particles in personal care products the methods have been listed at FTIR and ATR. Further elaboration can be done on this point. For example size, shape and color of these polymers were also analyzed using an open-source particle analysis software named ImageJ 1.51 and composition of polymers was identified using Thermo Scientific Nicolet 6700 FTIR Spectrometer.\nThank you Best wishes Harsh Priya\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-390
https://f1000research.com/articles/12-388/v1	12 Apr 23	{ "type": "Research Article", "title": "Experiences of domestic violence prevention interventions and gender equality promotion work: a qualitative study of Nirdhar Groups in rural India", "authors": [ "Mark Hayter", "Amanda Lee", "Anuprita Dixit", "Sandeep Rasalpurkar", "Heidi Fewings", "Parveen Ali", "Clare Whitfield", "Sneha Giridhari", "Pratyush Kabra", "Kranti Rayamane", "Pavel Ovseiko", "Anand Ahankari", "Mark Hayter", "Amanda Lee", "Anuprita Dixit", "Sandeep Rasalpurkar", "Heidi Fewings", "Parveen Ali", "Clare Whitfield", "Sneha Giridhari", "Pratyush Kabra", "Kranti Rayamane", "Pavel Ovseiko" ], "abstract": "Domestic violence and abuse (DVA) against women in India is a serious concern. To address this, community-based interventions to facilitate sustained change at local levels have been implemented in 37 villages by the Halo Medical Foundation (an NGO working in the Maharashtra state of India). This qualitative study of community led alliances (known as Nirdhar Groups) evaluates the experiences of these ‘community citizen groups’. Participants from 12 villages from the project area participated in qualitative focus group discussions. Nirdhar Groups used local interventions to address priorities such as safe and equal access to education and healthcare. They offered education to address financial oppression and used community pressure and ‘Responsible Couples’ as mechanisms for change, to instil ‘fairness, gender equality, kind and compassionate community values’ and a variety of techniques to reduce DVA. The drivers for change included role modelling, empowerment of women in communities and a focus on changing cultural practices for present and future generations. This paper reveals how local interventions empower communities to address gender disparities and DVA.", "keywords": [ "Gender equality", "Domestic Violence and Abuse (DVA)", "Women", "India", "Community Intervention." ], "content": "Introduction\n\nMany Indian women face domestic violence and abuse (DVA) from their male partner (Jejeebhoy & Santhya, 2018; Borah et al., 2017). The causes of gender inequality and violence varies with local culture and norms, meaning local, community driven interventions play an important role (Desai et al., 2017) to reduce sufferings and promote change. In India, many rural communities face problems around women and girls’ safety (Ghosh & Choudhuri, 2011). Cultural pressures and norms often lead men into gender discriminatory practices (Ram et al., 2019). This may result in financial, emotional, or physical gender-based violence and discrimination (Anitha, 2019). As such, gender inequality severely hampers progress towards the Sustainable Development Goals (SDGs), particularly towards the fifth SDG, Gender Equality, and it is also important to achieve several others SDGs where girls/women involvement in health, economic and sustainable progress are imperative (United Nations Statistics Division, 2017). Rural and remote areas across India have inadequate resources such as limited judicial infrastructure, poor policing and environmental problems like frequent drought affecting agricultural yield which is the primary source of livelihood for many (Mahapatro et al., 2012; Showalter et al., 2020; Stephenson et al., 2013). Alcoholism and poverty are prevalent in these communities (Ram et al., 2019; Mshweshwe, 2020; Gunjal et al., 2020), fuelling men to dominate and engage in DVA (Daruwalla et al., 2019). Top down (governmental or socially led) strategies lack the capacity to address such issues faced in rural areas, which have their own social structures, community cultures, and related challenges.\n\nThere is evidence that the most effective gender equality work can be done within and with the cooperation of the community itself (Daruwalla et al., 2019). Policies and laws are only effective when enacted in the community setting (Freudberg et al., 2018). Furthermore, engaging influential community members and ensuring that both women and men are actively involved in community-based gender equality interventions are markers for impact (Mahapatro et al., 2012). Targeted and localised approaches are deemed effective means for change (Kalokhe et al., 2017). With engagement from local communities, NGOs and community, volunteers are more readily accepted and recognised by those who need it most (Nair et al., 2020).\n\nIn partnership with and support from the SWISSAID India, the Halo Medical Foundation (HMF) initiated the ‘Responsible Couples’ project. The project involved developing local community level groups called Nirdhar Groups to address and reduce gender inequality, DVA and provide village-based support structures for women affected by gender inequality. Nirdhar in a local language, Marathi, means determination. Nirdhar Groups consisted of male and female community health workers, village police representatives, teachers, and elders. The NGO (HMF) convened, trained, and empowered such Nirdhar Groups with information, support, and resources so that they could support women’s rights within their own communities. The ‘Responsible Couples’ project involved the following key activities: (a) establishing village level gender equality groups known as Nirdhar Groups; (b) conducting training, education and media campaigns to educate people across project areas on the importance of gender equality; (c) motivating men (men’s involvement & engagement) to participate in household chores which are often shouldered by women, helping them understand the importance of gender equality and improving relationships at family levels; (d) advocating and working towards equal rights for women, such as joint bank accounts, joint property registrations for house and agricultural land (including ownership rights & decision making); (e) providing access to gender equality and domestic violence counselling services through HMF’s registered counselling centre (known as SAVALI Kendra, registered with the government authorities to provide support services & justice to victims of violence). The SAVALI Kendra receives referrals from village level Nirdhar Groups where specific support and expert advice are required to address DVA cases. Nirdhar Groups have now been in existence in 37 villages located in the Osmanabad district of the Maharashtra State of India since 2017. The overall project scope and objectives were focused on reducing any violence against women/girls and improve gender equality in rural communities, which is informed by SWISSAID and HMF’s strategies and work priorities to improve wellbeing of marginalised groups, children, and women in local communities. HMF has been working in community health and development sectors since 1990’s in rural areas of Maharashtra, particularly in Osmanabad district, which is one of the marginalised regions in the country.\n\nAll outlined project activities were designed and delivered by a dedicated team at HMF, through financial support from the SWISSAID. Team members had a range of backgrounds, primarily in social work with experience and interest to work on community development initiatives. The project structure offered each staff member an opportunity to work with a specific set of villages over time to conduct field-based meetings, training, educational tours, and also help communities develop village level groups (Nirdhar Groups) to improve gender equality and reduce domestic violence in communities. SWISSAID provided technical support through consultants and subject experts to train staff and village groups along with an intensive monitoring approach to track project progress. The support was also extended to design project activities such as initiatives to involve men in household activities, develop training manuals, and village/community level activities. Village groups were involved on a voluntary basis, and all project staff were full-time (~10 members) or part-time (~37 members) employed by the HMF (funded by the SWISSAID).\n\nThe purpose of this research was to explore the experiences of village level ‘gender equality’ Nirdhar Groups and to: (a) reveal how they developed processes and structures which impacted gender discriminatory practices and domestic violence & abuse (DVA) prevention across their local communities; (b) identify the issues and challenges they faced; (c) identify interventions which they used to combat gender inequalities in rural communities. The paper is presented in line with the COREQ guidelines (Tong et al., 2007).\n\n\nMethods\n\nThe study has been approved by the Faculty of Health Sciences Ethics Committee, University of Hull, UK (approval reference number- FHS125), and also by the ethics committee of the Ashwini Rural Medical College, Hospital and Research Centre, Solapur, Maharashtra, India (approval reference number- ARMCH/IECHR/03/2019).\n\nThe study involved 12 focus group discussions (FGDs) in 12 villages, which were part of the ‘Responsible Couples’ project. The groups were selected from a pool of 37 villages, where village groups were actively working in the ‘Responsible Couples’ project at the time of data collection. Each village had one group and each group had 15 and 20 members. The outlined 12 groups/villages were randomly selected from project area by members of the UK research team, Professor Mark Hayter and Dr Anand Ahankari, who were not aware about individual group level performance, to avoid any selection biases. All 12 village groups were provided information about the study in advance and once they agreed to participate in the study, then FGD (data collection) date and timings were confirmed. Based on our series of consultations with partners, field visits, interactions with beneficiaries, and literature evidence, the proposed 12 FGDs were deemed sufficient to achieve data saturation.\n\nA semi-structured discussion guide comprised of open-ended questions was used to collect qualitative data (see Extended data (Ahankari et al., 2019). FGDs explored the following topics having several sub-questions; how the village level group was formed, what trainings the group attend, what they understand by gender equality, what work they undertake as a group in their communities, impact of their work and what should be done more to ensure replication and success of such work elsewhere in Maharashtra/India. FGDs were deemed suitable for this work considering village level issues related to gender inequalities and domestic violence were identified by one or more group members, who then discussed those within the group during monthly meetings, and then they decided on their strategy to address identified issues. As a result, identification, intervention, and delivery was agreed through group discussions involving several members along with necessary support and advice provided by project staff. Thus, the proposed FGD method was appropriate to capture relevant data to achieve study objectives. The interview guide (see Extended data (Ahankari et al., 2019)) was prepared and reviewed several times by research team, partner agencies, and relevant staff to ensure its relevance and application.\n\nAll 12 FGDs were conducted in community spaces such as a village/community hall, school, health centre, or other appropriate community location to ensure necessary meeting arrangements, where accessibility, confidentiality, and privacy were in place. Willing participants were requested to arrive at an agreed time to avoid disturbances or interruptions during the discussion. The village groups worked together to deliver ‘Responsible Couples’ project activities, thus members were aware of roles and work undertaken by others. While as a group participants knew each other, confidentiality was maintained about discussions undertaken during data collection session, where participants were requested not to discuss any sensitive topic(s) outside their Nirdhar Group. Participants were informed about research procedures to obtain a written group consent prior to the data collection session. The FGDs were conducted in local language (Marathi), audio recorded, transcribed, and translated into English for analysis. Time duration for FGDs varied between 90 and 120 minutes. Participants varied in each FGDs from eight to 12 and included both men and women members. The interviews were conducted by two qualitative researchers (Mrs Anuprita Dixit and Mr Sandeep Rasalpurkar), who were not involved in any capacity in the ‘Responsible Couples’ project in the past, and did not work previously in any capacity/role for organisations involved in delivering the ‘Responsible Couples’ project. Both qualitative researchers were bilingual (Marathi and English), and were native speakers of Marathi language with over 20 years of work experience in research and development sectors. They had backgrounds in health sciences (MSc), recruited as data collection consultants, and were given necessary resources to conduct in-depth FGDs. The combination of one male and one female data collector (gender balanced approach) was preferred considering the research topic, scope, and study participants background to create a suitable environment for all participants to engage in data collection sessions. Both data collection team and participants were aware about the study aims and objectives. Two key members of the UK research team, Professor Mark Hayter and Dr Anand Ahankari, provided in-person trainings to these qualitative researchers in India to inform about study procedures and ethics (Ahankari et al., 2019). Staff members of the ‘Responsible Couples’ project, funding agency (SWISSAID) did not attend FGDs to ensure that participants and research team members will have necessary autonomy and privacy to conduct in-depth data collection. FGDs data files (in Marathi/English) were accessible to research team members only, and were not shared with study participants. Each FGD was conducted only once, and study findings were communicated with village and project representatives through three dissemination events, where participants had opportunities to provide verbal feedback. All field based research work was completed between January 2019 and July 2019. Further details on the study procedures are available online (Ahankari et al., 2019).\n\nAnalysis was informed by Braun and Clarke’s (2006) six phase deductive reasoning methodology, which was felt to reflect the linearity of this project. All FGDs data files were stored using Microsoft Office Word Software for analysis purposes. No other software tools were used. The thematic approach started with researchers identifying individual coded elements within the data and then iteratively developing explanatory themes to generate a final thematic overview of the phenomenon under investigation. Themes, therefore, were separated into: (1) how groups identified problems and worked together to prioritise interventions; (2) short-term interventions to inform and engage communities; and (3) long-term interventions which reflected an embedded transformation across communities. Table 1 lists major themes and sub-themes. No field notes were taken/used in the analysis. Data analysis was conducted by two researchers (Ms Heidi Fewings and Dr Amanda Lee), and was supervised by senior qualitative researcher (Prof Mark Hayter).\n\n\n\n• Discourse of gender equality\n\n• Safe access to education and healthcare\n\n• Legislation and home ownership\n\n• Misogynistic rituals- power, community, and family dynamics\n\n• Domestic Violence and Abuse (DVA)\n\n• Substance misuse\n\n\n\n• Ensuring safe and equitable access to education and healthcare\n\n• Creation and establishment of ‘safe spaces’\n\n• Facilitating equal access to finance, grants, and home ownership\n\n• Addressing and challenging misogynistic rituals through family communication\n\n• Acts to reduce DVA\n\n\n\n• Role modelling\n\n• Empowerment (through access to information)\n\n• Empowering communities (by instilling kindness, care, and compassion)\n\n• A sustained change for future generations\n\n\nResults\n\nThis theme captures how Nirdhar Groups identified and defined their boundaries – using the norms and customs within their communities to assert the discourse of gender equality, and set the social transformation priorities within their communities. Group members highlighted several priorities, including addressing unequal access to education and healthcare, male dominated legislation (used as means to financial oppression and control), misogynistic rituals (dowry, child marriage), and family power dynamics with potential to increase ‘burden’ and disempower women. Nirdhar Groups highlighted confounding factors towards DVA and gender inequalities, such as substance misuse and the practice of hysterectomy to improve work productivity.\n\nDiscourse of gender equality\n\nAll groups began by discussing what they meant by ‘equality’ and then identified common practices, which impacted women’s rights and social status within communities. On defining gender equality, many of the groups acknowledged that men and women should be treated equally.\n\n“Gender equality means how a husband should behave with his wife” (FGD10).\n\n“Consider both of them similar” (FGD6).\n\n“Boys and girls are equal. Both have similar rights” (FGD7).\n\nSome groups identified physical burden on women running households, and culture-based lack of knowledge on their rights as women.\n\n“Household work should be shared … Men and women should help each other in their work” (FGD9).\n\n“[Men] should not harass … women … not treat them as slaves… [or] think that they are lesser than us” (FGD6).\n\n“There is need to increase female participation. A female must be aware of her rights, her likings, or she should think how to increase her say in the family, what changes she should adopt. When she is aware of her rights, she will demand for it [rights]. We must increase this awareness through self-help group meetings” (FGD7).\n\nSafe access to education and healthcare\n\nAccess to basic healthcare was identified as ‘unequal’ between men and women, where decision making power to access services remains with men. In our study area, women are known to neglect their personal healthcare issues and prioritise their children, husband, and family, especially in-laws.\n\n“A major issue is that the women don’t go to the doctor because they are thinking of money. If the problem is simple, they take some tablet [medicines] and wait till the problem gets worse, besides no one from family tells them to go to the doctor. They themselves never talk about their health problems” (FGD6).\n\n“When we tried to take pregnant woman [to] hospital [for antenatal care] her mother in-laws used to say, ‘I gave birth to 10 children but never saw [been to a] hospital’. Then we have to explain in-laws also about past and current situations” (FGD7).\n\nLocal cultures appeared to prevent women from accessing education. In particular, FGDs uncovered some practices that made women feel ‘unsafe’ when travelling to access education. These were mainly due to verbal harassments by young boys/men.\n\n“We used to blow whistle, to see the girls travelling on the bus or tease them in groups. Now we have stopped such things. Atmosphere is also changed” (FGD9).\n\n“Now since I joined this group, I am completely a different person. I can tell other boys not to do such things. I can tell them definitely that I used to do all these bad things and now stopped doing them. And of course, they listen to me” (FGD9).\n\nLegislation and home ownership\n\nGender equality in legislation was raised, with a focus on home, or land ownership and after being widowed or divorced. In the past, home ownership and finances were registered in the husbands’ name. New legislation has meant that women can now be placed on deeds for home ownership, financial institutes such as bank accounts, and such registration is also required to access government schemes and funding.\n\n“We never thought about our bank accounts. It was only in names of our in-laws and husband” (FGD9).\n\n“Women … had to sit behind and with dress code of saree … [they] never visited the bank” (FGD11).\n\n“In past, property belonged to men only” (FGD12).\n\nMisogynistic rituals – power, community, and family dynamics\n\nMisogynistic rituals and cultures were highlighted as critical reminders of the oppression of women in local communities. Thus, certain behaviours and practices were identified as priorities for intervention. These included addressing child marriage, gynaecological health, and female foeticide.\n\n“Child marriage should be banned. We should marry the girls after 18 years of the age” (FGD6).\n\nThe tradition of dowry was identified as problematic but noted to be reducing across societies.\n\n“[Dowry] is going on – but secretly” (FGD5).\n\n“A similar case that happened in [nearby village]. A girl was killed for dowry. I think this may be effect of this … this tradition of dowry… should be stopped. And most of the victims of these traditions are women” (FGD2).\n\nOne priority for Nirdhar Groups was identified as family planning. Smaller families were seen to decrease the burdens on women.\n\n“Now, they have started doing family planning following two children, even if they have two daughters” (FGD8).\n\n“I used to go to people those who have two children and tell them about the operation [family planning], but people used to say that you are not married and why you are telling this to us? Since Nirdhar Group is formed, these things are working out. Committee is influencing the villagers slowly. So we will do a good work. Even people from other villages … we inform them about our work” (FGD8).\n\nDomestic Violence and Abuse (DVA – formerly known as ‘intimate partner violence’)\n\nReducing DVA and aggression was a priority across most Nirdhar Groups. Many groups identified a need to address DVA and offer interventions to reduce addiction amongst ‘violent men’, educate men about these issues and impacts on women, which may help improve equality and wellbeing of women. These were primary project objectives set by SWISSAID and HMF.\n\n“We have a law against domestic violence … [but] women are unaware about it” (FGD11).\n\nThere were many examples of incidences of DVA.\n\n“he [used to] beat his wife” (FGD7).\n\n“a boy [man] used to fight with his wife … then send her back to her maternal home” (FGD4).\n\n“One drunken husband was beating his wife with a stone” (FGD1).\n\nSubstance misuse\n\nIn some communities, substance misuse with alcohol and betel nut were often identified as a causative factor. Betel nut, also known as areca nut, is the seed of areca palm tree. It is widely cultivated in South Asian countries. It contains psychoactive ingredients, and has many harmful effects on humans due to its carcinogenic nature (Arora & Squier, 2019). In our study area, it is known as ‘supari’ in the local language.\n\n“I was vicious. I ate tobacco and used to harass girls” (FGD9).\n\n“The movement of prohibition of alcohol is the most important issue, while it [alcohol] rises, women don’t get together because if they do, their drunkard male counterparts fight with them at home. The next day women came together and discussed about it. This is the biggest problem in our village” (FGD6).\n\n“If we say the violence is due to addictions, then we observed that addict beats only spouse [wife], doesn’t beat sarpanch [village chief], if he sees sarpanch, then he salutes [shows respect to] him. He says, save me, I from your group only and the same person beats the spouse on reaching home or can do anything as he wishes, like abusing, so we understood that violence is not only due to addiction” (FGD5).\n\nThis theme contains examples of how Nirdhar Groups interacted with specific families and groups, to instil personal-level change in culture and behaviours. Through regular communication and involvement with their community citizens, the Nirdhar Groups were able to anchor themselves as agents of change. Their work involved a range of initiatives in line with priorities identified in their communities as explained in the theme 1.\n\nEnsuring safe and equitable access to education and healthcare\n\nMany Nirdhar Groups implemented strategies for equal access to education and healthcare for both daughters and daughter in-laws/wives. Education opportunities are limited in our study region due to school structure, where most of the villages have schools until 10 or 13 years of age, and for middle school many need to travel to nearby towns. Due to preference given to male children, females often drop out from the education system (Psaki et al., 2022), therefore, access to education is important for young girls in our study area. Furthermore, such inequalities prevent women/girls from accessing healthcare where females/women do not prioritise their health and are often neglected by their families due to gender based discrimination.\n\n“What is the use of girl’s education; ultimately, she has to get married and work for family only. She is not going to be a collector [high level civil service post in India] or do any grand work. She won’t live at her mother’s place, so what’s the use of her success to us. This was our view. But after joining the Nirdhar Group we realised our mistake. We thought, what we were doing was correct. This is our culture, and it is right, nice. But now we came to know that we were misusing our good culture” (FGD9).\n\n“Some time ago we were also used to take permission [referring to permission from husband/in-laws]. Now as we are members of Nirdhar Group, we have all information. We come across all the points, so we don’t take any permission and directly go to hospital and take treatment. This change is happened because we have much information now” (FGD6).\n\nThis suggests that women are looking after their health after being empowered through education and project activities delivered through ‘Responsible Couples’ initiative.\n\nCreation and establishment of ‘safe spaces’\n\nGirls and young women often discussed concerns about access to public transport facilities, so that they felt safe to attend education. The number of college-going girls increased over time in these communities. Women and girls received a platform and also a support system through Nirdhar Groups initiative to share their concerns with policy makers and influential people in their communities. This provided opportunities to voice their concerns/problems during village meetings and demand for necessary actions to improve safety and transport facilities. Females were present at village meetings, which used to be traditionally attended by men in the past. Village government office then formally requested district authorities to re-start public transport services, which will benefit young girls and women to access education institutes. This also improves their safety during travel.\n\n“Girls used to stay home and prepared for exams, the number [of college going girls] has now increased to 25-30. Girls are not only going to colleges where college bus is available, but [also] to colleges where bus facility is not available. Number is increased to such colleges also” (FGD1).\n\n“This year iron tablets were given to women for improving health. Almost 200 women received medicines. Those who have low Hb level [Haemoglobin], they were taken to the HMF at Andur for further check-up. Madam [doctor and social workers at the hospital] had called, we had sent many girls there [for medical consultation]”(FGD8).\n\n“Not even ladies’ members were there before [on village government committees]. Now their seats are reserved, due to law. They get elected. Being members, they go with them [men]. They take their female friends with them. They understand work procedure, nowadays water supply is under their control …. women in bachat gat [self-help/microfinance group] will fight for it” (FGD5).\n\nFacilitating equal access to finance, grants, and home ownership\n\nNirdhar Groups used a variety of means to inform women of their rights and responsibilities and offered education sessions on legislation, so women could work towards a financial freedom. Groups were trained and equipped with resource by project staff, who also provide support during such activities. Previously, finances were in the male domain, with accounts held in the husband’s name, however women are now participating in financial activities.\n\n“Every woman can spend money from her own account … .no more dependent on men for expenditure … [men] are not reliable, so [some] government grants or subsidy goes directly in [bank] accounts of women” (FGD9).\n\n“They [women] have names on lands, on houses, on ration cards [government document to access food and essential supplies], they have bank accounts, means we have upgraded women” (FGD10).\n\nAddressing and challenging misogynistic rituals through family communication\n\nNirdhar Groups used a ‘community presence’ to access homes and communicate across family groups to instil change.\n\n“If a child marriage is taking place, we go to that home for convincing them to stop the marriage” (FGD8).\n\n“If we change ourselves, whole environment in family changes, once family environment changes, changes occur on our street [in our area], finally changes occur in our village” (FGD7).\n\n“It happened in a house. It was a matter between parents and son. We have Nirdhar Group in our village, so the mother came to us and told their problems. It is our system that if there is a complaint, we all, along with the president, secretary, and members [of Nirdhar Group] go and try to solve quarrels. If we go without complaint, there is always a risk of a question ‘who told you’. We wait for the family members to come to us. In this case as per our procedure we brought about, face to face meeting. We listen to both sides, we asked for their opinions, and then tried to convince them [to resolve issues]. Now everything is solved, and they are living peacefully” (FGD6).\n\nActs to reduce DVA\n\nCommunity pressure and communication also facilitated issues with gender-based violence and oppression. Nirdhar Groups played crucial role while working as a team to intervene and provide support to victims. Capacity building trainings and continuous support by project staff (HMF) was offered considering a range of project activities and varying requirements and complexity of issues across project areas.\n\n“Suppose there is violence at one place, we go there with some other reason. Have a talk with mother-in-law when daughter-in-law is not there. Similarly, talk with daughter-in-law when mother-in-law is not around” (FGD8).\n\nGroup members spoke of “visiting again and again” (FGD7) and putting pressure on issues to enable change: “People are afraid of doing wrong things because Nirdhar Group is active in our village” (FGD6).\n\nThis theme identifies how Nirdhar Groups worked towards a sustained, cultural agenda for change. It presents how people worked together, celebrating their successful outcomes, and ingraining the culture of gender equality across their communities. Thus, this theme presents higher level examples of cultural transformation, such as role modelling, empowerment, instilling kindness, care, and compassion, with a sustained power to change the next generation.\n\nRole modelling\n\nGroups modelled their work through a “willingness” and “responsibility” to have to work for the village (FGD7). Men took more responsibility for preparing meals and hosting visitors. Nirdhar members acknowledged that to present change, they had to act as role models. They acknowledged that, to instil change, they had to be a part of that change. Some Nirdhar members identified significant changes they had experienced, driven by a desire to be a social transformer, an activist in the ‘Prabhodhan’ ethos (Prabhodhankar was an ‘enlightened good citizen, working for the good of communities).\n\n“The villages worked together, role modelling gender equality.” “Household work should be shared between both male and female” “Both male and female can go out of the house for work. Female should have equal rights” “Both can go to market for shopping”, “Men and women should help each other in their work” (FGD9).\n\n“Before brining any change in society, we must change ourselves. In our village among the youngsters, I was the top drunkard, vicious since last year or two I have given up all vices. I used to eat supari [beetle-nut and tobacco preparation]. roam in the village; … I stopped riding bike and passing time uselessly. I stopped chewing supari, completed studies and joined a job. So others follow my footsteps. Now I am respected in the society. The people call me a good person now. You should change yourselves before telling people” (FGD11).\n\nRole modelling was achieved through dissemination of knowledge received from education and training provided by the HMF. A Nirdhar member spoke of the impact of training on their motivation to disseminate the new knowledge.\n\n“If the Samajdar Jodidar [‘Responsible Couples’] behaves like ideal couples then only other people will follow them. Sometimes when women come to my place and sit in our hall [living room]. He [her husband] prepares tea for them without saying anything. Then women start thinking her husband is doing such things then why not mine” (FGD9).\n\n“We are role model for them they observe us, how we will behave they will adapt it. This work is big [important], all groups should work together. Everyone must work for it” (FGD7).\n\nEmpowerment (through access to information)\n\nSeveral Nirdhar Groups facilitated health related information exchange – empowering adolescent girls with health and vaccine related information through social media. Nirdhar Groups helped to organise ‘health clinics’ for women in their communities. These were used as fora and means to instigate vaccination strategies and engagement in health literacy activities, as adjuncts to basic medical and maternity care.\n\n“Use of WhatsApp” (FGD9) “Women came out and got further information. Information technology coupled with it. And so it has speed in this field” (FGD5).\n\n“On vaccination day, we conduct tests [medical check-up] for adolescent girls. On that day we share information with them” (FGD5).\n\nEmpowering communities (by instilling kindness, care, and compassion)\n\nSome Nirdhar Groups used their forums to assert kindness, care, and compassion towards all members of the communities. This also shows a sense of responsibility group members developed towards individuals in their communities who needed help. The ability to provide help as well as intervene in such situations as a group is much more feasible in our study area rather than working individually.\n\n“We would neglect orphans … Now we take them to an orphanage…we told a few [neighbours] If we see any orphan, then we advise the neighbours to take care of that child … a farmer … early in the morning .. heard noise of a child crying. He found a small baby having soil on face.. He informed sir [director of HMF] and took that baby to his home, cleaned him, and afterwards took that baby to an orphanage” (FGD3).\n\n“We, the members of Youth Group started helping orphan children on the occasion of 15th August and 26th January from last year [Independence and Republic Day celebrations in India]. There are 24 such children in our village. We gave them educational material. We help them as per our capacity. We will continue doing this till it is possible for us. We collect whatever rupees we get-20, 30, 50 rupees, whatever people give. We do not give direct money to the children. Instead of that we give them material useful for school like bag, pencils, or notebooks” (FGD9).\n\nA sustained change for future generations\n\nThe underlying ethos of any sustained cultural change is to offer guidance and support to the next generation. Many Nirdhar Groups highlighted exemplars of ‘good citizenship’ which also helped in building confidence in girls and women in their communities.\n\n“It is our duty to set good examples for them to follow, subjects like equality, child marriage prevention, education for girls, must be instilled in the minds of younger generation. Then they will automatically start working. They will come forward and take initiatives. The work will continue” (FGD2).\n\n\nDiscussion\n\nThis qualitative evaluation of Nirdhar Groups’ experiences in community level interventions highlights the power of local citizens in embedding change within local areas. NGO leadership and community rural interventions have been proven as effective in previous studies (More et al., 2017; Krishnan et al., 2012; Jejeebhoy & Santhya, 2018). Involving residents who provide services within these communities (such as lawyers and police, healthcare professionals and other key stakeholders) is key to positive outcomes for addressing gender-based inequalities (Macdonald 2015; Daruwalla et al., 2019; Nair et al., 2020). The Nirdhar Groups within our study included multidisciplinary community citizens of ‘high standing’ such as ASHA workers (Accredited Social Health Activists), healthcare professionals and police representatives. The qualitative analysis for this study drew from the work of Braun and Clarke (2022) who propose that true thematic analysis develops themes as patterns of meaning’ rather than descriptive summaries of the phenomena under investigation. This systematic approach provides a richer account of the phenomena and produces findings that provide a more detailed and conceptualised account of the data (Braun & Clarke, 2022). This approach enabled the study to capture the complexity of gender-based oppression and DVA in a rural Indian setting to understand how individuals, families and communities interact and engage with one another on issues related to gender inequalities and DVA. To capture the physical, social, cultural, and historical contexts as well as attributes and behaviours of both Nirdhar Groups’ and their communities, an ecological perspective (providing a complete perspective of the factors that affect specific health behaviors, including the social determinants of health) offered a lens to present the collective values and perceptions of ‘norms’, and how these were challenged through reflection on ‘stereotypes, roles, and attitudes’ within the communities.\n\nThus, Nirdhar members used their own community ‘norms’ and identified the discourse of gender equality, so that they could shape and identify their own transformation priorities. These priorities were consistent across all Nirdhar Groups and focused on access to education and healthcare, misogynistic rituals (such as disproportionate burdening on women and hysterectomy as means to increase work productivity), internal family power dynamics, male dominated legislation, DVA and all confounding factors (such as substance misuse). Each of these foci have been cited in previous studies conducted in India (Daruwalla et al., 2019; Freudberg et al., 2018; Kalokhe et al., 2017). In creating ‘safe spaces’, women were able to access education and basic healthcare without victimisation. By informing women of their legal and economic rights, and ensuring communities engaged to a discourse which was unsupportive of physical and emotional violence, Nirdhar Groups were able to act as advocates. They worked with groups of women and individual families to assert the stance that DVA was not acceptable.\n\nA recent Cochrane review has highlighted the power of advocacy as a key intervention managing and reducing DVA, revealing the potential mediating factor of education as means to empower (Vigurs et al., 2019). With education and knowledge, women could then set their own goals and become an ‘expert’ on their own ‘life and self’. However, this review also noted limitations of ‘advocacy interventions’, and asserted that women’s needs were interwoven by the complexities of coercion, control, power, and economic/emotional control (Vigurs et al., 2019). Thus, it must be assumed that any interventions by Nirdhar Groups should be sustained and effective.\n\nGovernmental driven initiatives – such as the legislation which allows females to open bank accounts, to have home ownership and to access public distribution funds were already in place. Yet rural and remote communities were neither aware nor willing and thus less engaged in such key national schemes. The Nirdhar Groups were used as a conduit to offer guidance and education, so that women had the prerequisite knowledge and understanding of their rights to finances. There are several studies which support economic empowerment of women and link increased control of finances with both a reduced risk of DVA, and also increased access to reproductive and maternal healthcare services (Raj et al., 2018; McDougal et al., 2019; Singh et al., 2019; Dhar et al., 2018). Our study found several Nirdhar Groups applied legal pressure to ban sales of liquor (alcohol) across their villages. Misuse of substances has been directly linked to higher incidences of DVA (Jeyaseelan et al., 2004; Ragavan et al., 2014; Wagman et al., 2018). Groups chose to mitigate risks by the banning of liquor sales in local areas and shops. This has the potential to drive sales of alcohol to backstreet illegal trade routes, which could be even worse for populations. America’s prohibition period in the 1920s proved that blanket bans from senior leaders cannot replace personal responsibility, as they discovered ‘top down’ enforcement and censorship can cause disarray within social networks. Child marriages and dowry as means of gender-based oppression are reducing in India, perhaps due to the reduced number of females within the demographic population profile (United Nations Children’s Fund and United Nations Population Fund, 2018). However, dowry and early marriage were still raised across the focus groups for prioritisation. There were several interventions highlighted, which illustrated how Nirdhar Groups applied peer pressure and in-home advice to prevent dowry and child marriage. There was also community interest to lead such work to avoid dowry and child marriages for the greater good of girls/women considering its wider societal benefits. Interventions were supported, and celebrated, by hailing individuals who were identified as ‘samaj’ – or ‘good citizens’, (Samajdar Jodidar – Responsible Couples). The philosophy of Samaj – These ‘Responsible Couples’ and ‘enlighteners of society’ were reported amongst communities with pride. This role modelling shows a congruent relationship with Nirdhar Groups members and their communities. Changes are being made at personal levels as well as in the communities. These ‘Samaj’ uphold principles of goodness and respect and act as ‘pillars’ within the communities.\n\nThe good citizens (Samaj) were able to exert peer pressure and uphold principles of care, compassion, and kindness. Some of the Nirdhar Groups reported community level changes aimed at helping those identified as less fortunate. Others identified mechanisms by which the men would show how they took responsibility for cooking, childcare and home duties, demonstrating changes at individual and family levels breaking barriers to reduce discrimination, and also challenging existing social norms and misogynistic views. This philosophy was further entrenched as women in the communities could access ‘outside’ media influences. These included ‘WhatsApp’, Instagram, and Facebook (online forums) which enabled them to consider how women were viewed across the globe. As digital expansion has seen ‘millennial India’ adopt the second largest global internet use, the country is becoming democratized – and this is through a more geographically accessible public participation (Udupa et al., 2020). Thus, women are now more empowered with information on health, behaviours, politics, use and access to services, political participation and this has significant effects on their social mobility potential (Kumar et al., 2021). This movement is positively influencing women across rural India (Udupa et al., 2020).\n\nOur research findings showcase a number of positive achievements of the ‘Responsible Couples’ project. It is possible that there may been positive impacts on communities due to other project activities outlined in the introduction such as campaigns, which may have created a suitable environment for Nirdhar groups to establish themselves in communities and engage in gender equality work. We also acknowledge that HMF’s role is crucial considering their track record of over 30 years of work in Maharashtra state and their social influence may have helped to challenge existing ‘social norms’ while engaging men and communities in gender equality and DVA related discussions. However, our study design and data were not sufficient to analyse such effects or influences. The ‘Responsible Couples’ project has evolved over time through collaboration between HMF and SWISSAID, and their previous learning in Maharashtra, collaborations, and trainings from other NGOs. Their project design and delivery was more organic, which has undergone changes over time to identify and implement suitable activities to engage communities effectively. Therefore, our study approach is similar to process evaluation (Jejeebhoy & Santhya, 2018) to understand the core component- Nirdhar groups, which were crucial for their programme success. Nevertheless, we could not capture the entire process of development, training, and implementation of Nirdhar Groups initiatives (to achieve all objectives of the process evaluation), but rather conducted research once groups were active and well established. This prevented us from understanding fully what works, and what does not work in Indian rural communities. For example, HMF aimed to develop Nirdhar Groups in 40 villages, but managed to achieve that in 37 villages, which suggests possible resistance and lack of engagement from communities in three villages.\n\n\nConclusion\n\nThis study reveals the wide scope and influence that community gender equality groups can have. They provide a platform for women at the highest level in the villages and encourage dialogue confronting of a wide array of gender equality issues. The groups help raise awareness – particularly among men – but also help develop more practical and tangible change such as access to DVA prevention services and methods to address economic disparity. It also sheds light on the types of gender inequality women face in rural India and the types of challenges needing to be overcome. These groups are hopefully part of the solution – empowering women and educating men to provide safer and more equitable environments for women. They also perform the vital function of providing positive role models for the younger generation. Therefore, implementation of similar projects and its evaluation in other parts of the Maharashtra state as well as in other Indian states will provide valuable evidence. It is important to note that evidence from our research is from rural Indian communities, and considering constantly increasing migration to urban areas, further work is necessary to understand about its application in cities: how similar community groups could be formed in urban areas, which will be accessible to girls and women from all social classes and background. This will help design future community interventions to inform development work as well as policies to address DVA against women and girls in India and also in other similar settings.", "appendix": "Data availability statement\n\nData used in this study are confidential and are not available in the public domain. Ethics approvals provided permissions to use the dataset by specific individuals of the research team for stated purposes. Data from this project may be used further for future research by researchers/organisations involved in this work to conduct additional analyses.\n\nIf you wish to access this data, then please contact the corresponding author by email with the following information: a copy of your CV, research purpose/purpose for requesting data, and additional documents to support your request. Once these are reviewed by the researchers concerned, then you will receive a preliminary decision, and you will then be required to seek ethical approval from an ethical review board.\n\nFigshare: ADolescents GEnder SurVey, REsponsible CoupLes EvaluatiOn, and Capacity Building Project in India (DEVELOP): A study protocol https://f1000research.com/articles/8-958 (Ahankari et al., 2019).\n\nThis project contains the following extended data:\n\n• Supplementary Files 1 to 5.pdf\n\n• DEVELOP_Survey questionnaire Marathi.pdf\n\n• DEVELOP_FGD Guide Marathi.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\nAcknowledgments\n\nWe acknowledge the support received from the SWISSAID India and its Switzerland office. We thank the Halo Medical Foundation (HMF) for offering access to their project area and sharing their experiences to conceptualise this research project. 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Reference Source\n\nVigurs C, Cameron J, Yeo L: A realist view of which advocacy interventions work for which abused women under what circumstances. Cochrane Database Syst. Rev. 2019; 6. Art. No: CD013135.\n\nWagman JA, Donta B, Ritter J, et al.: Husband’s alcohol use, intimate partner violence, and family maltreatment of low-income postpartum women in Mumbai, India. J. Interpers. Violence. 2018; 33(14): 2241–2267. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "185418", "date": "30 Aug 2023", "name": "Thilini Chanchala Agampodi", "expertise": [ "Reviewer Expertise Public health", "qualitative research", "women's health." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article addresses a very important research gap on human empowerment interventions in low and middle income countries. However, the article need major revisions as embedded, in the introduction and results sections to level it up to a reasonable standard to be published. I’m sure the authors if concerned, can improve the quality of presentation so that the manuscript stands strong. I have given my detailed comments embedded in the manuscript. The manuscript need language editing specifically in translating the verbatim excerpts.\nPlease find the annotated pdf version of the article here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "197935", "date": "28 Sep 2023", "name": "Claire Powell", "expertise": [ "Reviewer Expertise Qualitative research", "domestic abuse", "child and family 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 interesting research study that addresses a gap in understanding about community interventions. However revisions are needed, particularly to strengthen the thematic analysis, tighten the structure and clarify the language throughout. My comments are below for each paragraph/subheading.\nAbstract – It would be good to have a clear summary of the key findings, there is a lot of detail in the abstract.\nIntroduction – it would be helpful to state with the problem you are addressing in the research and intervention before going into the wider context of gender inequality.\nCommunity-based gender equality interventions – ‘most effective gender equality work’, this is a very broad statement and only one reference is cited, which is theory of change development for an intervention in Mumbai. There need to be more references to back this up and possibly more detail on what effective gender equality work means and looks like in India specifically. Is there any wider literature or evidence on Nirdhar Groups that could be cited?\nLast sentences on project scope - these need moving to the beginning. The project could be described more succinctly with full details in an appendix. Finally, there could be a bit more development into why this study is needed.\nPurpose of the study – I think this could be more tightly described and it needs to match what your thematic analysis uncovers. My impression is you are exploring the perspectives of Nirdhar Group attendees with the aim of understanding how and why participants think they work.\nMethods – There needs to be a paragraph on overall study design and approach – is this a process evaluation? It is not really clear what the overall design is.\nEthics – Were there any other ethical issues you considered or reflected on that it would be worth reporting?\nParticipants – We do not need full names of researchers who selected the groups. But it would be helpful to have more detail on how the 12 groups were randomly selected. It is not clear why 12 was the number. Data saturation – a contested concept anyway – is something you reach through doing the research itself, you can not really decide this in advance. Were there other reasons for why 12 was chosen, for example did you try and include a diverse range of villages with different demographics/characteristics? Can you cite any relevant literature to support your sampling approach?\nStudy setting – this doesn’t describe the setting but the interview schedule/materials. This section could be more clearly described. How were questions developed – was it based on previous studies? It is not clear from this section how the focus groups worked, who facilitated them. It is not clear what this sentence is referring to ‘As a result, identification, intervention, and delivery was agreed through group discussions involving several members….’ This section could be more clearly described in general, perhaps separating out the rationale for focus groups from the development of the interview schedule might help.\nData collection – It is not clear whether all group participants consented or whether participants were recruited from existing groups. There are no details on recruitment. It would be helpful to know who discussed the study with potential participants, how was consent explained, how people agree to take part etc. Again this section could be more clearly described in shorter paragraphs going through each stage step by step. The order is a bit confusing and jumps to dissemination before you have discussed analysis. As previously full names are not needed.\nAnalysis – Braun & Clarke have written a lot more about thematic analysis since their 2006 paper, it would be better to use a more recent reference. Also thematic analysis can be either deductive or inductive, so you need to be clear that you decided to do it deductively, rather than it being specific to their approach. I also do not understand your reasoning ‘to reflect the linearity of this project’ – can you explain this a bit more and give examples of themes you were looking for based on theory/literature. This section also needs a bit more detail – how did the two researchers code the data? Did they both code all of the data or sections? Was there a codebook? Were there discussions to develop the themes? Were wider members of the team/research study consulted? What you go on to describe sounds more like inductive thematic analysis because you are developing themes based on the codes, rather than pre-defined themes. Another thing to consider is that Braun & Clarke’s approach is reflective so there needs to be some reflection on the roles of the researchers and the analysis. Or perhaps you could cite a different thematic analysis approach which is more descriptive.\nResults – You need to start with an overview of the participants and their demographics. Then follow with a brief summary of the themes and how they relate to each other.\nMy other main comment is that there are too many subthemes and many of them seem very descriptive, far too brief, and do not always really reflect the quotes you use. I would go back to your themes and think about how you describe them, and which are the really key subthemes (max 2 or 3) you would like to focus on. There are also too many quotes that are just listed and repeat the same idea. Each quote should be introduced and then explained and really encapsulate the theme or subtheme. Full quotes do not always need to be used – key words can be embedded in a paragraph. But overall the quotes mostly need to be paraphrased so we have much fuller sense of the nuance and detail of what people were saying.\nIt would also be helpful for participant IDs to have some information, at least whether they are male or female. A key with the list of the groups and how many participants and their demographics would also be helpful.\nI think the results section needs more of a focus on the research questions so there’s a clear sense on what the findings say, at the moment there are a lot of small details, but it is hard to build a full picture of the findings.\nDiscussion – this needs to start with a summary of the key findings and then widen out to the wider literature, both Indian based and global.\nThe focus on advocacy seems really important and there is a lot more relevant advocacy literature that could be cited in support of this. I think you could develop the limitations of advocacy interventions that you identify and discuss them specifically in the Indian context. I would hesitate to make such a strong statement as ‘it must be assumed any intervention by Nirdhar Groups should be sustained and effective’.\nThe paragraph that starts ‘governmental driven initiatives’ needs splitting where it starts with ‘Child marriages’ as these are two separate points. You’re making a lot of important points here, but they could be more clearly organised to make it easier for your readers to follow.\nYou briefly mention limitations at the end of the penultimate paragraph, but these need developing and to be in their own section with a subheading.\nConclusion – this is quite long. It would be helpful to focus on the two or three key messages you would like readers to take away from this 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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-388
https://f1000research.com/articles/11-1137/v1	05 Oct 22	{ "type": "Software Tool Article", "title": "A2TEA: Identifying trait-specific evolutionary adaptations", "authors": [ "Tyll Stöcker", "Carolin Uebermuth-Feldhaus", "Florian Boecker", "Heiko Schoof", "Carolin Uebermuth-Feldhaus", "Florian Boecker" ], "abstract": "Background: Plants differ in their ability to cope with external stresses (e.g., drought tolerance). Genome duplications are an important mechanism to enable plant adaptation. This leads to characteristic footprints in the genome, such as protein family expansion. We explore genetic diversity and uncover evolutionary adaptation to stresses by exploiting genome comparisons between stress tolerant and sensitive species and RNA-Seq data sets from stress experiments. Expanded gene families that are stress-responsive based on differential expression analysis could hint at species or clade-specific adaptation, making these gene families exciting candidates for follow-up tolerance studies and crop improvement. Software: Integration of such cross-species omics data is a challenging task, requiring various steps of transformation and filtering. Ultimately, visualization is crucial for quality control and interpretation. To address this, we developed A2TEA: Automated Assessment of Trait-specific Evolutionary Adaptations, a Snakemake workflow for detecting adaptation footprints in silico. It functions as a one-stop processing pipeline, integrating protein family, phylogeny, expression, and protein function analysis. The pipeline is accompanied by an R Shiny web application that allows exploring, highlighting, and exporting the results interactively. This allows the user to formulate hypotheses regarding the genomic adaptations of one or a subset of the investigated species to a given stress. Conclusions: While our research focus is on crops, the pipeline is entirely independent of the underlying species and can be used with any set of species. We demonstrate pipeline efficiency on real-world datasets and discuss the implementation and limits of our analysis workflow as well as planned extensions to its current state. The A2TEA workflow and web application are publicly available at: https://github.com/tgstoecker/A2TEA.Workflow and https://github.com/tgstoecker/A2TEA.WebApp, respectively.", "keywords": [ "plants", "crops", "adaptation", "evolution", "stress", "workflow", "software" ], "content": "Introduction\n\nWhile genomic resources are being expanded in crop species, with more and more high-quality reference genome sequences and transcriptome datasets becoming available, the lack of integrated trait and functional information limits the ability to interpret genomic-scale datasets and discover genotype-phenotype associations. While many efforts have focussed on individual (model) species, the availability of omics data for many more or less related genomes opens opportunities to explore genetic diversity through multi-genome comparisons. While differential expression analysis has led to the discovery of many candidate genes involved in tolerance to stresses, we aim to prioritize genes that were targets of evolutionary adaptation, thus facilitating application in crop improvement. To identify genomic footprints of adaptation, we use comparative genomics to identify protein family expansions. Gene duplication is a major driver of molecular evolution,1,2 and in plants, whole-genome duplication events are frequent (reviewed in Ref. 3), but tandem and transposon-mediated duplications also play a role (reviewed in Ref. 4). Most of the duplicates are lost or silenced,5 but which duplicates are retained hints at some evolutionary advantage. These may be targets of adaptation.6 Especially for the adaptation of regulatory networks, duplication allows for neo- or subfunctionalization.7 These form an evolutionary scenario that can be observed based on our integration of phylogeny and differential expression under stress data. However, we also consider differential expression under a given stress in any species as a functional link to the given stress, even if there is not sufficient data to confirm neo- or subfunctionalization. This allows us to filter for gene family expansions functionally linked to the given stress, as not all adaptations and thus retained duplicates in a genome need to relate to tolerance to the given stress, other traits not under analysis will also show adaptation and thus protein family expansions (Figure 1).\n\nThe challenge for this multi-genome approach is the cross-species integration of multiple types of omics data, which requires several software tools and various custom steps of transformation and filtering. To promote the exploration of genomics and transcriptomics data and the association of genotype with phenotype data in order to address adaptation, we developed A2TEA (Automated Assessment of Trait-specific Evolutionary Adaptations). Our software aims at identifying candidate genes for stress adaptation in plant species and enables GUI-based exploration of the results, but is suitable for gene family expansion analysis integrated with differential expression data in any set of genomes. It is composed of a Snakemake workflow and an R (Shiny) package working in tandem to automate and ease all bioinformatics & analysis tasks involved.\n\nThe A2TEA.Workflow functions as a one-stop processing pipeline, integrating the prediction of gene families in form of orthologous groups (OGs) with the analysis of their phylogeny, protein function, and expression, using RNA-seq data from all species. It allows the user to formulate adaptation hypotheses as specific scenarios of gene family expansion in one or several of the genomes, for example, based on a classification of species as stress-tolerant or sensitive, or to identify clade-specific adaptations. As input, the workflow requires for each species a protein FASTA file for orthologous group prediction and RNA-seq reads suitable for differential expression analysis (control vs. treatment), together with either a genomic FASTA file with appropriate gene annotation or a transcriptomic/cDNA FASTA file. Functional information for each species can be provided by the user or can be optionally inferred by our tool AHRD (https://github.com/groupschoof/AHRD) during runtime. The single compressed output is ready for analysis with the R programming language8 as we took care to create well-structured objects and easy-to-parse outputs. In addition, in order to facilitate immediate and easy exploration and visualization of the results, we created the A2TEA.WebApp written in R Shiny,9 which allows exploring, highlighting, and exporting the results interactively.\n\nThe A2TEA.Workflow combines state-of-the-art bioinformatics software with custom integration steps to combine inferred gene family expansion events with expression results and functional associations.\n\nThe workflow is designed as a complete solution starting with raw data and performing upstream quality controls and data transformations automatically. We also took care to allow for a high degree of customizability - e.g. RNA-seq analysis can be performed either alignment-based or using pseudoalignment, and tool-specific parameters can be tweaked in one central config file. Importantly, the workflow is designed to answer biological questions and as such requires the definition of hypotheses in form of combinations of the species of interest. For each hypothesis, the user needs to adjust parameters related to the definition and cutoffs of expansion events. This allows computation of results for several combinations in parallel and facilitates the investigation of many hypotheses downstream e.g., expansion in all tolerant species, in only a specific species, or in all species of a clade.\n\nThe A2TEA.WebApp provides an interactive web interface to explore, filter, and visualize the previously generated results via a straightforward tab-structured dashboard design. We took care to create a user-friendly mouse-controlled experience in order to extend the usability from bioinformaticians to experimentalists. The user first uploads the output file of the workflow and chooses the specific “hypothesis” to investigate. This generates a general information tab providing an overview of phylogeny, expression, and set sizes of orthologous groups (OGs) passing the thresholds. The user is then able to switch to dedicated analysis tabs relating to 1) filtering and analyzing OGs with associated data, 2) set size comparisons and tests, and 3) gene ontology (GO) term enrichment analyses. Reactively rendered tables and visualizations are dynamically populated with links to databases such as Ensembl10 and AmiGO11 to allow for an immediate follow-up exploration of interesting genes. Tables and graphs can be exported in a variety of formats. The web application also provides a bookmarking system that facilitates the collection and export of the most interesting genes and OGs.\n\nTo extend the usability of the workflow by allowing for further species-specific exploration of gene and geneset functional enrichments we integrated the creation of GeneTonic input data files into the A2TEA.Workflow. GeneTonic is a web application that serves as a comprehensive toolkit for streamlining the interpretation of functional enrichment analyses from RNA-seq data.12 As our workflow is built on Snakemake,13 the addition of further analyses or outputs allows for modular expansion of its current state. We also intend to add further analyses and features to the A2TEA.WebApp web application.\n\nA2TEA combines best practices in both choice of tools as well as reproducibility and offers a one-stop solution for the integration of genome comparisons with expression and functional data to unravel candidate genes for natural adaptation, e.g. in stress-tolerant plant species. The web application empowers users to explore stress-specific gene family expansions combined with transcriptomic data from their own or published stress experiments by providing interactive visualizations, statistical tests, and dynamically generated database queries.\n\nBoth the A2TEA.Workflow and A2TEA.WebApp are freely available at https://github.com/tgstoecker/A2TEA.Workflow and https://github.com/tgstoecker/A2TEA.WebApp, respectively, and archived in Zenodo.51,52 For demonstration purposes, we also made a public instance of the web application available at https://tgstoecker.shinyapps.io/A2TEA-WebApp.\n\n\nMethods\n\nThe A2TEA.Workflow is written in Python and makes use of the Snakemake workflow framework. It leverages the bioconda project channel14 of the conda package manager to handle software installation and dependency management. Another tool from our lab, AHRD, is integrated as a Git submodule and can be optionally used to infer protein function annotation for any of the species under investigation.\n\nThe typical use case for running the workflow consists of cloning the GitHub repository, configuring it to specific needs, and then starting the analyses with either installation of software and dependencies during runtime or usage of a Docker/Singularity container (Figure 2). Modification of the workflow is performed by changing dedicated configuration files controlling samples, species, hypotheses, and tool-specific options. With “hypotheses” we refer to the definition of “gene family expansion” in the set of species under investigation. Several hypotheses can be run in parallel. This multi-hypothesis structure permits the investigation of several defined biological questions, for instance, gene family expansion in stress-tolerant compared to stress-sensitive species. For each hypothesis, we always require the definition of a set of one or more species that should be checked for expansion compared to a second set of one or more species that should not show expansion. For each hypothesis, the user is able to set several options, such as the ratio or the minimal number of genes in a species, to qualify as an expanded OG. The hypotheses.tsv file is structured column-wise with both an index number and a “name” variable used to identify the choices throughout the workflow. Generally, the connection between files and workflow rules is achieved by the species names (e.g., “Arabidopsis_thaliana”). Many hypotheses can be computed in a single workflow with a single final output object that contains all results. This facilitates easy comparisons in the downstream web application, which is especially useful to check the parameter choices for the definition of gene family expansion or when it is necessary to work with unclear trait classification of some species.\n\nThe final output generated by the workflow is a single .RData file that can be loaded into an active R environment with the load() command. This provides several separate objects containing all results in a compact form factor:\n\n• HYPOTHESES.a2tea - List object with one S4 object per hypothesis. Each S4 object contains several layers of nested information. E.g. HYPOTHESES.a2tea$hypothesis_2@expanded_OGs$N0.HOG0001225 refers to a specific expanded OG and S4 data object that contains:\n\n– blast_table (complete BLAST/DIAMOND results for OG genes & extended hits)\n\n– add_OG_analysis (includes multiple sequence alignment (MSA), phylogenetic tree, and gene info for expanded OG and additional OGs based on best BLAST/DIAMOND hits)\n\n• HOG_level_list - List object with one tibble per hypothesis. Information includes OG, number of genes per species, boolean expansion info, number of significant differentially expressed genes (DEGs), and more. The last N list element is a non-redundant superset of all species analyzed over all formulated hypotheses. This makes it easy to create a comparison set e.g., conserved OGs of all species to which the hypothesis subset can then be compared.\n\n• HOG_DE.a2tea - Tibble of DESeq2 results for all genes + additional columns.\n\n• A2TEA.fa.seqs - Non-redundant list object containing corresponding amino acid FASTA sequences of all genes/transcripts in the final analysis (this includes those of expanded OGs + those in additional BLAST hits & additional OGs based on user-chosen parameters).\n\n• SFA/SFA_OG_level - Gene/transcript level tables that contain functional predictions (human readable descriptions & GO terms inferred by AHRD).\n\n• hypotheses - A copy of the user-defined hypotheses definitions for the underlying workflow run.\n\n• all_speciesTree - Phylogenetic tree of all species in the workflow run (a non-redundant superset of hypotheses) as inferred by Orthofinder/STAG/Stride.\n\nThe .RData output can be investigated inside an R session or via the A2TEA.WebApp, which was specifically designed to allow for interactive inspection, visualization, filtering, and export of the results and subsets. We feature a tutorial for its usage and details on how to work with the results of an A2TEA.Workflow analysis run in the Use Case section and in the project’s pkgdown site.\n\nThe A2TEA.WebApp is written in the R programming language8 and uses the Shiny9 framework to facilitate interactivity with the data. It expects the user to upload an .RData file created by the A2TEA.Workflow. The web application comes with a test dataset that can be loaded with a single click so that interested users can try out its functionality before having to finish an A2TEA.Workflow run.\n\nWe developed the web application following community standards and have set up a continuous integration system with GitHub actions that performs build checks of both the package itself and the associated pkgdown site15 hosted via GitHub pages.\n\nThe A2TEA.WebApp is written in the R programming language and uses the Shiny framework to facilitate interactivity with the data. It expects the user to upload a .RData file created by the A2TEA.Workflow. The web application comes with a test dataset that can be loaded with a single click so interested users can try out its functionality before having to finish an A2TEA.Workflow run. We developed the web application following community standards and have set up a continuous integration system with GitHub actions that performs build checks of both the package itself and the associated pkgdown site (Wickham et al., 2022)15 hosted via GitHub pages.\n\nThe interface is structured in tabs with shinydashboard16 and shinydashboardPlus,17 providing the layout infrastructure (shown in Figure 3). The main functionality includes a selector to choose which hypothesis to display (Figure 3B), a sidebar menu that enables the user to switch between different analysis types (Figure 3C), and tool-specific options for parameters, visuals, and export (Figure 3F).\n\nWe designed the interface to allow the focus to be put on an individual analysis or plot to gain insight from the data. Plots and tables are contained in collapsible boxes, leaving it up to the user to decide how much information should be displayed at once. Additionally, we tried to separate important parameters from purely aesthetic choices in the plot options, with main options always visible at the side and aesthetic choices reactively displayed after the user switches a box toggle.\n\nSince the exploration of data can be a lengthy process with many iteration cycles, we looked for ways of aiding the user in storing the observations made. Following the example set by the GeneTonic web application,12 we integrated a bookmarking system that temporarily stores interesting genes/transcripts and OGs. For this, the user needs to mark the respective ID in one of the tables and then click the dedicated bookmarking button displayed at the top of the interface (Figure 3D). All bookmarks are rendered in two reference tables, both in a dedicated tab as well as a pop-up window that can be displayed on every analysis tab. This quick reference is convenient when performing filtering operations in the tables or choosing an interesting OG to display. While these tables can of course, be downloaded, we also implemented a subsetting feature on the bookmarks tab that creates a smaller .RData file with information only pertaining to the bookmarks and associated data. These smaller subsets are fully functioning complete inputs and can be loaded into the application again at a later time - for re-plotting purposes for instance. With this feature, it is straightforward to extract, store and share all information about interesting genes, transcripts, or OGs.\n\nWorkflow diagram of the A2TEA.Workflow displayed as Snakemake rulegraph. After computation of expanded orthologous groups (OGs) (rule expansion - marked with A) the directed acyclic graph (DAG) is re-evaluated since the results are not known beforehand. This Snakemake checkpoint then uses the reciprocal best hits computed by Orthofinder to find the N most similar additional OGs per OG, where N is a variable set by the user. For each OG and additional set of 1 to N additional OGs, multiple sequence alignments and phylogenetic trees are built and used in the downstream steps.\n\nThe A2TEA.Workflow has been primarily designed for use within Linux and requires a standard bash environment with working installations of Snakemake13 and conda/mamba (Ref. 14, https://github.com/mamba-org/mamba). The former facilitates compatibility with common cluster setups such as SLURM or LSF. Instructions for a minimal setup are described in the project’s README.\n\nAt the moment, for each species, the A2TEA.Workflow requires as input RNA-Seq reads (both paired-/single-end possible) suitable for differential expression analysis (control vs. treatment), either a genomic or transcriptomic FASTA file, annotations, and a peptide FASTA file. The user can provide functional information per species, or it can be optionally inferred by our tool AHRD during the workflow. Control of the workflow is handled by several configuration files, which the user needs to adapt to their specific inputs and scientific questions.\n\nThe samples.tsv table needs to list all RNA-seq FASTQ files with the columns providing additional information based on which the workflow can infer associations such as species, replicate, and the correct steps to perform. For instance, by leaving out the column for the second paired sample, it is automatically inferred that single-end options have to be used (trimming, mapping, etc.). Operations such as recognizing that files are gzipped and need to be handled appropriately are performed automatically as well. The species.tsv table functions similarly and needs to provide per species information on the FASTA and annotation files, the ploidy of the species, and the location of a file providing the functional information, in the form of GO terms, per protein. If no functional information can be provided the user can choose to add “AHRD” instead of a file path which will trigger a sub-workflow during computation that will create an appropriate file via our functional annotation tool AHRD. Based on whether the user provides a genomic or cDNA FASTA file for a species, the workflow will perform either traditional alignment with STAR18 or pseudoalignment with kallisto.19\n\nThe config.yaml controls parameters such as thread usage for individual steps, tool-specific parameters, and parameters relating specifically to the A2TEA.Workflow. Two other very important choices that have to be considered are whether or not automatic filtering for the longest representative isoforms of the peptide FASTA files should be performed and whether gene or transcript level quantification is wanted. Choosing automatic isoform filtering will create a subset peptide FASTA file with only the longest isoform per gene; the header will be shortened to just the gene name identifier. This option must be used in conjunction with gene level quantification since otherwise matching both types of data is not possible.\n\nThe notion behind the hypotheses.tsv table is outlined in the Implementation section due to its central importance to the expansion calculation. Here, we briefly want to present some of the available choices the user can consider. Besides defining sets of species that should be analyzed for expansion compared to other sets of species, the user is able to specify the required numerical differences between the two and which OGs to disregard immediately. For instance, “Nmin_expanded_in” takes as input an integer value that defines the minimum number of the investigated species that need to fulfill the expansion criteria in order for the gene family to be called “expanded”. These criteria are for instance “min_expansion_factor” and “min_expansion_difference” with which either the minimum factor of expansion or the minimum number of additional genes compared to the non-expanded set of species can be defined. To complement these broad cutoffs, the workflow also integrates a hypothesis-specific CAFE analysis, with which changes in gene family size are analyzed in a way that accounts for phylogenetic history and provides a statistical foundation for evolutionary inferences.20\n\nAfter all choices have been made, the workflow can be started with a single Snakemake command. A2TEA.Workflow will then perform all previously listed steps and merge results into the final output file described in the Implementation section (Figure 2). The user is then able to investigate the integrated and condensed results.\n\nWe offer several ways of starting an A2TEA.WebApp instance for downstream investigation of the data: 1) installation with R devtools from our GitHub repository, 2) a docker container with the latest release installed, and lastly, 3) a demo instance hosted on shinyapps.io (https://tgstoecker.shinyapps.io/A2TEA-WebApp/). As the A2TEA.WebApp is an interactive tool with an explorative focus and no strict work order, we illustrate its core operative features in the dedicated Use cases section of this manuscript.\n\n\nUse cases\n\nIn this section, we will illustrate the functionality of the A2TEA.WebApp, using the A2TEA.Workflow results of a three-species analysis of Hordeum vulgare (barley), Zea mays (maize), and Oryza sativa japonica (rice) that investigates adaptive processes in barley to drought stress. Details on the files used as well as their respective publication and SRA accession numbers are listed in detail in both GitHub repositories and the Source data section.48,49,50\n\nWe integrated this dataset into the workflow and the web application to illustrate the software’s setup and to allow for a quick exploration of the tools’ functionalities. After cloning the A2TEA.Workflow repository, an additional script can be run (get_test_data.sh) that quickly sets up the experiment by downloading the required input files. Peptide FASTA files are reduced to 2000 proteins; the transcriptomic data is subsampled to 2M reads to allow for a quicker runtime. The functional annotations are precomputed by AHRD. The differential expression analysis is set to be performed on the gene level and two comparisons are performed as defined in the hypotheses.tsv table. These are “Expanded in barley compared to rice and maize” and “Expanded in barley compared to maize”. For both, expansion is defined as \"number of genes species A ≥ 2 × number of genes of species B”.\n\nThe final output produced by the workflow is also integrated into the current release of the A2TEA.WebApp and can be loaded via clicking the “Try a demo A2TEA.RData file” at the top of the interface.\n\nThe user can either decide to load the included test dataset or upload a .RData result object (A). Other options in the sidebar menu are a selector of the hypothesis (meaning: species comparison) to display or change to another analysis tab (C). Genes, transcripts, or orthologous groups (OGs) can be marked in tables or boxes ticked and then bookmarked with a dedicated button (D). Bookmarks have their dedicated tab but can also be displayed as a sidebar window anywhere for quick reference purposes (E). Analysis- or plot-specific parameters (F) are displayed to the left of the visualization, and a box-specific sidebar window for aesthetic parameters can be opened by clicking the gears icon (G). The underlying dataset investigates drought tolerance among four Brassicaceae species. Displayed here are the maximum likelihood phylogenies of a gene family showing potential subfunctionalization of Eutrema salsugineum homologs (top two genes in the tree). Blue bars show log2 (fold change) of gene expression between drought and control conditions, stars mark adj. p ≤ 0.1 significance cutoff, and the multiple sequence alignment of amino acid sequences is displayed to the right. We provide this particular OG as a bookmarked subset .RData file (see Underlying Data).\n\nThe general analysis tab is the default view inside the A2TEA.WebApp. Once input is loaded, reactive information boxes display the number of species, the number of expanded OGs, and the number of DEGs for the currently selected hypothesis. Changing the hypothesis (Figure 3B) e.g., to the second hypothesis in our test set (“Expanded in barley compared to maize”), changes the statistics and all other sets/plots to reflect only the species considered in the hypothesis. Two tables display gene-level differential expression results and functional annotation information (human readable descriptions and GO terms), which allow, for example, the exploration of genes related to a particular function. Also displayed are an inferred phylogenetic tree of the species in the hypothesis subset and an intersection plot (Venn/UpSet) which displays the number of conserved (OG with ≥1 gene from every species), overlapping, or species unique OGs and singleton genes. Importantly, a table describing the details of the currently displayed hypothesis is also displayed. All of this facilitates a broad overview of the data and allows the user to spot errors such as faulty hypothesis definitions or cutoffs that are too strict.\n\nThe main feature of the TEA (trait-specific evolutionary adaptation) tab is a comprehensive toolkit for the visualization of maximum likelihood phylogenies of expanded OGs and associated information such as the log2(fold change) of the displayed genes and an MSA of the respective protein sequences (Figure 3). The MSA can be added as a geometric layer to the tree plots or displayed separately with additional options such as a conservation bar (Figure 3A). To make an informed decision of which OGs are most worthwhile to investigate closer, a table showcasing the total and significantly differentially expressed genes per OG is also provided. With this, the user is enabled to apply several filters, for example, to select all expanded OGs that possess at least 1 DEG and more than 4 genes from Hordeum vulgare. The last table on the tab provides insight into the reciprocal BLAST/DIAMOND hits for the currently chosen OG and the additional most similar OGs. Notably, this table also provides the identifiers given to the proteins by Orthofinder,21 making it easy to relate insight gained in the web application back to other outputs created by Orthofinder in the A2TEA.Workflow, such as the list of putative xenologs.\n\nTo describe adaptive processes at a larger scale, we also integrated functionality to visualize distributions of user-defined OG sets and test for their over-representation; e.g., “What is the frequency of OGs that show expansion and at least 1 DEG in Hordeum vulgare in all conserved OGs?” and “Is this set over-represented within the background distribution of conserved OGs with at least 1 DEG from any species?”. We took care to make answering such questions very accessible by providing the user with text-based choices of which sets to plot or compare. Currently integrated are an enrichment analysis suite allowing for Fisher-Tests and a corresponding circular set plot (Figure 4B) that visualizes the chosen sets. Also provided is a tool for comparing the size distributions of the OGs (Figure 4C) with which group size effects can be checked; e.g., “Do we see differences in the number of DEGs in OGs of a certain size range between the set of interest and the background set?”.\n\n(A) Multiple sequence alignment of an expanded orthologous group (OG) + additional most similar singletons or OGs. Bars at the bottom represent the degree of conservation. (B) Visual representation of a hypergeometric test for over-representation - colors/layers represent sets in the urn model. The outer ring shows the background set (light blue; “complete background”) and the subset in the background set (light orange; “success in background”). The inner ring displays the set of interest (blue; “sample size”) and the subset in the set of interest (orange; “success in sample”). (C) Barplot of the total number of OGs per group size (number of genes) between all OGs (blue) and only those OGs that are conserved among the analyzed species (orange). (D) Dotplot of the top over-represented biological processes in the subset of OGs of interest compared to the background; dot size indicates the number of OGs with the respective GO term; color displays negative log [base 10] of the p-values from the enrichment test.\n\nThe last analysis tab provides options for performing GO term over-representation analysis based on the topGO R package.22 Functions that occur more often than expected can be identified by setting several parameters that specify the set of OGs the user wants to analyze. With our test data, the user could, for instance, be interested in enriched molecular functions of OGs that are expanded in Hordeum vulgare and also possess at least 2 DEGs of Hordeum vulgare. Once computed, a table is displayed that shows the top significantly enriched GO terms and also contains dynamically created links for these to AmiGO2.11 A second table contains information on the corresponding OGs and genes so that the user can follow up on a particular enriched GO term and inspect the underlying data. We also provide two visualizations that summarize the results. The first is a GO enrichment dotplot (Figure 4D) straightforwardly showcasing the overall results, and the second is a GO subgraph of selected top N enriched GO terms. With the latter, we provide the user with an insightful way of investigating how the significant GO terms are distributed over the GO graph.\n\nTables can be downloaded as .tsv files, and plots are exportable into various formats, such as.pdf, .png, or.svg, allowing the user to easily save and share the observations and results. However, even a relatively small set of species, like the three Poaceae species in our test data, lead to several OGs that are worthwhile to investigate, substantiating the need for the bookmarking system outlined in the Implementation section. It quickly becomes very valuable to bookmark, e.g., all OGs annotated with the top 5 enriched BP GO terms in the OGs expanded in Hordeum vulgare if the intention is to return to the analysis later or to generate a list to use with another tool quickly. Relating this to the previous sub-sections, we want to emphasize that bookmarking is integral to using the A2TEA.WebApp and is fully featured on all analysis tabs except the “Set analyses” since here individual genes or OGs are not the focus. To further aid users in the bookmarking process, we also added informative pop-up messages to indicate for instance, that all selected genes/OGs have already been saved. Since the bookmarks can also be used to export a completely functional .RData subset file, only the most relevant information is kept while the processing speed is increased, and all relevant results of the integrative effort are kept. If, for instance, during the analysis, it turned out that hypothesis 2 in our example data (“Expanded in barley compared to maize”) is, in fact, not of interest anymore, subsetting the .RData file to interesting OGs of hypothesis 1 completely removes the unneeded “bloat” of hypothesis 2. Similarly, the user could create 2 .RData files (one for each hypothesis) and run a custom script on each separately, efficiently producing hypothesis-specific results.\n\n\nDiscussion\n\nClassic transcriptomic studies produce large lists of gene regulatory information for which, traditionally, pathway or GO term analyses are used to discover the overall molecular trends caused by the experimental treatments.23 We propose that we can identify novel genes relevant for stress adaptation by comparing same-stress experiments of several plant species with different levels of stress adaptation in combination with evolutionary footprints in the form of protein family expansion. As illustrated in the Methods & Use Cases sections, our novel software tool A2TEA facilitates the identification of genes associated with the evolution of a trait in a species or a group of related species. Based on the rediscovery of known genes related to the trait, we believe that also novel genes discovered through A2TEA are related to the trait, but experimental verification is in progress, see below. As an example, Figure 3 presents a possible subfunctionalization of gene duplicates in Eutrema salsugineum, discovered from data of drought tolerance among four Brassicaceae species (details see Underlying Data). The A. thaliana homolog is involved in drought stress response.24\n\nSeveral approaches have been employed to identify potential candidate genes that could provide a genetic basis for more resilient crops. This includes forward genetics approaches such as identifying causative genes for advantageous mutant phenotypes,25 finding common regulators for several stresses via traditional transcriptomics,26 usage of Quantitative Trait Locus (QTL) mapping and Genome-wide association studies (GWAS) incl. potential integration with expression data,27 the combination of expression data with functional information and clustering methods28,29 and also machine learning based approaches that employ transcriptomic or phenomic data as the basis of their candidate gene predictions.30,31\n\nThe underlying methods are manifold and include approaches such as Bulked-Segregant analysis,32 k-means clustering,33 WGCNA,34 co-expression networks35 and set analyses of DEGs often in combination with pathway or GO term enrichment analyses.23 While most studies share the approach of reducing a list of regulated genes via secondary criteria, to our knowledge, A2TEA is the first openly available tool that specifically combines stress-specific expression data from several species with gene family expansion to unravel candidate genes for stress adaptation in stress-tolerant species.\n\nWith A2TEA, we present software that simplifies the complex bioinformatics workflow for the user and provides an interactive web interface for analysis of the results. By using Snakemake as a bioinformatic workflow manager, we remove the need for step-by-step handling of raw data (including software setup and dependencies necessary for computations) and ensure FAIR (findable, accessible, interoperable, and reusable) computational analysis standards.36 The downstream analysis and visualization framework makes the navigation of the resulting large sets of tabular data faster, more intuitive, and more practicable for scientists without programming skills.12,23 It offers a variety of summary statistics on the levels of gene family expansion, differential expression, and functional enrichment to ensure quality control. The Shiny framework provides interactivity regarding the visualization and the analysis of the results, and this interactivity highly facilitates the exploration of scientific questions.\n\nBased on user experiences with the web application, we have included analyses and visualizations to allow detecting problems in the bioinformatic predictions, e.g., of orthologous groups (OGs). In order to spot potential misassignments of Orthofinder, close homologs to members of an OG are detected by similarity search and displayed with phylogenetic trees and multiple sequence alignments. A typical case is the non-inclusion of a singleton gene of a species due to a significant portion of protein sequence missing in the annotation, caused e.g., by gaps or sequencing errors in the genome sequence or errors in gene prediction. Similarly, false expansions based on a putative paralog that has only very limited alignment overlap with other members of the OG can be detected. These could be actual duplicates but degenerated through pseudogenization or partial duplication, e.g., the action of transposable elements.\n\nWe designed A2TEA with extendability in mind. Both the Snakemake-based A2TEA.Workflow and the A2TEA Shiny App can be easily expanded in a modular fashion to integrate novel features. We are currently testing several additional visualization and testing options. This includes the option for positive selection tests concerning a particular OG, e.g., by calculating the ratio of non-synonymous amino-acid substitutions over synonymous amino-acid substitutions (dN/dS)), distribution comparisons between random and actual DEG-containing OGs, and visualizations for the analysis of general gene/transcript regulation trends. The GO term enrichment functionality is aimed at discovering general trends in the adaptation to the particular stress under investigation. At the moment, the implemented enrichment tests provide options for single over-representation analysis as implemented in the R topGO package.22 It will be interesting to evaluate and potentially implement further options for functional enrichment analysis in A2TEA, such as modern ensembl approaches37 or simplification strategies that aid in summarization.38 Lastly, we intend to implement the option to download a comprehensive RMarkdown/Quarto report summarizing plots and statistics for all bookmarked genes and OGs. This has been demonstrated to be a significant step forward in guaranteeing the portability of results once an interactive session is concluded.12\n\nWhile our research focus is on crops, from a software perspective, A2TEA is entirely independent of the underlying species and can be used with any set of species. This opens the question of how feasible applying the A2TEA methodology to species from other kingdoms might be. Our motivation for developing A2TEA is primarily rooted in the notion that genome duplication played a major role in the evolutionary past of plants.4 Plant comparative genomics research has shown that gene families are mostly conserved across great evolutionary timescales, comprising even the diversification of all angiosperms and nonflowering plants.39,40 Fascinatingly, this conservation of gene families is combined with lineage-specific fluctuations in gene family size, which are frequent among taxa.4,39–42 This suggests that since comparatively few novel gene families arose, much of the great diversity and phenotypic variation seen in land plants may have arisen primarily due to duplication and adaptive specialization of already existing genes.40\n\nWhile whole genome duplication events are expected and reported less frequently in the animal kingdom and thus gene duplication as a driver of protein family expansion does not play as prominent a role in animals as in plants,43,44 protein family expansion is still an important driver of adaptation.45 We expect that A2TEA will be useful in non-plant species, even if protein family expansion only represents a small portion of adaptive changes, with other sources of variation, like alternative splicing playing a potentially more important role.46\n\nCurrently, we are investigating several publicly available genomic and transcriptomic datasets from various groups of plant species with A2TEA. While we expect to detect candidate genes relevant to adaptation to the stress being investigated, this assumption is based on the rediscovery of known genes. One important follow-up step is thus to experimentally verify the impact of selected candidate genes in vivo. To this end, we perform stress experiments in plants bearing knockout mutations in candidate genes predicted by A2TEA, using sequence-indexed mutant collections such as BonnMu.47 This will allow us to assess the phenotypic impact of these mutations and, thus, the role of these genes in the tolerance of the stress. While testing all candidates will not be feasible, the rate of genes relevant to the trait under investigation among tested candidates will represent an estimate of the prediction performance.\n\n\nConclusions\n\nWith the availability of multiple genome sequence and RNA-seq data sets, it is now possible to combine comparative evolutionary analyses, in our case protein family expansion, with differential expression to predict genes involved in adaptive traits. However, running the required bioinformatics analyses and data integration tasks as well as summarizing and visualizing the results, remains challenging. A2TEA only requires standard data files as input, follows best practice software standards for both reproducibility and portability, and provides a user-friendly web application for interactive exploration and selection of the most promising candidate genes. We show that genes known to be involved in stress tolerance can be detected in datasets of stress-tolerant and stress-sensitive plants, but we expect A2TEA to be useful in a broader scope when analyzing protein families and their expression in multiple genomes as the parameters for selecting interesting families are very flexible. A2TEA follows a positive trend in modern research software development that provides easy installation and execution through the use of container and workflow technologies as well as interactive visualization and exploration tools for the generated results. Combined, this facilitates better reproducibility, communication, and shareability of comprehensive analyses.\n\n\nData availability\n\nPoaceae test data:\n\nTranscriptomics:\n\nHordeum vulgare: SRR6782243, SRR6782247, SRR6782257, SRR6782249, SRR6782250, SRR6782254\n\nZea mays: SRR2043219, SRR2043217, SRR2043190, SRR2043220, SRR2043226, SRR2043227\n\nOryza sativa japonica: SRR5134063, SRR5134064, SRR5134065, SRR5134066\n\nThese correspond to the following studies relating on drought stress:\n\nHordeum vulgare: https://doi.org/10.1186/s12864-019-5634-0\n\nZea mays: https://doi.org/10.1104/pp.16.01045\n\nOryza sativa japonica: https://doi.org/10.3389/fpls.2017.00580\n\nAssemblies & annotations hosted on EnsemblPlants:\n\nHordeum vulgare: cDNA FASTA, GTF, Peptide FASTA\n\nZea mays: Genome FASTA, GTF, Peptide FASTA\n\nOryza sativa japonica: cDNA FASTA, GTF, Peptide FASTA\n\nAn archived version of the complete grasses test data (reduced as used in the examples) is deposited here:\n\nhttps://zenodo.org/record/708902248\n\nData used in the Brassicaceae example:\n\nTranscriptomics:\n\nEutrema salsugineum: SRR7624684, SRR7624685, SRR7624692, SRR7624687, SRR7624721, SRR7624722\n\nArabidopsis lyrata: SRR7624680, SRR7624702, SRR7624703, SRR7624732, SRR7624733, SRR7624742\n\nArabidopsis thaliana: SRR7624694, SRR7624696, SRR7624697, SRR7624710, SRR7624714, SRR7624723\n\nBrassica napus: SRR12429701, SRR12429702, SRR12429703, SRR12429698, SRR12429699, SRR12429700\n\nThese correspond to the following studies on drought stress response: Eutrema salsugineum, Arabidopsis lyrata, Arabidopsis thaliana: https://doi.org/10.1111/nph.15841\n\nBrassica napus: PRJNA656507\n\nAssemblies & annotations hosted on EnsemblPlants:\n\nEutrema salsugineum: Genome FASTA, GTF, Peptide FASTA\n\nArabidopsis lyrata; Genome FASTA, GTF, Peptide FASTA\n\nArabidopsis thaliana: Genome FASTA, GTF, Peptide FASTA\n\nBrassica napus: Genome FASTA, GTF, Peptide FASTA\n\nThe results generated by the A2TEA.Workflow which are also used for demonstrating the A2TEA.WebApp’s functionality presented in this work are available at https://zenodo.org/record/708960849 and https://zenodo.org/record/7089606.50\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\nBoth the A2TEA.Workflow and the A2TEA.WebApp are available as MIT licensed open source softwares.\n\n• Software available from: https://tgstoecker.github.io/A2TEA.WebApp\n\n• Source code available from: https://github.com/tgstoecker/A2TEA.Workflow, https://github.com/tgstoecker/A2TEA.WebApp\n\n• Archived source code at time of publication: https://zenodo.org/record/7086290,51 https://zenodo.org/record/708628252\n\n• License: MIT", "appendix": "References\n\nOhno S: Evolution by gene duplication. 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Publisher Full Text\n\nMurat F, Van de Peer Y, Salse J: Decoding plant and animal genome plasticity from differential paleo-evolutionary patterns and processes. Genome Biol. Evol. 2012; 4(9): 917–928. PubMed Abstract \| Publisher Full Text\n\nDemuth JP, De Bie T, Stajich JE, et al.: The evolution of mammalian gene families. PLoS One. 2006; 1(1): e85. PubMed Abstract \| Publisher Full Text\n\nKim E, Magen A, Ast G: Different levels of alternative splicing among eukaryotes. Nucleic Acids Res. 2007; 35(1): 125–131. PubMed Abstract \| Publisher Full Text\n\nMarcon C, Altrogge L, Win YN, et al.: Bonnmu: a sequence-indexed resource of transposon-induced maize mutations for functional genomics studies. Plant Physiol. 2020; 184(2): 620–631. PubMed Abstract \| Publisher Full Text\n\nStöcker T: A2TEA.Workflow test data (v1.0.0) [Data set]. Zenodo.2022. Publisher Full Text\n\nStöcker T: A2TEA.Workflow Poaceae reduced example data (v1.0.0) [Data set]. Zenodo.2022. Publisher Full Text\n\nStöcker T: A2TEA Brassicaceae example data (v.1.0.0) [Data set]. Zenodo.2022. Publisher Full Text\n\nStöcker T: tgstoecker/A2TEA.Workflow: First release (v1.0.0). Zenodo.2022. Publisher Full Text\n\nStöcker T: tgstoecker/A2TEA.WebApp: v1.0.0 (v1.0.0). Zenodo.2022. Publisher Full Text" }	[ { "id": "154237", "date": "25 Nov 2022", "name": "Manuel Aranda", "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\nGeneral comments:\nThe manuscript submitted by Stöcker et et al. describes an integrated pipeline for the identification of genes potentially involved in adaptation by analyzing gene duplications and gene family expansions. The pipeline combines RNA-Seq expression analyses via DESeq2, identification and phylogenetic analysis of orthologous groups with OrthoFinder, statistical analyses of gene family changes taking into account phylogeny inference with Cafe5, and gene ontology enrichment analyses with TopGO. The results of the pipeline can be visualized using the R shiny package, which allows for easy exploration of the results even for experimentalists with little bioinformatic background. A2TEA is a simple yet useful tool for the visualization and integration of multiple analyses and datasets. A few things need to be revised in the manuscript, such as a corrupted Figure 2, repeated paragraphs, and the addition of missing references. The implementation of a “Download All Results” option in the general tab of the shiny app would be helpful. Allowing the pipeline to run without the addition of RNA-Seq could be quite useful to allow analyses of species without available RNA-Seq data from the same condition. Additionally, it would be appropriate to put an emphasis on the broader use of the app instead of focusing on plant stress, as it would make the app more appealing to a broader audience.\nSpecific comments\nAbstract: “It functions as a one-stop processing pipeline, integrating protein family, phylogeny, expression, and protein function analysis”. Change analysis to analyses.\n\nAbstract: “The pipeline is accompanied by an R Shiny web application that”. Jump in line, fix editing.\n\nIntroduction: “Most of the duplicates are lost or silenced….”. Maybe change it to something like: Most of the gene duplicates are lost or silenced, but retained duplicates may hint at some evolutionary advantage, which may be targets of adaptation.\n\nIntroduction: The first paragraph of the introduction needs some work. It is unclear if the authors are explaining the different approaches that can be used to identify genes related to adaptation or if they are referring to their pipeline, as they use “we” and “us”. It would be appropriate to first describe the different approaches and their benefits and then the need to combine them. The second and third paragraphs already properly address what the pipeline is doing.\n\nIntroduction: Change & for and at: “working in tandem to automate and ease all bioinformatics & analysis tasks involved.”\n\nIntroduction: Change “gene families in form of” to gene families in the form of…\n\nFigure 1 (and the whole article). While the tool was designed for the identification of genes for crop improvement, it is not limited to that application. As the name of the application indicates, it is designed for trait-specific evolutionary adaptations. It might be a good idea to generalize the possible applications of this tool rather than focus only on plant stress-specific responses. The authors can specify that an example of its use is in crop improvement while making sure to maintain a general tone for the use of A2TEA for a broader audience.\n\nCite DIAMOND, DESeq2, BLAST.\n\nCite Cafe in the main text (only found in the reference list).\n\nThis paragraph is repeated: “The A2TEA.WebApp is written in the R programming language and uses the Shiny framework to facilitate interactivity with the data. It expects the user to upload a .RData file created by the A2TEA.Workflow. The web application comes with a test dataset that can be loaded with a single click so interested users can try out its functionality before having to finish an A2TEA.Workflow run.”\n\nFigure 2 is full of typos like orthoander instead of orthofinder and many of the boxes don’t have text but empty squares, probably an error while formatting.\n\nFigure 3. Italicize Eutrema salsugineum.\n\nFigure 4. Increase the resolution or the font size, as words in panels B, C, and D are blurry.\n\nComments on the A2TEA Shiny App.\nGeneral tab: Differential expression and Functional annotation require a “Download Full Results” button and not only a download current page.\n\nGO term analyses tab: At GO term analyses, changing the algorithm does not change the column name at GO term set choices. It always says classicFisher. Same in enrichment plots. Also, in the enrichment plots, it might be better to plot the cut-off lines after plotting the terms, as sometimes the terms are small due to a low number of annotated genes belonging to them and might be covered by the line. Changing the x-axis label for the bar plot to the number of significant orthogroups might be better.\n\nDiscussion: “We propose that we can identify novel genes relevant for stress adaptation by comparing same-stress experiments of several plant species with different levels of stress adaptation in combination with evolutionary footprints in the form of protein family expansion”. Again, it would be better to keep the usability of this tool as general as possible, not only focusing on plat stress, as it could be used for any species and conditions. Also, using the word condition or treatment instead of stress would be better.\n\nAdditional comments. It could be beneficial if the App could also be run without RNA-Seq data for the development of a general hypothesis when comparing species for which RNA-Seq data under the same conditions is not available.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "9505", "date": "12 Apr 2023", "name": "Tyll Stöcker", "role": "Author Response", "response": "We thank all reviewers for providing valuable input to the article that helped us to improve it further. We have tried to address their comments and made efforts to incorporate their insightful criticisms and suggestions. Similar suggestions and criticisms have been grouped together in order to address them concisely. Abstract: “It functions as a one-stop processing pipeline, integrating protein family, phylogeny, expression, and protein function analysis”. Change analysis to analyses. Abstract: “The pipeline is accompanied by an R Shiny web application that”. Jump in line, fix editing. Introduction: “Most of the duplicates are lost or silenced….”. Maybe change it to something like: Most of the gene duplicates are lost or silenced, but retained duplicates may hint at some evolutionary advantage, which may be targets of adaptation. Introduction: Change & for and at: “working in tandem to automate and ease all bioinformatics & analysis tasks involved.” Introduction: Change “gene families in form of” to gene families in the form of… We thank the reviewer very much for taking the time and point out several minor formatting and phrasing errors, all of which we have corrected. Introduction: The first paragraph of the introduction needs some work. It is unclear if the authors are explaining the different approaches that can be used to identify genes related to adaptation or if they are referring to their pipeline, as they use “we” and “us”. It would be appropriate to first describe the different approaches and their benefits and then the need to combine them. The second and third paragraphs already properly address what the pipeline is doing. On re-reading the first paragraph of the introduction with the reviewer's comments in mind, we strongly agreed that a more logical separation was necessary. We have restructured and rewritten parts of it to more clearly separate the prior approaches and then – in a second step – the need to combine them as we do with our software. Cite DIAMOND, DESeq2, BLAST. Cite Cafe in the main text (only found in the reference list). We thank the reviewer for pointing out important missing citations in our text and have added these as well as additional ones. We have repositioned our citation of CAFE5 immediately next to the software’s name in the text (prior it was at the end of the sentence). This paragraph is repeated: “The A2TEA.WebApp is written in the R programming language and uses the Shiny framework to facilitate interactivity with the data. It expects the user to upload a .RData file created by the A2TEA.Workflow. The web application comes with a test dataset that can be loaded with a single click so interested users can try out its functionality before having to finish an A2TEA.Workflow run.” We thank the reviewer very much for pointing out this mistake that occurred during our editing process. Figure 2 is full of typos like orthoander instead of orthofinder and many of the boxes don’t have text but empty squares, probably an error while formatting. Unsure of the cause, we have uploaded the figure in a different format which seems to have solved the garbled text. Figure 3. Italicize Eutrema salsugineum. We italicized this species in the figure text and took care to check the manuscript for similar formatting flaws. Figure 4. Increase the resolution or the font size, as words in panels B, C, and D are blurry. We re-exported the image in higher resolution to increase the overall legibility. Comments on the A2TEA Shiny App. General tab: Differential expression and Functional annotation require a “Download Full Results” button and not only a download current page. The reviewer was correct in pointing out the need for this feature. For all tables, we have now implemented the possibility of downloading all results and not just the displayed page via the suggested buttons. If the user did not perform any filtering, the complete table is downloaded; if the user performs filter operations (e.g. \| log2FC \| > 1) the complete, filtered table is downloaded. GO term analyses tab: At GO term analyses, changing the algorithm does not change the column name at GO term set choices. It always says classicFisher. Same in enrichment plots. Also, in the enrichment plots, it might be better to plot the cut-off lines after plotting the terms, as sometimes the terms are small due to a low number of annotated genes belonging to them and might be covered by the line. Changing the x-axis label for the bar plot to the number of significant orthogroups might be better. The choice of the topGO algorithm concerns how the GO graph is analyzed (independence vs. consideration parent terms, etc.) and is a separate option from the downstream test statistic (https://bioconductor.org/packages/release/bioc/vignettes/topGO/inst/doc/topGO.pdf). In the current release, we only allow classicFisher as the test statistic. As such, the column does not need to change in our opinion. However, we agree that information of the used algorithm should be kept. We have now implemented that the name of the algorithm is part of the output tables that the user is able to download. We have changed the order of the plotting in the enrichment plot – now, the lines plot behind the terms, which ensures clearer visualizations. We have also adapted the reviewer's suggestion for a better x-axis label in the enrichment bar plot. Figure 1 (and the whole article). While the tool was designed for the identification of genes for crop improvement, it is not limited to that application. As the name of the application indicates, it is designed for trait-specific evolutionary adaptations. It might be a good idea to generalize the possible applications of this tool rather than focus only on plant stress-specific responses. The authors can specify that an example of its use is in crop improvement while making sure to maintain a general tone for the use of A2TEA for a broader audience. Discussion: “We propose that we can identify novel genes relevant for stress adaptation by comparing same-stress experiments of several plant species with different levels of stress adaptation in combination with evolutionary footprints in the form of protein family expansion”. Again, it would be better to keep the usability of this tool as general as possible, not only focusing on plant stress, as it could be used for any species and conditions. Also, using the word condition or treatment instead of stress would be better. We agree with the reviewer that the tone and focus of our manuscript can be somewhat loosened to emphasize the general technical feasibility of applying our approach to research outside of the plant kingdom. We have 1. altered parts of the manuscript to incorporate the words treatment/condition instead of the word stress (to underline broader context applicability) and 2. added an additional sentence to the end of the first paragraph emphasizing the general technical applicability in other organisms than plants. However, as we point out in two paragraphs near the end of the discussion, our particular approach stems from the support of previous research that genome duplication was a decisive factor in the evolutionary history of plants and much of the phenotypic variation in land plants may have arisen primarily due to duplication and adaptive specialization of already existing genes. Since our method specifically focuses on the analysis of protein family expansion events, it might be much less applicable in other groups of species where other evolutionary processes, such as alternative splicing or pathway/network rewiring, could play a proportionally much more important role. These considerations made us specifically target the \"Plant Computational and Quantitative Genomics\" collection because the feasibility of our software for research in other kingdoms is unclear. It could be beneficial if the App could also be run without RNA-Seq data for the development of a general hypothesis when comparing species for which RNA-Seq data under the same conditions is not available. We agree with the reviewer's notion of not requiring RNA-seq data for all species in a workflow run. The latest release of the A2TEA.Workflow and WebApp now allow for this. The README of the Workflow explains that if the user does not possess transcriptomic data for a species, they can leave the genomic/cDNA fasta and annotation positions in the species.tsv file empty. We updated the WebApp to handle such cases of missing data. Therefore our whole pipeline has become considerably more flexible." } ] }, { "id": "156575", "date": "01 Feb 2023", "name": "Rahul Siddharthan", "expertise": [ "Reviewer Expertise Computational biology: regulatory genomics", "chromatin", "algorithms" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe software tool A2TEA is well described and motivated. The idea of identifying genes involve in adaptive traits, such as stress-tolerance, using inter-species comparisons, is sound. A2TEA integrates the bioinformatics (RNA-seq, orthologous group computation, functional information inference, etc.\nI do not work in this field so have not tested out the tool. The webapp looks well laid out and user-friendly but I did not try it on actual data.\nSeveral boxes in the flowchart in figure 2 have garbled text (text replaced with boxes). I also suggest highlighting a couple of possible paths through this figure in an actual workflow (eg, a single workflow would not use both kallisto and STAR?).\nMinor comment: one block of text, starting with \"The A2TEA.WebApp is written in the R programming language...\" (two paragraphs) is repeated (as a single paragraph immediately after).\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "9506", "date": "12 Apr 2023", "name": "Tyll Stöcker", "role": "Author Response", "response": "We thank all reviewers for providing valuable input to the article that helped us to improve it further. We have tried to address their comments and made efforts to incorporate their insightful criticisms and suggestions. Similar suggestions and criticisms have been grouped together in order to address them concisely. Several boxes in the flowchart in figure 2 have garbled text (text replaced with boxes). I also suggest highlighting a couple of possible paths through this figure in an actual workflow (eg, a single workflow would not use both kallisto and STAR?). We have made sure that the figure is now without the noted garbled text. Regarding the paths through the diagram: The reviewer is correct that in a normal differential expression analysis one would not choose to process some of the samples with one tool and the rest with another. However, in our field of plant/crop research, we have encountered many cases of early assembly and annotation versions, with many cases of only a transcriptome or a genome assembly being available. Due to this, the workflow does support running one species through a pseudoalignment and another through classic alignment-based quantification to provide more flexibility when combining data from different sources. In our Workflow README, we point out that for runtime and resource purposes, we recommend kallisto/pseudoalignment, and it should always be preferred if possible. Minor comment: one block of text, starting with \"The A2TEA.WebApp is written in the R programming language...\" (two paragraphs) is repeated (as a single paragraph immediately after). We thank the reviewer very much for pointing out this mistake that occurred during our editing process." } ] } ]	1	https://f1000research.com/articles/11-1137
https://f1000research.com/articles/11-1133/v2	26 Jan 23	{ "type": "Research Article", "title": "Evaluation of antimicrobial susceptibility of Escherichia coli isolated from contaminated areas of Majengo slum in Meru County, Kenya", "authors": [ "Jared Ombuya", "Kennedy Gachoka", "Kagendo Dorothy", "Naomi Mutea", "Kennedy Gachoka", "Kagendo Dorothy", "Naomi Mutea" ], "abstract": "Background: Antimicrobial drug resistance is of great concern today. Infections by the antimicrobial resistant strains of Escherichia coli, including enteropathogenic as well as enterotoxigenic strains have been reported as a major cause of deaths, especially among young children in low- and middle-income countries. This has been augmented by antimicrobial misuse, over the counter availability and poor sanitation especially in low income areas. This study aimed at characterizing antimicrobial resistant strains of Escherichia coli isolated from sanitation environments of the Majengo slum in Meru County, Kenya Methods: A cross-sectional study was conducted on 61 samples from soil, water and drains swabs. These were tested against five antimicrobial drugs by the Kirby disk diffusion method. Results: A total of 42 (69%) of the samples had Escherichia coli. These recorded antimicrobial drug susceptibility as follows: Out of the five antimicrobial agents used, ceftazidime 28 (66.67%) showed the highest sensitivity followed by ciprofloxacin 26 (61.90%) and imepenem 25 (59.52%) respectively. cefotaxime and cefoxitin showed least sensitivity at 14 (33.33%) and 13 (30.95%) respectively. In intermediate imepenem and ciprofloxacin were the highest with 12 (28.57%) followed by cefotaxime 10 (23.81%). The least intermediate was observed in ceftazidime and cefoxitin both at 7 (16.67%). The highest resistance was observed in cefoxitin 22 (52.38%), followed by cefotaxime at 18 (42.86%). Ciprofloxacin, imepenem and ceftazidime had the lowest resistance 4 (9.52%), 5 (11.91%) and 7 (16.67%) respectively. The p-value <0.05 was considered significant to the study.\n\nConclusions: This study showed that Escherichia coli isolated from Majengo is pathogenic and resistant to antibiotics. Detection of Escherichia coli poses a great risk in the spread of resistant strains in human. Proper sanitation and hygiene awareness practices should be provided through education to the residents of this area.", "keywords": [ "E. coli", "Susceptibility Testing", "Antimicrobial Resistance", "Multidrug Resistance", "Ciprofloxacin", "Ceftazidime", "Cefotaxime", "Imepenem", "Cefoxitin" ], "content": "Introduction\n\nInfectious organisms are currently the major cause of diseases worldwide. A number of newly recognized pathogens and strains are now emerging. These organisms have resulted in high morbidity and mortality globally.1 The emergence of these pathogens has been attributed to microbial evolution, high mutations as the organisms try surviving in different environments as well as creation of new environments. This microbial evolution and high mutations has resulted to drug resistance, especially due to deposition of drug residues released in different environments, and more especially in areas with poor drainages and poor sanitation systems. As a result, there has evolved a need for surveillance and monitoring systems with emphasis on sanitation and water management. This will help curve the spread of emerging and re-emerging infectious diseases.\n\nHuman population growth currently has faced great challenges in accessing proper quality and quantity water resources. This has led to an increase in the number of water borne infectious diseases recently.2 An estimated 30% of the bacterial population has been reported to be emerging because of wastewater, agricultural practices, and poor sanitation systems. Water management could act as a barrier to prevent the spread of pathogens. Prevention of pathogens could reduce environmental contamination, antibiotic misuse and eventually mutation of microorganisms.\n\nIn different environmental set ups, studies have indicated increased multidrug resistance strains which has led to increased mortality and morbidity resulting from exposure to infection causing organisms. Improved sanitation facilities is one of the most important interventions needed in order to stop the spread of resistant bacteria.3 A study on typhoid among young children,4 stressed the need for careful monitoring of antimicrobial resistance in a view to prevent increase of resistance strains and resultant infections to vulnerable communities.\n\nDiarrheagenic E. coli has demonstrated a significant resistance to beta lactams antibiotics that are commonly prescribed,5 with contributing factors to diarrhea cases attributed to poor quality of foods, poor water systems, lack of proper hygiene often due to lack of water and poor sanitation. Studies have shown that human and animal waste are the main sources of contamination on the environment that hosts strains that are antimicrobial resistance.6 The study indicated that contamination resulted in formation of biofilms that supported bacterial resistance.\n\n\nMethods\n\nApproval to carry out the study was done by Meru University of Science and Technology MIRERC (Meru University Institutional Research and Ethics Review Committee). Approval Ref NO: MU/1/39/28 VOL.2(31). Date 17th February 2022.\n\nThe study was conducted in the Majengo area, an informal settlement in Meru County within the Eastern region of Kenya. As with other slum areas, Majengo has congested households with poor sanitation and limited access to safe toilet facilities. It is located at Imenti North sub-county and Ntima west ward. River Kathita passes along the slum.\n\nThe study employed a cross-sectional study design with an aspect of laboratory analysis. Environmental samples were collected from water collection points, soils near latrines, and open drains in the Majengo area. Samples that were collected included water, soils, and swabs of open drains. The samples were then transported to the laboratory for analysis. The study did not involve households or interactions with residents of the Majengo area.\n\nTo identify sites for sample collection, the study utilized a stratified sampling scheme, where the Majengo area was stratified by its constituent villages using available maps. In each village, areas of sampling were randomly selected based on availability of the water collection points, soils near latrines, and open drains. The number of sampled areas or type of samples in each village was dependent on accessibility and spatial/temporal distribution of the sample types. The sampled areas were marked using Google map to pin sample collection points. Figure 1 shows sampled areas in the Majengo slum. The area was divided into two strata A and B as shown on the map.\n\nTo determine the required sample size, the following formula was used at a 95% confidence interval.7\n\nA 500 ml river water sample was collected into 500 ml sterile bottles by laying the bottle in water facing upstream. Tap water was collected into sterile 500 ml bottles. To do this, the individual tap was sterilized using a flamed alcohol cotton swab, a little water was allowed to flow, before getting collected into the sterile sample bottle. Approximately 10 g of soil samples were collected near toilets and dumping areas using a sterile spoon. In the swab collection, the cover was first untwisted to remove the swab and the sample collection was done by moving the swab in a clockwise circular motion. The swab was then returned aseptically into the vial containing transport media. It was labelled and packed in a cooler box containing an ice pack for transportation to the laboratory for analysis.\n\nSoil sample\n\nA sample of soil weighing 1g was dissolved in sterile distilled water in a 250 ml conical flask.8 A serial dilution was performed up to 105 for E. coli all dilutions were cultured into MacConkey agar (oxoid CM007) and incubated (Biobase) China at 37°C for 24 hours using microbiological standards of culturing. After this duration pink colonies of target organisms were sub cultured in Tryptone Bile Glucuronide (TBX) CM 0964 (Techno Pharmchem, India) to detect E. coli. Confirmation of E. coli was done on indole test.\n\nDrain swabs\n\nIn the laboratory, drain swabs samples were cultured in MacConkey agar (Oxoid CM007) to isolate E. coli. The media was incubated at 37°C for 24 hours. After 24 hours’ growth pink colonies of E. coli in MacConkey agar (Oxoid CM007) were sub cultured in TBX to detect E. coli. Growth of green colonies was an indicator for the presence of E. coli.\n\nThe most probable number method was used to analyze water samples using MacConkey broth (Oxoid CM0505CM 0505) UK. Each bottle with sample was appropriately labelled. The water was mixed thoroughly by inverting the bottle several times. The cap of the bottle was removed and the mouth of the bottle flamed. The water samples were then inoculated by arranging the bottles in an incubator independently inside the incubator grill.9 After inoculation the bottles were incubated at 44°C for 24 hours and the samples were examined by observing the color change and gas formation. Table 1 shows water analysis set for untreated and treated water samples.\n\nIn the laboratory, samples were cultured in MacConkey agar to isolate E. coli. The cultures were incubated at 37°C for 24 hours. After 24 hours’ growth of target organisms in MacConkey agar were sub cultured in TBX to detect E. coli. Growth of green colonies was an indicator for the presence of E. coli.\n\nIsolates were subjected to biochemical identification using the indole test for E. coli. The test organism was inoculated in a bijou bottle containing 3 ml Tryptone water (Oxoid CM0087) UK and incubated at 37°C for 48 hours. The test for indole was done by adding one drop of Kovac’s reagent (Himedia) and the formation of a red ring within 10 minutes indicated presence of E. coli.\n\nGram staining is a technique used in differentiation of gram-positive and gram-negative bacteria. The technique is important since it helps in selecting the correct antibiotic during microbial sensitivity. A smear was prepared from the colonies to confirm if it was gram-negative rods using gram staining procedure. The procedure required gram stain reagents, slides, oil immersion and a microscope.\n\nColonies of the test organism were emulsified on a glass slide using a wire loop. A drop of normal saline was added to make a smear. The slide was allowed to air dry. The slides were passed through a flame to fix them. The smear was covered with crystal violet solution for 30 seconds. Then the smear was rapidly washed off to remove the excess stain. The smear was covered with Gram’s Iodine (Pro-Lab Diagnostics Netherland) for 30 seconds and washed off using tap water. The smear was decolorized by covering with acetone for a few seconds and washed off. The smear was then covered with a counter stain neutral red for 30 seconds. The smear was allowed to dry before being examined at power ×100 on a microscope (Olympus CX22). Presence of gram-negative rods confirmed the organism.\n\nAntimicrobial susceptibility testing was done using Mueller-Hinton Agar. Before inoculation of the organism to the Mueller-Hinton (CMO337 Oxoid) UK, single colonies were used to make 0.5 ml McFarland standard.10,11 Colony suspension was first prepared by picking single colonies and suspending them in a vial containing 1ml normal saline. The suspension was compared with McFarland’s standard in order to obtain the required concentration of the isolate. Using a sterile cotton swab, the isolate was picked and spread evenly on Mueller-Hinton agar. Once all the Mueller-Hinton agar was completely inoculated by suspension, antimicrobial discs were inserted on the swabbed Mueller-Hinton agar.12 The discs were well spaced to prevent them from overlapping. They were pressed on the media to ensure complete contact. They were incubated for 24 hours at 37°C. After 24 hours of incubation, the susceptibility and resistance was determined by measuring the diameter of the zone of inhibition using Vernier Calipers. The measurement was recorded in millimeters. This enabled classification of isolates of E. coli as either sensitive, intermediate, or resistant.11,13 ATCC 25922 E. coli was used as a control organism during the study. The microbial agents used were as follows in their corresponding concentrations; imepenem (10 μg); ceftazidime (30 μg); cefotaxime (30 μg); cefoxitin (30 μg) and ciprofloxacin (5 μg).\n\nLaboratory data was entered into Microsoft Excel v 2010 and analyzed using SPSS version 26.0. Descriptive analysis was done using Microsoft Excel. The analysis included, frequencies and graphs. Data was compared between each study strata and between each sample type using Kruskal-Wallis tests, between the two drugs using the Wilcoxon Signed Rank test and findings were presented as figures tables and graphs. A p-value of 0.05 or less was considered significant between comparison in susceptibility and resistance between the strata A and B.\n\n\nResults\n\nA total of 42 isolates were tested for antimicrobial susceptibility in both strata. Out of these antimicrobials, 5 (50.48%) were susceptible, 5 (22.86%) were intermediate and 5 (26.67%) were resistant. Out of the five antimicrobial agents used, ceftazidime 28 (66.67%) showed the highest sensitivity followed by ciprofloxacin 26 (61.90%) and imepenem 25 (59.52%) respectively. cefotaxime and cefoxitin showed least sensitivity at 14 (33.33%) and 13 (30.95%) respectively. In intermediate imepenem and ciprofloxacin were the highest with 12 (28.57%) followed by cefotaxime 10 (23.81%). The least intermediate was observed in ceftazidime and cefoxitin both at 7 (16.67%). The highest resistance was observed in cefoxitin 22 (52.38%), followed by cefotaxime at 18 (42.86%). ciprofloxacin, imepenem and ceftazidime had the lowest resistance 4 (9.52%), 5 (11.91%) and 7 (16.67%) respectively. Figure 2 shows a bar plot of the percentages of susceptibility patterns of different drugs under classes of response to E. coli.\n\nIMP-imipenem; CAZ-ceftazidime; CXT-cefoxitin; CTX-cefotaxime; CIP-ciprofloxacin.\n\nA total of 16 isolates from drain swabs were tested for antimicrobial susceptibility. Out of these antimicrobials, 5 (61.25%) were susceptible to the microbial, 5 (23.75%) were intermediate and 5 (15%) were resistant. The highest resistance was shown to be cefotaxime 6 (33.34%), followed by cefoxitin at 4 (23.34%), while imipenem and ceftazidime were the least resistant at 2 (10%) respectively. ciprofloxacin was 0% resistant to the isolates tested. Table 2 shows antimicrobial susceptibility patterns from drain swabs in both strata. Out of the five drugs ceftazidime showed the highest susceptibility at 13 (85%) while imipenem followed at 13 (81.67%) respectively. ciprofloxacin was the highest in intermediate at 8 (46.67%) while imipenem and cefoxitin were the least intermediate at 2 (13.34%). Figure 3 shows antimicrobial susceptibility pattern from drain swabs.\n\nIMP-imipenem; CAZ-ceftazidime; CXT-cefoxitin; CTX-cefotaxime; CIP-ciprofloxacin.\n\nA total of 23 samples of soil were tested for antimicrobial sensitivity using diffusion disc method of the five antimicrobials of which imepenem resistant were 4 (17.39%), 8 (34.78%) intermediate while 11 (48.83%) susceptible. Out of the five antimicrobials used in this study in ceftazidime resistant were 6 (26.09%), intermediate 5 (21.74%), susceptibility 12 (52.17%), cefoxitin resistant were 15 (65.22%), intermediate 5 (21.71%), susceptible 3 (13.04%), cefotaxime resistant were 9 (9.13%), intermediate 5 (21.74%), susceptible 9 (39.13%) and ciprofloxacin resistant were 4 (17.39%), intermediate 4 (17.39%), susceptible 15 (65. 22%). Table 3 shows E. coli antimicrobials susceptibility drug patterns from soil samples in both strata. In both strata ciprofloxacin showed the highest susceptibility at 65.22%, followed by ceftazidime at 52.17%, while cefoxitin showed the least susceptibility. Imepenem showed the highest intermediate of all the drugs. Cefoxitin showed the highest resistance. Figure 4 shows the comparison in susceptibility patterns of the samples.\n\nIMP-imipenem; CAZ-ceftazidime; CXT-cefoxitin; CTX-cefotaxime; CIP-ciprofloxacin.\n\nIn the first strata, imepenem at 9 (77.78%) showed the highest susceptibility, followed by ciprofloxacin at 6 (66.67%) and cefoxitin was the least sensitive at 22.22%. Ceftazidime showed the highest intermediate at 33.33% while cefoxitin and cefotaxime were least intermediate. Cefoxitin showed the highest resistance while imepenem showed no resistant. In strata two ciprofloxacin 64.29% showed the highest susceptibility, followed by ceftazidime 50%. Cefoxitin showed highest resistance at 64.29%, followed by cefotaxime at 42.86%. Imepenem showed the highest intermediate of all the drugs.\n\nOut of the three water samples analyzed it was observed that, ceftazidime and Ciprofloxacin showed 100% sensitivity while imipenem showed the least sensitivity Figure 5 shows the comparisons in % between the drugs. The highest resistance was observed in both cefoxitin and cefotaxime at 100%. Imipenem was the most intermediate at 66.67%. Table 4 shows E. coli antimicrobials susceptibility drug patterns from water samples from strata 1 and 2.\n\nIMP-imipenem; CAZ-ceftazidime; CXT-cefoxitin; CTX-cefotaxime; CIP-ciprofloxacin.\n\nThe resistance from this study was statistically significant since the p value=0.0005275354994284686 and the statistic=19.87938408896494. The p-value is less than 0.05, and so we reject the null hypothesis. This means that the resistance is not the same across the different drugs. To know which drug is more resistant, the Wilcoxon Signed-Rank test was used. This study found that cefoxitin and cefotaxime are statistically significant with a p-value 0.0156 which means they have highest resistance among the rest of the drugs. Table 5 shows p-values between compared drugs.\n\nImipenem, ceftazidime and ciprofloxacin are not significant to this study since the p value is greater than p>0.05. This p-value correlates to what was found in a study conducted by14 who recorded a p-value of 0.36 towards E. coli.\n\nStatistical analysis summary using plot box. Figure 6 shows a box plot showing summary of descriptive statistics of the means and standard errors of the antimicrobials.\n\nSensitivity from this study was significant to the study since the p value=0.01171770477419787 between the strata. This means that the sensitivity was not the same across the different drugs. To know which drug is more sensitive, the Wilcoxon Signed-Rank test was performed. It was observed that, ciprofloxacin, ceftazidime and imipenem were the most sensitive drug with a p-value>0.05.\n\nTable 7 shows the p-values of the antimicrobials in the study.\n\n\nDiscussion\n\nThis study reported a range of multidrug resistance to E. coli from all the samples analyzed. Therefore, there is high chance that E. coli isolated from this study area emerges from the surrounding poor unhygienic conditions and inadequate sanitation facilities from the residents. This findings agrees with a study on contamination of street food in Burkina Faso that enteric organisms emerge from cross contamination.15 The same observation was recorded by.16 A study conducted on antimicrobial profile in Juja and Kibera found cefotaxime, ceftazidime and ciprofloxacin recorded a greater than 30% of antimicrobial resistance17 this result is much lower than the one reported in the current study that recorded 25% increase. A study conducted by18 observed that this increase in resistance is due to use in antibiotics in the treatment of diseases associated with poor unhygienic conditions. This study found the highest resistance on soil where it was it was noted that cefoxitin 65.22% an indication that soil was contaminated in this area. These findings correlate with study on urban informal settlement on antimicrobial resistance on the environment. It was noted from this study that less attention is being given on this contamination and pose a great risk to antimicrobial resistance.19 Another study noted that soil is a hotspot carrier of resistant genes.20\n\nThe current study recorded 34% towards cefotaxime, imepenem10% and zero resistance to Ciprofloxacin. In a similar study,21 recorded 0% resistance to Ciprofloxacin and 1.68% to imipenem and 8.94% cefotaxime. This finding on cefotaxime and imepenem is much lower than the ones from the current study. However, both studies recorded 0% to ciprofloxacin. According to Ref. 22 multidrug resistance to E. coli was noted on cefotaxime at 79.7% resistance. A related study23 recorded 93% resistance to E. coli. The current study recorded a lower figure of 42.86% resistance to cefotaxime.\n\nThe current study showed the least resistance towards ceftazidime (16.67%) which is much lower that the resistance obtained from24 100%.\n\nThe current study showed 100% resistance to water samples analyzed during the study. This resistance is a likely indication that there is high contamination of wastewater that is in return used for home use by the residents. The current study only focused on river water samples passing through the study area and not water samples from the source area. This calls for a study in order to determine if there is contamination. The findings from the current study was higher by 20% from a related study conducted by Ref. 25 on antidrug resistance on Indian rivers. A study done by Ref. 26 while investigating resistance from water found that ceftazidime and ciprofloxacin had almost the same resistance; 1.7% and 1.8% respectively. These findings are higher than the current study which recorded 0% resistance on the drugs. According to Ref. 27, E. coli in wastewater plants was 60% resistant towards ciprofloxacin, which was higher than the current study at 0% resistance.\n\n\nConclusion\n\nAntimicrobial resistance noted from this study will result in an increase of infections caused by treatment failure hence, high mortality rates.\n\nProper sanitation and hygiene awareness practices should be provided through education to the residents of this area. In the future, molecular methods should be used to look in more detail at the resistance genotype of the isolate from the study.\n\n\nAuthor’s contribution\n\nJO: Developed the concept, wrote the project proposal, collected the research data, analyzed the data, and wrote the thesis.\n\nKG: Corrected the concept, provided necessary guidance, and corrections at the proposal writing, data analysis, and thesis writing.\n\nNM: Corrected the concept, provided necessary guidance, and corrections at the proposal writing, data analysis, and thesis writing\n\n\nData availability\n\nFigshare: EVALUATION OF ANTIMICROBIAL SUSCEPTIBILITY OF ESCHERICHIA COLI ISOLATED FROM CONTAMINATED AREAS OF MAJENGO SLUM IN MERU COUNTY, KENYA. https://doi.org/10.6084/m9.figshare.20325345.28 This project contains the following underlying data:\n\n• my data.xlsx (The data from this study represents data using measures of central tendency and the findings in percentages and bar graphs and statistical analysis involved using Kruskal Wallis and Wilcoxon from SPSS software.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nMany thanks to almighty God for enabling me throughout in doing this work. I thank everyone who supported me throughout this journey. In a special way I would like to thank my supervisors Dr. Kennedy Gachoka and Dr. Naomi K. Mutea for believing in me and guidance he has given me throughout this journey of research further with their authority allowed me to publish this work. I appreciate my mentor Dorothy Kagendo for guiding me in my work till the end.\n\nI am very grateful to Sanitation Research Institute Director Dr Joy Riungu for the support in terms of laboratory reagents for carrying out the practical research.\n\nI also offer my appreciations to chairman school of biological sciences for acceptance to use the biological laboratory in carrying out my study.\n\nMany sincere appreciations to Mr. Sigido Mbogoh laboratory Technician Biological lab for laboratory for providing me with equipment and space to carry out the sample processing in the laboratory. Lastly I thank Mr Anampiu Kirimi for the guidance in carry out microbiology procedures during the study.\n\n\nReferences\n\nHealth P: Future Emerging Issues in waterborne diseases and microbial agents.2012; 34–36.\n\nPedley S, Pond K: Emerging issues in water and infectious disease. World Health Organization;2003.\n\nReACT: Addressing the antibiotic resistance threat-the role of water, sanitation and hygiene.2018; 1–4.\n\nTown G, Mohsin S, Aziz Q, et al.: Original article Burden of antibiotic resistance among children with typhoid in.2022; 3(1): 81–91.\n\nIseghohi MG, FA K: Escherichia Coli Encoding Extended-Spectrum Beta-Lactamases Isolated from Diarrheic Patients in Minna, Nigeria. Int. J. Biotechnol. 2021; 10(2): 52–68.\n\nHaenni M, Dagot C, Chesneau O, et al.: Environmental contamination in a high-income country (France) by antibiotics, antibiotic-resistant bacteria, and antibiotic resistance genes: Status and possible causes. Environ. Int. 2022; 159(December 2021): 107047. PubMed Abstract \| Publisher Full Text\n\nCochran WG: Cochran_1977_Sampling_Techniques__Third_Edition.pdf.1977; p. 448.\n\nAmoafo OY, Malekar V, Jones E, et al.: Antibiotic resistance and phylogenetic profiling of Escherichia coli from dairy farm soils; organic versus conventional systems. Curr. Res. Microb. Sci. 2022; 3(July 2021): 100088.\n\nYeboah SIIK, Antwi-Agyei P, Domfeh MK: Drinking water quality and health risk assessment of intake and point-of-use water sources in Tano North Municipality, Ghana. J. Water Sanit. Hyg. Dev. 2022; 12(2): 157–167. Publisher Full Text\n\nGhabban H, Alnomasy SF, Almohammed H, et al.: Antibacterial, Cytotoxic, and Cellular Mechanisms of Green Synthesized Silver Nanoparticles against Some Cariogenic Bacteria (Streptococcus mutans and Actinomyces viscosus). J. Nanomater. 2022; 2022: 1–8. Publisher Full Text\n\nZelalem A, Abegaz K, Kebede A, et al.: Investigation on Salmonella enterica, Escherichia coli, and coliforms in beef from Ethiopian abattoirs: A potential risk of meat safety. Food Sci. Nutr. 2022(December 2021): 1–11.\n\nmonicah cheesbrough: monicah cheesbrough part 2.2006; 137–140 p.\n\nKahlmeter G, Giske CG, Kirn TJ, et al.: Point-counterpoint: Differences between the European Committee on Antimicrobial susceptibility testing and Clinical and Laboratory standards institute recommendations for reporting antimicrobial susceptibility results. J. Clin. Microbiol. 2019; 57(9). PubMed Abstract \| Publisher Full Text\n\nMaina J: Multiple-drug resistances gram-negative bacterial strains recoverable from food and environmental samples in kibera informal settlements Master of Science (Medical Microbiology) Jomo Kenyatta University of.2020.\n\nNikiema MEM, De La Gandara MP, Compaore KAM, et al.: Contamination of street food with multidrugresistant Salmonella, in Ouagadougou, Burkina Faso. PLoS One. 2021; 16(6 June 2021): 1–13. Publisher Full Text\n\nTahri L, Hafiane FZ, Fekhaoui M: Prevalence and antibiotic resistance of the Escherichia coli in the groundwater (Tadla-Morocco). Groundw. Sustain. Dev. 2021 May 1; 13: 100572. Publisher Full Text\n\nMaina J, Ndung P, Muigai A, et al.: Antimicrobial Profiles of Selected Gram-Negative Bacteria Recoverable from Sewage and Sludge from Juja and Kibera Informal Settlements of the Larger Nairobi Metropolis.2019; 507–524.\n\nFleece ME, Nshama R, Walongo T, et al.: Longitudinal assessment of antibiotic resistance in fecal Escherichia coli in Tanzanian Children. Am. J. Trop. Med. Hyg. 2019; 100(5): 1110–1114. PubMed Abstract \| Publisher Full Text\n\nIskandar K, Molinier L, Hallit S, et al.: Drivers of antibiotic resistance transmission in low-and middle-income countries from a “one health” perspective—a review. Antibiotics. 2020; 9(7): 1–23. Publisher Full Text\n\nSamreen AI, Malak HA, Abulreesh HH: Environmental antimicrobial resistance and its drivers: a potential threat to public health. J Glob Antimicrob Resist. 2021; 27: 101–111. PubMed Abstract \| Publisher Full Text\n\nDivyashree M, Vijaya Kumar D, Ballamoole KK, et al.: Occurrence of antibiotic resistance among Gram negative bacteria isolated from effluents of fish processing plants in and around Mangalore. Int. J. Environ. Health Res. 2020; 30(6): 653–660.\n\nSonola VS, Katakweba AS, Misinzo G, et al.: Occurrence of Multi-Drug-Resistant Escherichia coli in. Antibiotics. 2021; 10(1137): 1–12.\n\nHope D, Ampaire L, Oyet C, et al.: Antimicrobial resistance in pathogenic aerobic bacteria causing surgical site infections in Mbarara regional referral hospital, Southwestern Uganda. Sci. Rep. 2019; 9(1): 1–10.\n\nNgene AC, Ohaegbu CG, Awom IE, et al.: High prevalence of multidrug resistant enterobacteriaceae isolated from wastewater and soil in Jos Metropolis, Plateau State, Nigeria.2021; 13(December): 22–29.\n\nBilal H, Khan MN, Rehman T, et al.: Antibiotic resistance in Pakistan: a systematic review of past decade. BMC Infect. Dis. 2021; 21(1): 1–19.\n\nMukami SN: phenotypic and molecular characterization of pathotypes, and antimicrobial resistance profiles of escherichia coli from food, water and livestock in selected informal settlements in nairobi.2021.\n\nPraveenkumarreddy Y, Akiba M, Guruge KS, et al.: Occurrence of antimicrobial-resistant escherichia coli in sewage treatment plants of south India. J. Water Sanit. Hyg. Dev. 2020; 10(1): 48–55.\n\nOmbuya J, Kennedy G, Naomi M: Evaluation of antimicrobial susceptibility of escherichia coli isolated from contaminated areas of majengo slum in meru county, kenya. figshare. [Dataset].2022. Publisher Full Text" }	[ { "id": "161608", "date": "17 Feb 2023", "name": "Eman S. Ibrahim", "expertise": [ "Reviewer Expertise Microbiology (bacteriology)", "Molecular biology", "Nanotechnology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 respected colleagues,\nI have read the manuscript entitled ‘Evaluation of antimicrobial susceptibility of Escherichia coli isolated from contaminated areas of Majengo slum in Meru County, Kenya ’ thoroughly.\nThe manuscript is discussing an interesting topic; Which is the danger of antibiotic resistance concerning E.coli and the linkage between antibiotic resistance and poor hygienic condition in rural areas and misuse of antibiotic treatment which in turn make it necessary to make antibiogram for choosing drug of choice before beginning treatment scheme. The work is clearly and accurately presented and cite the current literature.\nThe work is technically sound but the study design is partially appropriate as the study here made screening against only five antibiotic disc which is not enough to build on it a conclusion. Can you explain why was this antibiotic chosen over others? Also why was genotypic identification of antibiotic resistant genes not taken?\nIn the conclusions found in the abstract section you mentioned that the isolated E.coli is pathogenic. What is your practical support for this information as you didn't make detection for virulence genes? So it is preferable to remove the word pathogenic. The methods and statistical analysis and it's interpretation as well as result is well presented.\nIn discussion section concerning reference 15 this paper was discussing Salmonella isolates how are you comparing your result with this paper? It is preferable to remove it.\nConclusion section should include future scope of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9445", "date": "12 Apr 2023", "name": "jared ombuya", "role": "Author Response", "response": "The reason why I concluded that the organism is pathogenic is that I did serotyping of E coli strains and one strain was positive for the test The reason for using the 5 antibiotics is that those are the commonly prescribed drugs in the health centers in the study area Genotyping was not done as that could have quite expensive for me but that's the work to be done in my next level of study The reference included for salmonella was mistakenly included hence will not appear in revised version Conclusion will be amended accordingly as well in the revised version" } ] } ]	2	https://f1000research.com/articles/11-1133
https://f1000research.com/articles/11-395/v1	06 Apr 22	{ "type": "Research Article", "title": "Temporomandibular joint (TMJ) disorders prevalence and awareness of appropriate clinical practices, among Al-Madinah community in Saudi Arabia", "authors": [ "Albraa Alolayan", "Shayma S. Alsayed", "Ruwaa M. Salamah", "Khadija M. Ali", "Mashael Alsousi", "Shadia Elsayed", "Albraa Alolayan", "Shayma S. Alsayed", "Ruwaa M. Salamah", "Khadija M. Ali", "Mashael Alsousi" ], "abstract": "Background: Painful temporomandibular joint disorders (TMDs) are of musculoskeletal origin and are considered the most common cause of non-odontogenic pain in the orofacial region. The purpose of this study was to investigate the prevalence and awareness of temporomandibular joint (TMJ) disorders in Almadinah Almunawwarah community. Methods: An observational cross-sectional study with convenience sampling was conducted. A modified version of Fonseca's questionnaire was employed. The questionnaire asked about the participant's personal information, if they thought they had TMDs, and who to visit for therapy if necessary. These were followed by 10 items from Fonseca's questionnaire, each with a three-point scale. Results: The questionnaire was completed by 598 people. Females made up 57.1% of the participants. TMDs were present in 61% of the population, with varying degrees of severity. Males (44.3%) were less affected than females (55.7%). The difference, however, was not statistically significant (P = 0.354). Out of the 61% TMDs Positive patients, 74.1% had mild TMDs symptoms, while 20.8% and 5.1%, respectively, had moderate and severe TMDs symptoms (P = 0.05). The severity of the symptoms was unaffected by demographic data (P > 0.05). Only 40% seek care, with 64.6% selecting for a dentist and 24.6% preferring for an orthopaedic specialist (P= 0.008). Conclusions: Participants from Al-Madinah had a greater prevalence of mild TMDs. The majority of the participants had no idea who to go to for treatment. The findings of this study highlight the importance of educational activities to enhance public awareness. Fonseca's Anamnestic Index could also be considered as a useful instrument for early identification and measuring the severity of TMDs in the general population.", "keywords": [ "TMJ", "prevalence", "awareness", "severity", "Almadinah Almunawwarrah" ], "content": "Introduction\n\nTemporomandibular disorders (TMDs) is a term used to describe a set of musculoskeletal and neuromuscular diseases characterized by localized discomfort in the pre-auricular and face regions, as well as deviations or restrictions in mandibular joint movement, in addition to joint noises.1 TMDs are multifactorial, caused by stress, trauma and malocclusion.2 Painful TMDs are of musculoskeletal origin and are considered the most common cause of non-odontogenic pain in the orofacial region.3–5 The most prevalent symptom of TMD is tenderness of the masticatory muscles when palpated.6,7\n\nThe prevalence of TMDs was twice higher in women compared to men,8 also pain intensity was greater in women.9 According to a previous report, 75% of the participants exhibited one TMD sign and 33% had one TMD symptom.10 Another study concluded that 50–75% of the population had TMD signs at some moment in their lives.11 One study showed that only 39% participants had sought treatment for pain related to TMDs.12 Patients with TMDs symptoms tend to consult first with general medical practitioners due to the availability and financial feasibility in nearly six countries. 27% of children and adolescents in Saudi Arabia were found to have TMDs.\n\nFor our patients, temporomandibular joint (TMJ) disorders still cause difficulty in diagnosis and conflicts between different specialties. Some individuals seek treatment from different specialized clinics such as ENT (ear, nose and throat) or neurosurgery clinics, and therefore the goal of this study was to establish the prevalence, awareness, and severity of TMJ disorders in our Almadinah Almunawwarah community.\n\n\nMethods\n\nThis study was designed to be an observational cross-sectional study with convenience sampling technique. The Taibah University Dental College Ethical Committee approved the study protocol (TUCDREC/20200219/MIAlsousi), and all participants’ identities were kept anonymous. The study followed the Helsinki Declaration on Human Research Studies. The patients were selected from the dental college and the hospital at Taibah University in Al-Madinah, Saudi Arabia. Following an explanation of the study’s aims and objectives, patients who agreed to participate in the study signed an informed consent form.\n\nAll patients with systemic disease associated with TMJ like rheumatoid arthritis, regular use of medication such as anti-anxiety or antidepressants, history of TMJ surgery, wearing removable denture or splints, psychological problems and/or with incomplete questionnaire form were excluded from the study.\n\nResponse bias was reduced by keeping the questions simple and short, as in the verified Fonseca questionnaire, which has structured proper length and understandable language, and by limiting the patient experience to the previous three months.\n\nUsing a sample size calculator with a confidence level of 99% and estimated the entire Madinah population to be above one million, the sample size was calculated to be 523 participants. To avoid missing or insufficient data, we extend the total to 598. Patients seeking dental treatment were recruited through the Oral and Maxillofacial Surgery clinics at Taibah University, Almadinah Almunawwarah, in Saudi Arabia.\n\nIn order to estimate Fonseca’s Anamnestic Index (FAI), a modified Fonseca’s questionnaire was utilized. The original Fonseca’s questionnaire was proposed by Fonseca et al13 in Brazilian Portuguese.\n\nThe questionnaire included questions about participant’s demographic data, if the participant thought he/she is suffering from TMDs, and whom to seek for a treatment if he/she needed to. These questions were followed by 10 questions of Fonseca’s questionnaire with three points scale, and it was distributed to patients in an Arabic version. For each question, the participants were instructed that just one answer should be marked: “yes” (10 points), “no” (0 points), and “maybe” (5 points). Based on the sum of their points, the individuals were classified as TMD free (0–15), mild TMD (20–40), moderate TMD (45–60), and severe TMD (70–100). The FAI has been usually applied in Brazilian studies to measure the severity of TMDs.9,14 A copy of the questionnaire can be found in the Extended data.\n\nThe data was evaluated using a social science statistical software (SPSS version 25). Means, standard deviation, and percentages were used to show descriptive data. It was followed by Chi-square test for nominal outcomes and Student’s t-test for continuous study variables. Significance level was set at P value of 0.05.\n\n\nResults\n\nA total of 598 participants answered the questionnaire, based on the above-mentioned exclusion criteria, 80 (13.4 %) participants were removed from the study. Thus, leveeing a total of 518 (86.6%) recruited participants, females made up 57.1% of the participants. The age group distribution of participants and the level of education are presented in Figure 1 and Figure 2, respectively.\n\nBased on the participants’ answers on Fonseca’s questionnaire, 61% had TMDs with variable degree of severity. Male group (44.3%) was less than female group (55.7%). The difference, however, was not statistically significant (P = 0.354). On the other hand, there was a significant difference in the positive responses to the questions of whether or not the participants had TMDs (12.5 %), and the participants whom actually suffering from TMDs according to Fonseca’s questionnaire (P < 0.05). The level of TMDs in regard to gender distribution is presented in Figure 3.\n\nOut of the 61% TMDs positive patients, 74.1% had symptoms of mild TMDs, while the moderate and severe symptoms were 20.8% and 5.1%, respectively (P < 0.05). However, the demographic data showed no effect on the severity of the symptoms (P > 0.05).\n\nIn regards whom to seek for treatment, 51.6% of the sample chose a dentist while 41.8% chose an orthopedic specialist (P > 0.05). Based on participants who suffered or suffering from TMDs, only 40% sought out treatment, 64.6% of them chose a dentist while 24.6% chose orthopaedic specialist (P ≤ 0.008).\n\nThe symptoms mostly associated with TMDs ranged from behavioral changes and quick fatigue of the mastication muscles, to frequent headache or pain related to the TMJ or the neck, reaching to TMJ stiffness and clicking or even limitation in mouth opening. Table 1 summarizes the frequencies of the most common symptoms.\n\n\nDiscussion\n\nThe study evaluates the prevalence and awareness of TMD amongst the population of Al-Madinah to produce sufficient data on TMD, helps to find crucial part of planning programs to educate and raise awareness through the population. The study showed that TMDs were present in 61% of the population. Many studies employed the Fonseca questionnaire to diagnose and grade the severity of TMDs. TMD is divided into four categories: minor, moderate, severe, and non-TMD.15 It has a number of advantages, including quick application, low cost, minimal inconsistency, and self-administration.16 The Fonseca questionnaire was used in this study to assess the prevalence of TMD symptoms and manifestations in the Almadianh region of Saudi Arabia. It is used to gather data from a wider number of people in a short period. The most significant advantage of employing this questionnaire is that it has no bearing on the investigator and is straightforward.17\n\nMore than half of the participants had some level of TMDs, which has been associated with headache. In regard, frequent headaches were associated with 40% of the participant, patients with TMD are five times more likely to report headaches.8 The results are related to a study done in Brazil in 2018 which over two third of the adolescents had headache/migraine and 36 percent of them connected it with TMD.18\n\nThough TMD is suggested to have a higher frequency and severity in females than in males, and females were reported to have a greater sensitivity to pain19 which has been connected to hormonal, psychological, and even neurological changes,8,9,20 no conclusive results achieved to the date. In this study TMD male patients (44.3%) were less than female patients (55.7%). However, the difference is not significant, also in a similar study done in Al-Badar Dental College mentioned that men (49.14%) had higher free of TMD symptoms than women (44.78%) but this difference was also not statistically significant.20\n\nAlthough more than 70% of the TMD positive participants have mild TMD, only 51.6% of the sample knows who to see for TMD therapy, and 40% of TMD patients seek treatment. TMD treatment varies from one physician to another. It ranges from patient education and the development of new habits such as self-massage, hot and cold packs, diet and nutrition instruction, and muscle exercises, which have been shown to have a high level of success and improvement, to the need for invasive and high-technical or invasive surgical treatments.1,21,22 According to Reynaldo, 91.7% of patients responded well to conservative treatment. There was no TMD recurrence in the majority of patients who could be examined four to six years after treatment completion.23 The current study’s findings highlight the importance of public education and emphasize the limitations of generalizing the findings to other sectors of the health clusters in our community’s population.24\n\n\nConclusion\n\nBased on this study, participants from Al-Madinah showed higher prevalence in mild degree of TMDs. Most of the participants were lacking the knowledge of TMDs and whom to seek for a treatment. The results from this study emphasize the need for educating programs to raise awareness through the population, and to consider Fonseca’s anamnestic index as a valuable tool for early identification and severity assessment of TMDs in the general population.\n\n\nData availability\n\nFigshare: Data for TMJ disorders prevalence and awareness of appropriate clinical practices, among Al-Madinah community in Saudi Arabia.sav https://doi.org/10.6084/m9.figshare.1721594025\n\nThis project contains the following underlying data\n\n- Data for TMJ disorders prevalence and awareness of appropriate clinical practices, among Al-Madinah community in Saudi Arabia.sav (raw data in Arabic)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Data for TMJ disorders prevalence and awareness of appropriate clinical practices, among Al-Madinah community in Saudi Arabia.sav. https://doi.org/10.6084/m9.figshare.19086635.v226\n\nThis project contains the following underlying data\n\n- Data for TMJ disorders prevalence and awareness of appropriate clinical practices, among Al-Madinah community in Saudi Arabia.sav (raw data in English)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Questionnaire. https://doi.org/10.6084/m9.figshare.1929247427\n\nThis project contains the following extended data\n\nA copy of the questionnaire (in Arabic and the English version).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBahar AD, How QY, Tan XP: Temporomandibular disorder symptoms and their association with quality of life of dental patients in Malaysia. Cranio. 2021; 1–6. Publisher Full Text\n\nEttlin DA, Napimoga MH, Meira e Cruz M, et al.: Orofacial musculoskeletal pain: An evidence-based bio-psycho-social matrix model. Neurosci. Biobehav. Rev. 2021; 128: 12–20. PubMed Abstract \| Publisher Full Text\n\nAltaweel AA, Elsayed SAH, Baiomy AABA, et al.: Extraoral Versus Intraoral Botulinum Toxin Type A Injection for Management of Temporomandibular Joint Disc Displacement with Reduction. J. Craniofac. Surg. 2019; 30: 2149–2153. PubMed Abstract \| Publisher Full Text\n\nElgazzaz M, Khalifa F, Amin G, et al.: The Use Of Injectable Platelet Rich Fibrin In The Treatment Of Tempromandibular Joint Hypermobility. Al-Azhar Dent. J. Girls 2019; 6: 507–515. Publisher Full Text\n\nGauer RL, Semidey MJ: Diagnosis and treatment of temporomandibular disorders. Am. Fam. Physician 2015; 91: 378–386. PubMed Abstract\n\nWänman A, Agerberg G: Prevalence of functional disturbances and diseases of the stomatognathic system in 15-18 year olds. Acta Odontol. Scand. 1986; 44: 55–62. Publisher Full Text\n\nWänman A, Agerberg G: Gender differences in temporomandibular disorders in adult populational studies: A systematic review and meta-analysis. Acta Odontol. Scand. 1986; 45: 720–729. PubMed Abstract \| Publisher Full Text\n\nSchmid-Schwap M, Bristela M, Kundi M, et al.: Sex-Specific Differences in Patients with Temporomandibular Disorders. J. Orofac. Pain 2013; 27: 42–50. PubMed Abstract \| Publisher Full Text\n\nDe Melo PC, Lins Aroucha JMCN, Arnaud M, et al.: Prevalence of TMD and level of chronic pain in a group of Brazilian adolescents. PLoS One. 2019; 14: e0205874–e0205893. PubMed Abstract \| Publisher Full Text\n\nGray RJ, Davies SJ, Quayle AA: A clinical approach to temporomandibular disorders. 6 splint therapy. Br. Dent. J. 1994; 177: 135–142. PubMed Abstract \| Publisher Full Text\n\nEgermark I, Carlsson GE, Magnusson T: A 20-year longitudinal study of subjective symptoms of temporomandibular disorders from childhood to adulthood. Acta Odontol. Scand. 2001; 59: 40–48. PubMed Abstract \| Publisher Full Text\n\nAhmed S, Ali M, Rehan F, et al.: General Medical Practitioners’ knowledge of assessment and management of Temporomandibular joint disorders: A questionnaire -based survey in the UK. Br. J. Med. Pract. 2019; 12.\n\nFonseca DM, Bonfante G, Valle ALDFS: Diagnóstico pela anamnese da disfunção craniomandibular. Rev. Gaucha Odontol. 1994; 4.\n\nPoveda Roda R, Bagan JV, Díaz Fernández JM, et al.: Review of temporomandibular joint pathology. Part I: classification, epidemiology and risk factors. Med. Oral Patol. Oral Cir. Bucal. 2007; 12.\n\nKaraman A, Buyuk SK: Evaluation of temporomandibular disorder symptoms and oral health-related quality of life in adolescent orthodontic patients with different dental malocclusions. Cranio. 2019; 40: 55–63. PubMed Abstract \| Publisher Full Text\n\nYap AU, Zhang MJ, Lei J, et al.: Accuracy of the Fonseca Anamnestic Index for identifying pain-related and/or intra-articular Temporomandibular Disorders. Cranio. 2021; 1–8. PubMed Abstract \| Publisher Full Text\n\nKaynak BA, Taş S, Salkın Y: The accuracy and reliability of the Turkish version of the Fonseca anamnestic index in temporomandibular disorders. Cranio. 2020: 1–6. PubMed Abstract \| Publisher Full Text\n\nWänman A, Agerberg G: Mandibular dysfunction in adolescents II. Prevalence of signs. Acta Odontol. Scand. 1986; 44: 55–62. Publisher Full Text\n\nEmel DN: Prevalence of Temporomandibular Disorder in Turkish University Students: a Questionnaire Study. Balk J. Dent. Med. 2019; 23: 80–87. Publisher Full Text\n\nSyeda A, AKatti G, Ahmed Syed R, et al.: Prevalence of Temporomandibular Joint Disorders in Outpatients at Al-Badar Dental College and Hospital and Its Relationship to Age, Gender, Occlusion and Psychological Factors. J. Indian Academy Oral Med. Radiol. 2012; 24: 261–268. Publisher Full Text\n\nNokar S, Sadighpour L, Shirzad H, et al.: Evaluation of signs, symptoms, and occlusal factors among patients with temporomandibular disorders according to Helkimo index. Cranio. 2019; 37: 383–388. PubMed Abstract \| Publisher Full Text\n\nMatsuka Y, Yatani H, Kuboki T, et al.: Temporomandibular Disorders in the Adult Population of Okayama City, Japan. Cranio. 1996; 14: 158–162. PubMed Abstract \| Publisher Full Text\n\nMartins-Júnior RL, et al.: Temporomandibular disorders: a report of 124 patients. J. Contemp. Dent. Pract. 2010; 11: 71–78. Publisher Full Text\n\nNomura K, Vitti M, de Oliveira AS , et al.: Use of the Fonseca’s questionnaire to assess the prevalence and severity of temporomandibular disorders in Brazilian dental undergraduates. Braz. Dent. J. 2007; 18: 163–167. PubMed Abstract \| Publisher Full Text\n\nElsayed S: Data for TMJ disorders prevalence and awareness of appropriate clinical practices, among Al-Madinah community in Saudi Arabia.sav. figshare. Dataset. 2022. Publisher Full Text\n\nElsayed S: Data forTMJ disorders prevalence and awareness of appropriate clinical practices, among Al-Madinah community in Saudi Arabia.sav. figshare. Dataset. 2022. Publisher Full Text\n\nElsayed S: Questionnaire. figshare. Preprint.2022. Publisher Full Text" }	[ { "id": "129949", "date": "06 Apr 2022", "name": "Essam Ahmed Al‐Moraissi", "expertise": [ "Reviewer Expertise Oral and Maxillofacial Surgery" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nUpdate 07/07/2022 - We have added a conflict of interest statement that was not disclosed before this report was published. This statement can be found below this report under the heading ‘Competing Interests’.\nThank you for giving me this chance to review this paper. My comments are:\n\nAuthors completely failed to state the rationale of the study (why it's important to do this study) or show the size of the gap in the literature. Unfortunately, the indexing of this study will not add any new material to the existing literature.\n\nAuthors did not state the inclusion criteria completely, only mentioned the exclusion criteria. So authors should state inclusion criteria based on PICOS criteria.\n\nAuthors did not present adequately information about how they calculated the sample size.\n\nAuthors should explain why they chose a modified version of Fonseca's questionnaire.\n\nThe generalizability of this study is not applicable.\n\nAuthors should include others predictors variables to be analysed type of TMDs if myogenous or arthrogenous, chronicity, signs and symptoms of TMDs such as clicking, muscles tenderness, headache, limited mouth opening etc.\n\nAuthors did not clarify who performed the assessment.\n\nThis study is based on a survey so its mandatory to clarify the limitations of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-395
https://f1000research.com/articles/12-386/v1	12 Apr 23	{ "type": "Case Report", "title": "Case Report: Management strategy of rare case first-generation antipsychotic drug induced neglected femoral neck fracture in schizophrenia patients", "authors": [ "Rudiansyah Harahap", "Putri Anugerah Meisari", "Kevin Christian Tjandra", "Putri Anugerah Meisari", "Kevin Christian Tjandra" ], "abstract": "Background: The early onset of femoral neck fracture is commonly treated by bone grafting surgery. However rare cases of neglected femoral neck fractures in schizophrenic patients need to be treated by arthroplasty because of the rapid destruction of the femoral head within the acetabulum. Case presentation: We report a rare unique case of first-generation antipsychotic drug-induced neglected femoral neck fracture in a 41-year-old man presenting after a month complaining of not being able to walk due to falling in a ditch. X-ray examination findings revealed total destruction of the femoral head within the acetabulum with no acetabulum dysplasia detected. Ordinarily, it should be treated with a bone grafting procedure since no dysplasia was detected and it was an early onset case. However, arthroplasty was chosen because the femoral head was destructed. Three months post-surgery x-ray shows the acetabulum dysplasia and lytic lesion that confirm drug-induced bone destruction by the first-generation antipsychotic drug. Conclusions: We suggest that even though rare, first-generation antipsychotic drugs neglected femoral neck fractures in schizophrenia patients can be successfully treated by arthroplasty supported by the patient’s compliance. Bone mineral density (BMD) test is also suggested to monitor the bone density of schizophrenia patients.", "keywords": [ "drug-induced", "neglected femoral fracture", "hip arthroplasty", "first-generation antipsychotic drug", "schizophrenia", "case report" ], "content": "Introduction\n\nSchizophrenia is a long-term, complicated mental health condition that manifests in a variety of ways, including delusions, hallucinations, disorderly behavior or speech, and diminished cognitive function.1 The prevalence of the schizoaffective disorder from 2008 to 2015 was 6.2 per 1000 individuals aged 15 to 64 years, with a greater frequency in men than in women.2 Femoral neck fractures occur in 63.3 out of every 100,000 women, 27.7 out of every 100,000 men, and 5.54 out of every 1000 patients with schizophrenia. People under the age of 50 accounted for 23% of them.3,4 After controlling for potential confounders, a case-control study from the United Kingdom (n = 44,500) discovered that both current and prior antipsychotic use, as well as the duration of antipsychotic use, were associated with a small significant increase (OR 1.3, 1.3, r 2 = 0.88, respectively) in hip/femur fractures as the side.\n\nAt least 30 days after the fracture, a neglected femoral column fracture can be promptly identified with a history and X-ray examination.5 Bone grafting is frequently used to treat the early stages of untreated femoral neck fractures. However, arthroplasty may be an useful treatment option in unique circumstances that result in an early malformation of the femoral head or acetabulum.3\n\n\nCase presentation\n\nIn November 2021, an Austronesian unemployed 41-year-old man who had been found in a ditch complained to the emergency department that he had been unable to walk for four weeks. After an X-ray indicated schizophrenia, the patient's femoral neck fracture was determined to be the cause. After the patient acknowledged having schizophrenia since he was 25 and used haloperidol often, the diagnosis was changed to a first-generation antipsychotic drug-induced neglected femoral neck fracture.6\n\nPhysical examination, as shown in Figure 1, reveals that the patient is unable to lift the right leg but is able to move the right leg to the right and to the left. The right hip also exhibits signs of exhalation, has flexion in the knee and hip, and is erythematous and warm to the touch. Other physical examination findings include crepitus, pain and motion (+), and a limited range of motion (ROM) of six joint movements (flexion, extension, abduction, addition, rotation) and a physical examination revealed a body temperature of 37.6°C, a blood pressure of 134/85 mmHg, a pulse rate of 97 beats per minute, and a respiratory rate of 20 beats per minute. An evaluation by a psychiatrist indicated auditory hallucinations (+) and a speech disability.\n\nA right femoral head fracture was suspected after a radiological evaluation that can be seen in Figure 2, of the pelvis and the right femur's PA view revealed an osteochondroma in the middle third of the right femur. The discomfort and potential for falls prevented the adoption of the lateral perspective.\n\nRegistration for right femoral reconstruction and hemiarthoplasty as opposed to bone grafting is part of the management of femoral neck fractures. As depicted in Figure 3, a right femoral osteotomy and hemiarthoplasty was carried out in December 2021. This treatment was chosen because removing the femoral head from the acetabulum caused the femoral head and neck to rejoin.\n\nSeven days after the surgery, monitoring and follow-up were carried out before outpatient management was put into action. Following surgery, monitoring of the patient's general health, vital signs, surgical scars, and ROM was done. Along with educating patients and family about the disease (neglected femoral neck fracture along with his schizophrenia condition), treatment, post-operative problems, and prognosis, physiotherapists and rehabilitation professionals constantly monitored ROM patients after surgery. The patient was instructed in deep breathing relaxation techniques as well as how to immobilize the right femur (abduction and exorotation). In order to treat the patient's pain, analgesic therapy was also offered. However, despite being given schizophrenia medication, dislocation still happened because he frequently performed hip flexion and endorotation movements to their maximum extent, the outpatient was reluctant. Moreover, the effect of prolonged use of the drug may cause lytic lession that worsen the patient condition.5\n\nThree months post-surgery X-ray shows the acetabulum dysplasia and lytic lesion that provide drug-induced bone destruction by the first-generation antipsychotic drug.\n\nFigure 4 depicts the patient's state three months after surgery. Abduction and endorotation of the right hip joint in the image suggest a dislocation at that hip joint as well as a lytic lesion inside the acetabulum. The lytic lesion exhibits fast loss of bone density that may be brought on by chronic haloperidol use. The patient has a good prognosis if the hip immobilization and effective physical treatment are carried out effectively. However, if the aforementioned parameters are not met, the prognosis may become poor.\n\n\nDiscussion\n\nThis case report gives a presentation of a rare medical condition. The presentation included the history, pathogenesis, limitation of the treatment, and suggested treatment. This information may be useful to other practitioners. However, the lack of follow up is a main limitation of this case report.\n\nThe patient in this case study is a 41-year-old male who was identified having a rare and exceptional case of a femoral neck fracture brought on by the misuse of first-generation antipsychotic medications. Treatment with long-term haloperidol had begun at age 25 to treat schizophrenia.6\n\nIn comparison to healthy, sex-adjusted controls, schizophrenia patients are more likely to experience hip fractures and have lower hip bone mineral density after long-term therapy.7 The only effective treatment for a desired hip fracture is surgery.8 However, the course of treatment is determined by the patient's age, the severity of any pre-traumatic symptoms, the type of fracture, and its dislocation.9\n\nCompared to second-generation antipsychotics, first-generation antipsychotics (AGPs) seem to have a higher risk of fracture (AGKs). To determine whether there was a link between antipsychotic drug use and fracture, Lee et al (2017) conducted the study that revealed that 1.5 times risk of fracture increase in people with antipsychotics drug usage. The same study also found that the risk of first generation antipsychotic drug with odds ratio 1.67 higher than second generation antipsychotic drug with odds ratio 1.33.10\n\nAntipsychotics are prolactin secretion inhibitors and dopamine D2 receptor antagonists. This may prevent the hypothalamic-pituitary-gonadal axis from functioning properly, leading to low BMD and hyperprolactinemia. The release of gonadotropin-releasing hormone (GnRH) from the hypothalamus can be inhibited by an increase in serum prolactin. Low levels of GnRH consequently inhibit the pituitary gland's production of luteinizing hormone and follicle-stimulating hormone, lower levels of estradiol, progesterone, and testosterone, and cause improper bone metabolism and osteoporosis. Antipsychotic-treated schizophrenia patients experienced reduced BMD and more pathological fractures. Numerous epidemiological studies have shown a dose-response association between the use of antipsychotics and the incidence of hip fracture in nursing home patients as well as fracture risk in the Danish population.4,5,10,11 Thus, one effect of long-term antipsychotic medication is the increased risk of fracture development in treating schizophrenia patients.12\n\nThese studies' findings support this case and show that antipsychotic usage in schizophrenia patients carries a risk of hip fracture.\n\nAvascular necrosis (AVN) can result from femoral neck fractures, especially if they are not treated right away. The predominant blood supply to the femoral head is provided by the medial rotator cuff femoral artery, lateral radial femoral artery, and obturator artery. The femoral head is supplied by the obturator artery through the round ligament. Osteonecrosis is caused by fractures with dysplasia, which interfere with the artery's terminal branches at the level of the femoral head.3\n\nThe according-to-time type of neglected femoral neck fractures management can be differentiated by the Sandhu classification3:\n\n1. Neglected fracture of femoral neck less than four weeks\n\nInternal fixation and closed or open reduction are the primary therapy options for stage I. Consider bone grafting in a Non-Union (NU) site.\n\n2. Neglected fracture of femoral neck four weeks to three months\n\nStage II, open reduction and internal fixation, vascularized or nonvascularized bone grafting, or vulgarized osteotomy are the recommended therapeutic options.\n\n3. Neglected fracture of femoral neck three months to six months\n\nStage III, the headpiece was vascular, reduced opening, bone grafting at the NU site, fracture surface renewal, internal fixation with cannulated cancellous screw (CCS), and muscle-pedicle bone grafting (MPBG) can be effective methods. The surgeon can decide which muscle to use for MPBG and how to handle its side effects. Young patients may be thought to have hip arthritis. Patients and surgeons do not, however, favor this technique. Myoplastic surgery or hypertension may be required in situations of AVN Stage III that have been confirmed.\n\n4. Neglected fracture of the femoral neck more than six months\n\nHemiarthroplasty or total arthroplasty replacement is usually preferred.3\n\nThe main treatment option for early-stage neglected femoral neck fracture is the bone grafting procedure. However, hemi hip arthroplasty (HHA) is preferred in this case due to the rapid destruction of the femoral head bone and no acetabular dysplasia detected at first.3,4,13 In order to prevent post-surgery mechanical complications, longer hospital stays inpatient is preferred. This is also able to maximize the monitoring and evaluation surgery process.1,7,14 Arthroplasty reduces the need for repeat surgery. This procedure allows early load adaptation.15–17\n\nThe case's approach must take into account a number of factors. The initial decision is whether to fix the fracture or replace the joint. Joint replacement may be a sensible option if fracture repair is not practical. Hemi hip arthroplasty, which replaces only the proximal femur, and total hip arthroplasty, which replaces both the proximal femur and the articular surface, are the two basic forms of joint replacement.15,16 Hemi hip arthroplasty is indicated when there is no acetabular dysplasia and complete hip arthroplasty is preferred when there is acetabular dysplasia.8 Since there was still no acetabular dysplasia, the patient in this case study was finally treated with a hemi hip arthroplasty. However, three months post-surgery X-ray shows the acetabulum dysplasia and lytic lesion, so we suggest performing total hip arthroplasty (THA) with or without acetabular dysplasia to prevent repeated surgery.\n\nA single impression was used to create the proximal femoral hemiarthroplasty prosthesis, which had the femoral shaft joined to the femoral head and neck. The Austin-Moore prosthesis is a typical illustration.15,16\n\nAfter cutting the femur's neck to size and prepping the medullary canal, the proximal femoral prosthesis is put into the femur.15\n\nWhen doing an arthroplasty, many surgeons favor an anterior or posterior approach, with the patient laying on their side or back. In this instance, anterior capsule-conserving surgery is advised since it may lower the chance of prosthesis dislocation, particularly for joint dysplasia.15 Three months post-surgery X-ray shows the acetabulum dysplasia and lytic lesion that provide drug-induced bone destruction by the first-generation antipsychotic drug.\n\nTo lessen the threat of prosthetic hip dislocation for the duration of the primary 6 weeks, the patient has to avoid too many hip joint movements. Adduction throughout the midline, hip flexion extra than 80° to 90°, and inner rotation have to be averted particularly after treatment.16,17\n\n\nConclusion\n\nThe long-term use of the first-generation antipsychotic drug, haloperidol, tends to cause bone destruction, especially weight-bearing bone, in this case, the head and neck femoral bone. It may lead to early onset first-generation antipsychotic drug-induced neglected femoral neck fracture because the bone destruction was slowly progressive. This made the patient less aware of his disease until complete destruction occurred. It was shown in this case that there was a worsening condition that show acetabular dysplasia along with lytic lesions 3 months after the first hemi hip arthroplasty as the side effect of haloperidol long-term use. The suggested strategy for a similar case in the future will be arthroplasty rather than bone grafting, total hip arthroplasty is suggested rather than hemi hip arthroplasty in order to prevent repeated surgery.\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\nStubbs B, Gaughran F, Mitchell AJ, et al.: Schizophrenia and the risk of fractures: a systematic review and comparative meta-analysis. Gen. Hosp. Psychiatry. 2015; 37: 126–133. Publisher Full Text\n\nOrrico-Sánchez A, López-Lacort M, Munõz-Quiles C, et al.: Epidemiology of schizophrenia and its management over 8-years period using real-world data in Spain. BMC Psychiatry. 2020; 20: 1–9. Publisher Full Text\n\nJain AK, Mukunth R, Srivastava A: Treatment of neglected femoral neck fracture. Indian J. Orthop. 2015; 49: 17–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen CY, Lane HY, Lin CH: Effects of Antipsychotics on Bone Mineral Density in Patients with Schizophrenia: Gender Differences. Clin. Psychopharmacol. Neurosci. 2016; 14(3): 238–249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHugenholtz GWK, Heerdink ER, Van Staa TP, et al.: Risk of hip/femur fractures in patients using antipsychotics. Bone. 2005; 37: 864–870. PubMed Abstract \| Publisher Full Text\n\nLavretsky: Clinical Handbook of Schizophrenia. New York: Guildford Press; 2008.\n\nTseng PT, Chen YW, Yeh PY, et al.: Bone mineral density in schizophrenia an update of current meta-analysis and literature review under guideline of PRISMA. Medicine (United States). 2015; 94(47): e1967. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKenanidis E, Zagalioti S-C, Milonakis N, et al.: A Catastrophic Cascade of Postoperative Complications Following Hemiarthroplasty for Femoral Neck Fracture in a Middle-Aged Patient With Schizophrenia. Cureus. 2020; 12. Publisher Full Text\n\nLaursen TM, Nordentoft M, Mortensen PB: Excess Early Mortality in Schizophrenia. Annu. Rev. Clin. Psychol. 2014; 10: 425–448. Publisher Full Text\n\nLee SH, Hsu WT, Lai CC, et al.: Use of antipsychotics increases the risk of fracture: a systematic review and meta-analysis. Osteoporos Int. 2017; 28: 1167–1178. PubMed Abstract \| Publisher Full Text\n\nWu H, Deng L, Zhao L, et al.: Osteoporosis associated with antipsychotic treatment in schizophrenia. Int. J. Endocrinol. 2013; 2013: 167138. Epub 2013 Apr 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGalivanche AR, Kebaish KJ, Adrados M, et al.: Postoperative Pressure Ulcers After Geriatric Hip Fracture Surgery Are Predicted by Defined Preoperative Comorbidities and Postoperative Complications. J. Am. Acad. Orthop. Surg. 2020; 28: 342–351. Publisher Full Text\n\nOwen MJ, Sawa A, Mortensen PB: Schizophrenia. Lancet. 2016; 388: 86–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiao CC, Shen WW, Chang CC, et al.: Surgical adverse outcomes in patients with schizophrenia: a population-based study. Ann. Surg. 2013; 257: 433–438. Publisher Full Text\n\nHopkins J: Hip Replacement Surgery.2022. Accessed 17 Oct 2022. Reference Source\n\nZachwieja E, Butler AJ, Grau LC, et al.: The association of mental health disease with perioperative outcomes following femoral neck fractures. J. Clin. Orthop. Trauma. 2019; 10: S77–S83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlorida Orthopaedic Institute: Hip Hemiarthroplasty.2022. Accessed 17 Oct 2022. Reference Source" }	[ { "id": "251180", "date": "25 Mar 2024", "name": "Samo K Fokter", "expertise": [ "Reviewer Expertise hip", "knee", "arthroplasty", "osteoporosis" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI appreciate the opportunity to review the manuscript titled \"Case Report: Management Strategy for a Rare Case of First-Generation Antipsychotic Drug-Induced Neglected Femoral Neck Fracture in Schizophrenia Patients\". This case report addresses an intriguing and underreported issue, shedding light on a complex clinical scenario. My comments are organized into major and minor points for clarity. Major Points:\nLanguage and Readability: The manuscript is currently hindered by significant language issues that impact its readability. Clarity of Objectives: The introduction lacks a clear statement of the manuscript's aim. It is essential to explicitly define the purpose and significance of the case report to guide the readers through the relevance and contributions of the study. Accuracy in Case Presentation: The statement “After an X-ray indicated schizophrenia …” appears to be a significant error. Schizophrenia is a psychiatric diagnosis, not detectable through X-ray. This likely represents a misunderstanding or typographical error and needs immediate correction to avoid misleading the readers. Image Quality: Figures 1-4 are of insufficient quality for publication. High-resolution images are crucial for the readers' understanding and interpretation of the clinical findings. Please improve the quality of these figures. Treatment Options Discussion: The discussion inaccurately identifies bone grafting as the main treatment option for early-stage neglected femoral neck fractures. In younger patients, the primary approach is typically open reduction and internal fixation. This section requires revision to reflect current clinical guidelines and evidence-based practices.\nMinor Points:\nSome references appear to be outdated. I recommend reviewing the latest literature to ensure the discussion is grounded in the most current understanding of the topic. The case report could benefit from a brief review of literature regarding antipsychotic-induced bone density changes or fractures, adding depth to the discussion. In several instances, the manuscript could benefit from tightening the prose to eliminate redundancy and improve the flow of information.\nIn conclusion, while the case presented is of significant interest and has the potential to contribute valuable insights into the management of femoral neck fractures in patients with schizophrenia,there are certain points to be noted. The manuscript should be well-written, clear, and easy to understand. Technical jargon should be defined or minimized, and the text should flow logically from one section to another. - The research should address an important question or issue in the field. The significance of the findings should be clearly articulated, highlighting how they advance understanding or practice. - Research involving human or animal subjects must adhere to ethical guidelines and obtain appropriate approvals. Authors should disclose any potential conflicts of interest or biases. - The study design, data collection methods, and analysis should be rigorous and appropriate for the research question. Any limitations or potential biases should be acknowledged and addressed. - Results should be reliable, reproducible, and supported by evidence. Conclusions should be drawn based on the data presented.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/12-386
https://f1000research.com/articles/12-228/v1	01 Mar 23	{ "type": "Research Article", "title": "Sugar content and erosive potential of commonly prescribed Orodispersible tablets- An in vitro study.", "authors": [ "Lahari Anand", "Kalyana Pentapati", "Revathi Shenoy", "Geethika Yelleti", "Saurabh Kumar", "Lahari Anand", "Revathi Shenoy", "Geethika Yelleti", "Saurabh Kumar" ], "abstract": "Background: Dental caries is a major non-communicable disease of public health concern caused due to freely available dietary sugars. We aimed to compare the sugar content and erosive potential with duration of use and drug classes of orodispersible tablets (ODTs). Methods: We conducted an in vitro evaluation of the total sugar content (TSC), Potential of Hydrogen (pH), solubility, and Titratable Acidity (TA) of commonly prescribed 62 ODTs. TA was measured by titrating the samples with known amount of. 0.1N sodium hydroxide (NaOH) with phenolphthalein indicator and pH was determined by digital pH meter. TSC was evaluated by phenol sulphuric acid. Solubility was assessed by filtration. Results: Out of the 62 ODTs, majority were Antimicrobials (n=30). One-quarter of the ODTs (26%) had a mean pH below ≤5.5. No significant difference was seen in the mean pH with respect to different drug classes (p=0.082) and duration of use of ODTs. A significant difference was seen in the mean percentage solubility with respect to drug classes (p<0.001). Antimicrobials had the least percentage of solubility as compared to other drug classes. Antiemetics and proton pump inhibitors (24.33 ± 17.34) had significantly higher mean percentage sugar content than Antimicrobials (23.25 ± 17.16). No significant difference was seen in the mean TSC with respect to various drug classes (p=0.718) and between the duration of use of drugs (P=0.568) respectively. No significant difference was seen in the mean percentage TA with respect to drug class (p=0.123) and duration of use of drugs (p=0.424). Conclusion: Overall, we can conclude that one in four ODT formulations had a pH below 5.5 (critical pH). Only one ODT formulation did not have a sugar content. No difference was seen in the mean pH, sugar content, and TA with respect to duration of use of drugs and drug classes.", "keywords": [ "Oral", "Dispersible tablets", "pH", "Sugar content", "Erosive potential" ], "content": "Introduction\n\nDental caries is a major and widespread non-communicable disease of public health concern.1 Freely available sugars in a person’s diet are a major risk factor in the etiology of caries which has a detrimental effect throughout the life (more information is available from the World Health Organisation (WHO) (https://www.who.int/news-room/fact-sheets/detail/sugars-and-dental-caries). Children have less immunity, thus making them vulnerable to chronic diseases which requires administration of medicines. Children who are unwell for an extended period may be more prone for developing caries, and more dietary sugars may increase the likelihood and severity of it.2\n\nMany liquid oral medications (LOM) contain readily available sugars like sucrose and glucose.3 The requirement for easily adjustable dosage forms is one of the most frequent issues in pediatric pharmacology.4 This issue has long been resolved by the development and use of liquid preparations.4 Because of anticipated stability problems with liquid media, like degradation and microbiological contamination, this formulation is not appropriate for all pharmacologically active ingredients.4 Patient-friendly dose forms such as Orodispersible tablets (ODT) were developed to overcome this.4\n\nODT are “uncoated tablets that rapidly disintegrate in the oral cavity before being swallowed, turning into a solution or a suspension using the existing saliva and rarely requiring the use of external liquids such as water.”4 Carbohydrates and polyols have been used in ODT formulations because they are highly soluble in water and have taste-improving properties. Polysaccharides and/or their corresponding alcohols are thus included in several excipient combinations developed for production. The ODT formulations rely on sugars and sugar alcohols.4\n\nPeople of all ages are affected by tooth wear. Dietary erosion is one such factor of tooth wear which is evident in those who consume acidic fruit juices and carbonated soft beverages as well as in patients on specific acidic oral drugs.5 Clinical studies have demonstrated the erosive detrimental effects of vitamin supplements in chewable and lozenge forms,6 and respiratory medications.7 Additionally, research conducted in vitro has demonstrated the demineralizing capability of iron syrups8, effervescent nutritional preparations,9 mouthwashes,10 and drugs used to treat phenylketonuria11 and kidney disease.12 The findings of these studies raised concerns about the adverse dental effects of taking these medicines with a potential of hydrogen (pH) ≤ 5.5 (critical pH).2,13\n\nHellwig and Lussi14 highlighted that those patients who are taking chewable and other dispersible forms of medications need to be aware of their erosive potential, which can cause drug-induced xerostomia. Pharmaceuticals typically employ acids as buffering agents to preserve stability, compatibility, enhance flavour and facilitate dissolution when they come into contact with water.15 To maximize effectiveness and patient acceptance of ODTs, the pH needs to be adjusted. Therefore, preparations with low pH are frequently required for dispersion for better patient compliance.16\n\nThe European Pharmacopoeia stipulates that ODTs must disintegrate in less than three minutes in the water to remain hassle-free17 In contrast, the “FDA defines ODTs as forms that disintegrate in under thirty seconds in an in vitro setting.” (https://www.fda.gov/regulatory-information/search-fda-guidance-documents/orally-disintegrating-tablets)\n\nOnly a few high-quality publications have addressed the role of quantity and frequency of sugar intake as an important factor in caries progression.18–20 The routine use of LOMs and its relation to caries have been linked in numerous studies.21–24 It is known that most of the common paediatric LOMs can cause dental erosion since their pH is below 5.5.13,15,25 But the literature on ODT's erosive potential and sugar content in the Indian pharmaceutical market is lacking. With this background, we aimed to compare the total sugar content (TSC), pH, solubility, and Titratable Acidity (TA) with duration of use of drugs and among different drug classes of various ODTs.\n\n\nMethods\n\nWe conducted an in vitro study to evaluate the TSC, pH, solubility, and TA of commonly prescribed ODTs.\n\nThe study protocol was approved by “Kasturba Hospital and Kasturba Medical College Institutional Ethics Committee” (IEC2 – 22/2022).\n\nThe study involved two main phases: identification of ODTs and laboratory analysis.\n\nPreliminary survey\n\nA preliminary survey was conducted in Udupi city among paediatricians (n=4), and associated pharmacies (n=5), and pharmacists (n=8), about their most prescribed ODT. The participants were contacted personally with prior appointment and briefed about the study followed by written informed consent. Participants were chosen by convenience sampling. Subsequently, they were asked to list generic or brand names that are commonly prescribed to get a comprehensive list of ODTs used. According to the interviews, 22 drugs belonging to five different classes were identified. Further information was gathered to identify different manufacturers, which yielded 62 most prescribed ODTs (Table 1). ODTs were further classified as long-term (anticonvulsants, anti-Tuberculosis, antivirals, antihistamines) and short-term medications (antibiotics, analgesics, and antiemetics).\n\nDetermination of TSC, pH, solubility, and TA was carried out among the above identified ODTs in the department of Biochemistry, Kasturba Medical College, Manipal.\n\nPreparation of sample solution from the ODTs2\n\nEach full dose of ODT was added to 50 mL distilled water (DW) and waited for three minutes for dissolution from which a 2 ml solution was pipetted into a test tube and further diluted with 40 ml DW. The solution was freshly prepared for each analysis.\n\nMeasurement of pH26\n\nA calibrated digital pH meter (“Eutec pH 700, Eutech Instruments Pte Ltd, Thermo Fisher Scientific India Inc, India”) accurate to 0.1 was used to determine the pH. The pH of the sample solution was displayed when the electrode was inserted into the 10 ml sample solution. The values were recorded in triplicate with three different samples and the average value was recorded.\n\nEstimation of TA27\n\nA 40 ml sample solution was pipetted into a 100 ml conical flask. A 50 ml solution of 0.1 N NaOH was taken in a burette. Four drops of phenolphthalein indicator were added to the sample and it was titrated manually against the NaOH solution until a pale pink colour was achieved. The titration was conducted three times using a different ODT. The TA value was determined by multiplying the total amount of 0.1 N NaOH solution needed to neutralize the sample solution by a correction factor of 0.0978. Percentage TA was calculated using the following formula27:\n\nEstimation of TSC28\n\nThe estimation of TSC was done by the colorimetric phenol sulphuric acid method which detects both reducing and nonreducing sugars. Stock sugar solution was freshly made by adding 50 mg of glucose and 50 mg of fructose in 100 ml of saturated benzoic acid (1mg/1ml) (0.42 gm of benzoic acid in 100 ml water). Working standard was prepared by diluting 10ml of stock solution in 100 ml of DW (1 ml = 100 mcg). To prepare dilutions with different concentrations the appropriate volume was taken in the series of test tubes. S1 – S6 (for ex: 0.2 ml = 20 μg of sugar concentration). A calibration curve was made using a series of standards (S1-S6) with concentrations of 20–120 μg. In two test tubes, 0.5 mL of a sample solution was taken. DW was used to fill each tube to a capacity of 2 ml. As a procedural control, 2 ml of DW was utilized as a blank. Each test tube received 0.05 mL of 80% phenol after which 5 mL of concentrated sulfuric acid was dispensed immediately into the solution. The test tubes were covered with parafilm (Bemis Company Inc, Neenah, WI, USA) and a 30-minute incubation period allowed the solution to develop its colour at room temperature. The optical density (OD) was read at 480nm by placing the samples in an auto colorimeter (LT-114, Labtronics, Haryana, India).\n\nPercentage solubility29\n\nA single ODT was added to 50 mL of DW. A pre-weighted filter paper (Grey kalpi filter paper, Saran hand-made paper industry, UP, India) was folded into a cone shape and placed in a glass funnel. The solution was poured into the funnel, and the filtrate was collected in a conical flask. The filter paper was air-dried for 24 hours until no moisture remained and then weighed. The difference between the weight of the filter paper before and after filtration gave the weight of the insoluble precipitate based on the below formula29\n\nAll the analysis was done using SPSS version 20 (IBM Corp. IBM SPSS Statistics for Windows, Armonk, NY) (RRID:SCR_002865). A p-value of ≤0.05 was considered statistically significant. The normality of the data was evaluated using the Shapiro Wilk test. Inter-group comparisons of continuous variables were done using analysis of variance (ANOVA) with post-hoc Tukey's test or Kruskal Wallis ANOVA with Post-hoc Dunn's test based on the distribution of data among various drug classes of ODTs. Similarly, independent sample t-test or Mann-Whitney U test was used to compare with duration of use of ODTs. Sugar content was compared using the Mann-Whitney U test between different pH categories. Underlying data for this study can be accessed at Mendeley Data.30\n\n\nResults\n\nOut of the 62 ODTs, four belonged to Antihistamines; five belonged to Anti-emetics and proton pump inhibitors; eight belonged to analgesics; 14 belonged to Anticonvulsants and antipsychotics; and 30 belonged to Antimicrobials (further divided into antibiotics, anti-tubercular, and anti-viral drugs).\n\nThe distribution of pH, percentage solubility, sugar content, and titratable acidity as per the generic content is listed in Table 1. The mean pH value for all the samples of medicines was 6.11±0.88 (Table 2). The lowest mean pH, with respect to drug class, was seen in Antimicrobials (5.84±0.6) and the highest mean pH was seen in Analgesics (6.71±0.68) (Table 3). There was no significant difference seen in the mean pH among the different drug classes (p=0.082) and between short-term (6.04±1.02) and long-term (6.22±0.61) ODTs (Table 4).\n\nThe mean percentage solubility for all the ODTs was 59.11±17.97 (Table 2). The mean percentage solubility was significantly different among various drug classes (p<0.001). The post hoc test showed least percentage solubility in Antimicrobials than other drug classes. No other significant differences were seen (Table 3).\n\nThe sugar content in the ODTs was reported in two ways. Initially, the TSC was calculated per tablet (as per the dose form), followed by the percentage of sugar present per 100 mg ODT. The total mean sugar content and percentage of sugar were 25.13%±19.5 and 13.28%±16.07, respectively (Table 2). The lowest percentage of sugar content was seen in Antimicrobials (6.67%±7.70), and the highest percentage of sugar content was seen in Antiemetics and proton pump inhibitors (24.33%±17.34). The mean percentage sugar content was significantly different among the five drug classes. The post-hoc test showed that Antiemetics and proton pump inhibitors (24.33%±17.34) had significantly higher mean percentage sugar content than Antimicrobials (23.25%±17.16). No other significant differences were seen. The lowest TSC was seen in antihistamines (23.08%±20.28), while the highest was in Analgesics (34.36%±24.86). (Table 3) The mean TSC for various drug classes did not significantly differ from one another (p=0.718). Similarly, there were no significant differences in the mean percentage sugar content and TSC between long-term ODTs and short tern ODTs (p=0.23 and 0.568) respectively (Table 4).\n\nThe overall mean percentage TA was 0.39% (Table 2). No significant difference was seen in the mean percentage TA between the five drug classes (p=0.123). Similarly, mean percentage TA did not significantly differ between the long-term (0.44%±0.35) and short-term (0.36%±0.31) ODTs (p=0.424) (Table 4).\n\nThe pH, solubility, percentage sugar content, and TA showed no significant difference with respect to duration of use of ODT (p=0.409, p=0.241, p=0.23, p=0.424). A moderate significant negative correlation was seen between the pH and percentage TA (rho=-0.342; p=0.015).\n\nThe distribution of various ODTs as per the drug classes and critical pH was listed in Table 5. One-quarter of the ODTs (26%) had a mean pH below ≤5.5. There was no significant difference in the mean TSC and percentage sugar content with respect to critical pH (p=0.224 and 0.368) respectively (Table 6).\n\n\nDiscussion\n\nWe conducted a comprehensive in vitro analysis of pH, solubility, sugar content, and TA among the commonly prescribed ODTs in the Udupi district, Karnataka, India. The spectrum of medications evaluated was quite broad. However, most of them were antimicrobials, which are less likely to be prescribed frequently and for a more extended period of time because their primary therapeutic indication is for short-term usage to treat acute infections.31 Many excipients are used in the formulation of ODTs to ensure a good taste profile because they have a longer oral residence than other dose forms.32\n\nThe overall mean pH for the total sample of ODTs was found to be 6.11 ± 0.88, which was less than that reported by Maguire et al. and Arora et al. (8.09 and 7.49, respectively).33,34 We found that the pH was lowest in antimicrobials which were similar to Maguire et al. and Arora et al. (4.29, 5.7).2,33 However, antimicrobials in liquid oral medications showed higher pH.3,35 Analgesics in our study showed the highest pH (6.71), which was more than that reported in LOMs.13,15,35,36 Antiemetics had a similar (6.03) pH as compared to Maguire et al. and Arora et al. (6.28 and 6.04, respectively).33,34 Antihistamines had a pH of 6.56, while Maguire et al., Arora et al., Siddiq et al., and Cavalcanti et al. reported pH less than the critical value (4.29, 4.8, 5.5, 4.1 respectively).26,33–35 There was no significant difference in the pH between the short-term and the long-term medication, similar to the previous study.35\n\nTA is indicative of the erosive potential of drugs.37 The overall TA of ODTs was 0.39 g/l, more than that reported by Maguire et al. and Arora et al. (0.04 and 0.06 g/l, respectively).33,34 Antiemetics and proton pump inhibitors showed the highest TA (0.65 g/l), which was higher than previous studies conducted by Maguire et al. and Arora et al. (0.13 and 0.24 g/l respectively).33,34 No significant difference was seen in the TA between long-term and short-term medications, similar to the previous study on LOM (Siddiq et al.).35 Previous in vitro research showed that an acidic drugs can decrease enamel hardness and increase the enamel roughness.38–41 Antimicrobials were the least water soluble seen among the different classes of drugs tested. A direct comparison could not be made as no previous studies were done on the solubility of ODTs.\n\nWe found that 98.37% (61) of the evaluated ODTs presented with sugar similar to previous studies on LOM.35,42 But few studies on LOMs, showed the presence of sugar in 40-70% of the formulations.13,43,44 Out of the 62 ODTs, only one medication in the anticonvulsant category (Lamitor DT) was sugar-free. Antiemetics and proton pump inhibitors showed the highest percentage of sugar content (24.33%), and the mean across all the drug classes was below 25%, which was lower than previous studies on LOM.45 Our study showed no significant difference in the amount of sugar content in long-term and short term ODTs which was like that reported by Siddiq et al.35\n\nThe long-term use of ODTs can be a potential risk for the development of caries especially among children.22,23 According to Passos et al. only 5% sucrose concentration is required to form the cariogenic biofilm.45 The majority of the ODTs contain carbohydrates as an excipient which plays a vital role in forming a tablet.46 Despite available artificial substitutes, like sodium cyclamate, sodium saccharin, aspartame,47 and sorbitol,48 carbohydrates are used widely since they are economical and renewable material and can form tasteless, odorless, and biodegradable tablets which can disintegrate rapidly.\n\nWhen metabolized by cariogenic microorganisms, fermentable sugars can induce a rapid pH drop below critical pH.49 With lower pH among the ODTs, there can be a prolonged acidic environment in the oral cavity. Due to these factors, there can be a potential risk of dental erosion, decreased enamel hardness, increased roughness of tooth surface leading to plaque accumulation, demineralization of enamel, and rapid initiation and progression of dental decay.38–41,50,51 Continuous usage of ODTs over a long time coupled with other risk factors like systemic comorbidities can cause the early onset and rapid progression of dentinal hypersensitivity, pain, rampant caries, and gum diseases. These can lead to poor aesthetics, functional limitations, and poor Oral Health-related Quality of Life.\n\nBuffering capacity and salivary flow rate can vary among individuals with the substantial influence of comorbidities, pre-existing caries, age, gender, hormonal levels, diet, and medications. Healthcare personnel should consider the above factors before recommending ODTs, especially for long-term usage.\n\nOverall, we can conclude that one in four ODT formulations had a pH below 5.5 (critical pH). Only one ODT formulation did not have a sugar content. No difference was seen in the pH, sugar content, and TA between the long-term and short-term ODTs. There were no differences seen in the pH, sugar content, and TA among drug classes. Antiemetics and proton pump inhibitors had higher sugar content than antimicrobials.\n\nThe exact contribution of the acidic potential of ODT on the tooth is unclear, especially in the oral cavity. The buffering capacity of saliva is a complex phenomenon and has the role of multiple host factors, which can vary significantly among individuals. Citric acid-containing ODTs with high TA and low pH can stimulate the salivary flow, thereby increasing the saliva’s buffering capacity.49 The effect of salivary buffering capacity and plaque pH due to ODTs needs to be explored in the clinical scenario.\n\nThe comprehensive list of ODTs was limited to this geographic area. Hence, formulations of ODTs can vary in different countries and guidelines. Regular and long-term use of ODTs with longer oral clearance can be detrimental to oral health. Reformulating the medications as sugar-free can reduce the risk of various dental conditions as compared to the sugar containing formulations.52 Manufacturers should consider reformulating these medications whenever feasible to minimize the potential risk of oral conditions.\n\nHealthcare professionals should be aware of the potential risks of oral conditions due to the extended use of ODTs. Hence prescription of these ODTs should be limited to those individuals where compliance for swallowing is poor. Health care personnel should advise comprehensive oral examination and treatment of existing oral conditions before initiating extended ODT therapy. Also, individuals should be advised to follow at-home oral hygiene measures like gargling with water immediately after taking ODTs, use of fluoridated toothpaste, and brushing twice daily with or without supervision. Frequent dental examinations to monitor the oral conditions and progression of the same should be recommended. Preventive measures like topical fluoride applications, preventive sealants, and remineralising agents can be recommended to minimize the above oral conditions. However, sugar-free ODTs may contain polyols which may have laxative effects over long-term usage, and certain polyols are not recommended in children.53", "appendix": "Data availability\n\nMendeley Data: Evaluation of sugar content and erosive potential of commonly prescribed Orally Dispersible Tablets- An in vitro study, https://doi.org/10.17632/w5z857k3cw.2. 30\n\nThis project contains the following underlying data:\n\n- Mendeley Data Dr Lahari.xlsx\n\n- Mendeley extended Data Dr Lahari 03-02-23.xlsx (interview responses)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nGiacaman RA, Fernández CE, Muñoz-Sandoval C, et al.: Understanding dental caries as a non-communicable and behavioral disease: Management implications. Frontiers. Oral Health. 2022; 3: 3. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "165215", "date": "27 Mar 2023", "name": "Tarakant Bhagat", "expertise": [ "Reviewer Expertise Oral health", "dental caries", "oral cancer", "oral health related quality of life", "musculoskelatal disorders", "burnout" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 compared the sugar content and erosive potential of commonly used orodispersible tablets (ODTs) in Udupi city of south India. Although the list of drugs was obtained from a single site, these are the most commonly used drugs throughout India. It was observed that one in four ODT formulations had a pH below 5.5 (critical pH); hence, regular and long-term use of ODTs with longer oral clearance can be detrimental to oral health. Recommendations like following up of at-home oral hygiene measures such as gargling with water immediately after taking ODTs, use of fluoridated toothpaste, and brushing twice daily with or without supervision, frequent dental examinations to monitor the oral conditions, preventive measures like topical fluoride applications, preventive sealants, and re-mineralising agents to minimize the oral conditions are important.\n\nThe article is well written. This research will certainly be helpful to all the practitioners while prescribing such drugs, especially to children.\nIn the results section, it is mentioned that a moderate significant negative correlation was seen between the pH and percentage TA, but the test used is not mentioned in the methods section. Please mention the name of the correlation test used in the relevant section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9566", "date": "13 Apr 2023", "name": "Kalyana Pentapati", "role": "Author Response", "response": "Query: In the results section, it is mentioned that a moderate significant negative correlation was seen between the pH and percentage TA, but the test used is not mentioned in the methods section. Please mention the name of the correlation test used in the relevant section. Response: We have added the information in the statistical analysis section of the manuscript. “Correlation between pH and TA was evaluated using Spearman’s rho.”" } ] }, { "id": "165216", "date": "04 Apr 2023", "name": "Harsh Priya", "expertise": [ "Reviewer Expertise Public health Dentisry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 innovative in nature. Congratulations to the authors.\nA few clarifications.\nThe mean TSC should not be expressed as percentage: “The total mean sugar content and percentage of sugar were 25.13%±19.5 and 13.28%±16.07, respectively (Table 2)”\n\nThe figure entered needs to be rechecked for antimicrobials: “the post-hoc test showed that Antiemetics and proton pump inhibitors (24.33%±17.34) had significantly higher mean percentage sugar content than Antimicrobials (23.25%±17.16).”\n\nThe pH number can be mentioned. This would help in better understanding the comparability: “However, antimicrobials in liquid oral medications showed higher pH.”\n\nEnglish correction is suggested for the following sentence, “Buffering capacity and salivary flow rate can vary among individuals with the substantial influence of comorbidities, pre-existing caries, age, gender, hormonal levels, diet, and medications.”\n\nA mention of confounding factors with suitable explanation is suggested.\n\nExcipients can be further explained in the manuscript. The following reference can be helpful: Hannan et al., (2016)1.\n\nNewer innovations are formulated for caries prevention due to ODTs2.\n\nThank you.\nBest wishes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9567", "date": "13 Apr 2023", "name": "Kalyana Pentapati", "role": "Author Response", "response": "Query: The mean TSC should not be expressed as percentage: “The total mean sugar content and percentage of sugar were 25.13%±19.5 and 13.28%±16.07, respectively (Table 2)” Response: Thank you. We have incorporated the change in the manuscript. Query: The figure entered needs to be rechecked for antimicrobials: “the post-hoc test showed that Antiemetics and proton pump inhibitors (24.33%±17.34) had significantly higher mean percentage sugar content than Antimicrobials (23.25%±17.16).” Response: Thank you for highlighting the typo. We have incorporated the change in the manuscript. “The post-hoc test showed that Antiemetics and proton pump inhibitors (24.33%±17.34) had significantly higher mean percentage sugar content than Antimicrobials (6.67%±7.7).” Query: The pH number can be mentioned. This would help in better understanding the comparability: “However, antimicrobials in liquid oral medications showed higher pH.” Response: Yes. Thank you. We have mentioned the same in the manuscript. (6.4 and 6.36) Query: English correction is suggested for the following sentence, “Buffering capacity and salivary flow rate can vary among individuals with the substantial influence of comorbidities, pre-existing caries, age, gender, hormonal levels, diet, and medications.” Response: We have revised the text in the manuscript. “Buffering capacity and salivary flow rate can vary among individuals with a substantial influence of comorbidities, pre-existing caries, age, gender, hormonal levels, diet, and medications.” Query: A mention of confounding factors with suitable explanation is suggested. Response: We have added this information. “The role of excipients and processing of ODTs can have substantial influence on the pH and sugar content. So ODTs of a particular drug may have different levels of pH and sugar content based on the dosage forms and can vary with different manufacturers. Hence, the current findings cannot be generalised to other similar available ODTs.” Query: Excipients can be further explained in the manuscript. The following reference can be helpful: Hannan et al., (2016)1. Response: We have added this information. “Excipients in ODTs have a superdisintegrant, a bulking agent, a lubricant and swelling agent, a permeabilizing agent, sweeteners and flavorings agents which may have potential influence on the pH and sugar content of ODTs.” Query: Newer innovations are formulated for caries prevention due to ODTs2. References 1. Hannan PA, Khan JA, Khan A, Safiullah S: Oral Dispersible System: A New Approach in Drug Delivery System. Indian J Pharm Sci. 2016; 78 (1): 2-7 PubMed Abstract \| Publisher Full Text 2. Kiniwa R, Miyake M, Kimura SI, Itai S, et al.: Development of muco-adhesive orally disintegrating tablets containing tamarind gum-coated tea powders for oral care.Int J Pharm X. 2019; 1: 100012 Response: Thank you for your insight. These innovative products are yet to be proven for their effectiveness for caries prevention." } ] } ]	1	https://f1000research.com/articles/12-228
https://f1000research.com/articles/10-1156/v1	15 Nov 21	{ "type": "Research Article", "title": "Does receiving a cash grant improve individual earnings in a war-torn country? Evidence from a randomized experiment in Afghanistan", "authors": [ "Fatema Kashefi", "Hisahiro Naito", "Fatema Kashefi" ], "abstract": "Background: In this study, the effect of receiving a non-trivial cash grant and mentorship for business practice on individual earnings in Afghanistan was assessed. Methods: This randomized control trial (RCT) included 2177 individuals (n=2177), with the age range of 18-35 years. The amount of cash grant (500 USD) was approximately equal to the gross domestic product (GDP) per capita in the country. By a process of lottery, eligible applicants were equally divided into the group that received the grant (treatment group) and the group that did not (control group). Results: Almost two years after this study, the percentage of increased annual income to the size of this cash grant was approximately 173 % and 69 % for males and females, respectively. This result was much higher than the estimated percentage reported in previous literature assessing the effect of microfinance loans and cash grants in other countries. The treatment group had 7% higher probability of having an officially registered business than the control group. The treatment group also increased labor supply and employed additional workers, compared to the control group. The male treatment group bought more capital equipment (e.g., trike and commercial carts) than the male control group, while the female treatment group bought more domestic animals. Conclusion: A non-trivial cash grant has a strong positive effect on the earnings of the recipient of the cash grant in Afghanistan. If the state of increased income continues for the next few years, the sum of increased income will be more than the sum of the administration cost, the interest cost and the amount of cash grant given. This implies that the project passes the standard criteria to determine the appropriateness of government program. Additionally, we did not find any economically and statistically significant positive effect of mentorship on income.", "keywords": [ "Cash Grant", "Income", "Poverty", "RCT", "Afghanistan", "microenterprise" ], "content": "Introduction\n\nThe Lack of adequately paying jobs has become a major challenge in Afghanistan. In 2016–2017, according to the Afghanistan Living Conditions Survey, 25% of the population were unemployed and 54% lived below the poverty line (Islamic Republic of Afghanistan Central Statistics Organization, 2018). Even among those employed, their employment is mainly informal and through microenterprises. World bank (2019) reports that 80% of those employed have unstable jobs by either being self-or family employed or having short-term (daily) employment in informal microenterprises.\n\nHowever, the prevalence of informal microenterprise is not unique to Afghanistan but common among the majority of developing countries (La Porta & Shleifer, 2014). Thus, the main question would be how the prevalence of informal microenterprises can be transformed into an opportunity for income growth and stable employment.\n\nIn the last decade, through randomized experiments, a growing number of studies have indicated the strong and positive effect of cash grants on income growth and microenterprise activities in developing countries (Blattman et al., 2014; Blattman et al., 2020; De Mel et al., 2008; De Mel et al., 2012; Fafchamps et al., 2014; Haushofer & Shapiro, 2016; McKenzie, 2017). In Afghanistan, however, there are several reasons as to why such cash grants might not be effective in increasing microenterprise activities and income. First, the lack of security is still a major issue in this country. According to the United Nations Assistance Mission to Afghanistan, the number of civilian death due to conflicts and explosives was over 3000 yearly, from 2014 till 2017(United Nations Assistance Mission in Afghanistan, 2018). Second, the female unemployment rate (41%) is higher than the male unemployment (18%), which is mainly due to the negative attitude and lack of social acceptance and support for women in business (Allen et al., 2007; Beath et al., 2013). Third, Afghanistan is recognized as one of the most corrupted countries worldwide. (United Nations Office on Drugs and Crime, 2012). In the corruption perception index, which ranks 180 countries and territories based on their recognized levels of public sector corruption, Afghanistan was ranked at fourth from the bottom in 2017 (Transparency International, 2017). Several official documents report that the corruption in Afghanistan hinders the effectiveness of aid programs from enhancing enterprise activities (Special Inspector General for Afghanistan Reconstruction, 2018; Special Inspector General for Afghanistan Reconstruction, 2021). As such if weak microenterprise activities are mainly due to these three factors, then providing cash grants in Afghanistan might not be as effective as in other countries.\n\nIn this study, we have analyzed the effects of receiving a non-trivial amount of cash grant in Afghanistan for individuals aged 18–35 on their income growth.\n\nOur study has several unique aspects. First, most previous studies with the same topic were conducted in relatively peaceful countries, except for post-conflict studies in Uganda by Blattman et al. (2014) and Blattman et al. (2020). However, Afghanistan has been at war for the past 20 years, which ended recently (Wall Street Journal, 2021). Thus, knowledge on how to improve people's income in such a country will be useful for other fragile war-torn countries that need reconstruction. Second, Afghanistan is an Islamic country where Islam with a strong norm, is practiced. In the strictest interpretation of the religion, lending money with interest is prohibited (Nagaoka, 2012). Therefore, it is important to evaluate whether receiving a cash grant, which does not involve the repayment of the principal and interest, would be effective in this country. Third, in this study, we provide a large amount of cash grant, which is almost equal to the gross domestic product (GDP) per capita. Fourth, most importantly, Islamic Republic of Afghanistan ended on August 15, 2021, without much resistance to Taliban (Wall Street Journal, 2021). The fact that Taliban took over the country quite easily may indicate that people had already lost trust in the government even before 2021, because the aid programs were not working well due to bad designs and implementation of those programs (BBC, 2021; Special Inspector General for Afghanistan Reconstruction, 2018). Thus, it is important to evaluate to what degree is this specific government program effective.\n\n\nMethods\n\nHigh population growth rate of Afghanistan along with the lack of security in the country creates a challenging environment for reasonable job opportunities. Weak security conditions make it difficult to conduct business, while reducing the incentive for new businesses.\n\nAccording to Afghanistan Living Conditions Survey 2016–2017 (Islamic Republic of Afghanistan Central Statistics Organization, 2018), unemployment rate defined as the ratio of people who are seeking a job in the labor force but do not have one, was 25% in this country. In particular, the male unemployment rate was 18%, whereas the female unemployment rate was 41%, in that year. Even among those who were employed, 19.7% of males and 23.7% of females stated that they were seeking additional work. Among those employed, 17.2% were salaried employees (including public sector). Specifically, unpaid male family employees were 24.5%, and daily labor employees were 15.7%. It was reported that 40.1% were business owners.\n\nIn 2017, during this experiment, the Special Inspector General for Afghanistan Reconstruction reported that approximately 59.7% of the country's 407 districts were under Afghanistan government control or influence; 11.1% of total districts were under insurgent control or influence; 29.2% of all districts were contested districts. (Special Inspector General for Afghanistan Reconstruction, 2017). According to the United Nations Assistance Mission to Afghanistan, the number of civilian death due to conflicts and explosives was 3701, 3565, 3510, and 3428, in 2014, 2015, 2016, and 2017, respectively (United Nations Assistance Mission in Afghanistan, 2018). Meanwhile, the number of injuries caused by these activities for the same years was 6384, 7419, 7924, and 7015, respectively.\n\nIn terms of education, there is a substantial disparity between males and females, depending on the area. For example, in urban areas, 66.9% of males and 40.8% of females are literate, whereas in rural areas these values decrease to 45.6% and 13.1% for males and females, respectively. The poverty rate is also severe in Afghanistan. In 2016, it was reported that 54% of households had expenditure less than the poverty threshold.\n\nThe program \"Promoting Entrepreneurship among the Youth in Afghanistan\" (PEYA) was a Randomized Control Trial (RCT)-based project funded by the Afghan Reconstruction Trust Fund. It was launched in August 2015 and ended in July 2018. The PEYA program was implemented by the Ministry of Labor and Social Affairs of Afghanistan in Balkh, Kabul, and Nangarhar provinces. The program's objective was to help establish new microenterprises or expand existing businesses by providing a cash grant of USD 500 (approximately 33,500 Afghani (Afs)), which is approximately equivalent to 2017 GDP per capita. The amount of this grant can be considered significant in a country with high levels of unemployment and illiteracy. In this study, 3490 eligible applicants were chosen depending on their business proposals and social economic background. Based on a lottery process, half of the study participants (1745) were assigned to the treatment group where they received a cash grant, whilst the other half who were assigned to the control group did not. The lottery and the baseline interview were conducted in August 2016. Due to the potential lack of sufficient numbers of business mentors in all provinces, mentorships were randomly given to half of the individuals who received the cash grant and lived in Kabul.\n\nAs presented in Table 1, the key eligibility criteria for the applicants were male and female aged between 18–35 years, who were illiterate or had a minimum education, and who resided in one of three provinces: Balkh, Kabul, and Nangarhar. The selected applicants had to have a national ID (Tazkira) and a bank account. This project was implemented through the collaboration of four teams: i) the Non-formal Approach to Training, Education, and Jobs in Afghanistan team (NATEJA), ii) the Provincial Directories of the Ministry of Labor and Social Affairs, iii) the Employment Service Centers (ESCs) (set up for better coordination of the intervention), and iv) the implementing team of the Ministry of Labor and Social Affairs.\n\nSource:Narrative Progress Report (1st Quarter: Jan to Mar 2016) for subcomponent 3.2 (Promoting Entrepreneurship among Afghan Youth) August 8 2016, Non-formal approach to training education and jobs in Afghanistan(NATEJA)\n\nFigure 1 shows the procedure of the experiment. The selection process of the grantees started by providing information about the intervention and eligibility criteria. Four districts in each province were specified. The operation teams were assigned to mobilize the communities in these areas. This process lasted for 30 days, and each community was visited twice. During the initial briefing (week 1) and refresher briefing (week 2), information about the objective and selection criteria was also shared with local authorities, such as the governor's office staff, provincial councils, and community leaders, to have their support.\n\nSource:Narrative Progress Report (1st Quarter: Jan to Mar 2016) for subcomponent 3.2 (Promoting Entrepreneurship among Afghan Youth) August 8 2016, Non-formal approach to training education and jobs in Afghanistan(NATEJA).\n\nThe application form was a three-page questioner containing 19 questions that covered six sections (skills, support, and investment; product/services; price; place; promotion; customers). The answers to most questions were closed-ended; however, there were several questions that had open-ended options to provide space for detailed answers. Each question and option had equivalent marks. Completion of the questioner on average took 15–20 minutes. Quality assurance was made by the training and moderating committee at the local level. Collectedly, the project had 8238 applications from the three provinces (Non-formal approach to training education and jobs in Afghanistan(NATEJA), 2016).\n\nConsidering the low level of literacy within the communities, application forms printed on large banners were also provided in addition to conventional brochures and posters. Subsequently, the applicants were selected based on an evaluation of their initial submitted business plan, which included information, such as education, age, demographic characteristics, income, work experience, business type, and their plans on how they would spend the grant. In many cases, the applicants were assisted by other family members, neighbors, etc., who had a higher level of education and were able to convey the information in the application form. The applications with a higher score than the threshold level of the score become qualified for the lottery process. After the qualified applicants were selected, scheduled meetings for baseline interviews and lottery were announced to be held in each province. At the meetings, professionally trained interviewers conducted the baseline interviews. After which each qualified applicant drew a lottery number that determined the receipt of the cash grant.\n\nGiven the amount of the cash grant and concern about corruption in the process of choosing the recipients, it was important that all applicants understood that the recipients of cash grants were chosen in a transparent manner. Thus, the process of lottery took place in the presence of representatives from provincial councils, provincial governors, the Implementing Partner Agency (IPA), ESCs, and representatives of provincial directorates of labor and social affairs. After determining the recipients of the grant, a three-member panel verified the information provided, and the grant agreement was signed. Finally, the full grant was transferred in one transaction to the grantees' submitted bank account.\n\nAt the endline, the evaluation teams started to interview individuals among those who participated at the baseline, from June 2018 till July 2018. During this period a total of 3005 individuals were interviewed. However, due to miscommunication between the Ministry of Labor and Social Affairs and the external teams the study ID numbers and national ID numbers for some surveyed individuals in the endline that could match their endline with the baseline data, was not recorded by the external teams. To ensure that correct matching was implemented, we included only individuals whose study ID number and national ID number were matched with the baseline and the endline data in the final analysis sample. As a result, the final sample size was 2177 individuals (n=2177) in this study, with the number of the individuals in the treatment group (1146) slightly higher than that of the control group (1031) at the endline (Table 2), implying 38 % attrition rate.\n\nNotes: For the endline data, due to administrative erros, the matching id that matches the endline data with the baseline data were lost due to miscommunication between the ministry of labor and social affairs and the data collecting company. As a result, the sample size of the cleaned data set, which matches the baseline data set with the endline data set is much smaller than the endline data set surveyed.\n\nIn order to address the potential bias that can occur due to the high attrition rate, several robust bounding methods have been used in this study (Lee, 2009; Manski, 1990) to control this high attrition rate, as presented in the robustness checks.\n\nAs the PEYA program is a RCT experiment, we estimate the effect of the policy by estimating the intent to treat (ITT) with the following analysis of covariates specification:\n\nwhere i is the index of an individual, Yi,A is the outcome variable in the endline period (e.g., monthly earnings in the last month) for individual i, Yi,B is the outcome variable at the baseline of individual i, and Granti is the dummy variable which is equal to one if individual i received the grant and zero if not.\n\nAs discussed, the grant was randomized at each individual level. Thus, to calculate the standard error, we used the robust standard error. β1 captures the impact of the cash grant; Xi,B is a set of control variables at baseline, which includes age group dummy, province dummy, years of schooling, and zero education dummy; and εi refers to the individual error term. We estimated the above equation using ordinary least squares (OLS). In order to address the potential bias that can occur due to the high attrition rate, several robust bounding methods were applied in this study (Lee, 2009; Manski, 1990). Stata version 16 was used for the study analysis.\n\n\nResults\n\nTable 3 shows the summary statistics of baseline variables and the balancing test of the sample used in the study analysis. Panel A shows that of the 2177 participants, 72% of the individuals were male, and 42% were in 18–23 age group. Moreover, 54% of the participants were married at baseline. Approximately 47% of them were heads of the household at baseline.\n\nNotes. The total sample size is 2177. The sample size of the male sample is 1562 and the and sample size of the female sample is 615. All variables are from the baseline survey. The joint significant test regresses the treatment dummy on baseline covariates and then testing the estimated coefficients are all equal to zero. For regresses the treatment dummy on baseline covariates, we eliminated age 30-35 dummy, Nanghar dummy, and other langue dummy due to multi-colinearity. * p<0.01, p<0.05, * p<0.1.\n\nPanel B shows the geographic distribution of the three provinces and their native language. The geographic distribution is evenly apportioned between the treatment and control groups. This panel shows that approximately 51% of the participants have stated Dari language as their mother tongue, 46% of them were Pashto native speakers, and 3% were minority language speakers (i.e., Turkemni, Uzbeki, and Pashai).\n\nPanel C shows income-related information at the baseline. The results indicate that 92% of the individuals in our study were new to business, implying that they had not setup their businesses at the baseline. Specifically, the average income in the last month at the baseline was approximately 4,000 Afs. The P-value (0.33) of the joint significant test as shown in Table 3, shows that our analysis sample was balanced.\n\nFigure 2 shows the histogram of monthly income for those who had received cash grants and those who had not at baseline. According to estimates by the National Statistical Office in a 2016–2017 study, the poverty line threshold was 2,064 Afs per person per month (Islamic Republic of Afghanistan Central Statistics Organization, 2018). Thus, the average income in our study sample was two times higher than the income at the poverty line. However, as shown in Figure 2, a substantial percentage of the population had zero income. Figure 2 also shows that the distribution of income was quite similar between those who received cash grants and those who did not.\n\nFigure 3a and 3b display the histogram of monthly earnings for the male and the female groups, respectively. Figure 3a and 3b show that the distributions of earnings for the male and the female samples were quite different. For example, less than 20% of the male sample have zero earnings. However, more than 70 % of the female sample have zero earnings.\n\nTable 4 shows the mean and balancing test of the key variables of the baseline variables in the male group. About 42% of this group were without education, and 57% were married. The average monthly income for this group was 5130 Afs, although 17% were without income.\n\nNotes. The total sample size of the male sample is 1562. The joint significant test regresses the treatment dummy on baseline covariates and then testing the estimated coefficients are all equal to zero. For regressing the treatment dummy on baseline covariates, we drop age 30–35 dummy Namghar dummy and other langue dummy due to multi-collinearity. * p<0.01, p<0.05, * p<0.1C.\n\nTable 5 shows the mean and balancing test of the key variables in the female group. About 47% of this group were without education, and 48% were married. The average monthly earnings of this group were 5130 Afs; however, 76 % were without income.\n\nNotes. The total sample size of the female sample is 615. The joint significant test is regressing the treatment dummy on baseline covariates and then testing the estimated coefficients are all equal to zero. For regressing the treatment dummy on baseline covariates, we drop age 30–35 dummy, Nanghar dummy and other langue dummy. * p<0.01, p<0.05, * p<0.1.\n\nTable 6 displays the balancing test regarding the mentorship program. The p-value of the joint significant test shows that the data is balanced between the group who received the mentorship and who did not.\n\nNotes. The sample is individuals who received the grant and lived in Kabul. The joint significant test is regressing the mentorship dummy on baseline covariates and then testing the estimated coefficients are all equal to zero. For regressing the treatment dummy on baseline covariates, we eliminated age 30–35 dummy Nanghar dummy and other langue dummy. * p<0.01, p<0.05, * p<0.1.\n\nTable 7 displays the index of different types of assets between the control and treatment groups. These assets were divided into Group 1: productive durable, including animal ownership, and local transportation equipment (e.g., cart, three-wheel/truck, and bike), and Group 2: consumer durable, including household goods (e.g., home appliances and mobile phones. To construct household asset-related indices, the values of each variable were standardized to ensure that the value of each variable represents its difference from the mean. Then, the variables are compiled into indices using factor analysis.\n\nNotes: The level of assets is standardardized including the baseline and the endline. Thus, many values are negative in the baseline. * p<0.01, p<0.05, * p<0.1\n\nIn our analysis sample, according to Table 7, the p-value of the joint significant test was 0.49, which shows that the mean was balanced between the control and treatment groups.\n\nDespite the high attrition rate, overall, the balancing tests shown in Table 3 – Table 7 show that the balance between the treatment and control groups was maintained in the analysis sample.\n\nTable 8, column 1 shows the effectiveness of receiving cash grants on monthly earnings of the entire group. These results show that receiving cash grants increased the treatment group's monthly earnings by approximately 4000 Afs. As such, the increased annual income will be 48,000 Afs, which is more than the amount of the grant that an individual in the treatment group received. Considering the 33,500 Afs cash grant, the percentage of the increased annual income to the amount of the cash grant would therefore be:\n\nNotes. Robust standard errors in parentheses. OLS estimation is applied. Control variables are, the receipt of mentorship dummy, monthly earnings at the baseline, years of schooling, no education dummy, gender dummy (full sample), marital status, household size, head of household dummy, age group dummies, province dummies, language dummies, new to business dummies, zero earnings dummy, number of plots, the house ownership dummy, non-agricultural business dummy, having animal livestock dummy. All control variables are from the baseline information. The estimated coefficients of control variables are shown in Table S1 of Extended Data * p<0.01, p<0.05, * p<0.1.\n\n(48000/33500) × 100 = 143%.\n\nColumn 2 displays the effect of receiving a cash grant on monthly earnings in the male group. This shows that receiving the cash grant increased the monthly earnings by about 4839 Afs, and therefore the percentage of the increased annual income to the amount of the cash grant for this group was 173%.\n\nColumn 3 displays the effect of receiving the grant on monthly income on the female group. The column shows that receiving the cash grant increased the monthly earnings by about 1900 Afs, which is less than half of the estimated effect on the male group. This implies that the percentage of the increased annual income to the amount of the cash grant for this group was 69%.\n\nThus, in either of these three cases, if the state of increased income continues for the next few years, the sum of increased income will be more than the sum of the administration cost, the interest cost and the amount of cash grant given. This implies that the project passes the standard criteria to determine the appropriateness of government program.\n\nIn columns 4–6, we added several important covariates and checked whether our estimated coefficients were sensitive to the inclusion of those control variables. These columns indicate that the estimated coefficients of the grant variable reported in columns 1–3 are not sensitive to the inclusion of various control variables (Extended data) (Naito, 2021b). Additionally, the results in Table 8 show that providing mentorship did not influence the rise in the monthly earnings.\n\nAs mentioned previously, the endline study sample size was much smaller than that of the baseline due to the administrative error during the data collection. One concern was whether such attrition of the sample affects the estimated coefficients. Therefore, we controlled for the attrition by applying the inverse probability weighted (IPW) estimation (Wooldridge, 2002; Wooldridge, 2010) (Table 9, panel A). To calculate the standard error correctly, we use the Generalized Moment Method estimation (GMM) with IPW.\n\nThe Effect of Receiving Cash Grant on Monthly Income.\n\nNotes: Robust standard errors in parentheses. Control variables are the same as the ones used in Table 8. * p<0.01, p<0.05, * p<0.1.\n\nTable 9, column 1 of Panel A shows the estimation results of applying IPW to the full sample. It shows that the effect of receiving the cash grant on monthly earnings was 3984 Afs, which was quite similar to the estimated coefficient in column 4 of Table 8 (i.e., 4036 Afs). Column 2 displays the estimated coefficient using IPW for the male group. The estimated coefficient was 4876 Afs, similar to the estimated coefficient displayed in column 5 of Table 8 (i.e., 4984 Afs). Column 3 of Panel A presents the estimated coefficient for the female group by using IPW. The estimated coefficient is 2062, which is close to the estimated coefficient in column 6 of Table 8 (i.e., 2070 Afs). Panel A shows that, when attrition is controlled by IPW estimation, the estimated coefficients of the cash grant were similar to the estimated coefficients obtained in the OLS estimation presented in Table 8.\n\nTable 9, Panel B shows bounding treatment effects. As demonstrated by Karlan & Valdivia (2011); Manski (1990), and Blattman et al. (2014), we first imputed the outcome variable for the unfound sample by assuming that the monthly earnings of unfound treatment group members were equal to the average monthly earning of the found treatment group minus 0.25 standard deviation of the monthly earnings of the found members. We also assumed that the monthly earnings of the unfound control group were the average monthly earnings of the found control group plus 0.25 standard deviation of the found members. After this imputation, we estimated the effects of the cash grant and other covariates using both the found and unfound samples. With this assumption, we found that receiving the cash grant increased the monthly income by 2000 Afs and 1200 Afs for the male and female groups, respectively. Thus, the cash grant is likely to have increased the monthly earnings.\n\nTo further correct for attrition bias, Lee's treatment effect bounds for receiving the cash grant on monthly earnings was estimated (Table 9, Panel C) (Lee, 2009). The lower bound of the effect of receiving the cash grant was 2328 Afs and 1637 Afs for the male and female groups, respectively. The OLS estimates were between the lower and upper bounds for each specification and the lower bounds, 2328 Afs and 1637 Afs for the male and female groups were statistically different from zero (p<0.01). Thus, even with conservative estimates, the percentage of the increased annual income to the amount of the cash grant would be 83% for the male group and 58% for the female group.\n\nOverall, analysis based on several estimation methods demonstrates that the effect of receiving the cash grant on monthly earnings was economically and statistically significant.\n\nTable 10 shows the effect of the cash grant on having an officially registered business, labor supply and asset levels. In this table, Panel A displays the effect of the cash grant on the probability of having an officially registered business at endline. Column 1 of Panel A shows that an individual who received the cash grant had an 8% higher probability of having an officially registered business on average. Column 2 and 3 show that male and female individuals who received the cash grant have 7% and 9% higher probability of having an officially registered business, respectively. As shown in columns 4–6, we added several important control variables. The columns also show that the estimated coefficients of the treatment dummy were similar to the estimated coefficient in columns 1–3.\n\nNotes: Robust standard errors in parentheses. OLS estimation is applied. Control variables are, the receipt of mentorship dummy, the outcome variable at the baseline, years of schooling, no education dummy, gender dummy (full sample), marital status, household size, head of household dummy, age group dummies, province dummies, language dummies, new to business dummies, zero earnings dummy, number of plots, the house ownership dummy, non-agricultural business dummy, having animal livestock dummy. All control variables are from the baseline information. The estimated coefficients of control variables are shown in Table S2-S5 of Extended Data * p<0.01, p<0.05, * p<0.1\n\nPanel B shows the effect of the cash grant on the number of household members working for a microenterprise. Panel B shows that an individual who received the cash grant increased the number of household members working for their microenterprise by 0.2 persons on average. Column 2 of Panel A, a male individual who received the cash grant increased the number of family members working for their microenterprise by 0.18 persons on average. Column 3 shows that a female individual who received the cash grant increased the number of family members working for their micoenterprise by 0.254 persons on average.\n\nPanel C shows the effect of the cash grant on the number of non-household members working for a microenterprise. Column 1 shows an individual who received the cash grant increased the number of non-family members who work for their microenterprise by 0.23 persons. Column 2 shows that, for the male group who had received the cash grant, the employment number of non-family members increased by 0.232 persons. The results in column 3 show that, for the female group who had received the cash grant, the number of non-family members who were employed for their microenterprise increased by 0.229 persons.\n\nWhen starting or extending a business, additional equipment, such as phone, truck, and cart, might be needed. Panel D shows the effect of receiving cash grants on the general asset index for production. To calculate the general asset index for production, we first standardized the level of each asset for production. Then, through the principal component analysis, we determined the weight for each asset and calculated the weighted index (Jolliffe, 2005). As shown in columns 2 and 3, we found that, receiving the cash grant increased standard deviations of the index of assets for production by 0.176 and 0.062 for the male and the female group, respectively. When the control variables were included, the cash grant increased the index of assets for production by 0.167 standard deviation for the male group and by 0.074 standard deviation for the female group (columns 5 and 6).\n\nResults in Panel E display the index of assets for consumption. Columns 2 and 3 show that receiving the cash grant increased the index of assets for consumption by 0.1 standard deviation for the male group, while decreasing it by 0.02 standard deviation for the female group. When the control variables were included in the estimation, the estimated coefficient and statistical significance did not change. Columns 5 and 6 show that the cash grant increased the index of assets for consumption for the male group by 0.094 standard deviation, while decreasing it by 0.073 standard deviation for the female group. Thus, for the male group, receiving the cash grant increased the index of assets for consumption. However, our results do not reflect the same for the female group.\n\nTable 11 shows the effect of receiving the cash grant on the levels of different types of assets. The results in this table show that, for the male group, receiving the cash grant increased the level of assets, e.g., bicycles, carts, three-wheel trucks, mobile phones, and cows. Conversely, the number of cows was the only asset that increased after receiving the cash grant in the female group.\n\nNotes. Robust standard errors in parentheses. OLS estimation is applied. Control varialbles are, the receipt of mentorship dummy, the outcome variable at the baseline, years of schooling, no education dummy, gender dummy (full sample), matrial status, household size, head of household dummy, age group dummies, province dummies, language dummies, new to business dummies, zero earnings dummy, number of plots, the house ownership dummy, non-agricultural business dummy, having animal livestock dummy.All control variables are from the baseline information.* p<0.01, p<0.05, * p<0.1.\n\n\nDiscussion\n\nThe government of Islamic Republic of Afghanistan collapsed on August 15, 2021 (Wall Street Journal, 2021). The minimal resistance to Taliban takeover, may indicate that all the efforts by the western societies for development of this country might not have been entirely effective (BBC, 2021). Several official documents by the western governments state that development aids to enhance enterprise activities in Afghanistan failed due to bad implementation of the programs (Special Inspector General for Afghanistan Reconstruction (2018); Special Inspector General for Afghanistan Reconstruction (2021)).\n\nIn contrast, the results of this study have shown that the effect of a non-trivial cash grant on income is substantial in Afghanistan. The percentage of increased annual income to the size of this cash grant almost two years after receipt of the cash grant was approximately 173% for males and 69% for females. The treatment group had a 7% higher probability of having an officially registered business than the control group. The treatment group also increased labor supply and employed additional workers, compared to the control group. The male treatment group bought more capital equipment (e.g., trike and commercial carts) than the male control group, while the female treatment group bought more domestic animals. Our results are consistent with the previous studies in other countries which similarly show a positive effect of cash grants on income (Blattman et al., 2014; Blattman et al., 2020; De Mel et al., 2008, De Mel et al., 2012; Fafchamps et al., 2014; Haushofer & Shapiro, 2016; McKenzie, 2017). However, our estimated coefficient is higher than the estimated coefficients in the previous literature.\n\nWe would like to emphasize, however, that this study had some limitations. First, the endline survey was conducted almost two years after the study. Thus, the short-term effect of cash grants was measured. Blattman et al. (2020) who studied the long-term effects of grants on poverty, have shown that cash grants are ineffective after 9 years. Thus, it is possible that the strong effect of the cash grant seen in this study might be less significant in the long term. Second, this PEYA program focuses on microenterprises, not small- or medium-sized enterprises. Those two factors might explain the difference between our results and the assessment of several official documents of the aid programs in Afghanistan. Third, as emphasized in the introduction, Afghanistan is a country with fragile security; thus, the strong effect might be due to this situation. For example, it is possible that, due to the security situation of the country, many existing businesses were destroyed. As a result, when individuals start businesses with cash grants, there might not be so much competition in the existing markets. If this is the reason for the high return from the cash grants, the applicability of our results can be limited to countries whose security situation is very weak.\n\nAs discussed in the introduction, initially there was a concern that giving cash grants may not enhance economic activities in Afghanistan due to negative perception toward women in business and the prevalence of corruption. First, our estimation results show that although the effect of the cash grant on female individuals was lower than the effect on their male counterparts, the effect on earnings of female individuals was strong enough to justify the cash grant. Second, despite the prevalence of corruption in Afghanistan, the effect of the cash grant was strong. One possible reason for the effectiveness of this program might be due to the transparency of the selection of the recipients of the cash grants. As such, the project paid special attention to the lottery process to ensure random selection of these individuals. This implies that although the lottery process prevents the project from selecting the best candidates, it also prevents a case in which the project chooses corrupted and incapable individuals, a case which often happens in the government contracts in Afghanistan (Special Inspector General for Afghanistan Reconstruction, 2018; Special Inspector General for Afghanistan Reconstruction, 2021). This may indicate that random selection might be effective in countries with high corruption rates.\n\nThird, in this study, randomization for the receipt of the cash grant was done at individual level instead of village level. This implies that if there is a positive/negative externality of the cash grant, and if treatment and control individuals tend to live in the same village, the effect of the cash grant is underestimated/overestimated. In our context, a positive externality is likely if the recipient of the cash grant starts a new business, employs other individuals, and increases the income of other individuals as well as his/her own income. A negative externality is likely if the recipient of the cash grant starts a new business in an existing market and decreases the profit of the control individuals. In an extended study (Extended data) (Naito, 2021b), Global Positioning System (GPS) information was also used to determine whether the distance of each individual to the nearest treatment individual affects the estimated coefficient of the cash grant dummy. We found that there is no systematic correlation between the distance and the estimated size of the coefficient of the cash grant dummy. This suggests that it is unlikely that the experimental design that uses individual level randomization induces a systematic bias.\n\nAccording to the International Monetary Fund (IMF), the lending interest rate in Afghanistan in 2016 was 15% (International Monetary Fund, 2018). Given the high percentage of increased income to the amount of the cash grant and the relatively low lending interest rate, a question arises as to why individuals do not borrow money for business investments. Plausible explanations could be capital constraints and/or limited risk-sharing. Finding the correct mechanism is important because policy implications depend on the correct mechanism, as different mechanisms imply different policy implications. For example, if the lack of a risk-sharing mechanism is the source of the strong effect of the cash grant, then a policy that enhances the income risk-sharing of the business activities, such as progressive taxation on income with subsidy on business investment, is likely to enhance the welfare. If the underlying mechanism is the inaccessibility to capital, then a policy that makes capital more available such as public lending, will improve welfare. Future studies are needed to examine the underlying mechanisms.\n\n\nConclusions\n\nNon-trivial cash grants had a substantial effect on the monthly earnings of the treatment group two years after this study. The percentage of increased annual income to the size of this cash grant two years after the experiment is approximately 173% for males and 69% for females. We did not find evidence that receiving mentorship on business increases monthly earnings.\n\nIt is still not clear as to why receiving a large amount of cash grant has such a significant effect on the monthly earnings. To investigate, further longitudinal studies are needed to not only confirm the results of this study but to utilise quantitative and qualitative strategies to assess how such a large cash grants can affect the economy of a household.\n\n\nData availability\n\nOpen Science Framework (OSF): The effect of receiving cash grant on income in Afghanistan, https://doi.org/10.17605/OSF.IO/JQHRP (Naito, 2021a)\n\nThis project contains the following underlying data:\n\nQuestionnaire_cash_grant_afghanistan.zip. survey questionnaires\n\nSummary_stat1.do: stata file for summary statistics\n\nF1000_regression.do: stata file for regression analysis\n\nF1000_sample_selection_model.do: stata file for generalized moment method with inverse probability weighted estimation and bounding estimation\n\nThe above data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication)\n\nThe RCT dataset used in this study is owned by the World Bank Group. This RCT dataset can be requested from the World bank group (infoafghanistan@worldbank.org).\n\nThe findings, interpretations, and conclusions expressed in this article are entirely those of the authors and should not be attributed in any manner to the World Bank, to its affiliated organizations, or to members of its Board of Executive Directors, or the countries they represent. The World Bank does not guarantee the accuracy of the data included in this publication and accepts no responsibility for any consequence.\n\nOpen Science Framework (OSF): The effect of receiving cash grant on income in Afghanistan: Extended Data, https://doi.org/10.17605/OSF.IO/QG49F (Naito, 2021b).\n\nThis project contains the following extended data:\n\nExtended_data.pdf: additional regression results\n\nData are available under the terms of the creative commons zero \"no rights reserved\" data waiver (CC0 1.0 Public domain dedication)\n\nOpen Science Framework (OSF): CONSORT checklist for 'The effect of receiving cash grant on income in Afghanistan', https://doi.org/10.17605/OSF.IO/JQHRP (Naito, 2021a).\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\nFatema Kashefi would like to thank the World Bank Group for their assistance and permission to use this dataset. The authors appreciate the comments from Kennnichi Kashiwagi, Yoshinori Kurokawa, Mohammad Abdul Malek, Mari Minowa, Yuko Nakano, and Zeng Fei Yu. The authors also appreciate the editorial assistance from the editorial staff of F1000, Dr. Azharian.\n\n\nReferences\n\nAllen IE, Elam A, Langowitz N, et al.: Global Entrepreneurship Monitor Report on Women and Entrepreneurship. Babson Park, MA: Babson College and London: London Business School, 2007. Reference Source\n\nBBC: Afghanistan: How the Taliban gained ground so quickly. 2021; (accessed in August 20, 2021). Reference Source\n\nBeath A, Christia F, Enikolopov R: Empowering women through development aid: Evidence from a field experiment in Afghanistan. Am Polit Sci Rev. 2013; 107(3): 540–557. Reference Source\n\nBlattman C, Fiala N, Martinez S: Generating skilled self-employment in developing countries: Experimental evidence from Uganda. Q J Econ. 2014; 129(2): 697–752. Publisher Full Text\n\nBlattman C, Fiala N, Martinez S: The Long-Term Impacts of Grants on Poverty: Nine-Year Evidence from Uganda's Youth Opportunities Program. Am Econ Rev Insights. 2020; 2(3): 287–304. Publisher Full Text\n\nde Mel S, McKenzie D, Woodruff C: Returns to capital in microenterprises: evidence from a field experiment. Q J Econ. 2008; 123(4): 1329–1372. Publisher Full Text\n\nde Mel S, McKenzie D, Woodruff C: One-time transfers of cash or capital have long-lasting effects on microenterprises in Sri Lanka. Science. 2012; 335(6071): 962–966. PubMed Abstract \| Publisher Full Text\n\nFafchamps M, McKenzie D, Quinn S, et al.: Microenterprise growth and the flypaper effect: Evidence from a randomized experiment in Ghana. J Dev Econ. 2014; 106: 211–226. Publisher Full Text\n\nHaushofer J, Shapiro J: The short-term impact of unconditional cash transfers to the poor: experimental evidence from Kenya. Q J Econ. 2016; 131(4): 1973–2042. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInternational Monetary Fund: International Financial Statistics Yearbook, 2018. 2018; (accessed September 10, 2021). Reference Source\n\nIslamic Republic of Afghanistan Central Statistics Organization: Afghanistan Living Conditions Survey 2016-2017. Kabul, CSO, 2018. Reference Source\n\nJolliffe IT: Principal Component Analysis. Encyclopedia of Statistics in Behavioral Science. 2005. Publisher Full Text\n\nNaito H: The effect of receiving cash grant on income in Afghanistan. 2021a. http://www.doi.org/10.17605/OSF.IO/JQHRP\n\nNaito H: The effect of receiving cash grant on income in Afghanistan: Extended Data. 2021b. http://www.doi.org/10.17605/OSF.IO/QG49F\n\nKarlan D, Valdivia M: Teaching entrepreneurship: Impact of business training on microfinance clients and institutions. Rev Econ Stat. 2011; 93(2): 510–527. Publisher Full Text\n\nLa Porta R, Shleifer A: Informality and development. J Econ Perspect. 2014; 28(3): 109–26. Publisher Full Text\n\nLee DS: Training, wages, and sample selection: Estimating sharp bounds on treatment effects. Rev Econ Stud. 2009; 76(3): 1071–1102. Publisher Full Text\n\nManski CF: Nonparametric bounds on treatment effects. Am Econ Rev. 1990; 80(2): 319–323. Reference Source\n\nMcKenzie D: Identifying and spurring high-growth entrepreneurship: Experimental evidence from a business plan competition. Am Econ Rev. 2017; 107(8): 2278–2307. Publisher Full Text\n\nNagaoka S: Critical overview of the history of Islamic economics: Formation, transformation, and new horizons. Asian and African Area Studies. 2012; 11(2): 114–136. Publisher Full Text\n\nNon-formal approach to training education and jobs in Afghanistan(NATEJA), Narrative Progress Report (1st Quarter: Jan to Mar 2016) for subcomponent 3.2 (Promoting Entrepreneurship among Afghan Youth). 2016.\n\nSpecial Inspector General for Afghanistan Reconstruction: Quarterly Report to the US Congress July 30, 2017. Arlington, VA, 2017. Reference Source\n\nSpecial Inspector General for Afghanistan Reconstruction: PRIVATE SECTOR DEVELOPMENT AND ECONOMIC GROWTH: LESSONS FROM THE US EXPERIENCE IN AFGHANISTAN. Arlington, VA, 2018. Reference Source\n\nSpecial Inspector General for Afghanistan Reconstruction: WHAT WE NEED TO LEARN: LESSONS FROM TWENTY YEARS OF AFGHANISTAN RECONSTRUCTION. Arlington, VA, August 2021. Reference Source\n\nThe World Bank: The World Bank in Afghanistan. 2019; (accessed December 20, 2019). Reference Source\n\nTransparency International: Corruption Perception Index 2017. 2017; (accessed in September 10, 2021). Reference Source\n\nUnited Nations Assistance Mission in Afghanistan: Afghanistan Protection of Civilians in Armed Conflict Annual Report 2017. Technical Report, 2018. Reference Source\n\nUnited Nations Office on Drugs and Crime: Corruption in Afghanistan:Recent patterns and trends. Viena Austria. 2012; (accessed in August 10, 2021). Reference Source\n\nWall Street Journal: Afghanistan Government Collapses as Taliban Take Kabul. online version of the Wall Street Journal August 16,2021, 2021; (accessed in Septmber 10, 2021). Reference Source\n\nWooldridge JM: Inverse probability weighted M-estimators for sample selection, attrition, and stratification. Portuguese Economic Journal. 2002; 1(2): 117–139. Publisher Full Text\n\nWooldridge JM: Econometric Analysis of Cross Section and Panel Data. MIT press, Cambridge, Massachusetts, 2010. Reference Source" }	[ { "id": "121770", "date": "01 Mar 2022", "name": "Ellina Samantroy Jena", "expertise": [ "Reviewer Expertise gender and labour market research", "unpaid work", "time use studies", "gender", "labour laws and international labour standards" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is based on an interesting issue pertaining to economic empowerment through micro enterprises. The gender inequalities in terms of access to income was apparent in case of women. The paper would have benefitted more from literature highlighting the gendered constraints of women .The paper has highlighted that the percentage of annual income to the size of cash grant was greater for males in comparison to females. The reason for this could have provided a fresh insight into the study, for example women's access to resources, finances etc. and the challenges they face.\n\nThe paper definitely raises certain questions that need more in depth research in future. On the whole, the paper is based on a very important topic and may be considered for indexing after checking the statistical part from trained statisticians.\nAs the authors have clearly indicated that further studies on qualitative aspects are required. I would suggest to explore the qualitative aspects with a focus on gender dimensions particularly contextualizing women in fragile contexts may be in another paper in future.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9443", "date": "12 Apr 2023", "name": "Hisahiro Naito", "role": "Author Response", "response": "Reply to the first referee Thank you for reading our manuscript, approving it, and giving constructive comments. As you suggested, in our future research, we are planning to explore the points you suggested, that is, the qualitative aspect of gender in Afghanistan. Thank you." } ] }, { "id": "137413", "date": "20 Jun 2022", "name": "Svetlana Vlady", "expertise": [ "Reviewer Expertise Cross-cultural differences", "education", "oil and gas industry", "sustainabilty." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 is interesting and timely; it relates in a coherent and cogent way to issues of real-world significance. The authors evaluated the effect of implementing a government program and conducted research on how the prevalence of informal microenterprises can be transformed into an opportunity for income and employment growth. In Afghanistan, where a quarter of the population is unemployed and more than half of the population lives below the poverty line, the government implemented a program that helped establish new microenterprises by providing a cash grant. The authors make an original contribution to knowledge by investigating the effect of cash grants on income growth in the context of Afghanistan.\nThe uniqueness of this research is that it focuses on a country that suffers from a lack of security, a high level of corruption, a low level of literacy, and gender inequalities. Afghanistan is an Islamic country with rigid social norms and a high female unemployment rate due to a negative attitude toward women empowerment and a lack of support for women in business. Nevertheless, research found that cash grant has a strong positive effect on the earnings of the recipients. However, this research found that the increase in annual income was 2.5 times higher for males than females.\nThe article could be enriched by a literature review of previous research on the effect of cash grants in other countries and a discussion on culture-specific differences in gender roles at home and in the business in Afghanistan.\nThe article demonstrates the applicability, relevance, and effectiveness of the program and will be useful for other fragile or war-torn countries that need reconstruction. Also, receiving a cash grant, which does not involve the repayment of principal and interest, would be effective in Islamic countries.\nConclusions are clear and insightful, as they are drawn from the data, but could be expanded by recommendations. Minor proofreading and check results by qualified statisticians are suggested.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9444", "date": "12 Apr 2023", "name": "Hisahiro Naito", "role": "Author Response", "response": "Reply to the second referee Thank you for reading our manuscript and providing constructive comments. Comment 1: “The article could be enriched by a literature review of previous research on the effect of cash grants in other countries and a discussion on culture-specific differences in gender roles at home and in the business in Afghanistan.” Reply: On page 3, we added a section regarding the literature on the effect of cash grants and provided a comprehensive literature survey. Furthermore, on page 2, we discuss culture-specific differences in gender roles at home and in business in Afghanistan. We hope that our literature survey and discussion on the role of culture in women’s lives are satisfactory. Comment 2: ”Conclusions are clear and insightful, as they are drawn from the data, but could be expanded by recommendations. Minor proofreading and check results by qualified statisticians are suggested.” Reply: We provide some recommendations in the Conclusion section. More specifically, we wrote, “The results could indicate that the cash grant is a useful tool to increase income in a country that suffers from a lack of security, a high level of corruption, a low level of literacy, and gender inequalities.” We also asked a professional company to proofread our revised manuscript. The research field of one of the authors is applied econometrics. In the past, Naito conducted theoretical research on econometrics and published a paper. He checked the econometric aspects of this paper thoroughly." } ] } ]	1	https://f1000research.com/articles/10-1156
https://f1000research.com/articles/12-382/v1	12 Apr 23	{ "type": "Method Article", "title": "Phylogenetic placement of whole genome duplications in yeasts through quantitative analysis of hierarchical orthologous groups", "authors": [ "Samuel Moix", "Natasha Glover", "Sina Majidian", "Samuel Moix" ], "abstract": "Background: Whole genome duplications (WGD) are genomic events leading to formation of polyploid organisms. Resulting duplicated genes play important roles in driving species evolution and diversification. After such events, the initial ploidy is usually restored, complicating their detection across evolution. With the advance of bioinformatics and the rising number of new well-assembled genomes, new detection methods are ongoingly being developed to overcome the weaknesses of different approaches.\nResults: Here we propose a novel method for detecting WGD in yeast lineages based on the quantitative and comparative analysis of hierarchical orthologous groups (HOGs) of duplicated genes for a given set of organisms. We reconstruct ancestral genomes to obtain evolutionary information for each phylogenetic branch. This reconstruction relies on the inference of HOGs from the selected species’ proteomes. To estimate WGD events, the number of HOGs of duplicated genes across all taxonomic ranges are adjusted according to the molecular clock hypothesis and by the average genome size. Branches with a significant increase in the adjusted number of duplicated gene families are kept as candidates for WGD placement. The developed method was tested on two real datasets and showed promising results in phylogenetic WGD placements on the yeast lineage.", "keywords": [ "comparative genomics", "orthologous groups", "whole genome duplications", "yeast" ], "content": "Introduction\n\nThe potential roles of gene duplication in driving evolutionary innovation have now long been recognized.1 Whole genome duplication (WGD) is a major event leading to duplicated genes, found several times over the course of evolution in many eukaryotic organisms,2 and leads to the formation of an organism (or a cell) with additional copies of its entire genome. This event is often followed by massive gene losses due to non-functionalization, however some duplicated genes might undergo subfunctionalization (gene partitioning) or neofunctionalization (acquire a novel gene function) which will favor their preservation.3\n\nTo understand the circumstances leading to WGD in eukaryotes, Marcet-Houben and Gabaldón proposed a hypothesis describing an ancient allopolyploidization event (hybridization event between two different species of the yeast prior to the genome doubling).4 In case of an autopolyploid scenario (genome doubling in a single species), benefits from neofunctionalization would only take effect in the long run. In contrast, an allopolyploid scenario would lead to an initial selective advantage, because of the fact that it brings together different physiological properties and isolates the newly formed lineage sexually.4\n\nElucidating the causes of WGD remains a non-trivial task, as such events are often ancient and leave few traces, since over the course of evolution polyploidy is usually not maintained.5–7 For the same reasons, dating these events becomes challenging. One type of remaining traces in available genomes are conserved paralogs (i.e. ohnologs) which are defined as descendants of an ancestral gene derived from a duplication event. Paralogs are also referred to as ohnologs when resulting from WGD.\n\nFinding homologous relationships (genes derived from the same ancestor) is a key concept in evolutionary biology used by researchers to describe genetic differences and similarities between different species. One way to categorize genes is by inferring so-called hierarchical orthologous groups (HOGs). HOGs are defined as “sets of genes that have descended from a common ancestral gene in a given ancestral species”.8 These nested groups of orthologs and paralogs, defined at each taxonomic level, can be used to reconstruct ancestral genomes and infer gene families. This also provides information on evolutionary events such as gene duplications, gains or losses between known or reconstructed genomes. These inferred relationships play a critical role for this study.\n\nOver the past few decades, multiple WGD events on the tree of life have been detected.9 In early vertebrate evolution, Susumu Ohno proposed that two rounds of complete genome duplication occurred (often referred as 2R). There is now strong evidence confirming this hypothesis.10 Additionally, a consensus has been reached in favor of a third round (3R) WGD occurring in the fish lineage and thus also referred to as the fish-specific genome duplication.11 As mentioned earlier, WGD events have also been established in other eukaryotic lineages. For instance, an ancestral genome duplication has been placed before the common ancestor of extant seed plants, followed by another in the common ancestor of extant angiosperms.12 Moreover, multiple WGD events have been estimated among eudicotyledons and monocotyledons.12 Over the course of evolution, another well-known case occurred just before the separation of the yeast species Vanderwaltozyma polyspora from the S. cerevisiae lineage.13\n\nAlthough found in vertebrates, plants, fungi, and invertebrates, WGDs remain relatively rare evolutionary events that have far-reaching repercussions on speciation and biodiversity dynamics.2,14 Being able to place them across the tree of life is crucial to our understanding of species’ evolutionary history. Additionally, polyploidy has important effects on human health. Belonging to the hallmarks of cancers, polyploidization events are found in roughly one third of human cancers. Besides cancer, genome duplications also occur in normal cells such as hepatocytes and in premalignant lesions, impacting other disease conditions.15 Thus, expanding knowledge of WGD is of great importance for cancer and other disease research.\n\nCurrently, the main existing approaches for detecting WGD using genomic data are based on the detection of large conserved syntenic regions, gene counts along phylogeny, and synonymous mutation rates.16,17 These methods all have their own drawbacks. For instance, investigations through synteny require high-quality genome assemblies providing gene order, and might be biased by small scale duplications or remaining layers of older WGD.18 In methods based on the number of synonymous substitutions per synonymous site (Ks), signal is lost in older WGDs due to Ks stochasticity. Moreover, these methods suffer from Ks saturation which might lead to the formation of artificial peaks.19 Such limitations could be mitigated by leveraging a combination of multiple techniques when trying to infer WGD events. Finally, to cope with the rapid increase in available genomic data, scalable methods need to be developed.\n\nThis study aims to propose a novel approach, based on hierarchical orthologous groups of duplicated genes (henceforth referred to as duplication HOGs), to locate WGD events on the given phylogenetic tree of a set of organisms. The method relies on the organisms’ proteomes, i.e. protein sequence annotations, for the detection of WGD resulting from allopolyploidy or autopolyploidy. This study is focused on the yeast lineage as it contains a single well-established WGD.13 Additionally, yeast species have relatively small and less complex genomes, facilitating computation and analysis, thus favoring this choice. Applying our proposed method on two datasets comprising yeast species allowed proper identification of previously reported WGD events (details in the Results section). It is worth mentioning that the method could be used for newly sequenced genomes as well.\n\n\nMethods\n\nOur method uses the HOGs in addition to the species tree as inputs. In the following section, we described how one can estimate HOGs for the genome of interest. Then, in the section “Detection of whole genome duplications (WGD)”, our method to detect WGD is presented in detail. Of note, we evaluated our method using 32 yeast genomes from the Saccharomycetales clade (details in the Results section).\n\nWe used the OMA Standalone software20 to infer HOGs and to reconstruct ancestral genomes. To obtain HOGs, OMA Standalone performs multiple steps. First, the data undergoes quality and consistency checks. Second, all-against-all sequence alignments are computed in order to infer pairwise orthologs. We exported precomputed all-against-all protein sequence alignments from the OMA browser.21,22 Using precomputed all-against-all alignments as the input of the OMA standalone software significantly lowers the needed computational resources. In contrast to most other orthology inference resources, during the all-against-all alignment phase, OMA compares multiple isoforms in order to identify and utilize the isoform with the best cross-species matches.21 Next, pairwise orthologs are used to build an orthology graph. Finally, the GETHOGs algorithm (“Graph-based Efficient Technique for Hierarchical Orthologous Groups”) utilizes this graph and the given species tree for HOG inference. If no species tree is given, OMA estimates it using a least-squares distance approach based on OMA groups23 (Figure 1). OMA standalone was run with the selected sets of yeasts to obtain an estimated species tree in Newick format and HOGs in an OrthoXML file.20\n\nThe main steps of the developed method are illustrated on this flowchart. (For the complete description of the method, see the Methods section).\n\nFor WGD analysis, the number of inferred duplicated genes at each taxonomic level is essential. To retrieve this information, the python library pyHam (“python HOG analysis method”)24 was used. This package facilitates the extraction of evolutionary information such as gene duplications or losses at specific taxonomic levels from OMA Standalone’s HOG inference output. The duplication events can be inferred by the “mapper” class by comparing evolutionary relationships between an ancestral node and any of its descendants.\n\nOur method to place WGD events is based on the number of HOGs which were the direct result of a duplication event (we call them as “HOGs of duplicated genes”) (Figure 1). The species tree was traversed from the root node and the evolutionary history, including duplication events, of all the genes between each ancestor node and its two closest descendant nodes were inferred using pyHam’s “vertical comparison” method.24 The number of HOGs of duplicated genes, rather than the number of duplication events was considered, as ohnologs should result from one large duplication event. Branches in the species tree with significantly higher numbers of duplication HOGs (after the adjustments described below) were considered as putative WGD events.\n\nIn order to have a fair comparison between branches on the species tree with different lengths, the number of duplication HOGs was adjusted by evolutionary distance and average genome size. Adjusting by distance is based on the molecular clock hypothesis, which states that DNA and protein sequences evolve at a somewhat constant rate throughout time and across organisms.25 With this assumption, we would expect more evolutionary events over a longer time period and thus need to adjust the number of duplication HOGs accordingly by dividing by branch length.\n\nNote that since there could be some small branches in the tree which make the number of duplication HOGs adjusted value misleadingly high, it is needed to use a pseudocount when dividing counts by distance. Depending on the dataset, the user of our code is able to use different values of this pseudocount (set to one as default). Finally, to balance among the different genome sizes, the average number of genes between the compared nodes is used based on the assumption that the ancestor and descendant share similar genome sizes. These adjustments can be summarized with the following formula:\n\nAfter obtaining the number of adjusted duplication HOGs for each branch, values and distribution were plotted to determine a significant threshold for selecting possible WGD candidates. Another way to find candidates is by computing adjusted HOG counts with different pseudocounts and considering branches that remain significant over the various adjustments. In order to find these high values (outliers), we set a threshold for each separate case which is defined based on the interquartile range.\n\nBenchmarking of this method was done by comparing results with known cases found in the literature.4,26\n\n\nResults\n\nA diverse range of yeast organisms were used in this project in order to assess the performance of our WGD placement method. We chose yeast for a proof of concept because this clade is well documented and the species have simple genomes relative to other eukaryotes. These genomes were extracted from the Orthologous MAtrix (OMA) database.20 This database currently contains 2,496 genomes (Nov. 2022) from across the entire Tree of Life and is continuously being updated. As part of method evaluation, we first consider a small dataset, “small yeast”, of five species including Schizosaccharomyces pombe, Kluyveromyces lactis, Zygosaccharomyces Rouxii, Vanderwaltozyma polyspora and Saccharomyces cerevisiae. In this dataset, the well-known model organism S. pombe acts as a rooting species. It is known that K. lactis and Z. rouxii are potential candidates of species that might have led to the WGD in the hybridization theory4 and thus branching off before the known WGD placed before V. polyspora’s branch. In other words, Vanderwaltozyma polyspora and Saccharomyces cerevisiae are known to have undergone polyploidization.4 Another dataset, “large yeast”, was used as proof of concept and is composed of 32 species from the Saccharomycetales order found on the OMA database in addition to the species Penicillium chrysogenum for rooting.\n\nAs described in the method section, once HOGs were inferred with OMA standalone, the estimated number of gained, duplicated, retained or lost genes on each branch was obtained and visualized with a python package pyHam (Figure 2A,B). We notice a striking gene loss on the branch leading to the extant Saccharomyces cerevisiae South African wine strain (strain AWRI796), which may be due to its domestication. However, many of the gene losses might be false positives, which can happen when the extant genome is of lower quality or incomplete. Branches leading to Kluyveromyces lactis and Zygosaccharomyces rouxii have slightly less duplication than the other branches, but this data on its own is not sufficient to discriminate against possible WGD candidates (Figure 2A,B).\n\n(A) Inferred phylogenetic tree of the “small yeast” dataset using OMA standalone pipeline. Numbers in black correspond to branch length and numbers in purple to the branch id found in the bar plots. Colored stacked bars on branches illustrate the non-adjusted number of gained, duplicated, retained or lost genes from one node to another (these species have roughly 5000 genes in average). The same information can be found as a barplot on panel B. The red-colored branch corresponds to the placement of the whole genome duplications (WGD) detected by our method. (B) Bar plots represent the number of gained genes, duplication events, duplication HOGs, retained genes, and lost genes for the “small yeast” dataset. Branch ids correspond to the ids found on the phylogenetic tree of panel A. (C) Same bar plots as panel B adjusted by the distance (branch length plus pseudocount of one) and the average genome size for each branch (id).\n\nHowever, when counting the number of duplication HOGs adjusted by genome size and branch length, a distinctive larger relative quantity (seen as an outlier on the distribution) appears on the branch of the known WGD (branch id 5; Figure 2C). The significance threshold is determined by adding the third quartile to the multiplication of the interquartile range by 1.5 (Figure 3A). This allows clear placement of the WGD event on the species tree. Note that this significant value remains with a pseudocount of five (see Figure 3B). Indeed, the next highest quantity (branch id 8) lies at only 52% of this significant value.\n\n(A) Bar graph of the relative (by min-max normalization) adjusted (by branch length plus one and average genome size) number of duplication hierarchical orthologous groups (HOGs) for each branch of the “small yeast” dataset (see Figure 2 for branch id correspondence). On the right side, the box plot represents the distribution of these values. The distribution outlier is considered as a significant value and thus as a WGD candidate. Red colored bars correspond to branches that are significant over a range of different pseudocounts. (B) Replication of panel A barplot with a pseudocount of five. (C) Adjusted number of duplication HOGs for each branch of the “large yeast” dataset. Pseudocount added to the distance changed to five. Red colored bars correspond to branches that remain significant across a range of different pseudocounts (e.g. 1,5 and 10). See Figure 4 for branch id correspondence.\n\nApplying the same approach on the bigger data set containing 32 species from the Saccharomycetales order resulted in two possible WGD candidates. The first corresponds to the aforementioned WGD branch with the branch id 25 (Figure 4). The second, with an adjusted number above 0.8, corresponds to the branch leading to the extant species Pichia sorbitophila with the branch id 42. Interestingly, it was shown that Pichia sorbitophila results from allopolyploidization and has a genome of about twice the size of its ancestor.27 In this data set, we can see smaller differences among adjusted HOG counts which makes it more difficult to set the threshold to distinguish WGD branches, i.e. those with the highest duplication HOGs (Figure 4).\n\n(A) Inferred phylogenetic tree of the “large yeast” dataset. Established post-whole genome duplications (WGD) species under the light-green box. (B) Graph represents the number of duplication HOGs adjusted by the distance (branch length plus one) and the average genome size for each branch (id). On the right side, the box plot represents the distribution of these values. Keeping significant values that remain with higher pseudocounts (Figure 3C) correspond to found WGD candidates (branch id 25 and 42). Filled blue colored circles on the phylogenetic tree correspond to remaining significant values with a pseudocount of one.\n\n\nDiscussion\n\nResults obtained on the two yeast datasets are promising as the branch with the maximum adjusted duplication HOGs corresponds to the known WGD.\n\nIt is important to note that despite adding a pseudocount, when adjusting by evolutionary distance, small branches are still slightly more pushed upward compared to longer branches. To account for this, it is recommended to consider different pseudocounts and compare the outcomes. With the “large yeast” dataset we see various WGD candidates depending on the used pseudocount. By repeating the analysis with different pseudocounts (e.g., integers between one and 20) two branches persist: the known WGD branch but also the branch leading to P. sorbitophila which has a very large number of duplicated genes. As mentioned earlier, it was shown that this species has undergone genome duplication through allopolyploidization.28\n\nOverall, the applied method seems to perform quite well on its own for WGD placement in the yeast lineage, assuming the user keeps a fairly stringent threshold or discriminates by applying different pseudocounts.\n\nAs the scope of this work was limited on yeast and witnessing clear results obtained on this lineage, extending the approach to find placement of WGD on plant datasets would be of great interest. This is quite challenging due to the fact that the plant dataset in the current release of OMA contains at the moment only few and relatively distanced species for many reported WGD. First of all, based on comparison, the method depends entirely on the characteristics of the species used as input. Only using species from post-WGD or opposingly, only ones that did not undergo WGD, would not lead to significant values of interest, as an average needs to be set. In such cases, results should probably not be considered on their own to place WGD events but could be combined with different approaches.\n\nIn addition, the number of input species also play an important role in setting the baseline to find abnormal duplication increases. While only a few well-selected species of the yeast lineage allowed proper WGD placement, a random selection might require a larger dataset to identify significant values. The size and variety of the set should also be considered. First, to infer ancient duplications, more extant information is needed for a better resulting ancestral reconstruction. Second, to find abnormal changes, normality needs to be defined through a sufficient amount of information and such changes might also result from different events than WGD. Moreover, in cases with multiple overlapping WGDs on the same branch, only one event could potentially be highlighted.\n\nIn the end, the proposed method indicates branches with an abnormal increase of HOGs of duplicated genes under the assumption of the molecular clock hypothesis and correct ancestral genome reconstruction. While these may not be WGD events per se, this information remains beneficial, or even sufficient on its own, to detect WGD. Like most approaches this method has its own strengths and weaknesses and should therefore be combined with other known or novel methods in order to place WGD in more complex datasets. A coherent next step would be to consider the HOG synteny of the found WGD candidates, as this information can be retrieved as a weighted network on the OMA platform and would likely improve the detection accuracy.\n\nWGD placement, despite the many existing methods and algorithms,27 remains a complicated task and is therefore an ongoing research field. Most recently, researchers tried to apply a supervised machine learning approach based on the known detectable signatures of WGD.18 The advance of computational and biotechnologies should lead to the development of such new or improved methods to resolve remaining questions around WGD evolution. Such findings would contribute to a better understanding of the related rise of evolutionary innovations throughout history and could also be expanded to the study of WGD as macro-evolutionary event occurring in tumorigenesis.", "appendix": "Data availability\n\nDatasets are publicly available:\n\n- species tree with Newick format https://omabrowser.org/All/speciestree.nwk\n\n- precomputed all-against-all alignments can be exported from https://omabrowser.org/oma/export/\n\n\nReferences\n\nOhno S: Evolution by Gene Duplication. Allen and Unwin; 1970.\n\nVan de Peer Y, Mizrachi E, Marchal K: The evolutionary significance of polyploidy. Nat. Rev. Genet. 2017; 18: 411–424. Publisher Full Text\n\nGlasauer SMK, Neuhauss SCF: Whole-genome duplication in teleost fishes and its evolutionary consequences. Mol. Gen. Genomics. 2014; 289: 1045–1060. PubMed Abstract \| Publisher Full Text\n\nMarcet-Houben M, Gabaldón T: Beyond the Whole-Genome Duplication: Phylogenetic Evidence for an Ancient Interspecies Hybridization in the Baker’s Yeast Lineage. PLoS Biol. 2015; 13: e1002220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWendel JF: The wondrous cycles of polyploidy in plants. Am. J. Bot. 2015; 102: 1753–1756. PubMed Abstract \| Publisher Full Text\n\nAlbertin W, Marullo P: Polyploidy in fungi: evolution after whole-genome duplication. Proc. R. Soc. B Biol. Sci. 2012; 279: 2497–2509. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCampbell MA, Ganley ARD, Gabaldón T, et al.: The Case of the Missing Ancient Fungal Polyploids. Am. Nat. 2016; 188: 602–614. PubMed Abstract \| Publisher Full Text\n\nZahn-Zabal M, Dessimoz C, Glover NM: Identifying orthologs with OMA: A primer.2020. Publisher Full Text\n\nWatts RL, Watts DC: Gene duplication and the evolution of enzymes. Nature. 1968; 217: 1125–1130. Publisher Full Text\n\nClear Evidence for Two Rounds of Vertebrate Genome Duplication. PLoS Biol. 2005; 3: e344. Publisher Full Text\n\nMeyer A, Van de Peer Y: From 2R to 3R: evidence for a fish-specific genome duplication (FSGD). BioEssays. 2005; 27: 937–945. Publisher Full Text\n\nJiao Y, et al.: Ancestral polyploidy in seed plants and angiosperms. Nature. 2011; 473: 97–100. Publisher Full Text\n\nScannell DR, et al.: Independent sorting-out of thousands of duplicated gene pairs in two yeast species descended from a whole-genome duplication. Proc. Natl. Acad. Sci. 2007; 104: 8397–8402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolfe KH: Origin of the Yeast Whole-Genome Duplication. PLoS Biol. 2015; 13: e1002221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatsumoto T, et al.: Proliferative polyploid cells give rise to tumors via ploidy reduction. Nat. Commun. 2021; 12: 646. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMabry ME, et al.: Phylogeny and multiple independent whole-genome duplication events in the Brassicales. Am. J. Bot. 2020; 107: 1148–1164. Publisher Full Text\n\nYang Y, Li Y, Chen Q, et al.: WGDdetector: a pipeline for detecting whole genome duplication events using the genome or transcriptome annotations. BMC Bioinformatics. 2019; 20: 75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKibben MTW, Barker MS: Applying Machine Learning to Classify the Origins of Gene Duplications.2021. Publisher Full Text\n\nVanneste K, Van de Peer Y, Maere S: Inference of Genome Duplications from Age Distributions Revisited. Mol. Biol. Evol. 2013; 30: 177–190. PubMed Abstract \| Publisher Full Text\n\nAltenhoff AM, et al.: OMA standalone: orthology inference among public and custom genomes and transcriptomes. Genome Res. 2019; 29: 1152–1163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltenhoff AM, et al.: OMA orthology in 2021: website overhaul, conserved isoforms, ancestral gene order and more. Nucleic Acids Res. 2021; 49: D373–D379. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDylus D, et al.: How to build phylogenetic species trees with OMA.2020. Publisher Full Text\n\nAltenhoff AM, Gil M, Gonnet GH, et al.: Inferring Hierarchical Orthologous Groups from Orthologous Gene Pairs. PLoS One. 2013; 8: e53786. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\npyHam: a python package to visualize and process hierarchical orthologous groups (HOGs) – Open Reading Frame - Dessimoz Lab. http\n\nThe Molecular Clock and Estimating Species Divergence \| Learn Science at Scitable. http\n\nKellis M, Birren BW, Lander ES: Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature. 2004; 428: 617–624. PubMed Abstract \| Publisher Full Text\n\nLallemand T, Leduc M, Landès C, et al.: An Overview of Duplicated Gene Detection Methods: Why the Duplication Mechanism Has to Be Accounted for in Their Choice. Genes. 2020; 11: 1046. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLouis VL, et al.: Pichia sorbitophila, an Interspecies Yeast Hybrid, Reveals Early Steps of Genome Resolution After Polyploidization. G3 GenesGenomesGenetics. 2012; 2: 299–311. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "171823", "date": "12 May 2023", "name": "Xinyi Guo", "expertise": [ "Reviewer Expertise Genome evolution" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 presents a simple method for WGD inference based on gene counts and the authors have shown its good performance in yeast lineages. While the authors recognized knowledge gaps in the field and presented both their own solution and its limitations, I found that the rationale and description of the method are still not very clear, and there is a lack of comparison with other existing methods based on gene count data. In addition, the presented method is a Python-based wrapper for extracting and processing HOG gene count information from an existing gene orthology inference method (OMA), which might exclude many potential users who prefer other pipelines.\nMajor comments:\nThe main contribution of the method presented by the authors is the use of the adjusted number of HOGs according to the molecular clock hypothesis and by the average genome size. While branch length is somehow expected to be associated with gene counts due to the impact of paralogs on phylogenetic inference, genome size might be a poor indicator for WGD or gene counts due to the amplification of genomic repeats or progressive diploidization. For both factors, the authors didn’t mention in their introduction why they decided to consider their impact on WGD and why to implement the parameter in their method.\n\nFollowing point 1, in their analyses, the authors presented adjusted values based on both branch length and genome size. Does this mean that both factors should be jointly considered in order to give the best performance? If so, I suggest providing comparative results based on individual factors as well.\n\nThe principle of the method is easy to understand and should be sufficient to replicate. I didn’t check if similar methods have been presented elsewhere, but I do realize that there are some existing methods based on gene count data, e.g., WGDgc (Rabier et al. 20141). Therefore, I recommend that the authors check more publications and compare the performance of their methods with others.\n\nIn their discussion, the authors presented a short but nice discussion on the development of WGD placing methods and their limitations. I like it. However, related to point 1, I found much dispensable information in their introduction. I would expect more focus on the rationale of the study and on developing the method.\nMinor points:\nIn Abstract:\n“After such events, the initial ploidy is usually restored” - The sentence is not very clear to me. What is the initial ploidy, do you mean diploid? And if you believe this usually happens, why consider genome size?\n\n“We reconstruct ancestral genomes” - I guess you mean you inferred the number of genes in ancestral genomes. Please check such wording carefully throughout the manuscript.\nIn Introduction:\nParagraph 2 (P2). “Marcet-Houben and Gabaldón proposed a hypothesis” - This sentence is misleading as you were talking about eukaryotes first and mentioned yeast only in the parenthesis.\n\nP3. “For the same reasons, dating these events becomes challenging. ” - If it’s for the same reason, why not mention both tasks in one sentence to make it more coherent?\n\nP3. “One type of remaining traces in available genomes are conserved paralogs (i.e. ohnologs) which are defined as descendants of an ancestral gene derived from a duplication event. Paralogs are also referred to as ohnologs when resulting from WGD.” - Please check these two sentences for redundancy (paralogs/ohnologs appeared twice). If you mention one type, I would expect some information on other types.\n\nP4. “One way to categorize genes is by inferring so-called hierarchical orthologous groups” - How about other ways? Why did you choose this way?\n\nP5. Ref9 is too old – Is a paper from 1968 well suited to represent something “over the past few decades”? Also, I don’t think it’s necessary to give too many details (nearly a whole paragraph) on other species.\n\nP6. Similar to P5, it seems too detailed. Both P5 and P6 are not so much related to the method itself.\n\nP7. I found synteny and Ks here. How about gene counts, which is the main focus of this study?\n\nP7. “scalable methods need to be developed” - This sentence seems to imply that something on the scalability of the methods is considered by the authors. But I didn’t see it in the remaining part of the manuscript.\n\nP8. “proteomes, i.e. protein sequence annotations” - Are you sure proteomes mean just annotations? You might want to say protein sequences and their annotations.\nIn Methods:\nP2. I would like to know why the authors consider OMA instead of many other orthology inference pipelines (orthofinder, orthoMCL etc.) to perform their analyses, although the authors mentioned some features of the OMA pipeline. Perhaps some comparison between pipelines will help.\n\nP3. “For WGD analysis, the number of inferred duplicated genes at each taxonomic level is essential.” - This sentence is not very clear. What kind of WGD analysis? And I would say it’s the reliable estimates of the duplicated gene number that is essential.\n\nP4. “The number of HOGs of duplicated genes, rather than the number of duplication events was considered, as ohnologs should result from one large duplication event. ” - I suggest some rewording as you already mentioned ohnologs resulted from WGD. Perhaps it’s better to say there are also many local gene duplication events besides large scale duplication events caused by WGD.\n\nP5. Why adjust genome size? This is not mentioned in the introduction or here.\n\nP5. “With this assumption, we would expect more evolutionary events over a longer time period” - What kind of events do you mean? Gene duplication? And again, how this is linked with the branch length of the phylogeny, which indicates the substitution rates?\n\nP6-7. Any recommendation to adjust the pseudocount?\nIn Results:\nP1. This should belong to part of the methods.\n\nP2. “which may be due to its domestication” - Any evidence or reference for that?\n\nP2. “However, many of the gene losses might be false positives, which can happen when the extant genome is of lower quality or incomplete.” - Again any evidence to support the low quality or completeness of the genomes?\n\nP3. I noticed that the value of 1.5 appears in the results but not in the methods (although in the formula).\n\nP4. What’s the difference between Fig3C and Fig4B?\nIn Discussion:\nP2. “To account for this, it is recommended to consider different pseudocounts and compare the outcomes.” - Again, it’s better to give more details. What does pseudocounts mean? How could the value vary across organisms?\n\nP5. “more extant information” - What kind of information, for example?\n\nP6. “correct ancestral genome reconstruction” - Again genome reconstruction is not proper here.\nExpanded answers to the mandatory questions:\nIs the rationale for developing the new method (or application) clearly explained? The explanation is not very clear enough. Please see my major comments #1-3\n\nIs the description of the method technically sound? Technically, the description is fine but some more details should be needed. Please see my major comment #1\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? The conclusions should be supported but the performance of the method needs to be further tested or compared. Please see my major comment #4.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "203842", "date": "28 Sep 2023", "name": "Gavin Conant", "expertise": [ "Reviewer Expertise Evolution of polyploid genomes" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview of Moix et al., “Phylogenetic placement of whole genome duplications in yeasts through quantitative analysis of hierarchical orthologous groups.”\nNOTE: I agree with much of what X. Guo has written in their peer review and notice that these points have not been addressed, which will be essential should this manuscript be published.\nOverview: In this manuscript, the authors propose a new approach to detecting whole-genome duplications from sequenced genomes using the concept of hierarchical orthologous groups (HOGs) and validate that method on genomic data from yeast.\nMajor comments: At the highest level, there is no harm in having new methods for detecting WGD events, and I think that there is some promise in the idea of using HOGs for such a problem. I nonetheless find it difficult to summon a great deal of enthusiasm for this tool and manuscript for a few reasons.\nMost importantly, if my understanding is correct, this method represents an approximation to existing approaches of modeling gene family evolution on a phylogeny using birth-death processes (this point was again noted by the first reviewer). Since there is at least one existing tool that uses just such models to infer the presence of WGD events (1, 2), the authors here need to explain the similarities and differences between their approach and the prior work. I will note that this existing tool does treat the WGD events in a slightly ad hoc way, so there is potentially room for improvement.\nMy second concern is that what the community actually lacks in not tools for detecting WGD events. We lack tools that can assess the statistical support for those inferences. The authors take some welcome steps in this direction, but I don’t find them to be especially well-explained nor particularly robust. It appears that the authors use the normalized distribution of HOG appearance across the branches of the tree as a null distribution and consider quartile outliers within this distribution to be potential WGD events. That approach gives the appearance of good behavior in the dataset considered here, which essentially has exactly one WGD. However, it is less clear that the method would reject the hypothesis of a WGD in datasets where in fact no WGDs occurred or could handle data like flowering plants with many WGD events on the tree. I would encourage the authors to better explain this section.\nMinor points: I am not sure “pseudo-count” is the right term for the scaling parameter the authors use, since those call to mind a more controlled procedure for accounting for unobserved data. Here what is being done is accounting for short branches, but the authors should differentiate between branches that are short but non-zero verses branches that the routines are estimating to be numerically zero. Doing so is tricky but important. For instance, some phylogenetic packages can estimate branch length confidence intervals, which might be a more rigorous approach to this problem.\nThe first paragraph of the abstract is rather badly written. I would say that polyploidy leads to WGD, not vice versa. More accurately, WGD is a shorthand for genomes with polyploidy in their history. Likewise “initial ploidy is restored” isn’t really correct: the chromosomes may return to a diploid pairing state in meiosis with the organism still retaining many more chromosomes that its ancestors had.\nThe first paragraph of the introduction could also mention the dosage balance hypothesis and how it impacts duplicate retention (3). (In contrast to the first reviewer, I would urge the authors not to eliminate this more general discussion of polyploidy here).\nI would not write that “allopolyploidy…lead[s] to an initial selective advantage…” because I do not believe this is invariably the case. Rather I would phrase this as “Allopolyploidy could…”\nThe first reviewer is also correct that the section of Marcet-Houben and Gabaldon’s work is not clear.\nReferences:\nTiley GP, Ané C, & Burleigh JG (2016) Evaluating and Characterizing Ancient Whole-Genome Duplications in Plants with Gene Count Data. Genome biology and evolution 8(4):1023-1037.\n\nRabier C-E, Ta T, & Ané C (2014) Detecting and locating whole genome duplications on a phylogeny: a probabilistic approach. Molecular biology and evolution 31(3):750-762.\n\nEdger PP & Pires JC (2009) Gene and genome duplications: the impact of dosage-sensitivity on the fate of nuclear genes. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology 17(5):699-717.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-382
https://f1000research.com/articles/11-175/v1	14 Feb 22	{ "type": "Review", "title": "Polygenic Risk Score in African populations: progress and challenges", "authors": [ "Yagoub Adam", "Suraju Sadeeq", "Judit Kumuthini", "Olabode Ajayi", "Gordon Wells", "Rotimi Solomon", "Olubanke Ogunlana", "Emmanuel Adetiba", "Emeka Iweala", "Benedikt Brors", "Ezekiel Adebiyi", "Yagoub Adam", "Suraju Sadeeq", "Judit Kumuthini", "Olabode Ajayi", "Gordon Wells", "Rotimi Solomon", "Olubanke Ogunlana", "Emmanuel Adetiba", "Emeka Iweala", "Benedikt Brors" ], "abstract": "Polygenic Risk Score (PRS) analysis is a method that predicts the genetic risk of an individual towards targeted traits. Even when there are no significant markers, it gives evidence of a genetic effect beyond the results of Genome-Wide Association Studies (GWAS). Moreover, it selects single nucleotide polymorphisms (SNPs) that contribute to the disease with low effect size making it more precise at individual level risk prediction. PRS analysis addresses the shortfall of GWAS by taking into account the SNPs/alleles with low effect size but play an indispensable role to the observed phenotypic/trait variance. PRS analysis has applications that investigate the genetic basis of several traits, which includes rare diseases. However, the accuracy of PRS analysis depends on the genomic data of the underlying population. For instance, several studies show\n\nthat obtaining higher prediction power of PRS analysis is challenging for non-Europeans. In this manuscript, we review the conventional PRS methods and their application to sub-Saharan African communities. We conclude that lack of sufficient GWAS data and tools is the limiting factor of applying PRS analysis to sub-Saharan populations.\n\nWe recommend developing Africa-specific PRS methods and tools for estimating and analyzing African population data\n\nfor clinical evaluation of PRSs of interest and predicting rare diseases.", "keywords": [ "Prediction medicine", "GWAS", "post-GWAS", "PRS analysis", "Africa population" ], "content": "Introduction\n\nGenome-Wide Association Studies (GWAS) can be used successfully to identify associations between hundreds of genomic variations with complex genetic traits.1 In general, GWAS report single nucleotides polymorphisms (SNPs) as statistically significant genomic variations associated with the trait of interest when their p-values are smaller than a cutoff value of 5e‐09 in the African population.2 This cutoff value statistically depends on the number of SNPs analyzed.2 The statistically significant SNPs reported by GWAS are used to understand the biomolecular mechanisms of many phenotypic traits including various human diseases. Due to the statistical threshold, GWAS might fail to detect SNPs that are associated with low or moderate risks.3,4 The limitation of filtering variants associated with low disease risk increases the GWAS false-negative rate. Also, conventional GWAS can not be used to integrate the polygenic nature of many complex traits.5 Therefore, several post-GWAS approaches have been introduced to overcome the above mentioned pitfalls.6,7 Due to privacy issues, such as access to the individual level of GWAS data sets, most post-GWAS approaches require only GWAS summary statistics. Some public resources for GWAS summary statistics include: the GWAS Catalog,8 GWAS Central,9 and the dbGaP database.10,11 A distinct approach of performing a post-GWAS analysis is known as Polygenic Risk Score (PRS) analysis. The PRS methods map genotype data from a GWAS summary into a single variable used to estimate an individual-level risk score for the phenotypic trait. PRS analysis is used to predict an individual heritability by incorporating all selected SNPs.12 However, it is important to consider that not all existing genomics technologies have the capabilities to capture the informative variants among trans-ethnic populations. Nevertheless, obtaining a precise PRS value from case-control studies can be used in personalized medicine. Challenges still exist when translating PRS values from clinical validity to clinical utility.13 To successfully perform conventional PRS analysis, two distinct GWAS summaries are required. The first data set (training sample) is used to select the SNPs for PRS analysis and the second data set (from the discovery sample) is used to evaluate the predicted value of PRS methods. The following traditional PRS approaches are discussed in this review: (i) weighted methods that consider the effect sizes derived from GWAS result; (ii) unweighted methods that consider the single marker analysis; (iii) shrinkage methods that consider multivariate analysis. This review focuses on the tools and methods that perform PRS analysis and their applications in understanding the predictive power of PRS analysis. The reviewed PRS tools are chosen based on the following criteria:\n\n1. The approach must perform PRS analysis based on “base” (GWAS) data (summary statistics) and “target” data set (genotypes and phenotypes in each of the target data set),\n\n2. The approach may involve linkage disequilibrium pruning, and\n\n3. The method or approach should be readily available as a tool or package so that it can be executed on any data set.\n\nBesides reviewing PRS methods, we aim to investigate the application of PRS analysis in the African population. It is worth mentioning that the term “African population” covers all those whose ancestors are Africans (including Africans in diaspora). Nevertheless, in this manuscript, the focus is on sub-Saharan Africa. It is important to note that when we searched PubMed for PRS publications on August 21, 2021, the query reported 194,203 hits in total (see Figure 1 and text Box 1 for the query terms). For this review, we included articles based on their underlying PRS methods.\n\nWe used the following terms for querying Pubmed:\n\n(((’Polygenic Risk score’) OR ((Polygenic) AND (PRS)) OR (’Polygenic score’) OR ((Polygenic) AND (PGS)) OR (’Genetic Risk Score’) OR ((’Genetic Risk’) AND (GRS)))\n\nAND\n\n((African) OR (Africa) OR ((Yoruba) AND (YRI)) OR ((Luhya) AND (LWK)) OR ((Mandinka) AND (MAG)) OR ((Mende) AND (MSL)) OR ((Esan) AND (ESN))))\n\n\n\n• For PRS terms (in blue color), we included the terms for Genetic Risk Score as some articles used them to refer to PRS.\n\n• For African populations (in red color), we included terms for Africans tribes based on 1000 genomes.\n\nRefer to,14 for the query syntax.\n\n\nClassification of PRS methods\n\nThe different conventional approaches under the umbrella of PRS analysis are presented in Figure 2 and Table 1. We can categorize PRS methods into two; Bayesian-based and non-Bayesian methods. PRS methods can also be classified using their usage of linkage disequilibrium (LD): PRS methods that incorporate LD and PRS methods which apply LD pruning. To ease the understanding of their underlying algorithms, we grouped the PRS analysis approaches into four (see Table 2). Those with;\n\n1. Clumping with thresholding (C+T)\n\n2. p-value thresholding\n\n3. Penalized regression\n\n4. Bayesian shrinkage\n\nThis is a typical polygenic risk score analysis workflow showing base data, target data and encapsulating different approaches. Using genotype and phenotype data,individual-level or summary statistics, approaches such as lasso/ridge regression, clumping and p-value thresholding can be employed to increase the predictive accuracy of PRS analysis. Furthermore, the results may be used to predict health or disease risk as well as give information for appropriate therapeutic approaches.\n\nIn practice, When the markers are LD pruned, the prediction accuracy of PRS analysis tends to improve. Thus, the absence of LD information limits the predictive accuracy of PRS analysis.15 For instance, the method of LD pruning and p-value thresholding (P + T) is commonly used, in the presence of LD patterns to improve the PRS prediction accuracy.16 For instance, LDPred (2.0.1) is a Bayesian approach that applies LD information in the presence of LD patterns. From this approach, the posterior mean effects of LD linked loci may be calculated analytically using a Gaussian infinitesimal prior, a non-infinitesimal model, in which only a portion of the markers is causative is perhaps a more realistic prior for effect sizes. For this reason, the following Gaussian mixture prior is considered:\n\nThe LD matrix within the LD region is denoted by Di and the estimated effects within the target region are represented by β˜l, which is estimated using the least-squares method. The approximation assumes that the heritability explained by the region is small and LD with SNPs outside of the region is negligible.\n\nThese PRS methods are non-Bayesian approaches that apply informed LD pruning (LD clumping) in PRS computation (Figure 2). Generally, they are known as pruning and thresholding (P+T) methods. We may apply p-value thresholding, for example, with a univariate regression coefficient (r2) and a threshold of 0.2. To achieve prediction accuracy in the validation data, we would ensure that the p-value thresholding method is optimized across a grid. LD pruning, in which the less significant marker is pruned first, may result in more accurate predictions than random marker pruning. For the p-value threshold selection, researchers should include only SNPs that are statistically significant in GWAS. This technique essentially shrinks all omitted SNPs to zero estimates and does not perform shrinkage on the effect size estimates of the included SNPs. The optimal p-value threshold is a priori unknown and the targeted phenotype is assessed for the chosen threshold, which is why PRS is commonly computed over several thresholds. This technique can be interpreted as a variable selection process that essentially executes the GWAS p-value forward selection based on the size of the increment in the p-value thresholds.\n\nBayesian techniques have been successfully applied to model pre-existing genetic architecture with a prior that accounts for the range of effect sizes and thus increases polygenic score accuracy. The Bayesian statistical approach computes a refined posterior distribution from prior probability distributions using available data such as functional annotations. It shrinks marker effects by using LD information from a reference panel.17 The key benefit of Bayesian-based PRS analysis is its ability to enhance PRS prediction accuracy from summary statistics by taking LD among markers into consideration.18 Bayesian approaches in PRS explicitly model pre-existing genetic architecture that accounts for the distribution of effect sizes. These approaches allow the introduction of prior probability that improves the prediction accuracy of a polygenic score.\n\nThe EB-PRS technique is an innovative method that relies on the Empirical Bayes theorem. It incorporates information across markers to strengthen prediction accuracy.19 By utilizing the predicted distribution of effect sizes, the EB-PRS technique tries to reduce prediction error. Suppose all the SNPs are independent, the optimum PRS value is given by:\n\nwhere m denotes to the number of the all genotyped SNPs. The matrix Xi stands for the genotypic value and βi is the log-odds ratio (OR) of the ith variant. The equation below can be used to measure the log-OR:\n\nThe actual values of effect sizes are generally unknown, thus they can be estimated empirically. Song et al.19 used the Empirical Bayes method to estimate β. The estimators can be equally derived from GWAS summary statistics. Unlike other improved genetic risk prediction methods which utilize effect size distributions for PRS computation, the EB-PRS does not require external panels.16,18,20,21 Also, the EB-PRS approach has theoretical superiority, resulting in a better PRS by lowering prediction error. The EB-PRS has recorded excellent performance in comparable to the other tool from following complex traits; Crohn’s disease, celiac disease, Parkinson’s disease, asthma, breast cancer, and type 2 diabetes.19 Furthermore, a significant improvement was recorded when tested against the unadjusted PRS method, P + T, LDpred-inf, LDpred.18 Although The EB-PRS approach has demonstrated that it can generate superior results without adjusting any parameters or relying on external data, studies have shown that further improvement is possible with a reference panel. For instance, the LD information as used in LDpred. Also, to increase the prediction accuracy, Song et al.19 suggested that other available datasets such as GWAS summary statistics focused on functional annotations and genetically correlated traits could further improve EB-PRS accuracy.\n\nThe PRS-CS is based on a Bayesian high-dimensional regression framework for polygenic modeling and prediction:\n\nSince SNP effects are calculated with uncertainty and not all SNPs have an impact on the traits, unadjusted effect size estimates of all SNPs can lead to a low-estimated PRS with high standards error.17 Two shrinkage methods have been implemented to solve these problems; shrinkage of the effect estimates of all SNPs by adapted statistical techniques and use of p-value filtering thresholds as the criterion for inclusion of SNPs.\n\nShrinkage of the effect estimates of all SNPs by adapted statistical techniques: Some PRS methods performs shrinkage of all SNPs. These methods are typically apply shrinkage/regularisation techniques such as LASSO/ridge regression28 or Bayesian approaches performing shrinkages by prior distribution specification.16 Varying degrees of shrinkage may be accomplished under different methods or parameter settings. The most suitable shrinkages to be implemented depends on the underlying mixture of distributions of null and true effect size. PRS estimation is usually tailored over several (tuning) parameters since the optimum shrinkage parameters are a priori unknown. For example, it includes a setting for a fraction of causal variant16 in the case of LDpred.\n\np-value filtering thresholds as the criterion for inclusion of SNPs: In this process, the PRS includes significant SNPs with a P-value below a choosen threshold (e.g. p-value < 23e-05). This method shrinks all omitted SNPs to an estimated effect size of zero and does not perform shrinkage on the effect size estimates of the included SNPs. Since the optimum p-value threshold is a priori unknown, PRS is computed over a range of thresholds associated with each of the tested target traits and optimized appropriately for the prediction. This is similar to optimizing parameters in the systematic shrinkage approach and regarded as a parsimonious method of variable selection. It is efficient in performing the forward selection of variables (SNPs) using GWAS and p-value with the sizes depending on the p-value threshold increment. Therefore, this forward selection method is the chosen’optimal threshold’. Furthermore, PRS derived from another subset of the SNPs may be more predictive of the target trait. Considering the fact that GWAS focuses on millions of SNPs, the number of subsets of SNPs for the study could be too large.\n\nUsually, association studies in GWAS are done individually.17 The power of GWAS can be enhanced by leveraging the results of several SNPs concurrently.29 Unfortunately, the raw data of all samples are not readily available. Researchers may need to take advantage of standard GWAS by considering either (i) SNPs are clumped such that the retained SNPs are almost independent of each other or (ii) all SNPs are included and the LD between them is adjusted. In the’standard’ polygenic scoring approach, option i is usually preferred and requires p-value thresholding. Option ii is commonly used in methods that incorporate conventional methods of shrinkage21,16 (see Table 2). As for option i without clumping, some researchers tend to apply the methods of p-value thresholding. Although breaking this presumption can lead to marginal losses in certain situations.21 Choi et al.17 suggested that clumping should be applied when GWAS estimates of non-shrunk effect sizes are available. The standard method tends to work when compared to more advanced approaches.21,16 It is possible that the clumping method captures conditionally independent effects. A critique of clumping for SNPs elimination in LD is that researchers usually use an arbitrarily selected correlation threshold.30 Thus, no technique is without arbitrary features. This could be an area for the potential development of the classical method.\n\nPRS based variant decomposition focuses on decomposing or factorizing suitable genetic variants matrix into different components. This approach is mainly based on the use of an appropriate matrix decomposition technique. Contrary to traditional methods that compute PRS for a trait as the sum of effects from several genetic variants, this technique uses genetic risk for a single component to approximate risk for a weighted combination of relevant traits. Although there are many approaches to genetic variants decomposition,31–33 only truncated singular value decomposition (TSVD) and singular value decomposition (SVD) have been used in the context of PRS.\n\nAguirre et al.34 and Chasman et al.35 are the first to use genetic risk decomposition to derive polygenic scores. They both applied TSVD and SVD respectively to compute polygenic risk scores from genetic components. While it is similar to the traditional PRS in predictive ability, it also enables an appropriate assessment of drivers of genetic risk for the phenotype. For example, Aguirre et al.34 applied this method to body mass index and classified polygenic risk factors into overall health indicators, including sleep duration, alcohol, water intake, fat mass, fat-free mass. Consequently, they encouraged modeling PRS from the components of the decomposition of genetic risk association.\n\nLet Wn×m be a sparse matrix of genetic associations with n rows and m columns, then TSVD can be performed on W to identify different genetic components. The decomposition will lead to factors of three matrices which approximates W:\n\n• A singular matrix for trait Un×c,\n\n• A singular matrix for variant Vm×c, and\n\n• A diagonal matrix Sc×c of singular values. i.e., W.\n\nUsing the individual-level genotype vector Gm×1, component polygenic risk scores (cPRS) can be computed by applying matrices U, S, and V, using the following formula\n\nFinally, PRS can be defined by summing through the component PRS, using cPRS for each component, then;\n\n\nPRS tools\n\nThe next section will provide examples of some PRS tools that are commonly used to perform PRS analysis.\n\nThis method estimates the posterior mean effect size of each marker of GWAS summary data using a priori effect sizes and LD information from an external reference panel.16 In this process, the inner products are re-weighted and the test-sample genotypes are the posterior mean phenotype. The posterior mean phenotype is an optimum predictor under the model assumptions and a point-normal mixed distribution is used as the effect size prior, allowing for non-infinitesimal genetic structures. Heritability explained by the fraction of causative markers and genotypes are the two parameters of the prior. The heritability parameter is calculated using summary statistics from GWAS and takes into account sample noise and LD.43\n\nIn an attempt to check the performance of LDpred in comparison to the method of pruning followed by thresholding, using five complex traits, including breast cancer, schizophrenia, muscular dystrophy, and coronary artery disease. GWAS summary statistics for large sample sizes ranging from 27,000 to 86,000 individuals and raw genotypes for an independent dataset validated, LDpred outperforms the other approach18 particularly at large sample sizes. For instance, the predicted R2 rose from 20.1 percent to 25.3% and from 9.8% to 12.0% in a large dataset of schizophrenia and multiple sclerosis, respectively. Although the accuracy of the predictive values were lower in absolute terms in another study to predict schizophrenia risk in non-European validation populations of African and Asian heritage, similar observations were made for other approaches.\n\nLDpred is a powerful tool that can be used for performing polygenic scores using summary statistics and LD information.44 However, one of its limitations is that its underlying algorithm assumes the existence of causal variants, which may result in limited predictive performance. In addition, its Gibbs sampler is sensitive to the model parameters for the large sample sizes. Moreover, LDpred can not predict PRS accurately for genomic regions with long-range LD, for instance, the human leukocyte antigen (HLA) region of Chromosome 6.23,25 However, long-range LD regions of the genome might contain many known disease-relevant variants.45,46 Privé et al. developed a new version of LDpred to address these shortcomings and improve its computational efficiency.38 This new version of LDpred has been implemented in the R package bigsnpr; see the next section.\n\nLDpred2 is the improved version of LDpred tool by introducing new options to learn the effect accurately. For instance, the option sparse can estimate the effects that are 0 while the option auto can estimate the parameters from data and computes values for hyper-parameters p and h2. Due to these improvements, LDpred2 has been widely used to generate polygenic models with good predictive performance.47 However, LDpred2 still has some issues regarding its stability.25,23 These issues contributed to the discrepancies in reported prediction accuracies.37,48 For instance, in contrast to LDpred, LDpred2 performs very well in the HLA regions but not for all traits as LDpred2 does not perform well for type 1 diabetes (T1D) and pure red cell aplasia (PRCA). LDpred2 performs poorly on T1D because T1D is mainly composed of large effects in the HLA region, while summary statistics typically have a small sample size. However, it is unknown why LDpred2 performs poorly, specifically for PRCA. Further studies are needed to understand why LDpred2 under-perform in these two cases.\n\nPRSice, developed by Euesden et al.36 in 2015, was the first specialized PRS analysis program. PRSice is built in R and includes wrappers for bash data management scripts as well as PLINK-1.9 to speed up computation (Table 1). Using a list of m SNPs and n individuals from the ‘target phenotypic’ dataset, here, thegenotypes have some influence on the ‘base phenotype’. If assessing the common genetic overlap of phenotype between samples/populations, the base and target phenotypes may be the same. A univariate regression on the base phenotype for each SNP, such as from genome-wide association research, can be used to estimate genotype effects (GWAS). For a SNP i, where i = 1, 2, …, m, a p-value, Pi, is computed for the association between the SNP and genotypes, Gi,j=0,1,2 for individual j where j = 1, 2, …, n and the phenotype. Under the standard additive assumption used in GWAS, a corresponding effect size for the effect of a unit increase in genotype Gij on the phenotype is estimated by βi. The degree of estimate is used to determine which SNPs should be included in a PRS value. SNP i will be included in in a PRS computation if Pi is less than a threshold, PT, based on the p-value for their association with the base phenotype in a GWAS. Typically, PRS values are calculated at distinct PT p-value thresholds.\n\nAt threshold PT, the PRS value for individual j can be calculated as:\n\nThe PRS value is computed across all individuals, yielding n scores per PT threshold value. A suitable regression model could be used to assess the relationship between these PRS values and the target phenotype. The PRSice tool was created to fully automate PRS analyses, significantly enhancing PLINK-1.9’s capabilities.49 Unless the genotypes have previously been imputed, there is generally some missing genotype data in real data. PLINK-1.9 fills in any missing data using mean allele frequencies. Nevertheless, it is not equipped to handle very large data sets. Hence a more memory-efficient approach is used in its advanced version, PRSice-2.\n\nPRSice-2 is an improved version of PRSice. It works with genotyped and imputed data, gives empirical association p-values that are free of overfitting inflation, supports numerous inheritance models, and analyzes numerous continuous and binary target traits at the same time.37 This technique simplifies the PRS analysis pipeline by eliminating intermediary files and doing all of the core computations in C++, resulting in a significant decrease in execution time and memory use. Furthermore, while computing the PRS value, PRSice-2 can immediately handle the BGEN imputed format and convert it to either best-guess genotypes or doses without producing a big intermediate file. While PRS values based on best-guess genotypes are produced using genotyped input, PRS values based on dose are derived using the following formula:\n\nWhere ωij is the probability of observing variant j, the value of j∈0,1,2, for the ith SNP/variant; m represents the number of SNPs/variants; and βi denotes the effect size of the ith variant estimated from the relevant base data set. A simulation study has been used to compare the performance of PRSice-2 to alternative polygenic score software lassosum21 and LDpred16 in terms of run time, memory usage and predictive power on servers equipped with 286 Intel 8168 24 core processors at 2.7 GHz and 192 GB of RAM.\n\nBased on a simulation study, PRSice-2 outperformed lassosum and LDpred in all circumstances. PRSice-2, in particular, can do full PRS analysis on 100,000 samples in 4 minutes, 179 times quicker than lassosum, which required 10 hours for the same task, and 241 times faster than LDpred, which took about 13 hours 27 minutes. Similarly, PRSice-2 uses substantially less memory than lassosum and LDpred, requiring less than 500 MB for 100,000 samples against 11.2 GB for lassosum and 45.2 GB for LDpred.\n\nIn another study to compare its predictive power for quantitative traits with a heritability of 0.2 and a base sample size of 50,000, and a target sample size of 10,000, PRSice-2 resulted in PRS values that are higher than LDpred but not as high as lassosum. The details about how it performs, inspection and analyses can be found (here). While the PRS values obtained by PRSice-2 do not fully optimize prediction accuracy, the straightforward technique and use of fewer SNPs allow for a clearer understanding of the results when compared to approaches that employ all SNPs.50\n\nLassosum is an alternative method that uses summary statistical data to estimate PRS and takes LD into account by using reference panels21 based on the commonly used LASSO and elastic net regression.51,52 Consider the linear regression given below:\n\nFor which X represents a data matrix of n-by-p, and y denotes a vector of the observed outcome. LASSO is a commonly used method for deriving β estimates and y predictors, especially in cases where p is high and where it is rational to conclude that many β are 0. By minimizing the objective function, LASSO also obtains estimates of β given y and X. To test the efficiency of lassosum relative to LDpred, simulation studies were carried out using summary statistics accounting LD and Phase 1 data from Welcome Trust Case Control Consortium (WTCCC) for seven diseases.44 The outcome of LDpred, lassosum and simple soft-thresholding (setting s = 1 in lassosum) was compared with most of the diseases in the WTCCC dataset, except for T1D where lassosum seem to outperform LDPred. The performance of LDpred and lassosum was comparable when the number of causal SNPs was 1,000 and the sample size was 11,200 for the simulated phenotypes, and both were superior to soft thresholding. Unlike lassosum, LDpred’s performance was considerably reduced when the sample size was halved. The lassosum was not influenced in the same way when reducing the sample size by half. All methods performed equally when the number of causal SNPs was 25,000 and the sample size was 11,200. The fact that summary statistics can be confounded by population stratification and population heterogeneity makes the real-life application of PRS difficult. These problems in the lassosum design were not considered. One possible issue with the use of meta-analytical summary statistics is that the original data produced by the summary statistics was an amalgamation of datasets around the world with corrections for population stratification. There is possibly no homogenous dataset suitable as a reference panel. Further research is required to explain the best approach.\n\nSchork et al.53 have demonstrated that different genome regions have different false discovery rates, thus have different chances of being causally correlated with a phenotype. Genome annotation information can be used theoretically to enhance the performance. Similarly, it is possible to utilize the fact that certain phenotypes have common genetic determinants (pleiotropy) to improve PRS.\n\nPLINK 1 is an open-source C/C++ toolbox for population genetics research and GWAS data analysis. The increasing rise of data from imputation and whole-genome sequencing research necessitated the urgent need for speedier and scalable implementations of its essential functionalities. Furthermore, genotype likelihoods, phase information, and multiallelic variations are commonly found in GWAS and population-genetic data. However, these features cannot be handled by PLINK 1 primary data format cannot accommodate any of these. For these reasons, Chang et al.42 developed a new version called PLINK 1.9. This version features heavy use of bit-level parallelism, O (pn)-time/constant-space Hardy-Weinberg equilibrium computation, Fisher’s exact testing, and a slew of other algorithmic enhancements. PLINK 1.9 speeds up most processes by 1-4 order of magnitude, allowing it to handle data sets that are too huge to store in RAM. The basic functional domains of PLINK 1.9 are identical to those of its predecessor, and it may be used as a drop-in replacement for existing scripts in most circumstances. Features, including the import/export of VCF, Oxford-format files, and fast cross-platform genomic relationship matrix calculators, have been included to facilitate easier interoperability with newer applications. Despite its computational advantages, PLINK 1.9 may still be an unsuitable tool for working with imputed genomic data due to the limitations of the PLINK 1 binary file format. To address this problem, the authors have developed PLINK 2.0, which features a new core file format capable of holding the bulk of the data generated by modern imputation systems.\n\nApplying PRS analysis for multi-ethnic groups is still limited. Novel PRS methods have been developed to address the applicability of PRS analysis across ethnic groups.\n\nMulti-ethnic PRS analysis: Multi-ethnic PRS analysis is a new PRS approach that combines PRS analysis based on two distinct populations.54 For instance, multi-ethnic PRS analysis could merge PRS analysis based on European training data with PRS analysis based on training data from another population. The multi-ethnic PRS approach computes PRS value given a target individual with genotypes g as follows:\n\nwhere M denotes the number of individual’s genetic markers, and the term b̂i is an estimate of effect sizes. For a multi-ethnic PRS analysis, this approach uses a linear combination of the two distinct PRS values and applying mixing weights parameters αi.\n\nLinear unbiased predictors (BLUP): PRS analysis can be molded using the well-known approach of best linear unbiased predictors (BLUP).55 BLUP is used to consider and linearly model both random effects and fixed effects. It is also known as genomic best linear unbiased prediction (gBLUP).56 BLUP/gBLUP estimates PRS values using the following formula\n\nWhere β represents a vector of the fixed effects, g denotes the total genetic effects in the base/training dataset, and ε are the normally distributed residuals. To evaluate the fixed effects, BLUP considers an individual GWAS indicator, the top 5 principal components (PCs) derived with all samples together and/or a list of the significant SNPs. The BLUP approach is a computationally efficient algorithm. Nevertheless, the limitation of BLUP arose due to its requirement of the Individual-level genotype data. BLUP has been implemented in GCTA software (Genome-wide Complex Trait Analysis) . Moreover, it has been extended to XP-BLUP to model PRS values for admixed populations.56 Also, BLUP has been extended to MultiBLUP to include multiple random effects.57\n\nGenetic Risk Scores Inference (GeRSI): GeRSI uses mixed models by combining fixed-effects models and random-effects models for controlling population structure.58 GeRSI performs Gibbs sampling to estimate individuals’ genetic risk score given the case-control study’s genotypes under a random-effects model. GeRSI proposed conditional distributions of the genetic and environmental effect using the standard liability-threshold model. One limitation of GeRSI is that it requires individual-level genotypes which are not available to many bioinformaticians.\n\nCross-population BLUP (XP-BLUP): XP-BLUP is an extension of the BLUP method that can be applied to trans-ethnic populations.58 XP-BLUP utilizes trans-ethnic information to improve PRS value predictive accuracy in minority populations. It combines the linear mixed-effects model (LMM) of the GeRSI method with the BLUP method.\n\n\nPRS analysis and population structure\n\nThe main cause of false-positive genotype-phenotype associations in PRS analysis is from population genetic structure.59,17 In African populations with population structure, GWAS analysis techniques provide a significant rate of false-positive results.60 These findings are influenced by the cohort’s relatedness rather than variations that have an effect on the trait or disease risk.60 In general, structures in mating patterns induce structures in genetic variation closely associated with geographic location. Furthermore, risk factors due to the environmental exposure may be creating the possibility for correlations between genetic variations. Sul et al.60 have noted some confounding issues that are unique to GWAS research, such as 1) genetic artifacts such as errors on SNP array chips; 2) phenotypic and environmental diversity in the participants, such as gender, ancestry, and age; and 3) strategic ignorance about disease risk.59 These confounding factors affect the genomic composition of populations and are difficult to calculate as they are not openly evident.17,59,60 The characteristics examined are confounded by example and location.61,62 Usually, this issue is resolved in GWAS by modifying the PCs61 or by using mixed models.63\n\nThe population composition in the PRS study presents a possible great issue since there are a significant number of null variants in PRS estimation. For example, allele frequencies are systematically different between the base and target data. These can be obtained from genetic drift or genotyped variants.64 In addition, there is a danger that variations in null SNPs may result in the correlation between the PRS and target traits if the distributions of the environmental risk factors for the phenotype vary in base and target data or highly probable in most PRS studies. Even if the GWAS had completely regulated its population structure, confounding is possibly reintroduced. Correlated variations between the base and target data in allele frequencies and risk factors are not taken into consideration.\n\nThe regulation of structure in the PRS study should be adequate to prevent false-positives, if the base and target samples are drawn from the same or genetically similar populations. Choi et al.17 advised that there are drastic variations between populations in the distribution of PRS.64–66 Such observations do not indicate many differences between populations in etiology. Genuine differences are likely to contribute to geographical, cultural and selection pressure variations. It challenges the use of base and target data from different populations in PRS studies that do not tackle problems of possible uncertainty generated by geographical stratification.65 Therefore, by exploiting large sampling sizes, the effect can be obtained using subtle confounding. The issues of population structures are as important as the variations between individuals in the base and target populations in genetics and the environment. In the coming years, the discussion of generalizability of PRS methods across populations can be an active field.66,54\n\n\nPopulation bias in available genotyping platforms\n\nThe PRS method that could be applied to diverse populations is still a challenging task.65 Many factors limit the application of PRS across diverse populations. These factors include:\n\n• The limitation in the current genomics technologies\n\n• LD distribution across diverse population\n\n• The minor allele frequencies (MAF) distribution\n\n• The distribution of the causal variants across diverse populations.\n\nCurrent sequencing technologies are based on the European reference genome. Hence, the current genomics technologies are still not robust enough to capture genetic diversity among trans-ethnic populations. Studying LD patterns across diverse populations showed that the distribution of LD patterns plays a critical role in the underlying PRS value.67,68 Incorporating the information of LD patterns across diverse populations would increase PRS utilities among trans-ethnic populations. Moreover, the utility of PRS across diverse populations has limited the MAF across diverse populations.65,67 The differences in MAF variants across diverse populations will result in different variant selection,69 which will reflect PRS in calculations. Furthermore, to improve the utility of PRS across diverse populations, researchers should investigate the causal variants shared across multi-ethnic groups.70 Type 2 diabetes and body mass index account for 70-80% of African ancestry. However, because of variations in LD and allele frequency, the accuracy of African-based PRS was lower than that of European-based PRS. Some studies showed that Europeans’ causal variants are also likely to be shared in African ancestry.71–73 Despite this, we can not generalize that the causal variants shared among trans-ethnic groups due to the limitation of representation of non-European populations, including sub-Saharan African communities. Previous approaches introduced to increase PRS accuracy in African populations prioritize the use of population-specific weighting and European discovered variants. However, due to the small sample sizes in African population, only moderate gains in accuracy are attainable. The example of a method that allows ethnic-specific weights to be included in their model is a two-component linear mixed model. In another study, Márquez-Luna et al.54 used Latino training data with limited sample size and publicly available large sample size European summary statistics to predict type 2 diabetes in a Latino cohort. When compared to previous methodologies, they achieved a relative improvement in prediction accuracy of more than 70%. This technique was also used to predict height using European and African training data in an African UK Bio bank.\n\n\nLimitations of current PRS algorithms\n\nThe methods for performing PRS vary based on two primary factors: (i) the list of SNPs to be used, and (ii) the weights to be used. Given the LD structure between SNPs, depending on the the trait’s genetic architecture and GWAS discovery sample size, the appropriate technique for determining what weights to apply and which SNPs to choose will differ between traits. The following tools LDpred, LDpred funct, SBLUP, P+T, LDpred-Inf PRS-CS, SBayesR, and PRS-CS-auto were employed in a comparative study to assess the PRS approaches in terms of their predictive potential.74 To accomplish this task, data from the major depressive disorder and Psychiatric Genomics Consortium working groups on schizophrenia were used. The results demonstrate that SBayesR outperforms the other tools in terms of speed and predicted accuracy. SBayesR, on the other hand, cannot produce converged solutions if the GWAS summary statistics have non-ideal features. While the benchmark P+T approach performed the least, the other tools achieved nearly the same level of accuracy. In addition to being the best approach in this study, SBayesR has been designed to learn the genomic architecture from the GWAS attributes. Some of these approaches, including LDpred, use tuning cohorts to specify parameters for the target cohort. When the length of the Markov chain Monte Carlo chain increases for example in LDpred, the prediction accuracy improves. One drawback of such strategy is that the user will have to tune the model parameters. Substantial effort is currently ongoing to expand GWAS sample collection across demographic groups. Most of the existing tools use only samples of European ancestry in the comparative PRS study. As a result, further study is needed to assess the accuracy of alternative techniques in other ancestries and across ancestries, taking into account probable differences in genomic architectures and LD.\n\n\nThe predictive power of PRS analysis\n\nMost articles within the current literature consider sample size as a milestone to power the PRS analysis. In 2013, Dudbridge estimated the predictive power of the polygenic score using results from several published studies.12 Dudbridge concluded that all published studies with a significant association of PRS values are statistically well-powered. In addition, Dudbridge pointed out that the accuracy of the PRS analysis depends only on the size of the initial data set (training sample). Furthermore, he provided a mathematical model to estimate the statistical power of PRS value as a function of sample size. In 2014, Middeldorp et al.28 suggested that PRS analysis on a sample size of 2000 individuals is good enough to obtain a statistically powered PRS value. However, Dima and Breen in 201575 demonstrated that a sample size of 1500 is enough to increase the predictive power to a statistically significant point. They stated that the predictive power of polygenic risk scores is not good enough for clinical utilities but it could be used as a biomarker for traits of interest within individuals. Recently, in 2017, Krapohl et al.5 introduced a multi-polygenic score that is capable of increasing the predictive power of PRS analysis. Regarding the relative accuracy of PRS values across ancestries, Yengo et al.76 proposed a theoretical model to estimate them. Their method utilized the frequencies of the minor alleles (MAF) in the two populations, the LD between the causal SNPs and the heritabilities. The authors assumed that causal variants are shared across ancestries however, their effect sizes might vary. Based on their model, Yengo et al.76 concluded that LD and MAF differences across ancestries explained 70-80% of the loss of relative accuracy of European-based PRS value in African ancestry.\n\n\nPRS clinical utility\n\nPRS analysis has been successfully applied to estimate and identify individuals with genetic risk for many biological traits such as type 2 diabetes, breast cancer, and prostate cancer (See the extended data109). Most of these studies provide significant evidence of the success of PRS analysis in identifying patients who are at high risk of developing disease complications. Additionally, the primary strength of PRS analysis is its capability of stratifying individuals based on their probability of developing a disease. The biological power of PRS analysis arose due to its capacity to identify therapeutic and genomic pathways for type 2 diabetes, breast cancer, and prostate cancer. Moreover, applying PRS analysis on these traits showed that the reproducibility of PRS results is in the European population.\n\nNonetheless, one weakness of applying PRS analysis on these traits is its limited ability in detecting false-positive results. It is observed that most PRS studies are only available for European ancestries. Therefore, we can not apply them to non-European communities. In addition, performing PRS analysis on sizeable multi-ethnic data is indispensable for obtaining more accurate PRS values across populations. Furthermore, the possibility of applying PRS outcomes for personalized medicine requires robust validation procedures before broad clinical applications for multi-ethnic communities.\n\nUnderstanding complex diseases and their clinical manifestations can be advanced significantly using accurate models for estimating PRS. The current PRS models can be used to forecast outcomes accurately. Disease subtypes and mechanisms that underpin within-trait diversity are not accounted for in PRS models, which might be important for analysis or therapeutic response.34,35,77,78 PRS models are used mainly to estimate clinical risk prediction for certain diseases, that can be extended to lifetime risk trajectories.79,80 Furthermore, PRS models can be implemented by clinical care authorities to decrease potential adverse health outcomes. Public health authorities can benefit from PRS models to control outbreaks of a particular disease by providing more efforts in high risk areas. PRS models can be used to define policies for administering the vaccination process. To use PRS accurately in clinical utilities as a personalized medicine tool, factors such as family history, rare monogenic mutations, ethnicity and ancestry, indirect genetic effects and gene-environment correlation should be considered. Refer to Table 3 for some commercial PRS kits that can be used for clinical utilities.\n\n\nPRS analysis on African populations\n\nThe approach of the PRS analysis is yet to be extensively applied to study traits in African populations. For instance, searching PubMed for PRS in African populations on August 21, 2021 (see Figure 1 and Box 1), only gave 8,843 hits. This number represents about 4.553483% of total hits that were obtained without using the term’for African populations’. The traits studied using PRS analysis in African populations include types 1 & 2 diabetes mellitus, depression, ischemic stroke, schizophrenia, sarcoidosis, Alzheimer’s disease, obesity, insomnia disorder, post-traumatic stress and cancer. Undermentioned are some selected PRS studies in sub-Saharan African populations.\n\nEkoru and his colleagues investigated the genetic risk scores for cardiometabolic traits in several African ancestries, including sub-Saharan African populations.81 They concluded that the predictive power of the risk score is limited in African ancestry populations. They stated that this limitation is due to the insufficient diversity among their samples of genomic discovery. Therefore, they adjusted the ancestry-derived principal components to obtain up to 5-fold and 20-fold higher predictive power. They observed that the predictive power of genetic risk scores was higher among African Americans (n=9139) and European Americans (n=9594) relative to the sub-Saharan African populations (n=5200). Based on this outcome, Ekoru and his colleagues concluded that PRS analysis performs poorly in sub-Saharan African populations. Furthermore, they recommended paying attention to the representation of the multi-ethnic population in genomic studies to improve the power of the genetic risk scores.\n\nIn 2020, Hayat and her colleagues investigated the genetic associations between serum low LDL-cholesterol levels and selected genetics variants in sub-Saharan African of four countries; Kenya, South Africa, Ghana and Burkina Faso.82 Using 1000 genomes data from the African populations, they selected four genes for their investigation (LDLR, APOB, PCSK9, and LDLRAP1). They performed genotyping of 19 SNPs using 1,000 participants in the Human Heredity and Health in Africa (H3Africa) AWI-Gen Collaborative Center (Africa, Wits-IN-DEPTH Partnership for GENomic studies). Although they used a limited number of variants, the outcome showed a significant association of these SNPs with lower LDL-cholesterol levels in sub-Saharan Africans.\n\nIn 2020, Cavazos and Witte proposed the inclusion of variants discovered from various populations to improve PRS transferability to diverse populations.83 They used both simulated data for the Yoruba group of the sub-Saharan African and European populations. They tested their findings on real data consisting of diabetes-free training samples of European ancestry (n = 123,665) and African descent (n = 7,564). They evaluated the performance of PRS analysis using genotype and phenotype data for a test (predictive) data set of European ancestry (n = 394,472) individuals of African origin from the UK Biobank (n = 5,886). Based on their findings, they concluded that incorporating variants selected from the European population will limit the accuracy of PRS values in non-Europeans populations including African communities. Also, they commented on the need for diverse GWAS data to improve PRS accuracy across populations.\n\nIn 2017, Marquez-Luna et al.54 proposed a multi-ethnic PRS analysis to improve risk prediction in diverse populations including African communities. To overcome the lack of enough training data for the African populations, the authors combined the training data from European samples and training data from the target population. We did not include their study because they did not state whether they used sub-Saharan African communities. This further highlights the challenge of performing PRS analysis in sub-Saharan African populations as a result of insufficient training data.\n\nIn 2017, Vassos et al. examined PRS values in a group of individuals with first-episode psychosis.84 For the control data set, they combined African-European (n= 70) and a sample of sub-Saharan African ancestries (n=828). Their finding showed that PRS value was more potent in Europeans, i.e. 9.4% discriminative ability, than in Africans, i.e. only 1.1% discriminative ability in Africans.\n\nPRS analysis is applied to investigate the risk score for prostate cancer. Prostate cancer is considered a complex genetic disease with high heritability which disproportionately affects men of African descent.85 A 1000 Genomes Project research that included seven African study sites and European males projected the risks of prostate cancer in urban African men. It was determined that the risks of prostate cancer are much more significant in African genomes than European genomes (p-value < 2.2 × 10e-16, Wilcoxon rank-sum test). This continental level pattern is consistent with public health data.86 A further investigation was done by the team of MADCaP (Men of African Descent and Carcinoma of the Prostate Consortium) to study sites that portrayed a substantial amount of overlap in the PRS distributions of different African populations. Based on their findings, the investigators of MADCaP observed within-continent heterogeneity for the predicted risk of prostate cancer. Their findings showed that individuals from Dakar, Senegal have the lowest predicted risks of prostate cancer than other African study sites while individuals from Abuja, Nigeria have the highest predicted risks. The MADCaP team concluded that allele frequency differences at common disease-associated loci can contribute to population-level differences in prostate cancer risk.\n\n\nChallenges of PRS analysis for the African populations\n\nMany PRS methods have been developed and applied to test the risk score of individuals. Nevertheless, PRS analysis has not been used in the clinical field for the African population. There are still many limitations and challenges regarding the application of PRS analysis in the African population. One of these challenges is lack of sufficient data to perform PRS analysis. For instance, querying the term “sub-Saharan” in the GWAS Catalog repository, the search resulted in only 70 publications out of 4,628 papers. Considering that several publications might use the same GWAS data, we affirm that more GWAS experiments need to be done in sub-Saharan African populations. Lack of African population genetic data might be due to the following reasons: (i) African populations are not well presented in the reference genomes for variant calling and genotype calling; (ii) There is insufficient genetic diversity to capture the African specific variations in the average observable African population, i.e. sample sizes and the number of sub-population representations; (iii) there is lack of infrastructure and funding to perform GWAS experiments in many countries in Africa. Infectious diseases like malaria, tuberculosis, and HIV might still be prioritized by African scientists due to their public health importance and funding opportunities. Providing funding priority for infectious diseases is necessary for African communities as they account for a higher mortality rate in the continent.\n\nDue to a lack of training and test data sets, some scientists choose to use training data from European samples that result in decreased PRS prediction accuracy. Therefore, PRS analysis is not widely applied for clinical utilities in Africa. The theory of genetics stated that when the genetic divergence in the target population and the original GWAS sample increases, the precision of the genetic risk prediction would decline. Several statistical discoveries are linked to this pattern: (i) The discovery of dominant genetic variations in the study population is favored by GWAS; (ii) even when the causative variants are the same, LD yields varied estimates of the marginal effect size for polygenic traits across populations; (iii) population-specific environmental and demographic differences. As a result, given the variety of the African population, the model developed elsewhere for PRS analysis does not fit for African sub-populations. Recent efforts to increase PRS accuracy in non-Europeans have prioritized the European discovered variants and population-specific weighting. Due to a limitation of GWAS studies in African populations, this technique might be utilized to construct an African-specific PRS method that incorporates diverse sources of information. While the African-specific PRS approach aims to improve PRS accuracy, the shortage of long-term funds for GWAS research is another major obstacle in conducting and applying PRS research in the African context. Understudied populations, particularly in Africa provide possibility for genetic research. The common variants in these populations but uncommon or lacking in the European population could not be discovered using European sample sizes. SLC116A11 and HNF1A genes, for example are linked to type 2 diabetes, whereas APOL1 is linked to prostate cancer and end-stage kidney disease in African-Americans. These issues are intractable with statistical techniques alone. Therefore, significant investment is required in African populations to yield similar-sized GWAS of biological traits.\n\nAs more data about genetic variation becomes available, the task of increasing the representation of African populations in the GWAS database has become increasingly essential.87,88 The inclusion of African multi-ethnic groups in GWAS analysis research is crucial for a more thorough, careful genetic variation and interpretation of the underpinnings of complex PRS analysis.87,88 In comparison to other under-represented populations, the average sample size of GWAS among Europeans continues to expand. PRS analysis in European populations has repeatedly failed to perform in African populations due to LD, confounding of environmental factors across populations and differences in allelic architecture.84,87–90, The frequency of causative, risk allele, correlated variants, and disease prevalence all show substantial-frequency variation between populations.16,88 The magnitude and frequency of disease-causing genetic variants differ greatly among different populations including African ancestry.91 Overcoming these obstacles might lead to an effective clinical management, and specialized therapy for individuals and populations impacted by these complex disease and risk factors all of which would improve the health of those affected.87,91,92 Moreover, it could help in decreasing genotype imputation error, increase levels of tag-SNP portability, GWAS design, and effectively addressed GWAS analysis and interpretation in Africa populations.88,91\n\nTherefore, African state authorities should be made aware of the challenges to make more funds available for genomic research. The funds should not be limited to the research institutes and principal investigators alone but they should equally provide scholarships (postgraduate programs like PhD) and financial aids for young African researchers. We have some promising African research consortiums like The Pan-African Bioinformatics Network for the Human Heredity and Health in Africa (H3ABioNet, h3abionet.org) and the Human Heredity and Health in Africa (H3Africa, h3africa.org) that are contributing in this regard. However, their funds come from outside Africa. There are new regional Africa efforts like the World Bank-funded Africa Center of Excellence (ACE). It is important to state that these initiatives consist of few genomic research projects. A follow-up project to the H3Africa, dedicated to data science health research, entitled Harnessing Data Science for Health Discovery and Innovation in Africa (DS-I Africa) will soon commence.\n\nMoreover, the lack of a pan-African genomic advisory board remains another challenge for genomic research in Africa. The existence of a research advisory board will help with transparency and establish ethical guidelines. These could open the window to get more grants from funding agencies such as the National Center for Biotechnology Information (NCBI). It is clear that without a rigorous ethical guide and transparency policies, it is hard to get long-term funds.\n\nOne more challenge of performing PRS for African populations is human migration. Environmental and social factors are the most critical drivers of disease risk than genetics in many cases so they must be effectively addressed. Benton et al.93 highlighted that early human migration out of Africa resulted in a higher genetic mutation rate, including disease-associated variants. Therefore, African populations do not carry the variants associated with disease at a higher frequency compared to non-African ancestries. As a result, given the genetic variation resulting from the diverse demographic history of the human populations, PRS prediction accuracy is still insufficient to generalize adequately across different populations, particularly for Africans.87,94 Furthermore, a lack of diversity in PRS development may contribute to existing health disparities among Africans.95,96 Therefore, consideration of environmental exposures and evolutionary histories must be key factors when performing PRS analysis.\n\n\nApplication of PRS analysis on type 2 diabetes in African populations\n\nDiabetes mellitus prevalence was projected in 2019 to be 463 million globally, 4% of which are in African populations.97 In addition, Africa will witness the world’s highest increase in diabetes prevalence by 2045.97,98 Currently, Africa has the most significant percentage of undiagnosed diabetics (59.7%) in the world. As a result, immediate policies and resources for developing surveillance and an early detection approach to help Africa combat this pandemic has been initiated.99 The use of PRS for the early detection of people who are genetically predisposed to type 2 diabetes could significantly reduce the diabetes burden. According to data from European nations, individuals in the top 90% of the population had a 5.21-fold higher likelihood of developing diabetes than those in the lowest 10%.100 Evidence has shown (coupled with a low GWAS study) that the transferability of polygenic scores developed in Europe decreases accuracy across diverse populations.87 Multi-ethnic PRS could be an alternative. However, the predictive performance of the African Americans and that of multi-ethnic PRS (who has about 80% African admixture) in continental Africans are yet to be examined.54,101 To ascertain this, Chikowore et al. aimed to see how well multi-ethnic, African-Americans, and European PRS would predict type 2 diabetes in Africans.99 For PRS development, the PRSice-2 software was used and the PRS with best result was chosen using area under the curve, i.e AUC and Nagelkerke R2. Finally, the results demonstrated that PRS derived from African Americans outperformed both multi-ethnic and European PRS in predicting type 2 diabetes. An earlier study of type 2 diabetes based on genetic risk score in Black South Africans used weight from Europeans (OR = 1.21, 95%CI).2 However, due to weights obtained from European-only studies, limited sample size, and use of only genotyped SNPs, this research was less predictive of Type 2 diabetes. Unlike previous work, this current study (Fatumo et al.99) took advantage of a larger sample size (1,690), improved genome coverage and a multi-ethnic discovery dataset GWAS. All of these factors worked together to improve the PRS predictive ability.2\n\n\nPRS analysis on breast and prostate cancers in the continent of Africa\n\nAfrica reportedly has the highest age-standardized death rate of breast cancer globally with sub-Saharan Africa having the highest prevalence rates. Although the occurrence in Africa was lower than in other continents, except for Asia, the mortality rate in Africa’s sub-Saharan region (for example in Nigeria) was the highest in the world.102 Men of African origin have a greater prevalence and mortality rate from prostate cancer than men of other ethnic groups. Uganda has one of the highest prostate cancer incidence rates of all African nations.103 Genetic contributions to this difference are supported by evidence of genetic heterogeneity across populations. Breast and prostate cancer research in African populations can contribute to the elevated disease burden within this population by genetic risk factors. As a result, policymakers, academics and the general public must become aware of the rising threat that breast and prostate cancer can pose to Africa’s growth. Early detection and stratification of women and men based on their risk of breast and prostate cancer using PRS could enhance screening and prevention strategies. Early detection of high disease risk individuals could also reduce the burden and threat to Africa’s development. The application of PRS for breast and prostate cancer allows for early detection and risk stratification for recommendations and monitoring.104 To date, most of the GWAS SNPs were found almost entirely in European ancestry populations. They also demonstrate distinct patterns of relationship among the African populace.15,104 In addition, variants found in one community often do not apply to other populations of African ancestry.105 These contradictory findings may be attributed to various factors, including variations in allele frequencies and LD and differences in population characteristics within one ethnicity. As a result, there is a risk of PRS transferring PRS across populations.106 Some studies investigate PRS developed using GWAS data from various ancestry groups.107,108 For example, Belsky et al.107 constructed an obesity-based PRS relying on GWAS from European ancestry and discovered that it performed poorly in African Americans but worked well in European ancestry.107 On the other hand, Fritsche et al.105 concluded that, to some degree, cancer based PRS obtained from large Europeans ancestry GWAS may still be employed for disease risk stratification in populations if the limitations listed below are properly addressed:\n\n• To accurately put an individual’s PRS within their reference PRS distributions, a matched ancestry cohort with large control sample sizes is required.\n\n• Non-European ancestry-derived PRS will be particularly useful for breast and prostate cancers because they have certain advantages over other traits: the high heritability is relatively high, normal in all ancestry groups, and publicity of summary statistics.\n\n• Unlike individuals of diverse ancestries from different populations, the participants in the UK Biobank are mostly from the same country and healthcare accessibility and other risk factors are similar.\n\nIf summary statistics and large GWAS are available, Fritsche et al.103 argued that PRS development based on the same ancestral group might increase its predictive ability if summary statistics and large GWAS are available. Several methods are now being investigated to increase PRS predictive accuracy in African populations. If a large-scale GWAS for non-European populations are unavailable, these methods might be employed to improve PRS. On the other hand, these methods may incorporate the fact that SNP selection based on European based GWAS is applicable when employing European based GWAS effect sizes in ethnically mismatched populations.71,103\n\n\nConclusion and future research\n\nThere are several approaches under the umbrella of PRS analysis. GWAS are conducted on finite samples extracted from particular subsets of the human population. Moreover, the SNP effect size estimates are some combination of true effect and stochastic variation, thus producing’winner’s curse’ among the top-ranking associations and the estimated effects may not be well generalized to different populations. Furthermore, the correlation complicates the aggregation of SNP effects across the genome. Therefore, linkage disequilibrium holds the key to apply PRS analysis across ethnic groups. Thus, critical factors in the development of methods for calculating PRS values are\n\n• The potential adjustment of GWAS estimated effect sizes e.g. via shrinkage and incorporation of their uncertainty.\n\n• The tailoring of PRS values to target populations.\n\n• The task of dealing with LD.\n\nAs members of the H3Africa consortium and the Associated Bioinformatics Consortium, H3ABioNet, (see h3abionet.org and https://sysbiolpgwas.waslitbre.org), we are working to extend existing methods to be applicable to African populations. Also, one future direction will be to develop an African-specific PRS method that combines the different sources of information. The information that we would consider to improve the current PRS methods include: (i) individual’s ancestry information to include the diversity within Sub-Saharan populations; (ii) environmental risk factors to include the environmental diversity in Africa. Due to the variation in genetic architecture among trans-ethnic groups, we will consider incorporating information at the transcriptome level in the Sub-Saharan populations. Thus, providing a new PRS method that performs individual ancestry estimation and transcriptome risk score would improve the predictive value of the PRS besides providing insights into the molecular determinants of phenotypic traits, including rare diseases.\n\n\nData availability\n\nNo data is associated with this article.\n\nDryad: Polygenic Risk Score in Africa Populations: Progress and challenges, https://datadryad.org/stash/share/btv5LlmVX0wVXdghkEOg1Thhua5VqQhugcG-82592lQ.109\n\nThis project contains the following extended data:\n\n• README file which provides information about the contents of the other file.\n\n• A table contains selected studies in 2020 that demonstrate the PRS methods applied to diabetes type II, prostate cancer, and breast cancer.\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\nAuthors acknowledge the logistical support of Mr. Babajide Ayodele. 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Reference Source" }	[ { "id": "124444", "date": "10 Mar 2022", "name": "Bingxin Zhao", "expertise": [ "Reviewer Expertise genetics and 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\nThis is an interesting paper on the review of PRS, with a focus on African populations. The research question is interesting and the writing is knowledgeable. I have the following suggestions that might improve the quality of this paper.\n\nMajor:\nAlthough the title states that this paper focuses on African populations, I feel the current version of this paper is a bit too focused on general PRS research in all populations. I encourage the authors to include more details and examples of PRS applications in African populations, especially sub-Saharan African communities. Then, the paper may be more balanced.\n\nFor example, in Section \"PRS analysis on African populations\": The traits studied using PRS analysis in African populations include types 1 & 2 diabetes mellitus, depression, ischemic stroke, schizophrenia, sarcoidosis, Alzheimer’s disease, obesity, insomnia disorder, post-traumatic stress and cancer. Undermentioned are some selected PRS studies in sub-Saharan African populations.\nThe citations of these studies could be provided in the paper or a supplemental table. Then, the authors could provide a general overview of all the findings of these papers in the main text or supplemental note.\nThis is just one example, the authors could consider improve other sections of the paper to extend the discussions of African populations.\n\nIn the \"The predictive power of PRS analysis\" section, the authors could discuss and include a few more recent studies on the mathematical properties of PRS, such as https://onlinelibrary.wiley.com/doi/10.1111/biom.13466 and https://arxiv.org/abs/1911.10142. Particularly, https://arxiv.org/abs/1911.10142 studies the cross-population accuracy, which may fit the topic and discussion paper well.\n\nMinor:\nIn the beginning of the \"PRS methods that incorporate LD\" section, the \"W\" in \"When\" should be lower case?\n\nIn the sentence \"Feng & Smoller 24presented the PRS-CS-auto method, a fully Bayesian approach that enables automatic learning of...\" The citation (24) of Feng and Smoller is wrong?\n\n\"For instance, searching PubMed for PRS in African populations on August 21, 2021 (see Figure 1 and Box 1), only gave 8,843 hits.\" What is the result for sub-Saharan African?\n\n\"This number represents about 4.553483% of total hits that\" 4.55% could be enough here.\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": "9220", "date": "11 Apr 2023", "name": "Ezekiel Adebiyi", "role": "Author Response", "response": "Answers from the authors to the reviewer’s comments Authors would like to thank the reviewer for valuable comments and suggestions. Below are the answers for each reviewer’s comments: Our Response to the Major Comments 1. Although the title states that this paper focuses on African populations, I feel the current version of this paper is a bit too focused on general PRS research in all populations. I encourage the authors to include more details and examples of PRS applications in African populations, especially sub-Saharan African communities. Then, the paper may be more balanced. For example, in Section \"PRS analysis on African populations\": The traits studied using PRS analysis in African populations include types 1 & 2 diabetes mellitus, depression, ischemic stroke, schizophrenia, sarcoidosis, Alzheimer’s disease, obesity, insomnia disorder, post-traumatic stress and cancer. Undermentioned are some selected PRS studies in sub-Saharan African populations. The citations of these studies could be provided in the paper or a supplemental table. Then, the authors could provide a general overview of all the findings of these papers in the main text or supplemental note. This is just one example, the authors could consider improve other sections of the paper to extend the discussions of African populations. The Authors thank the reviewer for this major comment and we agree totally with the reviewer’s point of view. Therefore, we have reviewed the content and also changed the title-heading “PRS Analysis on African Populations” to more specific title-header “PRS Analysis on Sub-Saharan African Populations”, page 15. More so, we have cited the key articles and summarized the findings in the main text of the manuscript while we included additional table content, pages 17-19. The following text is provided for the revised version of the manuscript. PRS Analysis on Sub-Saharan African Populations The PRS Analysis on Sub-Saharan African populations is limited due to lack of enough GWAS studies on traits associated with Sub-Saharan African populations. For instance, searches on PubMed for PRS on Sub-Saharan African populations on December 23, 2022 (see Figure 1 and Box 1) results in only 5 hits (4 research articles and 1 review paper). These four research articles performed PRS analysis mainly on traits associated with cardiometabolic disease such as heart attack, type 2 diabetes, and stroke. Other contributing risk factors including body mass index (BMI), waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), systolic blood pressure (SBP), diastolic blood pressure (DBP), triglycerides (TG), total cholesterol (TC), low-density lipoprotein(LDL), high-density lipoprotein (HDL), fasting plasma glucose( FPG), and type 2 diabetes (T2D), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TGs) and total cholesterol (TC) (Ekoru et al., 2021; Kamiza et al., 2022; Choudhury et al., 2022; Meeks et al., 2022). More so, the variance detected for Sub-Saharan populations in these studies has been summarized in Table 4. The general outcome of these five articles emphasize an urgent needs of GWAS research studies for Sub-Saharan African populations in order to continue to perform PRS analysis that would add more benefit to the use of PRS in precision medicine as well as an improved representation of multiple ethnic populations in GWAS to better reflect risk stratification, variabilities in genetic equitable, and translation of GRS in clinical setting . For instance, Ekoru et al (2021) demonstrated that several traits such as cardiometabolic have less predictive power of genetics risk score in Sub-Saharan Africans compared to others populations such as African Americans and European Americans. The less predictive power of cardiometabolic traits were as a result of underrepresented African populations based on GWAS data in the current reference genomes. However, Kamiza et al. (2022) studies showed an increase in PRS performance on lipid traits (such as, LDL-C) with dataset from Sub-Saharan populations, European, and multi-ancestry. Other lipid traits include HDL-C, TGs and TC. PRSs performance varies significantly even among the sub-Saharan African populations. This variation on PRS performance occurs due to variations on Africa population-specific genetic structure, such as minor allele frequencies and the population-specific associated environmental factors. It is worth reporting that there are several PRS studies that have been done using African populations. However, these studies are not restricted to sub-Saharan Africa's populations because the 1000 genomes reference panel data include samples from Africa populations. In 2020, Hayat and her colleagues investigated the genetic associations between serum low LDL-cholesterol levels and selected genetic variants (Hayat et al., 2020). Using 1000 genomes data from the African populations, they selected four genes for their investigation (LDLR, APOB, PCSK, and LDLRAP1). They performed genotyping of 19 SNPs using 1000 participants in the Human Heredity and Health in Africa (H3Africa) AWI-Gen Collaborative Center (Africa, Wits-IN-DEPTH Partnership for GENomic studies). Although they used a limited number of variants, the outcome showed a significant association of these SNPs with lower LDL-C levels in sub-Saharan Africans. In 2020, Cavazos and Witte proposed the inclusion of variants discovered from various populations to improve PRS transferability to diverse populations (Cavazos and Witte, 2020). They used both simulated data for the Yoruba group of the sub-Saharan African and European populations. They tested their findings on real data consisting of diabetes-free training samples of European ancestry (n = 123,665) and African descent (n = 7564).They evaluated the performance of PRS analysis using genotype and phenotype data for a test (predictive) data set of European ancestry (n = 394472) individuals of African origin from the UK Biobank (n = 5886). Based on their findings, they concluded that incorporating variants selected from the European population will limit the accuracy of PRS values in non-Europeans populations including African communities. Also, they commented on the need for diverse GWAS data to improve PRS accuracy across populations. In 2017, Marquez-Luna et al. (2017) proposed a multi-ethnic PRS analysis to improve risk prediction in diverse populations including African communities. To overcome the lack of enough training data for the African populations, the authors combined the training data from European samples and training data from the target population. We did not include their study because they did not state whether they used sub-Saharan African communities. This further highlights the challenge of performing PRS analysis in sub-Saharan African populations as a result of insufficient training data. In 2017, Vassos et al. (2017) examined PRS values in a group of individuals with first-episode psychosis (Vassos et al., 2017). For the control data set, they combined African-European (n= 70) and a sample of sub-Saharan African ancestries (n=828). Their finding showed that PRS value was more potent in Europeans, i.e. 9.4% discriminative ability, than in Africans, i.e. only 1.1% discriminative ability in Africans. References Badianyama, M., Mpanya, D., Adamu, U., Sigauke, F., Nel, S. and Tsabedze, N., 2022. New biomarkers and their potential role in heart failure treatment optimisation—An African perspective. Journal of Cardiovascular Development and Disease, 9(10), p.335. Ekoru K, Adeyemo AA, Chen G, Doumatey AP, Zhou J, Bentley AR, Shriner D, Rotimi CN. Genetic risk scores for cardiometabolic traits in sub-Saharan African populations. Int J Epidemiol. 2021 Aug 30;50(4):1283-1296. doi: 10.1093/ije/dyab046. PMID: 33729508; PMCID: PMC8407873. Kamiza AB, Toure SM, Vujkovic M, Machipisa T, Soremekun OS, Kintu C, Corpas M, Pirie F, Young E, Gill D, Sandhu MS, Kaleebu P, Nyirenda M, Motala AA, Chikowore T, Fatumo S. Transferability of genetic risk scores in African populations. Nat Med. 2022 Jun;28(6):1163-1166. doi: 10.1038/s41591-022-01835-x. Epub 2022 Jun 2. PMID: 35654908; PMCID: PMC9205766. Choudhury A, Brandenburg JT, Chikowore T, Sengupta D, Boua PR, Crowther NJ, Agongo G, Asiki G, Gómez-Olivé FX, Kisiangani I, Maimela E, Masemola-Maphutha M, Micklesfield LK, Nonterah EA, Norris SA, Sorgho H, Tinto H, Tollman S, Graham SE, Willer CJ; AWI-Gen study; H3Africa Consortium, Hazelhurst S, Ramsay M. Meta-analysis of sub-Saharan African studies provides insights into genetic architecture of lipid traits. Nat Commun. 2022 May 11;13(1):2578. doi: 10.1038/s41467-022-30098-w. Erratum in: Nat Commun. 2022 Aug 2;13(1):4474. PMID: 35546142; PMCID: PMC9095599. Meeks KAC, Bentley AR, Doumatey AP, Adeyemo AA, Rotimi CN. Mendelian randomization study reveals a causal relationship between adiponectin and LDL cholesterol in Africans. Sci Rep. 2022 Nov 8;12(1):18955. doi: 10.1038/s41598-022-21922-w. PMID: 36347891; PMCID: PMC9643497. 2. In the \"The predictive power of PRS analysis\" section, the authors could discuss and include a few more recent studies on the mathematical properties of PRS, such as https://onlinelibrary.wiley.com/doi/10.1111/biom.13466 and https://arxiv.org/abs/1911.10142. Particularly, https://arxiv.org/abs/1911.10142 studies the cross-population accuracy, which may fit the topic and discussion paper well. Authors thank the reviewer for his valuable suggestion. We added the text below to the manuscript, page 13, to improve it. “Zhao & Zou (2022) showed in their study that PRS predictivity can be improved based on SNPs selection. The process of SNPs selection depends on the genetic architecture, i.e, causal variants, and the sample size of the training data set. To select a set of SNPs that provide the optimal PRS prediction, the sample size of the training data set should be much larger than the number of potential causal variants. That is, performing PRS where the ratio of causal variants and sample size is large results in poor PRS prediction due failure in causal variants separations. Therfores, in the case of the ratio of causal variants to the sample size is large, i.e, small sample size is the training data set, Zhao & Zou recommended to include a large number of variants to get higher PRS prediction power. Moreover, Zhao & Zou recommended to include independent uncorrelated variants to improve PRS predictivity. Moverover, Zhao et al. (2022) demonstrated that accounting for correlation between causal variants, i.e, LD, will improve PRS predictivity and accuracy for heterogeneous populations. Furthermore, the performance of the PRS mathematical model can be assessed by evaluating the model's output using machine learning techniques, including area under the curve (AUC) of the receiver operating characteristic (ROC) (Janssens et al., 2007; Igo et al., 2019). The ROC can be visualized by plotting true positive rate against false positive rate for model’s thresholds. Janssens et al. (2007) recommend using a model that provides AUC >0.75 for PRS clinical utility, ie, screening of individuals who are at risk. Igo et al. (2019) has suggested using the proportion of trait variability explained by one or more variants as an indicator for PRS predictivity, for more details refer to Janssens et al. (2007). Igo, R. P., Jr, Kinzy, T. G., & Cooke Bailey, J. N. (2019). Genetic Risk Scores. Current protocols in human genetics, 104(1), e95. https://doi.org/10.1002/cphg.95 Janssens, A. C., Moonesinghe, R., Yang, Q., Steyerberg, E. W., van Duijn, C. M., & Khoury, M. J. (2007). The impact of genotype frequencies on the clinical validity of genomic profiling for predicting common chronic diseases. Genetics in Medicine, 9, 528–535. doi: 10.1097/GIM.0b013e31812eece0. Zhao, B., & Zou, F. (2022). On polygenic risk scores for complex traits prediction. Biometrics, 78(2), 499–511. https://doi.org/10.1111/biom.13466 Zhao, B., Zou, F., & Zhu, H. (2022). Cross-trait prediction accuracy of summary statistics in genome-wide association studies. Biometrics, 10.1111/biom.13661. Advance online publication. https://doi.org/10.1111/biom.13661 Authors responses to the Minor comment 1. In the beginning of the \"PRS methods that incorporate LD\" section, the \"W\" in \"When\" should be lower case? Authors thank the reviewers for the suggestions, and we applied the correction accordingly. 2. In the sentence \"Feng & Smoller 24presented the PRS-CS-auto method, a fully Bayesian approach that enables automatic learning of...\" The citation (24) of Feng and Smoller is wrong? Authors thank the reviewers for the suggestions, and we applied the correction accordingly. We cited it as Ge et a as given is the correct citation below Ge T, Chen C-Y, Ni Y, Feng Y-CA, Smoller JW. Polygenic prediction via Bayesian regression and continuous shrinkage priors. Nat Commun. 2019;10(1):1776. 3. \"For instance, searching PubMed for PRS in African populations on August 21, 2021 (see Figure 1 and Box 1), only gave 8,843 hits.\" What is the result for sub-Saharan African? Authors thank the reviewers for the suggestions, we updated our search terms and we have added the recent results, including PubMed hits for sub-Saharan African in Figure 1. 4. \"This number represents about 4.553483% of total hits that\" 4.55% could be enough here. We thank the reviewer, such small number demonstrate the lack of enough PRS studies on African populations." } ] }, { "id": "137670", "date": "22 Jun 2022", "name": "Cathryn M. Lewis", "expertise": [ "Reviewer Expertise Statistical Genetics", "Genetic epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral:\nAdam et al. provide an extensive review of the important topic of PRS methods, and their applications in African ancestry populations. The paper summarises the various approaches to calculating PRS and provides a fair assessment of the advantages and disadvantages of each method. It also describes the challenges associated with calculating PRS in African populations and the approaches currently being undertaken to address these. The paper is comprehensive and a useful addition to the literature, pulling together a large amount of information across methods for calculating polygenic scores, and their applications in African populations.\nMajor comments:\nTo make the application of polygenic scores more accessible the authors could summarise the findings from studies conducted in Sub-Saharan African populations. For example, a table that orders studies by outcome/disease type and summarises key study parameters: methods (cohort/populations, LD reference panel, method) and results (variance explained) would be useful to readers.\nWhen referring to predictive power being limited in African populations, additional detail as to what the AUC or equivalents are would be useful to contextualize the scores and provide comparisons to scores in non-African populations e.g. scores in EUR and AFR for a similar trait.\nWe suggest that the authors could also address the variability in transferability of scores not only between super-populations (eg. AFR and EUR) but also between SSA populations and the potential contributory role of environmental factors. Potential paper to do this includes Kamiza, A.B. et al. Transferability of genetic risk scores in African populations.1\nOne of the most recent useful advances in PRS development for ancestrally diverse populations is the PRS-CSx method.2 Although this new method was published after the date cut-off for the manuscript, a comment could be added in the Discussion.\n\nMinor comments:\nIntroduction:\nTable 1 is a comprehensive summary of PRS tools. How are these ordered? Given the authors have previously classified the methods into four groups (p3), it might be useful to use this information in structuring the table. In addition, what parameters/factors were used to determine whether an approach was user-friendly?\n\nP3 “PRS analysis is used to predict an individual heritability by incorporating all selected SNPs.” What do the authors mean here by the phrase ‘individual heritability’? PRS methods that incorporate LD. In practice, when the markers are LD pruned…\nPRS analysis on African populations:\nThis number represents about 4.553483% of total hits that - this number represents about 4.55% or 4.6% of total hits that\n\nThey observed that the predictive power of genetic risk scores was higher among African Americans (n=9139) and European Americans (n=9594) relative to the sub-Saharan African populations (n=5200). - Consistency in number formatting: They observed that the predictive power of genetic risk scores was higher among African Americans (n=9139) and European Americans (n=9594) relative to the sub-Saharan African populations (n=5200).\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": "9221", "date": "11 Apr 2023", "name": "Ezekiel Adebiyi", "role": "Author Response", "response": "Answers from the authors to the reviewer’s comments Authors would like to thank the reviewer for valuable comments and suggestions. Below are the answers for each reviewer’s comments: Major comments responses 1. To make the application of polygenic scores more accessible the authors could summarise the findings from studies conducted in Sub-Saharan African populations. For example, a table that orders studies by outcome/disease type and summarises key study parameters: methods (cohort/populations, LD reference panel, method) and results (variance explained) would be useful to readers. Authors thank the reviewer for his suggestions. Authors think that this point is quite similar to a comment raised by reviewer 1. Therefore, we address these two comments together by adding Table (4), pages 17-19. 2. When referring to predictive power being limited in African populations, additional detail as to what the AUC or equivalents are would be useful to contextualize the scores and provide comparisons to scores in non-African populations e.g. scores in EUR and AFR for a similar trait. Authors thank the reviewer for his suggestions, and we agree that adding AUC as a method for evaluating PRS method will be useful for readers who are not familiar with machine learning and model evaluation. Therefore, we addressed this point by adding a text about AUC in the main manuscript. 3. We suggest that the authors could also address the variability in transferability of scores not only between super-populations (eg. AFR and EUR) but also between SSA populations and the potential contributory role of environmental factors. Potential paper to do this includes Kamiza, A.B. et al. Transferability of genetic risk scores in African populations Authors accepted the reviewer's comment, we added the text below on page 19 of the revised version. We also added table 5 to give a summary of Transferability of PRS. in African populations. \"Previous studies suggested that PRS derived from individuals of African ancestry performed significantly better in sub-Saharan Africans than PRS derived from individuals of African Americans and Europeans and multiancestry (Duncan et al., 2019; Cavazos & Witte, 20210; Martin et al., 2019; Johnson et al., 2015). However, PRS might differ across sub-Saharan Africans populations due to differences in contributory role of environmental and genetic factors. For instance, Kamiza et al} reported that the differences in environmental and genetic factors play critical roles in transferability of PRS between the South African Zulu and individuals from Ugandan cohort Kamiza et al (2022), Table 5.. Finding from Kamiza et al} noted that the poor performance of PRS across populations has implementation impact in preventative healthcare. Therefore, applying PPRS to different ethnic groups, even within within sub-Saharan Africa, may lead to inaccurate result. This further suggests the need for more efforts to optimize polygenic prediction in Africa. For instance, Choudhury et al (2022) demonstrated that PRS transferability among African can be improved by sample size of the African cohort studies.\" References Duncan, L., Shen, H., Gelaye, B. et al. Analysis of polygenic risk score usage and performance in diverse human populations. Nat Commun 10, 3328 (2019). https://doi.org/10.1038/s41467-019-11112-0 Cavazos, T. B., & Witte, J. S. (2021). Inclusion of variants discovered from diverse populations improves polygenic risk score transferability. HGG advances, 2(1), 100017. https://doi.org/10.1016/j.xhgg.2020.100017 Martin, A. R., Kanai, M., Kamatani, Y., Okada, Y., Neale, B. M., & Daly, M. J. (2019). Clinical use of current polygenic risk scores may exacerbate health disparities. Nature genetics, 51(4), 584–591. https://doi.org/10.1038/s41588-019-0379-x Johnson, L., Zhu, J., Scott, E. R., & Wineinger, N. E. (2015). An Examination of the Relationship between Lipid Levels and Associated Genetic Markers across Racial/Ethnic Populations in the Multi-Ethnic Study of Atherosclerosis. PloS one, 10(5), e0126361. https://doi.org/10.1371/journal.pone.0126361 Kamiza AB, Toure SM, Vujkovic M, Machipisa T, Soremekun OS, Kintu C, Corpas M, Pirie F, Young E, Gill D, Sandhu MS, Kaleebu P, Nyirenda M, Motala AA, Chikowore T, Fatumo S. Transferability of genetic risk scores in African populations. Nat Med. 2022 Jun;28(6):1163-1166. doi: 10.1038/s41591-022-01835-x. Epub 2022 Jun 2. PMID: 35654908; PMCID: PMC9205766. 4. One of the most recent useful advances in PRS development for ancestrally diverse populations is the PRS-CSx method.2 Although this new method was published after the date cut-off for the manuscript, a comment could be added in the Discussion. Authors thank the reviewer for the comment and we agree that PRS-CSx method is on the key method that can be used for the application of PRS across multi-ethnic group. We did not include because we submitted our review before publishing PRS-CSx method. However, we have include an overview of the PRS-CSx method in our reviewed manuscript, page 11. We also cited the article for those are interested to know more about its underlying algorithm. PRS-CSx method PRS-CSx method is proposed to improve the accuracy of the application of PRS across multi-ethnic populations by using posterior inference algorithm (Ruan et al., 2022; Ge et al., 2022). PRS-CSx combines GWAS summary files from different population to increase the accuracy of PRS. PRS-CSx estimates population-specific effect size by incorporating the population-specific LD pattern, population-specific allele frequency information, and the information of shared continuous shrinkage prior across populations. For more details about the mathematical method underlying PRS-CSx, refer to Ruan et al., 2022). References Ruan, Y., Lin, YF., Feng, YC.A. et al. Improving polygenic prediction in ancestrally diverse populations. Nat Genet 54, 573–580 (2022). https://doi.org/10.1038/s41588-022-01054-7 Ge, T., Irvin, M. R., Patki, A., Srinivasasainagendra, V., Lin, Y. F., Tiwari, H. K., Armstrong, N. D., Benoit, B., Chen, C. Y., Choi, K. W., Cimino, J. J., Davis, B. H., Dikilitas, O., Etheridge, B., Feng, Y. A., Gainer, V., Huang, H., Jarvik, G. P., Kachulis, C., Kenny, E. E., … Karlson, E. W. (2022). Development and validation of a trans-ancestry polygenic risk score for type 2 diabetes in diverse populations. Genome medicine, 14(1), 70. https://doi.org/10.1186/s13073-022-01074-2 Minor comments: Table 1 is a comprehensive summary of PRS tools. How are these ordered? Given the authors have previously classified the methods into four groups (p3), it might be useful to use this information in structuring the table. In addition, what parameters/factors were used to determine whether an approach was user-friendly? The authors thank the reviewer for the comments. We determined whether an approach is user-friendly based on the installation process, the popularity of the methods among the users, the availability of an application manual (tutorial), the application user interface, and the number of options that should be considered and tuned by users. However, we have removed a column in the table (no.) that classifies the methods from the revised version based on four different approaches, such as (p3): clumping with thresholding (C+T), p-value thresholding, penalized regression, and Bayesian shrinkage, because some tools perform PRS analysis using more than one method. For instance, LDpred can perform PRS analysis either using clumping and thresholding (C+T) or the p-value thresholding method. We also cited et al . (2022), which performed a recent a comparison on some of these tools. References Wang Y, Tsuo K, Kanai M, Neale BM, Martin AR. Challenges and Opportunities for Developing More Generalizable Polygenic Risk Scores. Annu Rev Biomed Data Sci. 2022 Aug 10;5:293-320. doi: 10.1146/annurev-biodatasci-111721-074830. Epub 2022 May 16. PMID: 35576555. P3 “PRS analysis is used to predict an individual heritability by incorporating all selected SNPs.” What do the authors mean here by the phrase ‘individual heritability’? PRS methods that incorporate LD. In practice, when the markers are LD pruned… We mean by individual heritability here by the proportion of trait variance (phenotype) that is associated with genetic variants (genotype). We cited Privé et al (2020) & Vilhjálmsson et al (2015) that provided more details of individual heritability that can be explained by the genetic variants. References Florian Privé, Julyan Arbel, Bjarni J Vilhjálmsson, LDpred2: better, faster, stronger, Bioinformatics, Volume 36, Issue 22-23, 1 December 2020, Pages 5424–5431, https://doi.org/10.1093/bioinformatics/btaa1029 Vilhjálmsson BJ, Yang J, Finucane HK, Gusev A, Lindström S, Ripke S, Genovese G, Loh PR, Bhatia G, Do R, Hayeck T, Won HH; Schizophrenia Working Group of the Psychiatric Genomics Consortium, Discovery, Biology, and Risk of Inherited Variants in Breast Cancer (DRIVE) study, Kathiresan S, Pato M, Pato C, Tamimi R, Stahl E, Zaitlen N, Pasaniuc B, Belbin G, Kenny EE, Schierup MH, De Jager P, Patsopoulos NA, McCarroll S, Daly M, Purcell S, Chasman D, Neale B, Goddard M, Visscher PM, Kraft P, Patterson N, Price AL. Modeling Linkage Disequilibrium Increases Accuracy of Polygenic Risk Scores. Am J Hum Genet. 2015 Oct 1;97(4):576-92. doi: 10.1016/j.ajhg.2015.09.001. PMID: 26430803; PMCID: PMC4596916. PRS analysis on African populations: This number represents about 4.553483% of total hits that - this number represents about 4.55% or 4.6% of total hits that Authors thank the reviewer for this comment. We updated the Pubmed search terms that we used and formatted all numbers accordingly. They observed that the predictive power of genetic risk scores was higher among African Americans (n=9139) and European Americans (n=9594) relative to the sub-Saharan African populations (n=5200). - Consistency in number formatting: They observed that the predictive power of genetic risk scores was higher among African Americans (n=9139) and European Americans (n=9594) relative to the sub-Saharan African populations (n=5200). Authors thank the reviewer for this comment. We formatted the style of all numbers accordingly." } ] } ]	1	https://f1000research.com/articles/11-175
https://f1000research.com/articles/12-379/v1	11 Apr 23	{ "type": "Research Article", "title": "Distribution of sentence length and dependency distance in children’s compositions: Characteristics of natural language and variations in language development", "authors": [ "Mizuho Imada" ], "abstract": "This study analyzed the distribution of the sentence length and mean of dependency distances (MDD) in Japanese sentences, comparing data from random sources with that obtained from children's compositions, and identifying changes in distribution according to grade level. Findings reveal that the sentence length in random data is well suited to a geometric distribution, whereas MDD is well suited to a lognormal distribution. In contrast, data from children's compositions show a shift in the distribution of the number of clauses from a lognormal to a gamma distribution, depending on the school year, with MDD suiting a gamma distribution. Mean MDD increases exponentially with the logarithm of the number of clauses in random data, while it increases linearly in composition data, thus generally supporting previous findings that dependency distances are optimized in natural language. However, MDDs exhibit non-monotonic changes with grades, suggesting the complexity of children's language development.\n日本語の文における文節数および係り受け距離平均（MDD）の分布について、ランダムデータと児童作文データの比較、および学年による分布の変化を分析した。その結果、ランダムデータにおいては文節数は幾何分布、MDDは対数正規分布によく当てはまるのに対して、作文データにおいては文節数は学年によって対数正規分布からガンマ分布に推移し、MDDはガンマ分布によく当てはまることが分かった。また、MDDの平均はランダムデータでは文節数の対数に対して指数的に増大するのに対し、作文データでは線形に増大することが分かった。これは自然言語において係り受け距離が最適化されるという従来の知見を概ね支持する結果と言える。一方、MDDは学年に対して非単調な変化を示し、児童の言語発達の複雑さを示唆する。", "keywords": [ "sentence length", "dependency distance", "children's compositions", "probability distribution", "generalized linear mixed model", "文長", "係り受け距離", "児童作文", "確率分布", "一般化線形混合モデル" ], "content": "1. はじめに\n\n本稿では、日本語の文における文長（文節数）および係り受け距離の分布の特性について検討し、児童作文における分布の変異を分析する。文長は文の複雑性を代表する基礎的な指標と見なされており、文の可読性（リーダビリティ）を評価するためにしばしば利用される（建石ほか 1988、柴崎・原 2010、李 2016）。文長は文字、形態素、文節などを単位として測定されるが、おおむね右に裾野が長い分布を示すことが知られており、文字数については対数正規分布ないしガンマ分布（安本 1958、佐々木 1976、新井 2001）、形態素数についてはハイパーパスカル分布 (Ishida and Ishida 2007)、文節数については負の二項分布（古橋・早川 2012）の当てはまりがよいことが報告されている。\n\n一方、同じ長さの文であってもその統語的な構造は多様であり、それによって統語的な複雑性に違いがある。よく知られた指標としては文の構成要素間の依存関係の距離 (dependency distance) があり、一般に距離の長い依存関係は認知的負荷が大きいため、自然言語では長距離の依存関係は避けられる傾向があると考えられている (Gibson 1998)。依存距離の研究の多くは単語レベルの依存構造に依拠して行われており、その中には日本語を含む多言語における依存距離最小化の分析 (Liu 2008、Futrell et al. 2015、Ferrer-i-Cancho et al. 2022) や、日本語における文章ジャンルの位相差の分析（李ほか 2022）が含まれる。一方で、日本語の統語構造の表現としては文節単位の係り受け構造が広く用いられてきた経緯があり、係り受け距離の分布が Zipf 則に近いこと (Maruyama and Ogino 1993、金 1996)、日本語学習者の係り受け距離平均について学習者のレベルに応じた増大が確認できないこと (Komori et al. 2018) などが報告されている。日本語の構文情報についてはUD-Japanese BCCWJ（大村・浅原 2018）など単語単位の依存情報つきコーパスも開発されているものの、現在でも BCCWJ-DepPara（浅原・松本 2018）などの文節係り受け情報コーパスやCaboCha（工藤・松本 2002）などの文節係り受け解析器がよく用いられており、この構造の数学的特性を理解することは依然として有用性が高い。\n\nそこで本稿では、文節数に基づく文長の分布、および一文あたりの係り受け距離平均の分布という 2 つの観点から、文の統語的構造の数学的特性を検討する。検討のために、2 種類のデータを用いる。ひとつはランダム性を持つ文節列および係り受け構造であり、もう 1 つは児童作文における文節係り受け構造である。これらのデータを分析対象とすることには、いくつかの意味がある。第 1 に、ランダムデータと作文データを比較することで、自然言語に固有の数学的特性を知ることができる。第 2 に、作文データを分析対象とすることで、児童の言語発達と統語的複雑性の関連を調べることができる。分布の特性は自然言語の特性の研究においては強い関心が置かれている一方で、可読性や言語発達の研究ではそれほど関心が置かれていない。可読性研究の多くは平均文長を説明変数として利用しているが、文長の分布は特に問題とされていない。Komori et al. (2018) は日本語学習者のレベルと係り受けの距離や深さとの関連を調べているが、分布の正規性や等分散性を問題としない Brunner-Munzel 検定を使用している。今田 (2021)1 は児童の学年と係り受けの距離や深さとの関連を調べているが、ランダムデータに見られる対数正規分布をそのまま作文データに対して適用しており、自然言語に固有の特性を十分に考慮していない。本稿は、ランダムデータと作文データにおける文長と係り受け距離の分布の特性を詳細に検討し、その特性に基づいて児童の言語発達と統語的複雑性の関係を分析する。\n\n\n2. 方法\n\n「児童・生徒作文コーパス」（宮城・今田 2015）を使用する。このコーパスは小学校 1 年生から中学校 3 年生までの作文を電子化した係り受け構造タグ付きコーパスであり、自然言語の特性、および学齢による構造の変化を観察するために適している。コーパスの規模を表 1 に示す。\n\nこのコーパスを使用して、文長および係り受け距離の分布を調べる。文長については1文あたりの文節数をデータとする。係り受け距離については1文あたりの係り受け距離の平均 (MDD; Liu 2008) をデータとする。いずれも裾が長い分布を示すため、対数正規分布およびガンマ分布によるフィッティングを行い、当てはまりのよさを調べる。さらに文長については学年を固定効果、作文 ID をランダム効果とする一般化線形混合モデル分析、MDD については文長、学年および交互作用を固定効果、作文 ID をランダム効果とする一般化線形混合モデル分析を行い、文長、MDD、学年の関係を分析する。\n\n作文データの自然言語としての特性を明らかにするために、2 種類のランダムデータを対照データとして分析する。文長の分析においては、作文データの文節をランダムに並べ替えた文節シャッフル列を用いる。元データにおける文末文節をシャッフル列でも文末と見なし、ランダムな文節列における文長の分布を調べる。ランダムデータにおける文長は幾何分布に従うことが予測されるため、文長についてはデータの分布が幾何分布に従うことのみ確認する。\n\nMDD の分析においては、文節数 n の文における可能な全ての係り受け構造を列挙した係り受けパターン集合を作成し2、可能な全ての構造が等確率で生起する場合を仮定して、MDD の分布を調べる。可能な係り受け構造のパターン数はカタラン数で計算できるが、文節数に対して指数的に増大するため、文節数 2 から 10 の範囲まで作成する3。MDD は裾が長い分布を示すため、対数正規分布およびガンマ分布との当てはまりを確認し、さらに一般化線形モデルで MDD と文長の関係を分析する。作文データと異なり著者による個体差を伴わないので、混合モデルによる分析は行わない。\n\n全ての実験は R 言語を用いて行い、分布のフィッティングは MASS::fitdistr 関数、一般化線形モデル分析は stats::glm 関数、一般化線形混合モデル分析は lme4::glmer 関数を使用した。主なソフトウェアのバージョンを表 2 に示す。\n\n\n3. 文の長さの分布\n\n文がランダムに生成された場合、文長（文節数）の分布はどのようになるだろうか。本節では、作文データの文節をランダムにシャッフルした文節シャッフル列を用いて、文長の分布を調べる。元のデータで文末だった文節を、文節シャッフル列においても文末とする。文節シャッフル列は、長さ 598535 の文節列である。このうち文末文節の数は 77512 である。従って、文末文節は 77512/598535=0.1295029 程度の確率でランダムに生起することになる。\n\nこのような文節列において、文長の分布は幾何分布に従うと予想される。幾何分布は、確率 p で成功する事象が次に成功するまでの失敗数の分布とされる。確率 p で文末文節が生起する文節シャッフル列においては、次に文末文節が生起するまでの非文末文節の数x（=文長n-1）は次の幾何分布に従うと考えられる。\n\n図 1 は、文節シャッフル列における文長のヒストグラムと、p=0.1295029 の幾何分布曲線を重ね合わせたものである。ヒストグラムに曲線がよく当てはまることが確認できる。以上から、ランダムな文節列においては、文長は幾何分布に従うと考えることができる。\n\n曲線は幾何分布による予測曲線。分布の母数は文末文節の出現確率に基づく。\n\n文節シャッフル列における文長の分布が幾何分布に従うのに対して、実際の作文データにおける文長の分布は幾何分布とは異なり、対数正規分布ないしガンマ分布に類似する裾の長い分布を示す。図 2 は、作文データにおける文長のヒストグラムと、フィッティングによって推定した対数正規分布およびガンマ分布の確率密度曲線を重ね合わせたものである。対数尤度はガンマ分布が -213659.0、対数正規分布が -213695.8 で、ガンマ分布の方がわずかに当てはまりがよい。\n\n曲線はガンマ分布および対数正規分布によるフィッティング。フィッティングは R 言語の MASS::fitdistr 関数による。\n\n対数正規分布とガンマ分布のどちらの当てはまりがよいかは、学年によって異なる。図 3 は、学年ごとに 2 種類の分布のフィッティングを行い、対数尤度比 (LLR) をプロットしたものである。LLR が 0 より大きいとき、ガンマ分布の方が当てはまりがよい。学年別に見ると、小 1 から小 6 までは対数正規分布の当てはまりがよく、中 1 から中 3 まではガンマ分布の当てはまりがよい。このデータは、一文あたりの文節数の分布は、年齢が低いときには対数正規分布になるが、年齢が上がるに従ってガンマ分布に接近することを示唆する。\n\n0 より大きいときはガンマ分布の当てはまりがよく、0 より小さいときは対数正規分布の当てはまりがよい。\n\n従って全ての学年を単一のモデルで分析することには限界があるが、ここでは次の 2 つのモデルで一般化線形混合モデル分析を試みる。\n\nglmer(length ~ grade + (1\|document_id), family = Gamma(link = “identity”))\n\nglmer(length ~ grade + (1\|document_id), family = gaussian(link = “log”))\n\nどちらのモデルも被説明変数を文節数 (length)、固定効果を学年 (grade)、作文ID (document_id) を変量効果としたモデルである。第 1 のモデルは文長の分布をガンマ分布と仮定し、第 2 のモデルは文長の分布を対数正規分布と仮定している。学年はカテゴリ変数としている。作文 ID は、著者の個体差による効果を説明するためにランダム効果として加えている。結果を表 3 に示す。決定係数や AIC を見ると、全体としてはガンマ分布モデルの方が当てはまりはよいようである。\n\nR 言語の lme4::glmer 関数による。\n\n* p < 0.001.\n\nガンマ分布モデルについて、学年が文長に及ぼす効果の予測値を図 4 に示す。学年が上がるに従って文長が長くなることが確認できるが、一方で学年が上がるほど文長の増加速度は緩やかになる。\n\n髯は 95%信頼区間。R 言語の lme4::glmer 関数による解析結果を ggeffects::ggpredict 関数で可視化した。\n\n\n4. 係り受け距離平均の分布\n\n前節では文長（文節数）の分布について検討したが、同じ文節数の文でも係り受け構造には多様なパターンがあり、それによって文処理のための認知的負荷が異なることが考えられる。ここでは係り受け距離が文の複雑さの一側面を表すものと仮定して、ランダムな構造や自然言語の構造において係り受け距離がどのような分布を示すか検討する。\n\nなお、本稿では日本語の規範的な係り受け構造のみを検討対象とする。規範的な係り受け構造においては、文末以外の全ての文節は自分より後ろの文節に係り、文末の文節は係り先を持たない。1 つの文節は、最大で 1 つしか係り先を持たない（従って、1 文に含まれる係り受けの数は文末を除いた文節数と同じであり、文節数 n の文における係り受けの数は n-1 である）。また、係り受けは交差しない（例えば第 1 文節が第 3 文節に係り、第 2 文節が第4文節に係るというような交差はしない）。実際には語順の入れ替えによって後ろから前への係り受けが発生したり、係り受けが交差したりすることはあり得るが、本稿では検討しない。\n\n文単位で係り受け距離を数値化するために、係り受け距離平均 (MDD, Mean of Dependency Distances) を用いる。MDD は、ある文に含まれる全ての係り受けの距離の平均である。ある文に含まれる全ての係り受けの距離の和を SDD とすると、係り受けの数は文節数 n-1 なので、MDD=SDD/(n-1) になる。例として文節数4の文を考えると、可能な構造は 5 通りあり、それぞれの SDDとMDD は図 5 の通りである。\n\nSDD は係り受け距離和、MDD は係り受け距離平均。本稿では日本語係り受け文法の慣例に従って前の文節から後ろの文節にエッジを引くが、依存構造の研究では主辞 (governor) から依存要素 (dependant) にエッジを引くことが一般的であり、矢印の方向が逆になることに注意されたい。\n\n文節数 n の文における可能な構造の数はカタラン数 Cn−1で計算できる。カタラン数 Cn は次の式で定義される。文節数 4 の場合、可能な構造数は C3=5 である。\n\nMDD が最小になるのは全ての文節が直後の文節に係る場合であり、最大になるのは全ての文節が文末文節に係る場合である。文節数 n の文における可能な全ての構造の MDD の集合を MDDn とすると、MDDn の最小値と最大値はそれぞれ次の値を取る。\n\n可能な全ての構造が一様の確率で生起する場合の MDD の分布はどのようになるだろうか。可能な全ての構造を列挙した係り受けパターン集合を用いて MDD の分布を確認することにしよう。例として文節数 10 の文を考えると、可能な構造の数は C9=4862 である。これらの構造における MDD の最小値は 1、最大値は 5 である。ヒストグラムを書くと、MDD は裾が長い分布を示し、この分布は対数正規分布によく当てはまる。図 6 は、ヒストグラムとフィッティングにより推定した対数正規分布およびガンマ分布の曲線を重ね合わせたものである。対数尤度は対数正規分布が -4573.228、ガンマ分布が -4565.008 で、対数正規分布の方がよく当てはまる。\n\n曲線はガンマ分布および対数正規分布によるフィッティング。フィッティングはR言語の MASS::fitdistr 関数による。\n\n文節数 n が変化すると、MDD の分布はどのように変化するだろうか。図 7 は文節数に伴うガンマ分布と対数正規分布の LLR の推移と、MDDn の平均の推移をプロットしたものである。LLR の推移を見ると、文が長くなるほど対数正規分布の方がよく当てはまることが確認できる。また、文節数 n と MDDn の平均の推移を両対数グラフにプロットすると、ほぼ線形に分布することが確認できる。すなわち文長と MDDn の平均は冪関係にある（あるいは文長の対数に対して MDDn の平均は指数的に増大する）と考えられる。\n\n対数尤度比が 0 より大きいときはガンマ分布の当てはまりがよく、0 より小さいときは対数正規分布の当てはまりがよい。右図は両対数グラフ。\n\nそこで、次のモデル式で一般化線形モデル分析を試みる。このモデルは、被説明変数を mdd、説明変数を文節数 length の対数、mdd の分布を対数正規分布とし、mdd の期待値を対数変換すると log (length) と線形関係になると仮定している。\n\nglm(mdd~log(length), family=gaussian(link=”log”))\n\n結果を表 4 に示す。構築されたモデルによる予測値は、実測による MDDn の平均の分布とほぼ一致する。以上から、ランダムデータにおける MDD の分布は対数正規分布に従い、その平均は文節数の対数に対して指数的に増大すると考えられる。\n\nR 言語の stats::glm 関数による。\n\n* p < 0.001.\n\nところで、モデルの切片 -0.383 は、係数 0.554 に−log2 を乗じた値とほぼ一致する4。従って、可能な全ての構造がランダムに生起した場合の MDDn の平均は、おおよそ次の式に従う。\n\n作文データにおいても、MDD は裾の長い分布を示す。しかしランダムデータと同様に対数正規分布によく当てはまるかは自明ではない。図 8 は、作文データから文節数 10 の文のみ抽出し、MDD の分布をヒストグラムにしたもので、曲線は対数正規分布およびガンマ分布でフィッティングした予測曲線である。対数尤度は対数正規分布が -3389.641、ガンマ分布が -3374.439 で、ガンマ分布の方がよく当てはまる。\n\n曲線はガンマ分布および対数正規分布によるフィッティング。フィッティングはR言語の MASS::fitdistr 関数による。\n\n文節数に伴う LLR の推移と MDD の平均の推移を図 9 に示す。作文データでは、文節数が 20 を超える文の頻度が小さいため、文節数 20 までのデータを示す。LLR の推移を見ると、文が短いときにはガンマ分布の方がよく当てはまるが、文が長くなると両者の差は小さくなる。MDD の平均の推移を見ると、ランダムデータでは文節数と MDD 平均が両対数グラフで線形に分布するのに対して、作文データでは文節数と MDD 平均が片対数グラフで線形に近い分布を示す。すなわち、MDD の平均は文節数の対数に対して線形であると考えられる。\n\n対数尤度比は 0 より大きいときガンマ分布の当てはまりがよく、0 より小さいとき対数正規分布の当てはまりがよい。右図は片対数グラフ。\n\n以上から、2 つのモデルで MDD と文長nの一般化混合モデル分析を試みる。どちらのモデルも被説明変数を MDD、固定効果を文節数 (length) の対数と学年 (grade)、およびそれらの交互作用項、作文 ID (document_id) を変量効果としたモデルである。第 1 のモデルは文長の分布をガンマ分布と仮定し、第 2 のモデルは文長の分布を対数正規分布と仮定している。前者は mdd の期待値がlog (length) に対して線形であることを仮定しており、後者は mdd の期待値の対数がlog (length) に対して線形であることを仮定している。学年はカテゴリ変数である。学年によってlog (length) の係数が変わることが考えられるため、交互作用項をモデルに含めている。作文 ID は、著者の個体差による効果を説明するためにランダム効果として加えている。\n\nglmer(mdd~log(length)grade+(1\|document_id), family=Gamma (link=”identity”))\n\nglmer(mdd~log(length)grade+(1\|document_id), family=gaussian (link=”log”))\n\n結果を表 5 に示す。決定係数や AIC を見ると、ガンマ分布モデルの方がよく当てはまっている。この結果から、ランダムデータでは MDD が対数正規分布に従うのに対して、作文データでは MDD がガンマ分布に従い、また文長に対する分布の平均の推移も両者では異なると考えることができる。\n\nR 言語の lme4::glmer 関数による。\n\n* p < 0.05,\n\n p < 0.01,\n\n* p < 0.001.\n\nガンマ分布モデルの固定効果を見ると、学年の係数は小1から小3まで減少するが、その後は増加する。文長と学年の交互作用項は小学 1 年生から4年生にかけて増加したあと、中学 3 年生まで減少する。このうち、前者は文が短いときに MDD に強く影響し、後者は文が長いときに MDD に強く影響する。固定効果の予測値を見ると、実際にそのようになっていることが確認できる（図 10）。\n\n髯は 95%信頼区間。R 言語の lme4::glmer 関数による解析結果を ggeffects::ggpredict 関数で可視化した。\n\n\n5. 考察\n\n文長の分布は、ランダム列においては幾何分布に従い、作文データにおいては低学年においては対数正規分布に近いが、学年が上がるに従ってガンマ分布に接近することを確認した。またその平均は学年に従って増加するが、高学年になるに従って増加の仕方が緩やかになる。\n\nランダム列ににおける文長の分布が幾何分布に従う理由は容易に解釈することができる。ランダム列においては文末文節が一定の確率 p で生起するので、文長の分布は母数 p の幾何分布に従う。これに対して、作文データにおける文長の分布が対数正規分布やガンマ分布に従う理由は自明ではない。古橋・早川(2012) は京都大学テキストコーパスを用いて文節を単位とする文長の分析を行い、対数正規分布と負の二項分布（ガンマ分布の離散確率分布に相当するもの）では後者の方がよく当てはまることを報告している。また、2 つの分布の生成過程について検討し、対数正規分布の場合は依存構造木が乗算過程的に枝の数を増やしていくプロセスによって生成され、負の二項分布の場合は部分木の長さが幾何分布に従う場合にその加算過程によって生成される可能性を提示しているが、どちらのモデルもデータを説明するには不十分であると結論づけている。\n\n負の二項分布は p と r という2つの母数を持ち、母数 p の幾何分布に従う回数で成功する事象がr回成功するまでの回数の分布として解釈される。文長にあてはめて考えると、部分木の長さが母数 p の幾何分布に従うのであれば、r 個の部分木を持つ文の長さは負の二項分布に従うと考えられる（ガンマ分布と指数分布で考えても基本的には同じである）。だが自然言語の文では部分木の数は常に同じではなく、各部分木の長さも常に同じ母数の確率分布に従うわけではない。文がいくつの節を持つかは文ごとに異なり、節の長さもその種類によって異なる。例えば連体節は短く、並列節は長いといった違いがあり得る。また、著者やテキストの属性によって、使用される節の種類の内訳も異なる。例えば著者の学年が上がるほど連体節の割合は多くなる傾向があるし、話し言葉と書き言葉では後者の方が連体節の割合が多くなるかも知れない。さらに言えば、低学年ほど対数正規分布がよく当てはまり、高学年ほどガンマ分布がよく当てはまるという規則性が見られることは、どちらかの分布が正しいモデルで、偶発的に別の分布の当てはまりがよくなる場合があるというわけではないことを示唆する。しかしいずれにせよ、文節を単位とした場合にも文長の分布が右に歪んだ分布になることは明らかであり、多くの場合においてガンマ分布は（少なくとも正規分布などより）よい近似になると考えられる。\n\n学年を説明変数とする一般化混合モデル分析からは、学年に従って文長が長くなることが確認できる。これは学齢の上昇に伴って言語能力が発達し、より構成要素が多い複雑な文を生成することができるようになることを示していると解釈できる。一方で、文長の増大は学年が上がるほど緩やかになる。直感的には、文は原理的には無限に長くすることができるものの、我々は通常、特に書き言葉においては適切な長さで文を切る方略を用いるので、ある程度の長さの文が書けるようになると、それ以上は文を長くしないものと解釈できる。\n\nMDD の分布は、ランダム構造においては対数正規分布に従い、作文データにおいては概ねガンマ分布に従う。また文が長くなると、ランダム構造の MDD は文長の対数に対して指数的に増加するが、作文データでは線形に増加する。学年は、MDD と文長の線形関係の切片と係数に影響を及ぼすが、切片は学年が上がるほど大きくなり、係数は小学4年まで増加した後は減少する傾向が見られる。\n\n対数正規分布が乗算過程によって生成され、ガンマ分布が加算過程によって生成されることを考えると、前者が指数的、後者が線形的に増大することは自然な帰結かも知れない。しかしながら、なぜ MDD の分布が対数正規分布やガンマ分布に従うかは、文長の分布の場合と同様十分に明らかではない。対数正規分布が生成される過程については、ある程度の手がかりがある。ランダム構造においては、文節数n、係り受け距離和sの可能な構造のパターン数は次の漸化式で計算することができる5。MDD は係り受け距離和をn-1で除した値なので、Dn,n−1MDDとすれば同じ式で分布を計算することができる。本稿はこの式の代数的な解を示すことができないが、これを解くことでランダム構造における MDD の分布のより正確なモデルを得ることができるはずである。\n\n文節数の対数に従った MDD の増加が、ランダム構造では指数的であるのに対して、作文データでは線形であることは、前者よりも後者（すなわち自然言語）の方が MDD の増加が抑制的であることを示している。これは従来よく知られているように、自然言語が長い係り受けを避ける傾向があることの当然の帰結と言える。一方で、作文データの MDD は常にランダム構造の MDD より小さいわけではない。実データの分布（図 11）を見ると、文長が 5 文節未満のごく短い文においては、ランダム構造よりも作文データの方が MDD の平均が大きい。\n\n文法的な観点から見ると、自然言語における MDD の分布には少なくとも2つの原則が寄与しているように思われる。1 つは節や句の形成に伴う MDD の増大の原則で、例えば動詞句では主語、目的語、副詞などの構成要素が句末の動詞に係ることによって MDD が増大する。もう 1 つは複数の句による階層構造の形成に伴う MDD の最適化の原則で、長い句を前に、短い句を後ろに置くことで長い係り受けをなるべく少なくするように構造が作られる。文節が 4 つ程度までの短い文は階層構造を持たない単文が多く、全ての文節が文末の述語に係る。そのため、ランダム構造よりも MDD が大きくなる傾向が見られる。それより長い文になると文中に階層構造が現れ、最適化の原則によってランダム構造よりも MDD が小さくなる。\n\n2 つの原則は、回帰モデルにおける切片と係数の解釈にも関連する。MDD が log(length) と線形関係にあると仮定すると、MDD は文が短いときには切片の影響を強く受け、文が短いときには log(length) の係数の影響を強く受ける。そのため、切片は句における MDD の増大と強く関連し、係数は階層構造におけるMDD の最適化と強く関連する。同様に、学年の係数は MDD の増大と関連し、学年と文長の交互作用項の係数は MDD の最適化と関連する。一般化線形混合モデル分析の結果では、学年の係数は高学年ほど大きくなり、学年と文長の交互作用項の係数は小学4年まで増加した後は減少する傾向が見られた。前者は学齢の進行に伴って句の構成要素を増やすことで複雑な文を形成する能力が発達することを示唆し、後者はある程度の学齢に到達した後に長距離の係り受けを回避し、構造を最適化する能力が発達することを示唆する。\n\n\n6. まとめ\n\n日本語の文の複雑性について、文長（文節数）と係り受け距離平均（MDD）の観点から分析した。ランダムデータと作文データの比較では、前者の文長が幾何分布、MDD が対数正規分布によく当てはまるのに対して、後者の文長は対数正規分布ないしガンマ分布、MDD はガンマ分布によく当てはまることが確認された。また、ランダムデータにおける MDD の平均が文長の対数に対して指数的に増大するのに対して、作文データにおける MDD の平均は文長の対数に対して線形に増大することが分かった。この結果は、自然言語における係り受け距離の分布は無作為な構造と比べて有意に小さくなるという従来の知見を支持するものだが、本稿の分析からはその差が係数の違いに留まるものではなく、指数的か線形かという関数の特性のレベルで異なることが示された。また、文節数がごく小さい文においてはランダムデータよりも作文データの方が係り受け距離平均が大きくなることも分かった。\n\n作文データの分析においては、学年が上がるほど文長が長くなることが確認された。また MDD については、学年による効果は減少した後に増大し、学年と文長の交互作用は増大した後に減少することが分かった。MDD の非単調な変化は、児童における統語構造の構成能力の発達が、構造の複雑化と最適化の 2 つの側面を持つ複雑な過程であることを示唆する。児童はより構成要素の多い複雑な構造を構成する能力を発達させるのと並行して、係り受け距離の短い最適化された構造を構成する能力を発達させているかも知れない。特に前者の能力については、係り受け構造におけるノード次数の分布など、より直接的な証拠の分析が追加で必要だろう。\n\n他の残された課題の 1 つは分布を生成する過程の解明である。特に作文データにおける文長や MDD の分布がなぜランダムデータとは異なるロングテール分布になるかは十分に分かっておらず、そのためガンマ分布の当てはまりのよさも現時点では近似に過ぎないと言わざるを得ない。しかしながら、日本語の文節係り受け構造における文長や MDD の比較的良い近似が確認できたことは応用的研究のために有益である。今後、分布の生成モデルの検討とともに、文章の可読性の分析や文章ジャンルによる文構造の差異の分析への応用を進めたい。\n\n\nデータ可用性\n\nデータはすべてオープンサイエンスフレームワークで利用可能です。児童作文における文節数および係り受け距離の分布. https://doi.org/10.17605/OSF.IO/3YAQU (Imada, 2023).\n\nこのプロジェクトには以下の基礎データが含まれています。\n\n• sakubun_chunks.csv（「児童・生徒作文コーパス」ver.1.6 から文節情報のみを抽出したデータです。本文を含むオリジナルのデータは公開されていません。）\n\n• random_chunks.csv（文節数 2 から 10 までの範囲で、可能な係り受け構造を全て列挙したデータです。）\n\n解析コードはオープンサイエンスフレームワークより入手可能です。\n\nアーカイブされた解析コード（論文公開時）: https://doi.org/10.17605/OSF.IO/3YAQU\n\nデータは、Creative Commons Zero “no rights reserved” data waiver (CC0 1.0 Public domain dedication) の条件下で利用可能です。\n\n\n同意\n\n本研究では「児童・生徒作文コーパス」から取得した文節係り受け情報をデータとして使用している。このデータは作文本文を含まず、また公開にあたって同意を必要とする著者の個人情報は含まれていない。なお、「児童・生徒作文コーパス」はプロジェクト関係者のみが利用可能な非公開データであり、データ構築の時点で著者の特定に繋がるデータは作文本文中の固有名などを含めて全て被覆されている。", "appendix": "参考文献\n\n浅原正幸, 松本祐治: 『現代日本語書き言葉均衡コーパス』に対する文節係り受け・並列構造アノテーション. 自然言語処理. 2018; 25(4): 331–356. Publisher Full Text\n\n新井皓士: 文長分布の対数性規制に関する一考察: 芥川と太宰を事例として. 一橋論叢. 2001; 125(3): 205–223. Publisher Full Text\n\n今田水穂: 児童作文における係り受け距離と階層距離. 言語資源活用ワークショップ発表論文集. 2021; 6: 338–347. Publisher Full Text\n\n大村舞, 浅原正幸: UD Japanese-BCCWJ の構築と分析. 言語資源活用ワークショップ発表論文集. 2018; 3: 161–175. Publisher Full Text\n\n金明哲: 文節の係り受け距離の統計分析. 社会情報. 1996; 1996(2): 1–6. 10742/754.\n\n工藤拓, 松本裕治: チャンキングの段階適用による日本語係り受け解析. 情報処理学会論文誌. 2002; 43(6): 1834–1842.\n\n佐々木和枝: 文の長さの分布型. 計量国語学. 1976; 78: 13–22.\n\n柴崎秀子, 原信一郎: 12 学年を難易尺度とする日本語リーダビリティー判定式. 計量国語学. 2010; 27(6): 215–232.\n\n建石由佳, 小野芳彦, 山田尚勇: 日本文の読みやすさの評価式. 情報処理学会研究報告 (HCI). 1988; 25(1988-HI-018): 1–8.Reference Source\n\n古橋翔, 早川美徳: 文長分布型と係り受け関係に基づいた文構造の解析. 計量国語学. 2012; 28(7): 250–260.\n\n宮城信, 今田水穂: 『児童・生徒作文コーパス』の設計. 第7回コーパス日本語学ワークショップ予稿集. 2015; 223–232.\n\n安元美典: 文の長さの分布型について. 計量国語学. 1958; 4: 20–24.\n\n李在鎬: 日本語教育のための文章難易度に関する研究. 早稲田日本語教育学. 2016; 21: 1–16.Reference Source\n\n李文平, 劉海濤, 小森早江子: 統語依存関係に基づく位相研究: 文章ジャンルの位相差を対象に. 言語研究. 2022; 162: 47–62. Publisher Full Text\n\nFerrer-i-Cancho R, Gómez-Rodríguez C, Esteban JL: Almany-Puig Lluís: The optimality of syntactic dependency distances. Physical Review E. 2022; 105: 014308. PubMed Abstract \| Publisher Full Text\n\nFutrell R, Mahowald K, Gibson E: Large-scale evidence of dependency length minimization in 37 languages. Psychological and Cognitive Sciences. 2015; 112(33): 10336–10341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGibson E: Linguistic complexity: locality of syntactic dependencies. Cognition. 1998; 68(1): 1–76. PubMed Abstract \| Publisher Full Text\n\nImada M:児童作文における文節数および係り受け距離の分布. [dataset].2023. Publisher Full Text\n\nIshida M, Ishida K: On distributions of sentence length in Japanese writing. Glottometrics. 2007; 15: 28–44.\n\nKomori S, Sugiura M, Li W: Examining the applicability of the mean dependency distance (MDD) for SLA: A case study of Chinese learners of Japanese as a second language. Proceedings of the 4th Asia Pacific Corpus Linguistics Conference (APCLC 2018). 2018; pp. 237–238.\n\nLiu H: Dependency distance as a metric of language comprehension difficulty. Journal of Cognitive Science. 2008; 9(2): 159–191. Publisher Full Text\n\nMaruyama H, Ogino S: A Statistical Property of Japanese Phrase-to-Phrase Modifications. Mathematical Linguistics. 1993; 18(7): 348–352.\n\n\nFootnotes\n\n1 ここで引用する論文は「言語資源活用ワークショップ 2021」（2021 年、国立国語研究所コーパス開発センター主催学会）の発表原稿であり、DOI を付与して公開されているが、査読を経ていない。本論文は、同学会で発表された研究の一部を再検討し、内容や分析方法に相当の改変を加えたものであり、発展的研究の成果として位置づけられる。\n\n2 別のランダムデータの生成方法として、実際のデータの依存構造を保持したまま構成要素の線形順序をシャッフルしてデータを生成する方法が考えられる。Futrell et al. (2015) はこの方法で、語順をランダムに入れ替えたデータ、枝分かれの方向を固定して語順をランダムに入れ替えたデータなどをベースラインとして検討している。Ferrer-i-Cancho et al. (2022) はある構造 D の語順をランダムに入れ替えたときの最小値 Dmin と平均 Drla をベースラインとして使用しており、この構造では依存構造の交差も許される。本稿で使用するパターン集合は、枝分かれの方向は固定し（日本語の文節係り受け構造では、規範的には前の文節から後ろの文節に係り受けする）、交差は認めず、実データの構造に依らず可能な全ての構造を生成する。\n\n3 本研究では一般化線形モデル分析のためにランダムデータのセットを生成したが、単に MDD の分布を取得するだけであれば漸化式を用いて比較的小さなコストで計算することができる（5.2 節参照）。\n\n4 MDDn の最小値が 1 、最大値が n/2 であることから、平均も n/2x に従うことが考えられる。これを対数化すると xlogn−xlog2 であるため、平均が n/2x に従うのであれば切片は係数xに −log2 を乗じた値になると予想され、実際にその値が得られた。\n\n5 この漸化式は以下のアルゴリズムに基づく。文節数が 1 の文における可能な構造パターンは1通りのみである。この構造は係り受けを持たないので D1,0=1 である。\n\n文節数が 2 以上の文は、文を 2 つの部分構造に分割して考える。2 つの部分構造は、前方の部分構造の末尾文節から後方の部分構造の末尾文節（すなわち文末）への係り受けによって結合されているものとする（二分木型の句構造に置き換えるならば、最上位の句接点で 2 つの部分構造が分割されていると考えられたい）。 2 つの部分構造の長さを n1 、 n2 とし、SDD を s1 、 s2 とする。このとき、文全体の長さは n=n1+n2 である。また、文全体の SDD は部分構造の SDD の和に 2 つの部分構造を結合する係り受けの長さを足したものになる。2つの部分構造を結合する係り受けの長さは後方の部分構造の長さ n2 と同じなので s=s1+s2+n2 である。ここで n2=i 、 s2=j とすると、 n1=n−i 、 s1=s−i−j なので、文全体の可能な構造パターン数は Di,j×Dn−i,s−i−j となる。これを n2 および s2 が取りうる全てのi, jについて計算し、その総和を求めることで Dn,s の値を求めることができる。iの変域は[1, n-1]である。jの変域はiに応じて変化し、[i-1, i ( i-1)/2]である。従って Dn,s=∑i=1n−1∑j=i−1ii−1/2Di,jDn−i,s−i−j である。\n\nただし、文節数nの文におけるsの最小値は n-1、最大値は n(n-1)/2である。sがこの範囲を逸脱する構造は存在しないので、その場合には Dn,s=0 である。" }	[ { "id": "169125", "date": "19 May 2023", "name": "Teiko Arai", "expertise": [ "Reviewer Expertise 人文情報学" ], "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本稿は、日本語の文章の複雑性について、文長と係り受け距離平均(MDD)の観点から分析している。分析に際しては、ランダムデータと児童作文データを比較し、学年による分布の変化を検討する方法がとられている。その結果、ランダムデータと作文データを比較すると、作文データの文長とMDDの分布がランダムデータとは異なるロングテール分布になること、そして作文データでは学年が上がるほど文長が長くなり、MDDについては、学年による効果は減少した後に増大し、学年と文長の交互作用は増大した後に減少することが報告されている。　児童の言語発達という難しい課題に対し、義務教育段階の9学年の質の良い作文コーパスが構築されたことの学術的有用性と教育的意義は極めて大きい。学習者の著作権・プライバシー等の事情から、作文コーパスの数は現状多いとは言えず、その作文コーパスを使用した研究事例は未だ少なく、学習者の作文能力の発達過程の解明が待たれる。例えば本論文では、学年が上がるにつれ文長が長くなるが、その増加速度は緩やかになることが示唆されているが、学年に伴う変化の解釈の確定には未だ多面的な分析が必要になるだろう。　表1に示されるコーパス規模のデータから、小学校1年生の1作文あたりの平均文節数が約40であるのに対し、中学校3年生ではおよそ140にまで増加していることが分かる。一方で、これが作文課題や教授（指示等）、作業時間などの影響を受けているのかどうかは論文で説明されていないため、例えば課題が異なる作文群について、文長やMDDを一括して評価することについて懸念がある。この点について明確な説明があればありがたい。また、MDDの結果に関しては、これを児童の言語能力の発達における文構造の複雑化と最適化の2つの側面を持つものとする解釈は、直感的に有望であるが、学習者の追加的調査等の今後の分析によってこの解釈が裏付けられることが期待される。　本稿は、児童の構文に関する言語能力に焦点を当てた日本語の計量言語分析の研究事例であり、また、単純な学年（年齢）による発達過程の予想の困難を示唆する結果として評価できる。\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。一部該当\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。一部該当\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": "169127", "date": "23 Jun 2023", "name": "Masatoshi Sugiura", "expertise": [ "Reviewer Expertise 第二言語習得論、学習者コーパス" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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日本語を母語とする「児童・生徒作文コーパス」データにおける文長（文中の文節数）および係り受け距離平均（MDD）の分布について、小学校１年生から中学３年生までの学年間の変化を、一般化線形混合モデルで分析している。実際の作文データと作文データ内の文節をランダムにしたデータとを比較することにより、自然言語に固有の特性を明らかにしようとしている点に特徴がある。\n自然言語では文長およびMDDの分布がいずれもガンマ分布もしくは対数正規分布に当てはまることが観察されている。どちらの分布が当てはまりが良いかについて、対数尤度比（LLR）で分析し、文長は低学年は対数正規分布、高学年はガンマ分布に従い、MDDは概ねガンマ分布に従うことが観察された。\n自然言語の場合、MDDは、文長の対数と比例関係にあること、そして、文長と学年の交互作用があり、１年生から４年生までは増加するがそれ以降は減少する傾向が見られたと報告されている。文長自体も、学年とともに増加傾向にあるが高学年になると増加が緩やかになると報告されている。こうした分析から、文長もMDDも低学年の間は増加傾向が見られるが、高学年になるにつれ、MDDは文長と学年の交互作用のため必ずしも常に増加傾向にあるとは言えないと言えるであろう。\nこうした点について、言語発達が、統語構造の複雑化と最適化の二つの側面を持つという考察をしているが、これは卓見であるといえよう。人の認知処理能力（ワーキングメモリー容量等）に上限があるため、MDDは必ずしも上がり続けないというLiu (2008)の研究との関連性も議論できるのではないかと思われる。\n内容が高度で複雑であるため理解が難しいという面もあるが、もう少しわかりやすく書くこともできたのではないかと思われる点もある。例えば、「前者は」「後者は」という指示詞が複数個所で使われているが、話が複雑なため、それぞれが具体的に何を指すのかがわかりづらい。「前者」「後者」の内容を明示的に書いた方が読みやすくなると思われるため、この点は改訂していただきたい。\n細かい点であるが、確認していただきたい点がある。11ページ3行目と4行目に「文長の分布」とあるが、この二か所はいずれも「文長」ではなく「MDD」のことではないかと思われる。もう一点、14ページ第3段落2行目に「文が短いとき」が2回出てくるが、2つ目は「文が長いとき」のことではないかと思われる。確認をし、間違っていれば訂正をしていただきたいし、間違っていないとしたら、間違って読まれないように表現を工夫していただきたい。\n本論文は文長とMDD両方の分布の言語発達に伴う推移をGLMMを使って分析した論文として大変意義のある論文であるといえる。GLMMによる文長の学年進行に伴う予測値については図４で示されているが、MDDの学年進行に伴う予測値の図が掲載されていない。この論文にとってMDDのグラフは画竜点睛であり、是非とも掲載するよう改訂していただきたい。\nまた、これは今後の課題として位置づけてもよいが、トピックの影響についての議論、もしくは説明もあった方が望ましい。一般的に言って、トピックの違いにより作文データは影響を受ける。その影響について今回の分析ではどのように対処したのか、もしくは、何らかの理由で、しなかったのかという点の説明があった方が論文としての説得力を増すと思われる。また、データにつていて、小学校から中学校までのデータを連続した9年間として扱っているが、同じ小学校内であれば、1年生から6年生までの横断的データだとしてもほぼ等質であろうと想定されるが、小学校と中学校は通例、学校が違うので、それだけでも何らかの違いを生じさせることにならないかと懸念される。例えば、表1で作文データの規模が示されているが、小学校と中学校の間は連続的な変化ではなく、中学校の方が桁違いに多い。また、図３で文長の対数尤度比の変化が示されており、グラフとしては折れ線で連続しているかのように見えるが、小学校側はマイナスで中学校側がプラスで、小学校と中学校の境目でマイナスとプラスが逆転していると解釈される可能性がある。こうした、トピックの影響と小学校・中学校のデータの連続性という二点について説明があった方が望ましい。\n「児童・生徒作文コーパス」が、非公開データであるという点は理解できるが、「観察するために適している」（p3. 「2.1 作文データ」第一段落）と判断する根拠として、データとしてのコーパスの特徴（上記のトピック等）に関する具体的な情報をここで提示しておいた方が良いと思われる。\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。一部該当\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。はい", "responses": [] } ]	1	https://f1000research.com/articles/12-379
https://f1000research.com/articles/12-109/v1	30 Jan 23	{ "type": "Review", "title": "Studying ancient human oral microbiomes could yield insights into the evolutionary history of noncommunicable diseases", "authors": [ "Abigail S Gancz", "Laura S Weyrich", "Laura S Weyrich" ], "abstract": "Noncommunicable diseases (NCDs) have played a critical role in shaping human evolution and societies. Despite the exceptional impact of NCDs economically and socially, little is known about the prevalence or impact of these diseases in the past as most do not leave distinguishing features on the human skeleton and are not directly associated with unique pathogens. The inability to identify NCDs in antiquity precludes researchers from investigating how changes in diet, lifestyle, and environments modulate NCD risks in specific populations and from linking evolutionary processes to modern health patterns and disparities. In this review, we highlight how recent advances in ancient DNA (aDNA) sequencing and analytical methodologies may now make it possible to reconstruct NCD-related oral microbiome traits in past populations, thereby providing the first proxies for ancient NCD risk. First, we review the direct and indirect associations between modern oral microbiomes and NCDs, specifically cardiovascular disease, diabetes mellitus, rheumatoid arthritis, and Alzheimer's disease. We then discuss how oral microbiome features associated with NCDs in modern populations may be used to identify previously unstudied sources of morbidity and mortality differences in ancient groups. Finally, we conclude with an outline of the challenges and limitations of employing this approach, as well as how they might be circumvented. While significant experimental work is needed to verify that ancient oral microbiome markers are indeed associated with quantifiable health and survivorship outcomes, this new approach is a promising path forward for evolutionary health research.", "keywords": [ "ancient health", "oral microbiomes", "dental calculus", "NCDs", "frailty" ], "content": "Introduction\n\nNoncommunicable diseases (NCDs) (e.g., chronic conditions that are not the result of a singular infectious agent) are among the leading causes of death worldwide (WHO 2020). Specifically, seven of the top ten causes of death in 2019 were attributed to NCDs, and these accounted for 44% of all global deaths. Yet, despite the devastating emotional and economic impacts of these conditions, much remains unknown about the history of these diseases or how past dietary, lifestyle, and environmental factors modulated their prevalence and impacts in different populations. To gain a fuller understanding of why these conditions vary across individuals and populations today, it is imperative to understand the patterning of these conditions across space and time. Such research would benefit our understanding of disease origins, etiologies, and prevention strategies, especially for non-Western, traditionally marginalized groups.\n\nWhile infectious diseases with specific biological pathogens such as tuberculosis (Donoghue 2017) or bubonic plagues (Bos et al. 2011; Spyrou et al. 2019) have benefitted from recent improvements in the abilities of researchers to recover, authenticate, and analyze ancient DNA (aDNA), research into NCDs and their health impacts has lagged behind. This trend has emerged for multiple reasons. First, the vast majority of NCDs leave no distinctive skeletal traces, while individuals with infectious diseases can often be identified through distinctive mass-death burials or specific skeletal pathologies. Moreover, unlike with infectious diseases, aDNA cannot be directly used to establish the clear presence of a single pathogenic agent. As such, identifying the presence of NCDs in a population, let alone diagnosing NCDs in specific ancient individuals, is extremely difficult in comparison.\n\nThis challenge is one that may now begin to be addressed by examining the human oral microbiome. The oral microbiome consists of the microscopic organisms (e.g., bacteria, viruses, fungi, archaea, protozoa) that colonize the teeth, gums and other tissues of the mouth (Gomez and Nelson 2017). While a core microbiome exists across most individuals, significant variation can arise depending on an individual’s unique environment, lifestyle, and physiology (Deo and Deshmukh 2019; Verma, Garg, and Dubey 2018; Gomez and Nelson 2017; Weyrich 2021). A natural part of the human body, the oral microbiome performs several critical functions underlying systemic health including pathogen inhibition, immune system training and regulation, nutritional absorption, and enhancement of metabolic uptake (Wade 2013). In addition, through both direct and indirect pathways, the oral microbiome modulates the risks and severities of local and systemic human diseases. Indeed, over the last decade, it has become increasingly evident that the oral microbiome has important immunological and mechanistic functions associated with NCD risk. Specifically, the relationships between the oral microbiome and cardiovascular conditions, diabetes mellitus, rheumatoid arthritis, and Alzheimer's disease have been well-established. By curating the oral microbiome features associated with the presence of these NCDs and testing their association with quantifiable indicators of health and survivorship in ancient populations, researchers can now begin to explore the possibility of identifying facets of hidden, NCD-associated morbidity and mortality risk within these groups.\n\nDifferential health risks and trends associated with ancient oral microbiomes have already begun to emerge. Specifically, an analysis of over 127 Medieval and Post-medieval individuals from the city of London conducted by (Gancz et al. in-review) found clear associations between systemic health associated skeletal traits including non-specific periostitis, joint porosity, and osteophytic lipping and oral microbiome community features. These findings highlight that specific markers of the oral microbiome are indeed associated with disease risk. In the following review and theoretical perspective, we highlight future steps that should be taken to improve upon and utilize these associations for the benefit of ancient health research.\n\n\nBackground\n\nThe human microbiome encompasses several distinct communities of microbes that exist on nearly every surface of the body, most abundantly in the gut and mouth (Ursell et al. 2012). In recent years, researchers have explored the associations of these microbiomes with human migration, evolution, culture, and, importantly, health (as reviewed in Vangay et al. 2018; Sharma et al. 2018; Weyrich 2021). The relationship between health and the microbiome has been explored using animal models, examinations of close evolutionary relatives (i.e., comparisons across primates), and research on how native or indigenous lifestyles influence the microbiome differently from those in Western societies (e.g., Liddicoat et al. 2020; Dent, Berger, and Griffin 2020; Janiak et al. 2021).\n\nAlthough the gut microbiome is the most extensively studied microbiome, the oral microbiome has also begun to elicit considerable interest from researchers and the public for its connections with a number of systemic conditions. The oral microbiome comprises millions of microbes, including over 700 species of bacteria, known to colonize both the soft and hard tissues of the oral cavity (Kilian et al. 2016). The establishment of these microbes begins directly after a child’s birth and continues through early childhood. Initial colonization begins with pioneer species, and once tooth eruption begins, a more complex microbial community is established on the hard surfaces (Deo and Deshmukh 2019). While a core microbiome consisting of Streptococcus, Lactobacillus, Actinomyces, Neisseria, and Veillonella bacteria is common across individuals, significant variation can arise depending on an individual’s unique environment, lifestyle, physiology, and heritage (Deo and Deshmukh 2019; Verma, Garg, and Dubey 2018; Gomez et al. 2017; Weyrich 2021; Handsley-Davis et al. 2022). The oral microbiome performs several critical functions underlying systemic health. Specifically, human oral microbes are involved in pathogen inhibition, immune system training and regulation, nutritional absorption, and the enhancement of metabolic uptake (Wade 2013; Shaw, Smith, and Roberts 2017). These functions have evolved alongside humans over time with shifting environments, diets, and behaviors (Weyrich 2021).\n\nUnlike other microbiomes, the oral microbiome can also be reliably reconstructed in ancient populations from calcified dental plaque, also known as dental calculus. Calculus forms during life and shares similarities with a living individual's oral microbiome (Velsko et al. 2019). This biological substance accumulates over the lifespan as oral microbiota organize into complex biofilms macroscopically observable as plaque (Welch et al. 2016). Over time, salivary calcium phosphate salts cause the biofilm to calcify, thereby encapsulating microorganisms, food debris, proteins, and other materials within (Weyrich, Dobney, and Cooper 2015). Through ancient metagenomic (Warinner, Speller, and Collins 2015; Weyrich, Dobney, and Cooper 2015), proteomic (Jersie-Christensen et al. 2018; Hendy et al. 2018), isotopic (Eerkens et al. 2014), and other forms of analysis, dental calculus has been used to study human migrations (Eisenhofer and Weyrich 2018), subsistence practices (Adler et al. 2013) and disease (Yaussy and DeWitte 2019).\n\nThe oral microbiome is a major driver of both oral and systemic health. In the mouth, the oral microbiome is associated with two of the most common dental health conditions, specifically caries (cavities) and periodontitis. Caries are associated with tooth decay, which is caused by the breakdown of enamel, and are a major public health problem today (Heng 2016). Their etiology is associated with dietary, environmental, behavioral, developmental, and genetic factors. Several bacterial genera, including Streptococcus, Lactobacillus, Actinomyces, Fusobacterium, Porphyromonas, Selenomonas, Bifidobacterium, Veionella and Scarvoia have been associated with caries (Simón-Soro and Mira 2015; Tanner et al. 2011; Skelly et al. 2020; Handsley-Davis et al. 2020). The state of disease is not solely linked to the composition of the microbiome but also to its functional activities (Solbiati and Frias-Lopez 2018) and the ways that microbes interact with each other (e.g., suppressing colonization by competitors) (Sharma et al. 2018). Periodontitis is a form of gum infection that is associated with both soft and hard tissue destruction, as well as eventual tooth loss. Some common examples of bacterial species in the periodontal microbiome include Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Eikenella corrodens, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans (Pritchard et al. 2017). Like caries, there are also key functional traits (e.g., upregulation of virulence factors) associated with periodontitis (Solbiati and Frias-Lopez 2018).\n\nWhile these local health outcomes of the oral microbiome are of significant research interest in and of themselves, these oral diseases have been shown to be strongly correlated with, and at times specific risk factors for, systemic disease (Handsley-Davis et al. 2020). For example, caries have been used as an indicator of systemic diseases (Dashper et al. 2019), and periodontitis has been linked to a multitude of other conditions and systematic effects likely mediated by inflammation (Hajishengallis 2015; Genco and Borgnakke 2020). In fact, systemic inflammation and periodontitis are known to form a positive feedback loop by which one exacerbates the other (Akcali et al. 2013). Despite these associations, there are also direct mechanisms by which the oral microbiome can contribute to systemic diseases independently of oral diseases. Three main mechanisms have been proposed for how the oral microbiome is able to impact the rest of the body: (1) the translocation of oral microbes into other regions, (2) the translocation of oral microbiome metabolites, and (3) the instigation of immunological and inflammatory modulations that have systemic effects (Hajishengallis 2015; Thomas et al. 2021; Kleinstein, Nelson, and Freire 2020; Park et al. 2022; Bowland and Weyrich 2022). Via these mechanisms, various NCDs have been shown to be directly caused by the oral microbiome; these interactions are described in greater detail in the following sections.\n\nThe observation that oral health and systemic health outcomes are related has already been utilized for research into ancient human health. Specifically, macroscopically observable differences in oral health and calculus formation have been studied by archeologists as a marker of frailty (i.e., heightened susceptibility to different diseases and stressors and their risks of death) (DeWitte and Stojanowski 2015). Dental calculus and other indicators of oral health have been effectively used as overall indicators of general health and disease risk in past populations (DeWitte and Bekvalac 2010; Yaussy and DeWitte 2019; Hakeem, Bernabé, and Sabbah 2019), although these data have not yet been directly linked to specific microbial or immunological mechanisms. By analyzing the microbiome of individuals, it may be possible to further identify the specific markers of frailty within ancient populations and define some of the microbially-modulated mechanisms by which health outcomes occur. This technique would offer novel approaches to addressing the osteological paradox, a major challenge of paleoepidemiological research, specifically the issue of heterogeneity in frailty, as described by DeWitte and Stojanowski (2015).\n\nIn the following sections, we review some of the most common NCD in modern populations and curate oral microbiome markers that could be applicable to ancient populations (Figure 1).\n\nThese relationships and the consequences they have for oral and overall health may be critical for shedding light on noncommunicable disease risk and impacts in past populations.\n\n\nSystemic disease and the oral microbiome\n\nCardiovascular disease (CVD) is a broad category of conditions that includes congestive heart failure, cardiac arrhythmias, valvular heart disease, stroke, coronary heart disease, atherosclerosis, myocardial infarctions, and strokes. Today, CVD is a leading cause of death both worldwide and in the United States (Lin et al. 2020). In the US alone, CVD accounts for almost one in every four deaths (Benjamin et al. 2017). While many risk factors exist for CVD, such as obesity, hypercholesterolemia, sex, age, hypertension, smoking, and genetic predispositions, many patients do not exhibit these predispositions (Frias-Lopez and Duran-Pinedo 2020). Multiple studies have demonstrated the linkages between oral health and CVD risk (Buhlin et al. 2002; Meurman, Sanz, and Janket 2004; Jansson et al. 2001; Mattila et al. 1989; Dietrich et al. 2017). For example, the prevalence and incidence of coronary heart disease is significantly increased in periodontitis according to a meta-analysis of five cohort studies and 86,092 patients (Bahekar et al. 2007). Moreover, periodontal interventions have been shown to reduce the risk of CVD (Roca-Millan et al. 2018).\n\nWhile the links between oral health and CVD are more established than that of CVD and the oral microbiome, several studies have still demonstrated clear associations between these factors. For example, in 2011, Figuero and colleagues scraped atheromatous (arterial) plaque from the carotid arteries of patients and used 16S rRNA sequencing methods to evaluate bacterial DNA (Figuero et al. 2011). The researchers found periodontitis-associated bacteria within the plaque, including P. gingivalis, A. actinomyctemocitans, T. forsythia, E. corrodens, F. nucleatum, and Campylobacter rectus. In 2011, Koren and colleagues used the same approach to look at bacteria in athersclerotic plaque, oral, and gut samples in a case control study of 15 patients (Koren et al. 2011). They found that the abundances of Villanella and Streptococcus species were correlated between the oral cavity and atherosclerotic plaques. Both of these studies are important because they demonstrate a possible mechanism linking oral microbes to CVD, wherein oral microbes invade the bloodstream and result in inflammation, atherosclerosis, and other risk implications. Animal models also support the linkage between CVD and oral microbes. In 2014, Velsko and colleagues published on hyperlipidemic mice that were infected with P. gingivalis and T. denticola (Velsko et al. 2014). The researchers demonstrated that infection was associated with alveolar bone loss, aortic atherosclerosis, and an induced immune response (Velsko et al. 2014). Within all of these studies, the differences observed in Streptococcus are especially remarkable, as specific Streptococcus species are thought to be directly associated with CVDs (Shi et al. 2021; Chen et al. n.d.; Nomura et al. 2020). Specifically, certain species of Streptococcus have been directly observed to bind to host cardiovascular tissues and contribute to diseases such as endocarditis. In addition, other species of Streptococcus, specifically Group A (GAS), can lead to rheumatic fever, which also contributes to CVDs (Coffey, Ralph, and Krause 2018).\n\nAs a whole, this research indicates that there are strong associations between the oral microbiome and CVDs. These relationships are facilitated by two major mechanisms. The first of these is bacterial escape from the oral cavity, and the second is via inflammatory pathways. Specifically, oral microbes associated with periodontitis can reach the vascular system, invade local cells, and be associated with CVD (Reyes et al. 2013). Via inflammatory pathways, microbes inhabiting dental plaque can stimulate cytokine production and elevate their levels in the blood, leading to inflammatory responses (Dietrich et al. 2017). These mechanisms suggest that there are both specific and nonspecific oral microbiome traits associated with CVDs. Nonspecifically, oral microbiome taxa and functions associated with increased periodontitis or systemic inflammation may serve as a proxy for CVD risk. Specifically, the presence or a specific abundance of Streptococcus species capable of binding to cardiovascular tissues may serve as a direct risk factor. Indeed, it may even be possible for ancient health researchers to sample dental pulp chambers as a way to investigate whether CVD-associated taxa were present in the circulatory system of an individual. As such, these oral microbiome features suggest that it may well be possible to curate a proxy for CVD-risk using microbial traits.\n\nDiabetes mellitus (DM) describes a set of metabolic disorders associated with blood sugar dysregulation. Approximately 451 million adults (5.9% of the global population) in 2017 were estimated to be living with DM, with the disease burden increasing annually (Lin et al. 2020). Individuals living with DM contend with both reduced quality of life and increased rates of mortality (Yang et al. 2019). Type one DM (T1D) and type two DM (T2D) are the most common and are caused by pancreatic β-cells insulin-production failure and acquired insulin resistance, respectively (Menezes-Silva and Fonseca 2019). In T1D, this is caused by the self-destruction of insulin producing pancreatic cells by the immune system (Boerner and Sarvetnick 2011). In T2D, bodily tissues such as the muscles and fat also develop insulin resistance, in addition to often co-occurring underproduction of insulin. Both T1D and T2D are believed to have genetic, environmental, and lifestyle associated risk factors.\n\nOf NCDs, DM is amongst those most closely associated with oral health. Oral health, particularly gingivitis and periodontitis, has long been studied in relation to both T1D and T2D. While the causal nature of the relationship (i.e., whether DM causes poor oral health, or vice versa) is challenging to deduce, it is clear that the disease is closely linked to oral microbiota composition and diversity (Graves, Ding, and Yang 2020). A number of studies indicate that gingivitis and periodontitis rates are higher in diabetic individuals (Genco and Borgnakke 2020; Lamster et al. 2008; Novotna et al. 2015), potentially up to three or four times for periodontitis (He et al. 2015). These conditions are associated with fundamental changes in the functions of oral tissues, including bone loss, attachment degradations, and increased inflammatory cytokine expression (Graves, Ding, and Yang 2020). These DM-associated changes impact the entire host immune system, as well as local microbes. Some research even suggests that the treatment of oral conditions is associated with glycemic control and therefore reduced DM symptoms (Bharti et al. 2013; Moeintaghavi et al. 2012).\n\nRecent studies have identified several aspects of the oral microbiome that are associated with DM. Specifically, Shillitoe and colleagues used RTq-PCR to analyze specific microbiota in patients with T2D before and after gastric bypass surgery (a type of weight loss procedure) (Shillitoe et al. 2012). They found that the T2D patients had low rates of Bifidobacterium in the mouth, and that these rates increased after the procedure. Using 16S ribosomal RNA (rRNA) sequencing technology, Long et al. examined 98 patients with T2D, 99 patients who were obese and did not have DM, and 97 normal weight patients (Long et al. 2017). The researchers found that Actinobacteria was significantly less abundant among diabetics, and that within this phylum, five families and seven genera were significantly less abundant. Kampoo and colleagues also harnessed 16S to examine T2D patients in Southern Thailand (Kampoo et al. 2014). These researchers found that in the supragingival plaque of the diabetics, total levels of Streptococci and Lactobacilli were higher. In 2020, Matsha and colleagues conducted a similar study in South Africa and found that Fusobacteria and Actinobacteria were more abundant in T2D patients and Proteobacteria less abundant (Matsha et al. 2020). Most recently in 2021, Balmasova and colleagues used 16S rRNA sequencing to find markers associated with three groups: those with chronic periodontitis associated with T2D, those with chronic periodontitis alone, and healthy controls (Balmasova et al. 2021). The T2D group was associated with a lower abundance of Streptococcus and Pasturellacaea and a higher abundance of Leptotrichicacea. Those with T2D differed from non-T2D periodontitis patients in terms of lower abundance of Veillonellaceae and higher Neisseriaceae. Although these studies all indicate that periodontitis and a shift in oral microbiota are associated with T2D, the specific oral microbiome markers of T2D remain unclear, likely reflecting biases in methods used and populations studied.\n\nFor T1D, significant differences have also been found in the oral microbiome. In 2006, Lalla and colleagues examined 50 T1D patients from the Columbia University Diabetes Center with age, gender, and periodontal disease matched controls and found that Eubacterium nodatum was elevated in diabetic patients, although mostly their subgingival infection patterns were similar (Lalla et al. 2006). Meanwhile, de Grot and colleagues matched 53 T1D patients with healthy controls and investigated their oral and fecal microbiota, finding that the oral microbiota were markedly different, with a high abundance of Streptococci and differences in composition (de Groot et al. 2017). T1D individuals had higher abundances of Actinobacteira and Firmicutes, including taxa within Streptococcus, Actinomyces, and Rothia genera, while Bacteroidetes and Proteobacteria were increased in the controls. In 2021, Jensen and colleagues found a link between glycemic control, T1D, and the complexity and richness of plaque microbiota. This was associated with an inflammation response on a cellular level, possibly due to glycemic control interactions with the microbiome (Jensen et al. 2021). From these studies, it appears that T1D is associated both with different composition as well as some alterations in taxa. More research is needed to determine whether these differential markers are consistent across populations, especially those that underwent different evolutionary selective pressures in the past.\n\nSeveral mechanisms specifically linking DM and the oral microbiome have been hypothesized in the literature. The most commonly cited mechanism is systemic inflammation, which serves as a link between periodontitis, gingivitis, the oral microbiome, and systemic immune responses related to DM (Makiura et al. 2008; Aemaimanan, Amimanan, and Taweechaisupapong 2013; Hyvärinen et al. 2015; Thorstensson, Dahlén, and Hugoson 1995; Preshaw et al. 2012; Levine 2013). Specifically, researchers postulate that oral microbiome in the mouths of individuals with DM, especially those in periodontal areas, causes chronic inflammation and can even trigger insulin resistance by influencing the body’s immunity. In support of this theory, Blasco-Bacque et al. found that mice with periodontitis had increased insulin resistance that was mediated by an adaptive immune response against oral infection (Blasco-Baque et al. 2017). Glycemic control is another proposed factor, especially related to bacteria associated with periodontal disease (Lamster et al. 2008). Taylor et al. found support for this theory in their longitudinal study of residents at the Gila River Indian Community, where they discovered that severe periodontitis was associated with the increased risk of poor glycemic control (Taylor et al. 1996).\n\nThese studies demonstrate that the microbiome of patients with DM are different than those without it. However, excluding the association with periodontitis, the observed differences vary within studies. This result is not necessarily surprising, as these studies focus on several very different human populations with distinctive evolutionary histories that impact the composition of their oral microbiomes. As such, while certain changes in the abundances of taxa may be DM-associated within a specific population, these trends may not hold true in other groups. As such, DM-associated oral microbiome traits may need to be curated from modern populations related to the specific past populations researchers seek to study in order to be useful markers of DM-associated frailty. Alternatively, research into whether specific microbial functions (as opposed to taxa) are related to DM in modern groups may offer more generally applicable DM-markers.\n\nRheumatoid arthritis (RA) is a chronic autoimmune disorder that impacts the joints. RA is thought to be caused by a combination of genetic, behavioral, immunological, and environmental factors (Aho and Heliövaara 2004) The disease is more common among women than men and impacts more than 1.3 million individuals in the United States (Rheumatoid Arthritis: Causes, Symptoms, Diagnosis & Treatments 2021). Today, RA is amongst the most prevalent of chronic inflammatory diseases (Smolen, Aletaha, and McInnes 2016). Individuals with RA suffer from declined physical function, increased comorbidity risks, and reduced work capacity. Although RA has relatively similar prevalence in many populations, some marginalized communities have much higher incident rates (Silman and Pearson 2002). Additional risk factors for RA include smoking, low socioeconomic status, and genetic histories (Smolen, Aletaha, and McInnes 2016). Similar to the previously discussed NCDs, RA has been shown to be linked to periodontal disease, and numerous studies have explored the risks of RA onset and progression in relation to the oral and gut microbiomes (Bingham and Moni 2013). While it remains unclear whether the onset of periodontal disease is a causative or correlative factor for RA, this pattern suggests strong links to the oral microbiota.\n\nIndeed, researchers have demonstrated that there are microbial markers of RA. A good potential discriminant of RA was found in 2018 by Lopez-Olivia and colleagues. The researchers looked at 22 RA and 19 controls subgingival plaque and used 16S rRNA sequencing. PICRUSt, a tool used to predict the functional composition of a metagenome, demonstrated that arachidonic acid and ester lipid metabolism might explain clustering patterns in communities. C. curtum, another organism capable of producing large amounts of citrulline, emerged as a robust discriminant of the microbiome in individuals with RA (Lopez-Oliva et al. 2018). In another study in 2015, Zhang and colleagues used shotgun sequencing to look at 105 oral microbiomes in RA and control patients (Zhang et al. 2015). The researchers found that Veillonella were elevated in dental plaques of RA patients, as were Haemophilus, Aggregatibacter, Cardiobacterium, Eikenlla, Kingella and Rothia dentocariosa. Among the anaerobes found to be different were Lactobacillus salivarius, Atopobium spp., and Cryptobacterium curtum (enriched in RA), as well as Neisseria spp. and Rothia aeria (decreased in RA). As a whole, the study concluded that there were differences between RA and control microbiomes. Further, Cheng et al. looked at subgingival plaque in healthy and diseased sites in early RA and healthy individuals. Microbial community differences were found at phylum, genus, and species levels (Cheng et al. 2021). Specifically, Capnocytophaga, Cardiobacterium, Neisseria, and Streptococcus genera were all associated with RA. Likewise, Chen and colleagues found that eight oral bacterial biomarkers differentiated RA from osteoarthritis (OA) and that the microbial composition of RA, OA and healthy subjects did differ at the phylum and genus levels (Chen et al. 2018). In another study, Milkuls and colleagues profiled 260 RA and 296 osteoarthritis control patients by collecting their subgingival plaque (Mikuls et al. 2018). Using 16S rRNA sequencing, the researchers found that 10 different OTUs were less abundant in RA patients, including Peptostreptococcus, Porphyromonas, Prevotella and Treponema species. However, the researchers did not find associations with previously identified RA-associated oral microbes such as A. actinomycetemomitans or P. gingivalis. As with the previously discussed diseases, these differences could be associated with differences among the populations investigated.\n\nThe mechanisms linking oral microbiomes to RA center on antibodies and other immunological factors circulating through the body and setting off an inflammatory response (Lopez-Oliva et al. 2018). The translocation of microbes from the oral cavity into the bloodstream is another likely mechanism (Huang et al. 2016). Another possible mechanism is the specific production of metabolic products by microbes that lead to RA formation. For example, a paper by Konig et al from 2016 suggested that microbes such as A. actinomycetemcomitans could induce hypercitrullination in host neutrophils and thus cause RA (Konig et al. 2016). Studying the microbial associations between RA and the oral microbiome in the past could shed lights on these mechanisms and the specific microbial functions associated with them.\n\nTo conclude, RA has a number of possible specific microbial biomarkers, such as the presence of specific microbial functions (e.g., citrulline production) and taxa (e.g., Veillonella, Haemophilus, Aggregatibacter, Cardiobacterium, Eikenlla, Kingella and R. dentocariosa). However, it is important to note that several of these studies demonstrate contrasting results, possibly due to their focuses on populations with different evolutionary histories and microbiome structures. Therefore, more research is needed to investigate why different groups and studies return different microbes as significant biomarkers. One possibility is that different populations have different microbial structures that lead to the disease, in which case modern references for model construction should be carefully chosen by similarity to ancient samples of interest.\n\nAlzheimer’s disease (AD) is a serious condition that currently impacts 6.3 million Americans and leads to about 120,000 deaths a year (2021 Alzheimer’s Disease Facts and Figures 2021). It is among the most common causes of dementia and is the sixth leading cause of death in the United States (Weller and Budson 2018). AD is caused by the accumulation of amyloid beta plaque deposits and neurofibrillary tangles in the brain. These processes can be exacerbated by immune dysfunction caused by systemic inflammation (Heneka et al. 2015). A number of studies have linked oral health to AD (Harding et al. 2017; Kamer et al. 2020; Chen, Wu, and Chang 2017; Liccardo et al. 2020). For example, Chen and colleagues used a retrospective matched cohort study in Taiwan to demonstrate that chronic periodontitis and AD were risk-correlated, with patients with periodontitis having higher rates of disease than controls (Chen, Wu, and Chang 2017).\n\nMultiple studies have also linked the oral microbiota and AD progression. For example, in 2012, Stein and colleagues examined immunoglobulin G antibody levels for seven oral microbes (A. actinomycetemcomitans, P. gingivalis, C. rectus, T. denticola, F. nucleatum, T. forsythia, and P. intermedia) in relation to AD onset and progression (Stein et al. 2012). The researchers concluded that elevated antibodies associated with periodontitis could contribute to AD. A more direct investigation was conducted by Jiao and colleagues in 2019. The researchers examined 39 patients with AD and 30 healthy controls and used 16S to compare the salivary microbiome. They found a lower richness and diversity in AD patients with a relatively higher abundance of Moraxella, Leptotrichia, and Sphaerochaeta, while Rothia was reduced (Jiao et al. 2019). Another recent study by Wu and colleagues found that AD individuals have lower microbial diversity, increased number of Lactobacillales, Streptococcaceae, Firmicutes/Bacteroidetes, and a significantly decreased number of Fusobacterium (Wu et al. 2021).\n\nThe oral microbiome can influence AD outcomes through two mechanisms: the instigation of systemic inflammation and the introduction of oral microbes and their virulence factors directly into the brain through the blood-brain barrier (Thomas et al. 2021; Sureda et al. 2020; Singhrao and Olsen 2019; Harding et al. 2017). Directly, oral microbes can enter brain tissue via the blood or lymphatic system and damage the neural system (e.g., Singhrao et al. 2015). Both these and microbial byproducts that enter the neural system can trigger inflammatory and other antibacterial responses that can promote AD (Weaver 2020; Narengaowa et al. 2021). Indirectly, oral microbes can exacerbate systemic inflammation. However, the relationship between AD and the microbiome may also be bidirectional, as patients with AD are known to have limited mobility and poorer oral hygiene (Peng et al. 2022).\n\nThese findings suggest specific bacterial biomarkers, such as lower diversity and the relative abundance of certain species, could be used for risk modeling of AD within populations. However, the occurrence of AD in mostly older individuals necessitates improved age estimations for archaeological applications. Specifically, point age estimations with confidence intervals, such as those acquirable via transition analysis, would be extremely invaluable (Milner and Boldsen 2012). While individual AD-diagnosis in ancient individuals may be beyond reach, it is conceivable that the bacterial biomarkers described above could be used to detect differences in risk amongst populations.\n\nIn addition to the NCDs described above, there is mounting evidence linking the oral microbiome to several other morbidities. Specifically, various types of cancers (Frias-Lopez and Duran-Pinedo 2020; Peng et al. 2022), adverse pregnancy outcomes (Frias-Lopez and Duran-Pinedo 2020), mental health disorders (Wingfield et al. 2021), and other conditions have been recently explored. While this paper has focused on CVD, DM, RA, and AD, additional NCDs could be added as morbidities of interest for ancient health research.\n\n\nApplying the present to the past\n\nWith a number of specific microbial risk factors associated with NCDs evident in modern populations, an emerging frontier of ancient metagenomic research involves utilizing this information in past populations. By developing predictive risk models from the NCD data of modern populations, researchers may be able to quantify the risks associated with either specific or nonspecific NCDs in the human past (Figure 1). Even a constrained ability to discern differential NCD-associated risk in past populations would enable researchers to ask new questions about how changing environments, lifestyles, and behaviors impacted human health. These advances are critical to pursue in order to advance the goals of the osteological paradox and improve paleoepidemiological research (Wood et al. 1992; DeWitte and Stojanowski 2015). While this research is unlikely to lead to diagnostic capabilities of NCDs in past individuals, it does have the potential to reveal previously cryptic elements of frailty that are associated with the same microbial mechanisms as NCDs. That is, by identifying microbiome markers associated with higher risks of inflammation and NCDs, researchers would be better able to differentiate between the health experiences of both populations and individuals.\n\nTo implement this proposed methodology, several steps must be pursued. First, using data from modern populations (and ideally non-industrialized modern populations descended from the ancestral population of interest), specific NCDs markers, such as the ones discussed in this paper, should be curated via a meta-analysis. These markers would not need to be specific, but rather contain additive risk information regarding the likelihood of an individual or community to express NCDs. These markers may include specific taxa (e.g., species), relative diversities (e.g., low beta diversity), community structures (e.g., co-occurring groups of taxa), or microbial functions (e.g., functions related to inflammation). Next, these markers will need to be incorporated into a statistical model of risk for a given individual or population as previously done for modern individuals (Zheng et al. 2020; Aryal et al. 2020). This would also allow researchers to explore how microbial taxa and functional profiles translate into physiological effects. Moreover, markers beyond those explored in this paper could also be utilized to explore the associations between microbes and frailty. For example, ancient metabolomics and metaproteomics could also be integrated into risk models to test if specific markers impact health and survivorship.\n\nIn ancient populations, NCD-risk models could then be validated against skeletal markers of health including age-at-death and prevalence of nonspecific skeletal stress markers. These comparisons between the oral microbiome and skeletal markers of health and survivorship would enable researchers to validate whether the oral microbiome is indeed informative about measurable aspects of frailty. Should these relationships prove valid the ancient oral microbiome approach described in this paper would not just enable risk assessment for ancient populations, but potentially provide insights into the mechanisms via which environments, behaviors, and health are linked in the modern day.\n\n\nChallenges and limitations\n\nWhile this is a promising new area of exploration, we acknowledge that there are a number of challenges and limitations. For instance, archeological metadata must be improved for samples for whom ancient dental calculus is analyzed (Gancz, Wright, and Weyrich 2022b). Specifically, improved age estimations, demographic data, and standardized paleopathological information must be collected in order for the morbidity and mortality risks associated with systemic diseases to be assessed. Without these data, it would be difficult to control for enough health-related factors to enable differences in health associated with systemic conditions be found. To elaborate on this point, the traditional age categories assigned to ancient individuals (e.g., ‘young adult’, ‘middle adult’, ‘juvenile’) are almost certainly insufficient to detect differences in survivorship associated with systemic diseases in populations. This is especially true as many of these conditions selectively impact older individuals, whose ages in the archeological record are often underestimated (Milner and Boldsen 2012).\n\nA deeper and more fundamental challenge of this analysis is that all the risk factors for the conditions of interest are available only for modern populations. It is known that oral microbiota changes over time (Deo and Deshmukh 2019; Yates et al. 2021), and microbiota in ancient populations may not be linked with disease in the past in the same ways they are today. Moreover, these diseases may not be highly prevalent in ancient populations, which would necessitate large sample sizes. For example, RA has a prevalence of approximately 0.41–0.54 among US adults, meaning that the likelihood of obtaining a sufficient number of ancient individuals exhibiting this disease from a single tempo cultural context is low (Hunter et al. 2017). Even for more widespread conditions in industrialized societies, such as obesity and diabetes, it is difficult to estimate what percentage of the population may have exhibited these diseases in the past.\n\nAdditionally, there are a number of challenges involved in the reconstruction of ancient oral microbiomes that may limit the resolution with which the suggested approach may be implemented. First, aDNA is highly fragmented and prone to contamination (Llamas et al. 2017; Salter et al. 2014). When aDNA methodologies are applied to ancient microbes, there are additional constraints in the ability of researchers to identify specific taxa at high resolution, often due to damage as well as limited or inaccurate references (Velsko et al. 2018). However, these challenges are key issues currently being addressed in the field of ancient metagenomics, and so they are likely to be mitigated as analytical procedures and bioinformatic pipelines are improved.\n\n\nConclusions\n\nThe oral microbiome is a promising tool for understanding the mechanistic pathways via which environmental factors translate into health impacts, both today and in the past. In this paper, we suggest a novel approach for identifying NCD-associated frailty in ancient populations. The benefits of this approach are numerous as NCDs could be studied in and of themselves in association with a variety of environmental and behavioral changes over time. Moreover, the health and environmental impact of historical shifts help researchers examine why differences in disease manifestations exist in modern populations and what might be effective ways of approaching them. While substantial model development and refinement is necessary before these methods can be added into the ancient health research methodological repertoire, we consider it a promising application of ancient metagenomics.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\n2021 Alzheimer’s Disease Facts and Figures. Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association. 2021; 17 (3): 327–406. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeller J, Budson A: Current Understanding of Alzheimer’s Disease Diagnosis and Treatment. F1000Res. 2018; 7(July): 1161. F1000 Faculty Rev-1161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWarinner C, Speller C, Collins MJ: A New Era in Palaeomicrobiology: Prospects for Ancient Dental Calculus as a Long-Term Record of the Human Oral Microbiome. Philos. Trans. R. Soc. B Biol. Sci. 2015; 370(1660): 20130376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeyrich LS, Dobney K, Cooper A: Ancient DNA Analysis of Dental Calculus. J. Hum. Evol. 2015; 79(February): 119–124. Publisher Full Text\n\nWeyrich LS: The Evolutionary History of the Human Oral Microbiota and Its Implications for Modern Health. Periodontology. 2021; 85(1): 90–100. PubMed Abstract \| Publisher Full Text\n\nWingfield B, Lapsley C, McDowell A, et al.: Variations in the Oral Microbiome Are Associated with Depression in Young Adults. Sci. Rep. 2021; 11(1): 15009. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWood JW, Milner GR, Harpending HC, et al.: The Osteological Paradox: Problems of Inferring Prehistoric Health from Skeletal Samples [and Comments and Reply]. Curr. Anthropol. 1992; 33(4): 343–370. Publisher Full Text\n\nWu Y-F, Lee W-F, Salamanca E, et al.: Oral Microbiota Changes in Elderly Patients, an Indicator of Alzheimer’s Disease. Int. J. Environ. Res. Public Health. 2021; 18(8): 4211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang JJ, Danxia Y, Wen W, et al.: Association of Diabetes With All-Cause and Cause-Specific Mortality in Asia: A Pooled Analysis of More Than 1 Million Participants. JAMA Netw. Open. 2019; 2(4): e192696. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYates JA, Velsko IM, Aron F, et al.: The Evolution and Changing Ecology of the African Hominid Oral Microbiome. Proc. Natl. Acad. Sci. 2021; 118(20). 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "161874", "date": "14 Feb 2023", "name": "Sarah Schrader", "expertise": [ "Reviewer Expertise Bioarchaeology", "Archaeological Science", "Paleopathology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this review contribution, Gancz and Weyrich present a compelling argument for how the oral microbiome might elucidate non-communicable diseases in the past. The authors draw on data from modern studies and couch this within potential applications to archaeological studies. More specifically, they address how cardiovascular disease, diabetes, rheumatoid arthritis, and Alzheimer’s disease interact with the microbiome and skeletal/dental lesions, such as periodontitis. The authors argue, and I agree, that this approach could significantly contribute to our understanding of frailty and the osteological paradox. A more thorough understanding of non-communicable diseases—a major culprit of morbidity and mortality around the world—through time is essential to tackling this problem. In the Challenges and Limitations sections, Gancz and Weyrich acknowledge that the diagnosis of specific non-communicable diseases in the past is unlikely; however, identifying microbiome markers associated with inflammation and non-communicable diseases is attainable. This is a very thought-provoking contribution that highlights the cutting edge of microbiome research and will certainly directly contribute to a better understanding of non-communicable disease through time and space.\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": "9539", "date": "06 Apr 2023", "name": "Abigail Gancz", "role": "Author Response", "response": "We thank the reviewer for this thoughtful summary of our research." } ] }, { "id": "162486", "date": "23 Feb 2023", "name": "Taylor van Doren", "expertise": [ "Reviewer Expertise Infectious diseases", "tuberculosis", "influenza", "co-morbidities", "biocultural anthropology", "syndemics", "demographic transitions", "epidemiological transitions" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reviews and synthesizes the literature linking the oral microbiome to various non-communicable diseases, and additionally provides some intriguing paths forward on how to study these connections in skeletal remains to illuminate another dimension of NCDs in past human populations. I really enjoyed this interesting, well-written, and well-researched paper, and I think the authors have significant contributions to make to the body of literature on ancient human health.\nI have listed some of my more detailed thoughts below on the paper, some small and some more significant. Some of these comments are comments on mere spelling/style issues, but I have also included a couple larger items that I hope the authors consider, if not in this revision, then as the research progresses.\nFirst paragraph of introduction: Consider listing at least a couple of the seven of the top ten causes of death in 2019. Also, I do not suspect that the top ten causes of death would have changed substantially from 2019 to 2023 (other than the inclusion of COVID, of course), but do you have an updated source? I know that WHO has just a little bit of a lag, but there should be updated numbers from 2019 by now.\n\nSecond paragraph of introduction: One of the reasons listed for why the trend of studying NCDs through aDNA is that “infectious diseases can often be identified through distinctive mass-death burials or specific skeletal pathologies”. Certainly, for major epidemic/pandemic events, the mass death burials will be essential, but I do not agree that infectious diseases appearing in skeletal remains would be a major contributor to this. Overwhelmingly, acute infectious disease progression is too short to leave any real traces on skeletons. Infectious diseases like tuberculosis and leprosy are (sometimes) identifiable with some difficulty in skeletal material, but only after a very long period of infection and disease progression. Skeletal pathogolies like markers of frailty could suggest potential susceptibility to acute infectious diseases, but infectious diseases overwhelmingly do not manifest in the skeleton. Because of the previous mention of Donoghue (2017), I suspect tuberculosis and leprosy are top of mind here, but this should be more carefully stated in the text.\n\nEnd of first paragraph of background: consider capitalizing “native” and “indigenous.”\n\nLast full paragraph on page 4: The osteological paradox is mentioned very fleetingly, and although it is a popular concept and likely well known to your audience who studies skeletal material, it might be a good idea to define it briefly. Additionally, the citation of DeWitte & Stojanowski (2015) is good, but if you do dedicate space to the osteological paradox, it would be good practice to reference the original paper (Wood et al., 1992) as well.\n\nFirst paragraph on page 5 under “Cardiovascular disease”: in the list of conditions that make up CVD, “stroke” is listed twice.\n\nFirst paragraph, 3rd line on page 7: “Actinobacteira” should be “Actinobacteria”\n\nFirst paragraph in “Applying the present to the past” page 9: I see here that you do go ahead and cite the Wood et al. (1992) paper in reference to the osteological paradox! Just a small semantics thing in this sentence: I am not sure that the osteological paradox as a concept has “goals” per se, it would be more like the goals of understanding the nuances of the osteological paradox and the reality of the lived experiences of past populations.\n\nSecond paragraph in “Applying the present to the past” on page 9: It occurred to me while reading this paragraph (which I really like, because it provides some clear steps forward), that the issue of the prevalence of NCDs in historical and/or prehistoric populations will be really dependent on how industrialized/urbanized/modernized a particular population is. I would not recommend going into a detailed discussion of epidemiological or health transitions in this paper, but briefly: there were major shifts in the proportionate mortality of NCDs and infectious diseases that align pretty well with major transitions in human behavior and culture in lots of places throughout the world, roughly aligning with the agricultural revolution, industrial revolution, and globalization. Before the industrial revolution in lots of places, NCD mortality was relatively small, although we don’t know that much about the prevalence. There is some big message in here for you to think about in your research and potentially acknowledge here, in which the ability to actually study NCDs via the oral microbiome to uncover the nature of NCDs in historical/pre-historic populations will be a major contribution to our understanding of health in those populations, since we have these preconceived notions about their relative prevalence using mortality as a proxy—which is probably not going to be as good of a proxy as actual biological data.\n\nSecond paragraph on page 10: You discuss some limitations of using aDNA, specifically in that it is often fragmented and prone to contamination. Pathogens like Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, etc. have been studied using molecular clock calibration with aDNA. Given the fact that you are discussing the bacteria that make up the microbiome throughout this article, would that method be useful for what you are describing here? Could it improve upon the fragmentation and contamination issues that plague other aDNA methods?\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": "9540", "date": "06 Apr 2023", "name": "Abigail Gancz", "role": "Author Response", "response": "Reviewer 2 This paper reviews and synthesizes the literature linking the oral microbiome to various non-communicable diseases, and additionally provides some intriguing paths forward on how to study these connections in skeletal remains to illuminate another dimension of NCDs in past human populations. I really enjoyed this interesting, well-written, and well-researched paper, and I think the authors have significant contributions to make to the body of literature on ancient human health. We thank the reviewer for this summary of our research and their thoughts on its contribution to the field. I have listed some of my more detailed thoughts below on the paper, some small and some more significant. Some of these comments are comments on mere spelling/style issues, but I have also included a couple larger items that I hope the authors consider, if not in this revision, then as the research progresses. First paragraph of introduction: Consider listing at least a couple of the seven of the top ten causes of death in 2019. Also, I do not suspect that the top ten causes of death would have changed substantially from 2019 to 2023 (other than the inclusion of COVID, of course), but do you have an updated source? I know that WHO has just a little bit of a lag, but there should be updated numbers from 2019 by now. We thank the reviewer for this astute comment. First, we have added examples (“Specifically, seven of the top ten causes of death in 2019 were attributed to NCDs (ex. heart diseases, diabetes, and cancers), and these accounted for 44% of all global deaths.” We have also replaced the 2019 citation with the WHO 2022 report (although this still cites the same data from 2019, as that is the most up to date due to pandemic delays). Second paragraph of introduction: One of the reasons listed for why the trend of studying NCDs through aDNA is that “infectious diseases can often be identified through distinctive mass-death burials or specific skeletal pathologies”. Certainly, for major epidemic/pandemic events, the mass death burials will be essential, but I do not agree that infectious diseases appearing in skeletal remains would be a major contributor to this. Overwhelmingly, acute infectious disease progression is too short to leave any real traces on skeletons. Infectious diseases like tuberculosis and leprosy are (sometimes) identifiable with some difficulty in skeletal material, but only after a very long period of infection and disease progression. Skeletal pathologies like markers of frailty could suggest potential susceptibility to acute infectious diseases, but infectious diseases overwhelmingly do not manifest in the skeleton. Because of the previous mention of Donoghue (2017), I suspect tuberculosis and leprosy are top of mind here, but this should be more carefully stated in the text. We thank the reviewer for these remarks. To address them, we have modified our phrasing to state “while individuals with severe cases of infectious diseases can sometimes be identified through distinctive mass-death burials or specific skeletal pathologies”. We completely agree that it is difficult to diagnose infectious diseases in ancestors using skeletal pathology unless individuals had long-lasting infections, and that this biases the observable record towards more severe cases. When speaking of the benefits of aDNA to infectious diseases, we are referring to the capacity of researchers to analyze genomes and traits associated with specific infectious agents, and we have now made that more clear via the following sentence: “Moreover, unlike with infectious diseases, aDNA cannot be directly used to establish the clear presence of a single pathogenic agent nor to study the genomes and traits associated with it”. End of first paragraph of background: consider capitalizing “native” and “indigenous.” We thank the reviewer for this comment and have implemented the suggested changes. Last full paragraph on page 4: The osteological paradox is mentioned very fleetingly, and although it is a popular concept and likely well known to your audience who studies skeletal material, it might be a good idea to define it briefly. Additionally, the citation of DeWitte & Stojanowski (2015) is good, but if you do dedicate space to the osteological paradox, it would be good practice to reference the original paper (Wood et al., 1992) as well. We thank the reviewer for this observation and have done the following: First, we have included the original paper (Wood et al 1992) in our citation of the osteological paradox. In addition, we have incorporated a definition: “the observation that deceased skeletal populations do not directly reflect the health or demographic characteristics of living populations”. First paragraph on page 5 under “Cardiovascular disease”: in the list of conditions that make up CVD, “stroke” is listed twice. We thank the reviewer for this observation and have removed one instance of it. First paragraph, 3rd line on page 7: “Actinobacteira” should be “Actinobacteria” We thank the reviewer for catching this typo and have addressed it accordingly. First paragraph in “Applying the present to the past” page 9: I see here that you do go ahead and cite the Wood et al. (1992) paper in reference to the osteological paradox! Just a small semantics thing in this sentence: I am not sure that the osteological paradox as a concept has “goals” per se, it would be more like the goals of understanding the nuances of the osteological paradox and the reality of the lived experiences of past populations. We thank the reviewer for this insightful comment and agree with the reviewer’s point. As such, we have modified our phrasing as follows: These advances are critical to pursue in order to advance approaches to the osteological paradox and improve paleoepidemiological research” Second paragraph in “Applying the present to the past” on page 9: It occurred to me while reading this paragraph (which I really like, because it provides some clear steps forward), that the issue of the prevalence of NCDs in historical and/or prehistoric populations will be really dependent on how industrialized/urbanized/modernized a particular population is. I would not recommend going into a detailed discussion of epidemiological or health transitions in this paper, but briefly: there were major shifts in the proportionate mortality of NCDs and infectious diseases that align pretty well with major transitions in human behavior and culture in lots of places throughout the world, roughly aligning with the agricultural revolution, industrial revolution, and globalization. Before the industrial revolution in lots of places, NCD mortality was relatively small, although we don’t know that much about the prevalence. There is some big message in here for you to think about in your research and potentially acknowledge here, in which the ability to actually study NCDs via the oral microbiome to uncover the nature of NCDs in historical/pre-historic populations will be a major contribution to our understanding of health in those populations, since we have these preconceived notions about their relative prevalence using mortality as a proxy—which is probably not going to be as good of a proxy as actual biological data. We thank the reviewer for this comment and their insights. Indeed, a major reason we are pursuing this work is to enable a closer examination of health transitions across lifestyle shifts in different populations. Members of our team are currently working on the industrial revolution, agricultural revolution, and globalization in specific populations. To address your comment, we have modified the conclusion of our paper to touch upon these topics in more depth. Second paragraph on page 10: You discuss some limitations of using aDNA, specifically in that it is often fragmented and prone to contamination. Pathogens like Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, etc. have been studied using molecular clock calibration with aDNA. Given the fact that you are discussing the bacteria that make up the microbiome throughout this article, would that method be useful for what you are describing here? Could it improve upon the fragmentation and contamination issues that plague other aDNA methods? We thank the reviewer for this comment and have added the following clarification into the text: “While the genomes and evolutionary rates of well-studied microbes, such as those associated with pathogenic infections (ex. Mycobacterium tuberculosis, Mycobacterium leprae), are often better understood, much less information is available about understudied, commensal microbes such as those of the microbiome (Arriola et al. 2020).” To elaborate on this point, ancient microbiome studies differ from organism-specific studies in several ways, from the ways in which the DNA is captured to how it analyzed. For instance, while contamination is less of an issue for specific organism since researchers can (1) examine reads mapping directly only that organism and (2) authenticate it via DNA damage patterns against a reference, microbiome studies encompass both known and unknown organism. As such, contamination from modern sources is a far bigger issue. There are ways of addressing this (controls, decontam, source tracker, reference free authentication), and more are being developed. So, while ancient-pathogen studies share some of the same limitations as ancient microbiome studies, in some ways, microbiome studies can be more prone to these issues. However, much work is being done to improve methodologies related to these topics, including from researchers in our lab." } ] } ]	1	https://f1000research.com/articles/12-109
https://f1000research.com/articles/12-377/v1	06 Apr 23	{ "type": "Research Article", "title": "Criss-crossing the Sahara: New dangers to the ambitions of West African migrants", "authors": [ "Olusegun Paul Adesanya", "Oluwatomilade Olominu", "Olusegun O. Idowu", "Oluwatomilade Olominu", "Olusegun O. Idowu" ], "abstract": "Background: This study investigates new dangers that West African migrants travelling through the Sahara Desert to Europe contend with. COVID-19 has left an unprecedented impact on all aspects of global politics, migration inclusive. In turn, limitations to migration have drastically increased, especially the stricter visa application measures set by European countries. This intensified attempts at crisscrossing the desert to Europe. In turn, several migrants used the Sahara as the highway to Europe. While some persons successfully travelled through the desert-scape, several others were unsuccessful. Notwithstanding the negative experiences of previous migrants, several other nationals across the region have plans to travel through the desert to Europe. Methods: As a descriptive study, mixed method of qualitative and quantitative approaches to data gathering and analysis were employed. Primary data for the study were gathered through key informants’ interviews with some emigrants, and electronic copies of an open-ended questionnaire were administered to the staffers of certain international non-governmental organisations (INGOs) that are focused on migration. In addition, the chain-referral sampling technique was employed. Through this, certain former migrants who were interviewed suggested other persons with whom interview sessions were conducted, in all 15 persons were interviewed while 85 persons responded to the questionnaire for the study. Results: The findings of the study indicated that there are new dangers to the lives of the migrants travelling through the Sahara Desert. Interesting however is the fact that migrants prefer being in harm’s way in their attempts to access European states illicitly through the Sahara Desert than to remain safe in their home countries in West Africa. Conclusions: The study holds that there are new dangers to the aspirations of West Africans travelling to Europe through the Sahara and the Mediterranean Sea.", "keywords": [ "Migrants", "Sahara Desert", "West Africa", "Crisscrossing", "Dangers", "Irregular migration" ], "content": "Introduction\n\nTravelling from West Africa through the Sahara Desert is done not for the vertical journey to Europe only. Rather, those crossing sometimes go to horizontal destinations since several intra-African or interregional relations are ongoing within the continent. For instance, various nationals of the countries of West Africa travel within the region and sometimes cross to several other countries in Central Africa, East Africa, North Africa and South Africa. At least, not everyone that travels to North Africa embarks on the voyage across the Mediterranean Sea so as to access Portugal, Spain, or Italy for an onward journey to Western Europe or elsewhere. De Hass (2008) describes the notion that those crisscrossing the Sahara are in transit to Europe as a misconception and insisted that there are possibly more sub-Saharan Africans living in the Maghreb than in Europe. An estimated 65,000 and 120,000 sub-Saharan Africans enter the Maghreb yearly overland, of which only 20 to 38 percent are estimated to enter Europe. The Sahara thus serves as a travel route for Europe-bound individuals and those with no intentions of going as far as Europe. In West Africa for instance, 80% of migrants are strictly engaged in intra-regional travelling (cited in ICMPD, 2022, p. 2).\n\nIndeed, citizens across West Africa engage in migration, either to countries of Europe, North America, and Asia; as well as to other regions of Africa. Nonetheless, consequent upon unpalatable societal anomies and/or perilous living conditions, which comprises the groundswell of human insecurity, and climate change and its effects on food security (Diagana, 2021; Sky News, 2022) in particular, which West African countries are contending with at various levels, the number of migrants, especially those engaged in irregular migration surged. Whereas the Moroccan security services arrested 74,000 irregular migrants in 2019 (Migrant Project, 2022), they foiled two irregular operations between December 30 and 31, 2021 that led to the arrest of several irregular migrants who were attempting to get to the Canary Islands through the Atlantic (Toutate, 2022). In turn, the International Organisation on Migration (IOM) (2022) has it that\n\nirregular migration from sub-Saharan Africa towards Europe, and between West and Central Africa, has substantially increased. States in the region, previously considered countries of origin, are becoming transit and destination countries, with irregular migration featuring prominently in the political agendas of most of the governments concerned.\n\nIn spite of increasing efforts that citizens are making to migrate to elsewhere within or outside the region, the governments of the region are yet to do anything substantial to address the trend. This becomes worrisome and engenders concerns about the pace and rate of exit, which represents a huge demographic alteration to the West African population and severe haemorrhage of brain and strength. It further raises suspicion of what might likely become of the states of the region at future dates. Put differently, the region is gradually getting sapped of the youthful capacity that would be needed ahead in [re] ordering the spate of penury, carnage, crimes and underdevelopment bedevilling the region. For instance, 74% of Nigerians desired to relocate (cited in Babalobi, 2020). While 45% of Nigerian adults, according to Connor and Gonzalez-Barrera (2019), planned to move to European countries or the US within five years; and a substantial percentage of Senegalese, Ghanaians and Nigerians planned to have relocated by 2022 (PRC, 2017). Several push and pull factors are responsible for this. For example, the global economic pressures as triggered by eventualities and intensified development in certain aspects of the developed economies are some of the pull factors; some others include aging populations in rich countries (Van Agtmael, 2007), the incidence and fatality of the COVID-19 pandemic, which represents “a deep crisis with profound repercussions” (Jensen, 2021, p. 66), and the need to fill employment vacuum. These pull factors and several others are shaping the direction of economic and demographic management. The push factors however include personal ambition, incidence and burden of poverty, desire for better education, unemployment, haphazard governance conditions, as well as the spate of insecurity across the domain.\n\nIndeed, the ambitions of the potential migrants pose negative and positive implications to the respective home countries in particular, and the region in general. The problem concerns the brain and energy drain of these countries. Several of the real and/or prospective emigrants are trained in one discipline or the other, while several others possess artisanal skills that could be of immense advantage to their respective countries. Such educational capacities and skills were cheaply acquired compared to the amount such training would have cost in the better climes that are attractive to the potential migrants. In view of this, the home states are the ones losing hugely. The positive dimension concerns the remittances the emigrants would make to their relatives in their home countries. Equally, crime is likely to reduce, which might have been encouraged by the prevalence of poverty and peer pressure within the home countries.\n\nWest African citizens would have migrated from their home countries to elsewhere within the region, but the prevalence of the same governance style and standard of living across the region, the intra-regional migration reduced drastically. In the 1980s, intra-regional emigration, particularly to Nigeria, assisted in tackling poverty and crime in some of the neighbouring countries. In as much as such opportunity and space are no longer available, outright exit from the region gained currency, especially among the youths. A similar situation occurred in the 1980s when highly qualified professionals from Ghana and Nigeria, as Adepoju (2005) posits, emigrated to South Africa and elsewhere outside Africa. [This] intensified in the 1980s and 1990s, with qualified persons moving to Europe, North America and the oil-rich Middle East. Shortly after the collapse of Nigeria’s oil-led economy in the mid-1980s, there was massive emigration of highly skilled Ghanaians and Nigerians, in particular the paramedical personnel, teachers, engineers, nurses, scientists, medical doctors and technologists to developed climes because of relatively higher salaries and better living conditions. Many Ghanaian professionals, as Adepoju (2005) remarked, also moved from Nigeria in a step-wise pattern to South Africa’s then ‘homelands’, as well as to Swaziland and Lesotho.\n\nSince this trend happened about a generation ago, we acknowledge that the current emigration trend is the continuum of a pattern. For legal emigration to take place, however, there are serious hurdles to cross. Such include an application for and approval of a visa, and availability of funds for self-care and to finance the purpose of the journey. Lacking these items, several people decided to illicitly access Europe en route the Sahara Desert-North Africa-Mediterranean Sea. In a way, the route is legal; and this concerns possession of the legal travel documents that permit access to the North African states through the Sahara. This permission might however be dated, hence deadline for exiting such a state is set in the travel document. If the deadline is violated or the approved exit instructions, one of which might be the ‘exit route’ is violated, the traveller then becomes a persona non grata; hence the status of an irregular migrant. Nevertheless, European and [North] African states, as De Haas (2008) posits, seem not so interested in the outright stoppage of migration into their confines, especially because their economies have come to rely more on migrant labour, while their home states appreciate the remittances respectively. This explains\n\n“why increasing border controls have rather led to the swift diversion of migration routes and an increase in the risks, costs, and suffering of the migrants involved rather than a decline in migration. As long as no more legal channels for immigration are created to match the real demand for labour, and as long as large informal economies will exist, it is likely that a substantial proportion of this migration will remain irregular” (De Hass, 2008, p. 10).\n\nInvolved in all these issues are the dangers that the migrants contend with on the journeys. Previously, such persons contended with the challenge of being abandoned in the hot desert by human smugglers (Dockery, 2017), and the likelihood of dying as a result of thirst and weariness. Presently, such migrants have new dangers to contend with, hence this study. The study thus investigates the new dangers that West African migrants travelling through the Sahara Desert to Europe (though the Mediterranean Sea poses its own challenges too) have to contend with. In addition, it examines the implications of the new dangers to the emigrants’ desires for the states within the domain.\n\n\nTheoretical framework\n\nWhereas there is a litany of theoretical lenses for understanding the numerous gamut of migration and the politics it throws up, the push-pull theory is however preferred. This is in spite of the critique that the models amount to little more than lists of factors that lack a framework to bring them together in an explanatory system (Skeldon, 1990). And that in its original form, push-pull failed to account for changing motivations, altered circumstances or modified decisions en route (De Haas, 2011). In its simplest format, the push-pull model suggests that migrants were pushed by low incomes in their home states or regions and pulled by the structure of better prospects in affluent states or regions (Lee, 1966; Harris and Todaro, 1970). In his seminal study, Ravenstein (1885) presented the first systematic principles that nuance the dynamics of migration. The rationale that underpins migratory processes, as Ravenstein remarks, is an individual rational decision hinged upon calculations of costs and benefits of migration. The economic situation of the place of origin thus constitutes the push force, while the structures supporting affluence and improved living conditions in the destination are the pull factors.\n\nThe questions nudging the mind at this juncture thus become: Why do West Africans migrate from the region to elsewhere across the world, particularly Europe? Why would several migrants from the region decide to travel through the Sahara Desert at the expense of life for the purpose of resettling in Europe? They are mostly seeking job opportunities and security. Obviously, the states within the domain have myriads of challenges they are contending with; and of no mean implications are those tied to economic and safety issues bedeviling the citizens. In view of this, the push-pull perspective, according to Ley (2005, 60), serves as a prism through which migrants are regarded as rational economic [wo]men. On the basis of human want, emigrations from the region are patterned on economic opportunities. In turn, the theory remains valid for understanding the thrust of the study. After all, the weather condition of the region is relatively conducive, and not responsible for the ongoing hollowing of people, especially the middle-class. On the basis of time and space of occurrence, this study herein proposes the West African variant of the push-pull theoretical framework. The ongoing mass emigration of West Africans is not only to Europe but rather to several countries in other regions of the world. Any location that provides a modicum of improvement in human security is preferred to West Africa for several of the emigrants.\n\nHuman mobility, as expressed by the Global Commission on International Migration (GCIM) (2005), is an inherent and desirable component of development process. For the host society, the immigrant population would introduce new energy and innovation; particularly to the industry and education. This situation would enhance the filling of pension gap, especially for new young workers who would be paying taxes. All these would only be possible since the host states have spaces available to be filled because of their birth rate, relatively low population and demographic pattern. For the countries of provenance, it portends ‘Janus-faced’ situations of (under)development. The underdevelopment is hinged upon how a low-density population may not inspire the remaining citizens to display and actualise many developmental initiatives for their countries. Rather, several others are likely to desire emigration too. The development however would be partly hinged upon the remittances from the nationals of such countries that are in diaspora. In as much as the above is apt, howbeit that West African countries have yet to create conducive spaces that could attract migrants? In lieu of this, more citizens from the zone have consistently berated their home countries, especially as is the case in Nigeria presently, and have been making efforts to emigrate elsewhere across the world. It thus means that the living conditions (standard and cost), security and economic situations in most of the states within the region are appalling, even as the demographic rate is increasing.\n\nThe above scenarios thus reveal the essence of and reinforce the need to use the push and pull framework for this study. In addition, the need for people to improve their living conditions substantiates the choice of the framework. For the European Asylum Support Office (EASO) (2016):\n\n“human mobility is resultant from specific factors that either attract an individual to migration (pull factors) or that repel the individual from a continued stay in his/her place of habitual residence (push factors). These factors may relate to different levels of characteristics or systems (e.g. micro-, meso-, macro-) and may or may not interact with one another to shape mobility outcomes.”\n\nOne of the elements of ‘push’ in the theory concerns the deteriorating socio-economic and politico-security-scape of West African states, which started in the late 1970s and 1980s, to the perception of a dismal future in the 1990s; as well as the fast-paced descent into the abyss of untoward circumstances of insecurity and low or no development to mention some. This has caused several citizens to regard emigration as the last hope for improving their living standards. The yawning gap in exchange rates of the several currencies across the world vis-à-vis those of West Africa is alarming. The situation thus trivialises the value of and/or for the effort and labour that several workers within the domain commit to their jobs; thereby demeaning (on the basis of the exchange rate) the contributions of the workers to the national or regional development as irrelevant. The worsening element in this scenario is the fact that the salary of the workers hardly increases as the inflation rate and prices of goods and services increase. Thus, the current pace at which West African states are sinking into irrelevance, and the abyss of underdevelopment and crimes has intensified the resolve of more persons at various strata of socioeconomic status to seek greener pasture and stable residence abroad.\n\nSome other factors responsible for the intentions of West Africans to embark on irregular migration across the Sahara Desert/Mediterranean Sea route to Europe include socio-economic crises of social inequality, market economic challenges, and politico-security challenges like instability, conflicts, wars, discrimination, natural catastrophe, and climate change; as well as the wider processes of transformation. It is to this end that push-pull theory would assist in deepening knowledge of the irregularity of journeys across the sandy-sea route.\n\n\nMethods\n\nThis study received ethical approval from the Afe Babalola University Research Ethics Committee under approval number ABUADBREC 887359. Data protection, objectivity and privacy rights of respondents are all catered for in the study. Efforts were made to make the instrument anonymous as the survey instrument did not require the participant to include their names, telephone number, nor contact details. Effort was also made to protect all the key informants; they were assured that the information provided and identity is protected at the point of collecting and reporting their submission. This process was embarked on to maintain the confidentiality of all participants in the study.\n\nRespondents were informed that there are no direct and immediate benefits for participation in the study. However, the importance of their submission in the study was made clear, including the purpose of the study. The decision to participate was left entirely to the potential participant without any influence. The research was designed to guarantee that all personal data are safe from unforeseen, unintended or malevolent use. The researcher ensures that respondents/interviewees were voluntarily engaged in the study with full knowledge of relevant risks and benefits. This implies that, prior to consenting to participation, participants were clearly informed of the research goals, possibilities to refuse participation or withdraw from the research, at any time, and without consequences. The respondents/interviewees were adults above 18 years, they all understood the information, and were fully aware of the consequences of their consent in relation to the study. However, there was no physical risk associated with the respondents in the study. Written informed consent was obtained using a consent form from all participants, distributed in English (Adesanya and Idowu, 2022c).\n\nThe study falls within the descriptive design. A mixed method of qualitative and quantitative approach in gathering and analysing data was employed. Primary data for the study were gathered through key informants’ interviews with migrants, especially some of those that returned from Libya and Morocco to Nigeria and Benin Republic. Equally, between April 9, 2022 and June 20, 2022, electronic copies of open-ended questionnaire, which was developed in-line with the objectives of the study, were circulated via Google form in English language, amongst the officials of the International Organisation for Migration (IOM), and international non-governmental organisations (INGOs), for example, Migration Policy and the Mixed Migration Centre, which are focused on the numerous aspects of migration. The participants were contacted through their email addresses, which were accessed on the websites of their respective organisations. 250 copies of the questionnaire were circulated, out of which we received 85 responses (Adesanya and Idowu, 2023a). In addition, the snowball sampling method, also regarded as a chain-referral sampling technique, was employed. To do this, we identified and interviewed some initial responders that suggested other persons that were interviewed using snowball sampling. The interviewee was visited in their various offices. Two of these persons are resident in Lagos, while two other persons are residents of Ogun and Ekiti States respectively. These four people suggested other people that were interviewed for our study (Adesanya and Idowu, 2023b). The study, also, relied on extant literature on diverse dimensions of vertical irregular migration to Europe and the numerous processes involved in it, as well as the horizontal migrations happening within the West African region. These include textbooks, journal and web articles, online news tabloids, and electronic news magazines. This was done between April and June 2022. The data gathered from both primary and secondary sources were subjected to thematic and descriptive analysis and content analysis respectively.\n\n\nResults\n\nA total of 85 persons responded to the questionnaire and 15 persons were interviewed (Adesanya and Idowu, 2022a). We found that the reasons for several irregular migrants’ decision to embark on the illicit journey through the Sahara Desert are economic, serious limitations to obtaining valid traveling documents, and considerations of improved living conditions for families.\n\nEqually, it is observed that aside from challenges such as sexual exploitation/prostitution, organ harvesting, human trafficking, slavery, torture, hard labour, and financial exploitation, the emigrants traveling through the Sahara Desert have new dangers like COVID-19, terrorism in sub-Saharan Africa and the Sahel, and changing topography and environmental conditions as a result of climate change to contend with. In addition, it was realised that irregular migrants possess a huge dose of endurance and are very resilient.\n\n\nDiscussion\n\nIn view of Homer-Dixon’s (1999) claim that analysts must trace the long and tangled chains of causation, we believe that Van Hear et al. (2017) made a concerted effort at describing the drivers of migration as factors leading to migration and responsible for the continuity of migration. Drivers, therefore, are limited to factors that may make up the structural elements shaping the decision space for those considering migration (Van Hear et al., 2017). They proposed a drivers’ complex framework which suggests that outflow-migration and inflow-migration are often determined by a complexity of factors encompassing the economic, social and political environment. For Scheffer (2021), five factors account for the movement of people. These include demographic changes marked by growth or decline in population growth, climate change characterised by varying degrees of rising sea levels, famine, drought, etc.; the wealth gap between the North and the South, and the tendency for existing migrant networks to attract those that are still in the home country.\n\nThe above analysis suggests that economic factors are not the ultimate reasons for the South-North migration; especially when a majority of those who emigrate fall in the category of middle or higher class. Synonymous with this is the finding of Van Hear et al. (2017) that the poorest of the poor are not typical migrants. Urbanski (2022) further put these factors into categories of economic, social and political factors. The study insists that economic pull factors such as better employment, increased income and standard of living weigh more as the determinant of migration in Poland and Romania compared to the other pull factors. While political push factors such as war, unfair legal system, and bad governance formed a strong determinant of migration in these countries.\n\nThis study is consistent with the research of Kirwin and Anderson (2018) who investigated the factors that necessitate migration from West Africa, using Nigeria as a case study. The study affirms that economic factors remain the major pull factor for emigrating, followed by weakness of the political system and lack of trust in the security apparatus. These findings are affirmed by Duru (2021). Using 100 Nigerians as the study population, respondents ranked job opportunities as the leading cause of migration, succeeded by unemployment, wealth prospects, safety and security, and better conditions of living. This clearly shows that economic factors and political factors rank high as the drivers of migration. However, while there is an abundance of research and theoretical backing for economic drivers of migration, Giménez-Gómez et al. (2017) projected human security as another serious driver of migration. These authors used datasets from 51 African countries and 21 European countries between 1990 and 2014 and found that despite the income gap being an important driver of out-migration, it is not the only factor.\n\nArguably, Europe's proximity to Africa, and the fact of colonial history, make it a target for intending migrants. Substantiating this, IOM (2021) explains that in Q2 of 2021, a total of 35,338 immigrants and refugees were registered arriving through the three Mediterranean routes and the Western African Atlantic route to Europe, which is more than three times higher than the 10,334 arrivals registered in the same period in 2020, and 56 percent more than the 22,626 registered in Q2 of 2019. This reinforces Attali’s remark that:\n\n“migration is set to increase in the future and the dimension of this growth cannot be predicted, especially migration from the global south to the north. Statistically, more than half of international migrants live in countries of the global North as 35% of international movement is from the south to the north” (cited in Scheffer, 2021).\n\nIn turn new factors and events are bound to surface as determinants of these moves. That is more people are bound to risk migration in an attempt to get to safety or a better life. In recent times, the international community has faced several migration challenges ranging from the aftermath of the Taliban takeover in Afghanistan, influx of immigrants fleeing economic hardship from global south, calls for human right protection of immigrants, refugee crisis, the effect of the COVID-19 pandemic, internal displacement caused by extreme natural disasters, perceived pressure on host countries resources, and many others. What all these suggest is that more people are bound to migrate from their home country in the future. Therefore, despite the fact that most of global south, Africa in particular, are witnessing rapid economic growth, the income gap will continue to widen in the next decades, causing migration to the European Union (EU) to be pushed to a new level. Conflicts and climate change are also significant drivers of migration from Africa to the EU, and it is fair to submit that they will continue to play a role in the future. Environmental reasons may become a larger motivator for migration in the future than they are now (Grieveson et al., 2021).\n\nNotably, as diverse as the drivers of migration are, so also are the routes involved in the migration process. The circumstances surrounding migration might sometimes determine the routes to be taken and the nature of the migration. War, conflict, political tensions and instability often result in increased smuggling of people across land and sea borders, as well as diversification of the migration routes, as seen with migrants from Egypt, Tunisia and Libya as the Arab Spring swept through North Africa (Massari, 2015). Irregular migrants (smuggled and trafficked) often take both land routes to cross the Sahara and then sea routes to get to Europe. As in 2015, among the first five top countries that are taking the sea route to Europe, three African countries - Eritrea, Somalia, and Nigeria - were inclusive (Ogu, 2017).\n\nAdeniyi (2019) identified four major exit points from Nigeria. The first one is the Northern route characterised by less risk and the most commonly used. Migrants would travel either to Kano or Sokoto and then from the border, cross either by arranged vehicles or motorcycles. While the risk of this route is minimal, the financial cost is often humongous as there would be a need to bribe government officials if they do not have the required documents. The second route is a long established route between Lagos and Benin as old as the origin of trade between both countries. Migrants move from Lagos to Benin, even though this is not often a preferred route because more people often enter into Nigeria joining Nigerians who travel north to cross the Nigeria/Niger border into Niger. The north eastern border into Cameroon which is the third exit route is not a regular way for migrants. Only forced migrants like those escaping Boko Haram insurgents follow this route. So, the most prominent route to Europe via the Central Mediterranean is through Niger Republic via North Africa. Niger is home to Agadez, the notorious smuggling city on the African continent, and an integral part of the journey to Europe.\n\nIn Agadez, many migrants obtain the services of the intermediaries required to continue the journey. They are often presented with two routes, one through Libya and another through Algeria. The former from Dirkou to Seguidine, Madama, Tummo and often end in Sebhais, according to Benattia et al., (2015), is considered the expensive route involving the use of a vehicle to convey a migrant between $150 and $200. While the latter takes the migrant to Arlit, Assamaka, and Guezzam, and ends in Tamanrasset at $100- $120. This route often involves walking in the desert and mountains and puts migrants at risk of death, robbery and rape.\n\nFrom Tamanrasset, migrants travel in the company of smugglers through the desert to the Libyan border or up north to Morocco. The journey to Morocco passes through Ghardaia and then Oran, Maghnia and over the border into Oujda and Nador. Meanwhile, the Mali route is a substitute to the popular Niger Republic route. This is the route frequently travelled by Malians and migrants from Senegal, Gambia, and Guinea before Nigerians began to travel through that route. The route runs from Gao to Bor Mokhtar (Algerian border) and ends up in Tamanrasset. Along the transit routes, bandits often waylay the travelers, stealing from them, raping several male and female migrants (Molenaar and Kamouni-Janssen, 2017), and in the process, some are also introduced to crimes for survival. Despite the differences in the travelling routes, the common denominator for these risk takers - majority of whom died or languish in some detention centres in Chad, Niger, or Libya - is the desperation for a better life for themselves and their families (Adeniyi, 2019; Massari, 2015).\n\nMassari (2015) further explains that Khartoum is another route to Libya. Several migrants made it a transit city on their journey to Europe, or a destination, for a while. Like the western route described above, this route involves several alternatives based on how safe they are. Depending on the country of origin, the routes to Khartoum differs for migrant. Once in Khartoum, people are transported out of Khartoum in small cars, and once in Omdurman, they are moved to the edge of the desert where the journey continues in full-sized freight trucks, sitting on top of piles of packaged goods. The initial drivers are generally Sudanese, but when the migrants change vehicles in the middle of the desert, they are usually replaced by Libyans or Chadians. There are two main threats for migrants travelling along this route: one is crossing the Darfur region, where there is the risk of encountering armed rebels or bandits; the second is extortion by the Libyan and Chadian drivers who very often stop in the desert and ask people to pay additional sums of money. Drivers are also known to leave migrants who fall from the trucks to die in the desert (Massari, 2017; Kuschminder et al., 2015).\n\nFlowing from the foregoing, it is evident that safety and conflict along the routes; weather conditions; border surveillance and push-back policies; and changes in countries’ political status or visa regimes, as Kuschminder et al. (2015) insist, are factors that determine the routes that migrants choose to follow.\n\nThe process involved in crossing the Sahara to the Mediterranean Sea and Europe is often characterised by immense risks, which include sexual exploitation/prostitution, organ harvesting, human trafficking, slavery, torture, hard labour, financial exploitation, boat capsizing and drowning in the Mediterranean Sea; as well as death (Ogu, 2017; Tjaden, 2021). Acknowledging this, Adeniyi (2019) writes perceptively about the dangers of crisscrossing the Sahara and noted that every stage of the journey is fraught with dangers. In spite of these dangers, some new ones are evolving, and getting very sinister; particularly because of their proclivities and the emergency situation they create owing to current realities within the international system. While crossing the Sahara has its dangers, crossing the Mediterranean Sea into Europe poses a huge challenge to irregular migrants. This part of the journey is the most dangerous as the end of it is usually death for some migrants. Notable however is that several irregular migrants are well-aware of the danger involved. What is obvious, as Tjaden (2021) remarks, are the variegated reactions of those involved to the consequences of their actions. While the awareness of risk can affect the decision to migrate, often time it in no way deterred the participants. Irregular migrants often conduct the cost/benefits analysis of travelling through the desert. However, the role of migration risk perceptions may vary depending on the context and the individual or group involved.\n\nBeyond these risk factors, it was discovered that the emergence of COVID-19, terrorism in sub-Saharan Africa and the Sahel, and changing topography and environmental conditions as a result of climate change are serious dangers to those traversing the Sahara. Migrants, from our findings, would rather put themselves in harm's way to get to their safe haven in Europe than remain in their home country. Such is the case of a Ghanaian returnee who spoke about how he and his co-travelers had to add packets of panadol to the gallons of water they had, and because the water was bitter to taste, they drank very little per time. The little water drank however served as medication as they travelled from Mauritania to the Moroccan town of Tagine as reported in literature by Okertchiri and Turlue (n.d.).\n\nThe discovery of COVID-19 has since changed how we examine mobility and engage in it. COVID-19 has impacted migration by slowing down the migration process as a result of lockdown and restricted movement in many countries of the world, increased the possibility of stricter immigration regimens, the emergence of rigorous visa processing and issuing, reemergence of vaccine passports, and draconian border control regimes. In that, the spread of the virus was made possible by the process of migration and mobility of goods, services and people. Its continued spread is also made possible by the agents of globalisation. With the nature of irregular migration and no medical check or evidence of vaccination required/requested at the point of crossing or accessing the transit country and/or destination, the risk of contracting and spreading the virus increases. This means that migrants are at risk of dying from undiagnosed symptoms exacerbated by the strenuous journey of crossing the Sahara. This not only creates problems, especially for transit countries, but it also increases the death risk factor and outbreak of pandemics/epidemics in such countries. The restriction brought forth by COVID-19 further creates a situation of heightened desperation among migrants to get to their destination by all means. Thus, Sanchez and Achilli (2020) find that coupling these restrictions and migration enforcement regulations are compounding the challenges and risks already faced by those travelling irregularly.\n\nBorder closure initiated as a result of the pandemic, according to Sanchez and Achilli (2020), caused smugglers to use new, less explored and riskier routes as a result of border closures, often putting migrants’ lives at greater risk since many of these routes are beyond the reach of any humanitarian assistance. This change in route as a response to COVID-19 pandemic outcomes means that existing risk factors are heightened. Specifically, 1,442 migrants, as Varrella (2021) explains, lost their lives while voyaging on the Mediterranean Sea in 2020, even as 1,369 died in 2021. The pandemic restrictions also increased the exposure of unaccompanied women and children to trafficking. Notably, traffickers and smugglers have moved with the trend of online business thereby recruiting intending migrants through different online platforms, including grooming and exploitation, particularly of girls, during the pandemic (UNODC, 2021). This, therefore, shows that COVID-19 is not only a new danger, rather, it also exacerbates the existing dangers; hence, making the control of irregular migration difficult.\n\nSignificantly, the COVID-19 restrictions at the height of the pandemic in 2020 left migrants stranded. Emigrants who had decided to return to their home countries were unable to do so because of the closure of borders, camps, containing areas or even detention facilities. This led to tension in many of such centres as seen in Niamey, Niger. IOM Niger (2022a) reported increased tensions at its centres in Niamey given the inability of migrants to return home as a result of the closing of borders, even though many of them had tickets to leave the country under IOM's Voluntary Return and Reintegration Assistance Programme (AVRR) (Sanchez and Achilli, 2020). Given this, another factor that presents a new danger for irregular migrants, especially those taking the sea route to Europe, is the prevalence of pushbacks by destination countries. Pushbacks of migrants refer to the apprehension of immigrants after they illegally access the border of a country and were summarily repatriated to a neighbouring country prior to the conduct of individual protection and health screenings. This practice is drastically increasing, especially in the second quarter of 2020. For example, in April 2020, both Italy and Libya announced their ports unsafe for the landing of boats carrying rescued people (Tondo, 2020; Reliefweb, 2020). Libya subsequently prevented the 280 migrants intercepted by the Libyan Coast guards from disembarking from the ship (IOM, 2020). This means that migrants can be sent back to their home country, organised criminal activities are left uninvestigated and assessing the case-by-case protection needs of irregular migrants is avoided. While pushbacks are reported on the basis of the violation of the principle of nonrefoulement, it is expedient to understand that for the irregular migrant, it means back-to-zero point and perhaps starting the rigorous and dangerous journey all over again (UNODC, 2021).\n\nTerrorism within the region poses a serious threat to lives; hence constituting a new danger to the irregular migration through the Sahara. For those who attempt to reach Maghreb countries, just as Micallef (2017) remarked, ongoing conflicts and instability pose serious dangers. In the desert regions across Libya’s South, tribal rivalries, primarily between the Tebu and Tuareg tribes, have been marked by violence and general lawlessness, though smuggling remains prominent. This however remains one of the old dangers since these tribal groups have always had conflicts between them. Nonetheless, their conflict still permits the smuggling of goods, a situation that bespeaks the period of amity and entente cordiale. Terrorism is however graver than the conflict between tribal groups, irrespective of the extent of violence. This is more so since terrorism is spreading across the Sahel, particularly around Mali, Burkina Faso, Niger and Nigeria. Really, terrorist groups in West Africa, as explained on a British page online (Gov.UK, n.d.), have been demonstrating their capabilities and intents by mounting attacks in Côte d’Ivoire, Burkina Faso and Mali since late 2015 and 2016. In as much as the Sahel where non-state actors of al-Qaeda in the Maghreb (AQIM), Boko Haram, and Islamic State West Africa Province (ISWAP) have their strongholds, is part of the Sahara, it logically follows that the terror groups constitute a major part of the new dangers to the migrants crossing the Sahara. ISWAP is regarded as the deadliest terrorist group in the world, particularly because each of its “numerous attacks took at least 15 lives in Niger; hence taking terrorism deaths from 257 in 2020 to 588 in 2021” (GTI, 2022, pp. 4-5).\n\nTerror groups move from place to place and are likely to pitch tents around migration corridors from West Africa through the Sahel into the Sahara Desert. An instance is the Agadez route in Niger [a spill over-country], which is bounded by countries [excluding Algeria and Benin] regarded as epicentre countries [Libya, Nigeria and Mali] and spill-over countries [Burkina Faso and Chad] of terrorism (GTI, 2022). A similar development is the en masse drift of arms-bearing nomadic Fulani herders and Touaregs, which pose a significant threat to the migrants. Indeed, the bandits, especially in Nigeria, have been labelled as terrorists, while the Touaregs were the ones that sacked Bamako, thus causing grave instability in that country. By and large, the routes for herding cows by the forebears of the Fulani herders and the Touaregs have varied greatly compared to the present routes. Much more, houses have been constructed on such routes. In turn, these routes have been altered significantly. This in part is responsible for the clashes between the herders and the farmers.\n\nIn as much as such groups intend to run over or take over the command and control of the states of West Africa, they can seize some of the [irregular] migrants, forcefully integrate such individuals into their operations and deploy them for nefarious activities against their home states or other countries within the domain. The other side of this coin is that such persons get killed or arrested in the process. If killed, an old danger would have been replayed, however, the process of death is different from the previous forms of death in the Sahara, which result from lack of supplies or sickness. When arrested, such individuals experienced so much distress. Either way, however, they would have become victims of circumstances unplanned by them.\n\nClimate change poses a great challenge to the migrants travelling through the Sahara Desert. The topography of the desert constitutes one of the most dangerous topographies in the world. This is because of its features like hard rocky plateaus, rough mountains, extensive plains covered with sand and gravel, salt flats, high dunes, and huge oasis depressions (World Atlas, n.d.). The average depth of the sand, according to an estimate cited by Busiello (2013), is approximately 150 metres, while the top of the dunes can reach a height of 320 metres from the bedrock (170-180 metres high measured from ‘sand-level’). The Sahara Desert, according to Pausata et al. (2020, p. 235):\n\n“owes its existence to a particular combination of regional continental geography and atmospheric circulation. In the last decades, the active role played by the Sahara in the climate system has been increasingly recognised: changes in Saharan climate, beyond local impacts, are also coupled to changes in regional atmospheric circulation and can have remote impacts on far-afield regions, such as the equatorial Pacific or the Arctic. In particular, the Sahara Desert is currently the largest source of airborne mineral dust on Earth”.\n\nThus, in view of the above, the journey must have been very worthwhile for anyone to think of travelling through this route at all. This reinforces economic differences between West Africa and Europe, which entail differences in earnings, livelihoods and living standards defined by the unfolding global political economy and its inequities (Van Hear et al., 2017) as push-pull factors encouraging irregular migration from West Africa.\n\nCreated by wind erosion of soil particles (Zender, et al., 2004), mineral dust is the most abundant atmospheric aerosol substance. Whereas aerosol is found in the air that we breathe, the atmosphere in the Sahara is laden with it. Aerosol affects the climate, human health, cloud formation, weather, visibility, corrosion, and the chemistry of the atmosphere in several ways. The Canadian Ministry of the Environment and Climate Change (2016) noted that smaller aerosol particles can cause severe health effects, including premature death, aggravation of asthma, cardiovascular disease, and lung disease. Given the sensitivity of mineral-dust emissions in the Sahara to changes in regional hydroclimate and land surface (Pausata, et al. 2020, p. 235), it is very likely that the acute alteration to the climate would cause more dust to be in the air. This is more so because of global warming caused by the depletion of the ozone layer and more sun rays hitting the atmosphere heats up the earth’s surface to the extent that the frozen Arctic and the great ice shield jutting off Antarctica melt; thereby raising the sea levels. As Watts (2018) put it:\n\n“the oldest and thickest sea ice in the Arctic has started to break up, opening waters north of Greenland that are normally frozen, even in summer. This phenomenon – which has never been recorded before – has occurred twice this year due to warm winds and a climate-change-driven heatwave in the northern hemisphere.”\n\nGiven the above, climate change constitutes another new danger to crossing the Sahara. Global warming is intensifying heat waves. Events worse than the current one, as Carrington (2021) claims, are set to strike every year by the 2040s. The dryness that comes with global warming definitely has implications for the desert where the temperature is up to 560 Celsius (GTC, 2021). With global warming, the Sahara sand is drier; making it easier for the mineral dust to rise as the migrants pass by, either in vehicles or on their legs. Irrespective of how covered their noses and mouth were, dust still entered their nostrils. This often causes severe health challenges for the migrants as they drift in the desert where treatment can never be accessed.\n\nAnother issue concerns the drying up of the few oases that used to be in the desert. The environmental condition in the desert has always been an issue, but currently, it has worsened. The Sahara Desert previously periodically witnessed wet durations, mostly regarded as African humid periods (AHPs) (Larrasoañ et al., 2013; Ségalen et al., 2007; Skonieczny, et al., 2019; Ziegler, et al., 2010). With global warming, the oases that ordinarily provided succour in the desert dried up. In Bryce’s (2016) terms, from Morocco to Libya, the oases of the Sahara’s Maghreb region are disappearing as temperatures rise and rainfall decreases.\n\nThe threats of climate change that the Maghreb is contending with were the thrust of the United Nations climate conference (UNCC) in Marrakech in 2016 (UN, 2016). In turn, the oases became a major point of discourse on the impacts already playing out within the desert-scape. Although there are collaborations ongoing at the UN, however, the drying up of the oases renders climate alternation to be a new danger to migration through the Sahara. This is more so since some of the conveyors are not so conversant with the route. Indeed, there are conspicuous differences in how the route looks consequent upon sandstorms that change the shape of the desert. In turn, drivers that are not conversant with the route, and their passengers get lost. Whenever this happens, as Mahamadou (cited by Kingsley, 2015) remarked, such a group run out of fuel and water. And if there is no water, you would not survive for more than three days. With the oases gone, the impact of climate change becomes severely biting.\n\nThe rapid population growth across West Africa has increased manpower supply, a high percentage of whom are unemployed or under-utilised youths; hence several of the population are living and/or languishing in poverty. Really, as the United Nations Conference on Trade and Development (UNCTAD) (2021) explains, the “2021 poverty headcount rate is estimated to have increased by three percentage points.” Much more is the growing corruption, which enables the oligarchs within the region to possess much of the commonweal of their respective countries. Most of the socio-economic infrastructures within these states are yet to be fully optimized while several are in distasteful conditions or have fallen into outright disrepair. In turn, several citizens live like scum. Living in West Africa is, as a respondent remarked, living in hell. To live at all, it is better to die trying to exit the region for elsewhere, especially Europe, than to remain in my father’s land (personal communication, April 12, 2022). The growing population creates a pool of [un] skilled labour willing and desperate to emigrate from their countries to elsewhere. This is mostly for better living conditions, while some others are fleeing from political persecution and insecurity. The living condition within their respective home states and the restrictive visa processes pushed some of these people into desperation, hence the choice of travelling through the Sahara Desert. When faced with limitations like restrictive immigration laws within the perimeters of the North African states, the migrants mostly prefer to remain as irregular migrants than return to their countries. This signals that the new dangers in the Sahara and any other limitations to migrants’ ambitions pose dire consequences for the migrants, their families, home countries and West Africa. Subsequent subsections are thus used in explaining some of the challenges thrown up by limitations to migration through the desert.\n\nBy and large, several migrants explored official channels to be on the safe side of the law. The restrictive procedures incorporated into the visa processes of the different European countries however rendered the efforts of some migrants futile. Thus, several persons are routinely denied visas. Given this, several West African migrants explore the alternatives of tourist visas, hiding in vessels without the crew's consent, and false documents when available; as well as travelling through the Sahara Desert - Mediterranean Sea route. As Carling and Schewel (2018) explain: the restrictive immigration policies make it difficult for individuals to access legal migration channels. In turn, this has led several migrants into ‘involuntary immobility’; hence the exploration of alternative migration ‘modes’ such as irregular migration. Despite the herculean efforts, several of these migrants did not make it to their aspired destination. Some of those that made it across the Sahara Desert or through the Sea got arrested, detained, or massacred. For instance, between 23 and 37 persons, who were part of a group of 2,000 irregular sub-Saharan migrants lost their lives in their attempt to enter Melilla, Spain from Nador, in Morocco’s Oriental region (Kasraoui, 2022; Benazizi, 2022; Brito, 2022). There however were reprisals from the migrants. About 140 security officers on the Moroccan side and 60 National Police and Civil Guard officers on the Spanish side, as Brito (2022) claimed, were injured. A situation like this signals that the migrants already have developed suicidal ideation, borne of migrants’ disinterest to continue with prolonged uncertainty in their home states, traumatic experiences and an overwhelming sense of burdensomeness to oneself and others. As a result, the new challenges to migrating through the desert, and/or restrictions to ease of passage by North African governments and southern Europe merely pushed the migrants into psychological disturbance and distress; thus, causing some returnees to witness worsening mental health (Sundram and Lo, 2012).\n\nBecause of the desperation that consigned youths to uncertain and highly hazardous journeys through the desert for the confines of very unfriendly North African countries and the Mediterranean Sea, we believe that several migrants must have felt trapped within the confines of their countries. Describing situations synonymous to this, Procter et al. (2017, p. 4) hinted that many people must have experienced “advanced stages of feeling mentally trapped, figuratively boxed in, and especially hopeless and helpless. The picture is one of lethal hopelessness.” Since we are in a world that transcends borders, and the fact that the migrants are seeking, as rational economic persons, to explore the pull factors readily available across Europe for the improvement of their economic status, the risk involved in travelling through the Sahara Desert becomes worthwhile. With interests and intentions so heightened, therefore, the new dangers identified above posed severe psychological challenges to the migrants. Returning to their countries without accessing their el dorado or repatriated shortly after getting into Europe is likely to make the returnees get depressed or become psychologically distressed.\n\nMigration from West Africa to Europe was to be a golden opportunity for the migrants, through which they might accomplish the purpose(s) peculiar to them. This is more so because the “individuals’ ability to act depends’, as Van Hear et al. (2017, p. 3) remarked, ‘on the social milieu in which their action is performed.” The termination of such aspirations however sustained the status quo ante in the home countries of the migrants. This must have had severe consequences on the migrants, their families and communities at large. For these migrants, the necessity of resettling into a ‘living’ pattern they were so elated to depart from while embarking on a Sahara-ward journey and the ‘indignity’ of reverting to prior living conditions and milieu must have been disturbing for their mental health. Getting a job is pertinent to resettlement. The addition of such a marginal population to that of the unemployed citizens might be onerous for West African countries to handle. Such a pool of unemployed youths that have socioeconomic responsibilities to satisfy constitutes a complex recipe for crimes and crises across the region. This is more so as such a population exerts itself on the already stretched job market and socio-economic infrastructures available.\n\nThe rate of unemployment in West Africa is a ticking time bomb. This scenario is not farfetched since unemployment is one of the factors responsible for and deepens poverty within the zone; these evil twins were responsible for the efforts made by several persons towards emigrating from their home states. The following table presents the population of West African countries and the percentage of unemployment.\n\nPopulation of labour force (Population ages 15-64, total), unemployment, total (% of total labour force) (modelled ILO estimate). International Labour Organization (ILO), ILOSTAT database, June 2022. World Development Indicators\n\nFrom Table 1, it is realised that the population of the unemployed is already overwhelming for each country than for those of the returnees to be added, marginal though, to the existing number. For instance, the population of the unemployed in Nigeria in 2020 and 2021 respectively are 9,835,863 and 9,949,084; while Cote d’Ivoire had 4,203,061 and 4,345,451 for the same years. This situation triggered horizontal migration within ECOWAS for jobs in the informal market and thereby expanded the informal economies across the domain. Over time, therefore, the conventional regional growth poles of the region - Côte d’Ivoire and Nigeria, according to Karagueuzian and Verdier-Chouchane (2014), have remained important receiving countries of returning migrants from across the region. An extension to this however is that some of the migrants share ethnic ties with citizens of the host country, thus rendering the identification or categorisation of migrants difficult. In view of this, the host state often found it difficult to identify the population of its citizens that are jobless. An instance of this is a Nigerian Yoruba man repatriated from the US or Canada, who for shame decided to stay in Benin Republic. He definitely must mingle with the Yoruba community. In turn, his identity will become difficult to prove by the Beninoise government. Such a situation has precluded the state from providing adequate job opportunities as necessary. Given the situation of the states within the region, the situation has even served as a guise for the states’ leaders from performing their statutory responsibility of job creation.\n\nSource: IOM, 2017 (This figure has been reproduced with permission from International Organisation for Migration (IOM), 2017).\n\nIn the bid to cross into Europe, some youthful West African irregular migrants flung themselves against the steel fence surrounding the Spanish enclaves of Ceuta and Melilla in Morocco to gain passage (Jones, 2018). This happened because of their desperation for ‘the golden accesses’ to their European ‘el dorado’! This action, however, was necessitated, as a respondent (personal communication, March 15, 2022) claimed, by the push factors of bad governance, rife poverty and insecurity in their home countries, and pull forces like economic empowerment and freedom, secured future, peace of mind and thought of good life in their desired haven. As long as youths in West Africa regard their prospects for securing improved standard of living, work and achieving lives aspirations as slim in their home states, they will continuously seek to migrate to better climes through available means; the alternative to this is crime. A combination of factors: new and old dangers to crossing the Sahara Desert, the prohibitive border policies of North African countries as well as nudges from the host government and IOM encouraged some migrants, particularly irregular and transit ones, to return to their home states through its AVRR and Return of Qualified Nationals (RQN) that entail administrative, logistical or financial support, including reintegration assistance to migrants that voluntarily decide to return to their home country (IOM, 2022b).\n\nNonetheless, some returnees might take to crime as an alternative to the aborted ambition of residence in Europe. The reasons for this opinion are not farfetched. While the IOM programmes are lofty and indispensable to migration management (Lehmann, 2019), the resettlement of the returnees as the West African governments and their agencies promised is yet to be properly done. In Nigeria for instance, a returnee indicated that the government did nothing towards their reintegration except for unending “promises of assistance and the 5,000 Naira given as transportation fare to the returnees” (Okwuosa, 2019). This is in spite of over €120 million provided by the European Union’s ‘Emergency Trust Fund for Africa’ in partnership with the IOM to Nigeria for projects focused on addressing irregular migration like job creation and conflict prevention (European Commission, n.d.; Adesanya and Idowu, 2022b)\n\nThe above perception that irregular migrants might take to crime is borne out of the fact that while abroad, they are mostly employed in the informal economies of the host states. This is because their illegal status hindered them from having their data documented with the host governments. Hence, they have to contend with legal and practical challenges that are not unconnected, as Karagueuzian and Verdier-Chouchane (2014) argued, with access to shelter, health, education, fair income and justice in respective host states in Europe. In turn, their earnings in no way assist them to break away from low income. Thus, they hardly were able to remit many funds to their relatives in their home states for tangible projects that they could rely on at return.\n\nThe returnees, like other citizens, have bills to settle, but the opportunity for employment is very constricted; thereby rendering crime a choice. After all, Aristotle (cited in Anon, 2015) insisted that poverty is the parent of revolution and crime. On return to their respective countries, most of the returnees, if not all would, depending on how judicious they are with personal funds and financial assistances from their home governments and any other entity, have had to depend on relatives for daily needs. Such persons might have competed with several other youths for the few job opportunities – formal or informal – available in their countries with very slim chances of getting employed. The relatives they might have been depending on could be stretched beyond capacity, and might put up distasteful and irritable disposition; hence compelling the returnees to decide what to do with the ‘Self’, perhaps go into crime for survival or stay off crime and live abjectly.\n\nThe dearth of employment opportunities and increasing cost of basic needs, which were some of the factors responsible for the migrants’ journey through the Sahara Desert in the first place, constitute the vortex pulling the returnees into crime. For example, a respondent explained the case of a Ghanaian returnee that impersonated a certified fabricator/technician of a firm that deals in the construction and/or fixing of Cold Storage rooms. The returnee defrauded a businesswoman of N500,000:00K in Ado-Ekiti, Ekiti State, Nigeria; and subsequently evaded her calls until he was arrested in Atan-Ota, Ogun State, Nigeria, which is 351.7 kilometres away from Ekiti State (personal communication, August 10, 2022). In another instance, as a respondent in Lagos explained, a 27-year-old young man returned from illegal migration to Europe and became dependent on his family since he could not get employed. He subsequently attempted to sell some of his father’s properties at Alagbole, Akute in Lagos State so as to start a business that would take care of his bills. Afterwards, he engaged in the illicit trafficking and usage of psychotropic substances, especially cannabis. He was later arrested with a supposed automobile mechanic in a ‘stolen Toyota Camry’ car and transferred to Police headquarters. After his release from Police custody, he was observed to have become mentally challenged and relocated to Abeokuta (personal communication, August 2, 2022). Another account concerns several female returnees from Italy that became sex workers. The sex workers mostly attempted to steal from their new customers to meet up expectations (personal communication, June 15, 2022). Whereas some cases were the fallouts of unemployment, others were either because of dissatisfaction with the jobs gotten or that the earnings could not satisfy the desired standard of living; while some other cases are habitual.\n\nCrime, particularly the organised type, is complex! Really, state-embedded actors were identified as the foremost group that enabled organised crimes, they however recruit lieutenants that perpetrate numerous crimes within a locale per time. Some of these include cybercrimes, robbery and its variants, terrorism, human and psychotropic substance trafficking, sex work, piracy, gun running, and assassination and its variants. In 2019, for instance, on a scale of one to 10, while Africa as a whole had a crime average of 4.97, only East Africa, with a regional average of 5.51 surpassed West Africa’s 5.29 on the organised crime index. Equally, with 5.22, West Africa had the highest criminal market score (Enact, 2019). With a 7.70 score, Nigeria took the lead among the highest-scoring countries on the criminality index. Cote d’Ivoire ranked eighth with a score of 6.23, whilst Mali ranked ninth with a 6.20 score (Enact, 2019). None of the West African states is on the list of the lowest-scoring countries. In view of the myriad criminal markets, the presence of numerous criminals, and the ample reasons for people to commit crimes within West Africa, we are not in doubt, as the available accounts have shown, that some of the returnees to the region are involved in some of the crimes committed daily.\n\n\nConclusion\n\nThis study examined the new dangers confronting West African irregular migrants travelling through the Sahara to Europe. Similarly, the study analysed the fallouts that the new dangers to the migrants’ aspirations pose to the migrants and their home states. Without much ado, we were able to point out the drivers of irregular migration, which range from the push-pull factors of widespread anomies in West Africa; and the enviable societal structures available in Europe. The yawning societal and developmental gaps compelled most people to drift from the gloomy landscape of West Africa to the bright scenery in Europe. With the surge in the immigrant population, however, European states introduced stringent policies for getting visas. The stringent policies, as well as lack of credible reasons, documentation and funding however made several persons seek alternate irregular approaches to European destinations. The alternatives pose their own challenges, which sometimes jeopardised the lives of the irregular migrants and are well noted in extant literature. While there were doubts about the existence of new dangers to journeying through the desert-scape, we identified COVID-19, growing terrorism in the Sahel, and the increasing challenges of climatic situations as the new dangers to migrating through the Sahara Desert. Hence, we concluded that these new dangers are intensifying across the desert and getting severe for the lives of irregular migrants.", "appendix": "Data availability\n\nThe underlying data for this study are restricted for security reasons. The privacy rights of our respondents can be compromised. Data that are shareable have already been included in the manuscript. The restrictions on the data are part of the ethical approval for the study by Afe Babalola University- Bogoro Research Ethics Committee (ABUADREC). The extended data of the questionnaire and interview guide have been deposited in a publicly accessible repository. Readers who wish to access the restricted underlying data should contact segunpaul@yahoo.com or idowu.olusegun@abuad.edu.ng and provide a request letter stating interest and purpose for access, which would have to be sent to all authors and interviewee respondents as required for permission.\n\nFigshare: README.xlsx. https://doi.org/10.6084/m9.figshare.21602502.v3 (Adesanya and Idowu, 2022a).\n\nThe project contains the following underlying data:\n\n• README.xlsx (description of the data in the file).\n\n• README.doc ( Table 1 – population of labour force and percentage of unemployment across West Africa).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nFigshare: Research Main findings on Criss-Crossing, 2022.docx. https://doi.org/10.6084/m9.figshare.21728018.v2 (Adesanya and Idowu, 2022b).\n\nThe project contains the following underlying data:\n\n• Research Main findings on Criss-Crossing, 2022.docx. (Summary of the main findings 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\nFigshare: Consent Form. https://doi.org/10.6084/m9.figshare.21664019.v3 (Idowu and Adesanya, 2022c).\n\nThis project contains the following extended data:\n\n• Consent form for Criss Crossing the Sahara.docx. (Blank English consent form used in 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\nFigshare: Questionnaire for Criss-Crossing Shahara. https://doi.org/10.6084/m9.figshare.21921897.v2 (Adesanya and Idowu, 2023a).\n\nThis project contains the following extended data:\n\n• Questionnaire Criss-crossing the Sahara Questionnaire 2022.docx. (Blank English questionnaire used in 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\nFigshare: Criss Crossing Structured Interview Guide. https://doi.org/10.6084/m9.figshare.21922428.v1 (Adesanya and Idowu, 2023b).\n\nThis project contains the following extended data:\n\n• Criss-cross Interview Guide.docx. (Blank English interview used in 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\nAdeniyi O: From frying pan to fire: How African immigrants risk everything in their futile search for a better life in Europe. Bookcraft; 2019.\n\nAdepoju A: Review of research data on trafficking in sub-Saharan Africa. Int. Migr. 2005a; 43(1 and 2): 75–98. Publisher Full Text\n\nAdepoju A: Perspectives on migration within and from sub-Saharan Africa. 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Reference Source\n\nWorld Atlas: Sahara desert. World Atlas; n.d.Reference Source\n\nZender CS, Miller RL, Tegen I: Quantifying mineral dust mass budgets: Terminology, constraints, and current estimates. The American Geophysical Union. 2004; 85(48): 509–512. Publisher Full Text\n\nZiegler M, Tuenter E, Lourens LJ: The precession phase of the boreal summer monsoon as viewed from the eastern Mediterranean (ODP Site 968). Quat. Sci. Rev. 2010; 29: 1481–1490. Publisher Full Text\n\nAdesanya O, Idowu O: Research Main findings on Criss-Crossing, 2022.docx. Dataset. figshare. 2022b. Publisher Full Text\n\nAdesanya OA, Idowu O: Questionnaire for Criss-Crossing Shahara. Dataset. figshare. 2023a. Publisher Full Text\n\nAdesanya O, Idowu O: Consent Form. Dataset. figshare. 2022c. Publisher Full Text\n\nAdesanya O, Idowu O: README.xlsx. Dataset. figshare. 2022a. Publisher Full Text\n\nAdesanya O, Idowu O: Criss Crossing Structured Interview Guide. Dataset. figshare. 2023b. Publisher Full Text" }	[ { "id": "183296", "date": "17 Jul 2023", "name": "Marco Mogiani", "expertise": [ "Reviewer Expertise Migration", "Borders", "Citizenship" ], "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 aims at investigating supposedly new dangers that West African migrants face en route to Europe using a mixed method approach. Despite the good intentions, the article has serious theoretical, methodological, and analytical flaws: it is very general and descriptive; it does not advance or challenge theory, and it barely employs the empirical findings.\nFirst, the article has no clear focus. Most of the initial discussion is about the drivers of migration from West Africa, which are merely described as push factors and are not analysed in depth nor with a critical angle. Sometimes, the authors also mention the pull factors that attract migrants to Europe, but similarly there is no analytical engagement with them. The new dangers of migration, which are supposed to be the main topic of the article, are briefly (and poorly) mentioned half way through the article, with no reference to the empirical data. Then, the discussion goes on talking about the supposed consequences of such dangers to the aspirations of returnees (and not of migrants in transit, as one would have thought). However, the relation between the dangers and returnees’ lives cannot be sustained, compelling the authors to make bold claims.\nSecond, the theory. The authors use push-pull theory, despite acknowledging that it “failed to account for changing motivations, altered circumstances or modified decisions en route”. Using relatively old and out-of-fashion sources, the authors unproblematically claim that migrants are rational actors that are simply pushed away from their countries due to general socio-economic circumstances, although a whole literature on migration intentions and aspirations is ignored. It is also claimed that \"push-pull theory would assist in deepening knowledge of the irregularity of journeys across the sandy-sea route”, although no further explanation on the relation between push-pull factors and illegality is provided.\nThird, the methodology. The research employs a mixed method of qualitative and quantitative approach, with purposive and snowball sampling techniques to reach the target group. However, it is not clear who the target group is: throughout the text, the authors make various references to irregular migrants and returnees, without a theoretical clarification nor a practical explanation of who they actually interviewed. The open-ended questionnaire employed (presumably) for the quantitative part contains very general and broad questions, which might allow ample responses. Besides, there is no explanation on how the data from the survey and the interviews were analysed.\nFourth, the overall analysis of data. Regarding the findings, the data from the interviews (which could enrich the article) are used only sporadically, and without connection to the theoretical framework or support to the argument. Secondary data are sometimes brought into the discussion, but these do not enrich the debate, or are of little significance (such as the absolute number of unemployed people in two countries). This is probably the reason why throughout the text the authors make bald claims that cannot be supported by their data nor by the literature. Just to make some example: in discussing the findings, the authors claim that “it was realised that irregular migrants possess a huge dose of endurance and are very resilient”, but this does not emerge from the data, nor is it clarified further. Similarly, when discussing the consequences of the dangers, the authors claim that these new challenges push migrants into worsening mental health or into crime, but this strong relations of causation are not proved.\nLast but not least, the prose is often unclear, redundant, or exaggerated. Sentences are sometimes colloquial (“The returnees, like other citizens, have bills to settle”) and other times correlated with obsolete or archaic words, making the reading quite difficult.\nFor these reasons, I believe that the article is theoretically, methodologically, and analytically weak, and needs to be substantially rewritten.\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?\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": [] }, { "id": "193755", "date": "06 Sep 2023", "name": "Adeyemi Saheed Badewa", "expertise": [ "Reviewer Expertise International migration", "public policy", "Area Studies: (Africa and the Middle East)", "International relations", "international development", "history", "and transformative justice" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 endeavors to interrogate the “new dangers” encountered by West African migrants attempting to reach Europe through the Sahara Desert and the implications such a perilous adventure portends for states within the transit routes. Employing a mixed method analysis of descriptive data, its analysis is conceived within the framework of push-pull migration theory. Hence, the authors’ intention re-echoes the growing voices elicited concerning the threats of irregular migration, and affected individuals’ vulnerability to inhuman conditions in transit, bearing in mind migrants’ decision-making amidst uncertainties. Below are my comments after a thorough review.\nFirst, the article's view of the economic situation in the migrant's source as the push factor, and the structures of improved living standards in the destination as the pull factors illustrate the narrow underpinning of its arguments. This is against the backdrop of the failure of push-pull theory to account for changing motivations, and altered circumstances of migrants' modified decisions to travel abroad. Using the push-pull perspective as a prism to generalize migrants' rationality appears narrow, incipient, and inadequate given the changing patterns of the scope of migration practices and decision-making processes. These and the layers of actors involved in facilitating migrants' mobilities across the source, transit and destination, as the array of policies and mechanisms that enhance irregular migration, including the outsourcing of migration control, militarization of migrant flow, and geopolitics have provided diverse perspectives inherent in human mobility in (West) Africa and all migrants transitions which include non-Africans across the Sahara-Europe route. While I agree with the authors about the myriad of factors responsible for West African migration, I do not agree with that as factors for the so-called intentions. These are quite different things, and the authors’ reference to \"the wider processes of transformation\" as a factor in migrant's intentions appears redundant and overstated. Hence, the push-pull theory provides a limited understanding of irregular migration across the Sahara-(Mediterranean) sea route. I would conclude that this is mostly a question of migrant's decision-making, experiences of past successful migrants, social capital, and the transit route as an enabler of irregular migration, however, the push-pull factors provide a tangential angle to the story.\nSecondly, the authors' choice of Mixed method research in data collection and analysis was appropriate including its descriptive presentation, however, descriptive is not a research design. The study's use of Primary data collected from key informants’ interviews with migrant returnees and officials of key organizations is relevant. The target was clear, and the snowball sampling method was justified for this kind of research. Nevertheless, explanations on how the survey and interview data were analyzed were not obvious. While the study is largely qualitative, which is in order, the research hypotheses that both the qualitative and quantitative data speak to could be well distinguished, and how they both reinforce each other could also be properly established to enhance the reliability and validity of the data.\nThirdly, on the study's findings, results, and discussion, the starting point could have been the data, further reinforced with arguments from extant studies or literature, but this was not the case, as the analysis appears sporadic and a rehash of others' views, leading to the conclusions the authors try to draw its audience attention to. If the focus of the study is the experience of migrants in transit, the focus should be the enablers of irregular migration in transit and the changing patterns of geopolitical landscape in the source, transit, and destination regions vis-a-vis the policies/practices and their after-effects. The authors' realization that \"irregular migrants possess a huge dose of endurance and are very resilient\" is valid but did not emanate from the data. I am certain, the phrase is a truism that underlies the question of choice and rationality in migration decisions, and the reality of inhuman circumstances or dangers experienced by migrants amid uncertainties. These could have been elaborated from the respondents/interviewed accounts and reinforced with scholarly arguments. Meanwhile, several generalizations and overstated/verbose expressions could be concisely shrunk to make a coherent analysis. If the authors devoid of using colloquial/informal expressions, summarize convincing arguments from current literature to buttress the stories from the data, in a manner that integrates the theoretical assumptions, its contributions to scholarship would be more. Hence the study's unique contribution to migration scholarship would not have been questioned. Furthermore, the study's analysis of the new dangers to migrants’ aspirations and the effects on them and their home states seems novel and provides avenues for future research on migration challenges from the South to the North. Having identified the surge in the immigrant population, stringent visa policies, and the threats of inhuman practices en route, suggestions for policy improvements and perhaps future research could have better enhanced the study's contribution.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "193756", "date": "06 Sep 2023", "name": "Doudou Dieye Gueye", "expertise": [ "Reviewer Expertise Migration", "Mobility", "Migration industry" ], "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 submitted for review examines the new hazards that stand in the way of irregular land and sea migration by West Africans crossing the Sahara desert to Europe. The authors start from the observation that the pandemic epidemic linked to COVID-19 is not only a danger for migrants crossing the Sahara, but also a factor exacerbating existing dangers.\nUsing a mixed qualitative/quantitative methodological approach, the authors use a descriptive conception of the new dangers of migratory routes, interviewing 100 people, 85 by questionnaire and another 15 by interview. This wealth of data collected is not reflected in the article, as the author could at least have extracted a few interview extracts to help understand these new dangers in comparison with the dangers already known (widely described in the literature; see Taye, 20221). In the same way, this could have helped to understand how COVID-19 contributes to exacerbating existing dangers and, above all, how the epidemic will make migrants crossing the Sahara even more vulnerable. In fact, the failure to list new dangers and the exacerbation of existing dangers are major shortcomings of the article. In addition, the main and central idea (new dangers of migration) is relegated far down the text, which may cause the reader to lose interest.\nThe methodology lacks clarity. All we know is that an adult population over the age of 18 was interviewed. Are they return migrants? Repatriated migrants? Potential migrants? This leads me to point out the lack of theoretical clarification of certain notions and concepts used by the author, such as potential migrants, repatriated migrants, irregular migrants, etc. Similarly, was the questionnaire open-ended or closed? Finally, how were the survey and interview data analyzed?\nDespite the captivating title, which focuses on the \"new dangers\" of irregular West African migration, the article teaches us nothing new, being evasive and overly general, and above all failing to focus on its original objective. Overall, the article lacks clarity and a clear objective. There is also a lack of analysis and in-depth discussion of the determinants of migration in West Africa, and similarly a lack of analysis of the attractive factors of emigration to Europe. From an analytical point of view, everything seems to indicate that the interviews are poorly exploited and, above all, do not feature in the author's argumentation.\nOverall, the article cannot be indexed in its current state, and I suggest a thorough and rigorous rewrite.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-377
https://f1000research.com/articles/12-375/v1	06 Apr 23	{ "type": "Research Article", "title": "Doing things: reconstructing hominin cognitive evolution from the archeological record", "authors": [ "Fabian Seitz" ], "abstract": "Following Pain’s (2021) critical assessment of the prospects of minimal capacity inferences within cognitive archeology based on ‘classical’ cognitive science, I elaborate on the chances of these inferences within so-called embodied, embedded, extended, and enacted (4E) frameworks. Cognitive archeologists infer the cognitive abilities of past hominins from the remains found in the archeological record. Here they face the problem of choosing a theory from the cognitive sciences. Results vary considerably, depending on one’s cognitive theory, so choice matters. Where classical views conceive cognition as mainly involving representations and computing, more recent 4E approaches focus on interactions between environment, body, and brain: hence the same trace, like a stone tool, might require capacities like a mental ‘blueprint’ according to the former, but only environmentally guided perception according to the latter. Given this crucial choice of theory, what are the prospects of 4E then? I present a model of cognitive hominin evolution based on 4E and niche construction theory. Based on this model, I argue that we should be guardedly optimistic: contrary to first impressions, minimal capacity inferences work well within the 4E framework, and adopting 4E might give us a methodological advantage, too.", "keywords": [ "cognitive evolution", "4E cognition", "niche construction", "cognitive archaeology", "minimum capacity inferences" ], "content": "Introduction\n\nCognitive archeology is the brave attempt to infer the cognitive abilities of past people from their material remains, thereby exploring how the cognitive evolution of hominins - and eventually humans - has unfolded. This is no easy task. To explain the evolution of human cognition requires understanding which forces shaped and re-shaped over and again the cognitive underpinnings of hominin lifeways from several million to only a few thousand years ago. To yield such explanations, cognitive archeologists have a varied toolkit of inferences (cf. Abramiuk 2012; Currie 2018), but regarding how our ancestors thought, two kinds of inferences stand out: minimum capacity and cognitive transition inferences (cf. Pain 2021; Currie and Killin 2019; Killin and Pain 2021). The former is for inferring cognitive capacities from material remains (often stone tools): what cognitive ability enabled the behavior required to produce the material trace we found in the record? The latter is to infer a change in cognitive abilities from a transition within the material record.\n\nYet the material remains aren’t sufficient to license an inference from stone to mind. Cognitive archeologists must interpret them considering a cognitive theory or model. Here, choice matters, for the results vary enormously with one theory or the other. Take the two ‘grand’ families of theories currently discussed in the cognitive sciences: the classical or ‘orthodox’ (Wheeler 2005; cf. Malafouris 2020) information-processing approach views cognition as mainly involving representations and computing; more recent embodied, embedded, extended, and enacted (4E) approaches focus on interactions between environment, body, and brain to yield cognitive abilities instead. So, the same trace in the record, say a specific stone tool, might require capacities like a mental ‘blueprint’ or sophisticated long-term memory according to classical theories, but only environmentally guided perception and simple learning according to ideas from the 4E family.\n\nCognitive archeologists have no choice but to decide. Only by using a theory or model, can they make inferences from artifact to cognitive capacity. And the choice of theory or model comes first. It is useless to switch between those ‘grand’ families of theories case by case or run through a trace from A to Z for which theory would yield which result. Particular artifacts might fare better with one approach, but these theories are about general aspects of the human mind. So we can’t switch from 4E to ‘orthodox’ views from one artifact to another. The human mind is either one or the other (or something altogether different, but we all have to await how the history of cognitive science will unfold). So, hominin cognitive evolution is a narrative about the unfolding of a mind as described by either 4E or classical theories. (It’s a different story what cognitive archeologists could contribute to the current understanding of human cognition. For example, they could show that, by and large, we yield better explanations of hominin cognitive evolution if we prefer 4E over ‘orthodox’ views (or vice versa). Such an endeavor could be - quite literally - an inference to the best explanation from the (best explanation of the) material record to which of the ‘grand’ theories on extant human minds gains more plausibility. But this is not the theme of this paper.)\n\nThere is no way to elude: Cognitive archeologists must make their best guess about theory first and then get to work. So, if we want to yield evolutionary trajectories of hominin cognitive abilities, established (mainly) by the above inferences: Which theory should one choose then?\n\nThis paper shall not decide this question. Instead, I want to elaborate on how inferences from the archeological record yield evolutionary trajectories of hominin cognition. Section 2 introduces this ‘inferential engine’: I present the early cognitive archeology approach, which by and large used ‘orthodox’ cognitive science, along with its problems. It will be a reference for later sections. In Sections 3 to 5, I discuss this ‘inferential engine’ within the context of 4E. Section 3 introduces the 4E cognitive framework; Section 4 provides an example from the emergence of numeracy in the neolithic as a ‘proof of concept’ for this framework. It shows the applicability of the 4E’s but raises the question of how to infer ‘thinking from things’ within this framework. Section 5 thus will revisit these inferences. I argue that we should be guardedly optimistic: not only do these inferences work well within the new framework, but adopting 4E might give us a methodological advantage, too.\n\nI start by characterizing the explanation we aim for.\n\n\n1. Lineage explanations\n\nCognition is a complex trait. Like any such feature, it unlikely evolved in just one step. Instead, one must reveal how ‘baseline’ organisms (without the trait in question) could turn into organisms with the trait. Any evolutionary trajectory of such lengths consists of many steps, and the selection of all these steps couldn’t happen in just a single event, either. There must have been an ongoing interplay between events, which selected for adaptions, and these adaptions then being starting points for the next step toward the trait of interest.\n\nTo account for such trajectories, we need so-called lineage explanations (Calcott 2008; Sterelny 2012b). Such explanations must make plausible how every single step along the line could have emerged. Each step must start from a platform: a scenario that sets the stage with a certain selective pressure and already includes a possible precursor to an adaption to this pressure. The step itself consists of only a small transformation of this precursor then: slightly so modified as to function as adaptive variation (This requires, of course, that there indeed is selective pressure for such a transformation; and that this adaptive response was within reach for the organism). Once selected, the new stage will afterward have to perform as a platform for the next transformation.\n\nThe process becomes iterative, so a modified platform becomes a new stage, which functions as a platform for the following modification (and so on). Each step must be the transformed version of the previous setting, and each stage must be selected in its scenario. So to explain the emergence of a complex trait, we need an idea of a baseline and information about a series of small steps, where each step, in turn, consist of a platform, an event selecting for new variation, and how a transformation of the platform could function as such a required variation (i.e., as an adaption to specific selective pressures). Only with these ingredients might we yield an evolutionary trajectory - from one state (without the trait) to a series of other states (with precursors of the trait in question) down until a state with the trait emerges.\n\nTake the manifold evolution towards complex eyes as an example (I follow Nilsson’s (2013, 2009) exposition here). Eyeless animals did not turn into animals with complex eyes overnight. In a nutshell, it started with cells only “monitoring the ambient light intensity” (Nilsson 2013, 6; i.e., non-directional photoreception), which evolved into pigment cells capable of directional photoreception, followed by eyes capable of low-resolution vision. Finally, descending from this stage, a compound lensed eye capable of high-resolution vision for “detection, pursuit, and communication with other animals” (Nilsson 2013, 6) evolved. Each of the stages enumerated is a step toward complex eyes. Furthermore, each of these steps had to be selected for to be a platform for the next step.\n\nLikewise with hominin cognition. To be sure, the evolution of the vertebrate eye covers a period of 600 million years (Lamb, Collin, and Pugh 2007). The specific characteristics of the modern human mind evolved much more quickly. Yet granted the main explanandum of cognitive archeology are “single individual components of cognition” (Wynn 2019, 501), say memory, spatial cognition, or language, and that their complete unfolding “occurred at several points in hominin evolution” (2019, 6): Such traits unlikely evolve without precursors over such long-time spans. Hence intermediate stages of precursors must meet likely occurred selection pressures to bring forth the (preliminary) final stage of the trait in question. Again, there must be a series of platforms transforming into new adaptions due to new selective pressures once and again until the trait in question could arise. In short, we need lineage explanations for hominin cognitive traits, too.\n\nHow does cognitive archeology achieve such an objective? In the remainder of this paper, I elaborate on how inferences made from the archeological record shall yield such lineage explanations. To start with, I present the early cognitive archeology approach.\n\n\n2. Early cognitive archeology and its inferences\n\nBrains alone enable cognition, according to classical cognitive science. If so, brains are the sole basis of cognitive capacities. These capacities, in turn, cause behavior, and behavior causes artifacts. If so, one can read off from an artifact first the behavior required (think Chaîne opératoire). One can then ask in a second step: what cognitive capacity is needed to produce this behavior?\n\nThis makes artifacts in the archeological record ‘markers’ for cognitive change within the hominin lineage. Artifacts function as indirect evidence of the occurrence of a particular mental capacity of its producer in the past. This first use of the archeological record is complemented by a second one: evidence about hominin brain evolution, as they manifest from the interpretation of fossilized skulls. Evidence about increasing brain size seems to correspond to the emergence of more complex artifacts - at least in a general pattern and over long timescales. Another example not based directly on artifacts is the well-known research by Robin Dunbar, which relates estimated growth in group size with increasing relative neocortical volume within the hominin lineage (Dunbar 1998). Bigger groups increase social complexity, a cognitive challenge one must cope with. Hence, if only brains realize cognition, increasing cognitive demand should manifest itself with changing brain size or structure (or an increase in neocortical volume, as in this case).\n\nSo, if we see a change in material culture and a corresponding change in brain size or structure, everything seems to match. Artifacts come into existence through the behaviors of their producers, and these behaviors become possible due to certain cognitive capacities they entertain - eventually because their brains realize these capacities. Since skulls and stone artifacts both fossilize, we have the in-principle possibility to track change in the archeological record and hence the evolution of cognition. Two kinds of inferences are at play here.\n\nThe first inferential practices here are so-called minimum capacity inferences. This kind of inference is best characterized as inference to the best explanation (cf. Pain 2021, 248): the assumed cognitive capacity functions as the best explanation for how an agent could bring forth the behavior required to produce the desired artifact as an outcome (Although it is rather common to frame these inferences as a deductive modus ponens-inference, cf. Abramiuk 2012, 143ff.). One must be parsimonious here: no more cognitive capacities than absolutely required shall be assumed. And eventually, which capacities are assumed to be necessary hinges on one’s cognitive theory. Two different theories might explain differently how the same behavior is brought forth. For example, to execute the behavior required to yield something sophisticated as an Acheulean hand axe might invoke something as entertaining ‘mental blueprints’ according to one theory, but only perception and learning of associations to another (cf. Killin and Pain 2021, An excellent illustration of this is the case studies given in Killin and Pain 2022; as well as the discussion on the changing interpretation of Oldowan stone tools by Wynn 2023). So, the right choice of cognitive model matters (pace the overall tenet of Killin and Pain 2021), yet I will discuss the details later on (see Sections 4 and 5).\n\nOn an abstract level, such an inference runs as follows:\n\nAnalysis: Artifact A requires behavior B to be produced.\n\nInference to the best explanation: Assumed a cognitive capacity C, this would be the best explanation for how an agent could bring forth B (and so A).\n\nHence: The agent (as the producer of A) likely was capable of C.\n\nThis kind of inference is a building block for tracking the chronology of cognitive change.\n\nMinimum capacity inferences allow us to identify the single stages within the trajectory, where the transition between these stages into one another is argued for by cognitive transition inferences, which are compounds of single minimum capacity inferences. Pain sketches them as follows:\n\n\n\n(1) Run a minimal-capacity inference on a technology, Y.\n\n(2) Run a minimal-capacity inference on a technology X, where the appearance of X predates the appearance of Y in the record.\n\n(3) Identify a capacity, C, that is the best explanation for Y but not X.\n\n(4) Infer that:\n\na. C was absent during the period X was produced;\n\nand\n\nb. Y signals the emergence of C. (Pain 2021, 249f.)\n\nNote that (3) is already included in (1), something imprecisely depicted by Pain. A more sophisticated account would also add something like this for (2):\n\n(3’) Identify a capacity, C-1, that is the best explanation for X (but not Y);\n\nthereby changing the conclusion into:\n\n(4) (b’) The transition from X to Y signals the transition from C-1 to C.\n\nIn any event, the application is simple: A minimum capacity inference gives us an idea about the cognitive capacity required to produce a particular artifact. So two of these inferences on either side of a transition in the archeological record from one artifact to another should signal a change in underlying cognitive capacity.\n\nFrederik Coolidge and Thomas Wynn are an example of this strategy (Coolidge and Wynn 2018; also discussed in detail by Pain 2021, 250ff.; cf. also Sterelny 2017). They argue that one major leap in the cognitive development of Sapiens occurred due to enhanced working memory, caused by “an additive genetic mutation or an epigenetic event that affected the neural organization of the brain” (Coolidge and Wynn 2018, 233; also cited by Pain 2021). Coolidge and Wynn posit enhanced working memory as the basis for abilities like abstract reasoning, contingency planning, or symbolic thinking. After identifying such a sophisticated cognitive capacity as an underlying cause for modern human cognition, the question arises when this capacity emerged during our evolutionary history. Despite arguing for this transition in a more complex manner than I illustrate here, at one point within their argumentation, Coolidge and Wynn turn implicitly to a cognitive transition inference.\n\nConsider the well-known figurine from Hohlenstein-Stadel, South Germany, ca. 40 kya. Since it depicts a lion-headed human, Coolidge and Wynn infer that the maker of this artifact must have been, at minimum, capable of abstract reasoning. The concepts of ‘lion’ and ‘human’ had to be combined into one abstract concept to yield a ‘mental blueprint’ for making this artifact. (As I only want to illustrate the ‘inferential engine’ at work here, I won’t discuss this step in their overall argument.) Since abstract reasoning, according to their line of thought above, requires enhanced working memory, the Hohlenstein-Stadel figurine indicates the occurrence of this capacity. The latter leads to whether another artifact, which predates this figurine, also requires something like abstract reasoning. Coolidge and Wynn discuss the also well-known Blombos beads from South Africa (ca. 77 kya) as a possible candidate but infer that for these beads learning simple associations suffices (where the latter does not need any enhanced working memory)(cf. 2018, 262f.). Their cognitive transition inference in a sketch:\n\n(1) Minimum capacity inference: (An artifact like) The Hohlenstein-Stadel figurine requires enhanced working memory.\n\n(2) Minimum capacity inference: (An artifact like) The Blombos beads requires learning of simple associations (only); where the appearance of the beads predates the appearance of the figurine in the record.\n\n…\n\nHence:\n\n…\n\n(4)(b’)The transition from (an artifact like) Blombos beads to (an artifact like) Hohlenstein-Stadel figurine signals the transition from learning of simple associations (no enhanced working memory needed) to enhanced working memory.\n\nOf course, the groups associated with the beads and the figurine weren’t directly related, so to mark the abstract nature of the inference above, I added ‘an artifact like’ in brackets to clarify this issue; yet this does not change the implied chronology. Coolidge & Wynn eventually locate the emergence of enhanced working memory somewhere between 70-40 kya. To conclude, cognitive transition inferences allow one to sketch a chronology of the arrival of cognitive capacities, in principle allowing one to aim for lineage explanations of the capacity in question.\n\n(Note that rather often, there is no information about the selective regime(s) contained, thus no argument about what caused the adaptions. Yet this is needed to explain a trait’s complete lineage. The (imagined) story in early cognitive archeology, therefore, rather often seems to be that as soon as new cognitive abilities arose by genetic variation, hominins explored and exploited new opportunities with them - including the new things they were allowed to do. This way, positive selection for new possibilities drove hominin evolution into new directions.)\n\nFrom a theoretical perspective, all this is good practice. The problem arises with the details. Three different kinds of issues are to be identified here. First, the fossil record of brain sizes and structures doesn’t fit the fossil record of material culture as straightforward as it once appeared (for a summary of the discussion see Sterelny 2021, 8557ff.; cf. also Kuhn 2020). The first appearance of stone tools is now associated with hominins 3.3 mya, long before brain size increased. Likewise, despite some coincidence of brain evolution in Erectines and Acheulian hand axes 1.7 mya, innovations such as the Levallois technique is now dated to 500 kya, with no corresponding change of brain size in the record. In general, the record does not reveal a unidirectional shift from simple to complex tools, along with increasing encephalization in the hominin lineage, but rather a fragile appearing and disappearing of tool technology with seemingly no direct link to brain evolution.\n\nSecond, a related problem is that we can track changes in a material culture where it is relatively safe to assume that no significant brain size changes occurred. Two causes for this are discussed, the first one being demographic. Group size might affect change in material culture with no change in the underlying cognitive capacities of its members. One possibility would be inhibiting innovation due to small group sizes. For example, Premo and Kuhn (2010) have argued that Middle Paleolithic hominins might have lacked the ability to innovate due to high extinction rates within their groups, causing “the demographic fragility of the small social groups in which they lived” (Premo and Kuhn 2010, 8; also discussed by Pain 2021). High extinction rates lead to loss of information, which hinders the transfer of required techniques - in the end, no resources to innovate remain. In the same vein, amongst others, Sterelny and Hiscock (2014) argue that bigger groups would accelerate change in material culture due to a higher ability to store and transmit information. An example would be an onset of diversity in the material record in Middle Stone Age Africa 100 kya and the Upper Paleolithic (once so-called) ‘cultural revolution’ in Europe 40 kya, both associated with an increase in (local) population size, long after the speciation of Homo Sapiens and their brains 300.000 kya. If true, demographic factors might explain a change in material culture without an underlying change in brain size or structure.\n\nThe second cause could be environmental in nature. Different environments might ask for different toolkits to be deployed. Pain (2021) discusses the work of Torrence (2001) here: For example, variation in food availability should select for more various toolkits to secure these resources (for further discussion, see also Sterelny 2017, 238f.). So environmental factors could play a role in the sophistication of tool kits, making them a possibility to be considered. Eventually, the problems sketched so far undermine the direct inference from (change in) material record to (change in) cognition, at least given the assumption that brain size or structure is the sole unidirectional cause of cognitive capacity.\n\nSo, finally, the third problem might be a change in plausible accounts of cognition itself. As mentioned above, minimum capacity inferences depend on one’s choice of cognitive framework; at first glance, they work well only with ‘orthodox’ cognitive science, assuming that there is a unidirectional causal link from brain structure to mental capacities, and from there to the artifacts uncovered in the archeological record. Yet within the last 30 years, the cognitive sciences have witnessed an alternative framework of the so-called 4E’s (embodied, enactive, extended, and embedded). In line with at least some of these E’s, new research also indicates high neuronal plasticity of human brains during both onto- and phylogeny (Malafouris 2013; Anderson 2014; Overmann 2017). If true, then brains (alone) don’t equal cognition, and tools (alone) no longer indicate cognitive capacities. Then, how can we infer cognitive abilities and track change within these abilities along evolutionary time scales? In the next section, I elaborate on this alternative framework before discussing its application in the remainder of this paper.\n\n\n3. A new cognitive framework\n\nCognition isn’t a complex feature only; it’s also always the feature of someone. Turning this insight into a new framework of cognition has consequences. Such an agent has a body, and the physical make-up of this body matters to their abilities, including cognitive ones; hence cognition is embodied. Often, these abilities become possible only thanks to things external to one’s brain; thus, cognition is extended. Likewise, interaction with one’s environment, in general, plays an important role here; hence cognition is embedded. Even more radical, abilities aren’t just stored as mental representations within one’s brain, as something (previously) thought about and then executed: they only come into being through active engagement with the environment; hence cognition is enacted. And finally, if the new cognitive sciences are on the right track, cognition is a skill: something to be learned in a socially scaffolded environment. Cognition isn’t inherited by genes alone but by developmental resources culturally transmitted. Given that this framework is correct, it will change how we tackle lineage explanations. Let me introduce it in some detail.\n\nThe body shapes cognition in at least two ways. First, one’s physical make-up is not only controlled by cognition. Rather the other way round, bodily parts do constitute cognitive processes as well. The typical example is walking here: balancing during locomotion requires constant feedback from physiological structures in direct contact with the ground (plus the inner ear), not cognitive control over physiological structures via amodal representations of how to walk. So, cognition emerges by feedback loops via one’s body with the environment, not by top-down control of the extra-cranial environment (including one’s body) - an idea to be explored further below. Second, a body constitutes the way it can use opportunities to interact with its environment; the latter changes how cognition unfolds. For example, as Ben Jeffares (2010, 2013) argues, transforming a body from a predominantly obligate bipedal (think Ardipithecus) to a habitual bipedal like the Australopithecines also changes their cognitive biases. Since there is not much evidence about brain structure and material culture, Australopithecines get much neglected in accounts of cognitive evolution. Most often, they work as a ’chimp-like’ baseline only. However, if Jeffares is right, bipedalism allowed them to interact more with the world. Their activities become more autonomous, and range size increases while constraints on their behavior cease. For example, they can carry things over more considerable distances now or exploit materials differently through a new way of hand-eye coordination. All “(t) hese behaviors set up the potential for new cognitive skills” (Jeffares 2013, 12f.). So due to a change in their bodily form, Australopithecines were open to new cognitive forms: a changing physical make-up allows them to exploit new opportunities, where such a changing set-up realizes new cognitive make-ups.\n\nThis also changes how the external environment can be used for cognitive purposes in a small but significant subset of these activities. Suppose one makes marks on a sheet of paper to visualize the number of paragraphs still to write, then scratches out one after another. It is an example of using artifacts to accomplish a cognitive task: “physical objects made by humans for the purpose of aiding, enhancing or improving cognition” (Hutchins 1995, 126). Here two aspects stand out. First, some of the artifacts humans use aid for cognition in the strong sense that without these aides, the cognitive process would be impossible. Hence the artifact is part of this ability. (Although, for our purposes here, we shouldn’t descend into metaphysical debates about minds.) Second, such cognitive artifacts do not always foster or ‘externalize’ a preexisting ‘internal’ cognitive ability — sometimes, the task at hand gets solved by changing the required cognitive abilities to cope with them. Hutchins (1995) discusses Cole and Griffin (1980), which pointed out that ‘remembering’ something by writing it down means that one transforms the task into a “different set of functional skills” (ibid.). In this case, writing it down involves visual skills once used for face recognition (reading) rather than memorizing complex themes, for example. Further examples of cognitive artifacts are calendars, writing systems, and mnemonic devices (items to help to remember things, like a string on your finger). Some have even argued that cultural practices like proverbs or rules of thumb are cognitive artifacts, too (Norman 1993). So we’re surrounded by all kinds of cognitive artifacts which form part of our mental life.\n\nGenerally, the environment one is embedded in facilitates one’s cognitive possibilities. For hominins, these environments have already been a long time both social and self-created. I discuss both of them in turn.\n\nSelf-created environments play a massive part in hominin cognitive evolution. Here Niche Construction Theory enters. In a nutshell: Organisms modify their environments, thereby changing the selective pressures on themselves. So organisms, to some extent, created their selective regime (which they had to adapt to) themselves. A prime example is a selection for lactose tolerance in those Homo Sapiens groups (and only those) which engaged in dairying (cf. Boivin 2008, 200f.). In niche construction theory, the term ‘niche’ refers to just this: so a niche is always selective.\n\nFurthermore, niche construction can result in evolutionary feedback: “organisms drive environmental change and organism-modified environments subsequently select organisms” (Laland and Brown 2006, 96). The latter again alter their environments anew for their descendants. Take Osvath and Gärdenfors (2004), who deploy niche construction to analyze the cognitive evolution of early hominins. According to them, a changing hominin niche 2.5 mya caused the coevolution of transport and planning. Transportation of raw materials and tools expanded at that time. Hence behavior sequences stretched more and more. Finally, according to Osvath and Gärdenfors, anticipatory planning became mandatory. For hominins planned these behavior sequences. Yet to handle these ever longer transport, they had to plan at a certain level in anticipatory mode. So here we have a case where a self-created niche of stone tool production and transportation imposed a cognitive challenge to which subsequent hominins had to adapt.\n\nYet it isn’t only selection pressures that get culturally transmitted via niche construction. One final idea in 4E cognition is that of cognition being enacted. Cognition doesn’t start with the passive reception of information, then acting on it to inform decisions, and finally becoming behavior in the world. Rather, cognition is already doing things: it is through ongoing interactions with one’s environment that cognition gets realized. This way, cognitive abilities come into being by doing. Interactions with one’s environment (with or without artifacts) become “transformational and not merely informational” (Paolo, Rohde, and Jaegher 2010, 39; cited by Overmann 2019, 432). A famous example (to be elaborated in the next section) is the idea that manipulating mathematical symbols is already the cognitive part, not just an external representation of pre-established inner lines of thought. (In this case, being enacted makes the mind also embedded or extended, since without the outer material, the kind of cognition considered wouldn’t be possible.)\n\nSo, cognition isn’t just doing: for humans - and most likely many hominins before us - it is doing with things (cf. Baggs, Raja, and Anderson 2020). Malafouris (2013) has applied this line of thought even to knapping, arguing that no mental ‘blueprint’ guides the making of a hand axe, for any “decision about where to place the next blow, and how much force to use, is not taken by the knapper in isolation; it is not even processed internally.” (Malafouris 2010, 17). Rather: “The flaking intention is constituted, at least partially, by the stone itself … (as) an integral and complementary part of the intention to knap” (2010, cf. also his 2013, 175ff.). If so, cognition turns out to be a skill, an activity often involving transforming material, turning them into artifacts, or operating with already produced (cognitive) artifacts. Such activities have to be learned.\n\nIn the context of hominin cognitive evolution, this also has important implications. As Karola Stotz (2010, 2017) argued, it is not only selective niches we inherit; humans also inherit the resources to deploy and develop answers to these self-imposed challenges. Humans can construct new variations and pass them on to later generations via scaffolds of teaching and learning (Sterelny 2003, 2012a). These teaching and learning scaffolds are required for the “robust and reliable development of species-specific traits” (Stotz 2017, 5). Humans, and very likely even hominins much earlier in our lineage, are no exception here. Even more important for our context is that these scaffolds enable “developmental plasticity” (2017, 5), which allows for an adaptive response to selective pressures - also the self-imposed ones of niche construction theorists. Here 4E, especially the idea of enactive cognition and ‘materiality’ (extended), comes into play: both developmental niche construction and 4E-cognition in tandem can explain rapid, theoretically even within less than one generation, adaption to selective pressures. As mentioned above: some have argued that cultural practices like proverbs or rules of thumb are cognitive artifacts. To repeat: these artifacts do not work because of their material properties only, but “are always embedded in larger socio-cultural systems that organize the practices in which they are used” (Hutchins 1995, 127). One has to embody rules for making these items, which makes social learning a prerequisite of their use. So not only selective regimes but also variations, some of them adaptions-to-be, including cognitive artifacts and skills, can come into being and be inherited ‘self-made’.\n\nTo get down to the basics: A selective niche - or rather: the selective part of a niche - fosters cognitive skills required to cope with them. Sometimes a cognitive task can be solved by exchanging one ability with another and adding a material medium. Both production and use of this material, however, get established by preexisting cognitive skills. (Preexisting cognitive skills allow one to create new cognitive artifacts, which - in tandem with (maybe) other preexisting skills - allow for the cognitive skill required. The latter explains the emergence of new cognitive skills out of only preexisting ones.) Once acquired, new cognitive skills allow one to change things already existing, adapt to what is coming next as future opportunities, or re-direct and channel slowly accelerating developments in otherwise impossible ways. And all these abstractly sketched doings are instances of a down-to-earth biological process: niche construction in both its selective and developmental manifestation.\n\nTheories of 4E cognition are still far from becoming orthodox in the field. Yet granted they are on the right track, how does this change how we should offer lineage explanations on hominin cognitive evolution? One advantage is high phenotypic plasticity: if cognitive skills depend on and are formed by social environments and technologies, they can come into being quickly and change rapidly (cf. Jeffares 2013). It also allows for high adaptability to the evolving niches: if cognitive skills don’t have to wait for a generation (or more) to come into existence, a population can meet their challenges quicker. Hence, given we have enough archeological information about changing (selective) niches, and information about their inhabitants’ potential resources to form new cognitive skills, cognitive change becomes more accessible to track than before. (I will elaborate on this in Section 5 below.)\n\nIn contrast, within early cognitive archeology, as sketched in Section 2 above, change was caused by a relatively slow variation on the genetic base of neural structures. Also, given the cognitive transition inferences discussed above, they potentially allowed for tracking cognitive change but couldn’t give a detailed answer to which selection pressure they were an answer. Regarding the use of the archeological record to identify cognitive skills, however, things aren’t as straightforward as they were before. For one thing, if cognition is distributed and scaffolded, the archeological record of the size and organization of hominin brains no longer serves as direct evidence of corresponding cognitive change (Sterelny 2017, 243). For another, if there is no longer a clear causal arrow from cognition to artifact, artifacts no longer straightforwardly equal cognitive capacities. Hence we also lose another source of evidence.\n\nIf so, how to construct reasonable lineage explanations based on 4E-cognition and Stotz’s conception of developmental niche construction? Showing this new framework in action works best with cognitive history, where we have relatively clear evidence of cognitive artifacts. I sketch an example from the early neolithic to make a case in point.\n\n\n4. An example: the cognitive life of clay\n\nWhen cognition is a compound of skills, its emergence must be the stepwise assembling of its components, either by transforming previously existing elements or recombining old ones to yield novel outcomes. Given a certain niche as the baseline for a lineage explanation, the existing skills must be sufficient to function as a platform. A minor transformation or recombination must yield a new cognitive skill as a step from one niche to the other. This iterates, and skills already existing within this new niche must suffice as a platform for the next step. (Granted that there is indeed a need, and thus selection for, these skills.) This way, skill after skill accumulates till everything is in place to explain the trait in question, be it a cognitive (compound of) skill(s) such as language, planning, mind reading - or numeracy.\n\nApplying this scheme might look like the following case study on the emergence of numerical cognition in the Ancient Near East (ANE), 10.000-5.000 before present (BP). Well-known to any cognitive archeologist, introduced by Denise Schmand-Besserat (1992, 1999, 2009, 2010), also discussed in detail from an enactivist viewpoint by Lambros Malafouris (2013; for a critical stance see Johnson and Everett 2021); and further elaborated by Karenleigh Overmann in recent years (2016, 2017, 2019). I rephrase this account on numeric cognition in the remainder of this section, adding the critical dimension of the selective regime(s) here and there to yield a complete lineage explanation.\n\nHunter-gatherers stroll around; but around 10.000 BP, some human groups in the Eurasian region became evermore locally bounded. Agriculture emerged for the first time in the Fertile Crescent of modern-day Syria to Iran. The reasons for this are multiple, scaffolded by other occurrences, and evolved over a more extended period as the once-used term ‘neolithic revolution’ might suggest. In any event, farming produced a surplus of food, with the need for storage, eventually yielding a new redistribution economy. This engendered social upheavals and cognitive ones as well.\n\nA need arose to control these streams of goods. This fosters trivial but challenging problems (cf. Schmandt-Besserat 2009, 152). Vast amounts of grain cannot be pushed around easily to ‘see’ who should get what. Unruly animals are no more accessible goods in this regard. Sometimes, grain is still on the field and not yet delivered; thus, the actual transaction will occur in the future. This likewise fostered new cognitive challenges. First, keep track of all the goods redistributed, while it is not always easy to account for them. Furthermore, the number of goods soon exceeded any human ability to memorize them. So because of these new soil-bound activities, the early farmers required a new technique to cope with their reshaped way of life.\n\nThe ability to solve this problem was soil-bound, too. Although in a slightly different sense: the new cognitive ability required was made possible by using small things made of clay: so-called tokens. They were counters used to administer the redistribution of various community needs (cf. Schmandt-Besserat 2009, 147). Albeit used for counting, they worked differently from our abstract numerals (where numbers are comprehended as detached from any particular object to be counted). In the beginning, there were about 12 different shapes to describe various goods, like cones, spheres, cylinders, disks, tetrahedrons, etc. Furthermore, each kind of token stood in a one-to-one correspondence to units of goods. For example, to account for three jars of oil, three ovoid tokens had to be selected to represent this amount of goods. Even for different quantities of the same good different counters were used: for large and small units of grain, for example, cones and spheres, respectively.\n\nTokens are cognitive artifacts made of clay. They functioned differently to our way of counting, yet despite these differences, we have here the emergence of a new cognitive skill: counting and keeping track of items, realized by preexisting cognitive abilities, joined by a material artifact designed to fulfill this very task. Overmann (2019, 440) argues that counting with so-called ‘restricted’ numbers (up to 20, but very often only one, two, many) might have been there already by either using finger counting or a tally (basically something like stick or bone with marks on it). ‘Artifacts’ to memorize items likely also have been in play at that time as the Late Pleistocene hunter-gatherer certainly already used narratives to remember things (see Section 3 above). Yet tokens are abstract in a much more direct manner and hence communicate the information contained (number of goods) much more directly. We also see how one skill (i.e., memorizing by oral communication) might have been replaced by another one to fulfill a new cognitive task. Tokens are a case in point here: they ‘build’ a model with a straightforward one-to-one correspondence, thereby visualizing what to know rather than remembering it. Thereby it is making access to information in some sense much easier (cf. Schmandt-Besserat 1999, 25).\n\nYet another interesting feature of the token system is that one must learn how to use it to decode the information contained (cf. Schmandt-Besserat 1999, 25). The cognitive skills attached to it remain elusive without being initiated into this system. This is a prime example of the developmental niche argued for by Karola Stotz. Once a challenge comes up, and so the selective regime attached to it, a combination of already existing skills might solve the problem. But these skills - or the recombination thereof - must be taught, learned, and transmitted to become a staple adaption to the challenge in the long run.\n\nOnce established for counting and keeping track of goods, this new material artifact of tokens initiated new complex cognitive operations. For example, they quickly allowed for “patterning, the presentation of data in a particular configuration” (Schmandt-Besserat 1999, 25). So the ancient accountants could arrange the units according to a type of good, respective value, date of entry or expenditures, and so on. Furthermore, “just by moving or removing tokens(,) they could add, subtract, multiply, and divide” (Schmandt-Besserat 2009, 153). The latter operations are a new cognitive skill made possible by engaging with the material (Malafouris 2013; cf. Overmann 2016, 2017, 2019). Compared to finger counting, for example, tokens allow for higher numbers counted and thus increase the likelihood of such complex algorithms like multiplication to become deployed (cf. Overmann 2019). The materiality of tokens, i.e., sets of freely arrangeable and combinable objects, fosters exploring the possible ways of combining, separating, bundling, and de-bundling them (cf. Overmann 2019, 446). Here, from an enactive viewpoint, “(b) rains can focus on what they do best: managing interactions with the world” (Anderson 2014, 232), thus eventually realizing these arithmetical operations. For “(c) ognitive processing emerges from - is indeed identical to - these iterated interactions” (2014, 232). This includes interactions with cognitive artifacts like tokens. As this new skill “stretched human cognition to cope with new levels of complexity” (Schmandt-Besserat 1999, 25), it also allowed for further regional development.\n\nBy 5.500 BP, the region’s economies became more extensive. Tokens, in turn, became more ‘complex’, and their numbers rose to 350. As urban workshops entered the production of goods, there were now tokens for raw materials like wool and copper, as well as crafted products, for example, bread, beer, garments, jewelry, or textiles. Some are iconic; they ‘picture’ the good they stand for. This is evidence that specialists have crafted them for their purpose. Shortly after that, emerging urban settlements got involved in redistribution processes. The token system is still well-functioning despite multiplying both kinds and numbers of exchanged goods.\n\nFor the accountants of the urban settlements could cope with the new cognitive demand still using tokens, although the skill changed from mere tracking and counting to basic arithmetic operations. But again, looking back at the previous niche, all the basic skills required to realize this new skill were already there to meet this demand (as described above). Hence it would have been only a comparatively small step within our lineage explanation. This is not the only change, however.\n\nAbout 5.300 BP, temple officials administered a redistribution of goods. By now, failing to deliver goods would lead to penalties for producers. Transactions between different producers and consumers became more complex, too. Any exchange is by now officially regulated under contracts, with severe consequences for breaches. To keep track of these debts, tokens were put into hollow clay balls, so-called ‘envelopes’, until the debt was paid. And to keep track of the content of these ‘envelopes’, the same tokens were pressed on the outside of them while still wet, leaving marks on the surface of the clay.\n\nThis practice of pressing tokens into clay turned out to be a momentous event. Gradually three-dimensional tokens standing for specific amounts of good became two-dimensional signs. For example, cones and spheres for grain became wedge-shaped and circular signs. Within a century, the envelopes were replaced by solid clay tablets; yet the impressions of the tokens on the surface remained. This yielded the next step, for “(b)y innovating a new way of keeping records of goods with signs, the envelopes created the bridge between tokens and writing” (Schmandt-Besserat 2009, 149). This new skill was in dire need.\n\nWith ever bigger getting city states, around 5100 BP, the kinds and numbers of goods reached an unexampled level. This challenged the administrative officials to transform their formerly used cognitive artifacts. They still needed new ways to keep track of all goods. The first change was brought about by no longer pressing tokens into the tablet. Instead, officials were now using a pointed stylus to sketch the former forms directly on the surface. This opened up an essential possibility. For now, the officials no longer record the ever-higher number of goods to be accounted for by repeating the token assigned to the good as often as necessary, for this would have produced hardly cognizable long rows. Instead, they used a specific sign for the good, preceded by signs to represent the numbers of this good. These are the first (proto-)numerals in Mesopotamia, and eventually, they made “obsolete the use of different counters and numerations to count different products” (Schmandt-Besserat 2009, 153). Interestingly, no new signs were invented, but the old signs for a small amount of grain (the wedge) and a large amount of grain (the circle) were now assigned with arithmetical values, for example, ‘1’ for the former and ‘10’ for the latter. A quantity of ‘33 jars of oil’ hence was presented as three circles (10 + 10 + 10), followed by three wedges (1 + 1 + 1), again followed by the sign for ‘jar of oil’ (based on the former token used for this good).\n\nThis new cognitive artifact involving (proto-)numerals was still needed for the same function, but it had to be adapted to the changing niche. Concretely, it had to adapt to ever more data to be manipulated to redistribute ever bigger streams of goods between ever more participants in the whole process. But again, all skills required for this were already there: recombined with this new artifact, this unique compound became a platform for the next step. Schmand-Besserat summarizes this well:\n\n“When these cognitive skills had been internalized for several millennia, the human mind was ready for new strides in abstraction. Concrete counting with tokens was the necessary foundation for the invention of writing.” (Schmandt-Besserat 2009, 153)\n\nIn the long run, the transition from tokens to tablets leads to both writing and mathematics. A change in material (and hence cognitive) artifact fostered new ways of abstraction and allowed for new behaviors (cf. Schmandt-Besserat 2009, 153; cf. also Overmann 2019, 447). As for mathematical operations, the split between (proto-)numerals and accounted commodities allowed them to explore further possibilities on what one can do with numbers. First used only for “recording and communicating numerical information” (Overmann 2019, 457), the sheer volume of numerical information recordable in a concise way via the new medium (e.g., multiplication tables) allowed the scribes of this information to eventually come up with new and more abstract ways to calculate with numbers than possible within the token system. As Overmann states:\n\n“This gave scribes more options for calculating than using tokens: They could additionally use information from tables or memory, a factor in developing new, complex algorithms for manipulating relations between numbers.” (Overmann 2019, 457)\n\nThis niche then provided the next platform, for “numbers are a cognitive technology that enables the management of complexity, allowing for even greater complexity to emerge.” (Overmann 2019, 450). So, in turn, this allowed for the next step within our lineage explanation toward complex numerical cognition.\n\nCurrent ‘enactivist’ neuroscience might add evidence (cf. Anderson 2014, 232f.). As indicated above, Anderson has argued that ‘doing math’ is “a sensorimotor skill characterized by the iterative interactions with external symbols” (2014, 233). Following enactive cognition, getting engaged in mathematical reasoning is a case of practices with symbols, whereas these symbols function not as “abstract indicators of some mathematical statement to be reproduced in an inner language of thought but rather as icons” (2014, 233; cf. also Malafouris 2013, 115 interpretation of the transition from tokens to tablets here). The spatial manipulation of these icons would then be part of mathematical reasoning, where changing the spatial properties likewise changes mathematical content (cf. also De Cruz 2012; De Cruz and De Smedt 2013; Menary 2015). If so, Anderson claims, mathematical reasoning could be disrupted when the spatial manipulation of these symbols is disrupted; this way, showcasing the underlying sensorimotor processes realizing this cognitive skill.\n\nAnd indeed, there is evidence for this. Anderson (2014, 234–36; like Menary 2015, 14f.) discusses several experiments from Landy and colleagues, all indicating that cognitive processing varies with the spatial properties of the external artifacts used. For example, Landy and Goldstone found that spacing around operators in mathematical equations matters. In one study (Landy and Goldstone 2007a), participants were asked to write down equations by hand: interestingly, they grouped numbers to be added less close together than numbers to be multiplied - thus indicating that spacing might be a cue for processing the content of an equation. In the next step, Landy and Goldstone (2007b) asked participants to judge whether addition and multiplication equations are true or false. In one set of these equations, incorrectly doing addition before multiplication led to false judgments. Also, in this set, the spacing around ‘+’ was closer than around ‘’ (contrary to the way the participants above would have written down the equation by hand, at least regarding spacing). The results matched the prediction that spacing is a cue. In this set, only 55% of the participants gave a correct judgment, compared to 80-90% with two other sets of equations, where spacing was either even (thus ‘neutral’) or ‘consistent’ (tighter around ‘’ than ‘+’). This suggests that “episodes of formal reasoning are indeed typically organized by attention-based interactions with external environments” (Landy and Linkenauger 2010, 2168; cited by Anderson 2014, 237). The latter is also fostered by yet another experiment where the illusion of motion interferes with the way notations need to be moved on a page to solve the equation (for details, see Landy and Goldstone 2009), displaying again that problem-solving in mathematics appears to be a matter of how we arrange symbols on the page (cf. also Menary 2015, 14).\n\nManipulation is here of mathematical notations, not concrete objects like tokens (the former being ‘marks on paper’, whereas tokens rather resemble something like an abacus). However, interestingly, both consist of spatial manipulation of objects in some sense or the other. If true, this might be additional evidence from current neuroscience (although given under an ‘enactivist’ interpretation of brain anatomy and activities) that the new cognitive framework is the right one to give proper explanations of why and how cognitive skills unfold in human populations (and history).\n\nInterim conclusion. The new cognitive framework works best when cognitive artifacts are preserved and we have rich knowledge of (changing) selective regimes. In this case, we have enough information to make a convincing lineage explanation; and the idea that cognitive change is realized by acquiring new skills neatly explains the stepwise transition from one state to another. (Note, however, that no minimum capacity inferences were deployed in this example, and hence the characterized cognitive transitions here do not depend on compounds of them. Probably because the ‘artifacts’ under consideration for these inferences would be streams of goods like goats or grain, but in principle, such inferences would be possible here, too.)\n\nIndeed, the specific case of ANE numerical cognition might be an inference to the best explanation for the new framework, in contrast to its ‘orthodox’ alternative. Early cognitive archeology runs into a ‘representational puzzle’ here: When the first (proto-)numerals were used, the same sign could change its value depending on the good accounted for; only a millennia later, the first ‘real’ abstract numbers seem to appear in the record (cf. Damerow, Englund, and Nissen 1988). But if genetically induced numerical cognition came first, why do we find a ‘misuse’ of (proto-)numerals in the beginning, not outright abstract numbers, as soon as there is a switch from tokens to tablets? This indicates that the new framework might be the better model of the human mind to work with.\n\nThings become more complicated with the deep past, however. Already 75-80.000 kya, with artifacts like the already mentioned ‘Blombos beads’, possible stages of a lineage explanation become ambiguous. In principle, these beads could be a cognitive artifact for counting just like tokens were - and hence form part of an explanation of the evolution of numerical cognition. There is reason to assume that hominins at that time would have been capable of this skill, and the materiality of the beads might allow for concepts like “one more” or “as many as” (another string of beads) (cf. Overmann 2019, 448). However, other uses are also possible (like being a social ornament, for example). Furthermore, in this specific case, a selective regime as an incentive to assume counting is ambiguous, too. For groups have been rather small, so any need for the subsistence of the group might not necessitate counting - unlike in the case of the ANE-people 10.000 BP discussed above, where a redistribution economy had to be accounted for. As Overmann (2019, 438) convincingly argues, to go beyond restricted numbers, a group’s “demographic density” must generate an according need first. So interpreting these beads as a cognitive artifact for counting wouldn’t be the most likely candidate explanation. One has to be careful since even if the materiality of an artifact allows for being a cognitive artifact, this doesn’t imply that this possibility has also been realized. Yet ambiguities like this don’t rule out any use of lineage explanations for the deep past (including our inferences discussed in Section 2). I discuss their prospects within the 4E framework in the final section.\n\n\n5. Inferences revisited\n\nCognitive history might provide a proof of concept for the new framework, but persistent challenges remain regarding the deep past. Cognitive artifacts, if they existed, virtually never ‘fossilized’. Language skills, the ability to follow norms, causal reasoning, teaching complex behavior sequences to others, and the like don’t leave direct evidence in the archeological record. Likewise, traces indicative of such abilities are fragmentary. They leave much room for speculation.\n\nHere, minimum capacity and cognitive transition inferences enter. Yet Malafouris (2020) locates archaeologists using them in the ‘orthodox’ camp. Historically, this is right, and even the philosophers discussing these inferences (again Pain 2021; Killin and Pain 2021; Currie and Killin 2019) tend to representationalism or even computationalism. However, this ‘inferential engine’ can be deployed usefully by an enactivist archeologist, too.\n\nFor minimum capacity inferences are still a valuable tool, also within the 4E framework. First of all, the presence of a particular artifact tells us something about the minimum behavioral requirements at least some members of the group in question had to be capable of. They still show us with sufficient certainty which behavior sequences the maker of an artifact had to realize. The link between the behavior required to produce an artifact and the cognitive requirement to bring forth this same behavior is affected by the choice of cognitive theory in the background, but it leaves the former behavioral part of the inference unimpaired. As such, we get a ‘base’ from which to determine the cognitive demand for executing the required behavior sequences. (How this demand is formulated, however, might vary with one’s choice of cognitive theory; see below.) So, the function of some artifacts as ‘markers’ for cognitive abilities remains intact. Furthermore, extracting behavior from artifacts yields enormous potential for future studies. Miriam Haidle’s cognigram-account is a prime example here. To name just three, she has worked out meticulously the behavior sequences required to produce such different artifacts as a folded leaf (to scoop water) by Pan troglodytes, wooden spears by Heidelbergensians or a bow-and-arrow set by anatomically modern Sapiens, among many others (Haidle 2009, 2012; Lombard and Haidle 2012).\n\nThings also look good concerning selective regimes. There are traces of the complexity a group had to cope with: in foraging, regarding their range size, ecological variety, and so on. These likely made up much of the selective niche. Furthermore, in terms of the overall complexity of a niche, they tell us something additional to minimum capacity inferences on the cognitive demand of this very niche. In sum: both the cognitive demand at a given niche and the selective regime of the previous niche, which had to select for the ability to fulfill this demand, can, to some extent, be inferred from the material record.\n\nHowever, this only displaces problems. The question is how exactly a specific cognitive skill for an earlier identified demand was realized at a given niche. Within early cognitive archeology, the assumption was that a change in cognitive capacity equals a change in brain size or structure. Hence any change in the archeological record on fossilized skulls could be an argument for any cognitive change identified earlier within the archeological record on artifacts. Within the 4E framework, this simple covariance can’t be assumed anymore.\n\nEven more daunting, as argued in Sections 3 and 4, most advanced cognitive skills are compounds of many ‘soft’ cognitive artifacts and other skills. So given a specific cognitive demand is identified via an artifact, one must first analyze how a skill meeting this demand might have been realized out of other cognitive skills. Since ‘soft’ cognitive artifacts and skills generally didn’t fossilize - like clay tablets with mathematical inscriptions - there is much indirect information to be obtained from the material record, with all uncertainties of such an endeavor attached. Nevertheless, even if fragmentary, attempts to construct a lineage explanation have their virtues: they order the available evidence in the most coherent way possible, and they build the base for further investigations by allowing to formulate new hypotheses afterward (cf. Currie and Sterelny 2017).\n\nThis gives us the following road map:\n\nGiven 4E-cognition:\n\nA composite of cognitive skills that meets the cognitive demand must be realized.\n\nThis means:\n\nGiven an artifact at Niche N, infer the cognitive demand at N (by conducting a minimum capacity inference on this artifact).\n\nAnalyze which cognitive skill could meet this demand; analyze which single components this skill could have been made of.\n\nThen:\n\nTry to infer these single skills from the material record of a niche previous to N; i.e.: identify the single components of a previous niche and whether they could create in a small step the new cognitive skill (given the previously identified cognitive demand).\n\nFinally, try to identify the selective regime of the previous niche and whether this regime selected for the cognitive demand in question.\n\nThis would be the reconstruction of one step within a lineage explanation: from a platform - the single components of a previous niche - and a selective regime within this previous niche towards a new skill (given the previously identified cognitive demand by a minimum capacity inference of a particular artifact) at the niche under consideration (see Figure 1).\n\nCognitive transition inferences also remain part of the ‘inferential engine’. Given two artifacts A and A’: If A predates A’ and both are indicative of different cognitive demands, where the demand of A’ exceeds the demand of A, an inference based on both these artifacts still would demonstrate a cognitive change from A towards A’. However, much information must be obtained to yield a complete lineage explanation within the 4E framework. Again, the cognitive skills realizing both the demand required to make A and A’ need to be analyzed. Being composites, based on which other skills were they realized? Regarding the cognitive skill to produce A, did the single components of this skill already exist in a niche prior to A? Did within the niche of A already exist all components required to realize the skill necessary for A’? Do we have evidence in the material record of A’s prior niche that makes selection towards A (or the cognitive demand associated with A) likely? Do we have evidence in the material record of A’s niche that makes selection towards A’ (or the cognitive demand associated with A’) likely?\n\nGiven these pieces of information can be obtained, complex lineage explanations become possible as well, based on sequences of cognitive transition inferences. The evolution of anticipatory planning based on Oldowan and Acheulean stone tool production has already been mentioned in Section 3. Other examples (by the same author) would include causal reasoning (Gärdenfors and Lombard 2018, 2020) or teaching (Gärdenfors and Högberg 2017). Here the authors apply a combination of different streams of evidence to reconstruct niches of certain hominins (e.g., from Homo habilis, Erectines, and so on), apply an analysis of the cognitive requirements to produce certain artifacts (e.g., that the Oldowan industry required “demonstrative teaching”), and eventually track cognitive transition through a change in the artifactual material record (e.g., the transition towards an artifact like bow and arrow as indicative of “Causal Network Understanding”; (cf. Gärdenfors and Lombard 2018, 3)). Gärdenfors and Lombard (2020) even highlight the importance of technology in forming hominin cognition during evolution, i.e., the apply the embedded mind thesis on their evolutionary trajectory.\n\nTo be sure, the approaches mentioned above are not enactivist. However, in principle, it would be possible to rephrase the ‘cognitive part’ of their explanations in terms of (the assemblage of) cognitive skills, roughly along the lines mentioned above and as exemplified in Sections 3 and 4. (Although this would also change the interpretation of the artifacts and require further information about possible precursors skills in their reconstruction of the hominin niches, of course). To conclude, lineage explanations based on the 4E framework are possible even in the deep past, and there is also an important methodological implication involved here.\n\nThinking of cognitive capacities as complex skills makes any evolutionary explanation of them an entangled affair. To give an example (albeit still a rather abstract one): Suppose a cognitive skill that requires more or less explicit norm following - say, argumentation perhaps. Internalizing or publicly announcing norms itself is a cognitive skill. In this regard, Jonathan Birch (2021a, 2021b) has argued that Acheulean-style hand axe production implies the capacity for norms. If probable, any lineage explanation of our cognitive skill must thus be in accordance with his account (or otherwise convincingly argue against it).\n\nThere is a lesson to learn here. The point just made iterates, and here entanglement becomes a methodological ‘check and balances’. The new way we would construct lineage explanations within any 4E framework - as skills composed out of other skills, which have to function at different times as different platforms for additional skills to emerge - has consequences: it fosters the alignment of the chronologies of all single components. Our account of a specific skill must fit with other lineage explanations, for it will have to be in accordance with any plausible account (itself constructed as lineage explanations) on all components of our skill under review.\n\nIn early cognitive archeology, following ‘orthodox’ cognitive science, a cognitive capacity is comprehended as a rather quasi-independent component of the human mind. In this picture, human cognition assembles a set of quasi-independent subsystems. Of course, interdependencies are considered, too: as mentioned above, Coolidge and Wynn argues that abstract reasoning depends on enhanced working memory; likewise, Ian Tattersall (2017) argues for an association of language with symbolic behavior (for an inference from the archeological record suggestive of symbolic behavior to the emergence of language). By and large, however, comprehending cognitive capacities as subsystems, initiated by genetically evolved brain structures first, make them appear to be relatively stable ‘units’. They give the impression of being examinable in relative isolation to other cognitive components of one’s overall cognitive make-up.\n\nCognitive skills are much more fragile in this regard (cf. Heyes 2018, 217f.). There are far more interdependencies to be considered. This fosters different accounts (which are, given the usual division of labor in our discipline, often worked out by various colleagues) to be in alignment with one another regarding their chronology of “cognitive events”: at which time (niche) which skill was realized and accessible to a population. In the end, this inter-connectedness of different cognitive skills makes any account of the ‘package’ of an enacted hominin mind much more coherent. We now have different chronologies of different skills, which in different lineage explanations must match each other. In other words, when constructing a lineage explanation for a specific cognitive skill, we meticulously must strive for “consilience” (Wilson 1998) with virtually any account on the single skills, which function as components of our skill. With so many checks and balances, we naturally progress to a much more balanced account.\n\n\nConclusion\n\nTo sum up the state of play: Minimum capacity- and cognitive transition inferences are used by both ‘orthodox’ and 4E cognitive archaeologists alike. Within an ‘orthodox’ framework, however, this implies that brains equal cognition and inferences made from artifacts are directly indicative of cognitive capacities. Hence any transition in artifacts should equal a transition in fossils indicative of changing brain encephalization – something not supported by the archaeological record anymore (as far as we know; see Section 2). Within a 4E framework, this problem vanishes: in principle, one could argue that any change in the material record on artifacts is accompanied by a change in cognitive skill. This might be interpreted as an epistemological incentive for the new framework. Furthermore, although contested by some (cf. Brown 2021), 4E cognition allows for quick cognitive changes, even within the lifetime of one generation. Hence it makes cognitive transitions much more likely, compared to cognition as assumed in the background of early cognitive archeology, where random genetic variations must cause new brain structures first. So given enough knowledge of the cognitive challenges of a group and their potential to create new cognitive skills, establishing lineage explanations becomes increasingly possible.\n\nSome problems remain. If cognitive demand in the past did not select for brain size and structure only, but compounds of brains and external scaffolds, then, also within the 4E-framework, it becomes more intricate to conjecture about why and how hominin brains evolved the way they did. Despite that, distinguishing between different candidate explanations for a given cognitive skill remains the most challenging problem (cf. Moore and Brown 2022). Only careful consideration of the archeological record (and hopefully new findings here and there) might make a change. In the end, given this endeavor’s speculative and fragmentary nature, we might have to live with the fact that, eventually, we will have a small class of candidate explanations for a given trait and no further means to decide between them. However, the more lineage explanations of different hominin cognitive skills we try to reconstruct, the more we are forced to strive for a coherent overall picture of all their cognitive abilities. 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Publisher Full Text\n\nWynn T: Redescribing the Oldowan.Wynn T, Overmann KA, Coolidge FL, editors. The Oxford Handbook of Cognitive Archaeology. 2023. Publisher Full Text" }	[ { "id": "169184", "date": "28 Apr 2023", "name": "Karenleigh Overmann", "expertise": [ "Reviewer Expertise I am an evolutionary cognitive archaeologist who specializes in post-Neolithic technologies (numbers", "writing systems)", "I view cognition as 4E and use descriptive pattern analysis." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverview. The article discusses two approaches in evolutionary cognitive archaeology (ECA). It describes them as taking, respectively, “orthodox” and 4E views of cognition. The 4E view is illustrated with a sustained discussion and analysis of Schmandt-Besserat’s view of numeracy in the ancient Near East. The example is misplaced: While Schmandt-Besserat’s views are widely known, she applied no theory, cognitive or otherwise, to guide her interpretation of artifacts associated with either numeracy or writing. This outdated work is a curious choice to illustrate the 4E ECA approach, especially when there are recent, well-developed 4E analyses of ancient Near Eastern numeracy and literacy to draw upon. Accordingly, a major revision is recommended. This reviewer is willing to review a revised manuscript that takes the comments below into consideration.\nIntroduction: The introduction contains several assumptions and claims that are misleading or inaccurate:\nP3: “Cognitive archeology is the brave attempt to infer the cognitive abilities of past people from their material remains, thereby exploring how the cognitive evolution of hominins - and eventually humans - has unfolded.”: The opening statement presents a narrow view of (evolutionary) cognitive archaeology (ECA), which is also concerned with the role of materiality in human cognition in 4E models of the human mind. Since the author expounds on 4E models, it is important to define cognitive archaeology accurately at the onset. (Ideational cognitive archaeology is concerned with inferring ideas from past material culture.) The term “brave” comes across as subtly disparaging.\n\nP3: “To explain the evolution of human cognition requires understanding which forces shaped and re-shaped over and again the cognitive underpinnings of hominin lifeways from several million to only a few thousand years ago.” (italics added): Not all ECA ignores that past few thousand years (assuming that brains and behaviors are essentially modern and thus unlikely to shed much light on evolutionary questions); nor is this period coextensive with cultural rather than evolutionary change in cognition. Given ECA’s concern with the role of materiality in cognition and the understanding that evolutionary change remains ongoing today (if difficult to discern), it is more accurate to say that ECA is concerned with cognitive evolution from the onset of tool use (currently understood as emerging 3.4 million years ago) to the present day and beyond.\n\nP3: “So, the same trace in the record, say a specific stone tool, might require capacities like a mental ‘blueprint’ or sophisticated long-term memory according to classical theories, but only environmentally guided perception and simple learning according to ideas from the 4E family.”: Rather than offering generic examples that imply a lack of familiarity with the literature, here the author might engage with ECA publications. For example, Coolidge and Wynn (e.g., Coolidge & Wynn, 2005; Wynn & Coolidge, 2007, 2019) argue from stone tools to constructs like working memory, expertise, and creativity, while Malafouris (2010, 2021) uses stone tools and mark-making to question intentionality and skill, the emergence of meaning, and where the boundaries of the mind should be drawn. Note that Coolidge and Wynn interpret patterns of change in the archaeological record using conventional psychological constructs, while Malafouris is a 4E theorist.\n\nP3: “Cognitive archeologists have no choice but to decide. Only by using a theory or model, can they make inferences from artifact to cognitive capacity. And the choice of theory or model comes first.”: The author seems to suggest that ECA practitioners must necessarily choose either a standard psychological paradigm or a 4E approach. However, this is misleading. Certainly, within the cognitive sciences, the first two Es (embodied and embedded) have become commonplace, if not mainstream. Granted, the cognitive sciences may still consider the second two Es (extended and enactive) to be radical positions, but even here, there is ongoing research and debate. The point is simply that ECA, like the cognitive sciences, cannot be accurately described as falling into two distinct camps that are easily bifurcated. As a relatively new discipline, ECA is still experimenting with theories and methods, and merging (what the author describes as) the two approaches is ongoing (e.g., Wynn et al., 2021b; Overmann’s work in numeracy and early writing systems).\nLineage explanations. For this section, the author might review work in ECA (e.g., Malafouris, 2013; Wynn et al., 2021a) for an orientation to its epistemology. That is, rather than explaining inferences in evolutionary biology generally, focus on the epistemology of ECA specifically.\nEarly cognitive archeology and its inferences. The author’s characterization of cognitive archaeology in this section as “early” is not explained. 4E constructs are not replacing the use of classical/orthodox psychological and neuroscientific constructs within ECA. Further, given Colin Renfrew (a 4E proponent) as an early pioneer of ECA, both approaches are equally as old within the discipline.\nP. 4: “So, if we see a change in material culture and a corresponding change in brain size or structure, everything seems to match.” One of the largest gaps in our understanding of human cognitive evolution is the lag between fossil indications of cognitive change (e.g., larger skulls indicating larger brains) and archaeological evidence of behavioral change (e.g., greater complexity of tool design). It the lag did not exist, it would be easy to say that bigger brains created better tools (when fossil change precedes archaeological change) or that tool use influenced brain change (when archaeological change precedes fossil change). Unfortunately, the pattern is unclear, as the author later notes. This ambiguity, however, does not argue in favor of one or the other; the relation is more likely to be highly intertwined and mosaic.\n\nP4: “Artifacts come into existence through the behaviors of their producers, and these behaviors become possible due to certain cognitive capacities they entertain - eventually because their brains realize these capacities.”: Here the author appears to subscribe to the idea that bigger brains make better tools. ECA also examines the reverse direction of influence, the idea that tools make minds (i.e., that tool use has an effect on cognition). See Malafouris (2013).\n\nP5: The author’s characterization of Coolidge and Wynn’s Enhanced Working Memory (EWM) hypothesis is more caricature and straw man than not, and the hypothesis deserves to be taken more seriously. Coolidge and Wynn draw on multiple technologies (not just the Blombos beads or Hohlenstein-Stadel figurine) to make their argument, and they also operationalize working memory so that its change can be identified archaeologically.\n\nP5: “Frederik Coolidge”: Coolidge’s first name is misspelled. It is Frederick.\n\nP6: The (imagined) story in early cognitive archeology, therefore, rather often seems to be that as soon as new cognitive abilities arose by genetic variation, hominins explored and exploited new opportunities with them - including the new things they were allowed to do.”: Coolidge and Wynn’s EWM hypothesis posits an inheritable genetic mechanism (which, as the author correctly notes, remains unidentified) precisely because working memory is a highly heritable genetic trait.\n\nP6: “Second, a related problem is that we can track changes in a material culture where it is relatively safe to assume that no significant brain size changes occurred.”: This is a problem only if brain size increases are assumed to precede, index, and cause material change; note that ECA does not generally assume this to be the case. Further, ECA does not generally assume that demographic change is the only other factor with explanatory power.\nA new cognitive framework. For this section, the author should appeal to the 4E literature to define the 4E terms: embodied (Lakoff & Johnson, 2008; Prinz, 2009; Varela et al., 2017); embedded/situated (Overmann & Malafouris, 2018; Robbins & Aydede, 2009; Smith, 1999; Wilson & Clark, 2009), distributed (Dunbar et al., 2010; Hutchins, 1995); extended (Clark, 2008; Clark & Chalmers, 1998; Sterelny, 2010); and enactive (Clark, 1997; Gallagher, 2013; Hutto, 2013; Hutto & Myin, 2013); representationalism (Chemero, 2000; Gallagher, 2008).\nP8: “First, some of the artifacts humans use aid for cognition in the strong sense that without these aides, the cognitive process would be impossible. Hence the artifact is part of this ability. (Although, for our purposes here, we shouldn’t descend into metaphysical debates about minds.)”: However, the debate about whether an artifact is part of a cognitive process is exactly what is at issue in the claim that minds are extended.\n\nThe discussion of extension is attenuated and unclear; instead of explaining and illustrating extension, the discussion is diverted into Niche Construction Theory, which as presented is not well tied to the concept of extension.\nAn example: the cognitive life of clay\nP9: “Showing this new framework in action works best with cognitive history, where we have relatively clear evidence of cognitive artifacts.” The author needs to define what is meant by the terms “cognitive history” and “cognitive artifacts.”\n\nP10: Rather than accepting Schmandt-Besserat’s (1992a, 1992b) account of Mesopotamian numbers at face value, the author should understand that her interpretation of clay tokens was performed in the absence of any guiding psychological or cognitive theory (beyond some tenuous and frankly indefensible appeals to the work of Lévy-Bruhl (e.g., his 1912 Les fonctions mentales dans les societes inferieures; see Schmandt-Besserat, 1982, p. 873), without any understanding of numeracy and contemporary number systems, and with no thought of a 4E approach. The author should consult the many criticisms of Schmandt-Besserat’s work (e.g., Chrisomalis, 2005, 2009; Englund, 1998; Friberg, 1994; Overmann, 2018, 2021; Zimansky, 1993). One of those criticisms is that there is little basis to conclude, as Schmandt-Besserat does, that numerical or counting tokens were used as far back as the 9th millennium BCE. Secure evidence of numerical tokens does not emerge until the mid-4th millennium BCE. Rather than explicating Schmandt-Besserat’s outdated hypothesis at length, the author should explain the 4E approach as articulated by Malafouris and Overmann.\n\nP11: ““When these cognitive skills had been internalized for several millennia, the human mind was ready for new strides in abstraction. Concrete counting with tokens was the necessary foundation for the invention of writing.” (Schmandt-Besserat 2009, 153)”: Schmandt-Besserat’s hypothesis regarding the invention of writing is as outdated and theoretically ungrounded as her work in numbers. For a 4E explanation of how scripts and literacy emerge, see Overmann (2016, 2022).\n\nP12: Manipulation is here of mathematical notations, not concrete objects like tokens (the former being ‘marks on paper’, whereas tokens rather resemble something like an abacus). However, interestingly, both consist of spatial manipulation of objects in some sense or the other.”: The author appears to have missed the fact that Overmann treats written notations (both numerical and non-numerical) as material objects. See Overmann (2016b, 2017, 2022).\n\nP12: “Interim conclusion. The new cognitive framework works best when cognitive artifacts are preserved and we have rich knowledge of (changing) selective regimes.”: As previously noted, the 4E approach is not “new” in ECA. Further, the conditions of the last 10,000 years are quite different from those of the 3.4 million years that precede them, and this should be acknowledged. Specifically, behaviors and brains in this period are effectively modern, so it is easier to generalize insights gained with extant brains to ancient ones (in the way that is true of generalizing from extant brains to erectines, habilines, or australopithecines). The archaeological record is also much richer, enabling a more detailed look at material change. Finally, interactions with materiality are likely to engender cultural change in cognition, rather than evolutionary (genetically inheritable) change.\n\nP12: “Indeed, the specific case of ANE numerical cognition might be an inference to the best explanation for the new framework, in contrast to its ‘orthodox’ alternative.”: The author provides very little support for or insight into the 4E framework, since the preponderance of explanation is focused on Schmandt-Besserat (who would fall into what the author calls the orthodox alternative, noting again that her interpretation of the archaeological record is ungrounded by any cognitive theories) and not Malafouris and Overmann (who fall within the 4E camp).\nInferences revisited\nP13: “Cognitive artifacts, if they existed, virtually never ‘fossilized’. Language skills, the ability to follow norms, causal reasoning, teaching complex behavior sequences to others, and the like don’t leave direct evidence in the archeological record.” The author needs to define what is meant by “cognitive artifact”; in the 4E approach, any artifact has the potential to play a role in cognition. A lot of work on causal reasoning and teaching based on the archaeological record has been done (e.g., Gärdenfors & Högberg, 2017a; Gärdenfors & Lombard, 2018; Haidle, 2017; Högberg et al., 2015; Osiurak, 2017; Osiurak et al., 2020; Osiurak & Reynaud, 2020b, 2020a).\n\nP13: Since there is no discussion of minimum capacity or cognitive transition inferences in Section 4, the author has not established grounds for concluding that they belong to the classical approach, rather than the 4E approach.\nConclusion: The interim sections (1–5) do not adequately support the conclusions as currently presented. After revising the interim sections, the author should revisit the conclusions.\nReferences Chemero, A. (2000). Anti-representationalism and the dynamical stance. Philosophy of Science, 67(4), 625–647. Chrisomalis, S. (2009). The cognitive and cultural foundations of numbers. In E. Robson & J. A. Stedall (Eds.), The Oxford handbook of the history of mathematics (p. 495‒517). Oxford University Press. Chrisomalis, S. (2005). Evaluating ancient numeracy: Social versus developmental perspectives on ancient Mesopotamian numeration. Paper Presented at the Annual Meeting of the Jean Piaget Society (Vancouver, British Columbia), 1–21. https://www.academia.edu/5622707/Evaluating_Ancient_Numeracy_Social_versus_Developmental_Perspectives_on_Ancient_Mesopotamian_Numeration Clark, A. (1997). Being there: Putting brain, body, and world together again. MIT Press. Clark, A. (2008). Supersizing the mind: Embodiment, action, and cognitive extension. Oxford University Press. Clark, A., & Chalmers, D. J. (1998). The extended mind. Analysis, 58(1), 7–19. Coolidge, F. L., & Wynn, T. (2005). Working memory, its executive functions, and the emergence of modern thinking. Cambridge Archaeological Journal, 15(1), 5–26. Dunbar, R. I. M., Gamble, C., & Gowlett, J. A. J. (Eds.). (2010). Social brain, distributed mind. British Academy. Englund, R. K. (1998). Review: Denise Schmandt-Besserat, How writing came about. Written Language & Literacy, 1, 257–261. Friberg, J. (1994). Preliterate counting and accounting in the Middle East: A constructively critical review of Schmandt-Besserat’s Before writing. Orientalistische Literaturzeitung, 89(5–6), 477–489. Gallagher, S. (2008). Are minimal representations still representations? International Journal of Philosophical Studies, 16(3), 351–369. Gallagher, S. (2013). The enactive hand. In Z. Radman (Ed.), The hand, an organ of the mind: What the manual tells the mental (pp. 209–225). MIT Press. Gärdenfors, P., & Högberg, A. (2017a). The archaeology of teaching and the evolution of Homo docens. Current Anthropology, 58(2), 188–208. Gärdenfors, P., & Lombard, M. (2018). Causal cognition, force dynamics and early hunting technologies. Frontiers in Psychology, 9, 1–10. Haidle, M. N. (2017). Development of teaching performance: Comment on P. Gärdenfors and A. Högberg, The archaeology of teaching and the evolution of Homo docens. Current Anthropology, 58(2), 202–204. Högberg, A., Gärdenfors, P., & Larsso, L. (2015). Knowing, learning and teaching—How homo became docens. Cambridge Archaeological Journal, 25(4), 847–858. Hutchins, E. (1995). Cognition in the wild. MIT Press. Hutto, D. D. (2013). Radically enactive cognition in our grasp. In Z. Radman (Ed.), The hand, an organ of the mind: What the manual tells the mental (pp. 227–252). MIT Press. Hutto, D. D., & Myin, E. (2013). Radicalizing enactivism: Basic minds without content. MIT Press. Lakoff, G., & Johnson, M. (2008). Metaphors we live by (2nd ed.). University of Chicago Press. Lévy-Bruhl, L. (1910). Les fonctions mentales dans les sociétés inférieures. Librairie Félix Alcan. Malafouris, L. (2010). Knapping intentions and the marks of the mental. In L. Malafouris & C. Renfrew (Eds.), The cognitive life of things: Recasting the boundaries of the mind (pp. 13–27). McDonald Institute for Archaeological Research. Malafouris, L. (2013). How things shape the mind: A theory of material engagement. MIT Press. Malafouris, L. (2021). Mark making and human becoming. Journal of Archaeological Method and Theory, 28(1), 95–119. Osiurak, F. (2017). Cognitive paleoanthropology and technology: Toward a parsimonious theory (PATH). Review of General Psychology, 21(4), 292–307. Osiurak, F., Lesourd, M., Navarro, J., & Reynaud, E. (2020). Technition: When tools come out of the closet. Perspectives on Psychological Science, 15(4), 880–897. Osiurak, F., & Reynaud, E. (2020a). The elephant in the China shop: When technical reasoning meets cumulative technological culture. Behavioral and Brain Sciences, 43, E183. Osiurak, F., & Reynaud, E. (2020b). The elephant in the room: What matters cognitively in cumulative technological culture. Behavioral and Brain Sciences, 43, 1–57. Overmann, K. A. (2016a). Beyond writing: The development of literacy in the ancient Near East. Cambridge Archaeological Journal, 26(2), 285–303. Overmann, K. A. (2016b). The role of materiality in numerical cognition. Quaternary International, 405, 42–51. Overmann, K. A. (2017). Thinking materially: Cognition as extended and enacted. Journal of Cognition and Culture, 17(3–4), 381–400. Overmann, K. A. (2018). Updating the abstract–concrete distinction in ancient Near Eastern numbers. Cuneiform Digital Library Journal, 1, 1–22. Overmann, K. A. (2021). A new look at old numbers, and what it reveals about numeration. Journal of Near Eastern Studies, 80(2), 291–321. Overmann, K. A. (2022). Early writing: A cognitive archaeological perspective on literacy and numeracy. Visible Language, 56(1), 8–44. Overmann, K. A., & Malafouris, L. (2018). Situated cognition. In H. Callan (Ed.), International encyclopedia of anthropology: Anthropology beyond text. Wiley-Blackwell. Prinz, J. (2009). Is consciousness embodied? In P. Robbins & M. Aydede (Eds.), The Cambridge handbook of situated cognition (pp. 419–436). Cambridge University Press. Robbins, P., & Aydede, M. (Eds.). (2009). The Cambridge handbook of situated cognition. Cambridge University Press. Schmandt-Besserat, D. (1982). The emergence of recording. American Anthropologist, New Series, 84(4), 871–878. Schmandt-Besserat, D. (1992a). Before writing: From counting to cuneiform (2 vols). University of Texas Press. Schmandt-Besserat, D. (1992b). How writing came about. University of Texas Press. Smith, B. C. (1999). Situatedness/embeddedness. In R. A. Wilson & F. C. Keil (Eds.), The MIT encyclopedia of the cognitive sciences (pp. 769–770). MIT Press. Sterelny, K. (2010). Minds: Extended or scaffolded? Phenomenology and the Cognitive Sciences, 9(4), 465–481. Varela, F. J., Thompson, E., & Rosch, E. (2017). The embodied mind: Cognitive science and human experience, revised edition. MIT Press. Wilson, R. A., & Clark, A. (2009). How to situate cognition: Letting nature take its course. In P. Robbins & M. Aydede (Eds.), The Cambridge handbook of situated cognition (pp. 55–77). Cambridge University Press. Wynn, T., & Coolidge, F. L. (2007). Did a small but significant enhancement in working memory capacity power the evolution of modern thinking? In P. Mellars, K. Boyle, O. Bar-Yosef, & C. B. Stringer (Eds.), Rethinking the human revolution: New behavioural and biological perspectives on the origin and dispersal of modern humans (pp. 79–90). McDonald Institute for Archaeological Research. Wynn, T., & Coolidge, F. L. (2019). The expert performance model of Neandertal cognition. In Y. Nishiaki & O. Jöris (Eds.), Learning among Neanderthals and Palaeolithic modern humans: Archaeological evidence. Springer. Wynn, T., Overmann, K. A., & Malafouris, L. (2021a). 4E cognition in the Lower Palaeolithic: An introduction. Adaptive Behavior, 29(2), 99–106. Wynn, T., Overmann, K. A., & Malafouris, L. (Eds.). (2021b). Special issue of Adaptive Behavior: 4E cognition in the Lower Palaeolithic (Vol. 29). SAGE. Zimansky, P. (1993). Review of Denise Schmandt-Besserat’s Before writing, Volumes I and II. Journal of Field Archaeology, 20(4), 513–517.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "175254", "date": "04 Jul 2023", "name": "Alex Aston", "expertise": [ "Reviewer Expertise Cognitive Archaeology", "Social Cognition", "Niche Construction", "Material Engagement Theory", "Enactivism", "Semiotics." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author provides an account of how 4E cognition can be synthesised with Niche Construction Theory in order to make minimal capacity inferences and discern cognitive lineages. Overall, I find the authors writing style very readable and the general structure of the argument to be coherent for the most part. However, though I am sympathetic to the authors goals, and believe that the paper has a great deal of potential, the paper is underdeveloped at present. More specifically, I think that the theoretical positions of the author are a bit muddied at times and I find the overall treatment of the various conceptual frameworks and the evidence to be a bit superficial. Thus, I recommend major revisions.\nFirst and foremost, the author appears to subscribe to a functionalist account of 4E cognition based on their terminology, framing of the issues and use of evidence, despite indicating to the contrary with some of their comments. For example, in the first paragraph on page 8 (of the pdf version of the article) the author gives an example of functional extended cognition in the vein of Clark and Chalmers 1998 and then alludes to how artefacts might constitute a cognitive process that would be otherwise impossible. The author then goes on to describe such relationships in terms of a functional reorganisation of cognitive capacities to generate novel behaviours, e.g., numerical cognition. In the midst of this paragraph, the author states, “Although, for our purposes here, we shouldn’t descend into metaphysical debates about minds.” Unfortunately, the author cannot so glibly put aside the fact they have placed themselves in the midst of a wide-ranging and robust debate about the ontology of the mind. For those of us that understand material culture as constitutive of cognition on a phenomenological level, this framing is wholly inadequate. The author is well within reason to argue for a functionalist 4E position; however, the problem resides in the fact that the author seems to assume that this is the default position in 4E cognition. Thus, the author makes numerous sweeping claims which more radically minded cognitive archaeologists would reject. For example, on page 13 the author states “Cognitive artifacts, if they existed, virtually never ‘fossilized’.” Any cognitive archaeologist that subscribes to frameworks such as Material Engagement Theory or Radically Enactive Cognitive Archaeology would reject this claim outright. From these perspectives, all artefacts are constitutive elements of distinct cognitive processes. Of course, it is completely valid to reject such positions, but the author seems unaware of the full range of perspectives and debates in cognitive archaeology. Correspondingly, despite the author’s engagement with 4E frameworks, they still appear to assume that material culture is an epiphenomenon of internal cognitive capacities, a position that most archaeologist employing 4E frameworks would reject. The author needs to engage with the nuances of these debates, and at least explain robustly why they choose a extended functionalism over both standard representationalist and more radical approaches to cognition. I shall include a list of readings at the end of my comments that I believe will help the author greatly improve their arguments.\nAt present, I shall focus on more specific issues I have with the paper. In general, I think that the first two sections need more effective signposting of the authors position, such as pointing out core axioms and assumptions and gesturing toward the critique they develop in section 3. For example, the discussion of “single individual components of cognition” in the fourth paragraph on page 4 could really use a bit more indication of the author’s position and direction of their argumentation.\nAs for section 3, I believe that the section needs a bit of restructuring, more thorough explication as well as more holistic integration of the various concepts employed. I personally recommend developing focused subsections dealing with the specific topics raised such as the 4Es and Niche Construction in more detail. At present, these concepts are dealt with in a fairly superficial manner, ignore a good deal of relevant literature and lack an effective synthesis, particularly in terms of connecting all 4Es into a robust niche constructing framework. I’ll return to this point with my recommended readings below. As for section 4 on numerical cognition, I shall largely defer to Dr Overmann’s comments on the topic as she is an expert on the topic. However, I think that a more detailed analysis of the materials, action-perception dynamics and developmental implications is warranted. I will also point out that the statement in paragraph 1 on page 10 that, “Hunter-gatherers stroll around; but around 10.000 BP, some human groups in the Eurasian region became evermore locally bounded.” This is a rather facile simplification, and I would direct the author to Graeber and Wengrow’s recent work for a solid overview of how this narrative is changing:\n\nWengrow, D., & Graeber, D. (2015). Farewell to the ‘childhood of man’: ritual, seasonality, and the origins of inequality. Journal of the Royal Anthropological Institute, 21(3), 597-619.\nGraeber, D., & Wengrow, D. (2021). The dawn of everything: A new history of humanity.\nA few final points, before I get into further reading recommendations. The author really needs to define their terminology more effectively, the use of scaffold and entanglement particularly stand out to me as needing explication. Furthermore, when arguing for minimal capacity inferences, the author needs to effectively address a number of issues, such as potential issues of equifinality and convergence in the mosaics of hominin evolution (issues that are particularly relevant considering the emergence, disappearance and re-emergence of similar artefact assemblages across large spans of time, e.g., Howiesons Poort). Furthermore, I think the author should explain why an artefact should be viewed as an epiphenomenon of underlying capacities (particularly given the ambiguities in the record between artefacts and encephalisation as well as the authors embrace of a niche construction framework). Finally, I think that authors discussion of cognition as skill has a great deal of merit, yet it is underdeveloped and needs to engage much more thoroughly with the existing literature, a few recommendations are:\nIngold, T. (2001). From the transmission of representations to the education of attention. The debated mind: Evolutionary psychology versus ethnography, 113-153.\nIngold, T. (2018). Five questions of skill. Cultural geographies, 25(1), 159-163.\nVan Dijk, L., & Rietveld, E. (2017). Foregrounding sociomaterial practice in our understanding of affordances: The skilled intentionality framework. Frontiers in psychology, 7, 1969.\nIn terms of the introductory sections, I highly recommend the following to help flesh out the nuances of the discourse:\nAdams, F., & Aizawa, K. (2010). The value of cognitivism in thinking about extended cognition. Phenomenology and the Cognitive Sciences, 9: 579–603.\nBarrett, J. C. (2013). The archaeology of mind: it's not what you think. Cambridge archaeological journal, 23(1), 1-17.\nCoolidge, F. L., & Wynn, T. (2016). An introduction to cognitive archaeology. Current Directions in Psychological Science, 25(6), 386-392.\nGallagher, S. (2017). Chapter 2: Variations of Embodied Cognition. Enactivist interventions: Rethinking the mind. Oxford University Press.\nGarofoli, D. (2019). Embodied cognition and the archaeology of mind: A radical reassessment. In Handbook of evolutionary research in archaeology (pp. 379-405).\nGarofoli, Duilio. \"RECkoning with representational apriorism in evolutionary cognitive archaeology.\" Phenomenology and the Cognitive Sciences 17, no. 5 (2018): 973-995.\nHaidle, M. N. (2011). Archaeological Aproaches to Cogntive Evolution. In D. B. Kronenfeld, G. Bennardo, V. C. de Munck, & M. D. Fischer (Eds.), A companion to cognitive Anthropology (pp. 450–467). Oxford: Wiley-Blackwell.\nIliopoulos, A., & Garofoli, D. (2016). The material dimensions of cognition: Reexamining the nature and emergence of the human mind. Quaternary International, 405, 1-7.\nMalafouris, L. (2016). Material engagement and the embodied mind. Cognitive models in Palaeolithic archaeology, 69-82.\nNewen, A., Gallagher, S., & De Bruin, L. (2018). 4E cognition: Historical roots, key concepts, and central issues.\nWard, D., Silverman, D., & Villalobos, M. (2017). Introduction: The varieties of enactivism. Topoi, 36, 365-375.\nFor section 2, I recommend that the author look at the following to help refine their approach to the epistemology of ECA:\nGarofoli, D. (2017). Holistic mapping: Towards an epistemological foundation for evolutionary cognitive archaeology. Journal of Archaeological Method and Theory, 24(4), 1150-1176.\n\nFor section 3, I recommend the following to help flesh out the relationship between 4E approaches and Niche Construction (apologies for recommending my own writing, but it explicitly engages with NCT):\nAntón, S. C., & Kuzawa, C. W. (2017). Early Homo, plasticity and the extended evolutionary synthesis. Interface Focus, 7(5), 20170004.\nAston, A. (2019). Metaplasticity and the boundaries of social cognition: Exploring scalar transformations in social interaction and intersubjectivity. Phenomenology and the Cognitive Sciences, 18(1), 65-89.\nBarona, A. M. (2021). The archaeology of the social brain revisited: rethinking mind and material culture from a material engagement perspective. Adaptive Behavior, 29(2), 137-152.\nBertolotti, T., & Magnani, L. (2017). Theoretical considerations on cognitive niche construction. Synthese, 194, 4757-4779.\nRiede, F. (2019). Niche construction theory and human biocultural evolution. In Handbook of evolutionary research in archaeology (pp. 337-358). Springer, Cham.\nRoberts, P. (2022). Metaplasticity, Not “Modernity”: Uniting the Search for Human Minds, Materials, and Environments. in The Oxford Handbook of Cognitive Archaeology\nSpengler, R. N. (2021). Niche Construction Theory in archaeology: A critical review. Journal of Archaeological Method and Theory, 28(3), 925-955.\nStutz, A. J. (2014). Embodied niche construction in the hominin lineage: semiotic structure and sustained attention in human embodied cognition. Frontiers in Psychology, 5, 834\nI think that at least some engagement with the above readings will then allow for a significant refinement of the arguments put forward in the final two sections of the paper and a far more convincing conclusion.\nA few final points:\nPg. 3 “Cognition is a complex trait.” From my perspective, I see this framing as a category error, akin to saying metabolism is a complex trait. Cognition is a general category that encompasses variable and distinct expressions amongst different organisms.\n\nPg. 7 “Cognition isn’t a complex feature only; it’s also always the feature of someone.” This is a vague and somewhat confusing sentence that needs more explication.\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 source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-375
https://f1000research.com/articles/12-372/v1	05 Apr 23	{ "type": "Research Article", "title": "The role of demographic factors on religious beliefs: Evidence from five countries", "authors": [ "Walton Wider", "Jem Cloyd M. Tanucan", "Xiaole Wu", "Christine Mutua", "Nicholas Tze Ping Pang", "Gabriel Hoh Teck Ling", "Charoline Cheisviyanny", "Jem Cloyd M. Tanucan", "Xiaole Wu", "Christine Mutua", "Nicholas Tze Ping Pang", "Gabriel Hoh Teck Ling", "Charoline Cheisviyanny" ], "abstract": "Background: Individuals' values, attitudes, and behaviours are significantly influenced by their religious beliefs. The present study examines the demographic factors (gender, age groups, religion, employment status, household income, marital status, self-perceived adulthood, and country of origin) that may influence religious beliefs. Methods: The data was collected from September to December 2022 using an online survey. A cross-sectional design was used to collect data from 885 respondents from five countries: Malaysia, the Philippines, China, Kenya, and Indonesia. The survey included questions on five variables related to religious belief: religious certainty, religious culture, religious importance, religious practice, and belief in God. Respondents were asked to rate their level of agreement or disagreement with statements related to each variable on a Likert scale. T-tests and one-way ANOVA were used to analyze the data and determine any significant differences in religious beliefs between the five countries. Results: The results revealed that female respondents held stronger religious beliefs than male respondents. Furthermore, respondents aged 30-45 rated themselves as having a higher religious culture and practice than respondents aged 18-29. Islam groups ranked highest in religious beliefs, followed by Christian, Roman Catholic, and Hindu groups. Full-time students rated themselves as less religious than full-time employed and other groups, while lower-income groups rated themselves as more religious than middle- and upper-income groups. Single groups rated themselves higher on belief in God than cohabiting and in-relationship groups. Conclusions: In conclusion, this study provides evidence that demographic factors influence religious beliefs among respondents from five different countries. These findings can contribute to a better understanding of how religiosity is shaped by various socio-demographic factors and can inform policies and interventions designed to promote religious tolerance and diversity. More research is required to investigate the complex relationships between demographic factors and religiosity, particularly in different cultural and religious contexts.", "keywords": [ "religious beliefs", "religious certainty", "religious culture", "religious importance", "religious practice", "belief in God", "demographic factors" ], "content": "Introduction\n\nReligious beliefs and practices are central to many people’s lives, and frequently play an essential role in shaping communities and individual identities (Schmitz et al., 2020). Christianity, Buddhism, Hinduism, and Islam are among the world’s largest and most widespread religions, and each has undergone significant changes and adaptations over time in response to social and cultural change (Yasin et al., 2020). Christianity has a long and complex history, with many changes occurring since its inception over 2,000 years ago (Crossley, 2008). While fundamental Christian beliefs, such as belief in Jesus Christ's divinity and redemption of humanity through His death and resurrection, have remained relatively unchanged (Bevins, 2019), different Christian denominations have developed different practices, rituals, and interpretations of the faith over time (Byaruhanga, 2015). The Catholic Church is one of the oldest and largest Christian denominations, and it has undergone significant changes and adaptations over the years (Lefebvre and Pérez-Agote, 2018). In response to modern-day challenges, the Catholic Church has taken more progressive positions on social justice, human rights, and environmental protection while still adhering to its core beliefs and traditions (Freston, 2016).\n\nBuddhism, like all other religions, has undergone significant changes and adaptations over time. This religion began over 2,500 years ago in India and has since spread throughout Asia and beyond (Raj, 2022). Today, there are numerous Buddhist schools, each with its own beliefs, practices, and interpretations of the Buddha's teachings (Lee and Tang, 2021). Despite these differences, Buddhism's core beliefs, such as the Four Noble Truths and the Eightfold Path, have remained unchanged and continue to guide the lives of millions of Buddhists worldwide (Mirola et al., 2022). Hinduism, like Buddhism, is an ancient religion that has evolved and changed significantly over time. This religion originated in ancient India and is distinguished by a diverse set of beliefs, practices, and rituals (Sengupta, 2021). Despite its complexity, Hinduism is united by a shared set of core beliefs, such as reincarnation, the existence of multiple gods and goddesses, and the ultimate goal of liberation from the cycle of rebirth (Chandratre and Soman, 2022). Hinduism has adapted and evolved in response to changing social and cultural conditions, developing new practices and interpretations of the faith while preserving its core beliefs and traditions.\n\nLike Christianity, Islam has been around for over 1,400 years and has undergone many transformations. This religion is distinguished by its monotheistic beliefs, strict adherence to the Quran's teachings, and emphasis on the importance of good deeds and community service (Donner, 2010). Today, Islam is one of the world's fastest-growing religions, with a significant presence in many countries (Fardila et al., 2020). Islam has evolved significantly in response to shifting social and cultural conditions, with some communities adopting more progressive stances on issues like human rights, social justice, and gender equality. In contrast, other religions have kept to more traditional interpretations of the faith (Galloway, 2014). In sum, due to interaction with new cultural norms and values, religious tenets and observances are constantly changing and developing (Sosis, 2020). Various factors drive this process, including demographic shifts, technological advancements, and shifting cultural attitudes (Becker et al., 2021). While each religion has a distinct history and set of beliefs, they are all shaped by the same forces of change and are constantly evolving and adapting to meet the changing needs and demands of their communities. Despite these changes, these religions’ core beliefs and practices have largely remained unchanged, and they continue to shape the lives and identities of millions of people worldwide.\n\nStudies that examine how demographic factors such as age, gender, race, and ethnicity influence religious beliefs and practices are scarce. This scarcity is due in part to the difficulty of measuring and defining religious beliefs and practices (Jensen, 2021), and the complexities of investigating the impact of demographic factors on such a sensitive and culturally diverse subject. Future research in this area, however, has the potential to provide valuable insights into how demographic factors shape religious beliefs and how these beliefs, in turn, shape individual and societal outcomes. Religious beliefs significantly impact individuals' and communities' beliefs and values (Van Buren et al., 2020). Understanding the relationships between demographic factors are critical for gaining a better understanding of religious beliefs (Algahtani et al., 2021; Dein et al., 2020; Saeedi et al., 2022; Ulhaq et al., 2020; Yoo, 2022). Public policies, social program, and community initiatives aimed at fostering religious harmony and understanding can benefit from this data. Therefore, it is critical to research religious beliefs and practices in response to demographic factors to advance our understanding of this complex and multifaceted aspect of human life.\n\nGender\n\nNumerous studies on the relationship between gender and religion have yielded mixed results. According to one study by the University of California, Los Angeles, women have a stronger belief in God and higher levels of religious participation than men (Bryant, 2007). This finding is primarily due to traditional gender roles that place women in nurturing and caring roles, which can lead to a greater reliance on religious beliefs for comfort and support. Trzebiatowska and Bruce’s (2012) study discovered that men are more likely to identify as religious leaders or hold positions of power within religious organizations. Other research has found that gender differences in religious beliefs and practices are not universal but rather are influenced by cultural and historical factors (Winkel, 2019). For example, a study conducted in Iran discovered that women play a more prominent role in religious practices than men because they are perceived to have a closer connection to God due to their maternal role (Betteridge, 2002).\n\nAge\n\nResearch has shown that people's religious views shift over time, often due to personal experiences (Butėnaitė, 2019). Older adults are generally more committed to their religious beliefs than younger adults (Meza, 2020). This finding is frequently due to more time spent reflecting on their beliefs and developing a deeper understanding of their religion (Hayward and Krause, 2015). On the other hand, the relationship between age and religious beliefs is more complex. According to some research, as people age, some become more flexible and open to different religious perspectives, while others become more rigid in their beliefs (Schaie and Willis, 1991).\n\nCountries\n\nReligious beliefs vary significantly between countries and regions (Behere et al., 2013). The levels of religious belief in the various religions vary among the five nations of Malaysia, Indonesia, China, Kenya, and the Philippines. It is necessary to examine nation's demographics, cultural influences, and historical backgrounds to determine which religion tends to have the highest-level religious belief.\n\nIndonesia and Malaysia are located in Southeast Asia, and they share many similarities because of their proximity in distance. Islam is the dominant religion in Malaysia and Indonesia, with most of the population identifying as Muslim (Amran et al., 2017; Lee et al., 2020). Approximately 61% of Malaysians identify as Muslim (Ng et al., 2020), with over 90% of the population identifying as Muslim in Indonesia (Lee et al., 2020). Despite the presence of diverse religions such as Christianity, Buddhism, and Hinduism in these countries, Islam significantly impacts the cultures of Indonesia and Malaysia because people of all religions are expected to adhere to specific Islamic rules. The high levels of religious belief in these countries can be attributed to various factors, including the influence of Islamic culture and tradition, and local communities' firm adherence to religious teachings and practices. Muslims, for example, are encouraged to attend the mosque regularly, observe religious holidays and rituals, and pray daily, all of which contribute to a strong sense of religious identity and belief (Hopkins et al., 2023).\n\nConversely, China is predominantly irreligious, with only 14% of the population adhering to religious beliefs (Yang and Huang, 2018). Nonetheless, religious freedom is guaranteed by the constitution. The laws protect individuals' right to believe in - or not believe in - any particular faith (Liang, 2021). It is often difficult to pinpoint what a Chinese person believes in religion. Some religions, such as Buddhism and Taoism, have deep roots in Chinese civilization (Luo and Chen, 2021). Because of China's polytheistic tradition, individuals or even believers may have blurred religious boundaries and worshipped both Buddhist and Taoist gods (Zhang and Lu, 2020; Chen, 2001). Apart from that, individuals who attend religious services may still see themselves as atheists as they do not perceive themselves as belonging to any particular religious institutions and never really associate their conduct with the term “religion”. In their study in Taiwan (China), Zhang and Lin (1992) discovered that while 40% of survey participants claimed they did not believe in God, 70% participated in religious events and rituals such as god-worship. According to Marighella (2020), Chinese millennials may be unaffiliated with forms of religion but are engaging with Buddhism in their lives, not as a religious belief but as a spiritual and cultural journey through life under pressure caused by rapid changes.\n\nKenya is an East African country with the equator running through it. The country shares borders with five other countries: Uganda, Tanzania, Ethiopia, South Sudan, and Somalia. Kenya is governed by the 2010 Constitution, which recognizes the supremacy of the Almighty God and, along with other laws, prohibits religious discrimination and protects religious freedoms. Religion pervades every aspect of African society, with nearly all political, economic, and social activities bearing a religious overtone (Alwala, 2022). Christianity was introduced to Kenya in 1844 by missionaries who settled near Mombasa, and the country now has several Christian denominations (Gathogo, 2020). Islam, on the other hand, is Kenya's second most widely practiced religion (Iyer and Weeks, 2020). Kenyans identify culturally with their tribe or ethnic group, and most members of the same ethnic group share religious beliefs (Lynch, 2006). There are churches in almost every town and village in the country, and most towns have at least one mosque where people can worship. This situation demonstrates the impact and spread of Christianity in Kenya.\n\nReligion is deeply ingrained in the culture and daily practices of many Filipinos in the Philippines. The Filipino people are the tenth most religious in the world, and nearly 87% place a high value on religion (Bagaoisan, 2016). This is because Spain exerted colonial rule over the country for three centuries during which time the dominant religion was Catholicism. The Philippines is one of only two Asian countries with a predominantly Catholic population (Hoh, 2018). Many Filipinos have long relied on various religious beliefs, practices, and attitudes to assist them in their daily lives or to help them cope with the difficulties and problems they face. Gonzalez (2009) stated that many people attend church and other religious events to live morally. Some people turn to religion for assistance, guidance, and miracles (Lagman et al., 2021). Pilgrimages, saints, and charismatic gatherings can also provide access to divine power (Brion et al., 2018). For example, it has become a tradition in many parts of the Philippines to celebrate patron saint feast days and Catholic holidays to celebrate bounty and protection, and to bring families and friends together. According to Bagaoisan (2016), many Filipinos use their religion to cope with issues and problems. When Filipinos have no other option but to turn to God, they usually say, “Ipagpasa-Diyos na natin,” or “they turn over their situation to God” (Cariñgal et al., 2022).\n\nIn conclusion, religious beliefs vary significantly across countries, but Islam and Christianity are more prevalent in Malaysia, Indonesia, and Kenya, while Catholicism is the dominant religion in the Philippines. Religious beliefs are generally weaker in China, with many people viewing religion as a personal choice rather than a necessary part of their daily lives. Finally, the strength of religious beliefs is determined by a complex combination of cultural, historical, and personal factors that cannot be readily determined by looking at statistics.\n\nEmployment status and household income\n\nStudies indicate that unemployed or underemployed people are more likely to have a stronger sense of religious beliefs (Christy et al., 2017). This could be because unemployment or underemployment can cause feelings of powerlessness, uncertainty, and existential anxiety, leading people to seek comfort and stability in religion (Zheng et al., 2020). On the other hand, those who are highly employed and have a higher income may be less likely to participate in religious activities and have a weaker sense of religious beliefs (Lipford and Tollison, 2003). This is because financial security and success can reduce the need for religious comfort and stability (Barber, 2013). Studies conducted during the COVID-19 pandemic revealed that Filipinos relied heavily on their religious beliefs as a means of coping. del Castillo et al. (2021) observed the practice of ‘Dungaw’, or the veneration of sacred images and placing them outside the window of a house or a church, as a coping strategy for difficulties. ‘Dungaw’ in Tagalog means ‘to look’ or ‘to peep out’, and in the context of religious belief, it is often used to describe the idea that saints or Jesus himself is watching over and protecting you from a house or window. Tanucan and Bojos (2021) also noted how Filipinos in slum areas use religion as a source of strength to survive the pandemic's hardships. It is important to note that these are general trends and that each person's experiences and beliefs are unique. However, these findings suggest that an individual's employment status and household income can significantly impact religious beliefs and behaviours.\n\nMarital status\n\nAlthough the effects vary depending on the individual and their cultural context, marital status can significantly impact religious beliefs. Marriage can increase religious involvement in some cases, as couples may attend religious services together and participate in faith-based activities (Kim and Dew, 2019). As partners support and encourage each other in their beliefs, this can form a shared religious identity (Alghafli et al., 2014). Nonetheless, there is evidence that marriage can have the opposite effect, leading to a decline in religious activity when spouses come from divergent religious backgrounds (Elmali-Karakaya, 2022). In these situations, the couple may compromise or take a more eclectic approach to their beliefs. Being single may impact on religious beliefs because one can have more time and freedom to explore different life perspectives or question their beliefs (Apostolou and Christoforou, 2022). Single people may be more likely to seek out supportive communities, including religious communities, to meet their social and spiritual needs (Himawan et al., 2018a, 2018b). Divorce can also significantly impact on religious beliefs because individuals may experience guilt, loss, and anger (Tuttle and Davis, 2015). To sum up, being married can profoundly affect religious beliefs, though these can be nuanced and unique to each individual. It is critical to consider the impact of various life events on our beliefs and be open to new perspectives and experiences.\n\nSelf-perceived adult status\n\nAdulthood is a period in life when people develop a sense of self-identity, self-sufficiency, and personal responsibility (Arnett, 1998; lowe et al., 2013). Perceiving oneself as an adult can significantly impact religious beliefs. Adults are more likely to question their previous beliefs, develop new spiritual perspectives, and engage in various religious experiences (Levenson et al., 2005). Through this process, they may gain a better understanding of their own beliefs, which can shape their religious identity and influence their future religious beliefs. Adults are more confident in their decision-making and less susceptible to external influences, which can significantly impact their religious beliefs (Arnett and Jensen, 2002). Individuals are more likely to make decisions as adults based on their own experiences and understanding of the world rather than blindly following their parents' or peers' beliefs and traditions (Barry and Nelson, 2005). Individuals may question the validity of their previous beliefs or embrace new ones that align more closely with their own values and experiences, which can lead to changes in religious beliefs. One way that seeing oneself as an adult can influence religious beliefs is by fostering greater independence from one's family or community (Bengtson, 2017).\n\nThe purpose of the present research is to investigate the demographic factors (gender, age groups, religion, employment status, household income, marital status, self-perceived adulthood, and country of origin) that may influence the five variables of religious beliefs (religious certainty, religious culture, religious importance, religious practice, and belief in God).\n\n\nMethods\n\nThe research was conducted following in accordance with the ethical principles outlined in the Helsinki Declaration. The research was conducted with the approval of the Scientific and Ethical Review Committee at INTI International University (ref no: INTI/UEC/2021/002). Written informed consent was obtained from all participants before taking part in the study.\n\nThis study used a cross-sectional design and survey methods to investigate the relationship between demographic factors and religiosity variables such as religious certainty, religious culture, religious importance, religious practice, and belief in God.\n\nRespondents for the study came from Malaysia, the Philippines, China, Kenya, and Indonesia. The inclusive criteria for the study include being at least 18 years old and residing in one of the five countries, while the exclusive criteria are respondents under 18, those who do not live in the mentioned countries, those who do not provide informed consent, and those who do not complete the survey. Convenience sampling was used to collect data for the study, as respondents were selected based on their availability and willingness to participate. Co-authors who collected the data were trained to explain the study's purpose and confidentiality, and to respond to questions from respondents. To ensure objectivity and anonymity, the researchers contacted the co-authors to discuss questionnaire administration before data collection. Co-authors were also required to let participants know they could drop out of the study if they did not feel comfortable answering any questions or providing personal information. The sample size was determined based on the guidelines proposed by VanVoorhis and Morgan (2007), which recommend a sample size of at least 30 for each group being compared. The questionnaire was distributed in the local language of each country and was translated as needed. Completed questionnaires were collected via an online survey platform, and missing variables were managed through mean replacement (Hair et al., 2021). Anonymisation of participants was ensured by assigning unique identification numbers to each respondent and using them to link their responses to the data set.\n\nWe adapted the religious belief questionnaires from Barry and Nelson (2005). The only adjustments implemented for the questionnaires were translations into Chinese and Indonesian, catering to participants originating from China and Indonesia, respectively. The English proficiency levels of citizens from Malaysia, Kenya, and the Philippines are relatively high, therefore participants from these countries were given the English version of the questionnaire. The questionnaire was initially translated from English to Chinese and then back-translated to English by three university lecturers proficient in English from Malaysia and China, ensuring consistency in content and terminology. The same process was applied to the Indonesian version of the questionnaire. The translation method employed aligns with the approach recommended by the World Health Organization for this purpose (WHO, 2020). Participants rated four items for religious culture on a scale of ‘not at all important’ (1) to ‘very important’ (4): ‘Suppose someone wanted to know all about you, how important would it be for them to know about your religious beliefs?’, ‘How important is it to you to have friends with the same religious background as you?’, ‘How important is it to you to date people with the same religious beliefs as you?’, and ‘How important is it to you to marry someone who shares your religious beliefs?’. These items were highly correlated (r’s ranging from.60 to.62, p < .01) and were combined to form an internally consistent composite (α = .87).\n\nFor religious practices, participants were asked to rate one item (‘How important is it to you to attend religious services regularly?’) on a scale of ‘not at all important’ (1) to ‘very important’ (4), and then another item (‘How often do you attend religious services?’) on a scale of from ‘about once or twice a year or less’ (1) to ‘at least three or four times a month’ (4). The items were significantly correlated (r = .56, p < 0.01), so they were combined to form an internally consistent composite (α = .71). Participants rated religious certainty on a scale of ‘very uncertain’ (1) to ‘very certain’ (4). Participants rated the following two items for religious importance on a scale of ‘not at all important’ (1) to ‘very important’ (4):’How important are your religious beliefs to you?’ and ‘How important is religious faith in your daily life?’. The religious belief items were significantly correlated (r = .83, p < 0.01) and then averaged to form an internally consistent composite (α = .91).\n\nParticipants rated a single religious belief item on a scale of ‘definitely do not believe this’ (1) to ‘definitely believe this’ (4): ‘To what extent do you believe that God or some higher power watches over you and guides your life?’\n\nThe data’s normality was first examined by referring to skewness and kurtosis. Kim (2013) proposed that skewness 2 and kurtosis 7 be absolute values. The collected data were analyzed using the IBM SPSS Statistic version 25.0 program (https://www.ibm.com/spss). GNU PSPP (https://www.gnu.org/software/pspp/), a freely accessible software, is also able to run the same analysis undertaken in this study. The statistical significance level was set at p < 0.05. Means, standard deviations, and ranges of religiosity variables were used to describe descriptive data. The independent t-test and one-way analysis of variance (ANOVA) were used to determine group differences in religious variables. In ANOVA, we test whether there is a significant difference between the means of three or more groups. If the F-test is significant, indicating that there is a significant difference between the group means, we use post-hoc tests like Tukey's Honestly Significant Difference (HSD) to determine which specific pairs of means are significantly different. Tukey's HSD test is used in ANOVA to provide additional information on where the significant differences between group means lie, allowing for more specific interpretations of the results. In addition, we defined household income as referring to the total incomes received (accumulated) by household members, whether in cash or in kind, that occur repeatedly within the reference period (within a year or more frequently). As household income is a specific measure and can vary widely within a country, and even within a particular region of a country, each co-author was advised to follow the latest government statistics guidelines from their own respective countries. For example, the most recent data on Malaysian household income is from the Department of Statistics' Household Income and Basic Amenities (HIS/BA) survey of 2019. This survey classifies Malaysian households into three income groups: Bottom 40% (B40; lower income), Middle 40% (M40; medium income), and Top 20% (T20; upper income). The B40 group consists of households with an income below RM4,850, while the M40 group includes households with an income ranging from RM4,851 to RM10,970. The T20 group represents households with an income exceeding RM10,970 (DOSM, 2020).\n\n\nResults\n\nA total of 885 participants took part in this study. Of the 885 participants, 52.2% were female, and 47.8% were male; most participants were between 18 and 29 (58.5%). The majority of participants were Buddhist (28.1%), followed by Christians (20.9%), Roman Catholics (17.4%), Muslims (16.5%), Hindus (5.0%), atheists (1.5%), and those who preferred not to answer (10.6%). Regarding employment, 45.6% were full-time students, 44.9% were full-time employees, and 9.5% were others. Regarding household income, most respondents, (61.1%) had a medium income, followed by a lower income (29.6%), and 9.3% had an upper income. In terms of marital status, the majority were single (50.2%), followed by married (27.5%), in-relationship (15.6%), cohabiting (5.1%), and divorced (1.7%). 68.7% of the respondents perceived themselves to be adults. In terms of country of origin, the majority of respondents were from Malaysia (43.1%), followed by the Philippines (20.2%), China (13.9%), Kenya (12.9%), and finally, Indonesia (9.9%). Table 1 summarises the study’s participants’ characteristics. The complete dataset can be found in the underlying data (Wider, 2023).\n\nThe descriptive statistics and normality results for the religiosity variables are shown in Table 2. The skewness and kurtosis values were less than two, indicating that the normality assumption was met for all religious variables. Religious practice had the lowest mean score among the five religiosity variables, followed by religious culture and religious certainty. At the same time, respondents reported high religious importance and belief in God. The mean item scores on our questionnaire ranged from 2.73 to 3.17, with corresponding standard deviations ranging from 0.88 to 0.99. The mean scores indicated that respondents’ religious beliefs were moderate.\n\nThe t-test analysis in Table 3 revealed that female respondents rated higher than male respondents in all religious belief variables, including religious certainty, religious culture, religious importance, religious practice, and belief in God.\n\nThe one-way ANOVA results in Table 4 revealed that only religious culture [F(2,882) = 6.98, p < 0.05] and religious practice [F(2,882) = 12.895, p < 0.01] differed across age groups. However, there were no age differences in religious certainty, religious importance, or belief in God. A post-hoc test using Tukey’s HSD revealed that respondents aged 30-45 perceived a higher level of religious culture and practice than those aged 18-29.\n\nTable 5 shows that religious certainty [F(6,878) = 55.96, p < 0.01], religious culture [F(6,878) = 43.21, p< 0.01], religious importance [F(6,878) = 90.35, p < 0.01], religious practise [F(6,878) = 90.67, p < 0.01], and belief in God [F(6,878) = 98.83, p < 0.01] differed across religions.\n\nA post-hoc test using Tukey’s HSD revealed that Islam groups rated themselves significantly higher than Buddha groups, and Atheist groups and preferred not to answer groups in all religiosity variables, including religious certainty, religious culture, religious importance, religious practice, and belief in God. In terms of religious culture and practice, Christian groups rated themselves higher than all other religions except Islam. On all religiosity variables, Roman Catholic groups rated themselves higher than Buddha groups. Hindu groups rated themselves higher than Atheist groups and preferred not to answer groups on all religious variables. Regarding religious importance, religious practice, and belief in God, Buddhist groups ranked higher than Atheist groups.\n\nThe one-way ANOVA results in Table 6 revealed that only religious practice [F(2,882) = 5.98, p < 0.05] and belief in God [F(2,882) = 4.60, p < 0.05] differed by employment status. However, there were no differences in religious certainty, religious culture, or religious importance based on employment status.\n\nA post-hoc test using Tukey’s HSD revealed that full-time students perceived themselves to have less religious practice than full-time employees and others. Meanwhile, other groups rated their belief in God as higher than the full-time employed groups.\n\nTable 7 shows that religious certainty [F(2,882) = 3.78, p < 0.05], religious culture [F(2,882) = 7.65, p < 0.05], religious importance [F(2,882) = 9.07, p < 0.01], religious practise [F(2,882) = 7.21, p < 0.05], and belief in God [F(2,882) = 6.61, p < 0.05] differed by household income.\n\nA post-hoc test using Tukey’s HSD revealed that lower-income groups rated themselves higher than middle- and upper-income groups on all religiosity variables, including religious certainty, religious culture, religious importance, religious practice, and belief in God. Meanwhile, upper-income groups rated themselves lower on all religiosity variables than middle-income groups.\n\nThe one-way ANOVA results in Table 8 showed that religious culture [F(4,880) = 2.52, p < 0.05], religious practice [F(4,880) = 2.92, p < 0.05], and belief in God [F(4,880) = 3.14, p < 0.05] differed by marital status.\n\nA post-hoc test using Tukey’s HSD revealed that married groups rated themselves higher on religious culture and practice than single and in-relationship groups. Meanwhile, single people rated their belief in God higher than cohabiting people and married people.\n\nThe t-test analysis in Table 9 revealed that respondents who perceived themselves adults rated higher religious culture and religious practice than respondents who perceived themselves not yet adults. In the meantime, there were no significant differences in religious certainty, religious importance, or belief in God based on perceived adulthood.\n\nAccording to the results of the one-way ANOVA in Table 10, religious certainty [F(4,880) = 78.88, p < 0.01], religious culture [F(4,880) = 48.78, p < 0.01], religious importance [F(4,880) = 120.24, p 0.01], religious practice [F(4,880) = 80.39, p < 0.01], and belief in God [F(4,880) = 139.94, p < 0.01].\n\nAccording to a post-hoc test using Tukey’s HSD, respondents from Indonesia and Kenya perceived themselves to have higher religious certainty, religious culture, religious importance, religious practice, and belief in God than respondents from the Philippines, Malaysia, and China. However, for all religiosity variables, there was no significant difference between respondents from Indonesia and Kenya. Furthermore, Chinese respondents scored the lowest in all religiosity variables.\n\n\nDiscussion\n\nThe primary goal of this study is to investigate the demographic factors that may influence religious beliefs, specifically religious certainty, religious culture, religious importance, religious practice, and belief in God. Our findings show that respondents from Malaysia, Indonesia, the Philippines, China, and Kenya have moderate levels of religiosity beliefs. Our findings revealed that female respondents have higher religious beliefs than male respondents in all five variables. Meanwhile, respondents aged 30-45 perceived a higher level of religious culture and practice than respondents aged 18-29. Furthermore, Islam groups rated themselves the highest in religiosity across religions, followed by Christian groups, Roman Catholic groups, and Hindu groups. In contrast, Buddhist and Atheist groups rated themselves the lowest, with ‘prefer not to answer’ groups in between. In terms of employment status, our findings revealed that full-time students rated themselves as having less religious practise than full-time employed and other groups, while other groups rated themselves as having more belief in God than full-time employed groups. Regarding household income, lower-income groups rated themselves more religious than middle- and upper-income groups. In terms of marital status, married groups rated themselves higher on religious culture and practice. In contrast, single groups rated themselves higher on belief in God than cohabiting and in-relationship groups. Respondents who perceived themselves as adults rated themselves higher in religious culture and practice. In terms of countries, Indonesian and Kenyan respondents perceive religiosity as being higher than respondents from the Philippines, Malaysia, and China. However, there is no significant difference between Indonesia and Kenya, with China scoring the lowest in all religiosity variables.\n\nThe low mean score of religious practice among respondents in the current study may be attributed to changing cultural and societal norms. People are becoming more secular in many modern societies, and their religious beliefs and practices are becoming less important to them (Jensen, 2021). This situation is especially true for younger generations growing up in a world where religion is no longer regarded as the dominant force it once was (Voas and Fleischmann, 2012). Many countries’ growing diversity of cultures and religions can be attributed to the lower mean score for religious culture. People are becoming more exposed to various beliefs and practices, which may make them less likely to identify with a specific religious culture. The low mean score of religious certainty among respondents may reflect the increasing scepticism and critical thinking in modern societies. People are increasingly sceptical of religious claims and seek evidence and rational explanations for their religious beliefs. The high mean score for religious importance is frequently interpreted as a reflection of religion’s continued relevance in people’s lives. Despite the decline in religious practice and certainty, many people still regard religion as an essential part of their lives and plays a vital role in their identity, values, and worldview. The high mean score of respondents’ belief in God may reflect religious belief’s resilience in the face of declining religious practise and certainty. Despite the challenges posed by secularism and critical thinking, many people continue to believe in God.\n\nAccording to our findings, females rate religious certainty, religious culture, religious importance, religious practice, and belief in God higher than males. This finding is mainly due to social and cultural factors that have traditionally shaped men’s and women’s roles in various societies. Traditionally, women have been encouraged to be more spiritual and less assertive, emphasizing nurturing, compassion, and community building (Wider et al., 2021). This has resulted in the formation of strong religious beliefs and a high level of participation in religious activities, as well as a stronger sense of connection to God.\n\nAccording to our findings, respondents aged 30-45 rated religious culture and religious practice higher than respondents aged 18-29. This phenomenon is caused by differences in life and developmental stages between the two age groups. Those between the ages of 30 and 45 are in the established adulthood stage, in which they have reached a level of stability in their lives and are looking for meaning and purpose (Mehta et al., 2020). As religion provides a sense of grounding and security, this frequently increases religious culture and practice. Individuals aged 18-29, on the other hand, are in the emerging adulthood stage, where they are still exploring their identity and are more focused on personal growth and self-discovery (Arnett, 2015). This period of self-discovery may result in a decreased emphasis on religion as people seek answers from other sources. Furthermore, cultural and societal factors may play a role. People may be exposed to more traditional and conservative religious practices as they age, resulting in a greater adherence to religious norms. Furthermore, as individuals settle into family life and raise children, family and community pressure to maintain religious beliefs may increase. The link between adult self-perception and religious culture and practise stems from a desire for structure, support, and meaning in one’s life. Adults can find comfort, stability, and a sense of community and purpose, through religious engagement. Religious culture and practice have long been viewed as fundamental components of adult life and identity, as they are frequently viewed as a means of finding meaning and purpose in their lives. Adults who consider themselves mature and responsible are more likely to engage in religious practices and identify with a religious culture because they need structure and order. Religious beliefs and practices frequently provide a moral code and a set of values that assist people in making decisions and navigating life’s challenges. This sense of security and guidance is essential for many adults in a chaotic and unpredictable world. Religious activities also provide opportunities for social interaction and support, which can benefit those struggling to find a sense of belonging or connection in other areas of their lives.\n\nVarious cultural, social, and personal factors influence religious beliefs and practices. Our findings show that different religious groups rate their level of religiosity, emphasizing the diversity of religious expression and identification. Unsurprisingly, Muslim groups ranked highest, given Islam’s emphasis on individual religious devotion and communal practice. Similarly, Christianity and Catholicism place a premium on religious practices and beliefs. Buddhism and Atheism, on the other hand, as well as groups that prefer not to respond, may place less emphasis on religious expression or have different interpretations of what it means to be religious. These findings imply that religious affiliation and expression are multifaceted and influenced by individual and group-level factors.\n\nBeing a full-time student can be a central part of one’s identity, competing with or even superseding religious identity. People’s religious identities may become more visible as they advance in their careers or other roles. Full-time students are frequently younger and in a different stage of life than employed people and other groups, and these differences impact on religious beliefs and practices. Younger people may be in a period of exploration and questioning, which causes them to rate their religious practice lower. Older people, on the other hand, may have had more time to solidify their beliefs and a stronger sense of commitment, resulting in higher ratings of belief in God.\n\nSeveral factors can be attributed to lower-income groups rating themselves as more religious than middle-and upper-income groups. Religion can provide comfort and hope to people facing difficult circumstances such as poverty, a lack of opportunities, and social inequalities. Religious beliefs and practices can provide stability and meaning for many people from lower-income groups, allowing them to cope with daily stress and challenges. Furthermore, lower-income groups are frequently more closely connected to their communities, including religious communities, which may contribute to their perceived religiosity. Religious communities can offer social support, a sense of belonging, and a common goal. Furthermore, these communities can provide practical assistance, such as food banks and housing programs, which can be especially helpful for those struggling to make ends meet. Finally, lower-income groups may see religion as a source of empowerment and a means to challenge established power structures. In the face of socioeconomic inequalities and discrimination, religion can allow many people to assert their beliefs, identities, and cultural heritage.\n\nThe difference in religious ratings between married and single groups may be due to societal norms and expectations surrounding marriage. Marriage is frequently regarded as a formal commitment, associated with religious and cultural traditions. As a result, married people may feel pressure to present themselves as more religious or to prioritize their religious practices. Single people, on the other hand, may not feel the same social pressure to conform to religious norms and thus feel freer to express their personal beliefs without the same level of social expectation. Individuals’ higher belief in God may also reflect a more individualistic approach to spirituality and religion. Meanwhile, cohabiting and in-relationship groups may have different attitudes toward religion and spirituality than married and single groups because they do not see their relationships as formal commitments like married people do.\n\nThe complex interplay of cultural, historical, socioeconomic, and political factors can explain the different perceptions of religious beliefs among our study’s respondents from different countries. For example, Indonesia and Kenya are predominantly Muslim and Christian, respectively, and these religions have deep roots in these countries’ cultural and historical fabric. In contrast, the religious landscapes of the Philippines and Malaysia are more diverse, with a mix of Christian, Muslim, and indigenous beliefs. Due to this diversity, individuals may not identify as strongly with a particular religion, which may result in a lower overall perception of religiosity. Furthermore, socioeconomic factors can influence religious perceptions. Individuals may prioritize other aspects of life, such as education and career, over religion in countries such as China, where there has been a rapid modernization and secularization process. This shift in values and priorities may result in a negative perception of religiosity. It is also worth noting that each country’s political and social context may influence religious perceptions. In some countries, for example, restrictions or limitations on expressing religious beliefs, may result in a lower overall perception of religiosity. Religious beliefs may be more openly displayed and incorporated into daily life in other countries, leading to a higher overall perception of religiosity.\n\nThis study proves that religiosity is a complex phenomenon influenced by various demographic factors. The study’s findings show that gender, age, religion, employment status, household income, marital status, and self-perceived adult status all contribute to one’s level of religiosity. The study’s results imply that religious beliefs are influenced by more than just an individual’s set of convictions and the quality of their spiritual experiences. The findings of this study have important practical implications for religious studies and religious communities. The study’s findings, for example, suggest that religious communities should consider the demographic factors that influence religiosity when developing programmes and initiatives to engage people in religious activities. Religious communities may need to tailor their program to appeal to specific age or gender groups. Furthermore, religious communities may need to consider the impact of employment status and household income on religiosity in order to design programmes that are accessible and appealing to people from various socioeconomic backgrounds. Important implications for policymakers and governments can also be drawn from the study’s finding. For example, governments may need to consider different demographic groups’ religious attitudes and practices when policies affect religious communities. Governments may need to consider the impact of education and employment policies on religious practices and ensure that policies do not discriminate against specific religious groups.\n\n\nConclusions\n\nThis study provides important insights into the impact of demographic factors on religious beliefs, which can help guide future research in this area. However, there are some limitations to this study. This study is limited to the five countries chosen and may not reflect the religious beliefs of people in other countries. Because different countries and populations have different cultural and religious beliefs, the findings cannot be generalized. The sample size used in this study for each country may also be considered small and uneven, potentially leading to bias in the results. To obtain a clearer and more comprehensive picture of religiosity beliefs, future research should consider expanding the sample size and including a more diverse range of countries and religious beliefs. Qualitative methods, such as in-depth interviews or focus groups, can also provide a more in-depth understanding of the participants’ religious beliefs and experiences. Overall, the findings of this study provide a preliminary understanding of the demographic factors that may influence religious beliefs in five different countries. Our results indicate that factors such as gender, age, religion, employment, household income, marital status, self-perceived adult status, and country of origin could influence religious beliefs. Further investigation is vital to examine religious beliefs’ underlying mechanisms and patterns in various cultural and social contexts.", "appendix": "Data availability\n\nZenodo: The Role of Demographic Factors on Religious Beliefs: Evidence from 5 Countries, https://doi.org/10.5281/zenodo.7758778 (Wider, 2023).\n\nThe project contains the following underlying data:\n\n• FULL DATA.sav (Raw data for the manuscript titled: The Role of Demographic Factors on Religious Beliefs: Evidence from 5 Countries).\n\nZenodo: The Role of Demographic Factors on Religious Beliefs: Evidence from 5 Countries, https://doi.org/10.5281/zenodo.7758778 (Wider, 2023).\n\nThis project contains the following extended data:\n\n• Questionnaire 1.docx (Blank English questionnaire for this study).\n\n• Questionnaire 2.docx (Blank Indonesian questionnaire for this study).\n\n• Questionnaire 3.docx (Blank Chinese questionnaire for 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\nAlgahtani FD, Alzain MA, Haouas N, et al.: Coping during COVID-19 pandemic in Saudi community: Religious attitudes, practices and associated factors. 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Psychol. 2020; 11: 1636. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "189240", "date": "26 Jul 2023", "name": "Ross Gore", "expertise": [ "Reviewer Expertise Data science", "predictive analytics", "modeling", "simulation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 uses data collected from an online survey in late 2022 from 5 non-western countries. The survey included questions on five variables related to religious belief: religious certainty, religious culture, religious importance, religious practice, and belief in God. The authors found that: (1) female respondents held stronger religious beliefs than male respondents; (2) respondents aged 30-45 rated themselves as having a higher religious culture and practice than respondents aged 18-29; (3) Islam groups ranked highest in religious beliefs, followed by Christian, Roman Catholic, and Hindu groups; (4) full-time students rated themselves as less religious than full-time employed and other groups; (6) lower-income groups rated themselves as more religious than middle- and upper-income groups and (7) single groups rated themselves higher on belief in God than cohabiting and in-relationship groups. The authors conclude that study provides evidence that demographic factors influence religious beliefs among respondents from five different countries.\nGeneral comments:\nThe methodology and the analysis of the paper seems technically correct. However, two things several comments come to mind.\nThe first is that this analysis appears very exploratory in nature. The authors collected some data analyzed it and found demographic differences. Given the large body of work on different demographic groups and religiosity this does not seem particularly novel or surprising. Furthermore, because the authors did not use any type of confirmatory analysis (i.e. confirmatory factor analysis or measurement of factor invariance) it is unclear to what extent some of these relationships are artifacts of a data collection exercise. The paper would be improved by either: (1) registering a number of hypotheses about the data set based in theory before the analysis and then testing them or (2) hypothesizing a structure of latent variables in the data and then confirming that structure and measuring its invariance analytically. Several studies below that have taken this tact on similar questions are below.\n\nNext, the paper would be improved by identifying to what extent the data collected for the 5 countries is different from all the religiosity data and analyses conducted on western countries. The data set the authors collected is interesting because of the non-western religions practiced in these countries. Identifying to what extent certain demographic views of religiosity (wether it be eastern or western religion) are cross-cultural in the east and west and which other demographic views depend entirely on if the religion being practiced is eastern or western.\n\nThe authors provide the raw data as well as their questionnaires. This is useful however, they do not provide a script that could be run to transform the raw data into the structure used for their analysis. Without providing the ability to see the data throughout the analytic process the study is not replicable. Providing a copy of the IRB or Ethical Review Committee documents would also help others repeat the study.\n\nThe paper would be improved by visualizing the analysis of the data and different demographic groups. This will help readers understand the shape and distribution of the results.\n\nThe authors provide a discussion of limitations in the conclusion section. They are to be commended for this. However, one limitation/bias that is not measured is that all participants were willing to take an online survey. Studies have shown that this can lead to recruitment of different demographic groups than a truly random sample. Stating this as an additional limitation would strengthen the paper. Several of these studies are identified below.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "211449", "date": "20 Oct 2023", "name": "L C", "expertise": [ "Reviewer Expertise Psychometrics", "Psychology of religion", "Religious Fundamentalism" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 want to express my gratitude for giving me the opportunity to review this manuscript. The time spent creating and submitting it is greatly appreciated. However, I believe that there are several areas that require improvement for manuscript’s potential publication.\n\nAbstract:\n\nPlease add the background, in order to understand the rationale of the study.\nIntroduction:\nPage 1-Line 1-34: If the paper's objective is to study the role of demographic factors in religious beliefs, it is not clear why the characteristics of the religions mentioned in the introduction are being examined.\n\nPage 1: “Studies that examine how demographic factors such as age, gender, race, and ethnicity influence religious beliefs and practices are scarce”. Which are these studies?\n\nPage 1: “Future research in this area, however, has the potential to provide valuable insights into how demographic factors shape religious beliefs and how these beliefs, in turn, shape individual and societal outcomes”. Which are these outcomes?\n\nPage 2: Demographic factors and religious beliefs regarding gender and age. Are there other recent studies?\n\nPage 3: “In conclusion, religious beliefs vary significantly across countries, but Islam and Christianity are more prevalent in Malaysia, Indonesia, and Kenya, while Catholicism is the dominant religion in the Philippines. Religious beliefs are generally weaker in China, with many people viewing religion as a personal choice rather than a necessary part of their daily lives. Finally, the strength of religious beliefs is determined by a complex combination of cultural, historical, and personal factors that cannot be readily determined by looking at statistics”. The authors could consider adding more adequate references to support their claims.\n\nPage 4: At the end of the introduction, the study's relevance should be better articulated before presenting the hypothesis\nMethods:\n\nInstrument: What are the original psychometric characteristics of the instrument used in the study? Moreover, is Cronbach's alpha referenced in this study?\n\nStatistical analyses: The statistical analyses must be consistent with the study's objectives outlined in the introduction and its corresponding hypotheses.\n\nObjectives: “The purpose of the present research is to investigate the demographic factors (gender, age groups, religion, employment status, household income, marital status, self-perceived adulthood, and country of origin) that may influence the five variables of religious beliefs”. If the authors are discussing variables that influence other variables, I would expect regression models or structural equation models in the statistical analyses.\nResults:\n\nThe statistical analyses about the adaptation of the instrument used in the study are missing.\nDiscussions:\nIn the discussion section, a comparison of its review with the initial contextualization is not made. Avoid introducing literature that has not been previously mentioned in the introduction, as it can create an incomplete review of the state of the investigated topic.\n\nIt would be interesting to create a personality profile based on demographic factors related to religious beliefs.\n\nCheck the format of the in-text references to ensure that it is consistent with the journal's criteria.\n\nI want to express my gratitude for giving me the opportunity to review this manuscript. The time spent creating and submitting it is greatly appreciated. However, I believe that there are several areas that require improvement for manuscript’s potential publication.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "211439", "date": "08 Nov 2023", "name": "Daniel Major-Smith", "expertise": [ "Reviewer Expertise Epidemiology", "anthropology", "psychology of religion." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study uses cross-sectional data collected from an online survey of five countries in Asia and Africa. The survey asked questions on a broad range of religious measures including: belief, certainty, culture, importance, and practice. These five factors were measured on a four-point Likert scale (with some religious measures a composite of multiple questions) and t-tests and one way ANOVAs were performed on the means of the data categorised by different demographics. The authors found that, amongst other things: female participants on average held higher levels of religious beliefs than male responders, those aged 30 – 45 were more religious than other age groups, and Muslim participants ranked themselves the highest in religious beliefs compared to other religions. We believe this paper contributes well to the understanding of under-studied religious groups in the wider literature, and appreciate that the authors shared their data – this will be incredibly helpful for readers, and was very useful for us when reviewing this manuscript to explore the data and analyses. However, there are some issues with the paper that we believe would need to be improved before the paper can be approved for indexing.\nMajor Recommendations:\n1) While the aim of the paper – to explore demographic associations with religiosity in 5 under-studied countries – is laudable, much more information on the samples from each country is necessary to know how representative these samples are. For instance, although the overall gender ratio is approximately 50:50 in the aggregated sample combining all countries together, when stratified by country some have a very biased response rate (e.g., 78% females from Indonesia). Similarly, in the full sample nearly 50% of respondents are students, which suggests that these samples are far from representative of these countries. More information on how sampling was conducted, the sample demographics of each country, and how similar these are to the demographics of the whole country (e.g., from census data) is necessary to help understand and interpret the study. As the study was conducted via an online survey (and in English for Malaysia, Kenya and the Philippines), this is likely to bias the sample to younger, more educated, individuals proficient in English, from richer and more urban areas with internet access. Non-representative sampling can result in biased estimates of religiosity, in addition to biased association of religiosity with these demographic factors of interest, so exploring this is essential for helping readers to understand and appropriately interpret the results.\n2) The aggregated results combining all countries together can result in misleading associations if there are differences in sample size, demographics and religiosity between countries (which there are in this study). This is an example of ‘Simpson’s paradox’ (see https://en.wikipedia.org/wiki/Simpson%27s_paradox). For instance, nearly half of the sample is Malaysian, and levels of religiosity are generally lower in Malaysia, which can bias and give misleading results. As an example, in table 6 there appears to be little/no association between religious certainty and employment/student status; however, if we repeat these analyses including ‘country’ as a covariate in a model (i.e., adjusting for differences between countries), then there is a strong negative association between being a student and religious certainty, compared to those in full-time employment. Similar results are found if we repeat these analyses separately within each country. This example is not confined to employment, as the interpretation of many results presented in this paper change when adjusting for, or stratifying by, country.\nAs these aggregated results can give misleading conclusions, we recommend repeating these analyses both with ‘country’ as a confounder, and separately within each country, and discussing if/how results differ from the original aggregated results. This will provide some assurance that these results are robust, while also highlighting some potential country-level differences which will be interesting for readers (e.g., some associations between gender and religiosity appear to vary between countries, which is very interesting and suggests that the female-bias of religiosity is not replicated in all countries). As these new analyses will alter some of the interpretation of existing results, some of the discussion may have to be re-written as well. We realise that this will require a fair amount of work, but it is important to ensure that the results presented in the paper are robust and accurate; many of these additional tables of these results can go in the supplementary information, where interested readers can find them.\n3) Another issue is the use of skewness and kurtosis to measure the normality of the data. As the number of values in the religiosity data is so low (from 4 to 13), these statistics are unlikely to be measuring anything valid. It is practically impossible for ordinal data with four (or even 13) categories to be normally distributed, and data that is categorised this way shouldn’t be subjected to tests that are designed for continuous data. We suggest inspecting the data visually before conducting tests and choosing appropriate analyses thereafter. For instance, see here for histograms of each of the 5 religiosity variables, and it is clear that none of these are normally distributed, regardless of the skewness and kurtosis values.\n\nRelatedly, we also disagree with the use of thresholds for p-values to dictate whether a result is ‘significant’ throughout the paper. This is especially problematic as the paper seems to be largely descriptive with few aims and no hypothesis. The use of p-values is not inherently a poor practice but the use of thresholds to arbitrarily decide whether a result is ‘significant’ or not is problematic. For example, in table 6 comparing employment groups across different religious variables, religious culture has a p-value of 0.055 which is essentially no different than a ‘significant’ value of 0.05. Yet the manuscript claims that “there were no differences in […] religious culture […] based on employment status”.\nThis leads onto the second issue surrounding the reporting of p-values in this paper; all p-values should be reported as the specific value rather than just saying that the value is lower than threshold (e.g., <0.05, or <0.01) and is significant. This makes it very difficult to compare p-values between models and further emphasises the reliance on arbitrary thresholds. P-values should always be reported unless they are equal to zero at your chosen number of decimal places i.e. <0.001.\nRather than choosing a p-value level to consider results “significant” at, we believe it would be more conducive to adding to the literature to consider the p-value a continuous measure of the evidence against the null hypothesis of no difference between categories (Sterne & Davey Smith, 2001) and interpret your results based on additional statistics as well (e.g., mean differences, 95% confidence intervals, etc.) which provide a more detailed and nuanced view of the data.\nMinor Comments:\nThe phrasing of different religious groups in some places in the text. When referring to the religion ‘Islam’ should be used and when referring to participants ‘Muslim’ or a variation should be used. Also when referring to those who follow Buddhism, ‘Buddhist groups’ makes more sense than ‘Buddha groups’ e.g. In the section of the results about table 5.\n\nOverall considering the aims and data used in this paper, it lends itself to a more descriptive paper rather than a causal one trying to find causal relations between variables in the data. This is reflected throughout most of the paper, although some areas could be improved upon in this aspect. For example, in the conclusion section of the abstract it says that “this study provides evidence that demographic factors influence religious beliefs”. However, as the study is cross-sectional it is difficult establish ‘influence’ (or ‘cause’) based on these associations; instead, these data simply show that there are differences in religious beliefs associated with these demographic factors. While it is possible that marital status (for instance) may cause differences in religion, it is also possible that religion may cause marital status (e.g., religious individuals being more likely to get and/or remain married); many these associations could also be explained by confounding factors (e.g., by age and/or gender). More cautious language could be used here and throughout the discussion to show awareness of these alternative explanations. Given that these data are purely cross-sectional and descriptive, it is difficult to say that “this phenomenon is caused by” for example in the fourth paragraph of the discussion. This is an issue throughout the discussion in the breakdown of each demographic factor as results are interpreted in causal terms, even though the results do not necessarily warrant such an interpretation.\n\nWhile these results are interesting, it is not clear how they can “inform policies and interventions designed to promote religious tolerance and diversity” (from the abstract, and mentioned again in the introduction and discussion). More information supporting this statement is needed, else it can probably just be removed from the paper.\n\nStudies that examine how demographic factors such as age, gender, race, and ethnicity influence religious beliefs and practices are scarce.” (Introduction, page 3). We are not sure that studies on this area are that rare, as there has been plenty of work on this (e.g., Crockett & Voas, 2006; Major-Smith et al., 2022; Levin et al., 1994, to name just a few). Also, some of this work is discussed in the subsequent paragraphs, meaning work in this area is not especially scarce. Although perhaps less research on these topics has been conducted in non-Western parts of the world (although see Vardy et al., 2022 for a cross-cultural study specifically on gender)? Acknowledging this existing research will not detract from this paper.\n\nWhen discussing and interpreting the associations between age and religion, keep in mind that as this research is cross-sectional it is not clear whether differences in age are due to people getting more religious as they get older or cohort effects due to comparing different generations (who may have differing levels of religiosity, but are stable with age). In Western countries it appears that these cohort effects may be stronger than changes with age within individuals (Crockett & Voas, 2006; Major-Smith et al, 2023).\n\nWhen discussing religion in China, the statement that “religious freedom is guaranteed by the constitution” may be technically true, it is far from always followed in practice and there is a history of religious repression in this country (https://www.pewresearch.org/religion/2023/08/30/government-policy-toward-religion-in-the-peoples-republic-of-china-a-brief-history/).\n\n“Finally, the strength of religious beliefs is determined by a complex combination of cultural, historical, and personal factors that cannot be readily determined by looking at statistics” (introduction, page 5). This seems a strange ending to this sentence, as this is precisely what the authors are trying to do in this paper! Perhaps more accurate to say “…and personal factors that can be difficult to separate”?\n\nMuch of the introduction (and discussion) seems to present associations between demographic factors and religion as if they were universal and do not vary by country. E.g., “Studies indicate that unemployed or underemployed people are more likely to have a stronger sense of religious beliefs” (introduction, page 5); this may be true in some populations, but in others (e.g., in the Southwest UK) lower socioeconomic position and income is associated with lower levels of religiosity (Major-Smith et al., 2022). Similarly, when discussing gender and religion, although in general women are more religious, this does vary by population (Vardy et al., 2022). Acknowledging these nuances would help situate your research better in this field.\n\n“Studies conducted during the COVID-19 pandemic revealed that Filipinos…” (introduction, page 5). Not clear how this part relates to the section heading of ‘Employment’?\n\nWe feel the ‘Objectives’ section could be strengthened by mentioning what this paper adds to the existing literature (e.g., most research has been on Western and Christian samples, so need to explore these questions in non-Western and non-Christian samples to see how generalisable these findings are).\n\nMean replacement of missing data (Methods, page 6). This is not a valid approach for imputing missing data and can lead to systematic biases in the data (van Buuren, 2018). Instead, please just describe the levels of missing data in each of the variables to readers. If the amount of missing data is substantial, discuss how this may impact your study.\n\nWhat online survey platform was used to conduct the research?\n\nWhy were the religion questions from Barry and Nelson (2005) used? E.g., Do they have high psychometric validity? There are a large number of validated scales of religiosity; please justify the choice of these specific questions. “Participants rated religious certainty on a scale of…” (Methods, page 7). Please provide the specific wording for this question in the text.\n\n“The religious belief items were significantly correlated…” (Methods, page 7). We think this is a typo and is supposed to be ‘religious importance’?\n\nFrom the descriptive statistics we don’t quite understand how 28% of the sample are Buddhist, seeing as Buddhism is not the main religion in any of these countries. When splitting religion by country, it appears that 60% of Malaysians are Buddhist, despite only around 20% of the Malaysian population being Buddhist (https://en.wikipedia.org/wiki/Religion_in_Malaysia). Similarly, rates of Islam (13%) are much lower in the sample compared to in the whole of Malaysia (~65%). Can the authors explain this discrepancy? E.g. was the Malaysian study conducted in an area with a majority Buddhist population? Or perhaps the coding of ‘Islam’ and ‘Buddhist’ have been accidentally swapped around during data processing?\n\nWhy are 'Christians' separate from Roman Catholics, as both are Christian? Would be helpful to justify this and say what Christian denominations are included in 'Christian'. Perhaps also rename to 'non-Catholic Christians', for clarity?\n\n“The mean scores indicated that respondents’ religious beliefs were moderate.” (results, page 9). It is not clear what ‘moderate’ means here. Compared to what? Relatedly, in the discussion the authors compare between these religiosity variables as ‘high’ and ‘low’, yet it is not clear that: i) the mean scores between these different items are directly comparable, as they based on different response scales; and ii) most mean values of all religiosity facets are around 3, so there doesn’t appear to be much difference between these.\n\nOught to report the results any of post-hoc tests for readers to view (this can go in the supplementary information).\n\nThe paragraph under the heading “differences of religiosity variables based on country of origin” is not a complete sentence.\n\nWe would suggest being cautious when discussing differences by religion, as these are difficult to separate from country-level differences (e.g., although Muslims were most religious on average, there were differences in religiosity between Muslims by country, with Malaysian Muslims being less religious than Indonesian Muslims).\n\nRather than embedded within the conclusion, limitations should be discussed in a separate 'limitations' section within the discussion (the conclusion should just summarise the key take-home messages). There also needs to be greater discussion of some of these limitations, particularly regarding the sample characteristics, how representative they are of the target country/population (large bias towards students, for instance, and online survey design likely misses important parts of the population), alternative explanations (e.g., reverse causality and confounding), and the implications of these for the authors interpretations.\n\nIt would also have been useful to discuss how these results compare to other countries (e.g., Western societies) and if there are any differences, to integrate these findings better within the wider field.\n\nThere is also some mischaracterisation of the data throughout the paper. Generally, t-tests and ANOVAs are best used on continuous data and may not give valid results when using ordinal data as there is no implication of distance between values. While this practice of analysing ordinal data with linear models is common, this can lead to bias and erroneous conclusions (Bürkner et al., 2019). This should at least be acknowledged in the methods and limitations sections, and perhaps some results with only 4 categories repeated using ordinal models to ensure that there is no change in interpretation.\n\nLiterature cited in review:\nBürkner, P. C., & Vuorre, M. (2019). Ordinal regression models in psychology: A tutorial. Advances in Methods and Practices in Psychological Science, 2(1), 77-101.\nCrockett, A., & Voas, D. (2006). Generations of decline: Religious change in 20th‐century Britain. Journal for the scientific study of religion, 45(4), 567-584.\nLevin, J. S., Taylor, R. J., & Chatters, L. M. (1994). Race and gender differences in religiosity among older adults: Findings from four national surveys. Journal of gerontology, 49(3), S137-S145.\nMajor-Smith, D., Morgan, J., Halstead, I., Tohidinik, H. R., Iles-Caven, Y., Golding, J., & Northstone, K. (2022). Demographic and socioeconomic predictors of religious/spiritual beliefs and behaviours in a prospective cohort study (ALSPAC) in Southwest England: Results from the parental generation. Wellcome Open Research, 7.\nMajor-Smith, D., Halstead, I., Morgan, J., Tohidinik, H. R., Iles-Caven, Y., Golding, J., & Northstone, K. (2023). Individual-level changes in religious/spiritual beliefs and behaviors over three decades in the parental generation of the ALSPAC cohort, UK. Religion, Brain & Behavior, 1-20.\nSterne, J. A., & Smith, G. D. (2001). Sifting the evidence—what’s wrong with significance tests?. BMJ, 322, 226-31.\nVan Buuren, S. (2018). Flexible imputation of missing data. CRC press.\nVardy, T., Moya, C., Placek, C. D., Apicella, C. L., Bolyanatz, A., Cohen, E., ... & Atkinson, Q. D. (2022). The religiosity gender gap in 14 diverse societies. Religion, Brain & Behavior, 12(1-2), 18-37.\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? 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/12-372
https://f1000research.com/articles/12-371/v1	05 Apr 23	{ "type": "Systematic Review", "title": "Benefit of Asian pigmented rice bioactive compound and its implication in breast cancer: a systematic review", "authors": [ "Wirdatun Nafisah", "Alexander Patera Nugraha", "Aditya Nugroho", "Andi Isti Sakinah", "Duano Sapta Nusantara", "John Philia", "Mohammad Iqbal Kurniawinata", "Wirdatul Aini", "Vika Tresnadiana Herlina", "Tengku Natasha Eleena binti Tengku Ahmad Noor", "Wirdatun Nafisah", "Aditya Nugroho", "Andi Isti Sakinah", "Duano Sapta Nusantara", "John Philia", "Mohammad Iqbal Kurniawinata", "Wirdatul Aini", "Vika Tresnadiana Herlina", "Tengku Natasha Eleena binti Tengku Ahmad Noor" ], "abstract": "Background: Utilizing the bioactive compounds found in pigmented rice might significantly reduce the risk of breast cancer. This study aims to systematically review existing literature on the benefit of Asian pigmented rice bioactive compounds and their implication in breast cancer. Methods: Searches of the literature were conducted in two databases (Scopus and PubMed) for a systematic review. The keywords resulted in a total of 407 articles, consisting of 103 PubMed and 304 Scopus articles. 32 manuscripts were excluded because the article was over 10 years old. After excluding book chapters and non-English languages, we had 278 potential articles to be reviewed. After checking and screening the title and abstract and eliminating duplicate articles, then 66 articles were obtained. After the selection and elimination of the full-text manuscripts, finally 10 of them which met the inclusion criteria. Result: The included studies in this review were entirely based in Asia. The year of publication ranged from 2013 to 2020. Half of included studies used black rice extract, two used red jasmine rice extracts, and three used Korean rice extracts (black, red, dark purple and brown rice). All studies were conducted in vitro and three studies were compared with in vivo tests on female mice. The pigmented rice is mainly black, red, and dark purple rice, and contains a variety of peonidin-3-glucoside, cyanidin-3-glucoside, γ-oryzanol, γ-tocotrienol, proanthocyanidin, cinnamic acid, and anthocyanins that may act as pro-apoptotic, anti-proliferative, and anti-metastasis of the breast cancer cells.\nConclusion: Pigmented rice is a beneficial food which possessed bioactive compounds that may have significant potential concerning a breast cancer.", "keywords": [ "medicine", "cancer", "non-communicable disease", "rice", "food" ], "content": "Introduction\n\nMost deaths throughout the world are now caused by non-communicable diseases (NCDs).1 The recognition of the challenges of NCDs has become a global spotlight as stated in the sustainable development goals (SDGs) on the target 3.4 by 2030, reduce by one-third pre-mature mortality from NCDs through prevention and treatment, and promote mental health and wellbeing.2 Cancer is one of NCDs that has become one of the leading causes of death worldwide with an estimate of 10.0 million deaths in 2020.3\n\nAn estimated 10.0 million people will die from cancer, a multifactorial illness, in the globe in 2020.4 Breast cancer is the most diagnosed cancer type found in women and poses a serious public health concern globally.5 An estimated breast cancer occurrence is 2.089 million in 2018 which is reported to increase continuously in all countries of the world.3 There are major risk factors for breast cancer, such as aging, family history, reproductive factors, and life style.6 Some of the factors are beyond individual controls and some are not. The factors that are within an individual’s control that can be altered to minimize the chance of getting breast cancer, include diet and lifestyle.7 Different food patterns may be one of the most fundamental factors for this.8 Rice is a staple food of dietary calories for half of humanity and has been widely demonstrated as a chemopreventive component.9 Whole-grain pigmented rice, in contrast to white rice, contains a higher concentration of bioactive phytochemicals with various health benefits.8\n\nDue to the high levels of polyphenol and anthocyanins in colored rice, it is regarded as a functional food in Asia.10 Many varieties of rice include colorful pigments, which are often concentrated in the pericarp or the bran of the rice kernel and give the grain hues like brown, red, purple, and black.11 The flavonoids anthocyanin and proanthocyanidin, which are recognized to have nutritional benefits, are responsible for these color changes. However, their efficacy varies depending on rice color or variety.12,13\n\nThe presence of polyphenols, which have been shown to have antioxidant, anti-inflammatory, and anti-adipogenic potential in in vitro and in vivo investigations,8,14 has been predominantly cited as the cause of the possible health advantages of this type of rice. Black, red, and brown rice bran's phenolic and flavonoid components, as well as their free and bound fractions, antioxidant and anti-proliferative properties, have all been documented.13,15–17\n\nThus, the aim of this study is to systematically review existing literature on the benefit of Asian bioactive compounds within pigmented rice and their implication in breast cancer.\n\n\nMethod\n\nArticles for the current review were acquired from two electronic databases (PubMed and Scopus), considering that they hold the most articles for biomedical and pharmaceutical studies. The database literature search was conducted from November 8 to November 10, 2022. We created the keywords for each database. For PubMed, we used the following keywords; (((((cancer) OR (“breast cancer”)) OR (metastasis)) AND ((((((rice) OR (“pigmented rice”)) OR (“black rice”)) OR (“red rice”)) OR (“purple rice”)) OR (“brown ice”)) AND ((“cancer therapy”) OR (“anti-metastasis”)). Meanwhile for Scopus, the keywords were \"Cancer\" OR metastasis OR “MCF-7” OR “Breast cancer” OR tumor AND “Pigmented Rice” OR rice OR paddy OR “Oryza sativa” AND “Cancer Therapy” OR medicine OR pharmaceutical OR physicochemical. During literature search, only free access articles were used.\n\nAll types of experimental and observational studies written in English language were included. All duplicate studies and literature reviews were excluded from the final selection. Study subjects focused on breast cancer, and any other objects of in vivo and in vitro studies. Study factors included in the studies were the types of pigmented rice, other interventions in the form of extracts, whole grain, bran and hull. The research results include cancer therapy, pharmaceutical, and bioactive compounds as anti-cancer.\n\nStudy selection, data extraction, and quality assessment\n\nThe aforementioned keyword search resulted in a total of 407 articles, consisting 103 and 304 articles from PubMed and Scopus, respectively. We excluded 32 manuscripts because the article were published more than 10 years ago. After excluding book chapters and papers not in English, we had 278 potential articles to be reviewed. After checking and screening carefully the title and abstract of the manuscript, the duplicate articles were eliminated, then 66 articles were obtained. The authors reviewed the full text of those studies and finally selected 10 of them which met the inclusion criteria. 46 articles did not meet the criteria because duplicated, a book chapter, not accessible, not in English and no full-text. The flow diagram of the selection of study is shown in.26,27\n\n\nResults\n\nThe included studies in this review were entirely of Asian. The year of publication ranged from 2013 to 2020. Five studies used black rice extract, two used red jasmine rice extracts, and three used a mixture of Korean rice extracts (black, red and brown rice). All studies were conducted in vitro and three studies were compared with in vivo tests on female mice. The summary of the included study were: tabulated based on the results, active compounds and sample breast cancer cell lines which are presented in Table 1.\n\nMethodological quality\n\nWe reviewed results from 10 studies, seven of which involved in vitro experiment and three of which involved both in vitro and in vivo experiment. Unfortunately, there are no specific studies that focus on in vivo experiments. In detail, we report 10 samples from in vitro studies and three samples from in vivo studies.\n\nIn vitro\n\nMany different cells have been used in in vitro experiments, including from human and murine breast cancer.13,18–21 Besides, there are several single-cell in vitro studies, among which are subcutaneous MDA-MB-453 cells,18,19 MCF-7 cells,13 MDA-MB-231 cells,20 and 4T1 cells.21 Furthermore, several studies used multiple cells, such as,15 which used HER2-positive (BT474, MDA-MB-453, and HCC1569 cells) and HER2-negative (MCF-7, SUM190, and MDA-MB- 231) breast cancer cell lines. The HT1080 fibrosarcoma and MDA-MB-231 cell lines were utilized in.22,23 The cells used by16 were MCF-7, B16F10, M-3, and YD-38. The cells used by24 were MCF-10A, MCF-7, and MDA-MB-453. In summary, the classification of breast cancer cells used in the 10 articles analyzed in this study were human triple-negative breast cancer (MDA-MB-231), human ERα-positive breast cancer (MCF-7), human HER2-positive breast cancer (BT474, MDA-MB-453, and HCC1569 cells), human HER2-negative (MCF-7, SUM190, and MDA-MB- 231), and murine breast cancer (4T1 and M3). There were also non breast cancer cell line used in this study, such as fibrosarcoma cell lines (HT1080), murine melanoma cell lines (B16F10), and human Squamous cell carcinoma cell lines (YD-38).\n\nIn vivo\n\nExperiments in vivo using same species of mouse, naked female mice, were carried out with different cancer cells. Two studies employed 4-week-old mice,18,21 while one used 6-7-week-old mice.15 In these experiments, several cells types were used and injected into the flank of mammary fat pad of mice to construct the breast cancer mice model. HER2-positive MDA-MB-453 cells were employed in one study.15 Besides, the previous study,18 found MDA-MB-453 (ErbB2-positive) was employed alongside MCF-7 (ErbB2-negative) and MCF-10A (normal) cells. In the last study, 4T1 cells were used to be transplanted to the mice.21\n\nPigmented rice cultivars differ in terms of both qualitative and quantitative characteristics.10–12 This rice variety is distinguished by its mostly red, black, or purple pericarps. Wide-color rice varieties have drawn more attention because of their many biological functions. The pigments serve as sources of phytochemicals when contrasting the nutritional value of colored rice with white rice brans. Anthocyanidins, ferulic acid, diferulates, anthocyanins, and polymeric proanthocyanidins are some of the phenolic chemicals.10,11\n\nPigmented rice residues are attractive sources of bioactive substances such as phenolic antioxidants and anticancer agents for the benefit of human health.12,13 Anthocyanin is found in the bran layers of the rice kernel, whereas phenolic acids are primarily present in the bran layers of rice in their free, conjugated, and bound forms.16 The two primary bioactive phenolic chemicals found in cereal grains, mostly found in the pericarp of colored types, are anthocyanins and proanthocyanidins.14 Anthocyanins, which are primary metabolites found in the bran layers of rice kernels, have been identified as functional dietary components that promote good health and have anticancer, antioxidant, hypoglycemic, and anti-inflammatory activities.11\n\nConsidering the numerous health benefits connected with functional components, such as anti-inflammatory, antioxidative, and anticancer properties, pigmented rice is considered an available food and food ingredient in many Asian countries.10–14 The anticancer effect is suggested partly through the enhancement of bioactive substances such as vitamin E, phytic acid, γ-aminobutyric acid (GABA), γ-oryzanol, and phenolics.9\n\nBlack rice\n\nA traditional and healthy cuisine, black rice is mostly grown in East Asia. Phytochemicals, tocopherols, polyphenols, B vitamins, and anthocyanins are only a few examples of the water-soluble bioactive substances abundant in black rice.21 These substances might help your health and shield you against long-term conditions linked to oxidative stress and antioxidant activity.10 A significant amount of phytochemicals are present in black rice bran, giving it strong medicinal properties. Black rice extract anthocyanins dramatically decreased the metastasis of breast cancer, according to seven research that were considered. The aleurone layer of black rice is where black rice anthocyanins (BRACs) are found. Human breast cancer cells that are ErbB2-positive can be prevented from metastasizing by BRAC.18 Additionally, Liu et al. (2013) demonstrated that black rice extract includes peonidin-3-glucoside and cyanidin-3-glucoside, which can diminish the tumor size and volume.15\n\nRed rice\n\nTwo studies found that red jasmine rice, which contains proanthocyanidins, inhibits breast cancer cell proliferation. Most of the phenolic chemicals in red rice, which are responsible for the red coloring of the pericarp, are proanthocyanidins and catechins.20,22 Proanthocyanidin from red rice (PRFR) contains procyanidins and prodelphinidins.22 Moreover, red rice extract also has a high concentration of γ-oryzanol and γ-tocotrienol in the Hex and DCM fractions.20\n\nPurple rice\n\nA variety of rice varieties, including glutinous rice, are referred to as purple rice and have historically been eaten throughout Eastern and Southeast Asia.23 The pigments are naturally occurring compounds belonging to the flavonoid family in which cyanidin, pelargonidin, peonidin, delphinidin, petunidin, and malvidin represent the most commonly occurring anthocyanin aglycone.11 One study found that purple rice hulls contained the highest phenolic compound.23 Cinnamic acid and its derivatives are the primary ingredients for breast cancer cell growth suppression and cell cycle arrest.13\n\nPhenolic and flavonoid chemicals act as an antioxidant to protect cells from oxidative damage, which is a common cause of aggressive diseases like cancer.16 However, the anticancer activities were not directly correlated with total phenolic or total flavonoid content values.16 So, phenolic and flavonoid from many types of pigmented rice have also been tested for apoptotic activity revealing that cell shrinkage and nuclear condensation occur in human breast cancer cell line MCF-7.16 The antioxidant activity of rice phenolic compounds is detectable in parallel with the inhibition of proliferation of MCF-7 cells through G1 cell cycle arrest and comparable to quercetin as an antioxidant standart.13\n\nProanthocyanidins are monomeric flavan-3-ol oligo- or polymers that are created as a byproduct of the flavonoid biosynthesis process.20 Proanthocyanidins, which are essentially concentrated tannins, are made by cytosolic multienzyme complexes acting along the phenylpropanoid route.22 By incorporating sugars, anthocyanidins are transformed into anthocyanins.19 Proanthocyanidins turn into anthocyanins when heated in an acidic medium. As byproducts of the flavonoid pathway, proanthocyanidins and anthocyanins have the same metabolic intermediates. Proanthocyanidins, in the form of procyanidin (catechin and/or epicatechin) and prodelphinidin (epigallocatechin and/or gallocatechin) showed anti-metastatic effects in cancer breast MDA-MB-231 and HT1080 cells.22 It reduces the cell invasion and migration in a dose dependent manner with IC50 at 7.52±1.42 μg/mL and 10.6±0.59 μg/mL respectively and as a molecular mechanism of metastasis it inhibits collagenase activity.20 Suppression of breast cancer metastasis including inhibition the mRNA expression of their signaling pathway has also been known.19\n\nThe anthocyanins pigment such as cyanin-3-glucosides or peonidin-3-glucosides known to dose-dependent inhibition of cell proliferation and suppress the tumour growth in HER2-positive cancer lines which associated with breast cancer.15 Inherent with the previously studied that both compounds inhibit cell human ductal breast carcinoma HS578T growth by blocking cell proliferation at the G(2)/M phase so that it cannot form a normal mitotic apparatus, lowering some cancer response proteins, and chromatin condensation leads to cell death.23 Anti-metastasis action prevents cancer cells from invading the blood or lymphatic vessel and spreading to other tissues or organs, among other benefits. The anthocyanins found in black rice are able to lower almost 40% of tumour nodules with in vivo studies and about 10% inhibits invasion of cancer breast MDA-MB-453 cells.18 Aprevious study reported a higher invasive inhibition of cells by 68% and also a decrease in the migration distance of MDA-MB-453 cells by 37% with anthocyanins compared to untreated cells after 24 hours.24\n\nSeveral bioactive substances with the potential to operate as anti-metastatic agents, including anthocyanins, phenolics, and flavonoids found in different kinds of pigmented rice, are among the articles employed. To ensure the efficacy of each bioactive chemical, however, more in vivo research or randomized clinical trials with multicenter investigations are required as the majority of them are in vitro studies.\n\nBreast cancer is a complex and multifactorial disease that involves many cellular and molecular pathways. Weinberg and Hanahan in 2000 stated 6 hallmarks of cancer, (1) continuous proliferative signaling, (2) escaping growth suppression, (3) countering cell death, (4) sustaining replicative immortality, (5) promoting angiogenesis, and (6) metastasis.25 To overcome this complex phenomenon, an effective therapy needs to be found. Due to the multi-target cancer therapy, natural product consumption is currently a major problem. As a result, this study investigated the bioactive chemicals found in Asian pigmented rice that were intended to target many pathways in the treatment of cancer (Figure 1).\n\nThis study found that bioactive compounds from the 10 articles reviewed, involved in several pathways on breast cancer, such as metastasis, cell growth and proliferative signaling, cell cycle progression and apoptosis.13,15–24\n\nBlack rice extract with unidentified compounds has anti-metastatis activity through the reduction of anti-metastasis related protein expression such as snail, vimentin, and E-cadherin.21 Besides, peonidin-3-glucoside, cyaniding-3-glucoside from black rice, and cinnamic acid from WD-3 varieties of Korean rice showed anti-proliferative activity on breast cancer.15 Peonidin-3-glucoside and cyaniding-3-glucoside of black rice also promoted apoptotic activity through the induction of caspase 3/7.15,16 Anti-metastatic pathway was mostly found to be involved in the implication of pigmented rice bioactive compounds against breast cancer.22 This study found that γ-oryzanol, γ-tocotrienol, and proanthocyanidin from red rice reduced MMP2 and -9 secretion.22 In addition, MMP9 along with ICAM, IL-6, and NFkB were reduced after treated by red jasmine rice that contained proanthocyanidin.20 Anti-metastasis also mediated by black rice anthocyanins through the reduction of FAK, cSrc and p130Cas phosphorylation. Black rice anthocyanins also posed anti-metastatic activity through the suppression RAF/MEK/ERK pathway and MMP2,-9 downregulation.19 Lastly, black rice anthocyanins can suppressed u-PA involved in tumor invasion.18\n\n\nConclusions\n\nThe pigmented rice covered by this study were black, red, and dark purple rice, and all contains a variety of peonidin-3-glucoside, cyanidin-3-glucoside, γ-oryzanol, γ-tocotrienol, proanthocyanidin, cinnamic acid, and anthocyanins that may act as pro-apoptotic, anti-proliferative, and anti-metastasis in breast cancer cells. Therefore, choosing whole-grain red, black, or purple is an excellent choice for health. Plus, these varieties are richer in disease-fighting antioxidants. Rice being a staple food for half of the world can be a source of energy for our generations only if it is accumulated with nutrition.", "appendix": "Data availability\n\nNo data associated with article.\n\n\nAcknowledgement\n\nThe author would like to thank Anjangsana Program Menuju Doktor Sarjana Unggul (PMDSU) 2022 from Human Resource Directorate, Ministry of Education, Republic of Indonesia for the support.\n\n\nReferences\n\nSullivan EJ: Global Action. ARTnews. 2020; 118(4): 114–117. Publisher Full Text\n\nDoran H: Bringing data to life. Biochem. (Lond). 2021; 43(5): 3–3. Publisher Full Text\n\nC. Agencies: Cancer 3.2020; 2020(no. March): pp. 1–7.\n\nNafisah W, Fatchiyah F, Hermawan M, et al.: AGRICULTURE AND as inhibitor of PD-L1/PD-1 interaction: An in silico study.2022; 56: 751–760.\n\nFeng Y, et al.: Breast cancer development and progression: Risk factors, cancer stem cells, signaling pathways, genomics, and molecular pathogenesis. Genes Dis. 2018; 5(2): 77–106. Publisher Full Text\n\nSun YS, et al.: Risk factors and preventions of breast cancer. Int. J. Biol. Sci. 2017; 13(11): 1387–1397. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarvie M, Howell A, Evans DG: Can Diet and Lifestyle Prevent Breast Cancer: What Is the Evidence? Am. Soc. Clin. Oncol. Educ. B. 2015; 35: e66–e73. Publisher Full Text\n\nXie M, Liu J, Tsao R, et al.: Whole grain consumption for the prevention and treatment of breast cancer. Nutrients. 2019; 11(8): 1–21. Publisher Full Text\n\nTan BL, Norhaizan ME: Scientific Evidence of Rice By-Products for Cancer Prevention: Chemopreventive Properties of Waste Products from Rice Milling on Carcinogenesis In vitro and In vivo. Biomed. Res. Int. 2017; 2017: 1–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeechamnanturakit V, Karrila TT, Sontimuang C, et al.: The Natural Pigments in Pigmented Rice Bran and Their Relation to Human Health: A Literature Review. KMUTNB Int. J. Appl. Sci. Technol. 2018; 11(1): 3–13. Publisher Full Text\n\nBhat FM, Sommano SR, Riar CS, et al.: Status of bioactive compounds from bran of pigmented traditional rice varieties and their scope in production of medicinal food with nutraceutical importance. Agronomy. 2020; 10(11): 1–15. Publisher Full Text\n\nMbanjo EGN, et al.: The Genetic Basis and Nutritional Benefits of Pigmented Rice Grain. Front. Genet. 2020; 11(March): 1–18. Publisher Full Text\n\nChung NJ, Choi KC, Lee SA, et al.: Rice hull extracts inhibit proliferation of MCF-7 cells with G1 cell cycle arrest in parallel with their antioxidant activity. J. Med. Food. 2015; 18(3): 314–323. Publisher Full Text\n\nCarcea M: Value of wholegrain rice in a healthy human nutrition. Agric. 2021; 11(8). Publisher Full Text\n\nLiu W, et al.: Selective anti-proliferation of HER2-positive breast cancer cells by anthocyanins identified by high-throughput screening. PLoS One. 2013; 8(12): 1–7. Publisher Full Text\n\nBaek JA, Chung NJ, Choi KC, et al.: Hull extracts from pigmented rice exert antioxidant effects associated with total flavonoid contents and induce apoptosis in human cancer cells. Food Sci. Biotechnol. 2015; 24(1): 241–247. Publisher Full Text\n\nGhasemzadeh A, Karbalaii MT, Jaafar HZE, et al.: Phytochemical constituents, antioxidant activity, and antiproliferative properties of black, red, and brown rice bran. Chem. Cent. J. 2018; 12(1): 13–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo LP, et al.: Anti-metastasis activity of black rice anthocyanins against breast cancer: Analyses using an ErbB2 positive breast cancer cell line and tumoral xenograft model. Asian Pac. J. Cancer Prev. 2014; 15(15): 6219–6225. PubMed Abstract \| Publisher Full Text\n\nChen XY, et al.: Black Rice Anthocyanins Suppress Metastasis of Breast Cancer Cells by Targeting RAS/RAF/MAPK Pathway. Biomed. Res. Int. 2015; 2015: 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPintha K, Yodkeeree S, Limtrakul P: Proanthocyanidin in red rice inhibits MDA-MB-231 breast cancer cell invasion via the expression control of invasive proteins. Biol. Pharm. Bull. 2015; 38(4): 571–581. PubMed Abstract \| Publisher Full Text\n\nTeng M, Zhou S, Zhang R, et al.: Extract Derived From Black Rice Functions as a Photothermal Agent for Suppressing Tumor Growth and Metastasis. Front. Bioeng. Biotechnol. 2020; 8(August): 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPintha K, Yodkeeree S, Pitchakarn P, et al.: Anti-invasive activity against cancer cells of phytochemicals in red jasmine rice (Oryza sativa L.). Asian Pac. J. Cancer Prev. 2014; 15(11): 4601–4607. Publisher Full Text\n\nBanjerdpongchai R, Wudtiwai B, Sringarm K: Cytotoxic and apoptotic-inducing effects of purple rice extracts and chemotherapeutic drugs on human cancer cell lines. Asian Pac. J. Cancer Prev. 2013; 14(11): 6541–6548. PubMed Abstract \| Publisher Full Text\n\nZhou J, et al.: Black rice-derived anthocyanins inhibit HER-2-positive breast cancer epithelial-mesenchymal transition-mediated metastasis in vitro by suppressing FAK signaling. Int. J. Mol. Med. 2017; 40(6): 1649–1656. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaha P, et al.: DNA polymerase eta: A potential pharmacological target for cancer therapy. J. Cell. Physiol. 2021; 236(6): 4106–4120. Publisher Full Text\n\nNugraha AP: PRISMA_2020_checklist Benefit of Asian Pigmented Rice Bioactive Compound and Its Implication in Breast Cancer: A Systematic Review. Dataset. figshare. 2023. Publisher Full Text\n\nNugraha AP: PRISMA Flowchart. figshare. [Data]. Figure. 2023. Publisher Full Text\n\nNugraha AP: Pigmented Rice Bioactive Compound Possible Mechanism from Breast Cancer. figshare. Figure. 2023. Publisher Full Text\n\nNugraha AP: The Summary of Studies Discovered Benefit of Asian Pigmented Rice Physicochemical Properties and Its Implication in Breast Cancer. figshare. Dataset. 2023. Publisher Full Text" }	[ { "id": "169255", "date": "13 Apr 2023", "name": "Kok-Yong Chin", "expertise": [ "Reviewer Expertise osteoporosis", "osteoarthritis", "evidence synthesis", "vitamin E", "men's health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this systematic review, the authors attempted to summarise the beneficial effects of Asian pigmented rice extract on breast cancer. However, the literature-gathering and evidence synthesis processes are flawed so the results and conclusion could not be judged fairly at this point. There is also no critical appraisal process, so it is hard to call this a systematic review.\nThe systematic review was not constructed based on PRISMA guidelines. The authors should follow the guidelines and rewrite the manuscript for it to be considered. The authors could also refer to any published systematic review during their writing.\n\nSearch string: Why was the bracket removed from the search string for the search in Scopus?\n\nKeywords: the inclusion of “cancer therapy” and “anti-metastasis” may restrict the search results. I recommend searching again without these terms.\n\nSetting open-access article as the inclusion criteria severely limits the search results.\n\nThe authors need to justify setting a time frame of 10 years in the search. How would older articles not be relevant anymore?\n\nWhy would the authors screen out items without original data (reviews, commentaries, letters etc) at the very last stage? This could be done earlier.\n\nOther necessary steps for deciding article inclusion are not illustrated: how many persons are making the decision, how was discrepancy resolved etc.\n\nThere is no PRISMA flow chart to summarise the entire literature search and article identification process.\n\nThe data extracted in the evidence table were not complete. There were no dose and treatment period data.\n\nThe Results and Discussion sections should be revamped. The results on the benefits of pigmented rice contain information that should appear in the introduction.\n\nThe limitation of review section discussed more on the limitations of the research field, not on the review itself.\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": [ { "c_id": "9580", "date": "21 Apr 2023", "name": "Alexander Patera Nugraha", "role": "Author Response", "response": "Dear honorable reviewer. First of all we would like to thank your valuable comments on our article. We will try to answer your review point per point as follows: This article has considered and involved critical appraisal process, such as Evaluation of the validity of the review The methodological quality was assessed through the following points: The question was clearly focused on breast cancer, study approach (in vitro and in vivo), intervention and outcome measures. Inclusion criteria have been indicated clearly in the article. Literature search method also has been clearly specified. Result interpretation The result interpretation was done according to the study approach, the type of rice and bioactive compounds role in breast cancer. This interpretation covered overall questions of this review. Applicability of the results of the review This review included possible mechanism and significance of finding that will be more applicable in health or pharmaceutical practice. This article was constructed based on PRISMA guidelines and checklist. This article also referred to one literature review. Seechamnanturakit V, Karrila TT, Sontimuang C, et al.: The Natural Pigments in Pigmented Rice Bran and Their Relation to Human Health: A Literature Review. KMUTNB Int. J. Appl. Sci. Technol. 2018; 11(1): 3–13. Hopefully you could understand and approve this manuscript. Thank you very much. Sincerely Yours" } ] }, { "id": "169259", "date": "17 Apr 2023", "name": "Ananto Ali Alhasyimi", "expertise": [ "Reviewer Expertise Orthodontic", "biomaterials", "biomechanic", "retention in orthodontic" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 your work. The paper is well-researched and written, with profound insights into the bioactive compounds of Asian pigmented rice and their role in breast cancer. Best wishes to all the authors. I believe it has merit and should be considered for indexing. Novelty alone is sufficient and has a significant impact. In order to improve its quality I think some minor issues should be noticed:\nPlease add the evaluation of the risk of bias in the included studies.\n\nThis systematic review was well-designed in accordance with PRISMA guidelines. It was included in the Reporting guidelines. Please incorporate the PRISMA flowchart into the primary text.\n\nSince this is a review article, it is expected that you describe some of your own premises and elucidate some novel findings from your literature studies.\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": "213312", "date": "16 Oct 2023", "name": "Mohammed Aljunaid", "expertise": [ "Reviewer Expertise Periodontontology", "Oral Medicine", "and Oral Hearbal medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have completed my evaluation and after reading this Systematic Review carefully I am of the opinion that this manuscript should be considered for indexing.\nNonetheless, the authors need to resolve some minor difficulties such as the following:\nIn general check the grammar.\nMethods:\nThe risk of bias needs to be added to the study or provide information on the quality assessment process. Add the critical appraisal process to the review.\n\nIn the result:\nIt is important to strike a balance between providing enough detail to support the conclusions and maintaining readability.\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": "213315", "date": "16 Oct 2023", "name": "Yuan Seng Wu", "expertise": [ "Reviewer Expertise Molecular pharmacology and therapeutics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented a systematic review on the Asian pigmented rice bioactive compounds and its implication in breast cancer. This manuscript is novel and has some values to the relevant community. Overall, the manuscript is acceptable in terms of writing and organisation. However, minor concerns as listed below should be addressed:\nTitle - the current title should be revised briefly to truly capture the content.\n\nThe authors should compare the included articles for the aspects in percentage. Like, how many articles (in percentage) presented or contained this compound or etc. Other than that, there were some missing words identified throughout the manuscript. Kindly check thoroughly.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-371
https://f1000research.com/articles/12-366/v1	04 Apr 23	{ "type": "Research Article", "title": "Health, psychology, economic resilience and wellbeing: Long-term effects on family welfare of early marriage", "authors": [ "Hery Ernawati", "Anni Fithriyatul Mas'udah", "Fery Setiawan", "Laily Isroin", "Hery Ernawati", "Fery Setiawan", "Laily Isroin" ], "abstract": "Background: Early marriage is a social problem that can have various impacts. This study aims to determine the long-term impact of early marriage compared to adult marriage on the aspects of health, psychology, economic survival and welfare in a comprehensive manner. Methods: This study used a cross-sectional study design. The sampling unit for this study was early marriage families from 3 sub-districts in Ponorogo district, namely Sawoo, Ngrayun and Pulung. The number of samples used were 104 families. The variables in this study consisted of health, husband's psychology, wife's psychology, economic resilience and well-being. The analysis to construct composite variables was polychoricpca analysis. Results: The long-term effects of early marriage can be seen comprehensively using polychoric PCA analysis. Based on the polyuchoric PCA analysis, a composite variable was obtained, namely Maternal Health (λ=2.79; CE 0.35); Infant health (λ=2.23; CE 0.45); Wife's psychology (λ=6.78; CE 0.56); Husband's psychology (λ=5.59; CE 0.56); Wellbeing (λ=2.07; CE 0.30); Economic resilience (λ=1.60; CE 0.40). The results of this study indicate that the long-term impact of early marriage compared to adult marriage is on the poorer aspects of maternal health, poorer infant health, poorer wellbeing and worse Economic Resilience. Conclusions: There needs to be special monitoring of cases of early marriage, so that it will suppress the bad impact.", "keywords": [ "adolescent marriage", "maternal health", "infant health", "psychology", "economic resilience", "wellbeing", "long-term effect" ], "content": "Introduction\n\nEarly married is a global problem.1–3 Globally, early marriage rates reach 12 million per 4 years.4 In Indonesia, data shows that over the last decade, the number of early marriages in Indonesia has fallen by 3.5%. However, this reduction will require systemic and integrated efforts to reach the target of 8.74% in 2024 and 6.94 percent in 2030.5 East Java is one of the regions with the highest early marriage rate. Early marriages in Ponorogo increased by 100% in 2020.6 Based on the area of residence, the rural area (16.87%) has a higher early marriage rate than urban areas (7.15%).7 The occurrence of early marriage in rural areas is a common practice. This is the the result of poor parenting environments, lack of maternal and paternal education, and lack of communication between parents and adolescents regarding reproductive and social issues.8 Rural communities assume that remarriage will not significantly affect their lives. Research has shown that the effect of early marriage includes maternal health, early school dropout, infantfeeding, early pregnancy, and economic well-being.9–11\n\nResearch is needed to determine the long-term effect of simultaneous early marriage on aspects of health, psychology, wellbeing and economic resilience. It can be used by advocacy groups as policy material to develop programs to reduce the impact of early marriage.\n\n\nMethods\n\nThis study used a cross sectional study design. The research was conducted in 3 sub-districts in Ponorogo district: Pulung, Ngrayun and Sawoo. Sampling of respondents was based on data from the office of religious affairs. Respondents in this study were couples who married in 2015-2017 based on the marriage certificates in the Ministry of Religion Office.\n\nWe used a probability sampling technique, namely by using cluster random sampling in each sub-district. The sample unit of this research is early marriage families who married in 2015-2017. The number of samples in this study were 104 families. The sampling selection was carried out in two stages: stratified random sampling for each sub-district. Furthermore, sampling was carried out using random cluster sampling based on villages in each sub-district.\n\nThis study's population was couples married in 2015-2017 based on marriage registration at the Religious Defense Office. A list of married couples registered at the office of religious affairs was used as the basis for sampling. Sampling took two stages: stratified random sampling in 3 sub-districts and cluster random sampling in villages. Respondents selected based on the 2-stage sampling then completed a questionnaire. The questionnaire consisted of 2 types for wives and husbands. The questionnaire used has been tested on 30 respondents. The results of the trials carried out have fulfilled the validation with the results R more than 0.361 (r table with 30 samples). The wife's questionnaire contained questions on maternal health, child health, maternal psychology, welfare and economic resilience. Meanwhile, the husband's questionnaire consisted of the husband's psychology. This study uses principal component analysis to construct index composites to measure each variable. The analysis used is polychoricpca. The variables constructed using the composite index are wife's health, children's health, husband's psychology, wife's psychology, economic resilience and wellbeing. Wife's health is built from the components Mother's age during childbirth, Antenatal care checker, Place of Antenatal care, Danger signs of pregnancy, Iron Tablet consumption, Birth attendant, Place of birth and Type of delivery. Infant's health is built from the components of Low Birth Weight (LBW), Exclusive breastfeeding, Immunization, Vitamin A, Anthelmintic. Wife's psychology and Husband's psychology were built from the components Satisfaction, Disappointment, Cries, Anxiety, Associations, Decision, Effort, Sleep quality, Feelings of offense, Concentration, Fatigue, Interest in sex. Wellbeing is built from the components Live with, Source of clean water, Toilet facility, Vehicle ownership, Agricultural land, Livestock ownership and Bank account ownership. Economic resilience is built from components of Financial Manager, Business ownership, Ownership of health insurance, Recipients of social programs.\n\nRespondents signed the informed consent form before proceeding with the questionnaire. Respondents were informed that their data would be used for research purposes only and would be deidentified before publication. Ethical approval was obtained from the Ethics Committee of the Faculty of Health Sciences, Universitas Muhammadiyah Ponorogo with the number 99/ER/KEPK/2021 on June 18, 2021.\n\nThis study uses polychoric PCA analysis to compile a composite index of wife's health, infant's health, husband's psychology, wife's psychology, economic resilience and well-being to compare the conditions of the effects of early marriage and adult marriage. The principal component analysis creates one index variable from correlated variables. Polychoricpca is used on the categorical scale items.\n\n\nResults\n\nThis research was conducted in a rural area in Ponorogo Regency, namely Ngrayun, Sawoo and Pulung sub-districts, with a total of 104 respondents of which 63.5% of respondents married in their teens (<19 years) and 37.5% of adults (≥19 years). The following is the frequency distribution of the respondents' characteristics (Table 1).\n\nIn this study, the table above shows that the wives when getting married were still adolescent, the husbands when getting married had been an adult, the wives' and husbands' last education was junior high school, the wives was unemployed after the marriage, the mothers' health was poor, the children's health was good, the psychological state of the wives and husbands was good, the level of family welfare of the early-married couple was in the prosperous category, and family economic resilience was in the non-survival category. The constituent variables are listed in Table 2.\n\nThe results of polychoric PCA analysis for each composite variable are shown in Table 3. In the Maternal Health variable, 35% of the variation can be explained by the variables Mother's age during childbirth, Antenatal care Checker, Place of antenatal care, Danger signs of pregnancy, Iron Tablet consumption, Birth attendant, Place of delivery and Type of delivery. In the infant health variable, 44% of the variation can be explained by the variables of low birth weight, breast milk, immunization, vitamin A, and worming. The wife's psychological variables 56% can be explained by the variables Satisfaction, Disappointment, Cries, Anxiety, Associations, Decision, Effort, Sleep quality, Feelings of offense, Concentration, Fatigue, and Interest in sex. On the husband's psychological variables, 56% can be explained by the variables Satisfaction, Disappointment, Anxiety, Associations, Decision, Effort, Sleep quality, Feelings of offense, Concentration, Fatigue, and Interest in sex. The 30% wellbeing variable can be explained by place of residence, clean water sources, toilet facilities, vehicle ownership, agricultural land, livestock, livestock ownership and bank account ownership. The 40% economic resilience variable, can be explained by the variables of financial manager, business ownership, ownership of health insurance and recipients of social programs.\n\nBased on Figure 1, it can be seen that there is a comparison of variables in the early marriage group with adult marriage. The variables include maternal health, infant health, wife's psychology, husband's psychology, welfare and economic resilience. The group of individuals who had early marriages (adolescents) had worse maternal health conditions, poorer infant health, poorer welfare, poorer economic resilience compared to the adult marriage group. This can be seen from the higher percentage of adolescents from each variable. However, the psychological condition variable of the wife and husband is better at the age of adolescence.\n\n\nDiscussion\n\nEarly marriage has a tremendous impact, not only on couples but also on children born to teenage mothers, and causes changes in the welfare and economic resilience of the family.\n\nMarried couples and their families often want to get pregnant soon after marriage, without acknowledging the dangers for young women who get pregnant under the age of 19 years. Adolescent pregnancy can also make the mother's health worse than adult pregnancy. Adolescent pregnancies come with a 2-4 times higher likelihood of causing death than adult pregnancies,12 can cause incidences of anemia and chronic energy deficiency caused by the growth period of adolescents who still require sufficient energy and high use of iron in the body.13,14 The impact of anemia causes bleeding and the condition of fetal development is hampered. Adolescents aged 15-19 years are 1.234 times more likely to experience pregnancy complications than women aged 20-24 years, such as hypertension and gestational diabetes, anemia.15,16 Adolescent pregnancy will also increase fertility rates. Women who had a delivery were 8.00 times more likely to have 3 children (lifelong fertility), compared with adult pregnancy (OR = 8.00; 95% CI: 7.52, 8, 46).17,18 Adolescents married before the legal age were 1.27 times as likely to have a terminated pregnancy, compared to women married at 18 years (OR = 1.27; 95%CI: 1.20, 1.34).17\n\nAdolescent pregnancy can cause the health of babies born to be worse than pregnancy in adulthood. Adolescent pregnancies are at risk of causing babies to be born with low birth weight (LBW), premature, and have abnormalities in the respiratory tract.12 Infants born to young mothers with repeated pregnancies had at least 0.15 (95% CI - 0.23, 0.08) lower LAZ and had a higher chance of stunting at least 40% (95% CI 1.19, 1,67) at the age of 12 and 24 months.19 Likewise, another study stated that the stunting prevalence was also higher in children under five years from adolescent pregnant women (44.4%) compared to mothers who were of sufficient age (35.6%). Adolescent pregnancy is associated with the incidence of stunting. A married teenage woman is 1.2 times at risk, and a pregnant woman who is less than 20 years old is 1.3 times at risk of having a stunting toddler.20\n\nAdolescent pregnancy also contributes to the wife's poor economic, emotional, social and health conditions, which are exacerbated by inadequate nutritional intake.16 In rural areas, the risk of age-related pregnancy is often compounded by malnutrition, poor socioeconomic conditions, low literacy rates and limited access to maternal health care.12 In this study, the psychological sub-variable of the wife in early marriage is better than in adult marriage. This contrasts with previous research which states the impact of early marriage on adolescents from a psychological aspect is anxiety and stress.21 Anxiety experienced by an adolescent wife who makes early marriages will feel afraid and anxious in dealing with problems that arise in the family.22 Most women who marry early do not have the power to make decisions. We found that the psychological condition of husbands in early marriage couples is better than that of adult marriages. This study's results differ from previous studies, which state that adolescents must have a higher commitment, so psychological conditions may be worse than in adult marriages.23\n\nThe welfare of the families of early married couples is worse than that of families of adult married couples. Low economic income had no effect on marital satisfaction.24 This shows that physical well-being is not always the main benchmark in a family. Efforts to strengthen the welfare of families with low economic income are expected to improve the ability to adjust to new roles with partners, with extended families, manage family stress and minimize vulnerable conditions.25 The risk of unemployment and poverty increases in couples who marry early21 and they often live in poverty and under pressure.26 In accordance with the results of previous studies that found women who marry young have a higher percentage of living in poverty when they are older, the results of this study imply that decisions women make early in life can have long-term consequences.27\n\nThis study identifies the impact of early marriage by collecting data directly from brands. The use of validated tools and enumerators improved data quality. However, this study was conducted at the local level thus limiting generalizability. In addition, the health of the mother and baby was assessed only by interview without directly checking the health condition of the mother and baby. Therefore, for further research, it is necessary to increase the scale of the study, carry out further analysis and carry out examinations for mothers and babies.\n\n\nConclusion\n\nThe long-term impact of early marriage compared to adult marriage is on the poorer aspects of maternal health, poorer baby health, poorer wellbeing and worse economic resilience There needs to be special monitoring of cases of early marriage, so that it will suppress the negative impact.", "appendix": "Data availability\n\nFigshare: Adolescent married. https://doi.org/10.6084/m9.figshare.22207741. 28\n\nThis project contains the following underlying data:\n\n- Data.xlsx\n\nThis project contains the following extended data:\n\n‐ Women’s and men’s questionnaires\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 author expresses his gratitude to the Ministry of Research and Technology/National Research and Innovation Agency which has funded research through Contract Number: 97/VI.4/PN/2021 and Universitas Muhammadiyah Ponorogo.\n\n\nReferences\n\nNasrullah M, Zakar R, Zakar MZ: Child Marriage and Its Associations With Controlling Behaviors and Spousal Violence Against Adolescent and Young Women in Pakistan. J. Adolesc. Health. 2014 Dec 1; 55(6): 804–809. Publisher Full Text\n\nParsons J, Edmeades J, Kes A, et al.: Economic Impacts of Child Marriage: A Review of the Literature. Rev. Faith Int. Aff. 2015 [cited 2021 Sep 15]; 13(3): 12–22. Publisher Full Text Reference Source\n\nSingh PK, Rai RK, Alagarajan M, et al.: Determinants of Maternity Care Services Utilization among Married Adolescents in Rural India. PLoS One. 2012 Feb 15 [cited 2021 Sep 15]; 7(2): e31666. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUNICEF: Child marriage - UNICEF DATA. UNICEF; 2021 [cited 2021 Sep 15]. Reference Source\n\nUNICEF Indonesia, BPS, PUSKAPA UI KPB: Perkawinan Anak di Indonesia\|UNICEF Indonesia. UNICEF; 2020 [cited 2021 Sep 15]. Reference Source\n\nPengadilan Agama: Pandemi Covid-19, Pernikahan Dini di Ponorogo Justru Naik 100 Persen. Jatimnow; 2020 [cited 2021 Oct 22]. Reference Source\n\nUNICEF: Pencegahan Perkawinan Anak.2020 [cited 2021 Oct 12]. Reference Source\n\nKassa GM, Arowojolu AO, Odukogbe AA, et al.: Prevalence and determinants of adolescent pregnancy in Africa: A systematic review and Meta-analysis 11 Medical and Health Sciences 1117 Public Health and Health Services. Reprod. Health. 2018; 15(1): 1–17.\n\nWodon Q, Male C, Nayihouba A, et al.: Economic Impacts of Child Marriage: Global Synthesis Report.2017 [cited 2021 Oct 21]. Reference Source\n\nPaul P, Chouhan P, Zaveri A: Impact of child marriage on nutritional status and anaemia of children under 5 years of age: empirical evidence from India. Public Health. 2019 Dec 1; 177: 95–101. PubMed Abstract \| Publisher Full Text\n\nBeattie TS, Bhattacharjee P, Isac S, et al.: Supporting adolescent girls to stay in school, reduce child marriage and reduce entry into sex work as HIV risk prevention in north Karnataka, India: protocol for a cluster randomised controlled trial. BMC Public Heal. 2015 Mar 25 [cited 2021 Oct 22]; 15(1): 212–292. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSychareun V, Vongxay V, Houaboun S, et al.: Determinants of adolescent pregnancy and access to reproductive and sexual health services for married and unmarried adolescents in rural Lao PDR: a qualitative study. Tetrahedron. 2018; 29(20): 3233–3236.\n\nApriliani FT, Nurwati N, Ilmu J, et al.: Pengaruh Perkawinan Muda terhadap Ketahanan Keluarga.2020; 90–9.\n\nSjahriani T, Faridah V: Faktor-faktor yang Berhubungan dengan Kejadian Anemia Pada Ibu. Jurnal Kebidanan: Jurnal Ilmu Kesehataan Budi Mulia. 2019; 9(2): 161–167. Publisher Full Text\n\nAdedokun O, Adeyemi O, Dauda C: Child marriage and maternal health risks among young mothers in Gombi, Adamawa state, Nigeria: Implications for mortality, entitlements and freedoms. Afr. Health Sci. 2016; 16(4): 986–999. PubMed Abstract \| Publisher Full Text\n\nLeftwich HK, Alves MVO: Adolescent Pregnancy. Pediatr. Clin. N. Am. 2017; 64(2): 381–388. Publisher Full Text\n\nYaya S, Zegeye B, Ahinkorah BO, et al.: Inequality in fertility rate among adolescents: evidence from Timor-Leste demographic and health surveys 2009 – 2016. Arch. Public Heal. 2020; 78: 1–10. Publisher Full Text\n\nErnawati H, Verawati M, Kesehatan FI, et al.: Kesehatan ibu dan bayi pada pernikahan dini. Media Ilmu Kesehat. 2014; 3(3).\n\nMaravilla JC, Betts K, Adair L, et al.: Stunting of children under two from repeated pregnancy among young mothers. Sci. Rep. 2020; 10(1): 14265–14269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimbolon D, Riastuti F, Jumiyati, et al.: Is there a Relationship Between Pregnant Women’ s Characteristics and Stunting Inci.2021; 16(3): 331–339.\n\nSchlecht J, Rowley E, Babirye J: Early relationships and marriage in conflict and post-conflict settings: Vulnerability of youth in Uganda. Reprod. Health Matters. 2013; 21(41): 234–242. PubMed Abstract \| Publisher Full Text\n\nSabbe A, Oulami H, Hamzali S, et al.: Women’s perspectives on marriage and rights in Morocco: risk factors for forced and early marriage in the Marrakech region. Cult. Health Sex. 2015; 17(2): 135–149. PubMed Abstract \| Publisher Full Text\n\nAstuti AW, Hirst J, Bharj KK: Indonesian adolescents’ experiences during pregnancy and early parenthood: a qualitative study. J. Psychosom. Obstet. Gynecol. 2020; 41(4): 317–326. PubMed Abstract \| Publisher Full Text\n\nJackson GL, Krull JL, Bradbury TN, et al.: Household Income and Trajectories of Marital Satisfaction in Early Marriage. J. Marriage Fam. 2017; 79(3): 690–704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrail TE, Karney BR: What’s (Not) Wrong With Low-Income Marriages. J. Marriage Fam. 2012; 74(3): 413–427. Publisher Full Text\n\nKnox SEM: How they see it: young women’s views on early marriage in a post-conflict setting. Reprod. Health Matters. 2017 Nov 9 [cited 2022 Nov 18]; 25(sup1): 96–106. Publisher Full Text Reference Source\n\nDahl GB: Early teen marriage and future poverty. Demography. 2010 [cited 2022 Nov 18]; 47(3): 689–718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMas'udah AF: Data. figshare. Adolescent married.2023. Publisher Full Text" }	[ { "id": "176672", "date": "12 Jun 2023", "name": "Margaret E Greene", "expertise": [ "Reviewer Expertise Demography", "child marriage", "gender inequality", "sexual and reproductive health and rights", "maternal health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nObjective This paper notes the need to \"determine the long-term effect of simultaneous (?) early marriage on aspects of ..wellbeing.\" Yet the analysis uses a cross-sectional design, including \"early marriage families\" who married in 2015-2017. Between the design, the selection of couples who married young, and the recency of their marriages, the study is not well set up to assess the long-term impact of marrying early, nor to compare the impact of marrying early to marrying later.\n\nThe terms describing the statistical method used, polychoric PCA analysis, are frequently mashed into one word, all lower case, which was initially confusing.\nThe description of the frequency distribution in Table 1 is strange and uses the majority distribution as describing everyone, e.g., \"the wives when getting married were still adolescent\" (63.5% were adolescents). What is more, the data collection suggests that the sample was collected from people who were young at the time of marriage, so this is doubly confusing.\nTo set up the polychoric PCA analysis shown in Table 3, a frequency distribution of the composite variables by age at marriage is needed.\n\nThe analysis is very thin, with tables and graphs presented and limited interpretation. Then the discussion seems to be based on a return to a literature review. I don't believe the studies cited substantiate the statement \"adolescent pregnancies come with a 2-4 times higher likelihood of causing death than adult pregnancies\" or cause energy deficiency. Those studies were not structured so as to show that relationship (reference numbers 12, 13, 14).\n\nNot sure what \"collecting data directly from brands\" means in the strengths and limitations section.\nOnce some of the substance of the paper is strengthened, it would benefit from English language editing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "195249", "date": "25 Aug 2023", "name": "Efehan Ulas", "expertise": [ "Reviewer Expertise Multivariate data analysis", "biostatistics", "clinical trials", "demography", "epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe submitted manuscript titled \"Long-term Impact of Early Marriage on Health, Psychology, Economic Survival, and Welfare: A Comprehensive Study\" addresses the important issue of early marriage and its implications on various aspects of individuals' lives. The study employs a cross-sectional design to investigate the long-term effects of early marriage compared to adult marriage on health, psychology, economic resilience, and well-being. The authors conducted the research in three sub-districts in Ponorogo district, namely Sawoo, Ngrayun, and Pulung. Using PCA analysis, composite variables were constructed to assess the multifaceted impacts of early marriage. The results indicate significant negative effects of early marriage on maternal health, infant health, well-being, and economic resilience. Although, the article addresses an important social issue – the impact of early marriage, I have several concerns about the study. Please find below comments for the manuscript.\nWhile the methods are briefly described, providing more detail on data collection procedures, survey instruments, and the selection criteria for the early marriage families would enhance the study's reproducibility.\n\nThe manuscript could benefit from a section discussing the limitations of the study. Addressing potential sources of bias, sample limitations, and other factors that might affect the validity of the findings would provide a more balanced presentation.\n\nThe findings suggest negative impacts of early marriage, however, a more nuanced discussion of the potential underlying reasons for these effects would enrich the interpretation. Additionally, providing potential mechanisms through which early marriage influences the studied aspects could contribute to the understanding of the observed outcomes.\n\nIncluding a discussion that relates the current findings to existing literature on early marriage and its consequences would help contextualize the study within the broader research landscape. Authors should update the literature part with the recently published studies related to early marriage. For instance; -Kamal, S. M., & Ulas, E. (2022). Child marriage and its association with Maternal Health Care Services utilisation among women aged 20–29: a multi-country study in the South Asia region. Journal of Obstetrics and Gynaecology, 42(5), 1186-1191. , - John, N. A., Kapungu, C., Sebany, M., & Tadesse, S. (2023). Do gender-based pathways influence mental health? Examining the linkages between early child marriage, intimate partner violence, and psychological well-being among young Ethiopian women (18–24 years old). Youth & Society, 55(6), 1155-1172. etc.\nIn summary, the manuscript contributes to the understanding of the long-term impacts of early marriage on health, psychology, economic resilience, and well-being. The comprehensive approach, along with the utilization of appropriate statistical methods, enhances the quality of the study. Addressing the suggested improvements would further strengthen the manuscript's clarity, robustness, and contribution to the field.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-366
https://f1000research.com/articles/12-32/v1	09 Jan 23	{ "type": "Research Article", "title": "Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen", "authors": [ "Imam Mustofa", "Suherni Susilowati", "Tri Wahyu Suprayogi", "Adeyinka Oye Akintunde", "Yudit Oktanella", "Djoko Agus Purwanto", "Suherni Susilowati", "Tri Wahyu Suprayogi", "Adeyinka Oye Akintunde", "Yudit Oktanella", "Djoko Agus Purwanto" ], "abstract": "Background and Aim: The Kacang goat (Capra hircus) is an indigenous livestock species in Indonesia that is at risk of extinction due to cross-breeding. Artificial insemination (AI) techniques are expected to increase the population of these goats. This study aimed to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI.\nMaterials and Methods: Fresh Kacang buck semen was diluted in SM–EY without or with the addition of 0.5, 1.0, 1.5, or 2.0 µg of EGCG CNPs/mL extender. Extended semen was packaged in French mini straws, frooze, and stored in liquid nitrogen at −196℃ for 24 hours. Six replicates from each treatment group were thawed for catalase, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, malondialdehyde (MDA), sperm intact plasma membrane (IPM), viability and motility analyses.\nResults: Post-thawed semen that was previously frozen without EGCG CNPs in the extender (control group) exhibited the lowest levels of catalase, DPPH, sperm viability, sperm motility, IPM, and the highest levels of MDA. However, the addition of EGCG CNPs at doses of 1.5 µg/mL extender increased post-thawed catalase, DPPH, sperm IPM, viability, and sperm motility and decreased MDA levels (p < 0.05) than those of control group.\nConclusion: This study was the first in which EGCG CNPs were used in SM–EY extender, and the addition of only 1.0 µg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen.", "keywords": [ "catalase", "extinction risk", "radical scavenging", "smallholder and farm", "sperm motility" ], "content": "Introduction\n\nSmallholder farmers raise Kacang goats (Capra hircus) to increase their financial income, reduce poverty, and prevent malnutrition. However, the pure breed of this indigenous livestock species in Indonesia is at risk of extinction due to cross-breeding and must be protected.1 Artificial insemination (AI) techniques involving freeze–thawed semen are expected to increase the population of these goats. However, goat sperm is sensitive to cold shock.2 Indeed, >60% sperm death was detected in post-thawed goat semen previously frozen in skim milk–egg yolk (SM–EY) extender without antioxidants,3 which does not meet the minimum requirements for AI, i.e., motility must be >40%.4 High sperm death and low motility occurs because the freezing and thawing process leads to an excess of reactive oxygen species (ROS) production, which damages the polyunsaturated fatty acids (PUFAs) in the plasma membrane of spermatozoa, increasing malondialdehyde (MDA) levels and ultimately reducing sperm viability and motility.5 Therefore, antioxidants are needed to counteract the effects of ROS and thereby increase post-thawed semen quality.6 In our previous studies, green tea extract was used to improve semen quality and decrease nucleotide mutations in mtDNA3 and protein-encoded mtDNA,7 presumably due to an increase in the antioxidant capacity.\n\nThe freeze–thawing process in goat semen causes lipid peroxidation in the spermatozoa membrane, which reduces semen quality.8 Semen contains endogenous antioxidants that help maintain the oxidant–antioxidant balance.9 However, the excessive production of ROS due to the freeze–thawing process cannot be overcome by endogenous antioxidants owing to the limitations of the spermatozoa cytoplasm and decreased antioxidant levels due to the addition of extenders.10 Epigallocatechin-3-gallate (EGCG) is a powerful antioxidant extracted from green tea.11 The nanoparticle extract has a large surface area to volume ratio, so it is expected to exhibit increased penetration into cells,12 including sperm cells, and improve the quality of post-thawed semen. The addition of EGCG chitosan nanoparticles (CNPs) to the extender when freezing Kacang goat semen has not yet been studied. Thus, in the present study, the addition of EGCG CNPs to SM–EY extender and their ability to increase antioxidant capacity was investigated by assessing catalase and 2,2-diphenyl-1-picrylhydrazyl (DPPH) levels. The aim of this study was to obtain the best possible quality of post-thawed Kacang buck semen for AI according to MDA levels, sperm intact plasma membrane (IPM), viability, and motility.\n\n\nMethods\n\nThe study was conducted in February to August 2022, at The Artificial Insemination Center of Airlangga University, Tanjung village, Kedamean, Gresik District, East Java, Indonesia, at coordinates 7° 19′ 25′′ S and 112° 32′ 54′′ E.\n\nThis study is part of a multiyear research project. The protocol was approved by the Animal Care and Use Committee of Airlangga University (number 520/HRECC.FODM/VII/2019).\n\nDried green tea (Camellia sinensis. Kuntze) leaves was obtained from Perkebunan Nusantara XII Malang, East Jawa Indonesia. Briefly, EGCG was isolated from C. sinensis using a thin-layer chromatography method and verified by a comparison with epigallocatechin gallate hydrate (Tokyo Chemical Co., Ltd., Japan).13 Subsequently, 5 mL of 0.1% chitosan solution [containing low molecular weight chitosan (Sigma-Aldrich) in 1% acetic acid] was added to 50 mL of EGCG solution [0.05% EGCG (Sigma-Aldrich) in distilled water], and the mixture was stirred at room temperature. Next, 0.5 mL of triphosphate (TPP) solution [0.025% TPP (Merck) in distilled water] was added drop-wise with stirring for 3 h at 112 × g. This solution was centrifuged at 21,952 × g for 10 min using a MC-10K centrifuge (Bio-Gener, Hangzhou, China) and washed three times with deionized water to obtain the EGCG CNPs, which were freeze dried for 48 h and stored at 4°C.14 The particle size of EGCG CNPs was measured using a Zetasizer Nano ZS (ZEN 3600, Malvern Instruments Ltd., Worcestershire, UK). A helium–neon ion laser at a wavelength of 633 nm was used as the incident beam at 25°C with a 90° angle.15\n\nSamples were collected from three of Kacang bucks aged two–three years and weighing 35–40 kg. These bucks were (owned by The Artificial Insemination Center, Airlangga University) fed approximately four kg of forage and 3.5 kg of concentrate (16%–18% crude protein) daily and provided with drinking water ad libitum. Semen was collected from the bucks using an artificial vagina twice per week to obtain six ejaculate samples to process as frozen semen.\n\nSkim milk powder (15 g; 115338; Merck) was dissolved in distilled water to a volume of 150 mL, heated for 10 min to 92°C–95°C, and then cooled to room temperature (25°C). Egg yolk (5 mL; derived from laboratory chicken eggs) was added to 95 mL of skim milk solution, then added with 1 IU/mL of penicillin (Meiji Seika Pharma, Tokyo, Japan) and 1 μg/mL of streptomycin (Thermo Fisher Scientific, Singapore).7 The solution was divided into five equal volumes without addition of EGCG CNPs for the control group (T0) and with addition of 0.5, 1.0, 1.5, and 2.0 μg of EGCG CNPs/mL extender for T1, T2, T3 and T4 groups, respectively.\n\nEach SM–EY extender group was divided into two equal volumes. The first volume was added to fresh semen to obtain 480 million spermatozoa/mL. The second volume was added with glycerol up to 16% concentration, which was in turn added to the first mixture to obtain 240 million spermatozoa/mL. The extended semen was cooled from room temperature (25°C) to 5°C for 1 hour, then filled in 0.25 ml French straws (I.M.V., France) and sealed. The filled and sealed straws were chilled in liquid nitrogen vapor from 5°C to − 140°C for 10 min, and immediately stored in liquid nitrogen (−196°C) for 24 hours before evaluation were conducted.7\n\nThe straws allocated to each group were thawed in sterile water for 30 s at 37°C. Six replicates randomly were used to assess sperm IPM, viability, progressive motility, MDA levels, catalase levels, and DPPH scavenging, respectively, according to methods reported in a previous study.7\n\nIPM\n\nA semen sample (0.1 mL) was added to 1 mL of a hypoosmotic solution [containing 7.35 g of sodium citrate (Sigma-Aldrich) and 13.52 g of fructose (Sigma-Aldrich) dissolved in distilled water to a volume of 1 L)] and incubated at 37°C for 30 min. The sperm IPM was assessed for 100 sperm under a light microscope (Olympus BX-53, Tokyo, Japan) at 400× magnification. Sperm with an IPM showed a curved tail, whereas those with a damaged plasma membrane showed a straight tail.7\n\nViability\n\nA drop of semen sample and a drop of nigrosine (Sigma-Aldrich) were mixed and smeared on a glass slide, after which the slide was dried over a flame. The slide was then examined under a light microscope (Olympus BX-53, Tokyo, Japan) at 400× magnification to evaluate the percentage of live sperm in 100 spermatozoa. Live sperm were identified by their brightly transparent heads, whereas dead sperm were colored red.7\n\nMotility\n\nAn homogenate of a semen sample (10 μL) and a 0.9% (w/v) NaCl solution (1 mL) was dropped onto a glass slide and covered. The number of progressively motile sperm was counted for 100 sperm at 400× magnification under a light microscope (Olympus BX-53, Tokyo, Japan) equipped with Linkam Warming Stages set at 37°C–38°C (Meyer Instruments, Texas, USA).7\n\nMDA levels\n\nMDA levels in semen samples were determined using the thiobarbituric acid (Sigma-Aldrich) method. A semen sample (100 μL) and MDA kits (0, 1, 2, 3, 4, 5, 6, 7, and 8 μg/mL of malondialdehyde) were added to 550 μL of distilled water and 100 μL of 20% trichloroacetic acid. These mixtures were then homogenized for 30 s, and 250 μL of HCl (1 N) was then added and homogenized. Subsequently, 100 μL of 1% sodium thiobarbiturate was added and homogenized. This mixture was centrifuged at 28 × g for 10 min, and the supernatant was incubated in a 100°C water bath for 30 min before being left to room temperature (25°C). The color absorption was determined at a wavelength of 533 nm using a spectrophotometer (Thermo Fisher Scientific). MDA levels (ng/mL) were determined by extrapolating the sample absorbance values using a standard MDA curve.7\n\nCatalase levels\n\nA 30 mM H2O2 phosphate-buffered solution [1 mL; comprising 0.34 mL of 30% H2O2 diluted in fresh phosphate buffer (50 mM and pH 7)] was added to a semen sample (2 mL) at room temperature and assessed against a blank (not containing an enzyme) solution. The UV spectrophotometric absorbance method was used to measure catalase activity at a wavelength of 240 nm.16\n\nDPPH radical scavenging\n\nA 5 mL of DPPH radicals (10 mM) in methanol were added to a cuvette containing 970 mL of mixed methanol. This mixture was incubated at 20°C for 3 min, and the absorbance was measured at 517 nm (A517) using a UV-Vis Spectrophotometer (Thermo Fisher Scientific). Next, 25 mL of each sample and 25 mL of an acetonitrile solution (9.5 M; used as negative control) were added and mixed, and the mixture was incubated at 20°C for 3 min. Subsequently, the A517 decrease related to DPPH radical decomposition was measured. All experiments were performed in duplicate, and the mean DPPH scavenging effect was calculated according to the following formula: DPPH scavenging effect (%) = (1 − A517 sample/A517 negative control) × 100.17 IC50 values were calculated using a relationship curve of RSA versus concentrations of the respective sample curve.18\n\nANOVA followed by the Tukey Honestly Significant Difference test were used to analyze these data with a significance level of p ≤ 0.05. SPSS (Version 23, IBM Corp., Armonk, NY, USA) was used for data analysis.\n\n\nResults\n\nThe diameters particles of EGCG CNPs was in range 41.31 – 388.36 nm with the averages as presented in Table 1 and size distribution curves of EGCG CNPs as seen in Figure 1. The progressive motility of sperm indicates the quality of fresh semen. Based on the criteria for the motility of individual spermatozoa of >70%, the obtained semen ejaculate of the Kacang goats met the requirements for freezing (Table 2).\n\nPost-thawed semen that was previously frozen without antioxidant EGCG CNPs in the extender (T0 group) exhibited the lowest levels of catalase and DPPH and the highest levels of MDA (p < 0.05). The addition of EGCG CNPs in extender resulted in higher catalase and DPPH levels and lower MDA levels (p < 0.05) in optimum dose of 1.5 μg/mL of extender (T3) than those in the T0 group (p < 0.05) (Table 3).\n\nSemen that was frozen without antioxidant EGCG CNPs in the extender (T0 group) exhibited lowest sperm IPM, viability, and motility both in the pre-freezing and post-thawed conditions (p < 0.05). The addition of EGCG CNPs at 1.5 μg/mL of extender (T3) was optimum dose for increasing of sperm viability, motility and IPM (p < 0.05) than those in the T0 group (Table 4).\n\n\nDiscussion\n\nNanotechnology techniques have been used to produce particles with a size scale of 0.1–1000 nm.19 The smaller particle size of nanoparticles than the microparticles, causes the NPs have larger surface areas to volume ratio and the opportunities for chemical reactions and biological activities also increase. Bioavailability is the ability of NPs to penetrate cells. The effect of NPs on the target site depends on their chemical composition, shape, surface structure, surface charge, catalytic properties, and aggregation ability with other materials.20 The NPs size causes the active compound to spread in the medium and reach the target with increased accuracy.21 One of the safest materials used in NP encapsulation technology is chitosan.22\n\nEGCG possesses metal-chelating properties that provide antioxidant functions. The two structures that give EGCGs metal chelation properties are the ortho-3′,4′-dihydroxy moiety and the 4-keto, 3-hydroxyl, or 4-keto and 5-hydroxyl moiety. Catechins prevent the generation of potentially damaging free radicals via the chelation of metal ions. Through their ability to chelate transition metal ions, flavonoids can complex and inactivate iron ions, thereby suppressing the superoxide-driven Fenton reactions that are thought to be a crucial route to forming ROS. Electron transfer from catechins to ROS-induced radical sites on DNA and the formation of stable semiquinone free radicals are other mechanisms by which catechins exert their antioxidant effects,23 which are more pronounced than those of vitamins C and E.24\n\nSemen has antioxidant enzymes, namely catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX), which maintain the oxidant–antioxidant balance25 and play fundamental roles in protecting biological systems against free radical attacks. The scavenging activity of SOD is accomplished via catalase, which reduces hydrogen peroxide to water and molecular oxygen.26 Indeed, catalase activates the decomposition of hydrogen peroxide into water and oxygen, thereby blocking the ROS-generating pathway and reducing oxidative stress.27 The addition of catalase to a commercially medium increased the total motility, membrane integrity, and viability of postliquid goat semen28 and ram semen.29 Optimal catalase levels in the extender also reduce detrimental effects on post-thawed motility, viability, plasma membrane, and acrosome integrity.30 In humans, catalase is used as a molecular target for diagnosing and monitoring male infertility31 and in strategies for optimizing sperm parameters.32 This study’s result is consistent with that of Papas et al.,33 who demonstrated that the specific activities of catalase, GPX, and glutathione reductase during stallion semen cryopreservation were similar between effective and ineffective freezing of ejaculates. However, SOD activity was found to be higher in ejaculate following effective freezing than in ejaculate subjected to poor freezing power.34 In stallions, the total and specific activity of catalase in seminal plasma is high; however, no correlation was observed between total catalase activity in stallion seminal plasma and sperm kinematic parameters.33\n\nA higher value of DPPH in the T3 than those of T0 group represents more effective free radical scavenging.35 A DPPH measurement has an acceptable reproducibility for determination of radical scavenging activity in several samples.36 The DPPH assay, a popular method for evaluating the kinetics and stoichiometry of antioxidative reactions, is used widely because it is easy to use, rapid, and sensitive. The assay is based on the reduction of the purple chromogen DPPH· via a hydrogen atom or electron transfer from the scavenging molecule, i.e., an antioxidant, which causes the formation of pale yellow hydrazine (i.e., DPPH).37\n\nThe highest levels of MDA in the control group indicate highest lipid peroxidation. Oxidative stress may result in an imbalance between ROS generation and endogenous antioxidant activities. Higher ROS levels cause cell damage through the peroxidation of PUFAs in the sperm plasma membrane. Lipid peroxidation generates toxic lipid aldehyde species, including MDA,38 and higher MDA levels indicate free radical attacks39 and plasma membrane damage.40 The lower MDA levels in post-tahwed semen of T3 group indicated the decreasing of oxidative stress, followed by an increase in membrane fluidity and a lower percentage of acrosomal damage owing to the rearrangement of membrane lipids and proteins.27\n\nGoat semen is sensitive to cryopreservation. Freezing semen leads to excessive ROS production41 followed by lipid peroxidation of the membrane, resulting in MDA production,42 which in turn markedly reduces sperm IPM, viability, motility, and DNA integrity.43\n\nIntactness of plasma membrane is essential for protecting the organelles of sperm and molecular transportation; thus, it is crucial for sperm viability and sperm motility.44 Post-thawed semen previously frozen without antioxidant EGCG CNPs in the extender showed the lowest IPM, sperm viability, and sperm motility levels, whereas adding EGCG CNPs to the extender increased each of these levels. Damage to the plasma membrane of the spermatozoa reduces the quality of post-thawed semen because the integrity of this membrane is essential for the survival and motility of sperm.45 Excessive ROS production in semen due to the freezing–thawing process causes lipid peroxidation in the sperm membrane,8 disrupting the structure and function of this membrane and leading to the death of the spermatozoa.43 Lipid peroxidation also damages axonemal and mitochondrial proteins, resulting in the loss of sperm motility, even though the spermatozoa remain alive.46 Membrane damage due to lipid peroxidation also results in higher MDA levels.47 Furthermore, ROS cause the opening of bonds between disulfide chains in proteins, thereby destabilizing the DNA structure and leading to DNA fragmentation.48 The ejaculate defense system, including antioxidant enzymes such as GPX, catalase, and SOD, deals with ROS.49 However, the smaller volume of the sperm cytoplasm than those of commonly cells is the challenging for the antioxidant.50 The addition of semen extender might also lead to decreases of endogenous antioxidants. Insufficient antioxidant levels to combat oxidative stress during cryopreservation can have multiple negative effects, including decreased sperm viability, sperm motility, and plasma sperm integrity. Thus, the addition of antioxidants to the extender prior to semen freezing is necessary.\n\nThe EGCG is an antioxidant that can reduce lipid peroxidation, protein carbonylation, and sperm DNA damage.51 The NPs form of EGCG increases the ratio of surface area of membrane to volume of particles, which allows EGCG to penetrate sperm cells efficiently.52 The presence of antioxidants reduces lipid peroxidation in the plasma membrane of spermatozoa, which in turn increases sperm viability, motility, and acrosome integrity.53 Adding EGCG CNPs at 1.5 μg/mL of SM–EY extender improved semen quality compared with that of the control group. These results are consistent with those of previous studies in which adding ethanol green tea extract in extender maintained the motility, viability, IPM, and DNA integrity of Simmental bull sperm.54\n\n\nConclusion\n\nIn this study, EGCG CNPs were used for the first time in SM–EY extender to improve the antioxidant capacity and quality of post-thawed semen. Indeed, the addition of 1.5 μg/mL EGCG CNPs to the SM–EY extender increased the antioxidant capacity and quality of Kacang buck post-thawed semen. Future studies are required to validate this finding for AI under farm conditions.\n\n\nAuthor’s contributions\n\nImam Mustofa (IM), Suherni Susilowat I (SS), Tri Wahyu Suparyogi (TWS), Adeyinka Oye Akintunde (AOA), Yudit Oktanella (YO), Djoko Agus Purwanto (DAP)\n\nIM, SS and TWS conceived the idea, designed the mainframe of this study, and conceived in detail the manuscript. IM, TWS collected, prepared extender and freezing semen process. DAP: extracted EGCG from green tea leaves. YO: processing EGCG nanoparticles and measuring the particle size of extenders. SS, YO: evaluated semen quality. IM: interpreted the data and statistical analysi. AOA, YO, DAP: read critically and revised the manuscript for intellectual content. All authors read and approved the final manuscript.", "appendix": "Data availability\n\nBiostudies. Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang Goat semen. DOI: https://www.ebi.ac.uk/biostudies/studies/S-BSST924 55\n\nThis project contains the following data:\n\n- This study aimed to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI\n\nARRIVE checklist. Figshare. DOI: https://doi.org/10.6084/m9.figshare.21531213.v1 56\n\n\nAcknowledgments\n\nThe authors thank Dikky Eka Mandala Putranto, DMV, M.Sc., the Chairman of the Insemination Center, Airlangga University, Subchan Aziz, and Agus Purwanto for technical support.\n\nApproval of support_EGCG CNPs, DOI: 10.6084/m9.figshare.21532326. https://figshare.com/articles/poster/Approval_of_support_EGCG_CNPs/21532326.\n\n\nReferences\n\nSuswono SDecree of the Minister of Agriculture of the Republic of Indonesia Number 2840/Kits/LB.430/8/2012.2012. Accessed June 17 2022.Reference Source\n\nHahn K, Failing K, Wehrend A: Effect of temperature and time after collection on buck sperm quality. BMC Vet. Res. 2019; 15: 355. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMustofa I, Susilowati S, Wurlina W, et al.: Green tea extract increases the quality and reduced DNA mutation of post-thawed Kacang buck sperm. Heliyon. 2021; 7: e06372. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen M, Liu W, Li Z, et al.: Effect of epigallocatechin-3-gallate (EGCG) on embryos inseminated with oxidative stress-induced DNA damage sperm. Syst. Biol. Reprod. Med. 2020; 66: 244–254. PubMed Abstract \| Publisher Full Text\n\nHill EK, Li J: Current and future prospects for nanotechnology in animal production. J. Anim. Sci. Biotechnol. 2017; 8: 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaramola JO, Adekunle EO, Oke OE, et al.: Effects of pyridoxine in combination with different antioxidants on viability and oxidative stress parameters of cryopreserved goat buck semen. Arch. Zootec. 2017; 66: 15–21. Publisher Full Text\n\nSusilowati S, Sardjito T, Mustofa I, et al.: Effect of green tea extract in extender of Simmental bull semen on pregnancy rate of recipients. Anim. Biosci. 2021; 34: 198–204. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMustofa I, Susilowati S, Suprayogi TW, et al.: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang Goat semen. BioStudies. 2022; S-BSST924.Reference Source\n\nMustofa I, Susilowati S: Author Checklist - Full_EGCG CNPs.pdf. figshare. Poster.2022. Publisher Full Text" }	[ { "id": "161557", "date": "07 Feb 2023", "name": "Muhammed Duman", "expertise": [ "Reviewer Expertise Semen quality and additives" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would like to suggest some additions to the manuscript, including the introduction and discussion sections. When I read the article, it sounds much more scientific and understandable. I do not see any methodological error, but I think additions for improving post-thawed semen quality in goats are popular subjects. In this updated area, the authors need to add some updated additives to improve semen quality in the introduction section and need to discuss them in the discussion. Why Epigallocatechin-3-gallate chitosan nanoparticles are well comparing other antioxidants and freezer protectors in goat semen.\n\nI am suggesting some minor points below:\nIntroduction should be enlarged\n\nThere are some other studies about additions increase post-thawed semen quality such as fish seminal plasma, egg yolk, curcumin, inositol and carnitine. Authors should mention other articles in the introduction section some samples given below\nInformation about post-thawed semen quality in goats is very critical subject and authors need to mention what is the updated researches on this subject. For example:\nhttps://doi.org/10.1016/j.anireprosci.2015.11.017 https://doi.org/10.1016/j.smallrumres.2009.11.015 http://zsp.com.pk/pdf47/119-124%20(16)%20PJZ-1915-14%2011-12-14%20page%20proof%20PJZ.pdf http://www.pvj.com.pk/pdf-files/34_3/347-350.pdf https://doi.org/10.1111/and.13555\n\nThe mentioned studies in introduction section, should be discussed here\n\nThe reference (56) is not relevant to the study and should be discarded\nAfter revisions, the manuscript will be high impact and mostly cited because of comprising an updated subject.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9520", "date": "04 Apr 2023", "name": "Imam Mustofa", "role": "Author Response", "response": "Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have added some other studies about additions substances in extender to increase post-thawed goats semen quality in the Introduction and Discussion section. The relevant references were added." } ] }, { "id": "160298", "date": "17 Feb 2023", "name": "Maria Madalena Pessoa Guerra", "expertise": [ "Reviewer 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 article aims to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. The work is well written and can make a great contribution to the freezing of goat semen. However, you need to make a lot of corrections before it's indexed.\nThe term \"viability\" is not appropriate, as it does not define well what was evaluated in the spermatic cell. I suggest replacing it with \"living cells\"\n\nWith the exception of IPM, the addition of EGCG CNPs at doses of 1.5 and 2.0 μg/mL extended post-thawed catalase, DPPH, viability, and sperm motility and decreased MDA levels (p < 0.05) than those of control group.\n\nThe conclusion of the abstract is not consistent with the results, as 1.5 μg/mL and 2.0 μg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen.\n\nWhat is the methodology used to cool semen from room temperature (25°C) to 5°C for 1 hour?\n\nResults: Part of the first paragraph can transfer to the discussion.\n\nThis statement “The addition of EGCG CNPs in extender resulted in higher catalase and DPPH levels and lower MDA levels (p < 0.05) in optimum dose of 1.5 μg/mL of extender (T3) than those in the T0 group (p < 0.05) (Table 3). “ is not correct, because the significant difference occurred at doses 1.5 and 2.0 μg/mL.\n\nThis statement “The addition of EGCG CNPs at 1.5 μg/mL of extender (T3) was optimum dose for increasing of sperm viability, motility and IPM (p < 0.05) than those in the T0 group (Table 4).” also does not agree with the results, since there was a difference between analyses: MPI was better from T4 (2.0 μg/mL), while live and motility were better with T3 (1.5 μg/mL), and T4 (2.0 μg/mL).\n\nThe discussion needs to be modified. Little of the results obtained were discussed with the literature data. Looks like a literature review.\n\nThis statement “The lower MDA levels in post-thawed semen of T3 group indicated the decreasing of oxidative stress, followed by an increase in membrane fluidity and a lower percentage of acrosomal damage owing to the rearrangement of membrane lipids and proteins.” should be modified, because groups T2, T3 and T4 did not differ from each other and were inferior to the T) (control).\n\nThe statement that \"A higher value of DPPH in the T3 than those of T0 group represents more effective free radical scavenging.\" should also be modified, since T4 did not differ from T3.\n\nThe statement that “Adding EGCG CNPs at 1.5 μg/mL of SM–EY extender improved semen quality compared with that of the control group.” should be modified, as T3 and T4 did not differ from each other.\n\nThe statement of the conclusion that “Indeed, the addition of 1.5 μg/mL EGCG CNPs to the SM–EY extender increased the antioxidant capacity and quality of Kacang buck post-thawed semen.” must be modified.\n\nThe title of the tables should explain the analyses performed, in the same order in which the results are presented below.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "9521", "date": "04 Apr 2023", "name": "Imam Mustofa", "role": "Author Response", "response": "Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have revised the narration in the Abstract, Introduction, Results, Discussion, and Conclusion based on data and suggestion of reviewers." } ] } ]	1	https://f1000research.com/articles/12-32
https://f1000research.com/articles/12-363/v1	04 Apr 23	{ "type": "Method Article", "title": "An economic case for small scale solar-wind-augmented agriculture in exurban localities", "authors": [ "Charles Thangaraj" ], "abstract": "Background: In addition to energy security and independence, infrastructure development, food production, agricultural resource management, and food security are increasingly becoming principal concerns for local and global affairs with public policy deliberations for economic, environmental, and new business initiatives. Furthermore, the COVID-19 pandemic has exposed the frailty of global supply chains and hastened the acceptance of remote work culture, motivating an urban exodus to exurbs and nearby rural communities. Globally, climate change has led to food scarcity and food supplies have been weakened by ongoing hostilities, commodity inflation, and political nationalism in banning exports. A potential solution for this is distributed small scale agriculture; which can increase food self-reliance, lower global demand, and can help achieve more equitable food availability. Methods: This paper presents a framework for designing an automatable farm for exurban locations with new population growth, identifying and quantifying the bounds on additional long-term economic benefits on an assumptive framework based model. The framework is based on localized, small scale modified pumped storage and gravity fed irrigation, where solar and wind provide the energy requirement. An economic case is made by contrasting capital cost to estimated reduction in food wastage and revenue from excess energy generation. Results: Using multidimensional US agricultural, energy, and economic data, a non-conventional economic analysis estimates that a 20% reduction in food wastage (due to use of exurban agriculture) over 10 years equates to USD $31.8 billion. This capital applied to exurban agriculture can be recovered in 10 years, plus setting up the infrastructure for long term agriculture and or local energy production for local consumption or export for additional revenue. Conclusions: Exurban agriculture, located closer to demand centers, leads to reduction in overall agriculture energy needs, improving food security and wastage. Additional energy production can generate revenue or reduce local grid-energy demand.", "keywords": [ "Agri-business", "energy and demand offset", "food security", "exurb economics" ], "content": "1. Introduction\n\nEnergy demand is generally considered to be relatively price inelastic. Which is to say that irrespective of price per unit of energy, the demand remains relatively unchanged; except during artificial periods of abnormal demand. On the supply side, intentional surplus production is generally counterproductive to profitability and intentional scarcity leads to loss of market share or markets all together. The delicate global supply-demand equilibrium is achieved via special economic and trade measures. Simply put, global energy trading is not a simple marketplace where open market economics can prevail. However, within a single political entity or partnership governed by common rules, open market principles and markets exist. Understanding this basic fact of our globalized economy is crucial to discussing energy usage and demand offset.\n\nAccording to Webber (2019), in the US 10% of all energy consumed is for the entire food supply chain i.e., 1% in calorific content and 9% expended as energy for processing, storing, refrigeration, transportation, and cooking etc. Therefore, in 2021, the total energy consumption for food in the US was roughly 9.7 quadrillion British thermal units (Btu). Of which upwards of 40% or 3.9 quadrillion Btu is lost due to food waste according to Gunders (2012). To quantify that loss, it is equal to USD $21.6 Billion per year (3.9 quadrillion Btu * USD $5.54/1 Million Btu) in natural gas equivalent as of July 6, 2022. Geographically, food waste occurs closer to areas where consumption is also high. Consequently, dense urban and suburban areas have the highest food demand.\n\nA 2020 study from Tufts University (Kurtz et al., 2020) found that local food production is feasible and a potential solution for food availability optimization. However, the study also found that not all urban regions are suitable for local food production. While the study’s conclusions are based on sound data, it is also based on traditional farming techniques and land use practices. This paper, however, explores the idea of mixed use of exurban lands i.e., both for agriculture and non-agricultural use. Also, the focus is on small scale farms that can be well integrated into exurban or even suburban landscapes, thus bringing the farm closer to the urban centers and reducing the farm-to-fork energy consumption and food waste. In addition to energy reduction due to proximity (lower transportation needs), the idea of further reducing energy demand by powering farm operations through wind and solar augmentation, is explored, analyzed, and justified.\n\nThe remained of this paper is organized in the following sections. The basic structure, dimensions, and features of the smallest unit field for the proposed exurban farm is described in Section 2. Section 3 discusses the capital needs, operational aspects, energy storage and offsetting capabilities of a unit farm; followed by comments and concluding remarks in Section 4.\n\n\n2. Proposed design\n\nThe proposed exurban farm is described and illustrated in this section. The basic unit of exurban agricultural parcel is defined as a unit field (UF) and described in Section.\n\nEach UF is served by two towers and three tanks for irrigation and other operations. The tower design and tank layout are presented in Sections 2.2 and 2.3, respectively. Sections 2.4, 2.5, 2.6, and 2.7, presents wind and solar energy augmentation, demand offsetting, space utilization improvements, and local food benefit aspects, respectively.\n\nFigure 1A shows the parameterized (variable x) dimensions for a UF. Where T1 and T2 are the two towers located along the center line of the UF. Let us consider the following two cases for the UF i.e., x is 50 and 100 meters.\n\n\n\n(A) A unit field (UF) with parameterized dimensions.\n\n(B) A unit field (UF) with regions of influence shown.\n\nThe smallest average farm size in any US state according to the United States Department of Agriculture Farm Labor Survey (USDA FLS, 2022) is 55 acres. Hence, the case described by Equation 1B (i.e., when x = 100) for a UF area of ≈5 acres, offers a reasonable multiple (10-11) to represent the smallest average US farm size. Therefore, this case will be considered as the standard UF dimensions for analysis throughout the rest of this work. Area of influence is defined as the region of a UF an individual tower is expected to serve, this is illustrated in Figure 1B. If the land available is larger than a single UF, then multiple UFs can be fit into the available land as shown in Figure 2 for analysis purposes.\n\nThere are two identical towers per UF. These towers are placed on circular concrete pads with a radius of (x/20). Each tower will have an above ground steel structure that will be angled to support a water storage tank. The tower base will consist of an underground concrete structure with built in water tank and contains usable space for additional utilities, if desired. The conceptual top view and side view of such a tower is shown (not to scale) in Figures 3 and 4, respectively.\n\nThe two tanks will play a significant role in irrigating the influence area, provide drinkable water to any life stock and for use as a pumped storage energy harvesting/demand offset system (more in Section 3). The steel structure also can serve as mounting frame for:\n\n• anchoring and securing any shading applications if livestock are raised along with food crops\n\n• water faucets for life stock drinking solutions\n\n• irrigation sprinklers or for drip irrigation distribution plumbing\n\n• solar panels and or wind generators\n\nIn every UF there are two towers; T1 and T2. At tower T1 there are two tanks, TT1 as the top tank at T1 and BT1 as the bottom tank at T1. Similarly, at tower T2, TT2 is the top tank at T2 and BT2 is the bottom tank at T2. In addition to these, there is a deep buried common tank (CTk) which will be located centrally to both the towers (T1 and T2). Figures 5 and 6 show the top and side views of the tank locations, respectively. Figure 6 also shows the water head definitions used in potential energy offset capability calculations done in Section 3.\n\nThe towers can be equipped with wind and solar power generation systems to en- able grid energy offsetting. Here the focus is on using multiple relatively inexpensive vertical or horizontal axis wind turbines (VAWT or HAWT) along with similarly in- expensive flexible mount solar photovoltaic panels (PV). Using multiple inexpensive units can improve serviceability and improve long term reliability, however other components such as power conditioners can become more complex and thus expensive. Consequently, achieving a balanced mix of sub-units will be critical. Also, a critical concern for using wind generation, particularly in suburban or exurban locations is its impact fatality of wild and specifically avian life. A simple solution is to use a mesh surround or shroud to shield the spinning parts from direct bird or wildlife hits, as illustrated in Figures 7 and 8.\n\nPeak efficiency and maximizing energy generation are not the key motivators in this application, rather, a lowering capital and maintenance costs, increasing replace-ability and serviceability, and reducing downtime cost of the subsystems are paramount. Therefore, any losses that may result as a consequence of including wild-life protection features should be considered reasonable and acceptable. Figure 9A shows the top view of an UF tower where two sets of wind generators are used to capture winds in both perpendicular (north-south and east-west) directions. Winds at any angles can be captured by a VAWT, however when a HAWT is desired (due to simplicity) it should be mounted on a swivel to capture winds in any directions. When photovoltaic panels are included, they can be easily mounted on the top surface of the top tank, as shown in Figure 9B.\n\n(a) Top view of a tower showing wind generators and wind capture directions.\n\n(b) Top view of a tower showing wind generators, wind capture directions and photovoltaic panels.\n\nEnergy demand offsetting can be achieved with in an UF when the energy requirements for operations within the UF is met partially or fully with energy that has been generated or (previously generated) and stored within the UF for later use. The primary and key feature the UF based farming proposes is the use of simple water storage tanks to store the excess energy production. The proposed scheme is therefore a modified pumped (potential energy) energy storage scheme where the energy generated is minimally conditioned and used directly to power pumps to pump water from the deep common CTk to each of the bottom tanks (BT1 and BT2). Then once they are full, it will continue to pump from the bottom tanks to the top tanks (TT1 and TT2) respectively. When the top tanks are full, gravity assist is used to irrigate, supply drinking water, or for other utility purposes. A small battery backup can be included for remote operation communication system, but batteries are not used for bulk energy storage.\n\nThe proposed scheme has the following distinct advantages;\n\n• Irrigation is a normal and necessary farm operation powered entirely by local energy generation.\n\n• Drinking use, particularly filtering, purification, and or heating/cooling is also a normal and necessary farm operation powered by local energy generation.\n\n• Even without wind or solar generators, when connected to the electric grid, UF tanks can serve as one way distributed grid storage to buffer any generation surplus (constant load for solar and wind producers) and help regulate the grid metrics. There is no need to idle generations facilities or use dump loads to dissipate generation surplus, which has both short and long term maintenance and economic benefits.\n\n• Unlike battery storage, there is no cyclic storage capacity loss. That is, the tanks can be filled and emptied infinitely without any loss of capacity, multiple times a day.\n\n• Unlike battery storage, operating temperature range, cold and heat tolerance is much higher.\n\n• Unlike battery storage, there are no toxic or hazardous chemicals involved.\n\n• Unlike battery storage, replacement cost and end of life cost are minimal.\n\n• Unlike battery storage, life cycle cost, environmental impact and carbon footprint for the entire system during construction, operation and decommissioning are smaller.\n\nThe proposed UF based approach to farming is mainly focused on exurban cultivation, although it can be applied to suburban and even urban agriculture (UA) under the right conditions such as land availability and zoning laws permitting. At the present, UA has gained support among non-governmental organizations (NGOs), community developers, urban planners, and politicians, as several examples both in formal academic and informal online literature can be found. Some examples are (Precker, 2021; Siegner et al., 2018; UGC, 2017; McCauley, 2021; Sustainability, 2019). However exurban agriculture (EA) concept as its own entity is not well developed and it is lumped together with UA.\n\nNotwithstanding the minutia, an UF based approach to EA will improve land utilization by;\n\n• Encouraging mixed use of lands for both traditional farming and energy offsetting and load stabilization applications.\n\n• Reclaiming contaminated land or land not suitable for agriculture, for food cultivation using more (than traditional) energy and labor intensive green house/hydroponics based techniques where the farm is mostly or fully energy self-sufficient.\n\n• Repurposing off-grid wet and coastal lands to become energy self-reliant aqua- phonics farms for oysters, seafood and fish.\n\nA food desert (FD) is defined as a neighborhood that lacks supermarkets, grocery stores, or other local food outlets offering healthy and affordable options (Dutko, Ploeg, and Farrigan, 2012). Criteria for designating food deserts include low-income and low-access census tracts, where food establishments are more than one urban or 10 rural miles away. Figure 10 shows the areas in the US designated as food deserts using this criteria (USDA FARA, 2010). When inspecting Figure 10, it is clear that FD (Rhone, Williams and Dicken 2022) are spread through the US. Large area FD in the southwest and western regions also coincide with low population density regions but are sufficiently close to densely populated urban regions. This means there is an abundance of cultivatable exurban land available and is underutilized for food production. According to Kaili (2021) economic opportunity zones (OZs) are located throughout the US, however there is only a 30% overlap between OZs and FDs regions. This means there are federally administered programs that can assist in setting up new businesses in these overlapped regions. For example, a newly setup downtown food co-op or a community restaurant can source all of their produce from a just-outside-the-OZ exurban farms. Such partnership will help address both food insecurity through local farming and economic insecurity through local jobs creation. Sustained access to nutritious food would likely result in better health outcomes (Gundersen and Ziliak, 2015), high school completion rates and regional economic outlook (USHHS, 2030).\n\nThis figure has been reproduced with permission from (USDA FARA, 2010).\n\n\n3. Capital, operation, and energy analysis\n\nEstimating the capital needed to set up a new farm with or without using the proposed UF approach, is generally considered difficult. Mainly due to opportunity cost for the capital used towards farm setup and the expected return from the farm both cannot be predicted accurately and the price for land varies based on location. However, we can attempt to quantify the present worth of the cost of energy saved using the proposed UF design approach over a period of time. This amount would serve as a baseline for setting the boundaries for investment in long term infrastructure i.e., setting up an UF.\n\nAs land is not considered a depreciating asset, we will not consider the cost of land in capital investment calculations. All the rest of the UF equipment such as towers, tanks, wind, solar generators and power electronic systems, irrigation systems, pumps, and motors are considered to be 10 year depreciating asset (as classified by the US tax agency, the internal revenue service - IRS) and so the calculations will be done over a 10-year period. Interest rate and inflation rate will be fixed for the entire duration and the calculations done on a monthly basis. Fixed interest and inflation rates are used to simplify the calculations, and it is a standard technique in economic analysis. Monthly time interval is chosen as most consumer and or small business (small farms) level economic activity is represented in monthly time frames (rents, loan payments etc.). The calculations are shown below in steps #1 through #6.\n\nStep #1: Assumptions\n\nWater head from Figure 6.\n\nStep #2: Estimating irrigation water usage and tank sizing\n\nAccording to the UDSA’s 2017 Census of agriculture, total irrigated acreage and water use per acre are 55.9 million acres and 1.5 acre-foot, respectively (USDA AgCensus, 2019). Acre-foot is the amount of water required to cover one acre to a depth of one foot. This is equivalent to 43,560 cubic feet or 325,851 gallons of water for one acre of land. In Figure 11, using data from Chiarelli et al., (2020), the relative water usage per acre of cultivated land is shown. It can be inferred that crops intended for food relatively consumed close to the average. Thus, average water usage is assumed for calculations.\n\nTotal available area in a UF is;\n\nTotal cultivatable area in a UF is, Auf tot - (area lost to tower concrete pads) here X = 10 m.\n\nTotal water consumption for an UF will be,\n\nIf the irrigation is done over a period of 300 days/year, the water consumption per day for an UF is;\n\nThe tower tanks (TT1, TT2, BT1, and BT2) should at minimum have a radius of 2.5 meters and height of one meter, for a cylindrical tank shape. A wider tank shape will be beneficial for stability purposes and also for providing surface are for the photovoltaic panels. Therefore, the tank volume with these dimensions is;\n\nWhen the top two (TT1 and TT2) tanks are full the entire UF can be irrigated and then the excess can be used for life stock drinking, cleaning, or for other utilities. The tank dimensions are well within the radius of the concrete pad size for the towers. The total overhead storage capacity is;\n\nThe buried common tank (CTk) should at minimum have a radius of 3.6 meter and a height of one meter.\n\nStep #3: Estimating potential energy storage capacity\n\nThe total water head available was shown in Figure 6 and the values assumed for h1 and h2 in Equation 2d. Energy needed to raise 39.26 m3 of water from the common tank CTk to the two bottom tanks BT1 and BT2 are;\n\nTotal energy storage capacity assuming an 80% pump efficient (η = 0.8) is;\n\nStep #4: Estimating size and cost of wind and solar generators\n\nThe total energy needed to irrigate a fully gravity fed UF a day is 2.4 kWh (Equation 12). If drip irrigation or pressurized irrigation are used, then the energy needed will be higher. Let us assume an irrigation overhead γ of 25%, and power electronics (for wind and solar energy conditioners and converters) efficiency ηpe of 90%, these are reasonable values sources from several manufacturer’s product documentation.\n\nLet us assume that pressurized irrigation is used and the duration of irrigation is one hour i.e., the tanks are already full and only the irrigation pumps are operating. This means that the solar and wind generators have to generate at a minimum 535 W of power, combined. Also, the time taken to fill the tanks can be several hours (but must be done within 23 hours). Therefore, with a generating capacity of 535 W, the tanks can be filled in 2.4 kWh/0.54 kW = 4.5 hours. The tank can be filled and ready to irrigate every single day if needed, completely free of any demand on the grid. Therefore, the total energy to irrigate an UF with pressurized irrigation is;\n\nStep #5: Estimating grid energy demand offset\n\nIf an UF is irrigated for 300 days a year, then the total energy demand on the grid would be;\n\nAt USD $0.15/kWh this means a total annual energy cost for an UF of;\n\nAccording to UDSA’s 2017 Census of agriculture, (USDA AgCensus, 2019), energy cost per farm on average was USD $15,289 for a total of 231,474 farms irrigating a total of 55.9 million acres.\n\nThis higher USDA figure is due to energy + transmission costs typically assessed by electric utilities and it included pumping from wells and pressurized irrigation systems. It is consistent with the estimates presented in this paper.\n\nStep #6: Estimating capital for setting up an UF\n\nUsing the estimate from USDA (Equation 18) and assuming the usage if uniform over the year, the monthly cost of energy for a UF is;\n\nAssuming the cost of electricity keeps with inflation and less than the interest rate, the present value of the cost of grid power can be calculated as;\n\nFor an UF or an UF sized farm, irrespective of the cost and source of energy used to operate it;\n\n• the cost of land is fixed\n\n• the cost of water for irrigation and farm use is fixed\n\n• the cost (some not all) of water storage tank(s) is fixed\n\n• the cost of irrigation sub-systems are all fixed\n\nThus, the equivalent present worth of the cost of purchasing grid power for a 10-year period, is sufficient (and greater than) the immediate cost of setting up wind and or solar generation systems. Even at twice the required generation capacity i.e., 1 kW (≈ 2× Equation 13), the cost of generating equipment needed is well under USD $2,500, at the time of writing this paper.\n\nHowever, the cost of materials and installation of the tanks, support structures, pumps, and concrete, should also be considered. Here the analysis is more nuanced. These structures have multiple purposes, they can be used for irrigation, for shading, as open barns, watering areas, and nursery etc. Therefore, the added cost of installing these structures should be considered as marginal or additional expenses to standard farm setup costs, which is difficult to estimate without detailed knowledge of the type and location of the standard structures.\n\nTo continue with the capital analysis, an unconventional approach would be to consider the cost of energy lost due to food wastage. Energy loss was estimated in Section 1 to be equal to USD $21.6 Billion per year in natural gas energy cost equivalent. Now, assume a portion of this loss is reduced due to local farming using UFs, let this loss reduction be 20%. That would imply an additional capital (monies not spent) availability of roughly USD $4.32 Billion in natural gas energy cost equivalent per year. The present value of total capital availability using the interest rate from Equation 2b, over a 10-year period is equivalent to USD $ 31.8 Billion. If this capital is made available as annualized capital infusion, loans or grants via some form of governmental program. For example, an initiative to promote 1,000,000 new exurban UF farms to solve food insecurity and reduce wastage, where each farm gets a voucher for USD $31,800 to be used and redeemed as capital investments are made within the 10-year period. Such an infusion of capital can completely reshape the national food landscape, realizing its many hidden health and economic benefits.\n\nThe calculation presented in Section 3 mainly focuses on irrigation to estimate tank sizing and making the economic case for solar wind augmentation at the smallest minimum dimensions, generation and storage capacities. However as mentioned earlier, infrastructure such as towers, concrete pads, irrigation lines etc. are fixed cost capital assets, irrespective of installed generation capabilities. Therefore, an UF or a collective of neighboring UFs can realize additional revenue by;\n\n• Installing additional and operating photovoltaic panels and or wind generators to produce excess energy that can be directly exported to the grid operator for additional revenue\n\n• Installing additional photovoltaic panels and or wind generators to produce excess energy that can be used to service loads with in the UF(s) such as living accommodations, grid backed refrigeration units, sterilization and food processing equipment etc; reducing overall grid demand, lowering operating costs and improving revenue of agri-businesses\n\n• Leasing the tank surface areas for third party virtual net-metering energy companies\n\n• Leasing the tank top for 5G network installations\n\n• Operating a private EV charging stations powered (partially or fully) by on premises wind and solar generation\n\nAll of the above mentioned activities inherently involve additional labor requirements for maintenance, operation and repair services. Thus, directly improving the farm economics and indirectly improving derived demand and local economic activity.\n\nIn 2021, 28% of the total energy used was for transportation of people and goods according to the U.S. Energy Information Administration (USEIA, 2022). Of the total transportation energy mix, biofuels accounted for a mere 5%. Which further breaks down as ethanol’s share around 4%; biodiesel, renewable diesel, and other biofuels making up the remaining 1%. Therefore, transport fuels are a major part of the total energy use mix and any reduction in the demand for fossil fuel derived transportation fuels will have a positive environmental and economic benefit.\n\nThe US Department of Energy (DOE), lists hydrogen H2 as an excellent choice for use as transportation fuel (USDOE H2, 2022). H2 has roughly 2.8 times the energy dense per kg weight of regular gasoline. However, due to its low volumetric efficiency (energy contained per unit volume), a centralized production model utilizing large storage containers (at very high pressure) and long distance transportation and distribution to end users (which is energy intensive); at the moment seriously limits the use of H2 as a mass transportation fuel.\n\nAt the same time, H2 is an excellent candidate fuel when its transportation and distribution is removed from the supply chain. This means H2 must be produced close to where it is needed and consumed. That is, H2 must be produced where it is needed almost on demand. Consequently, the US DOE recognizes the following as feasible pathways for local green H2 production (USDOE,2022).\n\n• H2 from Electrolysis: by splitting water into hydrogen and oxygen. Where the electricity is produced by excess solar or wind generation.\n\n• H2 from Renewable Liquid Reforming: by reacting a renewable liquid fuel, such as ethanol, with high-temperature steam to produce hydrogen gas.\n\n• H2 from Biomass Fermentation: by converting biomass into sugar rich feedstock for fermentation to produce H2 gas.\n\n• H2 from Biomass Gasification: by combining biomass with high-temperature steam and oxygen in a pressurized gasifier to generate H2 gas.\n\nOf these techniques, electrolysis is the most commonly used for low-volume H2 production. Making this pathway the most suitable for small scale production such as in the case of an UF. Also, this technique is relatively simple with least raw materials and equipment needed. However, it is more energy intensive and utilizes only one aspect of solar radiation i.e., photon intensity for H2 production. A commercial example of such an electrolysis based H2 production turn-key system is found here, (MRE LLC, 2022). Academically, a 2020 study quantified the benefits of using both solar and solar-heat for H2 production and found that the addition of heat improves the overall production volume and system efficiency (Gul and Akyuz, 2020).\n\nRenewable liquid reforming can also utilize energy from wind, solar photon intensity, and solar thermal energy to generate the electricity and heat needed in areas where alcohol production is readily available. While ethanol can be used directly as a transportation fuel, it had a limited shelf life due to its hydrophilic nature. Additional processing is required to extend its shelf life. Conversion of feedstock alcohol to H2 not only extends the shelf life and also improves energy density (per kg or per gallon) for use as a transportation fuel. The specific energy (energy per unit mass) of ethanol is approx. 25 MJ/kg, regular gasoline is approx. 46 MJ/kg and H2 is approx. 130 MJ/kg.\n\nElectrolysis and liquid reforming pathways both do not directly use or consume low-grade agricultural waste or biomass. Food waste is a major problem both for energy loss during production and transport phases, and during its final or post-consumption phase in terms of landfill use, decomposition and methane (landfill gas) escape into the atmosphere. Food waste can be considered biomass and it can be separated from landfill waste and used as feedstock for H2 production. Doing so further reduces overall energy wastage in the food cycle, in addition to the reduction realized through local farming.\n\nTherefore, up-cycling local food refuse, waste, farm refuse and byproduct (collectively biomass) and using it as the feedstock for biomass fermentation (Satinover et al., 2020) or gasification (Meramo-Hurtado et al., 2020) processes is an excellent way to minimize waste and capture otherwise wasted latent energy as useful transportation fuel. Furthermore, these processes can use the energy generated from wind, solar photon intensity, and solar thermal for H2 production. The produced H2 can then be used as transport fuel locally. Or a collective of local farms can join together to become an off-grid H2 producing center capable of satisfying and off-setting some regional transportation fuel needs. Such centers would greatly be benefiting their local environment and economy.\n\nAn example techno-analysis for a small to medium scale farm is presented in (Janke et al., 2020). They show that sharing production among farms can reduce H2 production costs by up to 28% and when local delivery vehicles were modified to run on H2, overall cost further reduced by 35%. Strengthening the case for local H2 production and consumption in agriculture, similar to the UF based approach proposed in this paper.\n\nBiomass fermentation/digestion pathway to H2 production can be further improved by the use of Microbial electrolysis cell (MEC) technology, where a selected electrochemically active bacteria oxidize organic matter to generate CO2, electrons and protons. MEC require external energy in the form of an electric current as an input to force ion movement in solution to produce H2. This current can be supplied by solar or wind energy generators making the process more energy neutral. The main challenges to use MEC technology are the reactor design and the need for membranes. Although, membrane free designs have been developed and are being tested currently; with large scale commercialization is still a few years away (Kadier et al., 2016; Dange et al., 2021). However, small scale reactors have been shown to be effective and can be readily incorporated into a distributed small scale farms for H2 production. Therefore, multiple successful demonstration of distributed small scale adoptions can bring about quicker large scale commercial adoption. Moreover, a favorable lending scheme or governmental assistance would also encourage commercial large scale adoption of such technologies earlier than when market economics could meaningfully support such adoptions.\n\nPotential future work\n\nThis paper presented a design methodology and framework for approaching ex- urban agriculture. The reader would notice that the proposed approach has not been implemented or tested. Doing so would require capital that the author does not currently possess. Moreover, local business and governmental support is also vitally needed. In order to obtain these and to begin the next phase of this research i.e., to setup and test the proposed approach, a publicly read, peer reviewed, and well flushed out proposal is essential. This is the desired fundamental outcome for the phase one theoretical work presented in this paper. The feedback obtained will guide the direction of subsequent work and to establish research partnerships, particularly in developing regions with acute food insecurity.\n\n\n4. Conclusion\n\nEnergy and food security are increasingly becoming principal concerns which can threaten economic and national security. With the COVID-19 pandemic exposing existing food supply chain weaknesses, pandemic driven exurban population growth, and ever increasing energy and food demands, local urban, suburban, and exurban agriculture have gained importance as a potential solution to both reducing grid energy demand and servicing local food needs via local production. In this paper, a modular land parcel i.e., unit field, based approach to exurban agriculture was presented. The unit field included a modified pumped storage design to reduce grid energy demand for standard farm operations by wind and solar augmentation.\n\nThe saving in energy costs over a 10-year period was shown to be economically equivalent to capital investment in solar and wind generation systems, even at minimal system size. When considering the cost of energy savings due to reduction in food waste over 10 years, it was determined that one million new farms could receive capital infusion of USD $31,800 to cover initial setup costs. Furthermore, additional avenues for revenue generation with and without new investments and capable of being operationalized in a short time, were also identified. Looking further into the future where new and renewable transportation fuels will be needed, several pathways which utilize the excess energy produced by the unit fields and the biomass generated locally to produce H2 were also identified. The produced H2, when used as transportation fuel, locally generates additional benefit both to the producers and the local energy and labor markets.", "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\nChiarelli DD, Passera C, Rosa L, et al.: Global Gridded Dataset of Crop-specific Green and Blue Water Requirements.2020. 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Reference Source\n\nMeramo-Hurtado SI, Puello P, Cabarcas A: Pro- cess Analysis of Hydrogen Production via Biomass Gasification under Computer- Aided Safety and Environmental Assessments. ACS Omega. 2020; 5(31): 19667–19681. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCauley DJ: Urban Agriculture Combats Food Insecurity, Builds Community. Reference Source\n\nMillennium Reign Energy LLC: Reference Source\n\nOffice of Energy Efficiency & Renewable Energy: Hydrogen Production. US Department Of Energy; 2022. Reference Source\n\nPrecker M: Food insecurity’s long-term health consequences. Reference Source\n\nRhone A, Williams R, Dicken C: Low-Income and Low-Foodstore-Access Census Tracts, 2015–19, EIB-236. U.S. Department of Agriculture, Economic Research Service; June 2022. Reference Source\n\nSatinover SJ, Rodriguez M, Campa MF, et al.: Performance and community structure dynamics of microbial electrolysis cells operated on multiple complex feedstocks. Biotechnol. Biofuels. 2020; 13: 169. Publisher Full Text\n\nSiegner A, Sowerwine J, Acey C: Does Urban Agriculture Improve Food Security? Examining the Nexus of Food Access and Distribution of Urban Produced Foods in the United States: A Systematic Review. Sustainability. 2018; 10(9): 2988. Publisher Full Text\n\nUSDA: FARA. Reference Source\n\nUSDA: Farms and Land in Farms 2021 Summary. Reference Source\n\nUSDA: Census of Agriculture. Reference Source\n\nUSEIA: Energy Explained. Reference Source\n\nHydrogen and Fuel Cell Technologies Office: DOE National Clean Hydrogen Strategy and Roadmap. Reference Source\n\nUrban Growers Collective: Reference Source\n\nWarshawsky D, Vos R: Governing at Scale: Successful Local Food Initiatives in the World’s Cities. Sustainability. 2019; 11(24): 7226. Publisher Full Text\n\nWebber ME: Power Trip: The Story of Energy. Basic Books; 2019. ISBN-13: 978-1541644397." }	[ { "id": "194318", "date": "08 Sep 2023", "name": "Ogheneruona Diemuodeke", "expertise": [ "Reviewer Expertise Renewable energy", "climate change mitigation technologies and deep decarbonisation." ], "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 is on an important research area, which focuses on food availability and reduction in energy intensity of agrifood production. However, the part of content seems to me like craftwork. The manuscript could be improved by considering the following:\nThe author could free space for the engineering design of all the equipment considered in the study, namely wind turbine, solar PV, power electronics etc.\n\nPresent the wind and solar resource assessments of the studied area. The author could use literature to support the resource availability.\n\nThe water needs analysis of the studied area to support the irrigation system.\n\nIs UFarea different from Auf tot?\n\nRegarding Equation 4, is the area covered by the buried tank considered a cultivatable area?\n\nRegarding Equation 5, where does 1.5 come from? If it is from water needs analysis, it should be explicitly presented.\n\nA potential leader would like to know the function of the buried tank. This should be presented explicitly along the justification in terms of energy storage and irrigation methods.\n\nEquation 10 should clearly show the conversion factor from Joule to Watt-hour.\n\nEquation 12, 2.4 Wh should read 2.4 kWh.\n\nOne wonders how 535 Wh suddenly became 535 W (approx. 0.54 kW). The author should be reminded that energy (Wh) is different from power (W).\n\nThe Section for hydrogen looks abrupt and does not connect with the research question, including the problem statement, of the study. Why do we need the hydrogen Section?\nIn general, the manuscript lacks the focus and rigour of a scientific paper. In addition, there were cases of measuring the volume of air with a syringe. The author could extend the work by computing emission reduction via the reduction in energy density of the agrifood.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? No\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-363
https://f1000research.com/articles/12-37/v1	10 Jan 23	{ "type": "Research Article", "title": "Comparative evaluation of dermatoglyphic patterns between skeletal class I and skeletal class III malocclusion", "authors": [ "Sonika Achalli", "U S Krishna Nayak", "Murali P S", "Keerthan Shashidhar", "Vinayak Kamath", "Sonika Achalli", "Murali P S", "Keerthan Shashidhar", "Vinayak Kamath" ], "abstract": "Background: Dermatoglyphics is the study of various dermal configurations on the fingers, palms, and soles. These appear during the 12th week of intrauterine life and develop completely by the 24th week. It is said that they remain constant thereafter. The aim of the present study was to compare and assess the association of dermatoglyphic patterns between skeletal class I and skeletal class III malocclusion. Methods: Finger and palm prints were collected using the ink and roller method from 604 subjects who were divided into skeletal class I, class III with maxillary retrognathism and class III with mandibular prognathism based on lateral cephalogram assessment. Results: Loop pattern was more predominant in skeletal class I malocclusion subjects and whorl pattern was more frequent in the other two groups. Total finger ridge count and atd angle also showed significant difference between the study groups. Conclusions: The present study attempted in assessing the association between dermatoglyphic patterns and skeletal malocclusion. Analysing dermal configurations may aid in indicating the type of developing malocclusion and thus help in interceptive and preventive orthodontics.", "keywords": [ "dermatoglyphics", "skeletal malocclusion", "fingerprint", "ridge count" ], "content": "Introduction\n\nThe study of dermal ridge counts and patterns on the fingers, palms and soles is referred to as ‘dermatoglyphics.’1 Cummins and Midlo coined the term dermatoglyphics in the year 19622 and defined it as the study of complex dermal ridge configurations on the skin covering the palmar and plantar surfaces of the hands and feet. It has been stated that studying the various dermal patterns on the fingers, palms and soles can help diagnose many diseases; mostly caused due to chromosomal abnormalities.3 The term dermatoglyphics has been derived from two Greek words; derma meaning skin and glyhe meaning carve.4\n\nLiterature says that the 12th week of intra-uterine life is characterized by the appearance of the dermal configurations and is completely established by the 24th week.5 It is believed that the configurations remain the same after that, except for the change in size. The teeth, alveolus and the palate develop during the same time as the dermal configurations, hence it is reported that there is an association between the two.5 Dermatoglyphics was initially studied in Down’s syndrome where abnormal dermatoglyphic patterns were noted. Later, various other medical conditions like Klinefelter syndrome, Turner’s syndrome, rubella syndrome, and leukaemia also have shown to exhibit abnormal or unusual dermal patterns.6 Expression of the gene is said to be the basis for the craniofacial growth and hence is accountable for skeletal malocclusions. Alterations in dermatoglyphic configurations might be a reflection of genetic or chromosomal abnormalities and hence may be used to study genetically influenced diseases.7 The dermal configurations are said to be unique for a particular individual; hence, studying these may help diagnose and treat certain genetic disorders in the individuals examined.8\n\nThe basic classification of the dermal patterns was given by Sir Francis Dalton in 1892.9 The fingerprint patterns have been mainly classified into three types i.e., arches, loops, and whorls. There may be two types of arches: simple or tented; two types of loops which may be ulnar or radial depending upon the direction they face and three types of whorls: symmetrical, spiral, and double loop (Figure 1).1,3 The atd angle is presented in the whole palm and this is formed by straight lines drawn from the digital triradius ‘a’ to the axial triradius ‘t’ and from this triradius to the digital triradius ‘d’ (Figure 2).3,10\n\nIn the present study we have evaluated the fingerprint patterns, total finger ridge count (TFRC) and atd angle and correlated them with skeletal malocclusion.\n\n\nMethods\n\nA convenience sample of 604 subjects including males and females reporting to the outpatient departments of Oral Medicine and Orthodontics, A B Shetty Memorial Institute of Dental Sciences, Karnataka, India were selected for the present observational study. Based on the previous studies;7 considering the effect size of 0.13, alpha error 5% and beta error 20%, a sample size of 192 was determined which was rounded off to 200 subjects per group. During the course of the study, 201 subjects in group I and II and 202 subjects in group III were included. The subjects were recruited between the period of September 2016 to January 2021. The subjects were aged between 21 and 30 years. Group I comprised of subjects with class I skeletal malocclusion where their SNA (sella tursica-nasion-point A) was 82±2 degrees and SNB (sella tursica-nasion-point B) was 80±2 degrees. Group II included subjects with class III skeletal malocclusion with maxillary retrognathism where SNA was less than 80 degrees and SNB was 80±2 degrees. Group III included subjects with skeletal malocclusion with mandibular prognathism where their SNB was more than 82 degrees with SNA 82±2 degrees. Cephalometric analysis was done by an experienced orthodontist and dermatoglyphic analysis was done by the principal investigator in order to avoid any bias. A pilot study was done initially with a sample size of 30 subjects in each group assessing the relationship between only fingerprint patterns and skeletal malocclusion.11\n\nSubjects with any one of the following conditions were excluded: malformation syndromes associated with maxilla and mandible; facial asymmetry and acquired skeletal defects; history of trauma/injury or surgical procedures done in the orofacial region; history of habits like mouth breathing, thumb sucking, lip biting, tongue thrusting; malformations of the fingers and palms which is congenital or acquired, amputated fingers, skin diseases, and wound or scars on the fingers.\n\nSubjects were informed about the method and procedure of the study following which written informed consent was obtained. Ethical approval was received from the central ethics committee of the university (NU/CEC/2016-2017/0078 dated 12/08/2016). Lateral cephalogram was assessed to identify the type of skeletal malocclusion. The finger and palm prints of both hands were recorded by ink and roller method as described by Cummins and Midlo.2 First, the subjects were asked to wash their hands with soap and water in order to remove any oily secretions, dirt and sweat and then dried with a towel. A small amount of ink was applied on the inking slab and a thin and even film of ink was obtained by rolling it thoroughly onto the entire surface of the slab. The palmar surface of the right hand was placed on the inking slab with a gentle press. The inked palmar surface was then gently pressed onto a clean, white bond paper and removed. The procedure was repeated for the left hand. In order to take the rolled impressions of the individual fingers, the bulb of the finger was placed at right angle to the surface of the inked slab and then rolled or turned until the bulb faced the opposite direction. The finger was then placed onto a clean, white bond paper and rolled in a similar manner. A clean, rolled impression of the fingerprint was obtained.2\n\nRidge count was calculated by drawing a line from the core to the triradius and counting the number of ridges the line touches or crosses (Figure 1) in each digit. The arch pattern has no triradius, there is one triradius in loop pattern and the whorl pattern has two triradii.3\n\nA palm print is required to measure the atd angle. Atd angle was estimated as an angle formed by joining the lines drawn from the digital triradius ‘a’ to the axial triradius ‘t’ and from this triradius to another digital triradius ‘d’ (Figure 2).10 Data was thus collected and analysed for various fingerprint patterns, total finger ridge count and atd angle of both hands (Figure 3).\n\nThe obtained data was then statistically analysed. The data was analysed using Statistical Package for Social Sciences software version 22 (IBM SPSS Statistics, Armonk, NY: IBM Corp). The distribution of different dermatoglyphic configurations on each hand in each class of malocclusion was calculated using percentages. The association between class of malocclusion and the dermatoglyphic pattern was tested using Fisher’s Exact test where p<0.05 was considered significant. Comparison of the variables between study groups was done using chi-squared and Kruskal-Wallis tests where p<0.05 was considered significant.\n\n\nResults\n\nThe study sample comprised of 604 subjects with 201 subjects in in group I and II; 202 subjects in group III aged between 21 and 30 years.12 Group I had 67 males and 134 females with a mean age of 24.02±2.99 years, group II had 109 males and 92 females with a mean age of 23.78±2.27 years and group III had 105 males and 97 females with a mean age of 24.09±2.57 years.\n\nThe obtained data was then statistically analysed. The distribution of different dermatoglyphic configurations on each hand in each class of malocclusion was calculated using percentages. The association between class of malocclusion and the dermatoglyphic pattern was tested using Fisher’s Exact test where p<0.05 was considered significant (Table 1). Loop pattern was predominant in group I subjects (i.e., class I skeletal malocclusion) and whorl pattern occurred more frequently in group II (i.e., class III skeletal malocclusion where maxilla is retrognathic) and group III subjects (i.e., class III skeletal malocclusion where mandible is prognathic). The frequencies of the dermatoglyphic patterns differed significantly between group I and the other two study groups but there was no significant difference between group II and group III subjects (Table 1).\n\n* Results are statistically significant where p<0.05.\n\nThe total finger ridge count in each hand was calculated and the mean value was determined. The atd angles of the right and left palm were also determined. Comparison of these variables between study groups was done using appropriate statistical tests (chi-squared, Kruskal-Wallis tests) where p<0.05 was considered significant (Table 2). TFRC differed significantly between group I and other two study groups whereas no significant difference was seen between group II and group III subjects. Mean TFRCs of both hands were the highest in group I subjects and lower in group II and group III subjects. Similarly, atd angle also differed significantly between group I and other two study groups with no significant difference between group II and group III (Table 2).\n\n[RT FRC=right finger ridge count; LT FRC=left finger ridge count].\n\n* Results are statistically significant where p<0.05.\n\n\nDiscussion\n\nDepending on the genetic background, the effect of an active environmental factor on a particular phenotype varies; this in turn will affect the structure developing during the same time.7,13 The facial structures and the dermal ridges of the fingers and palm develop from the ectoderm. Structures of the craniofacial complex and the epidermal ridges form during the same time i.e., second trimester of the intrauterine life. It has been deciphered that any hereditary or environmental factors causing various malocclusions may also trigger abnormalities in dermatoglyphic patterns.7,13 The study of dermal ridge configurations of the fingers, palms and soles can be a predominant diagnostic tool for many disorders particularly those with genetic abnormalities which may be associated with the deformation of fingerprint pattern.3 Hence, the present study was undertaken to evaluate and correlate the various dermatoglyphic patterns with skeletal class I and skeletal class III malocclusion.\n\nIn the present study, loop pattern was more predominant in both hands of group I subjects with skeletal class I malocclusion and higher frequencies of whorl pattern was observed in both hands of group II (subjects with skeletal class III malocclusion with maxillary retrognathism) and group III subjects (subjects with skeletal class III malocclusion with mandibular prognathism). The results were statistically significant.\n\nLoop pattern was more frequent in the skeletal class I malocclusion group in the studies done by Eslami N et al.14 and Gautham N et al.15 The present study also showed similar results. In the study conducted by Charles A et al.16 loop pattern was predominant in both skeletal class I and class III malocclusion. Some studies have contrasting results like in studies conducted by Reddy BRM et al.,7 Jindal G et al.17 and Tikare S et al.18 Nonsignificant results were obtained by Reddy BRM et al.7 pertaining to the class I malocclusion group, but a higher frequency of whorls was seen in class II and class III malocclusion. Jindal G et al.17 found in their study that dermatoglyphic pattern was not specific to any particular class of malocclusion, although higher frequency of whorls was seen in class II and class III malocclusion. The study by Tikare S et al.18 showed that the frequency of whorl pattern was equally distributed in all three classes of malocclusion. The contrast in results could be because in the above studies Angle’s classification of malocclusion was taken to classify the different malocclusion groups whereas in the present study skeletal malocclusion was considered.\n\nThe total finger ridge count of both hands was calculated. The present study showed that mean TFRCs were the lower in group II and group III subjects and highest in group I subjects. The results were statistically significant between group I and the other two study groups. This was in accordance with the studies done by Jindal G et al.17 and Eslami N et al.14 The study by Reddy BRM et al.7 showed an increase in TFRCs in all groups of malocclusions with no statistical significance. The variation in results in the present study could be due to the reason that Angle’s classification of malocclusion (molar relationship) was taken into consideration for grouping the subjects whereas in the present study skeletal malocclusion was considered.\n\nPalm prints of both hands were taken from all subjects to measure the atd angle. Mean atd angle was found to be higher in group I subjects when compared to the other two study groups. The results were statistically significant. The study done by Jindal G et al.17 also showed a significant difference in atd angle among the different groups of malocclusions. Other studies have shown varying results. The study done by Reddy BRM et al.7 showed that the mean atd angle was higher in the study groups when compared to the control group i.e., class I occlusion. The study done by Eslami N et al.14 showed no significant difference in mean atd angle between Angle’s skeletal class I, class II and class III subjects. The reason for the varying results could be because Angle’s classification (dental) was used to classify malocclusion whereas in the present study skeletal malocclusion was considered.\n\nIn the current study, racial, ethnic variations and hereditary factors were not considered which could be included in future studies. Recent advances like digital finger and palmprint method may be used instead of the ink and roller method used in the present study. Further, whether it can be relied as a sole factor for prediction of skeletal malocclusion is still questionable.\n\n\nConclusions\n\nThe current study strived to evaluate and correlate the various dermatoglyphic patterns with skeletal malocclusion. The results showed that a particular type of malocclusion may be specific to a certain dermatoglyphic pattern. Loop pattern was more predominant in skeletal class I malocclusion subjects and whorl pattern was more predominant in skeletal class III subjects. Mean TFRCs and mean atd angle was higher in skeletal class I malocclusion subjects when compared to skeletal class III subjects. Based on the results obtained in the present study, dermatoglyphic patterns may be used as an indicator of skeletal malocclusion. This shows that it could be used as a diagnostic tool at an early age with a fair degree of accuracy and may aid in intercepting and preventing the developing skeletal malocclusion.", "appendix": "Data availability\n\nFigshare: data sheet 1.xlsx. https://doi.org/10.6084/m9.figshare.21550662. 12\n\nThis project contains the following underlying data:\n\n• data sheet 1.xlsx (Data sheet showing the results obtained during the study)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nSir Galton F: Finger Prints. New York:Da Capo Press;1965.\n\nCummins H, Midlo C: The topographic history of the volar pads (walking pads) in the human embryo. Embryol. Carnig. Int. Wash. 1929; 20: 103–109.\n\nVerbov J: Clinical significance and genetics of epidermal ridges-a review of dermatoglyphics. J. Invest. Dermatol. 1970; 54: 261–271. Publisher Full Text\n\nGibbs RC: Fundamentals of Dermatoglyphics. Arch. Derrmatol. 1967; 96: 721–725. Publisher Full Text\n\nKanematsu N, Yoshida Y, Kishi N, et al.: Study on abnormalities in the appearance of finger and palm prints in children with cleft lip, alveolus, and palate. J. Maxillofac. Surg. 1986; 14: 74–82. Publisher Full Text\n\nTrehan M, Kapoor DN, Tandon P, et al.: A Correlative study of dermatoglyphic pattern with malocclusion. J. Indian Orthod. Soc. 2001; 34: 114–125.\n\nReddy BRM, Sankar SG, Roy ET, et al.: A comparative study of dermatoglyphics in individuals with normal occlusions and malocclusions. JCDR. 2013; 7: 3060–3065.\n\nKamboj: Dermatoglyphics, letter to editor. Br. Dent. J. 2008; 204: 51. Publisher Full Text\n\nGalton F: Finger prints. London:McMillan;1982.\n\nGupta V, Kumar P, Dupare R, et al.: Dermatoglyphics and dental caries: A review. Indian J. Forensic Odontol. 2011; 4: 33–37.\n\nAchalli S, Patla M, Nayak K, et al.: Assessment of dermatoglyphic patterns in malocclusion. J. Dent. Indones. 2018; 25: 104–107. Publisher Full Text\n\nAchalli S, Nayak USK, Murali PS, et al.:data sheet 1.xlsx. figshare. [Dataset]. 2022. Publisher Full Text\n\nMossey PA: The heritability of malocclusion: part 2. The influence of genetics in malocclusion. Br. J. Orthod. 1999; 26: 195–203. Publisher Full Text\n\nEslami N, Jahanbin A, Ezzati A, et al.: Can Dermatoglyphics Be Used as a Marker for Predicting Future Malocclusions? Electron. Physician. 2016; 8: 1927–1932. Publisher Full Text\n\nGoutham N, Shishira RJ, Shailaja AM, et al.: Malocclusion: An adjunctive aid in individual identification. J. Oral Maxillofac. Pathol. 2020; 24: 138–142. Publisher Full Text\n\nCharles A, Ramani P, Sherlin HJ, et al.: Evaluation of dermatoglyphic patterns using digital scanner technique in skeletal malocclusion: A descriptive study. Indian J. Dent. Res. 2018; 29: 711–715. Publisher Full Text\n\nJindal G, Pandey RK, Gupta S, et al.: A comparative evaluation of dermatoglyphics in different classes of malocclusion. Saudi Dent. J. 2015; 27: 88–92. Publisher Full Text\n\nTikare S, Rajesh G, Prasad KVV, et al.: Dermatoglyphics - A marker for malocclusion? Int. Dent. J. 2010; 60: 300–304." }	[ { "id": "159764", "date": "26 Jan 2023", "name": "Preethi Balan", "expertise": [ "Reviewer Expertise Oral health 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\nIn this study, the authors aimed to determine the association between dermatoglyphic patterns and skeletal malocclusion. The authors observed a predominance of loop patterns in skeletal class I and whorl patterns in skeletal class III. Total finger ridge count and atd angle showed significant differences between the study groups. The study has clinical relevance in terms of early identification and intervention of malocclusion. Please see below few suggestions.\nPlease provide the full form of atd in the abstract.\n\nPlease include values of statistical interpretation in the abstract.\n\nPlease cite the reason for rounding off the sample size to 200 after sample size estimation.\n\nThe authors mention that a pilot study was done initially with a sample size of 30 subjects. Was the data from the pilot study incorporated into the existing study?\n\n“Cephalometric analysis was done by an experienced orthodontist, and dermatoglyphic analysis was done by the principal investigator in order to avoid any bias”- Please remove the term principal investigator and mention the initials of the researcher who performed the analysis.\n\nAlso, please mention who performed the data collection on fingerprint patterns, TFRC and atd angle. Was any calibration exercise performed before data collection?\n\nPlease cite the criteria for the cephalometric classification of patients in this study.\n\n“During the course of the study, 201 subjects in group I and II and 202 subjects in group III were included”. Please rewrite this sentence for clarity in the methods and results section; Groups should be distinctly described.\n\nPlease include full forms of abbreviations at the first mention in the text-\n\nTFRC in the abstract and main text Provide legends for abbreviated terms in Table 1 10. Please cite or obtain permission to publish if Fig 1 and 2 are not from the existing original research\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9517", "date": "04 Apr 2023", "name": "Sonika Achalli", "role": "Author Response", "response": "Dear Dr Preethi Balan, Thank you for reviewing our article and giving us your expert suggestions. Reviewer 1: Please provide the full form of atd in the abstract. Comments from authors: Suggestion has been incorporated Reviewer 1: Please include values of statistical interpretation in the abstract. Comments from authors: Suggestion has been incorporated Reviewer 1: Please cite the reason for rounding off the sample size to 200 after sample size estimation. Comments from authors: Citation for the sample size calculation has been added. As suggested by the statistician the sample size was rounded off for calculation purpose. Reviewer 1: The authors mention that a pilot study was done initially with a sample size of 30 subjects. Was the data from the pilot study incorporated into the existing study? Comments from authors: Yes, the data from the pilot study has been incorporated into the existing study Reviewer 1: “Cephalometric analysis was done by an experienced orthodontist, and dermatoglyphic analysis was done by the principal investigator in order to avoid any bias”- Please remove the term principal investigator and mention the initials of the researcher who performed the analysis. Comments from authors: Suggestion has been incorporated Reviewer 1: Also, please mention who performed the data collection on fingerprint patterns, TFRC and atd angle. Was any calibration exercise performed before data collection? Comments from authors: Calibration exercise was not done but single examiner i.e principal investigator has performed the data collection and this has now been incorporated. Reviewer 1: Please cite the criteria for the cephalometric classification of patients in this study. Comments from authors: Suggestion has been incorporated Reviewer 1: “During the course of the study, 201 subjects in group I and II and 202 subjects in group III were included”. Please rewrite this sentence for clarity in the methods and results section; Groups should be distinctly described. Comments from authors: Suggestion has been incorporated Reviewer 1: Please include full forms of abbreviations at the first mention in the text- TFRC in the abstract and main text Provide legends for abbreviated terms in Table 1 Comments from authors: Suggestion incorporated Reviewer 1: Please cite or obtain permission to publish if Fig 1 and 2 are not from the existing original research Comments from authors: Suggestion incorporated" } ] } ]	1	https://f1000research.com/articles/12-37
https://f1000research.com/articles/12-362/v1	03 Apr 23	{ "type": "Research Article", "title": "Magico-religious beliefs, stigma and help-seeking for adolescent mental health at the community level – A multi-stakeholder qualitative exploration in southern India", "authors": [ "Vidya Prabhu", "Varalakshmi Chandra Sekaran", "Lena Ashok", "Brayal D'Souza", "Ravichandran Nair", "Vidya Prabhu", "Lena Ashok", "Brayal D'Souza", "Ravichandran Nair" ], "abstract": "Background: Almost half of all mental health disorders begin before the age of 14. However, several barriers exist to seeking help for mental health conditions among adolescents in the Indian context. The present study aimed at exploring multi-stakeholder perspectives on adolescent mental health and help-seeking behavior as well as referral. Methods: Our study adopted a qualitative design with focus group discussions (FGDs) and in-depth interviews (IDIs) conducted over a span of one year among 70 participants using purposive sampling. Participants included parents of adolescents, adolescents aged 14–16 years, high school teachers, and mental health professionals (MHP) and a pediatrician. The study was conducted in selected educational blocks of Udupi taluk. Parental written informed consent and assent from adolescents were obtained. Data was transcribed and thematic analysis was performed using Atlas.ti (version 8) to identify codes and code families following which themes were identified. Results: Our findings elicited community-held beliefs and attitudes indicating pathways and barriers to seeking care. Participants perceived that increased device use, accessing pornographic material, experiencing bullying, academic stress and negative perceptions surrounding mental health with the addition of magico-religious beliefs contributed to delayed help-seeking. Barriers to help-seeking included stigmatizing cultural perceptions. A considerable proportion of families found traditional healing more acceptable in the community. Conclusions: Stigma related to mental health continues to be one of the major barriers to early intervention. School counselors, teachers and even religious leaders in the community were found as effective liaisons in providing referrals to MHP. Recommendations for future studies and the way forward are discussed.", "keywords": [ "Adolescent mental health", "magico-religious beliefs", "stigma", "discrimination", "help-seeking behavior" ], "content": "Introduction\n\nCommon mental disorders such as depression affects an estimated 264 million individuals worldwide (WHO, 2019b). Over 197.3 million in India suffer from mental health conditions, with 457 million suffering from depression and 449 million suffering from anxiety respectively (Sharan & Sagar, 2008). Despite the high prevalence of mental disorders, a considerable majority of the population in poor and middle income communities do not seek treatment for mental health conditions (Rathod et al., 2017). Among adolescents, mental health disorders account for 16% of the worldwide burden of the disease. Half of all mental health disorders begin before the age of 14, however, the majority of instances go unnoticed and untreated (Patel et al., 2008; WHO, 2019a). Depression is one of the most common causes of illness in adolescents around the world with suicide recorded as the fourth highest cause of mortality among teenagers aged 15–19 years. According to the National Mental Health Survey 2015, 83% of people with mental illnesses in India do not seek treatment, resulting in a treatment gap that is larger than in any other health sector (Gururaj et al., 2016). This could have major ramifications for the person, their family, and their community. Religious beliefs and willingness to seek help are two elements that determine help-seeking behavior (Choudhry et al., 2016; Tesfaye et al., 2020). Religion and religious activities are major aspects of life in India, and individuals routinely seek help from faith healers for mental health conditions. Treatment seeking may depend on perceptions of family members, peer groups, relatives, and community members who may be involved in treatment decisions involving mental health help-seeking behavior. Help-seeking traditionally may impinge on a variety of reasons, including the decision-maker's belief that religious practices cure mental illnesses, ease of access, the stigma associated with psychiatric consultations, strong belief in the supernatural origins of mental illnesses, insufficient knowledge about mental illnesses, and relatively scarce mental health services(Padmavati et al., 2005; Sagar et al., 2020). According to the bio-psychosocial spiritual model, individuals have spiritual needs in addition to biological, psychological, and social requirements. These needs are enhanced during times of sickness (Koenig, 2012). Accessing religious and spiritual resources may also aid the individual and family to cope with mental illnesses.\n\nSocietal beliefs and attitudes that drive help-seeking for adolescent mental health conditions are not adequately explored in our setting and rarely include multiple stakeholders including adolescents. To bridge this gap, our study aimed at exploring diverse stakeholder perspectives on mental health risks among adolescents, prevalent magico-religious beliefs, and mental health help-seeking using qualitative methodology.\n\n\nMethods\n\nEthical clearance was obtained from Kasturba Medical College and Kasturba Hospital Institutional Ethics committee with IEC no:230/2015. The study areas included Udupi and Brahamavara educational blocks of Udupi taluk. Prior to data collection, approval was obtained from the Deputy Director of Public Instruction, Karnataka, and concerned school authorities. Written informed consent from parents of adolescents for their participation in the study and assent from the adolescents for audio recording was obtained prior to data collection. Participant information sheets (PIS) were provided as part of the informed consent process to the participants which clearly indicated that anonymized data from the study will be used for publication purposes.\n\nThe study employed a phenomenological approach to study the lived experiences of the participants. The study area included two educational blocks of Brahmavara and Udupi under Udupi taluk situated in coastal Karnataka, south India. Our study adopted a qualitative design with focus group discussions (FGDs) and in-depth interviews (IDIs) and was conducted over a span of one year and three months between October 2018 and December 2019 among 70 participants using purposive sampling. Participants included parents of adolescents, high school teachers, adolescents aged 14–16 years and mental health professionals (MHP) and a pediatrician. Parents of adolescents were identified at the community level through healthcare workers. Apart from contacting them for the study, the researchers had no prior relationships established with the participants. They were contacted face-to-face and an interview/FGD was arranged at a time convenient to them in a private space. Prior to conducting the interview or FGDs, the participants were provided details of the study and the reasons for conducting the study. The FGDs for parents were carried out at an anganwadi’s after school hours Anganwadis are part of the Integrated Child Development Scheme which is a government initiative at the community level. The Indian school system consists of government, private and aided schools. An attempt was made to include participants (adolescents and teachers) from all three types of schools. Data collection with adolescents and teachers happened at the school in a private room. All interviews were conducted face-to-face. Two adolescents refused participation as their parents had not provided consent. Apart from the participants and the primary researcher, there was a note-taker from the research team present during the FGDs and IDIs. The primary researcher and note taker were trained in qualitative methods. The interviewer facilitated the interview to achieve the study objectives and did not impose on how the interview proceeded.\n\nThe qualitative approach was adopted, and data collection spanned between 2018 and 2019. Interview guides for parents, teachers, adolescents and MHP and other health professionals were developed following literature review and quantitative data analysis of our larger study involving multiple phases. These were validated by a team of health professionals including clinical psychologists, a psychiatrist and social workers. FGD and IDI guides are publicly available (Chandra Sekaran, 2023b).\n\nIn all, three focus group discussions (FGDs) and six in-depth interviews (IDIs) each were conducted among parents and teachers; one FGD and six IDIs among adolescents and six IDIs were conducted among mental health and other professionals (Pham et al., 2021). The interviews were conducted by the primary researcher. Note-taking during the interviews was also performed. Each FGD lasted for about an hour to an hour and a half while interviews lasted about 45 minutes to one hour. Written informed consent was obtained for the audio recording of the interviews. Parents of adolescents were approached with the help of community health workers while teachers and adolescents were recruited through the schools. The interviews were conducted in a private room to maintain confidentiality. There were no repeat interviews conducted. Data was collected until saturation. Transcripts were discussed by the research team only and participants were not involved beyond data collection.\n\nPer participants' preference, the interviews were conducted either in the local language (Kannada) or in English. The Kannada transcripts were first transcribed verbatim and translated into English prior to analysis by native Kannada speakers well versed in English who were part of the research team. Thematic analysis was performed using Atlas.ti (version 8) to identify codes and code families following which themes were identified and are presented in Table 1. Open access qualitative data analysis software are available online such as QDA Miner for similar analysis. Several other software provide open access with restrictions for limited periods of time including Atlas.ti and MAXQDA. Thematic analysis involves reading each of the transcripts derived from both the interviews and FGDs are read through and coded. This enables identifying various meanings that may emerge across the data and how they may answer the objectives of the study. Coding was performed by three research team members with two repeated rounds of discussion to decide on the themes. Thematic analysis was both deductive and inductive. No feedback was sought from the participants.\n\n\n\n1. Perceived reasons for adolescent mental health conditions\n\n\n\n1. Internet and smartphone use\n\n2. Bullying\n\n3. Academic pressure\n\n4. Magico-religious beliefs\n\n\n\n2. Mental health help-seeking for adolescents\n\n\n\n1. Perceived Stigma, fear of discrimination from the community and labeling as barriers to help-seeking\n\n2. Traditional means of care\n\n3. Teachers and religious leaders as a bridge in the referral gap\n\n\n\n3. Promotion of adolescent mental health\n\n\nResults\n\nIn all, 70 participants were included in this qualitative study including 12 adolescents aged between 14–16 years and 58 adults. Among the 12 adolescents, eight lived in nuclear families while four lived in joint families. All adolescent participants except for one had at least one sibling. Two male adolescents and four female adolescents participated in the IDIs with five of them residing in nuclear families and one in a joint family. Only one of them was a single child. The number of family members ranged from two to nine in numbers. The parents of the adolescent participants were employed largely as unskilled workers (n=20 out of 24 parents of adolescent participants).\n\nOf the six IDIs among parents, three were conducted among fathers and three among mothers. Two FGDs were conducted among 15 mothers and one FGD among five fathers. A total of 26 teachers with teaching experience ranging from five to 22 years were recruited. Among the three FGDs with teachers, two were conducted in aided schools and one at a private school. The IDIs were conducted among three head teachers and three high school teachers and included one male and five female teachers from three private, two government and one aided school. Among MHP and other health professionals, the IDIs included two female and four male participants with a psychiatrist, a school counselor, a clinical psychologist, a pediatrician and two psychiatric social workers, all of whom were practicing at the time of the study. The major themes and sub themes that emerged from the study are listed in Table 1 and transcripts are publicly available (Chandra Sekaran, 2023a).\n\nInternet and smartphone use\n\nPerspectives of each of the stakeholder groups were gleaned on the reasons that adolescents were at risk for mental health conditions. Parents, teachers and adolescent participants largely cited the increased usage of smartphones, the use of internet, social media including accessing pornographic sites as major reasons for emotional disturbances among adolescents.\n\nThey are bombarded with so many things around them, especially today. I think it's the mobile. It is just disturbing children the utmost”. (IDI, Participant 2, Female)\n\nOne girl, she was not yet using social media. Then, her friend came to class and telling about these things. She was attracted by that and she also joined social media. Then, their academic progress came down. (FGD 5, Participant R4, Male)\n\nMental health professionals concurred with these life experiences and stated that reduction in communication between the parent and the child as well as developmental changes and other external factors as leading to mental health risks among adolescents.\n\nThere are the normal physiological changes, biological changes, physical changes, hormonal changes which are occurring. They don’t know what is right, what is wrong. So, there are chances that they get into the wrong hands or wrong websites or pornography and later pornography addictions. (IDI, Participant 3, Male)\n\nBullying\n\nBullying was also cited as a major problem in the school setting. The emphasis on understanding the phenomenon of bullying in addition to bully victims was evident from the data.\n\nBullying is a major problem, very major problem. There is something called bullying within cycle. So, today’s victims are tomorrow’s bullies. In a recent research which we did in our department, what we see is that most of the students are bully victims. Bully victims are adolescents who have been victimized and they have bullied also. (FGD 1, Participant R6, Male)\n\nThe stigma surrounding mental health also prevented students from seeking the help they could receive at the school level for fear of being bullied about it.\n\nWhen we go to school for interventions, if we call out one student for counselling that student is 100% bullied for coming for counselling. They are called ‘psycho’ or some other names. They are bullied just for coming to counselling. Without addressing these issues, I don’t think anything will be helpful. (FGD 1, Participant R6, Male)\n\nThe need to destigmatize mental health promotion and counselling at the school and community level is an important step in helping adolescents seek help at the right time.\n\nAcademic pressure\n\nExpectations from parents to perform better in academics was also cited by teachers.\n\nMaybe, the competitive parents, they always want their child to be toppers. So, that causes anxiety for the children. When they have that stress from their home that he/she has to get to a certain level, definitely anxiety will be more, isn't it? (IDI, Participant 2, Female)\n\nAdolescents were an important stakeholder group who raised the concern regarding inter-parental conflict or substance abuse, commonly alcoholism, by either or both parents which caused conflict. The added burden of academics, they stated, caused them to have ‘tension.’\n\nAt home, when parents fight and then there is pressure from teachers to study, students only take tension. (IDI, Participant 2)\n\nThis problem is, I think, the main thing is family issues, may be broken family, single parenting all these things. (FGD 1, Participant R2, Female)\n\nIn the Indian context, the emphasis on academics as an indicator of the child’s development was also discussed in that parents may observe poor academic performance of their child and deal with this rather than recognize possible underlying mental health conditions, hampering early identification and treatment.\n\nMagico-religious beliefs\n\nMagico-religious beliefs describe cultural beliefs that people hold surrounding mental health. The etiology is credited to supernatural activities. The belief that one’s past deeds, either of the individual or of the family, were thought to affect the present mental state which was evident from the interviews. Use of horoscopes or birth charts that predicted life circumstances, ‘Karma’ or the cycle of good and bad, and ‘fate’ was quoted often by community participants.\n\nThese beliefs, right from many centuries, we have been following, and it is deep-rooted in our society; we are not ready to change, particularly in poor families. (FGD 3, Participant R2, Female)\n\nIt is not our misdeeds but theirs. People who are mentally strong will have the ability to face it. But who are mentally weak will become victims easily. Sometimes it is fate. (IDI, participant 5, Female)\n\nInterestingly, adolescents were also able to recount instances of observing practices that they termed ‘black magic.’ These narratives indicated the intergenerational transmission of such societal beliefs.\n\nAstrologers cannot help. With sorcerers (magicians), it is possible to heal. They will do some black magic. It will cure mental illness. They will give something which they will keep in one place. If somebody crosses over that, the illnesses will pass on to that person. (IDI, Participant 3)\n\nAmong individuals with higher education and teachers, it was found that some participants questioned these beliefs.\n\nWe can see among the educated; they don’t follow such things because of media and awareness among people. (FGD 3, Participant R2, Female)\n\nAlmost all community stakeholders (98%), however, ascribed their belief in God to work things through for them.\n\nPerceived Stigma, fear of discrimination from the community and labeling as barriers to help-seeking\n\nAssociation with terms such as ‘mental’ or ‘mad’ are deeply stigmatizing. The belief that mental health help-seeking meant taking treatment for life and its associated side effects were barriers that were identified. As stated by a participating teacher:\n\nWhen we inform them (parents) to take help from the medical professionals, immediately they say that my child doesn’t have a problem. They never ever understand that, even mind will be sick. Immediate reaction is, they reject: “No, my child is healthy” (IDI, Participant 6, Female)\n\nOnce they start giving the pills and all, they will undergo depression and I don't know how much it will help them. (IDI, Participant 1, Female)\n\nHowever, participants stated that perceptions towards reaching out to professionals for mental health help were changing.\n\nBut now, I feel it is not like before. Earlier, people who are taken to the mental hospital were considered as ‘mad.’ Now, people’s approach has changed. Now, people have started calling it ‘psychological problem’ instead of madness. (IDI, Participant 5, Female)\n\nTraditional means of care\n\nHelp-seeking was found to be complex with community determinants and personal beliefs driving it. Participants from the community stated that deity belief, performing puja (religious rituals), or having their ‘horoscope’ checked would help in managing it.\n\nWe have our own deity in our house which belongs to our family and soon we are going to have puja which we perform every year. All that we have done, nothing is pending” (IDI, Participant 5, Male)\n\nMental health professionals stated strong beliefs surrounding supernatural entities led people to seek traditional healers, and that these magico-religious beliefs helped people cope.\n\nEffect of evil powers, effect of the stars, birth stars and effects of people who have died in the family, those things still exist. And these sorts of some magico-religious beliefs definitely will help psychologically.” (IDI, Participant 3, Male)\n\nTeachers and religious leaders as a bridge in the referral gap\n\nRegarding providing referral to adolescents, most schools it was observed, did not have counselors. Among those who did have counselors, they were important in the chain of referral.\n\nEven counselors themselves will refer. They work out from their side and refer. (FGD 5, Participant R2, Female)\n\nIn the absence of counselors, teachers largely took on the role of counselors themselves. Interviews with teachers brought about insights into their informal role as channels of liaison. As one teacher shared:\n\nHis (student’s) family members were saying that it is due to the possession of a spirit. Then, the headmaster called the parents and told them -‘I am not against your belief but he can be corrected if you take him for counselling.’ Family members agreed to take him and gradually, he improved. (FGD 3, Participant R2, Female)\n\nSome schools took initiative and also provided mental health awareness programs.\n\nWe have so many programs in the school. We call doctors or we call other organizations during special days. By different resource persons, we try to give more awareness to the children. Because of that, such problems are less in our school. (FGD 3, Participant R2, Female)\n\nIt was of interest that religious leaders also played the role of agents of referral in the community.\n\nI see a lot of them (mentally ill individuals) over there (at the temple). And they also have that main person (the priest). He supports professional help and in fact, he tells many people who go to the hospital. I have heard him recommending: “You go and speak to the doctor there. (IDI, Participant 2, Female)\n\nPromotion of adolescent mental health\n\nAt the family level, parents recognized that communication between family members was key. Family support with love and supportive school environments were thought to be important in nurturing adolescents.\n\nAffectionate talk has to be there; it should be by parents as well as family members, even teachers also should talk affectionately. They expect love and affection. (FGD2, Participant R2, Female)\n\nThe fear associated with stigma appeared to be a major barrier in seeking help. The MPH recounted that behavioral disturbances during adolescence were seldom brought to their notice. The knowledge that the first episodes of mental disorders can appear in early adolescence and were a risk for suicides was not common knowledge among parents and teachers.\n\nWhat we observe is that the initial episodes during adolescence is missed sometimes. Only when they come for help as adults, they will give a history that during his high school, he had these problems. We see mental illnesses are directly leading to suicide rates too. (IDI, Participant 3, Male)\n\nParents and teachers thus needed to be equipped with awareness on what could be considered normal versus abnormal behaviors among adolescents.\n\nPsycho-educate them regarding what is normal behavior and what is abnormal behavior in adolescents. (FGD 1, Participant R2, Female)\n\nImparting life-skills education (LSE) to adolescents through schools was seen as another important preventive measure. While some central schools did, this was not ubiquitously adopted by others.\n\nGiving full attention to adolescent health until they are 22 years or so, we are creating very strong and physically and mentally healthy adolescents. With life skills being taught to them, it becomes an armor to prevent them from future problems. (IDI, Participant 3, Male)\n\nThese findings indicate the perceptions at the community level on adolescent mental health and the need for promoting mental health at the school and community levels as opined by mental health professionals.\n\n\nDiscussion\n\nThis qualitative exploration was undertaken to explore facets of adolescent mental health from the viewpoint of multiple stakeholders including middle to late adolescents, high school teachers, parents of adolescents, MHP and a pediatrician. Since the study was conducted at the community level, our findings elicited community-held beliefs and attitudes indicating pathways and barriers to seeking care. Concerning risks for adolescent mental health conditions, most stakeholders including adolescents perceived that increased device or internet use, accessing pornographic material, academic stress, as well as supernatural causes, contributed. Literature supports the findings that unhealthy internet use can lead to addictions and can negatively impact physical and mental wellbeing (Camilleri et al., 2020; Chao et al., 2020; Pal Singh Balhara et al., 2019; Yu & Chao, 2016). These findings mirror that of Choudhry et al. in their meta-synthesis (Choudhry et al., 2016). Parental expectations and academic pressures (Jayanthi et al., 2015) as well as parenting factors(Sekaran, Ashok, et al., 2020; Sekaran, Kamath, et al., 2020) have been linked to a rise in mental health conditions among adolescents. Legislative measures to limit harmful internet exposures while reinforcing awareness on potential benefits and risks for adolescents is the need of the hour.\n\nCultural beliefs and help-seeking behaviors were found to be highly connected. Seeking help from MHP was not the initial recourse sought toward the mental health of adolescents. Barriers to help-seeking included stigmatizing cultural perceptions to labels such as the use of the term ‘mad.’ Kar (2008) describes similar belief systems prevalent in Orissa, India. A considerable proportion of patients and families asserted beliefs in ‘Karma’ or ‘fate’ and found faith-healing reassuring and more acceptable (Kar, 2008). Participating MHP recognized that seeking help from traditional providers was an important part of psychological coping. However, resorting to help seeking from traditional healers may also delay early intervention. Sensitivity to these belief systems is important for practicing mental health professionals.\n\nThough magico-religious beliefs appeared to be barriers in seeking early professional help, the integration of spiritual care in the context of health has been discussed in the literature (Kar, 2008; Roy et al., 2020). In the South Asian context, a study focusing on traditional healers and MHP in Nepal recommended integrating referrals and training traditional healers (Pham et al., 2021). Estrada et al. reported that integrating religious education can promote mental well-being among adolescents (Estrada et al., 2019). School counselors, teachers and even religious leaders in the community may act as effective liaisons in bridging adolescents to seek timely help from MHP as was comparable to findings in other settings (Long et al., 2017; Prabhu et al., 2021; Thomas et al., 2020). There is an urgent need to equipping the existing school system with competent counselors to aid mental health promotion among them.\n\n\nConclusion\n\nIn conclusion, our study elicited community stakeholders’ beliefs and attitudes towards the mental health of adolescents and help-seeking behavior. Increasing mental health awareness and equipping adolescents with the right coping strategies and establishing pathways to early referrals were important takeaways from this study. Targeting the community on sensitizing and destigmatizing issues surrounding mental health emerged as an important step in this direction. There is a need for further studies on interventions at the community and school levels targeting important stakeholders.\n\n\nAuthor contributions\n\nVidya Prabhu\n\nRoles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing\n\nVaralakshmi Chandra Sekaran\n\nRoles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing\n\nLena Ashok\n\nRoles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing\n\nBrayal D’Souza\n\nRoles: Formal Analysis, Methodology, Resources, Validation, Writing – Original Draft Preparation, Writing – Review & Editing\n\nRavichandran Nair\n\nRoles: Formal Analysis, Investigation, Methodology, Resources, Validation, Writing – Original Draft Preparation, Writing – Review & Editing", "appendix": "Data availability\n\nFigshare: Qualitative raw data. https://doi.org/10.6084/m9.figshare.22300426 (Chandra Sekaran, 2023a).\n\nThe project contains the following underlying data:\n\n- Qualitative Raw Data.docx.pdf\n\nFigshare: IDI and FGD guides.pdf. https://doi.org/10.6084/m9.figshare.21923664 (Chandra Sekaran, 2023b).\n\nThe project contains the following extended data:\n\n- IDI and FGD guides.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nCamilleri C, Perry JT, Sammut S: Compulsive Internet Pornography Use and Mental Health: A Cross-Sectional Study in a Sample of University Students in the United States. Front. Psychol. 2020; 11.\n\nChandra Sekaran V: Qualitative data. [Data]. figshare. 2023a. Publisher Full Text\n\nChandra Sekaran V: IDI and FGD guides. [Data]. figshare. 2023b. Publisher Full Text\n\nChao C-M, Kao K-Y, Yu T-K: Reactions to Problematic Internet Use Among Adolescents: Inappropriate Physical and Mental Health Perspectives. Front. Psychol. 2020; 11: 1782. Publisher Full Text\n\nChoudhry FR, Mani V, Ming LC, et al.: Beliefs and perception about mental health issues: a meta-synthesis. Neuropsychiatr. Dis. Treat. 2016; 12: 2807–2818. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEstrada CAM, Lomboy MFTC, Gregorio ER, et al.: Religious education can contribute to adolescent mental health in school settings. Int. J. Ment. Health Syst. 2019; 13(1): 1–6. Publisher Full Text\n\nGururaj G, Varghese M, Benegal V, et al.: National Mental Health Survey of India, 2015-16: Summary. NIMHANS Publication; 2016.\n\nJayanthi P, Thirunavukarasu M, Rajkumar R: Academic stress and depression among adolescents: A cross-sectional study. Indian Pediatr. 2015; 52(3): 217–219. Publisher Full Text\n\nKar N: Nilamadhab Kar (2008) Resort to faith-healing practices in the pathway to care for mental illness: a study on psychiatric inpatients in Orissa. Ment. Health Relig. Cult. 2008; 11(7): 720–740. Publisher Full Text\n\nKoenig HG: Religion, Spirituality, and Health: The Research and Clinical Implications. ISRN Psychiatry. 2012; 2012: 1–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLong KNG, Gren LH, Long PM, et al.: A picture of Indian adolescent mental health: An analysis from three urban secondary schools. Int. J. Adolesc. Med. Health. 2017;1–11.\n\nPadmavati R, Thara R, Corin E: A qualitative study of religious practices by chronic mentally ill and their caregivers in South India. Int. J. Soc. Psychiatry. 2005; 51(2): 139–149. Publisher Full Text\n\nPal Singh Balhara Y, Doric A, Stevanovic D, et al.: Correlates of Problematic Internet Use among college and university students in eight countries: An international cross-sectional study. Asian J. Psychiatr. 2019; 45(September): 113–120. PubMed Abstract \| Publisher Full Text\n\nPatel V, Flisher AJ, Nikapota A, et al.: Promoting child and adolescent mental health in low and middle income countries. J. Child Psychol. Psychiatry. 2008; 49(3): 313–334. PubMed Abstract \| Publisher Full Text\n\nPham TV, Koirala R, Kohrt BA: Traditional and biomedical care pathways for mental well-being in rural Nepal. Int. J. Ment. Heal. Syst. 2021; 15(1): 4–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrabhu V, Ashok L, Kamath VG, et al.: What predicts mental health literacy among school teachers? Ghana Med. J. 2021; 55(2): 141–146. Publisher Full Text\n\nRathod S, Pinninti N, Irfan M, et al.: Mental Health Service Provision in Low- and Middle-Income Countries. Health Serv. Insights. 2017; 10: 117863291769435–117863291769437. Publisher Full Text\n\nRoy D, Tripathy S, Kar SK, et al.: Study of knowledge, attitude, anxiety & perceived mental healthcare need in Indian population during COVID-19 pandemic. Asian J. Psychiatr. 2020; 51: 102083. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSagar R, Dandona R, Gururaj G, et al.: The burden of mental disorders across the states of India: the Global Burden of Disease Study 1990–2017. Lancet Psychiatry. 2020; 7(2): 148–161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSekaran VC, Ashok L, Kamath VG, et al.: Parental Involvement – Perceptions of Parents and their Adolescent Children. Indian J. Pediatr. 2020; 87(3): 200–206. Publisher Full Text\n\nSekaran VC, Kamath VG, Ashok L, et al.: Parenting attitudes and dimensions as predictors of adolescent behavioural problems in the Indian context: A community-based study. Asian J. Psychiatr. 2020; 50(December 2019): 101941. PubMed Abstract \| Publisher Full Text\n\nSharan P, Sagar R: The Need for National Data on Epidemiology of Child and Adolescent Mental Disorders. J. Indian Assoc. Child Adolesc. Ment. Health. 2008; 4(2): 22–27. Publisher Full Text\n\nTesfaye Y, Agenagnew L, Tucho GT, et al.: Attitude and help-seeking behavior of the community towards mental health problems. PLoS One. 2020; 15(11): e0242160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThomas E, Thomas E, Dey AM: Counseling Policy and Evaluation Role of School Counselors and the Factors that Affect their Practice in India Role of School Counselors and the Factors that Affect their Practice in India. Journal of School-Based Counseling Policy and Evaluation. 2020; 2(1): 22–28.\n\nYu T-K, Chao C-M: Internet Misconduct Impact Adolescent Mental Health in Taiwan: The Moderating Roles of Internet Addiction. Int. J. Ment. Health Addict. 2016; 14(6): 921–936. Publisher Full Text" }	[ { "id": "207415", "date": "19 Oct 2023", "name": "Suhad Daher-Nashif", "expertise": [ "Reviewer Expertise Sociology and anthropology of health and illness." ], "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 qualitative study aimed to explore perspectives of parents, adolescents and educators on adolescents' mental health experiences, perceptions and help seeking behaviours. The authors present an important topic and interesting findings, but major methodological and ethical issues are raised in this study.\n\nIntroduction: The introduction lacks literature about how mental health is framed by religion or different religions within the Indian context. The authors should give an idea about which religion/s they are discussing in their article. How do these religions frame mental illness and what does it advise for treatment? The authors talk about religion as a very general and vague category, separating it from other socio-demographic factors that have major impact on perceiving and managing mental illness.\nThere is minimal mentioning of previous studies on the topic, and how this research builds on these studies, and its findings.\nMethods:\nThe recruitment has several problems:\nHaving the health care providers (HCPs) as those to recruit patients is problematic due to the hierarchy issues. HCPs could promote the study, and ask their patients to contact researchers if they are interested to participate. Same regarding teachers and educators. They were recruited by the school, which is also problematic due to hierarchy issues.\n\nThe authors talk about purposeful sampling while they do not address their strategy to recruit participants. Although they mention the use of HCPs to help them, but still not clear how did they continued with those contacted by HCPs.\n\nThere’s no mentioning of inclusion and exclusion criteria, which is key factor in purposeful sampling.\n\nEthically: what strategies did they used to manage psychological distress through the interviews? Even if they did not have this, what strategies did they prepared to deal with this sensitive research?\nDid they include those who are experiencing a depressive episode at the time of the study?\nApplying focus group discussions/interviews and guided interviews do not fit at all with exploring the lived experience approach. What is the framework that guided the phenomenological study?\nConfidentiality within FGDs especially with adolescents: how did they manage that? How did they guarantee no use of any information by any adolescent? We know how at this age, persons might use these data to bully their colleagues. How did the researchers work to avoid that, if at all?\nBased on calculating the number of individual interviews and the number of focus groups, it seems that the focus groups included 12-14 people each, which is problematic by itself in phenomenology and lived experience research.\nAtlas.ti is used more for content analysis rather than thematic analysis that uses applies interpretation to identify patterns and abundance in the data.\nThe authors state that they used inductive and deductive: what is the theory that they have used in their deductive analysis? Deductive analysis is not something that is used in lived experience research. The results do not reflect deductive analysis.\nResults: Despite the big number of participants in this qualitative study, the content of the results does not reflect the number of the interviews, and diversity of participants. The results are listed in a very technical way, with quotes but lack narrative that reflects the lived experience of participants. Although, there is no differentiation between the different participants.\nDiscussion: very brief and this might be due to the content presented in the results section. It is very technical that does not reach the level of criticism and synthesis.\nConclusion: No mentioning of limitations and strengths 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? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-362
https://f1000research.com/articles/12-131/v1	03 Feb 23	{ "type": "Research Article", "title": "Increased growth temperature and vitamin B12 supplementation reduces the lag time for rapid pathogen identification in BHI agar and blood cultures", "authors": [ "Jawad Ali", "Mukund Joshi", "Asal Ahmadi", "Knut Olav Strætkvern", "Rafi Ahmad", "Jawad Ali", "Mukund Joshi", "Asal Ahmadi", "Knut Olav Strætkvern" ], "abstract": "Background: The rapid diagnostics of pathogens is essential to prescribe appropriate and early antibiotic therapy. The current methods for pathogen detection require the bacteria to grow in a culture medium, which is time-consuming. This increases the mortality rate and the global burden of antimicrobial resistance. Culture-free detection methods are still under development and are not used in the clinical routine. Therefore decreasing the culture time for accurate detection of infection and resistance is vital for diagnosis. Methods: In this study, we wanted to investigate easy-to-implement factors (in a minimal laboratory set-up), including inoculum size, incubation temperature, and additional supplementation (e.g., vitamin B12 and trace metals), that can significantly reduce the lag time (tlag). These factors were arranged in simple two-level factorial designs using Gram-positive (Escherichia coli and Pseudomonas aeruginosa) and Gram-negative (Staphylococcus aureus and Bacillus subtilis) bacteria, including clinical isolates with known antimicrobial resistance profiles. Blood samples spiked with a clinical isolate of E. coli CCUG17620 were also tested to see the effect of elevated incubation temperature on bacterial growth in blood cultures. Results: We observed that increased incubation temperature (42°C) along with vitamin B12 supplementation significantly reduced the tlag (10 – 115 minutes or 4% - 49%) in pure clinical isolates and blood samples spiked with E. coli CCUG17620. In the case of the blood sample, PCR results also detected bacterial DNA after only 3h of incubation and at three times the CFU/mL. Conclusions: Enrichment of bacterial culture media with growth supplements such as vitamin B12 and increased incubation temperature can be a cheap and rapid method for the early detection of pathogens. This is a proof-of-concept study restricted to a few bacterial strains and growth conditions. In the future, the effect of other growth conditions and difficult-to-culture bacteria should be explored to shorten the lag phase.", "keywords": [ "Lag time", "Growth temperature", "Vitamin B12", "Brain heart infusion", "Antimicrobial resistance", "Pathogen detection", "Blood culture" ], "content": "Introduction\n\nThe discovery of antibiotics was a breakthroughs in therapeutic medicines, which has enabled us to treat critical bacterial infections (Ali et al., 2020). But even before the introduction of the first antibiotic into clinical practice, some bacteria had already become resistant (D’Costa et al., 2006). The ability of bacteria to survive in the presence of antibiotics which were earlier used to treat the infections caused by these bacteria, is called “antimicrobial resistance (AMR)” (Kapoor et al., 2017). The misuse of antibiotics in clinical, agriculture, poultry, and aquaculture sectors is significantly increasing the emergence of AMR. The World Health Organization (WHO) has described AMR as a global health issue. Infections due to antibiotic resistant bacteria are rising. In 2019, 1.27M and 4.95M deaths were directly caused or associated with antibiotic-resistant bacteria, respectively (Murray et al., 2022). It is predicted that at the current rate, AMR will cause 10 million deaths per year and cost $100 trillion to the world economy.\n\nThe discovery void of new antibiotics also adds to the growing AMR problem. It has been more than 35 years since a new class of antibiotics was discovered, and bacteria are resistant to almost all the presently used antibiotics (Hoffman, 2020). The unavailability of new antibiotics further increases the need for rapid diagnostics of pathogens and their AMR. Early and appropriate antibiotic therapy is crucial in cases of severe bacterial infections and can significantly reduce mortality. Sepsis is considered a major health problem and accounts for an estimated 31.5 million cases with a mortality rate of 5.3 million deaths globally per year (Fleischmann et al., 2016; Taxt et al., 2020). In the case of sepsis, early treatment with appropriate antibiotics is highly vital for the patient’s survival (Bloos et al., 2017; Ferrer et al., 2014). In septic patients, delaying each hour the prescription of appropriate antibiotics can have lethal consequences (Avershina et al., 2022). Due to the lack of adequate and early diagnostics, clinicians are pushed to prescribe empirical antibiotics to the patients, which in most cases are not very effective and further disseminate antibiotic resistance (Khaledi et al., 2020).\n\nTo decrease the use of empirical and broad-spectrum antibiotics, optimizing the existing diagnostic methods and making them faster and more cost-effective is crucial. The current diagnostic procedures still rely on culture-based techniques where bacteria are cultured and then identified using different biochemical tests. After that, antibiotic susceptibility testing (AST) is performed, which gives an idea about the resistance or sensitivity of bacteria to different antibiotics. The culture-based methods have a turnaround time of two to four days in most samples. However, in the case of bloodstream infections where the bacterial load is lower, the identification and AST can take up to five days (Briggs et al., 2021; Metzgar et al., 2016). There are many emerging micro- and nanotechnologies for pathogen identification and AST, including both phenotypic (microfluidic-based bacterial culture) and molecular methods (PCR, hybridization probes, nanoparticles, synthetic biology, and mass spectrometry) (Li et al., 2017). Whole genome sequencing (WGS) using the Oxford Nanopore Technology’s (ONT) MinION has the potential to detect pathogens and their associated antibiotic resistance genes (ARGs) rapidly, but this technique is still in development, is costly, and not used in clinical settings (Avershina et al., 2022; Taxt et al., 2020). All these techniques are expensive, need dedicated infrastructure, or are time-consuming. Therefore, there is an urgent need to develop cost- and resource-effective methods that can speed up pathogen diagnostics and reduce the mortality from these infections.\n\nMesophilic bacteria are typically grown at 30 to 37°C in lab procedures. In rich growth media (i.e., Luria-Bertani, Terrific Broth), dense cultures are raised in a reasonably short time of two to six hours using inoculums 1-2% of overnight cultures (ca. 12-14 h). When growing bacteria from clinical samples in blood cultures, however, a far lower inoculum is experienced, i.e., <100 CFU or 0.5 McFarland units (MFU), and growth must be pursued for a minimum of 24 h up to several days to detect potential pathogens (Doualeh et al., 2022; Hardy et al., 1992; Waldeisen et al., 2011). The lengthy growth of fastidious bacteria in blood cultures to reach detectable titers is a significant bottleneck in the workflow of testing pathogens for AMR (Menchinelli et al., 2019; Somily et al., 2018; Verroken et al., 2015). Brain heart infusion (BHI) is a nutrient growth medium suitable for blood culture and to culture various fastidious organisms. BHI broth is often used in several application areas, including clinical microbiology testing, life science, biopharmaceuticals, food safety, water safety, and antibiotic sensitivity tests.\n\nThe bacterial growth curve shows four phases: lag phase, log (exponential) phase, stationary, and death phase. The lag phase is also called the preparatory phase, where the bacteria adapt to the new environment, and the cells are metabolically active. The log phase is where the bacterial cells double and increase in number until the depletion of nutrients. The log phase is followed by the stationary phase, where the bacterial growth rate equals the death rate due to the accumulation of toxic compounds and nutrient depletion. The final phase is called the death phase, in which the death rate of bacteria is greater than the growth rate.\n\nClearly, the time for a culture to reach detectable density, i.e., the length of lag phase growth, is a bottleneck in the diagnostic workflow. In this study, we wanted to investigate easy-to-implement factors (in a minimal laboratory set-up), including inoculum size, incubation temperature, and additional supplementation (e.g., vitamin B12 and trace metals), that can significantly reduce the lag time (tlag). These factors were arranged in simple two-level factorial designs using different Gram-positive (Escherichia coli and Pseudomonas aeruginosa) and Gram-negative (Staphylococcus aureus and Bacillus subtilis) bacteria, including clinical isolates with known AMR profiles (encoding methicillin resistance or extended-spectrum β-lactamases). BHI was used as the growth medium as it is suitable for use as a blood culture medium. Based on the results from experiments with pure bacterial isolates, blood samples spiked with a clinical isolate of E. coli CCUG17620 were also tested to see the effect of elevated incubation temperature on bacterial growth in blood cultures.\n\n\nMethods\n\nThe study focused on seven different bacterial strains covering four different bacterial species. Clinical bacterial strains E. coli NCTC13441 and S. aureus NCTC8325 were obtained from the National Collection of Type Cultures (NCTC), while S. aureus CCUG35600 and E. coli CCUG17620 were obtained from the Culture Collection University of Gothenburg (CCUG). Three non-clinical wild-type strains, including E. coli HB101K2, Pseudomonas aeruginosa INN, and Bacillus subtilis INN, were taken from the internal bacterial collection of Inland Norway University (INN). Additional details are available in Table 1.\n\n* Clinical strains.\n\nAll reagents were obtained from standard lab suppliers. Bacteriological agar and Brain Heart Infusion (BHI) broth for the media preparation were the products of HiMedia Laboratories (Mumbai, India; lot number: BCBH4004V), with BHI consisting of (g/L) calf brain infusion from 200 g (12.5), beef heart infusion from 250 g (5), peptone (10), sodium chloride (5), disodium chloride D (+) glucose (2), disodium hydrogen phosphate (2.5) at pH 7.4. Vitamin B12 or cyanocobalamin was from ThermoFisher, (Massachusetts, USA; number A14894). In addition, we also tested three clinical isolates using a trace metal solution (details in Supplementary Table 1, Extended data [Ahmad 2023]).\n\nBHI agar and liquid culture media were used to maintain cultures and to examine growth curves. Bacterial samples stored at -80°C were thawed gently at room temperature, inoculated to BHI broth, and incubated overnight at 37°C. BHI agar plates were prepared by adding 1.5% bacteriological agar powder to BHI broth. An overnight inoculum was streaked out, and plates were incubated at 37°C for 24 h. Single colonies were selected and inoculated into fresh BHI broth. The inoculated BHI broth was incubated at 37°C overnight and served as inoculum for culture experiments (See Supplementary figure S1 for an overview of the complete process, Extended data). Experimental liquid cultures were cultivated in shake flasks (500 mL broth in 2L Erlenmeyer bottles). Cultures within the same experimental design were inoculated from the same bacterial preculture suspension to ensure identical starting conditions.\n\nAll cultures in BHI broth were incubated at 37°C or 42°C under shaking conditions for up to 6 h, whereas blood cultures were cultivated for 12 h. During incubation, the optical density (OD) was measured at 600 nm every 20-30 minutes, starting from the time of inoculation (0 min) until the bacterial growth reached the stationary phase (approximately 4-5 h). Culture aliquots were immediately cooled and kept on ice until measurement. The OD 600 was plotted versus time (min) to obtain the growth curves.\n\nAs a practical response value to estimate the effectiveness of treatments, an endpoint of the lag time (tlag) was defined, the tlag being the time (min) from inoculation until the OD 600 value reached a net increase of 0.5. The net OD increase was obtained by subtracting the OD at t=0 from all OD values. The tlag was obtained by extrapolating at OD 0.5 from the zero-adjusted growth curve. Although this provisional end point is beyond the actual end of lag (Bertrand, 2019), an extended OD cut-off value was selected to reduce the estimation error when growth curves go from an undefined shallow rise to a steady exponential growth (Figure 1).\n\nThe 0.5-intercept represents the net increase in OD 600, i.e., the value subtracted from the optical density (OD) at time zero.\n\nA set of growth screening experiments examined the ability of selected variables to cause a reduced lag time. A structured two-level factorial design (Design of Experiments, DOE) was used to observe the effect of incubation temperatures (37°C and 42°C), low (0.05%), and high (0.5%) inoculum, vitamin B12 (±50 μM), and trace metal (present y/n) supplements. The design was tested on the wild-type strains (3) and the clinical isolates (4) but with a selection of two or three variables or factors. Table 2 shows the coded and decoded values of the factors tested (X1, X2, X3) at a low (-1) and high (+1) level. Thus, either four (22) or eight (23) growth experiments were performed for each strain. For a detailed overview of conditions for the individual strains, Table 1 is provided as well as in Supplementary Tables 2-4 (Extended data).\n\nThe estimated tlag values from DOE were presented in interaction effect plots, illustrating the impact of a variable (e.g., temperature) with connected experiments on another variable (e.g., vitamin B12 supplement). The main effects of tested variables are shown numerically in descending order in bar charts. The bars are the absolute effect values computed as twice the regression coefficients from fitted DOE models. They show the change in tlag when a variable goes from a low to a high level when other factors are kept at their average.\n\nClinical strain E. coli CCUG17620 was spiked with blood to mimic the sepsis infection. The blood was spiked with 40 CFU/mL from an overnight plate culture of the clinical strain. The lower CFU was used as an inoculum because, in the case of sepsis, the CFU/mL ranges typically from 10-100 (Doualeh et al., 2022). E. coli was grown overnight on a BHI agar plate and then suspended in saline to measure the OD. After measuring OD, the suspensions were plated on agar plates to calculate the CFU at different OD values. After optimizing the relation of OD with CFU counts, the desired concentration in the range of 10-100 CFU/mL was used for spiking with human blood. Fresh human blood obtained from healthy donors at Inland Norway University was used for this experiment. Human blood spiked with E. coli was mixed with BHI broth medium and incubated at the two study temperatures, i.e., 37 and 42°C. CFU/mL was calculated every 3 h for 12 h by plating out the samples on BHI agar plates.\n\nBacterial DNA from blood culture samples was extracted using QIAamp BiOstic Bacteremia DNA Kit from Qiagen (Germany). The concentration and purity of isolated DNA were determined using the Qubit system with a dsDNA HS assay kit (Thermo Fisher Scientific, USA) and the NanoDrop spectrophotometer. PCR was performed to confirm the presence of E. coli CCUG17620 DNA and human DNA in samples taken at 3 h, 6 h, 9 h, and 12 h of incubation at 37°C and 42°C. The primers used for identifying E. coli were uspA-F CCGATACGCTGCCAATCAGT and uspA-R ACGCAGACCGTAGGCCAGAT, and human DNA was β-actin (F-CGGCCTTGGAGTGTGTATTAAGTA and R-TGCAAAGAACACGGCTAAGTGT) (Hasan et al., 2016). The PCR reaction was set up in a total volume of 20 μL per sample containing 4 μL HOT FIREPol MultiPlex Mix Ready to Load master mix from Solis Biodyne (Estonia), 0.5 μL of 10 μM forward and reverse primers, 8 ng/μL of template DNA and nuclease-free water. The thermal cycling conditions for PCR were initial denaturation at 95°C for 12 min followed by 35 cycles of 95°C for 25 s, 61°C for 50 s and 72°C for 1 min, and a final extension of 72°C for 7 min. The PCR product was run on 1% agarose gel for 45 min at 100V and then visualized on the Gel doc system to determine the presence or absence of our desired bands.\n\nThe MODDE 13.0 Pro (Umetrics/Sartorius Stedim Data Analytics AB, Sweden) was used to generate the design matrix, regression coefficients, and statistical analysis. The significance testing (p < 0.05) of the DOE screening was performed with ANOVA. All the growth curve graphs are generated using GraphPad Prism 9.4.1.\n\n\nResults\n\nEscherichia coli\n\nE. coli HB101 K2\n\nIn the absence of vitamin B12 supplementation, the tlag for bacterial growth was 265 minutes and 255 minutes at 37°C and 42°C, respectively, when an inoculum size of 0.05% was used for culturing (Figure 2A). When using a higher inoculum of 0.5% in the absence of vitamin B12 supplement, the tlag recorded was 220 minutes and 145 minutes at 37°C and 42°C, respectively (Figure 2A). In the presence of vitamin B12 supplement and using an inoculum size of 0.05%, the bacterial tlag was 315 minutes (37°C) and 160 minutes (42°C). Similarly, when the inoculum size was increased to 0.5%, with vitamin B12 supplementation, the tlag was 200 minutes (37°C) and 190 minutes (42°C). The shortest tlag was recorded to be 145 minutes, which was observed using an inoculum size of 0.5% in the absence of vitamin B12 supplement and incubation at 42°C (Figure 2 and Supplementary Table 2 [Extended data]).\n\n(A) Growth curves of E. coli HB101K2 were obtained in three-factorial designs; at 37°C and 42°C, inoculum size 0.05% and 0.5 %, with and without vitamin B12. The dotted line indicates OD 0.5 for estimating the tlag from curves. (B) Effect plot of E. coli HB101 K2 in minutes (tlag) to reach optical density 0.5.\n\nUsing an inoculum size of 0.05% and a vitamin B12 supplement, we observed 95 minutes of reduction in the lag time at 42°C compared to 37°C (Figure 2B). In contrast, when the inoculum size was increased to 0.5% along with vitamin B12 supplementation, the tlag was reduced by 10 minutes at 42°C. Based on the effect plot, it is reported that the sample with no vitamin B12 supplement and 0.5% inoculum size showed the shortest tlag. But when the inoculum size was decreased to 0.05%, we observed that the sample incubated at 42°C in the presence of vitamin B12 supplement showed the shortest tlag (Figure 2B). The reduction in tlag was between 4% and 49% for the above mentioned eight tested conditions.\n\nE. coli CCUG17620 and E. coli NCTC13441\n\nBased on the results from the laboratory strain, we tested the growth of two E. coli clinical isolates at 37 and 42°C. E. coli CCUG17620 took 170 minutes when incubated at 42°C and 193 minutes at 37°C to reach OD 0.5 without adding vitamin B12. Similarly, E. coli NCTC13441 required 126 and 167 minutes to reach OD 0.5 at 42°C and 37°C, respectively (Figure 3A and Supplementary Table 4 [Extended data]). For the two clinical isolates, E. coli CCUG17620 and E. coli NCTC13441, the tlag was less at 42°C compared to 37°C.\n\n(A) Growth curves of clinical bacterial isolates at 37°C and 42°C (E. coli CCUG17620, E. coli NCTC13441), or with and without vitamin B12 supplement (S. aureus CCUG35600 at 42°C). The dotted line indicates OD 0.5 for estimating the tlag from curves. (B) Effect plot of clinical samples in minutes (tlag) to reach optical density 0.5.\n\nOverall, the effect plot of E. coli CCUG17620 and E. coli NCTC13441 at inoculum size 0.5% indicated that when the incubation temperature increased (42°C instead of 37°C), the lag time decreased by 15-25% in the tested strains (Figure 3B).\n\nStaphylococcus aureus\n\nS. aureus NCTC8325\n\nIn the case of the clinical isolate S. aureus NCTC8325 with no vitamin B12 supplements and 0.05% inoculum size, the time to reach OD 0.5 was 285 minutes and 210 minutes at 37°C and 42°C, respectively (Figure 4A and Supplementary Table 2). But in the presence of vitamin B12 supplementation and lower inoculum size of 0.05%, the tlag was 190 minutes (37°C) and 165 minutes (42°C). When the inoculum size was increased to 0.05%, the tlag decreased significantly at 42°C (135 minutes) in comparison to 37°C (215 minutes) in the presence of vitamin B12 supplementation (Figure 4A and Supplementary Table 2). We can conclude that incubation at 42°C, along with vitamin B12 supplement, reduces the tlag in S. aureus NCTC8325.\n\n(A) Growth curves of S. aureus NCTC8325 were obtained in three-factorial designs; at 37°C and 42°C inoculum sizes 0.05% and 0.5 %, with and without vitamin B12. The dotted line indicates OD 0.5 for estimating the tlag from curves. (B) Effect plot of S. aureus NCTC8325 in minutes (tlag) to reach optical density 0.5.\n\nThe effect plot of S. aureus NCTC8325 at 0.5% inoculum shows a reduction of 121 minutes in tlag at 42°C incubation in the presence of vitamin B12 supplement. At the same time, a reduction of 45 minutes in tlag was observed at 0.05% inoculum and 42°C incubation in the presence of vitamin B12 supplement (Figure 4B). The reduction in tlag was between 13% and 37% for the eight tested conditions.\n\nS. aureus CCUG35600\n\nBased on the above results, we tested the growth of another clinical strain of S. aureus. The effect plot of the clinical isolate S. aureus CCUG35600 at inoculum size 0.5% at 42°C co-cultured with vitamin B12 supplement showed a decline in lag time compared to without supplementation. Overall, the shortest tlag time was observed following the co-culture of bacteria with vitamin B12 supplement (Figure 3A and B). So, the reduction in tlag was 20%.\n\nPseudomonas aeruginosa\n\nP. aeruginosa showed a significant reduction in the tlag when vitamin B12 supplements were added along with 42°C incubation. It reached OD 0.5 in 185 minutes when vitamin B12 supplement was added along with 42°C incubation. On the other hand, 235 minutes was required with no vitamin B12 supplement and 37°C incubation (Figure 5A).\n\n(A) Growth curves of P. aeruginosa and B. subtilis were obtained in two-factorial designs, at 37°C and 42°C, with and without vitamin B12. The dotted line indicates OD 0.5 for estimating the tlag from curves. (B) Effect plot of P. aeruginosa and B. subtilis in minutes (tlag) to reach optical density 0.5.\n\nThe effect plot showed a reduction of 30 minutes in tlag when bacteria were cultured at 42°C in the presence of vitamin B12 supplementation (Figure 5B). The decline in tlag was between 9% and 20% for the four tested conditions.\n\nBacillus subtilis\n\nB. subtilis also showed the same pattern as other bacteria, with a tlag of 155 minutes with vitamin B12 supplement and 42°C incubation. On the other hand, the tlag was 210 minutes and 215 minutes to reach OD 0.5 when incubated at 37°C with and without vitamin B12 supplement, respectively (Figure 5A and Supplementary Table 3 [Extended data]). The effect plot showed a reduction of 45 minutes in tlag when bacteria were co-cultured at 42°C in the presence of vitamin B12 (Figure 5B). The reduction in tlag was between 5% and 28% for the four tested conditions.\n\nThe main effects on tlag from the variables tested in three- (E. coli and S. aureus) and two-factor (P. aeruginosa and B. subtilis) experimental designs are shown in Figure 6. We tested the time to reach OD 0.5 for S. aureus, E. coli, P. aeruginosa, and B. subtilis and noticed that incubating the bacteria at 42°C reduces the lag time. However, the clinical isolates (E. coli CCUG17620, E. coli NCTC13441, and S. aureus CCUG35600), which were tested for trace metal solution complement, adversely affected bacterial growth by prolonging the lag time (details in Extended data).\n\nBars represent the change in tlag (min) when a factor varies from its low to a high level (cfr. Table 1) when all other factors are kept at their averages. Calculated effects are given with 95% confidence. Other statistical features are given at the bottom.\n\nWe also observed that higher incubation temperature could increase bacterial growth in blood cultures as in pure bacterial isolates. The CFU of the blood samples, which were spiked with E. coli CCUG17620 and incubated at the two study temperatures, 37°C and 42°C, showed that bacteria could grow much faster in the early stages of incubation at 42°C compared to 37°C. When we calculated the CFU after 3 h and 6 h of incubation, we could see that the CFU/mL of bacteria were three times higher at 42°C than at 37°C. This higher growth of bacteria in the initial hours of incubation is critical for identifying the causative pathogen. This information is valuable for infection diagnostic purposes. Further incubation beyond 6 h, narrowed the gap between CFU/mL at 37°C and 42°C. At 12 h of incubation, we got the same CFU at both temperatures (Figure 7).\n\nWe performed PCR to confirm the presence of bacterial DNA versus human DNA at different incubation time points, as described in Methods. The PCR product run on 1% agarose gel was visualized for the presence of DNA and compared with a 100 bp DNA ladder, which was also run on the gel. We could see the DNA bands for the E. coli CCUG 17620 sample even after 3 h incubation. Although the CFU/mL after 3 h of incubation was low, i.e., 40 at 37°C and 120 at 42°C, it was still enough bacterial DNA to be amplified through PCR (Figure 8). For the samples which were incubated for a longer time and had greater CFU, the band signals for E. coli DNA appeared stronger in comparison to the human DNA (Figures 8 and 9). We can see from the PCR results that we obtained amplification at both the incubation temperatures (37°C and 42°C), but the DNA band strength and purity were higher at 42°C. This higher quality of DNA is beneficial for nanopore sequencing, which can be used to identify the bacteria and ARGs in real time.\n\nLane 1: 100 bp DNA ladder, Lane 2: 3 h sample (37°C), Lane 3: 3 h sample (42°C), Lane 4: 6 h sample (37°C), Lane 5: 6 h sample (42°C), Lane 6: 9 h sample (37°C), Lane 7: 9 h sample (42°C), Lane 8: 12 h sample (37°C), Lane 9: 12 h sample (42°C), Lane 10: Negative control (No DNA).\n\nLane 1: 100 bp DNA ladder, Lane 2: 3 h sample (37°C), Lane 3: 3 h sample (42°C), Lane 4: 6 h sample (37°C), Lane 5: 6 h sample (42°C), Lane 6: 9 h sample (37°C), Lane 7: 9 h sample (42°C), Lane 8: 12 h sample (37°C), Lane 9: 12 h sample (42°C), Lane 10: Negative control (No DNA).\n\n\nDiscussion\n\nEarly and appropriate detection of pathogens is crucial for treating bacterial infections. However, since the bacterial load is usually very low for infection detection, it is difficult to identify the pathogen and recommend appropriate antibiotic therapy. In the clinical routine, bacteria are grown overnight, requiring a longer time to grow in fresh culture media. Therefore, any method which increases bacterial growth in the initial hours of culturing can be extremely useful in treating patients with different bacterial infections. In the case of sepsis, early and appropriate antibiotic therapy in the first hour of infection can significantly reduce the mortality rate (Avershina et al., 2022; Bloos et al., 2017; Ferrer et al., 2014).\n\nThe most common method currently used for identifying bacterial pathogens in clinical samples is based on culture. Although these culturing methods, in most cases, provide information on the causative agent, they are slow and time-consuming. The routine culture-based identification takes around 48-72 h, and an additional 24-48 h for antibiotic susceptibility testing. Still, this method is most widely used in clinical diagnosis because it is cost-effective and does not require very advanced lab infrastructure. Many other methods, such as whole genome sequencing (WGS), fluorescent in-situ hybridization (FISH), PCR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS), has the potential for rapid identification of pathogens. However, most of these techniques still require bacterial culturing as the initial step and currently need to be well-developed to be used in clinical settings.\n\nIn this study, we have used different inoculation sizes (0.05% and 0.05%), incubation temperatures (37°C and 42°C), and bacterial growth supplements (vitamin B12 and trace metal solution) to study their effect on the growth of bacteria. We used BHI culture media for bacterial growth because it can support the growth of a wide variety of bacteria. BHI is also suitable for a blood culture medium and is similar to the BACTEC blood culture medium, which is extensively used in the clinical microbiology routine. Also, it has been previously used for short-term incubation of positive blood cultures to accelerate the identification of bacteria by MALDI-TOF MS (Torres et al., 2017). We have shown that incubating the bacteria at a higher temperature of 42°C instead of 37°C and adding vitamin B12 supplementation in the culture media can significantly reduce the tlag of bacterial growth. We observed the reduced tlag in the pure bacterial isolates and the E. coli blood culture using 42°C incubation. The current methods require the blood cultures to be incubated for 24 h or more before identifying pathogens using biochemical tests and then AST. Using this higher incubation temperature (42°C), we can see that after 3 h of incubation, the bacterial CFU/mL was 120, which is three times higher than the standard incubation temperature (37°C). Although the PCR results show bacterial DNA’s presence at both temperatures, we can see the better quality of DNA at 42°C by looking at the intensity of bands (Figure 8). This high-quality DNA can be beneficial for performing downstream analyses like PCR or real-time metagenomic sequencing for identifying pathogens and ARGs.\n\nThe duration of the lag phase is influenced by several factors such as pH, temperature, nutrients, physiological history of the cells, and size of the inoculum (Bertrand, 2019; Mellefont et al., 2003; Rolfe et al., 2012). Many studies have investigated the effect of environmental factors such as pH, temperature, and nutrients on the duration of the bacterial lag phase (Francois et al., 2006, 2007). However, so far, no studies have been done to investigate the effect of vitamin B12 supplements on the bacterial lag phase at different conditions, such as temperature (37°C and 42°C) in connection with inoculum size (0.05% and 0.5%). Other studies have also reported that bacterial lag time depends on the nutrient concentration, such as potassium bicarbonate (KHCO3), amino acids, ferrous sulfate, sodium pyruvate, and mucin (Berthelet and MacLeod, 1989). Jiang et al. have shown that adding 0.3% mucin to brain heart infusion horse serum (BHI-HS) medium reduced the lag time of Helicobacter pylori and enhanced its growth (Jiang & Doyle, 2000). These results are similar to our study, where we have also shown that vitamin B12 supplement in the BHI medium reduces the lag time of bacteria. Malani et al. reported that higher concentrations of sucrose, biotin and pantothenate, lower concentrations of cyanocobalamin (i.e., vitamin B12), CaSO4, and glutathione in a growth medium result in larger bacterial inclusion bodies and product expression (Malani et al., 2020). The microbial cell is composed mainly of carbon, hydrogen, nitrogen, and oxygen elements. Other elements such as phosphorus, sulfur, calcium, magnesium, potassium, iron, and sodium are equally crucial for the vital activities of bacterial metabolism (dos Santos et al., 2022). These micronutrients are essential for the enzymatic activities of the bacterial cell and act as prosthetic groups or cofactors for necessary metabolic enzymes (Hood and Skaar, 2012).\n\nIt has also been shown that an extended lag phase is advantageous for bacterial survival and helps in the regrowth of bacteria upon the removal of antibiotics (Bertrand, 2019). Li et al. evaluated the effect of 12 different antibiotics on the lag phase of different environmental bacteria. They observed that tetracycline and quinolone were the most common bacteria that resisted due to the extended lag phase of bacteria (Li et al., 2017). Therefore, reducing bacterial lag time is even more critical as it can contribute to drug resistance against the clinically relevant antibiotics used to treat bacterial infections. For all four tested bacteria (S. aureus, E. coli, P. aeruginosa, and B. subtilis at OD 0.5), we reported that incubating the bacteria at 42°C reduces the lag time (tlag).\n\nWith an increasing volume of cells in culture media, it takes less time for the bacteria to adapt to the new environment and therefore have a shorter lag time (Robinson et al., 2001; Aamir et al., 2015). In our study, we observed a short lag time for some of the bacteria by using a larger inoculum size of 0.5%, but in E. coli HB101K2 and S. aureus NCTC8325, we did not observe the same effect.\n\nIn this study, we have shown that using vitamin B12 supplements in culture media and incubation at a higher temperature of 42°C can significantly reduce the tlag. This reduction in tlag is significant for rapidly detecting pathogens and early and appropriate antibiotic therapy. We have also shown that incubating E. coli blood culture at a higher temperature of 42°C reduces the tlag and enhances the growth of bacteria. It will be beneficial in the future to enrich the culture media with different bacterial growth supplements, which can reduce bacterial lag time and thus allow rapid identification of pathogens. However, to increase the method’s reliability and for any potential future use in clinical microbiology, we need to increase the number of bacterial strains analyzed and focus on additional AMR profiles and clinically relevant pathogens. Moreover, the effect of other elements, such as phosphorus, magnesium, calcium, sodium, and iron, as a bacterial growth supplement will be interesting to explore in future research. These results demonstrate the potential that, unlike other molecular-based detection methods, these measures can be cost-effective and do not need any specialized instruments or expertise.", "appendix": "Data availability\n\nOpen Science Framework: Ali et al., 2023 F1000 Research, https://doi.org/10.17605/OSF.IO/HYNTQ (Ahmad, 2023).\n\nThis project contains the following underlying data:\n\n- Ali et al - Raw Data Blood culture Ecoli CFU.xlsx\n\n- Ali et al - Raw data_ growth curve OD and tlag.xlsx\n\n- Ali et al Raw_Gel_image_Blood_Culture_Ecoli_primers.tif\n\n- Ali et al Raw_Gel_image_Blood_Culture_human_primers.tif\n\nOpen Science Framework: Ali et al. 2023 F1000 Research, https://doi.org/10.17605/OSF.IO/HYNTQ (Ahmad, 2023).\n\nThis project contains the following extended data:\n\n- Supplementary_Material.docx\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 thank Anne Bergljot Falck-Ytter for her help in the laboratory.\n\n\nReferences\n\nAamir S, Ansari ZA, Fouad H, et al.: Effect of Inoculum Size and Surface Charges on the Cytotoxicity of ZnO Nanoparticles for Bacterial Cells. Sci. Adv. Mater. 2015; 7(12): 2515–2522. Publisher Full Text\n\nAhmad R:Ali et. al. 2023 F1000 Research. [Data set]. Open Science Framework. 2023, January 12. Publisher Full Text\n\nAli J, Awan MOU, Akca G, et al.: Prevalence of diversified antibiotic resistant bacteria within sanitation related facilities of human populated workplaces in Abbottabad. PLoS One. 2020; 15(8). Publisher Full Text\n\nAvershina E, Frye SA, Ali J, et al.: Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing [Original Research]. Front. Microbiol. 2022; 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerthelet M, MacLeod RA: Effect of Na+ Concentration and Nutritional Factors on the Lag Phase and Exponential Growth Rates of the Marine Bacterium Deleya aesta and of Other Marine Species. Appl. Environ. Microbiol. 1989; 55(7): 1754–1760. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBertrand RL: Lag Phase Is a Dynamic, Organized, Adaptive, and Evolvable Period That Prepares Bacteria for Cell Division. J. Bacteriol. 2019; 201(7): e00697–e00618. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBloos F, Rüddel H, Thomas-Rüddel D, et al.: Effect of a multifaceted educational intervention for anti-infectious measures on sepsis mortality: a cluster randomized trial. Intensive Care Med. 2017; 43(11): 1602–1612. PubMed Abstract \| Publisher Full Text\n\nBriggs N, Campbell S, Gupta S: Advances in rapid diagnostics for bloodstream infections. Diagn. Microbiol. Infect. Dis. 2021; 99(1): 115219. PubMed Abstract \| Publisher Full Text\n\nD’Costa VM, McGrann KM, Hughes DW, et al.: Sampling the antibiotic resistome. Science. 2006; 311(5759): 374–377. Publisher Full Text\n\ndos Santos V, Nizoli E, Galvan D, et al.: Micronutrient requirements and effects on cellular growth of acetic acid bacteria involved in vinegar production. Food Sci. Technol. 2022; 42. Publisher Full Text\n\nDoualeh M, Payne M, Litton E, et al.: Molecular Methodologies for Improved Polymicrobial Sepsis Diagnosis. Int. J. Mol. Sci. 2022; 23(9): 4484. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference SourceReference Source\n\nFerrer R, Martin-Loeches I, Phillips G, et al.: Empiric antibiotic treatment reduces mortality in severe sepsis and septic shock from the first hour: results from a guideline-based performance improvement program. Crit. Care Med. 2014; 42(8): 1749–1755. PubMed Abstract \| Publisher Full Text\n\nFleischmann C, Scherag A, Adhikari NK, et al.: Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations. Am. J. Respir. Crit. Care Med. 2016; 193(3): 259–272. PubMed Abstract \| Publisher Full Text\n\nFrancois K, Devlieghere F, Standaert AR, et al.: Effect of environmental parameters (temperature, pH and a(w)) on the individual cell lag phase and generation time of Listeria monocytogenes. Int. J. Food Microbiol. 2006; 108(3): 326–335. PubMed Abstract \| Publisher Full Text\n\nFrancois K, Valero A, Geeraerd AH, et al.: Effect of preincubation temperature and pH on the individual cell lag phase of Listeria monocytogenes, cultured at refrigeration temperatures. Food Microbiol. 2007; 24(1): 32–43. PubMed Abstract \| Publisher Full Text\n\nHardy DJ, Hulbert BB, Migneault PC: Time to detection of positive BacT/Alert blood cultures and lack of need for routine subculture of 5-to 7-day negative cultures. J. Clin. Microbiol. 1992; 30(10): 2743–2745. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHasan MR, Rawat A, Tang P, et al.: Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing. J. Clin. Microbiol. 2016; 54(4): 919–927. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffman PS: Antibacterial Discovery: 21st Century Challenges. Antibiotics-Basel. 2020; 9(5): ARTN 213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHood MI, Skaar EP: Nutritional immunity: transition metals at the pathogen–host interface. Nat. Rev. Microbiol. 2012; 10(8): 525–537. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang X, Doyle MP: Growth Supplements for Helicobacter pylori. J. Clin. Microbiol. 2000; 38(5): 1984–1987. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKapoor G, Saigal S, Elongavan A: Action and resistance mechanisms of antibiotics: A guide for clinicians. J. Anaesthesiol. Clin. Pharmacol. 2017; 33(3): 300–305. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhaledi A, Weimann A, Schniederjans M, et al.: Predicting antimicrobial resistance in Pseudomonas aeruginosa with machine learning-enabled molecular diagnostics. EMBO Mol. Med. 2020; 12(3): e10264. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Yang X, Zhao W: Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing. SLAS TECHNOLOGY: Translating Life Sciences Innovation. 2017; 22(6): 585–608. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalani H, Kumar D, Rathore AS: Effect of chemically defined growth medium components on characteristics of bacterial inclusion bodies. J. Chem. Technol. Biotechnol. 2020; 95(6): 1640–1648. Publisher Full Text\n\nMellefont LA, McMeekin TA, Ross T: The effect of abrupt osmotic shifts on the lag phase duration of foodborne bacteria. Int. J. Food Microbiol. 2003; 83(3): 281–293. PubMed Abstract \| Publisher Full Text\n\nMenchinelli G, Liotti FM, Fiori B, et al.: In vitro Evaluation of BACT/ALERT® VIRTUO®, BACT/ALERT 3D®, and BACTEC™ FX Automated Blood Culture Systems for Detection of Microbial Pathogens Using Simulated Human Blood Samples. Front. Microbiol. 2019; 10: 221–221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMetzgar D, Frinder MW, Rothman RE, et al.: The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood. PLoS One. 2016; 11(7): e0158186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurray CJL, Ikuta KS, Sharara F, et al.: Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022; 399(10325): 629–655. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson TP, Aboaba OO, Kaloti A, et al.: The effect of inoculum size on the lag phase of Listeria monocytogenes. Int. J. Food Microbiol. 2001; 70(1): 163–173. PubMed Abstract \| Publisher Full Text\n\nRolfe MD, Rice CJ, Lucchini S, et al.: Lag Phase Is a Distinct Growth Phase That Prepares Bacteria for Exponential Growth and Involves Transient Metal Accumulation. J. Bacteriol. 2012; 194(3): 686–701. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSomily AM, Habib HA, Torchyan AA, et al.: Time-to-detection of bacteria and yeast with the BACTEC FX versus BacT/Alert Virtuo blood culture systems. Ann. Saudi Med. 2018; 38(3): 194–199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaxt AM, Avershina E, Frye SA, et al.: Rapid identification of pathogens, antibiotic resistance genes and plasmids in blood cultures by nanopore sequencing. Sci. Rep. 2020; 10(1): 7622. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorres I, Gimenez E, Pascual T, et al.: Short-term incubation of positive blood cultures in brain-heart infusion broth accelerates identification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry. J. Med. Microbiol. 2017; 66(12): 1752–1758. PubMed Abstract \| Publisher Full Text\n\nVerroken A, Defourny L, Lechgar L, et al.: Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subculture. Eur. J. Clin. Microbiol. Infect. Dis. 2015; 34(2): 405–413. PubMed Abstract \| Publisher Full Text\n\nWaldeisen JR, Wang T, Mitra D, et al.: A Real-Time PCR Antibiogram for Drug-Resistant Sepsis. PLoS One. 2011; 6(12): e28528. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "162338", "date": "02 Mar 2023", "name": "Sarita Mohapatra", "expertise": [ "Reviewer Expertise Antimicrobial resistance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript compares the effect of growth temperature and VitB12 supplementation in reduction of lag time in pathogen growth. I do have the some comments which needs to be addressed:\nThe experiment has been conducted in only one isolate of four different genus. The difference of the bacterial count has been noticed in the initial period of incubation however, most of the experiments showing similar growth pattern over a longer incubatory period. Hence, more number of isolates need to be tested.\n\nPseudomonas aeruginosa is known pathogen and its growth at 42°C is taken as one of the identification characters for P. aeruginosa. It would be helpful if other Gram-negative bacilli, such as the ESKAPE group of pathogens, were included in the experiment.\n\nThe Introduction is too long, it should be reduced highlighting the limitations of conventional blood culture technique.\n\nHow was the CFU measured after 3hr and 6hr incubation?\n\nWas DNA quantified? Based on gel picture it is difficult to comment on quantity\nMinor comments\nCorrect the gram-positive and gram-negative distribution of bacteria in abstract and other places of manuscript e.g. gram-negative bacilli: E. coli & P. aeruginosa and gram-positive cocci: Staphylococcus aureus, gram-positive bacilli: Bacillus subtilis). Full form of CCUG1762 must be written where the term is used first\n\nIn Table 1 the Gram-stain interpretation should be correctly made as Gram-negative bacilli, or gram-positive bacilli, or Gram-positive cocci, etc.\n\nDegree symbol is missing in many places.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9491", "date": "03 Apr 2023", "name": "Rafi Ahmad", "role": "Author Response", "response": "We thank the esteemed reviewer for taking the time to provide us with feedback. The reviewer's comments have helped improve the manuscript. We hope that we have addressed the reviewer's comments sufficiently. The experiment has been conducted in only one isolate of four different genus. The difference of the bacterial count has been noticed in the initial period of incubation however, most of the experiments showing similar growth pattern over a longer incubatory period. Hence, more number of isolates need to be tested. Response: In this study, we tested three E. coli, two S. aureus, and one isolate of P. aeruginosa and B. subtilis. We agree that bacterial counts fluctuate more during the initial incubation period. However, this difference in the bacterial count during the initial incubation time can be advantageous for quickly detecting pathogens. In our unpublished work, we have observed that in blood cultures, pathogens with higher bacterial counts during the early stages of incubation allow for faster detection using nanopore sequencing. Sequencing at a timely stage enables the identification of pathogens and their antimicrobial resistance, allowing for swift therapeutic intervention. As this was a proof-of-concept study, we could only test available bacterial strains which we already possess – most of them known pathogens. Future studies should focus on testing more pathogens and using different nutrients to see their effect on bacterial growth. Pseudomonas aeruginosa is known pathogen and its growth at 42°C is taken as one of the identification characters for P. aeruginosa. It would be helpful if other Gram-negative bacilli, such as the ESKAPE group of pathogens, were included in the experiment. Response: In this study, we have included 3 E. coli isolates (Gram-negative bacilli), P. aeruginosa, and 2 S. aureus isolates from the ESKAPE group of pathogens, but we agree with the reviewer's suggestion that other pathogens from this group would be interesting to investigate. As this was a proof-of-concept study, we were not able to include all the bacterial strains and were restricted to those which we have in our lab, and some of them are clinically relevant antimicrobial resistant pathogens. In future studies, the focus should be on testing more pathogens with different conditions to have a broader knowledge of the bacterial growth response to different conditions. The Introduction is too long, it should be reduced highlighting the limitations of conventional blood culture technique. Response: The suggested changes have been incorporated into the revised version of the manuscript. How was the CFU measured after 3hr and 6hr incubation? Response: The samples were taken out from the incubator after 3 and 6 hours, and a small volume was cultured on BHI agar plates, which were then incubated overnight for the calculation of CFU using the formula: number of colonies × dilution factor/volume of culture plated. Text has been written in the last sentence of Blood culturing in the methods section. Was DNA quantified? Based on gel picture it is difficult to comment on quantity Response: Yes, the DNA was quantified, and the details are incorporated into the revised manuscript (Results section: PCR results). Minor comments Correct the gram-positive and gram-negative distribution of bacteria in abstract and other places of manuscript e.g. gram-negative bacilli: E. coli & P. aeruginosa and gram-positive cocci: Staphylococcus aureus, gram-positive bacilli: Bacillus subtilis). Response: The correction has been made in the abstract and introduction section of the revised manuscript. Full form of CCUG1762 must be written where the term is used first Response: The suggested change has been incorporated in the revised manuscript. In Table 1 the Gram-stain interpretation should be correctly made as Gram-negative bacilli, or gram-positive bacilli, or Gram-positive cocci, etc. Response: The table has been updated with the suggested changes included. Degree symbol is missing in many places. Response: Updated in the revised manuscript." } ] }, { "id": "162335", "date": "06 Mar 2023", "name": "Rashid Nazir", "expertise": [ "Reviewer Expertise Microbial interactions", "Antimicrobial resistance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled as “Increased growth temperature and vitamin B12 supplementation reduces the lag time for rapid pathogen identification in BHI agar and blood cultures” published on F1000 Research by Ali et al. is an interesting work of applied microbiology. The ms is a proof-of-concept study for future diagnostic improvements. Before its indexing, the following are some comments and observations which are recommended to authors for appropriate response during further revision of their manuscript:\nPlease explain the rationale for incubation temperature setting (37 to 42°C) and also provide potential reasons for lag phase changes with/without vitamin B12 in different bacterial stains.\n\nThe bacterial inoculum concentration showed different trends for the lag phase on different temperatures and vitamin B12 supplementation, please discuss different reasons to explain these shifts. Are there any other factors (except the ones studied here) to affect the bacterial lag phase?\n\nDue to the unique ways in which various bacteria behave and function, the responsiveness and functional capabilities fluctuate as the environment changes. When various environmental factors are activated, not all bacteria or pathogens behave the same way. Some act in opposition to the change, while others react positively. So, how likely is it to expand the observed modification for tens of bacterial pathogens? More precisely, the environmental changes may affect the bacterial physiological functions; in this context, do the tested incubation temperature and vitamin supplementation affect the microbes’ pathogenicity or efficiency as well?\n\nThe nutrients are necessary for the bacteria to grow; some are present in significant amounts, while others are scarce. What is the rationale, for authors, to test B12 out of several microbial nutrients?\n\nAs per the study-observations, increased incubation temperature (42°C) reduced the Tlag, may it do the same in real life body-temperature situations? Reduced lag time may enhance the pathogens’ survival in compromised immunity scenarios with raised body temperature. Please discuss this practical aspect as well.\n\nAs per the ms title and abstract, the 1st impression of the study is more like diagnostic improvements while the introduction start with AMR does not fit in appropriately. 2nd paragraph or even the 2nd sentence of 2nd paragraph might be something to start the introduction and whole AMR scenario may follow in later phase.\n\nAlmost half of the 2nd paragraph is on ‘Sepsis’, I think it narrates the examples of different fatal diseases, so please say it so.\n\nPage-1, 2 - abstract and last paragraph of introduction: Switch the Gram-positive (Escherichia coli and Pseudomonas aeruginosa) and Gram-negative (Staphylococcus aureus and Bacillus subtilis) bacteria in correct order. Table represented them correctly, though.\n\nEstablish a connecting link of 4th paragraph (start with Mesophilic bacteria…) with previous one.\n\nThe 1st sentence of the paragraph (starting with ‘The bacterial growth curve…) is sufficient, as the rest is quite basic for the microbiology readers.\n\nAs per the ms, bacterial growth reached the stationary phase (approximately 4-5 h), it would be interesting to see the doubling time of the tested bacteria. I am sure the authors would have calculated.\n\nFigure 8-9: In English, normal reading/writing is from left to write; please correct it ‘currently from right to left’.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9492", "date": "03 Apr 2023", "name": "Rafi Ahmad", "role": "Author Response", "response": "We thank the esteemed reviewer for taking the time to provide us with feedback. The reviewer's comments have helped improve the manuscript. We hope that we have addressed the reviewer's comments sufficiently. Please explain the rationale for incubation temperature setting (37 to 42°C) and also provide potential reasons for lag phase changes with/without vitamin B12 in different bacterial stains. Response: The incubation temperature (37 & 42°C) was used in this study because these temperatures can easily be maintained in standard laboratory settings and the most commonly used temperature range (37 to 42°C) for the majority of bacterial strains in microbiology. We used the higher (42°C) and lower (37°C) temperatures in this range to compare their effect on the bacterial growth (Gutierrez et al., 2018). The relevant text is added in the discussion section of the manuscript (Paragraph 3). The lag time for bacterial growth is dependent on many factors, such as temperature, bacterial strain, and growth medium (availability of nutrients). According to our study, vitamin B12 can affect the lag phase of bacterial growth, possibly because it can contribute to different metabolic activities in bacteria, such as the synthesis of nucleic acid and amino acids. Some bacteria can synthesize vitamin B12, while others require it to be present in the growth medium (LeBlanc et al., 2011). The text has been added in paragraph 4 of the discussion section). If the bacteria do not have vitamin B12 available in the growth medium, they require time to synthesize different enzymes, which is required for the production of vitamin B12 (Sultana et al., 2023). Therefore, the lag time of the bacteria, in that case, is longer. The relevant text is added in the discussion section of the manuscript (Paragraph 4). The bacterial inoculum concentration showed different trends for the lag phase on different temperatures and vitamin B12 supplementation, please discuss different reasons to explain these shifts. Are there any other factors (except the ones studied here) to affect the bacterial lag phase? Response: The reason we did not see any effect of the inoculum concentration on the lag phase in some of the bacteria may be due to several factors, such as inherent bacterial characteristics, culture growth phase, and adaptation to the environment. Some bacteria may have limited ability to utilize the available nutrients even when the inoculum size is larger. Therefore, the inoculum concentration in some of these bacteria may not have any significant effect on their lag phase (Bidlas et al., 2008). The relevant text is added in the discussion section of the manuscript (Paragraph 6). Many other factors besides the ones tested here can affect the bacterial lag phase. Some of them are the availability of different nutrients such as carbon, nitrogen, and phosphorus. The pH of the growth medium can also affect the bacterial growth rate and metabolism. Oxygen availability is also important for the optimal growth of bacteria as some bacteria require oxygen for their growth, and others are anaerobic (Bertrand, 2019; Mellefont et al., 2003; Rolfe et al., 2012). The relevant text is added in the discussion section of the manuscript (Paragraph 4). Due to the unique ways in which various bacteria behave and function, the responsiveness and functional capabilities fluctuate as the environment changes. When various environmental factors are activated, not all bacteria or pathogens behave the same way. Some act in opposition to the change, while others react positively. So, how likely is it to expand the observed modification for tens of bacterial pathogens? More precisely, the environmental changes may affect the bacterial physiological functions; in this context, do the tested incubation temperature and vitamin supplementation affect the microbes’ pathogenicity or efficiency as well? Response: The effect of higher incubation temperature and vitamin B12 supplementation on tens of bacteria is difficult to predict unless we have specific experimental data for each species. But in this study, we have shown that tested bacterial strains show reduced lag time in the presence of vitamin B12 and higher incubation temperature. This method can potentially be tested for other clinically important bacteria as well. The effect of higher incubation temperature and vitamin B12 supplementation on bacterial pathogenicity and efficiency is difficult to generalize because microbial pathogenicity and efficiency are complex phenomena involving many other factors, such as bacterial virulence factors, host-pathogen interactions, and immune responses. In some cases, higher incubation temperature and/or vitamin B12 can enhance bacterial pathogenicity by increasing its growth rate or virulence factor expression, while in other cases, it may have the opposite or no effect at all (Konkel & Tilly, 2000). We can hypothesize that faster growth will lead to the rapid spread of the pathogen and, therefore, a more serious infection. As the reviewer has stated, reduced lag time may enhance the pathogens’ survival in compromised immunity scenarios with raised body temperature, which will be more pathogenic. The text has been incorporated into the revised manuscript (Paragraph 5 - Discussion). The nutrients are necessary for the bacteria to grow; some are present in significant amounts, while others are scarce. What is the rationale, for authors, to test B12 out of several microbial nutrients? Response: Bacteria require several nutrients to grow, such as carbon, nitrogen, phosphorus, sulfur, etc. Out of all these nutrients, we selected vitamin B12 for testing because it is cheap and easily available (Fang et al., 2017). In our study, we have also used trace elements to see their effect on bacterial growth (Supplementary data). Also, in future studies, the effect of different nutrients on bacterial growth will be interesting to explore, as we have discussed in the manuscript. The text has been incorporated into the revised manuscript (Concluding paragraph - Discussion). As per the study-observations, increased incubation temperature (42°C) reduced the Tlag, may it do the same in real life body-temperature situations? Reduced lag time may enhance the pathogens’ survival in compromised immunity scenarios with raised body temperature. Please discuss this practical aspect as well. Response: An increase in the body temperature is usually called fever which is the body’s response to fight any pathogen or infection. In this study, we have shown that higher incubation temperature can reduce the tlag, which will enhance bacterial growth. However, it is difficult to replicate the identical effect as observed in the human body in the lab. According to some studies, bacteria have evolved to regulate their virulence factors due to changes in temperature when they enter from the external environment to the host. Bacteria recognize the signal provided by the temperature and enhance the production of their virulence factors accordingly within the host (Lam et al., 2014). The growth of bacteria at higher temperatures in the human body can be fatal. We can hypothesize that faster growth will lead to the rapid spread of the pathogen and, therefore, a more serious infection. The relevant text is added in the discussion (paragraph 5) to highlight this aspect. As per the ms title and abstract, the 1st impression of the study is more like diagnostic improvements while the introduction start with AMR does not fit in appropriately. 2nd paragraph or even the 2nd sentence of 2nd paragraph might be something to start the introduction and whole AMR scenario may follow in later phase Response: The text in the manuscript has been updated as per the reviewer's suggestion Almost half of the 2nd paragraph is on ‘Sepsis’, I think it narrates the examples of different fatal diseases, so please say it so. Response: Updated in the revised manuscript. Page-1, 2 - abstract and last paragraph of introduction: Switch the Gram-positive (Escherichia coli and Pseudomonas aeruginosa) and Gram-negative (Staphylococcus aureus and Bacillus subtilis) bacteria in correct order. Table represented them correctly, though. Response: Updated in the revised manuscript. Establish a connecting link of 4th paragraph (start with Mesophilic bacteria…) with previous one. Response: Updated in the revised manuscript. The 1st sentence of the paragraph (starting with ‘The bacterial growth curve…) is sufficient, as the rest is quite basic for the microbiology readers. Response: Updated in the revised manuscript. As per the ms, bacterial growth reached the stationary phase (approximately 4-5 h), it would be interesting to see the doubling time of the tested bacteria. I am sure the authors would have calculated. Response: The doubling time (t_d) for bacteria was calculated based on the steepest rise in the exponential phase of the growth curve. The OD (log) and time interval in the exponential phase were selected at two points, which showed a specific growth rate (µ). The following formula was used to calculate the bacterial doubling time. t_d = 0.693 / µ In the formula above, 0.693 is constant, and “µ” is the specific growth rate, which is calculated by using the following equation: µ= (Ln(OD2)-Ln(OD1)) / (t2-t1) where; Ln is the natural log OD2 is the OD at the endpoint of the exponential phase OD1 is the OD at the starting point of the exponential phase t2 is the time corresponding to OD2 t1 is the time corresponding to OD1 Relevant text has been added to the methodology (Pre-culture and growth procedures), table 1, and results (Bacterial doubling time). Figure 8-9: In English, normal reading/writing is from left to write; please correct it ‘currently from right to left’. Response: Figures are updated in the revised manuscript." } ] } ]	1	https://f1000research.com/articles/12-131

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